A Numerical Classification of the Genus Bacillus

Journal of General Microbiology (1988), 134, 1847-1882. Printed in Great Britain 1847 A Numerical Classification of the Genus Bacillus By F E R G U S G . P R I E S T , ’ * M I C H A E L G O O D F E L L O W * A N D CAROLE TODD2 Department of Brewing and Biological Sciences, Heriot- Watt University, Edinburgh EHl IHX, UK Department of Microbiology, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK ’ (Received 2 November 1987; revised 24 February 1988) Three hundred and sixty-eight strains of aerobic, endospore-forming bacteria which included type and reference cultures of Bacillus and environmental isolates were studied. Overall similarities of these strains for 118 unit characters were determined by the SSM, S, and D p coefficients and clustering achieved using the UPGMA algorithm. Test error was within acceptable limits. Six cluster-groups were defined at 70% SSM, which corresponded to 69% Sp and 48-57% SJ. Groupings obtained with the three coefficients were generally similar but there were some changes in the definition and membership of cluster-groups and clusters, particularly with the SJcoefficient. The Bacillus strains were distributed among 31 major (4 or more strains), 18 minor (2 or 3 strains) and 30 single-member clusters at the 83% SsMlevel. Most of these clusters can be regarded as taxospecies. The heterogeneity of several species, including Bacillus breuis, B. circulans, B. coagulans, B. megateriun, B . sphaericus and B . stearothermophilus, has been indicated and the species status of several taxa of hitherto uncertain validity confirmed. Thus on the basis of the numerical phenetic and appropriate (published) molecular genetic data, it is proposed that the following names be recognized; BacillusJlexus (Batchelor) nom. rev., Bacillus fusiformis (Smith et al.) comb. nov., Bacillus kaustophilus (Prickett) nom. rev., Bacilluspsychrosaccharolyticus (Larkin & Stokes) nom. rev. and Bacillus simplex (Gottheil) nom. rev. Other phenetically well-defined taxospecies included ‘B. aneurinolyticus’, ‘B. apiarius’, ‘B. cascainensis’, ‘B. thiaminolyticus’ and three clusters of environmental isolates related to B .firmus and previously described as ‘B. firmus-B. lentus intermediates’. Future developments in the light of the numerical phenetic data are discussed. INTRODUCTION Bacteria that produce heat-resistant endospores are classified in several genera in the family Bacillaceae. With the exception of the anaerobic, endospore-forming bacteria, the genus Bacillus is the largest and best-known member of this family, which also includes the genera Sporosarcina and Sporolactobacillus (Berkeley & Good fellow, 1981). Since endospore-formation is a universal feature of these bacteria, spore morphology has traditionally been given considerable weight in their classification and identification. The earlier taxonomy of the bacilli was very confused, yielding more than 150 named species, often described on the basis of single physiological or ecological features. In a comparative study of over 1000 strains, Smith et al. (1952) used spore shape, size and location within the sporangium as a means of differentiating groups within the genus and reduced the number of species to 19. These morphological divisions have remained in general use (Wolf & Barker, 1968; Hobbs & Cross, 1983), despite criticism (Gordon, 1981). Revised and supplemented descriptions o f common Bacillus species have also been published, together with information on 0001-4506 0 1988 SGM 1848 F. G. P R I E S T , M . G O O D F E L L O W A N D C . T O D D some unclassified strains (Gordon et al., 1973). However, it was appreciated that the criteria used for this classification were insufficient (Gordon, 1981) and that many strains could not be accommodated within it. Nevertheless, the descriptions of Gordon and her co-workers form the basis of the classification in Bergey's Manual of Systematic Bacteriology (Claus & Berkeley, 1986) and, together with strain histories, provide an invaluable framework for Bacillus taxonomists. The inadequacy of Bacillus classification has been emphasized by molecular studies. The wide range of base composition in chromosomal DNA indicates genetic diversity (Priest, 1981; Fahmy et al., 1985) and suggests that Bacillus species should be reclassified into several genera. Analysis of rRNA by partial oligonucleotide sequencing has indicated a close relationship between the genera Bacillus, Planococcus, Sporosarcina, Staphylococcus and Thermoactinomyces and revealed Bacillus as a fairly coherent taxon (Stackebrandt & Woese, 1981; Stackebrandt et al., 1987) equivalent in phylogenetic depth to the actinobacteria (Goodfellow & Cross, 1984) or the enteric bacteria-vibrio group (Stackebrandt & Woese, 198l), each of which encompasses several genera. Further, DNA homology studies have shown that many accepted Bacillus species, notably B. circulans (Nakamura & Swezey, 1983a), B. megaterium (Hunger & Claus, 1981), B. sphaericus (Krych et al., 1980) and B. stearothermophilus (Sharp et al., 1980), are markedly heterogeneous and in need of taxonomic revision. Taxometric studies using a wide range of characters have been shown to be effective for the taxonomic revision of large groups of related bacteria (Goodfellow & Dickinson, 1985; MacDonell & Colwell, 1985). The extensive data bases derived from such studies are increasingly being used for the construction of probabilistic identification matrices (Williams et al., 1985) and for designing media formulations that are selective for the isolation of industrially important bacteria (Goodfellow & Williams, 1986). Numerical taxonomy has been used to classify marine bacilli (Bonde, 1975; Boeyk & Aerts, 1976), and culture collection strains representing the genus Bacillus have been analysed for a small number of classical tests (Priest et al., 1981). However, in a more comprehensive study Logan & Berkeley (1981) concluded that further information was needed before Bacillus could be subdivided into 'three or more different genera', and 'spectra of strains', notably the B. JirmuslB. lentus and B. circulans groups, be unscrambled. Although much remains to be done, these and other studies indicated the value of the numerical taxonomic approach in helping to clarify relationships within the genus Bacillus. The primary aim of the current investigation was to establish the detailed intrageneric relationships of bacilli by examining representative strains for many properties using the numerical taxonomic procedure. It was also anticipated that the resultant data base would be used to construct a frequency matrix for the probabilistic identification of bacilli and for the formulation of media selective for specific bacilli of industrial importance. METHODS Strains and culture conditions. Three hundred and sixty-eight test strains were obtained from public and private collections (Table 1); 29 duplicate cultures were also included. Wherever possible type cultures were included. All cultures were stored on nutrient agar (Oxoid CM1) slopes at 4 "C, with the inclusion of 5% (w/v) NaCl for B. pantothenficusand adjusted to pH 6.0 with 1.0 M-HCl for B. coagufans strains. Suspensions of vegetative cells and endospores were stored in glycerol (20%, v/v) at - 20 "C. Each strain was examined for 118 unit characters (Tables 3-5). Thawed glycerol suspensions were used as inocula wherever possible but for sugar fermentation and organic acid utilization tests 2- to 4-d-old cultures grown on nutrient agar and suspended in physiological saline were used. All tests were done at least once on each strain but were repeated where ambiguous or clearly unexpected results were obtained. Inoculated media were usually incubated at 30 "C but thermophilic and psychrophilic strains were incubated at 50 "C and 15 "C, respectively. Morphological, degradation (with the exception of aesculin, allantoin, arbutin, hippurate and urea, which were done in test tubes), antibiotic sensitivity and physiological tests were done in Petri dishes. Replidishes (Sterilin) were used for 'spreading' organisms such as B. afvei and B. mycoides. They were also used for sugar fermentation and organic acid utilization tests. Petri and Replidishes were inoculated with a multipoint inoculator (Denley). Morphology and pigmentation. Colonial morphology was examined on isolated colonies grown on nutrient agar for 2-4 d. Cellular morphology was examined in Gram-stained smears of these cultures, and spores were stained using malachite green (Cowan, 1974). Spore morphology was examined on cultures from soil-extract agar (SxA) (Gordon et af., 1973) in cases where sporulation did not occur on nutrient agar (see Tables 3-5). Taxonomy of bacilli 1849 Degradative tests. The degradation of adenine and tyrosine (0.5%), elastin (0.3%), casein (1 %, w/v, skimmed milk), guanine (0.05%) and testosterone (0.1 %) was determined in nutrient agar after 7 and 14 d (2 and 5 d at 50 "C for thermophiles; 14 and 21 d at 15 "C for psychrophiles); clearing of the areas under and around the growth was scored as positive. Gelatin (0.4%) and starch (1 %) hydrolysis were detected in the same basal medium after 7 d (2 d for thermophiles; 14d for psychrophiles) by flooding plates with acidified HgCl, (Frazier, 1926) and iodine solution (Gordon et al., 1973) respectively. Hydrolysis of DNA (0.2%) and RNA (0.3%) was observed using Bacto DNase Test agar (Difco) and nutrient agar as nutrient bases, respectively. After incubation for 7 d (2 d for thermophiles; 14 d for psychrophiles) plates were flooded with 1 M-HCI and clear zones recorded as positive. Tweens 20 and 80 (1 %, v/v) were incorporated into Sierra's (1 957) medium and plates examined for opacity after 7 d (2 d for thermophiles; 14 d for psychrophiles). The hydrolysis of allantoin and urea was detected using the media and methods of Gordon (1966,1968). Aesculin and arbutin (both 0.1 %) degradation was determined by the methods of Williams et al. (1983) and examined after 7 d (2 d for thermophiles; 14 d for psychrophiles). Pullulan and pustulan hydrolysis was determined by the methods of Morgan et al. (1979) and Martin et al. (1980), respectively. Chitinolytic activity was observed after 14 and 21 d (3 and 5 d for thermophiles) as the appearance of zones of clearing in colloidal chitin agar (Hsu & Lockwood, 1975) and hippurate hydrolysis using the method of Gordon et al. (1973) after incubation for 14 d ( 5 d for thermophiles). Lecithinase activity was determined as opalescence in a medium comprising egg-yolk emulsion (5%, v/v; Oxoid) in nutrient agar incubated for 2 d (1 d for thermophiles; 5 d for psychrophiles). Pectin degradation was detected using the modified method of Williams et al. (1983); hydrolysis zones were detected after 7 d (2 d for thermophiles; 14 d for psychrophiles). Antibiotic resistance. Strains were examined for the ability to grow in nutrient agar supplemented with antibiotics (Sigma) at two concentrations (Table 3). The antibiotics used were benzylpenicillin, chloramphenicol, D-cycloserine, erythromycin, gramicidin, nalidixic acid, polymyxin sulphate, rifampicin, streptomycin sulphate and tetracycline. Growth was recorded after 7 d (3 d for thermophiles; 14 d for psychrophiles) and resistance scored as positive. Acidproduction from sugars and sugar alcohols. This was detected using the media and methods of Gordon et al. (1973). Replidishes were inoculated and examined after 7 d (3 d for thermophiles; 14 d for psychrophiles) for acid production. Organic acid utilization. The ability of strains to use organic acids was determined using the methods of Gordon et al. (1973). Replidishes were examined after 5 d (2 d for thermophiles; 10 d for psychrophiles) for the appropriate colour change. Tolerance tests. Nutrient agar was used as the basal medium. Growth at 5 "C and 17 "C was recorded after 14 and 21 d, growth at 37 "C after 3 d, and growth at 50 "C and 65 "C after 2 d. Growth at pH 4.5,6.0,8.0 and 9.5 was determined in media adjusted to the appropriate pH with HCl or NaOH and recorded after 7 d (3 d at 50 "C). Growth in the presence of NaCl (2, 5 and lo%, w/v) was recorded after 7 d (3 d at 50 "C). Miscellaneous biochemical tests. Anaerobic growth was determined according to Gordon et al. (1973) and gas production from glucose in glucose/peptone water containing Durham tubes. Production of dihydroxyacetone and indole, reduction of nitrate, deamination of phenylalanine, and the Voges-Proskauer test were determined using the standard methods for Bacillus strains (Gordon et al., 1973). Hydrolysis of o-nitrophenyl P-D-galactoside, the methyl red test, the oxidase reaction and presence of phosphatase were examined using the procedures of Cowan (1974). Ability to grow on MacConkey agar (Oxoid) was recorded after 5 d (2 d at 50 "C; 10 d at 15 "C). Coding of data. Nearly all the characters existed in one of two mutually exclusive states and were scored plus (1) or minus (0). Qualitative multistate characters were each scored plus (1) for the character state shown and minus (0) for the alternatives. Quantitative multistate characters such as tolerance to NaCl were coded using the additive method of Sneath & Sokal(l973). Characters which did not show any separation value or were poorly reproducible were deleted from the data matrix. The final n x t table, therefore, contained data for 368 bacteria ( t ) and 118 unit characters ( n ; Tables 3-5). Computer analysis. Data were analysed using the Clustan 1C package (Wishart, 1978) on a Burroughs B6370 Jaccard ( S , ) and pattern difference (Dp)coefficients (Sneath & Sokal, computer using the simple matching (SsM), 1973). Clustering was achieved using the unweighted pair group method with arithmetic averages (UPGMA) algorithm (Sneath & Sokal, 1973). Test reproducibility. Twenty-nine strains were tested in duplicate and an estimate of test variance calculated (formula 15; Sneath & Johnson, 1972) which was used to calculate the average probability ( p )of an erroneous test result (formula 4; Sneath & Johnson, 1972). RESULTS Test error Experimental test error was calculated from the data collected on the 29 duplicate strains. The average probability ( p ) of an erroneous test result was 3.90% calculated from the pooled 1850 F . G . PRIEST, M . GOODFELLOW A N D C . T O D D variance (S2= 0.0374) of all the unit characters for the duplicate cultures. The 29 pairs of duplicate strains showed a mean observed similarity of 93.86% SSM. Some groups of tests were highly reliable, particularly cellular morphology, degradation, acid from sugars, growth, and miscellaneoustests, all of which displayed a variance < 0.03. The most irreproducible tests were those involving organic acid utilization, in which the indicator change was difficult to read. Nevertheless, these results were included in the study because the variance (0.1 13) was only slightly greater than the generally accepted level of <0.1 (Sneath & Johnson, 1972). Gross taxonomic structure The data were analysed using the SSM, SJand Dp coefficients with the UPGMA algorithm. level (Fig. The S,, dendrogram was divided into six aggregate clusters at the 70% similarity (S-) 1 ; Table l), which corresponded to 69% Sp.The composition of the cluster-groups was slightly different in the S,, and Dp phenograms (Table 2) but the major and minor clusters were little affected. In the Sj/UPGMA analysis, five cluster-groupswere apparent but to delineate them a staggered line from 48 to 57% similarity was required. Given this relaxation of the generally accepted interpretation of dendograms, the composition of the cluster-groups showed good and Dpanalyses. The major variation was observed in congruence with those obtained in the SsM the distribution of the clusters of obligate aerobic strains within cluster-groups D and E. The SSM/UPGMA analysis most closely resembled classifications obtained in earlier studies of the genus (Logan & Berkeley, 1981; Priest et al., 1981) and it is presented here in detail. The composition of cluster-group A was largely unaffected by the coefficients used (Table 2). The bacteria encompassed by this taxon all produced acid from a wide range of carbohydrates, were facultative anaerobes with ellipsoidal spores that distended the sporangium, and hydrolysed a variety of polysaccharidesincluding starch and pullulan. Similarly, cluster-groupB encompassed bacteria that were aerobic or facultatively anaerobic and produced acid from a variety of sugars. They also formed oval spores which, with the exception of those of B. laterosporus and ‘B.psychrosaccharolyticus’, did not distend the sporangium. Strains assigned to cluster-group B hydrolysed casein and, with the exception of B. pumilus, starch. Cluster-group C was based on B.Jirmus, B. pantothenticus, marine strains and perhaps B. lentus, although in the S,,/UPGMA and Dp/UPGMA analyses this species was given clustergroup status. These bacteria were generally weak in their ability to form acid from sugars and grew poorly, if at all, under anaerobic conditions. They produced oval spores and were NaCl tolerant. Considerable affinity was found between cluster-groups C and D, which included ‘B. aneurinolyticus’ and B. sphaericus, but strains in the latter group were distinguished by lack of acid production from sugars ( B . psychrophilus was a very weak acid-former). These bacteria displayed a variety of spore morphologies. Cluster-groupE contained B. lentus and B. macquariensis but the weight of evidence (Table 2) suggests that these taxa might more appropriately be placed in cluster-groups D and A, respectively. Cluster-group F encompassed the two thermophilic taxa B. coagulans and B. stearothermophilus.These bacteria displayed heterogeneity of spore morphology and fermented a variety of carbohydrates. The full characteristics of the cluster-groups are given in Table 3. Composition and characteristics of major and minor clusters The strains were recovered in 31 major (four or more strains), 18 minor (two or three strains) and 30 single-member clusters at the 83% SsMlevel (Fig. 1). These clusters have been assigned names according to the distribution of type and reference strains. The characteristics of the major and minor clusters are given in Tables 4 and 5 , respectively. Within cluster-group A, cluster 1 contained 13 strains received as B. alvei. They formed a homogeneous phenon at 87% SsMand displayed typical motile micro-colonies (see Parry et al., 1983) and swollen sporangia containing oval, terminal spores. Cluster 3 comprised four strains of ‘B. thiaminolyticus’ that were morphologically similar to B. alvei but distinguishable by nonmotile micro-colonies and positive and negative reactions in the nitrate reduction and Voges- Taxonomy of bacilli 1851 Proskauer tests, respectively. Of the six strains assigned to cluster 4, four were originally labelled as B. circulans, one as B . alvei and the other as ‘B. sphaericus var. rotans’. These bacteria possessed motile micro-colonies typical of B . alvei but differed from the latter in failing to produce dihydroxyacetone and in being negative for nitrate reduction and the Voges-Proskauer reaction. Cluster 7 strains resemble B. pabuli (Nakamura, 1984a) and were named accordingly. The ten strains of B. macerans recovered in cluster 5 displayed the typical reactions of this species, in particular the production of gas from sugars, a property shared with B . polymyxa (cluster 8). However, the strains in the latter taxon fermented a less extensive range of sugars, hydrolysed casein and produced dihydroxyacetone. Related to B . polymyxa at 77.5% SsMwere five strains of B. circulans including the type strain (cluster 6). These bacteria did not produce gas from glucose. The heterogeneity of strains received as B. circulans was evident given their assignment to two major, three minor and four single-member clusters. The sole strain of ‘B. Jilicolonicus’ was recovered as a single member cluster in cluster-group A. Cluster-group B was numerically the largest in the study. Strains of B. cereus, B . mycoides and B. thuringiensis, assigned to cluster 11 within this cluster-group, were divided at the 89 to 92% SSM level into nine subclusters which approximated to the species and varieties represented. Subclusters 11A and 11B were heterogeneous and contained strains labelled B. thuringiensisand B. cereus. Subcluster 11C contained seven strains of B. cereus, some of which had been associated with food poisoning. Subcluster 11D also contained B . cereus strains, some of which were originally designated ‘B. cereus var. JEuorescens’ and ‘B. cereus var. albolactis’. B. thuringiensis strains were recovered in subcluster 11E and two B. cereus strains of serotypes 6 and 8 comprised 11F. Twelve strains of B . thuringiensis, including the type strain, formed subcluster 11G. Subcluster 11H was largely composed of B . cereus strains, and the final subcluster 111, contained four strains of B . mycoides. Bacillus cereus NCIB 8705 and a marine isolate representative of cluster IIC ( B . cereus) of Bonde (1975) formed single-member subclusters. Although the subclusters largely conformed to the designations B . cereus, B. mycoides and B. thuringiensis, consistent features that distinguished them, with the exception of the rhizoidal colony forms of B. mycoides, were not evident. Loosely associated with the B. cereus cluster were two marine isolates from group IIC of Bonde (1975), and two strains of ‘B. psychrosaccharolyticus’. Eight strains of B. laterosporus, including the type strain, were recovered in cluster 13. Their close affinity to B. cereus (76% SSM) may initially seem surprising, but if the unusual spore morphology is ignored, the taxa have many features in common. Both species contained facultative anaerobes that were largely methyl red positive and reduced nitrate; both degraded a variety of macromolecules and produced acid from a similar range of sugars. A single strain of ‘B. pycnoticus’ recovered within the B . laterosporus cluster at 86% S,, did not have the characteristic lateral spore position of B . laterosporus. The ‘B. subtilis group’, including B. megaterium, joined B . cereus at 72% SsM.Cluster 14 contained nine strains of which eight were authentic cultures of B . amyloliquefaciens or were strains labelled B . subtilis from amylase fermentations; one strain was a marine isolate. Although cluster 14 was distinct from B. subtilis (cluster 1 9 , consistent differential features were not evident. Fermentation of meso-inositol, lactose and xylose, and hydrolysis of DNA and Tween 80 provide some measure of distinction. Cluster 15 encompassed strains received as B. subtilis, including the type strain. Two strains received as ‘B. vulgatus’ and two designated as ‘B. aterrimus’ were recovered in this cluster. Two marine isolates, representatives of group IVA ( B . subtilis) and group IIB ( B . rnegaterium)of Bonde (1975), were assigned to this cluster as was a second ‘B.pycnoticus’ strain. Bacteria in cluster 15 conformed to the typical description of B . subtilis since they were obligate aerobes that were positive in the nitrate reduction and Voges-Proskauer tests and produced acid from a variety of sugars. Strains of B . pumilus formed a homogeneous cluster related to B. subtilis at 79 % SSM. Most of these organisms were received as B. pumilis, including two marine isolates, representing Bonde’s (1975) group IVB ( B .pumilus). However, representatives of his group IIB ( B . megaterium) and c Percentage similarity 80 90 70 '0 Cluster/ Cluster- No. of 100 subcluster group strains 00 cn Identity N IIA 60 Percentage similarity 70 80 90 100 Cluster No. of strains 1 IB L I IC I crl Identity Q IID 9 cd TIm B. uhei 1 13* S98 I B . circuluns 2 2 ' B. upiurius 3 4 'B. thiuminolyticus' 4 6 B . circulans 5 I o* B. maceruns 6 5* 7 2 B. circuluns sensu stricto B . puhuli SI 10 1 ' B. .filicolonicus ' v, 8 60 70 80 90 100 4 rl- 3 B . circuluns 10 3 B. circuluns S106 S107 S90 f 1 } IC 1 IF 2 1 IG 12 IIH 4 B . cereus1 B . thuringiensis r II* 9 I IE B . circuluns 1 I I I 60 70 80 90 I 100 111 5 S363 S65 1 1 S348 S366 I 12 2 B . psychrosacchurolyt icus 13 9* B . laierosporus Bacillus spp. 1 K 1853 Taxonomy of bacilli T I v I 1 I I 111 J -G J I I \o 0 Irl I ‘L - I I I Percentage similarity 60 70 80 90 No. of strains Identity 36 5 'B. aneurinolyticus' 31 11* B. brevis 38 6 B . azotoformans 39 4 100 Cluster 4 rc= S430 S299 1 I B . badiusl 'B.freudenreichii' B. globisporus B. sphaericus var. rotans S432 1 B. insolitus 40 5' B. psychrophilus 41 2 B. brevis 42 4 B. fusiformis 43 s349 s353 S295 S365 60 70 80 90 100 Clustergroup 9* B. sphaericus :> Bacillus spp. I I 60 Percentage similarity 70 80 90 1 I I 100 Cluster 1 Clustergroup No. of strains 44 1 B. lentus 45 2* 8. macquariensis B. coagulans sensu strict0 47 B. coagulans 48 B . stearothermophilus sensu stricro 49 J I I I 70 80 90 'B. cirroJagellosus' 46 s453 60 B . lentus S163 S I 13 'B. repens' Bacillus sp. 8* Identity I 100 Fig. 1 (continued). Simplified dendrogram showing the relationships between clusters recovered in the Ss,/UPGMA analysis. B. kaustophilus B. stearothermophilus Taxonomy of bacilli 1855 Table 1. Designation and source of strains assigned to cluster-groups (defined at 70% SsM, UPGMA) and cluster.9 (defined at 83% SSM, UPGMA) Binomials in inverted commas are not on the Approved Lists of Bacterial Names (Skerman et al., 1980) and have not been validly published since 1 January 1980. Type strains are marked with an asterisk (*). Cluster-group A s3-*s5 S648 s9 Sll-Sl6 Strains assigned’to cluster 1 (Bacillus alvei) B. alvei, NCIB 8212, NCIB 8199, NCIB 9371 B. alvei, NCTC 3324, NCTC 3349, NCTC 7583 B. alvei, J. R. Norris, Cadbury Schweppes Ltd, Reading, UK, BO 113 B. alvei, WR 2772, WR 2773, WR 3186 (E. Schreiner, SKG), WR 3187 (E. Schreiner, 4N), WR 3250 (E. Schreiner, A), WR 3251 (E. Schreiner, B) S414, S415 Strains assigned to cluster 2 (‘Bacillus apiarius’) ‘B. apiarius’, R. E. Gordon, Rutgers University, New Jersey, USA, NRS 1438 (H. Katznelson, BX3), NRS 1439 (H. Katznelson, BX5); bee larvae S327-S330 Strains assigned to cluster 3 (‘Bacillus thiaminolyticus’) ‘B. thiaminolyticus’, J. R. Norris, BO 286-BO 289 (J. Yamaguchi, Ml-M4) s10 S93, S94, S96, S103 S310 Strains assigned to cluster 4 (Bacillus circulans) B. alvei, J. R, Norris, BO 024 B. circulans, J. R. Norris, BO 030, BO 061, BO 197 (T. Gibson, 514), BO 319 ‘B. sphaericus var. rotans’, H. J. Somerville, Shell, UK, T216 S185 S186-Sl89, *S191 S192 S 193-S 195 Strains assigned to cluster 5 (Bacillus macerans) B. macerans, T. R. G. Gray, University of Essex, UK, NCIB 7588 B. macerans, NCIB 8160, NCIB 8210, NCIB 8930, NCIB 10443, NCIB 9368 B. macerans, H. J. Somerville, T521 B. macerans, WR 1013, WR 1014, WR 2614 (colonial variant of WR 1014) S89, S91 *S92, S95 S109 s101, s102 S247, S249, S280 *S251 S254S256, S2584261 S88 S97, S104 S99, S100, S105 S90 S98 S106 S107 s110 Strains assigned to cluster 6 (Bacillus circulans sensu stricto) B. circulans, P. A. Hartman, Iowa State University, Ames, USA, NRRL B-381, NRRL B-380 B. circuluns, J. R. Norris, BO 004 (NCTC 2610), BO 196 (NCTC 5849) B. circulans, NCIB 9555 Strains assigned to cluster 7 (Bacillus pabuli) B. circulans, J. R. Norris, BO 317 (T. Gibson, 261), BO 318 (T. Gibson, 287) Strains assigned to cluster 8 (Bacillus polymyxa) B. polymyxa, P. A. Hartman, IA 32 (J. C. Ayres, B-57-3B), ATCC 8523, IA 56 B. polymyxa, NCIB 8158 B. polymyxa, WR 1417, WR 1756, WR 1966, WR 2161 (banana skin), WR 2179, WR 2186 (garden soil), WR 2494 (potato) Strains assigned to cluster 9 (Bacillus circulans) B. circulans, P. A. Hartman, NRRL B-378 B. circulans, J. R. Norris, BO 266 (NCTC 7578), BO 320 (T. Gibson, 48) Strains assigned to cluster 10 (Bacillus circulans) B. circulans, J. R. Norris, BO 305 (T. Gibson, 137), BO 306 (T. Gibson, 255), BO 321 (T. Gibson, 38) Single-member clusters B. circulans, P. A. Hartman, NRRL B-395 B. circulans, J. R. Norris, BO 267; NCTC 9432 B. circulans, J. R. Norris, BO 322; T. Gibson, 92 B. circulans, J. R. Norris, BO 323; T. Gibson, 279 ‘B.filicolonicus’,J. R. Norris, BO 322; T. Gibson, 92 1856 F . G . PRIEST, M . G O O D F E L L O W AND C . T O D D Table 1 (continued) Cluster-group B 1 Strains assigned to cluster 11 (Bacillus cereus/Bacillus thuringiensis) Subcluster 1 1A (‘Bacillus thuringiensis var. Jinitimus’) ‘B.Jinirimus’, J. R. Norris, BO 308; T. Gibson, 1316 s120 ‘ B . thuringiensis var. alesti’, P. A. Hartman, BT-3 (serotype 3a) S332 ‘ B . thuringiensis var. Jinitimus’, P. A. Hartman, BT-2 s121 Subcluster 1 1B (Bacillus cereus) B. cereus, T. R. G. Gray, B20; NCTC 6474 S66 ‘ B . sotto’, J. R. Norris, BO 021 S297 Subcluster 11C (Bacillus cereus) B . cereus, B. Austin, Heriot-Watt University, Edinburgh, UK, SA 15; swan faeces S58 B. cereus, P. A. Hartman, IA 36 (Y. L. Quinn; ATCC 11778), NRRL B-344 S62, S63 B. cereus, T. R. G. Gray, B21; NCTC 7464 S67 B. cereus, R. J. Gilbert, Central Public Health Laboratories, Colindale, London, UK, S68 3502/73; fried rice (serotype 5 ) B. cereus, R. J. Gilbert, 4433/73; meat loaf S72 ‘B. cereus var. terminalis’, WR 7100 S86 Subcluster 11D (Bacillus cereus) B. cereus, J. R. Norris, BO 002; DSM 31 *S60 B. cereus, P. A. Hartman, IA 27, NRS 996 S61, S76 B. cereus, R. J. Gilbert, 4746/77; fried rice (serotype 1) s74 B. cereus, H. J. Somerville, TI 87; strain T s75 ‘B. cereus var. albolactis’, NCIB 5097 s77 ‘B. cereus var. Juorescens’, H. J. Somerville, TI 53; NCIB 2600 s79 S8 1 ‘B. cereus var. mycoides’, NCTC 2603 S470 ‘B. thuringiensis var. dendrolinus’, J. R. Norris, 10; H. Dulmage, 37 (serotype 4ab) Subcluster 1 1E (Bacillus thuringiensis) S64 B. cereus, NCIB 6349 s334 B. thuringiensis, WR 4138 S476, S477 ‘ B .thuringiensis var. aizawai’, J. R. Norris, 16 (H. Dulmage, 227), 17 (H. Dulmage, 137) (serotype 7) S471 ‘B. thuringiensis var. benyae’, J. R. Norris, 11 ; H. Dulmage, 136 (serotype 4ac) S472 ‘B. thuringiensis var. galleriae’, J. R. Norris, 12; H. Dulmage, 273 (serotype 5ab) S466, S467 ‘ B . thuringiensis var. kurstaki’, J. R. Norris, 6 (H. Dulmage, 187), 7 (H. Dulmage, 89) (serotype 3ab) S468 ‘B. thuringiensis var. sotto’, J. R. Norris, 8; H. Dulmage 5 (serotype 4ab) S478 ‘B. thuringiensis var. tolworthi’, J. R. Norris, 18; H. Dulmage, 125 (serotype 9) Subcluster 11F (Bacillus cereus) S70, S71 B. cereus, R. J. Gilbert, 4370/75 (serotype 6; barbecued chicken), 4431/73 (serotype 8; Indonesian rice dish) Subcluster 11G (Bacillus thuringiensis) s335 B. thuringiensis, W R 575 1 *S336 B. thuringiensis, T. R. G . Gray, B76; NCIB 9134 s337 B. thuringiensis, H. J. Somerville, T 537 (serotype 1) S464, S465 ‘B. thuringiensis var. alesti’, J. R. Norris, 4 (H. Dulmage, lo), 5 (H. Dulmage, 104) S331 ‘B. thuringiensis var. berliner’, P. A. Hartman, BT-1; Bonnefoi, BT-1 S469 ‘B. thuringiensis var. dendrolinus’, J. R. Norris, 9; H. Dulmage, 106 (serotype 4b) s474 ‘B. thuringiensis var. entomocidus’, J. R. Norris, 14; H. Dulmage, 9 (serotype 6) S463 ‘B. thuringiensis var. jnitimus’, J. R. Norris, 3; H. Dulmage, 3 s473 ‘B. thuringiensis var. galleriae’, J. R. Norris, 13; H. Dulmage, 29 S461, S462 ‘B.thuringiensis var. thuringiensis’, J. R. Norris, 1 (H. Dulmage, 39), 2 (H. Dulmage, 17) (serotype 7) Subcluster 11H (Bacillus cereus/Bacillus thuringiensis) S69, S73 B. cereus, R. J. Gilbert, 3605/73 (serotype 3; boiled rice), 4810/72 (serotype 1; vomit) S78 ‘B. cereus subsp. albolactis’, NCIB 8079 s475 B. thuringiensis, J. R. Norris, 15 Subcluster 111 (‘Bacillus cereus var. mycoides’) S80 ‘B. cereus var. mycoides’, NCIB 926 S83-S 85 ‘B. cereus var. mycoidex’, WR 1541, WR 2500, WR 2528 S482 ‘B. cereus var. mycoides’, H. J. Somerville, T193 Taxonomy of’bacilli 1857 Table 1 (continued) S438, S439 S145-S148, S150 S151 *S152, S154 S289 S65 S348 S363 S366 Strains assigned to cluster 12 (Bacillus psychrosaccharolyticus) ‘B. psychrosaccharolyticus’, J. L. Stokes, T25B, T27B; soil Strains assigned to cluster 13 (Bacillus laterosporus) B. laterosporus, J. R. Norris, BO 026, BO 115 (T. Gibson, 308), BO 116 (T. Gibson, 1066), BO 262, BO 309 (T. Gibson, 1080) B. laterosporus, W R 2 197 B. laterosporus, NCIB 8215, NCIB 11046 ‘B. pycnoticus’, J. R. Norris, BO 311 (T. Gibson, 51) Single-member clusters B. cereus, NCIB 8705 Bacillus sp., G. J. Bonde, 1 (cluster IIC; B. cereus) Bacillus sp., G. J. Bonde, 354 (cluster IIC; B. cereus) Bacillus sp., G . J. Bonde, 372 (cluster IIC; B . cereus) Cluster-group B2 s 21 S24 S23 S234 S312 S352 Strains assigned to cluster 14 (Bacillus amyloliquefaciens) B. amyloliquefaciens, F. E. Young, University of Rochester, NY, USA, F (L. L. Campbell, F), H (L. L. Campbell, H), K (L. L. Campbell, K) B. amyloliquefaciens, NCIB 10785 B. amyloliquefaciens, J. R. Norris, 30; ATCC 23843 B. subtilis, J. R. Norris, 29, T. Kaneko, N B. subtilis, ABM Chemical Ltd, Stockport, UK, B20; amylase fermentation B. subtilis, P. A. Hartman, JR 8; J. Robyt, amylase preparation Bacillus sp., G. J. Bonde, 50 (cluster 3A) S30 s22 S290 S31, *S316, S317 S339, S340 S311, S315 S321, S322 S32 S359, S362 Strains assigned to cluster 15 (Bacillus subtilis) ‘B. aterrimus’, J. R. Norris, BO 096; T. Gibson, 525 B. megaterium, J. R. Norris, 25; T. Kaneko, 203 ‘B.pycnoticus’, J. R. Norris, BO 322 B. subtilis, NCIB 2591, NCIB 3610, NCIB 8054 ‘B. uulgatus’, NCIB 8063, NCIB 8802 B. subtilis, P. A. Hartman, IA 5, W 23 B. subtilis, J. R. Norris, 6 (T. Gibson, 1115), 7 (T. Gibson, 1137) ‘B. aterrimus’, WR 2192; NCIB 8055 Bacillus sp., G. J. Bonde, 177 (cluster 2B; B . megaterium), 315 (cluster 4A; B . subtilis) S223, S224 S225 Strains assigned to cluster 16 (‘Bacillus subtilis var. niger’) ‘B. niger’, J. R. Norris, BO 099 (T. Gibson, 1208), BO 098 (T. Gibson, 1007) ‘B. subtilis var. niger’, E. Hemphill, Syracuse University, NY, USA, 1000 S87 S351 Strains assigned to cluster 17 (Bacillus circulans) B. circulans, P. A. Hartman, NRRL B-377 Bacillus sp., G. J. Bonde, 47 (cluster IIIA) S319 S323 Strains assigned to cluster 18 (Bacillus subtilis) B. subtilis, J. R. Norris, 2; T. Gibson, 636 B. subtilis, WR 2745 *S 18-S20 S208, S273, S274 S209, S275, S276 S278, *S279 S281, S282 S283, S284 S285-S288 S354, S355, S358, S361 S167 Strains assigned to cluster 19 (Bacillus pumilus) B. megaterium, B. Austin, SA 217, SA 232, SA 218; swan faeces B. megaterium, B. Austin, CGA 59, G-2-P, CGA 28; Canada goose faeces B . pumilus, T. R. G. Gray, B46 (NCTC 7576), B47 (NCTC 8241) B. pumilus, P. A. Hartman, NRRL B-3275, NRS 630 B. pumilus, NCTC 2595, NCTC 2596 B.pumilus, J. R. Norris, 10 (T. Gibson, 1130), 12 (T. Gibson, lo), 13 (T. Gibson, 47), 14 (T. Gibson, 67) Bacillus sp., G. J. Bonde, 86 (cluster IV, B. pumilus), 88 (cluster IVB; B. pumilus), 174 (cluster IIB; B. megaterium), 293 (cluster 2C, B. cereus) Strains assigned to cluster 20 (Bacillus licheniformis) B . licheniformis, P. A. Hartman, 9945A; C. B. Thorne, 9945A 1858 F . G . PRIEST, M . GOODFELLOW AND C. T O D D Table 1 (continued) S18 1-S 183 S314 S338 B. lichenformis, J. R. Norris, 17 (DSM 13), 18 (T. Gibson, 1174), 20 (T. Gibson, 1174), 22 (T. Gibson, 1160), 23 (T. Gibson, 5), 24 (T. Gibson, 1158) B. lichenformis, NCIB 6816, NCIB 7224, NCIB 8061, NCIB 8537, NCIB 8549, NCIB 8874, NCIB 9668 B. lichenformis, NCTC 962, NCTC 1097, WCTC 2120 B. subtilis, P. A. Hartman, R66-A B. subtilis, NCIB 9536 S356, S357 Strains assigned to cluster 21 (Bacillus sp.) Bacillus sp., G. J. Bonde, 127 (cluster V), 128 (cluster V) S205 S2164218 S215 S219 s220-*s222 S296 s379 Strains assigned to cluster 22 (Bacillus megaterium) B . megaterium, B. Austin, SA 174; swan faeces B. megaterium, J. R. Norris, BO 075 (T. Gibson, 386), BO 076, BO 077 (T. Gibson, 732) B. megaterium, P. A. Hartman, NRRL B-348 B. megaterium, J. R. Norris, BO 078, T. Gibson, 186 B. megaterium, NCIB 7581, NCIB 8291, NCIB 9376 ‘B. silvaticus’, NCIB 8674 ‘B. malabarensis’, NCTC 5637 *S168-S173 S174-S 180 s211, s212 Strains assigned to cluster 23 (Bacillusj e x u s ) B. megaterium, R. E. Gordon, NRS 602 (J. R. Porter; G. Brederman; ‘B. agrestis’), NRS 665 (B. S. Henry, ‘B.jexus’, 131) S412 s347 Strains assigned to cluster 24 (BacillusJirmus) B.Jirmus, WR 3389; R. E. Gordon, NRS 1147 Bacillus sp., A. BoeyC, Vrije Universiteit, Brussels, Belgium, VUB 231 (group Bl); North Sea sediment S137 S184 s202 s343 S346 Single-member clusters ‘B. globigii’, P. A. Hartman, IA 30 ‘B. longissimus’, J. R. Norris, BO 339 ‘B. maroccanus’, NCIB 10500 Bacillus sp., A. BoeyC, VUB 72 (group A2); North Sea sediment Bacillus sp., A. BoeyC, VUB 211 (group Al); North Sea sediment Cluster-group C S132 Strains assigned to cluster 25 (BacillusJirmus) B.Jirmus, NCIB 8162, NCIB 9366 (NRS 613) B.Jirmus, WR 3317 (R. E. Gordon, NRS 858), WR 3318 (R. E. Gordon, NRS 861), WR 3320 (R. E. Gordon, NRS 860), WR 3384 (R. E. Gordon, NRS 1070), WR 3385 (R. E. Gordon, NRS 1131) B.Jirmus, H. J. Somerville, T 544 S413 S483 Strains assigned to cluster 26 (Bacillus sp.) B. Jirmus, WR 3390 Bacillus sp., NRS 1151 S122, *S123 S126, S127, S129, S130, S131 S342, S344 S497, S492, S487, S494 S482, S495 S484, S4884490 S227, S228 S230 S231, S233-S237 Strains assigned to cluster 27 (Bacillus sp.) Bacillus sp., A. BoeyC, VUB 33 (cluster B4), VUB 73 (cluster B2); North Sea sediment Bacillus sp., R. E. Gordon, NRS 1574, NRS 1565, NRS 1570, NRS 1566; M. Turner, SM 34, SM 23, SM 29, SM24; salt marsh Strains assigned to cluster 28 (Bacillus sp.) Bacillus sp., R. E. Gordon, NRS 1147, NRS 1569 Strains assigned to cluster 29 (Bacillus sp.) Bacillus sp., R. E. Gordon, NRS 1329, NRS 1572, NRS 1575, NRS 1149 (H. W. Renszer, 1124) Strains assigned to cluster 30 (Bacillus pantothenticus) B. pantothenticus, J. R. Norris, BO 183, BO 184 B. pantothenticus, NCIB 8775 B. pantothenticus, WR 3019, WR 3023, WR 3024, WR 3026, WR 3028, WR 3043; soil Taxonomy of bacilli 1859 Table 1 (continued) S51-S55 Strains assigned to cluster 31 (‘Bacilluscarotarum’ sensu Gibson) ‘B.carofarum’,J. R. Norris, BO 079, BO 080, BO 081, BO 272, BO 303; T. Gibson, 148, 242, 511, 21 (NCIB 4821), 122 s210 S213 Strains assigned to cluster 32 (Bacillus simplex) ‘B. simplex’, R. E. Gordon, NRS 335 ‘B. feres’, R. E. Gordon, NRS 986 S498, S499 S485, S486 Strains assigned to cluster 33 (Bacillus sp.) B. megaterium, R. E. Gordon, NRS 608, NRS 828 Bacillus sp., NRS 1369, NRS 1370 S264, S265 S266 Strains assigned to cluster 34 (Bacillus pulvifaciens) B. pulvifaciens, WR 3622 (W. C. Haynes, NRS 1283), WR 3623 (W. C. Haynes, NRS 1285) B. pulvifaciens, H. de Barjac, Institut Pasteur, Paris, strain LES; human origin S417 Strains assigned to cluster 35 (‘Bacillus cascainensis’) ‘B. cascainensis’, ATCC 11968; R. E. Gordon, NRS 1471, NRS 1473, NRS 1474a, NRS 1475, NRS 1474b ‘B. cascainensis’, R. E. Gordon, NRS 1470 S56 S114 S341 S197 S226 S378 s493 S496 Single-member clusters ‘B. carofarum’, J. R. Norris, BO 314 ‘B. epiphytus’, J. R. Norris, BO 293 ‘B. loehnisii, NCTC 4825 ‘B. macroides’, J. R. Norris, BO 204; ATCC 12905 ‘B.paciJicus’, J. R. Norris, BO 291 Sporolactobacillus inulinus, NCIB 9743 Bacillus sp., R. E. Gordon, NRS 1562 Bacillus sp., R. E. Gordon, NRS 1573 S416, S418-S422 Cluster-group D S25-S29 s35-s37 S38, *S39 S40 S42, S44-S47 S423-S428 s33, s34 S133, S380 Strains assigned to cluster 36 (‘Bacillus aneurinolyticus’) ‘B.aneurinolyticus’,J. R. Norris, BO 205 (ATCC 12866), BO 206 (NRS 1448), BO 207 (NRS 1450), BO 208 (NRS 1450), BO 209 (NRS 1451) Strains assigned to cluster 37 (Bacillus brevis) B. brevis, J. R. Norris, BO 118 (T. Gibson, 539), BO 117 (T. Gibson, 442), BO 270 (NCTC 7096) B. brevis, NCIB 8803, NCIB 9372 B. brevis, T. R. G. Gray, B54; NCTC 7577 B. brevis, WR 2904, WR 2922, WR 2932, WR 2934, WR 3005; soil Strains assigned to cluster 38 (Bacillus azotoformans) B. azotoformans, F . Pichinoty, UER Scientifique de Huming, Marseilles, France, 1,2, 9, 32, 34, 36; garden soil Strains assigned to cluster 39 (Bacillus badiusy Bacillus freudenreichii’) B. badius, J. R. Norris, BO 180 (NCTC 10333), BO 201 (M. D. Appleman, NRS 1407) ‘B.freudenreichii’, J. R. Norris, BO 200 (ATCC 7053), BO 199 (T. Gibson, 68) s441 Strains assigned to cluster 40 (Bacillus psychrophilus) B. psychrophilus, J. L. Stokes, Washington State University, USA, W16A (soil), W3 (river water), W5 (soil), W70A Bacillus sp., J. L. Stokes, T75 S48, S49 Strains assigned to cluster 41 (Bacillus brevis) B. brevis, WR 3006, WR 3010 *S433-S436 S 1344136 S301 Strains assigned to cluster 42 (Bacillus fusiformis) ‘B.fusiformis’, J. R. Norris, BO 297 (T. Gibson, 1014), WR 2009, WR 2520 B. sphaericus, T. R. G. Gray, B22; NCTC 7582 1860 F . G . PRIEST, M. GOODFELLOW A N D C . T O D D Table 1 (continued) S298, *S300, S350 S302 S3034307 Strains assigned to cluster 43 (Bacillus sphaericus) B. sphaericus, NCIB 8216, NCIB 9370, G. J. Bonde, 13 B. sphaericus, P. A. Hartman, NRS 348 B. sphaericus, WR 1652, WR 2105, WR 2205, WR 2518, WR 2594 S430 S432 S295 S299 s349 s353 S365 Single-member clusters B. globisporus, T. L. Stokes, W8 B. insolitus, T. L. Stokes, W16B ‘B. repens’, J. R. Norris, BO 301 ‘B. sphaericus var. rotans’, NCIB 8867 Bacillus sp., G. J. Bonde, 6 (cluster IIA) Bacillus sp., G. J. Bonde, 52 (cluster I) Bacillus sp., G . J. Bonde, 453 (cluster IIAT) S156 S164 *S155 S158, S159 S160 S165, S166 Cluster-group E Strains assigned to cluster 44 (Bacillus lentus) B.Jirmus, R. E. Gordon, NRS 769 B.firmus, WR 3321, R . E. Gordon, NRS 769 B. lentus, NCIB 8773; NRS 670; T. Gibson, 165 B. lentus, R. E. Gordon, NRS 883 (T. Gibson, 165), NRS 1262 (T. Gibson, 258) B. lentus, J. R. Norris, BO 179; T. Gibson, 238 B. lentus, WR 3322 (R. E. Gordon, NRS 749), WR 3323 *S199, S201 Strains assigned to cluster 45 (Bacillus macquariensis) B. macquariensis, J. R. Norris, BO 188 (NCTC 10419), BO 190 (NCTC 10421) Single-member cluster S163 B. lentus, WR 2789 Cluster-group F S443, S447, S448 s445, s449 Strains assigned to cluster 46 (Bacillus coagulans sensu stricto) B. coagulans, J. Wolf, University of Leeds, UK, C77, C12, C88 B. coagulans, WR 2972, WR 2822; soil S444 S446 S450 Strains assigned to cluster 47 (Bacillus coagulans) B. coagulans, J. Wolf, C32 B. coagulans, unknown origin B. coagulans, WR 2974; mud S454S456 s459 Strains assigned to cluster 48 (Bacillus stearothermophilus sensu stricto) B. stearothermophilus, J. Wolf, T128, T168, T210 B. stearothermophilus, WR 4592 S451, S452, S457 S458, S460 Strains assigned to cluster 49 (Bacillus kaustophilus) B. stearothermophilus, T1, T39, T349 B. stearothermophilus, WR 4591, WR 4288 S113 s453 Single-member clusters ‘B. cirroBagellosus’, J. R. Norris, BO 290 B. stearothermophilus, J. Wolf, T76 IIC ( B . cereus)were also included in this cluster. B . pumilus strains are readily distinguished from others in the ‘subtilis group’ by being unable to hydrolyse starch or reduce nitrate. Several minor clusters contained organisms that shared a high overall similarity with both the B. subtilis and B. pumilus strains. Three strains of ‘B. subtilis var. niger’ formed a homogeneous cluster in both the S,, and Dp analyses. These organisms were not pigmented on nutrient agar and showed no consistent single features that allowed them to be distinguished from typical strains of B. subtilis. Similarly, a single strain of ‘B.globigii’, often considered to be closely related to either B. subtilis or B . licheniformis, was recovered as a single-member cluster in this area. Cluster 18 contained two strains received as B . subtilis; these organisms were unusual in being Taxonomy of bacilli 1861 unable to produce acid from xylose, salicin and mannose. A single marine isolate, a representative of cluster A2 of Boeyk & Aerts (1976), was recovered between the B. pumilus and B. Iicheniformis clusters. Cluster A2 strains were described as ‘B . pumilusfB. licheniformis intermediates’ in the original publication. Sixteen strains of B. licheniformis, which formed a tight group at 91 % SSM, fused with three additional strains to form cluster 20. These strains on the periphery of cluster 20 were B. subtilis NCIB 9536, originally deposited as ‘ B . tinakiensis’, B. licheniformis NCIB 9668 and a strain received as B. subtilis R66-A. Cluster 20 conformed to the standard description of B . licheniformis. Eleven strains of B. megaterium formed a fairly diffuse taxon (cluster 22) that showed a relatively close affinity with the ‘B. subtilis group’. This cluster included the type strain of B . megaterium, and strains labelled ‘B. malabarensis’ and ‘ B . silvaticus’. The cluster 22 strains formed large cells and conformed to the current description of B . megaterium sensu stricto, i.e. they were strictly aerobic, degraded a variety of polysaccharides, were predominantly urease positive and mainly Voges-Proskauer negative. Several minor clusters were associated with the B. megaterium taxon. Cluster 21 contained two marine isolates representing Bonde’s (1975) group V ( B . licheniformis); ‘B. longissimus’ S184 and ‘B. maroccanus’ S202 were recovered as single-member clusters and may represent new centres of variation. Two clusters which fused at 83% SSMwere peripherally associated with the B . megaterium cluster. Cluster 23 contained strains originally labelled ‘B.agrestis’ and ‘B.Jlexus’.Strains in these taxa have been considered to belong to the species B. megaterium (Gordon et al., 1973). They can be distinguished from B . megaterium sensu stricto as they do not hydrolyse aesculin or form acid from arabinose or xylose. .A strain of B . firmus and a marine isolate from group Bl of Boeyk & Aerts (1976), a cluster thought to be related to B.Jirmus, comprised cluster 24. Cluster-group C contained B. firmus,B . pantothenticus and a number of unnamed or poorly described strains. Eight strains of B.firmus were assigned to a tight taxon (cluster 25) which had the recognized characteristics of this species. These bacteria formed oval, central spores that did not distend the sporangium, were obligately aerobic, produced acid from a restricted range of sugars, and reduced nitrate. ‘B. epiphytus’ S114 was recovered on the periphery of the B.Jirmus cluster in the SsMand Dp analyses but seemed sufficiently dissimilar not to be included. Clusters 26 to 29 contained organisms described by Gordon et al. (1977) as ‘ B . firmus-B. lentus intermediates’. It is presently difficult to identify features that will distinguish these clusters, although acid production from sugars might be useful. Single-member clusters representing saltmarsh isolates of the so-called ‘B.firmus-B. lentus spectrum’ were also recovered in this area of the dendrogram, as was ‘ B . paczjicus’ S226. Cluster 27 included two marine isolates that were assigned to clusters B2 and B4, both equated with B.firmus, by Boeyk & Aerts (1976). Nine strains of B. pantothenticus comprised cluster 30. These NaC1-tolerant bacteria had a variable spore morphology but oval spores predominated. They grew anaerobically and produced acid from a restricted range of sugars. Strains labelled ‘Bacillus loehnisii’ are generally considered to belong to the species B . sphaericus but the single strain bearing this name in the present study was recovered as a single-member cluster near the B. pantothenticus taxon. Five strains received as ‘B.carotarum’ constituted cluster 3 1, with the sixth strain on the periphery of this cluster. All six strains contained oval central spores with some swelling of the sporangium and produced acid from a limited range of sugars; some were urease positive. ‘B.simplex’ and ‘B. teres’ are often considered to be closely related to B . megaterium. Strains bearing these names were assigned to cluster 32; they were distinguished from B . megaterium by reducing nitrate and failing to hydrolyse aesculin, pullulan or urea. A single isolate of ‘B. macroides’ was recovered adjacent to cluster 33; the latter contained two strains received as B . megaterium and two ‘B. Jirmus-B. lent us intermed iates’. Cluster 34 encompassed three strains of B . pulvifaciens which showed 77 % similarity (SsM) with the sole isolate of Sporolactobacillusinulinus examined. The B. pulvifaciens strains produced oval, central spores that distended the sporangium, and produced acid from a restricted range of sugars. Table 2. A comparison of the composition of cluster-groupsfrom several taxometric analyses of the genus Bacillus For clarity, only principal taxa in cluster-groups are given. For full composition of cluster-groups from the SSM/UPGMA analysis, see Table 1. The cluster-group designations used by Priest et al. (1981) and Logan & Berkeley (1981) are shown in parentheses. NI, Not included. Priest et al. (1981) DP sSM Cluster-group A B. alvei B. circulans ‘B. apiarius’ ‘B. thiaminolyticus’ B. macerans B. polymyxa B. alvei B. circulans ‘B. apiarius’ ‘B. thiaminolyticus’ B. macerans B. alvei B. circulans ‘B. apiarius’ ‘B. thiaminolyticus’ B. macerans B. alvei (6) B. circulans (6) Logan & Berkeley (1981) B. circulans (V) NI cr! B. macerans (6) B. polymyxa (6) B. macquariensis B. macerans (V) B. polymyxa (V) B. macquariensis (V) B. coagulans (6) Cluster-group B (I and 11) B. cereus B. laterosporus B. amyloliquefaciens B. subtilis B. pumilus B. licheniformis B. megaterium B. psychrosaccharolyticus Cluster-group C B. firmus B. pantothenticus ‘B. carotarum’ B. pulvifaciens ‘B. cascainensis’ Q m v1 B. cereus B. laterosporus B. amyloliquefaciens B. subtilis B. pumilus B. licheniformis B. megaterium B. psychrosaccharolyticus ‘B . cascainensis’ B. badius ‘B.freudenreicheii‘ B. pulvifaciens B. polymyxa B. cereus B. laterosporus B. amyloliquefaciens B . subtilis B. pumilus B. licheniformis B . megaterium B. psychrosaccharolyticus B. firmus B. firmus ‘B. carotarum’ ‘B. carotarum’ B. cereus (5A) B. cereus (I) B. amyloliquefaciens (5B) B. subtilis (5B) B. pumilus (5B) B. licheniformis (5B) B. megaterium (5B) B. amyloliquefaciens (IV) B. subtilis (IV) B. pumilus (IV) B. licheniformis (IV) B. megaterium (IV) NI B. pulvifaciens B. polymyxa NI B.jirmus (I) B. pantothenticus (I) NI ‘B. cascainensis’ (I) B. lentus (I) B. psychrophilus (I) B.jrmus (11) ‘B. carotarum’ (11) NI NI B. lentus (11) B. brevis (11) B. badius (11) ‘B. aneurinolyticus’ (11) B. alvei (11) B. laterosporus (11) ‘B. thiaminolyticus’ (11) 4 Cluster-group D ‘B. aneurinolytim’ B. brevis B. azotoformans B. badius B. freudenreichii B. psychrophilus B. sphaericus ‘B. aneurinolyticus’ B. brevis B. azotoformans B. sphaericus B. pantothenticus B. badius ‘B.freudenreichii B . psychrophilus ‘B. aneurinolyticus’ (4) B. brevis (4) NI B . badius (4) ‘B.freudenreichii (4) B . sphaericus (4) N1 ‘B.freudenreichir“ (111) B . psychrophilus (111) B. sphaericus (111) B. pantothenticus ‘B. thiaminolyticus’ B. lentus ‘B. cascainensis’ Cluster-group E B. lentus B. macquariensis B . lentus Cluster-group F B. coagulans B. stearothermophilus B. coagulans B . stearothermophilus ‘B. aneurinolyticus’ B. azotoformans B. sphaericus B. coagulans B. stearothermophilus B. stearothermophilus (2) B. coagulans (VI) B . stearothermophilus (VI) B. pantothenticus (VI) 1864 F . G . PRIEST, M. GOODFELLOW A N D C. T O D D Table 3. Percentage distribution of positive characters to cluster-groups defined at the 70% level (SsM) Cluster-group*. . . A B C D E F Number of strains. . . 59 154 52 46 10 17 Colonial morphology 1. Flatlraised 2. Smooth 3. Rhizoidal 4. Entire 5. Opaque 6. Pigmented 7. Motile colonies 90 97 0 36 61 3 30 74 66 5 32 97 4 0 61 100 0 86 96 27 0 76 100 0 74 89 0 0 60 100 20 80 0 0 0 94 94 0 59 35 0 0 Cellular morphology 8. Length > 3 pm 9. Diameter >0.9 pm 10. Ends round 11. Single 12. Vacuoles present 13. Gram-variable 14. Gram-positive 15. Spores oval 16. Spores round 17. Spores central 18. Spores terminal 19. Spores bulging 20. Sporulation 24 h 21. Sporulation 72 h 22. Sporulation 120 h 23. Sporulation SxAt 75 0 90 76 0 7 0 100 0 24 75 47 10 86 100 100 45 42 100 33 41 95 58 99 0 99 3 92 17 88 99 99 21 8 98 33 4 73 29 94 11 65 21 25 0 70 81 94 67 15 100 76 0 33 6 80 28 59 87 35 87 96 98 10 0 90 30 0 80 40 100 0 100 0 20 0 60 80 100 47 0 100 71 0 59 6 100 0 35 59 76 100 100 100 100 Degradation of: 24. Adenine 25. Aesculin 26. Allantoin 27. Arbutin 28. Casein 29. Chitin 30. DNA 31. Elastin 32. Gelatin 33. Guanine 34. Hippurate 35. Lecithin 36. Pectin 37. Pullulan 38. Pustulan 39. RNA 40. Starch 41. Testosterone 42. Tween 20 43. Tween 80 44. Tyrosine 45. Urea 0 100 7 100 68 28 68 10 90 0 57 52 15 83 12 80 98 85 100 73 12 20 15 99 16 100 100 38 95 53 100 1 21 88 28 64 100 75 1 100 80 32 21 2 65 10 38 100 0 94 13 100 13 52 71 4 60 0 81 46 2 98 86 27 4 28 7 15 15 61 0 100 17 72 0 39 28 0 0 0 67 0 50 72 65 28 26 0 100 0 100 0 0 0 0 0 0 80 0 0 100 0 0 100 30 80 80 0 80 18 94 0 100 41 0 100 0 71 0 100 0 0 53 0 88 100 29 100 47 0 0 3 20 15 19 73 86 37 66 42 52 25 35 35 56 30 34 11 11 21 29 13 50 35 23 0 6 13 24 65 85 37 30 20 30 0 0 10 30 80 80 0 0 0 0 0 0 6 6 Resistance to (pg ml - l ) : 46. Benzylpenicillin (8) 47. Benzylpenicillin (4) 48. Chloramphenicol (8) 49. Chloramphenicol (4) 50. Cycloserine (128) 5 1. Cycloserine (64) 52. Erythromycin (1) 53. Erythromycin (0.5) 1 50 Taxonomy of bacilli Table 3 (continued) Cluster-group*. . . A B Number of strains. . . 59 154 54. Gramicidin (64) 55. Gramicidin (32) 56. 57. 58. 59. 60. 61. 62. 63. Nalidixic acid (32) Nalidixic acid (16) Polymyxin (16) Polymyxin (8) Rifampicin (0-25) Rifampicin (0.125) Streptomycin (16) Streptomycin (8) 64. Tetracycline (2) 65. Tetracycline (1) Acid from : 66. Adonitol 67. Arabinose 68. Cellobiose 69. Dulcitol 70. Erythritol 71. Fructose 72. Galactose 73. Glucose 74. Glycerol 75. meso-Inositol 76. Lactose 77. Maltose 78. Mannitol 79. Mannose 80. Raffinose 81. Rhamnose 82. Salicin 83. Sorbitol 84. Sucrose 85. Trehalose 86. Xylose Utilization of: 87. Acetate 88. Citrate 89. Formate 90. Gluconate 91. Lactate 92. Malonate 93. Succinate Growth at: 94. pH 4.5 95. pH6.0 96. pH 7.2 97. pH 8.0 98. pH 9.5 99. 5°C 100. 17°C 101. 37°C 102. 50°C 103. 65°C Growth in (%, w/v): 104. NaCl (2) 105. NaCl ( 5 ) 106. NaCl (10) 1865 C 52 D 46 E 10 F 17 @ 0 73 83 22 17 80 88 22 34 49 66 46 49 61 73 7 22 89 90 14 43 35 52 43 72 27 35 54 65 6 27 25 31 0 4 0 0 78 83 72 83 50 72 4 11 59 61 20 37 10 30 90 100 0 10 0 0 100 100 0 0 100 100 6 6 0 0 0 0 0 0 36 49 97 0 2 75 85 98 90 30 73 100 60 63 97 7 76 25 100 93 63 0 35 96 0 0 94 0 0 2 0 0 24 99 94 35 23 93 42 59 48 4 94 26 80 98 43 0 0 40 2 11 40 25 79 71 2 19 60 63 17 21 11 19 13 75 75 0 0 0 0 60 0 0 30 60 80 20 0 80 50 70 70 60 10 60 10 40 10 50 6 0 35 0 18 71 23 100 41 29 0 88 47 71 23 0 18 0 71 88 35 19 12 34 49 24 5 32 55 74 77 47 62 10 41 48 63 30 50 4 37 4 50 40 10 20 30 20 0 10 12 59 18 6 35 6 29 0 78 100 100 95 12 78 100 27 0 15 96 100 100 99 3 97 100 34 0 23 98 100 100 100 6 94 100 25 0 0 87 100 100 80 9 72 89 43 0 10 100 100 100 100 20 100 90 0 0 12 94 82 35 0 0 6 94 100 53 83 100 99 52 100 96 79 87 70 0 100 80 20 47 29 6 58 m L 40 44 44 27 31 21 60 0 15 59 0 0 9 2 0 2 0 0 0 2 2 1866 F . G . PRIEST, M . GOODFELLOW A N D C . T O D D Table 3 (continued) Cluster-group*. . . Number of strains. . . A 59 B 154 C 52 D 46 E 10 F 17 98 36 41 27 41 78 75 100 72 0 37 36 52 0 40 2 68 76 75 57 29 29 71 80 23 0 0 0 77 6 52 48 6 32 13 0 0 0 4 2 63 0 67 13 41 6 20 0 20 0 0 0 80 20 30 100 80 0 20 0 57 0 0 0 35 47 100 47 0 0 65 18 Miscellaneous tests : 107. 108. 109. 110. 111. 112. 1 1 3. 114. 1 15. 1 16. 117. 1 18. Anaerobic growth Gas from glucose Dihydroxyacetone production Indole production Growth on MacConkey agar Methyl red test Nitrate reduction ONPGt Oxidase Phenylalanine deamination Phosphatase Voges-Proskauer reaction * For ease of computation, these cluster-groups do not take into account data for single-member clusters. t SxA, soil extract agar. $ ONPG, o-nitrophenyl Pa-galactoside. The final phenon in cluster-group C comprised seven strains originally described as ‘Krusella cascainensis’ (Castellani, 1954) but subsequently transferred to Bacillus as ‘B. cascainensis’ (Castellani, 1955). Most of these bacteria formed endospores that were oval and central but did not swell the sporangium; their other characteristics are given in Table 4. Cluster-group D contained the alkali-forming strains that have limited, if any, reaction in sugar-containingmedia. Five strains of ‘B.aneurinolyticus’formed a homogeneous group (cluster 36) that was closely related to B. brevis (cluster 37). Both of these taxa accommodated strains with oval central spores that distended the sporangium. They were obligate aerobes, reduced nitrate and with the occasional exception did not produce acid from carbohydrates. The two species were distinguished by the failure of ‘B.aneurinolyticus’ strains to grow in 5 % (w/v) NaCl or to hydrolyse casein, gelatin or hippurate. Six strains of B. azotoformans were recovered close to B. brevis in cluster 38. These species had many features in common, but B. azotoformans strains can be distinguished as they are unable to grow at 50 “C and fail to hydrolyse casein, gelatin, hippurate or RNA. In the SsM/UPGMAanalysis, cluster 39 contained strains of B. badius and ‘B.freudenreichii’ but these taxa were separated in the analyses based on SJand Dp coefficients. The B. badius strains were negative for nitrate reduction and urease production, but hydrolysed casein and gelatin; the ‘B.freudenreichii’ strains gave the opposite reactions. Five psychrophilic isolates were assigned to cluster 40, B. psychrophilus. These bacteria produced spherical spores that distended the sporangium and most grew at 5 “C but not at 37 “C. Cluster 41, which was recovered in all three analyses, contained two B. brevis strains. Morphologically similar to B . brevis sensu stricto, these strains differed by degrading adenine, allantoin and elastin, and were also urease positive and did not reduce nitrate. The B. sphaericus and ‘B. sphaericus var. fusiformis’ strains were recovered in two discrete clusters in all three analyses, suggesting that the latter should be given species status as B . fusiformis. The remaining strains in cluster-group D were recovered as single-member clusters and included ‘B. sphaericus var. rotans’ S299, B. globisporus S430, B. insolitus S432, ‘B. repens’ S295 and three marine isolates. Cluster-group E contained a single major cluster, B. lentus. The bacteria in this taxon had limited action on macromolecules, formed acid from few sugars other than glucose and produced oval central spores that did not swell the sporangium. Two strains of B. macquariensis were recovered in a minor cluster adjacent to B. lentus. The thermophilic bacilli were recovered in cluster-groupF. Eight strains of B. coagulans were recovered in two clusters, one of which, cluster 46, conformed to B. coagulans sensu stricto (Wolf Taxonomy of bacilli 1867 Type B; Wolf & Sharp, 1981). Similarly, the B. stearothermophilus strains were assigned to two major phena. Cluster 48 contained strains belonging to Groups 2 and 3 (B. stearothermophilus Donk) of Walker & Wolf (1971). These two groups of bacteria fused at 84% SSMbut were assigned to separate clusters in the S,analysis. Thus, the characteristics shown in Table 4 may not be typical for B. stearothermophilus sensu stricto. Cluster 49 equated with Group 1 of Walker & Wolf (197 1) (‘B. kaustophilus’). DISCUSSION It is encouraging that the three analyses presented here and the two previous comprehensive taxometric studies of bacilli (Logan & Berkeley, 1981; Priest et al., 1981) are essentially congruous despite the use of widely different data bases. Indeed, the assignment of species to cluster-groups seems to reflect a natural classification that is largely consistent with DNA base composition (Priest, 1981). The cluster-groups can be equated with genera in some groups of bacteria, but additional data derived from 16s rRNA sequencing or hybridization studies are needed before any dismemberment of the genus Bacillus can be proposed with confidence. For the present, the cluster-groups should be used as a framework for further taxonomic studies, and to this end their characteristics have been considered above. The ensuing discussion concentrates on species of Bacillus that currently present taxonomic problems. Cluster-group A . This study confirms the heterogeneity of strains currently classified as B. circulans. Gibson & Topping (1938) described B. circulans as a ‘complex’rather than a species, a view that persisted for some time (Proom & Knight, 1955; Wolf & Chowdbury, 1971;Gibson & Gordon, 1974). It is now apparent that the description of B. circulans encompasses a variety of genotypically unrelated bacteria. The mol% G C of 123 strains identified as B. circulans varied between 37 and 61 (Nakamura & Swezey, 1983a), and in DNA reassociation experiments nearly half of these bacteria were assigned to 10 homology groups, while the remaining 61 strains were unclassified (Nakamura & Swezey, 1983b). From these studies, four species names previously considered as synonyms of B . circulans, namely B. amylolyticus, B. lautus, B. pabuli and B. validus, were reintroduced (Nakamura, 1984~).Our numerical classification included few of the strains examined by Nakamura & Swezey (1983a, b) but it is possible to equate the two studies. Cluster 6 contained the type strain and has properties in accord with those of B. circulans sensu stricto (Nakamura, 1984~).Further evidence for homogeneity of this cluster is indicated by the inclusion of strain S109 (NCIB 9559, which originally bore the name ‘B. aporrhoeus’ but is now considered to be a synonym of B. circulans (Gordon et al., 1973) and shares 50 to 60% DNA sequence homology with the type strain of B . circulans (Nakamura & Swezey, 1983b). Cluster 7 is similar to B. pabuli in most respects. Similarly, strains assigned to cluster 9 have much in common with B . amylolyticus and cluster 10 can perhaps be equated with B. lautus, although it contains strains that hydrolyse Tween 80 and do not produce acid from rhamnose. The numerical classification also underpins the taxonomic integrity of B. alvei, B. macerans and B . polymyxa. It is also evident that strains of ‘B. apiarius’ and ‘B. thiaminolyticus’ form well-circumscribed taxa which may merit species status when DNA base composition data become available. + Cluster-group B. DNA reassociation studies support the view that B . cereus, B . mycoides and B. thuringiensis comprise a single species (Somerville & Jones, 1972; Seki et al., 1978). In this respect, it is interesting that crystal toxin synthesis is often plasmid-encoded and transmissible from B. thuringiensis to B . cereus by ‘conjugation’ (Gonzalez et al., 1982). The numerical phenetic data underline the close relationship between B . cereus and B. thuringiensis, although strains bearing these names were largely allocated to separate subclusters within cluster 11. Some strains of B. cereus are responsible for diarrhoeal and emetic types of food poisoning (Gilbert, 1979) and others for quite severe medical and veterinary pathogenic conditions (Turnbull et al., 1979); a serotyping scheme has been developed for the identification of these strains (Kramer et al., 1982). It has been claimed that strains of B. cereus responsible for the emetic form of foodpoisoning can be distinguished from diarrhoeal and non-food-poisoning strains by numerical 1868 F . G . PRIEST, M . G O O D F E L L O W AND C . T O D D Table 4. Percentage distribution of positive characters to major clusters defined at the 83 % level (SsM) 3 2 s H4 Cluster number. Number of strains. Colonial morphology 1. Flat/smooth 2. Smooth 3. Rhizoidal 4. Entire 5. Opaque 6. Pigmented 7. Motile colonies Cellular morphology 8. Length >3 pm 9. Diameter >0.9 pm 10. Ends round 11. Single 12. Vacuoles present 13. Gram-variable 14. Gram-positive 15. Spores oval 16. Spores round 17. Spores central 18. Spores terminal 19. Spores bulging 20. Sporulation 24 h 21. Sporulation 72 h 22. Sporulation 120 h 23. Sporulation SxA Degradation of: 24. Adenine 25. Aesculin 26. Allantoin 27. Arbutin 28. Casein 29. Chitin 30. DNA 31. Elastin 32. Gelatin 33. Guanine 34. Hippurate 35. Lecithin 36. Pectin 37. Pullulan 38. Pustulan 39. RNA 40. Starch 41. Testosterone 42. Tween 20 43. Tween 80 cd $ c d cried cricri 1 13 3 4 5 10 8 11 4 6 6 5 100 75 100 100 100 100 75 100 100 100 0 0 0 0 0 0 100 0 40 20 69 75 83 0 60 0 0 0 0 0 92 0 100 0 0 85 0 100 100 0 39 15 100 0 23 77 92 0 62 92 100 0 0 0 100 0 0 0 100 0 0 100 100 0 100 100 100 0 100 0 100 100 0 100 0 100 100 100 100 100 100 100 100 0 100 0 0 100 0 100 c r i c d 13 14 9 9 cd 15 15 19 20 82 98 44 56 91 76 100 1 1 0 0 0 9 46 0 89 22 73 100 89 100 0 0 0 0 0 0 0 0 67 73 0 47 93 0 0 75 55 0 65 95 0 0 32 26 0 21 100 0 0 100 80 0 100 80 100 0 100 0 0 7 87 100 100 0 0 100 25 0 90 15 100 0 100 0 0 10 80 95 100 0 0 100 16 0 90 42 100 0 100 0 0 0 100 100 100 60 100 0 100 100 0 93 100 100 0 27 0 27 60 0 100 100 0 loo 100 0 87 55 100 0 100 100 0 100 100 100 0 100 0 85 30 0 100 0 0 100 100 11 56 67 0 100 100 0 0 0 100 0 0 100 100 0 100 100 100 70 0 100 60 0 0 0 100 0 0 100 100 20 90 100 100 40 0 100 100 0 20 0 100 0 20 80 100 20 100 100 100 82 0 100 0 0 0 0 100 0 64 36 91 9 100 100 100 98 98 100 17 100 100 72 100 0 100 0 0 15 100 100 100 67 0 100 89 0 100 100 100 0 100 0 89 0 89 89 100 0 0 100 100 0 100 89 100 0 100 11 0 0 0 100 100 0 100 0 100 83 0 100 0 0 100 67 0 0 100 100 0 0 0 0 100 100 0 67 100 100 100 loo 100 0 0 67 100 100 100 46 0 50 0 100 0 100 0 100 0 0 100 0 50 0 0 50 0 0 100 0 100 100 0 100 0 100 0 0 100 20 100 0 0 0 0 100 0 100 100 20 100 80 0 100 0 100 100 0 55 0 100 0 0 0 6 100 0 100 100 0 100 100 0 100 0 100 100 47 100 9 100 0 2 100 4 0 95 2 100 86 0 100 98 0 67 83 100 100 100 100 67 100 0 67 55 0 0 0 100 0 0 100 100 0 100 0 100 100 0 33 100 100 0 11 0 0 33 0 100 100 0 cd cri 20 19 5 0 5 100 63 100 100 95 100 79 100 0 0 0 95 47 0 100 100 0 100 100 Taxonomy of bacilli 1869 Table 4 (continued) .?9 9 B LI tB E .a B $ E 1. 2. 3 4. 5. 6 . 7 4 3 ad fQi 22 11 25 8 4 27 6 73 88 17 73 100 100 . 0 0 82 <37 100 100 100 100 2 7 3 7 6 7 1 0 . 0 0 ad 3 29 4 30 9 31 5 33 4 35 7 75 100 0 100 100 0 0 0 100 100 0 100 100 0 0 0 100 0 100 100 0 0 0 100 0 100 100 5 0 0 100 100 0 100 43 0 0 8. 54 0 0 25 0 80 9.54 0 0 0 0 6 0 10. 100 100 100 100 100 100 11. 45 25 83 0 0 0 0 0 12.54 0 0 2 0 13. 100 25 100 50 78 100 14. 45 12 50 25 22 20 15. 100 100 100 100 100 100 16. 0 0 17 0 0 11 0 100 17. 100 88 83 100 18. 0 12 17 0 0 100 19. 0 0 17 0 0 100 20. 82 0 0 0 0 0 21. 100 88 67 75 67 60 22. 100 88 67 75 89 60 23. 100 100 100 100 100 100 24. 25. 26. 27. 28. 2 30. 31. 32. 3 3 34. 3 3 37. 3 39. 40. 41. 42. 43. 0 100 0 100 100 9 100 0 100 . 9 9 5 6 91 8 100 100 0 100 45 9 z 3 0 0 0 0 100 100 0 0 0 0 100 100 100 100 100 . 9 0 0 100 100 100 5 0 3 3 2 5 100 100 100 7 5 0 0 88 0 50 . 0 0 0 . 0 0 0 100 83 100 . 0 0 0 100 83 100 100 83 100 0 0 0 100 100 75 100 83 50 0 100 0 100 100 0 89 0 100 0 0 0 0 78 0 11 11 11 100 100 3 36 5 ad ad ad ad ad Qi ad ad ad ad 37 11 38 6 39 4 40 5 42 4 43 9 44 8 46 5 48 4 49 5 100 91 83 50 100 25 100 100 100 100 100 100 0 0 0 0 0 0 0 60 82 100 50 100 25 100 100 83 100 60 100 0 0 0 0 0 0 0 0 0 0 0 0 75 0 100 27 100 100 0 0 0 6 0 0 0 5 0 4 0 100 100 100 100 100 100 100 75 100 100 100 0 75 100 2 5 0 0 0 0 0 0 75 71 20 0 0 100 100 0 25 29 0 0 25 40 100 57 100 91 100 100 0 0 0 0 9 0 0 100 0 57 80 82 0 100 100 0 0 20 27 100 25 0 0 0 80 100 100 0 100 0 0 20 45 0 0 0 100 43 40 91 100 100 60 100 57 60 100 100 100 100 100 57 80 100 100 100 100 0 0 40 100 20100 60 100 100 100 0 0 100 100 0 0 100 100 0 0 100 100 0 0 0 0 0 25 0 0 100 100 0 40 0 0 100 100 100 100 100 0 100 0 0 25 0 0 100 50 50 100 50 100 100 100 56 63 100 75 100 100 100 100 100 80 0 0 0 0 ~0 67 75 80 100 0 100 0 80 25 0 0 0 0 0 0 0 0 0 0 0 89 0 100 100 0 33 0 0 100 11 89 89 67 100 100 100 0 0 100 50 0 100 63 100 0 100 0 0 0 88 100 100 1 4 0 0 0 0 0 1 0 0 7 8 0 100 0 27 0 0 20 0 0 100 0 0 0 0 0100 0 0 0 100 0 27 0 0 80 0 0 100 100 0 100 0 50 100 100 89 0 0 0 0 0 0 0 0 0 0 0 100 100 100 100 100 100 100 100 0 0 0 0 0 0 0 7 5 3 3 0 100 0 100 0 50 100 100 100 0 0 0 0 0 0 0 0 0 0 86 0 91 0 50 100 0 11 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 0 0 0 0 0 0 0 100 0 0 0 0 0 0 0 0 0 0 0 50 0 50 100 0 100 100 100 0 0 0 0 0 0 0 0 100 0 0 55 0 0 80 100 78 37 100 0 82 0 100 100 100 100 100 86 0 55 50 50 100 100 89 100 100 0 100 20 0 60 0 100 0 80 0 60 100 100 100 100 0 0 0 0 100 100 100 100 0 0 50 60 25 0 100 100 0 0 0 0 100 100 100 100 100 100 100 100 100 100 100 100 0 100 0 100 0 0 100 0 0 0 100 0 0 0 0 80 100 0 100 0 0 100 0 100 75 0 100 0 100 0 100 0 0 100 0 75 100 0 100 0 0 100 0 100 20 0 100 0 100 0 100 0 0 100 0 100 100 100 100 100 1870 F . G . PRIEST, M . GOODFELLOW A N D C. T O D D Table 4 (continued) Cluster number. . . Number of strains.. . 44. Tyrosine 45. Urea Resistance to (pg ml - I ) : 46. Benzylpenicillin (8) 47. Benzylpenicillin (4) 48. Chloramphenicol (8) 49. Chloramphenicol (4) 50. Cycloserine (128) 51. Cycloserine (64) 52. Erythromycin (1) 53. Erythromycin (0.5) 54. Gramicidin (64) 55. Gramicidin (32) 56. Nalidixic acid (32) 57. Nalidixic acid (16) 58. Polymyxin (16) 59. Polymyxin (8) 60. Rifampicin (0.25) 61. Rifampicin (0.125) 62. Streptomycin (16) 63. Streptomycin (8) 64. Tetracycline (2) 65. Tetracycline (1) Acid from: 66. Adonitol 67. Arabinose 68. Cellobiose 69. Dulcitol 70. Erythritol 71. Fructose 72. Galactose 73. Glucose 74. Glycerol 75. meso-Inositol 76. Lactose 77. Maltose 78. Mannitol 79. Mannose 80. Raffinose 81. Rhamnose 82. Salicin 83. Sorbitol 84. Sucrose 85. Trehalose 86. Xylose Utilization of: 87. Acetate 88. Citrate 89. Formate 90. Gluconate 91. Lactate 92. Malonate 93. Succinate Growth at: 94. pH 4.5 95. pH 6.0 96. pH 7.2 97. pH 8.0 98. pH 9.5 99. 5 ° C 100. 17°C 13 9 14 9 15 15 19 20 20 19 0 7 6 5 5 0 0 2 1 0 0 0 0 0 0 0 32 0 0 0 67 10 60 100 10 0 100 10 0 100 100 0 100 100 20 100 100 100 100 100 100 83 100 20 100 100 40 0 0100 100 17 0 100 17 100 80 100 100 83 100 80 0 83 30 20 70 20 83 0 0 100 100 0 0 0 100 100 0 100 100 100 100 100 100 20 0 9 5 4 4 1 1 0 0 98 89 11 7 0 3 11 0 0 9 15 22 11 0 91 64 11 33 47 91 98 33 0 67 0 20 0 89 0 0 64 23 33 100 73 32 0 78 93 100 48 0 89 100 0 0 2 0 0 13 7 67 0 0 100 100 100 100 100 100 100 100 100 100 21 0 11 27 27 0 22 60 60 55 26 67 89 73 18 89 11 73 60 64 0 53 81 11 36 97 67 100 67 36 0 0 21 84 90 0 0 68 100 95 100 42 100 100 100 5 32 42 95 37 58 0 75 100 0 0 100 100 100 100 50 100 100 0 0 100 100 0 0 100 91 100 91 0 91 100 91 82 100 0 100 18 100 100 91 2 0 84 0 0 86 3 100 91 2 2 100 0 21 3 0 91 3 65 98 0 0 67 0 100 100 0 0 100 100 0 0 0 44 0 0 100 0 0 18 0 18 20 82 60 36 40 18 0 1 8 0 46 76 80 91 43 79 5 14 89 11 67 0 0 0 99 3 4 4 6 5 10 6 5 8100 31100 0 0 0 0 0 0 1 13 15 0 31 0 0 0 0 0 69 100 100 100 39 100 69 100 100 100 100 100 0100 0 100 100 0 0 54 92 15 23 92 0 92 0 0 0 54 92 92 39 0 100 8 15 100 0 0 0 100 69 0 15 0 0 23 0 0 0 100 100 0 100 0 100 100 0 0 50 50 0 0 0 0 17 10 100 67 100 20 100 100 100 0 0 0 0 10 0 100 100 100 100 90 100 100 100 100 83 100 100 17 20 100 100 100 100 100 100 100 83 90 100 100 90 100 100 90 80 1 7 2 0 2 0 100 100 100 0 70 100 100 100 100 100 100 100 100 100 100 33 30 50 0 33 60 67 100 33 50 0 1 0 33 80 0 5 0 0 0 100 100 100 100 100 100 100 100 92 100 100 0 0 1 7 54 50 100 0 0 100 100 100 100 100 100 100 100 0 2 0 100 100 8 11 0 100 100 100 100 0 100 11 56 0 100 100 100 98 0 98 0 0 22 0 0 22 100 0 0 7 100 0 0 0 100 100 0 33 100 100 100 100 33 100 100 13 100 100 100 100 89 100 100 100 0 0 100 100 89 93 100 100 89 100 11 80 0 22 0 11 0 0 78 20 87 93 100 60 0 87 0 80 100 15 55 45 60 100 100 0 5 100 100 0 20 0 0 0 40 0 100 100 0 0 100 90 100 100 10 45 50 100 100 75 0 100 0 100 100 95 0 90 40 0 10 0 100 0 1 1 4 7 2 0 44 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 0 0 0 0 100 78 100 100 0 100 100 0 0 100 95 100 100 95 68 100 100 100 95 25 100 95 100 100 95 58 95 95 100 85 10 100 0 100 100 100 100 0 95 1871 Taxonomy of bacilli L. Table 4 (continued) c, s G 22 11 25 8 27 6 29 4 44. 45. 0 82 0 0 0 0 0 0 9 0 45 0 0 0 0 9 0 0 9 0 9 100 27 100 27 0 0 73 0 12 0 50 0 0 9 12 0 75 0 88 0 0 0 0 64. 0 0 65. 9 0 0 0 0 0 0 0 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 0 0 0 0 0 0 0 100 100 0 67 0 17 0 0 0 0 100 12 67 100 0 0 100 100 100 100 88 83 0 0 91 27 0 0 100 100 100 100 88 100 0 17 45 0 0 0 0 0 0 0 100 12 0 0 54 100 100 100 100 12 67 0 0 100 87. 91 100 88. 100 0 89. 100 100 90. 100 0 91. 100 50 92. 91 100 93. 100 100 94. 95. 96. 97. 98. 99. 100. 0 0 0 17 0 0 0 0 0 0 0 0 100 100 100 100 100 0 100 75 100 100 100 100 0 75 30 9 31 5 33 4 56 100 0 60 50 0 36 37 5 1 1 0 36 0 0 0 0 100 0 0 0 0 0 100 0 0 0 78 0 50 0 0 0 67 40 50 100 0 0 0 40 100 0 100 0 100 60 100 71 100 0 67 0 50 0 0 0 0 0 50 0 0 25 0 20 50 100 100 50 11 60 50 100 100 0 100 100 100 100 100 0 100 100 100 100 100 0 0 100 0 0 0 0 33 0 0 100 100 0 0 25 0 20 5 0 0 0 25 0 100 0 0 100 0 0 0 0 0 0 0 0 100 0 0 0 0 0 0 0 1 4 0 0 0 0 0 0 0 18 82 100 18 18 100 100 73 100 100 100 0 0 91 100 2 6 4 0 0 0 0 0 0 0 11 100 0 0 0 0 0 0 0 67 80 0 0 0 0 78 100 50 11 100 0 0 0 0 0 0 50 22 80 50 0 100 0 0 0 0 0 60 0 0 0 0 11 0 0 0 0 0 100 80 75 100 100 0 0 0 0 100 0 40 5 42 4 0 100 0 0 50 100 0 75 38 6 0 0 0 0 0 67 100 100 100 100 100 100 0 33 0 0 0 0 0 0 7 0 0 39 4 0 0 0 0 0 2 5 0 0 7 5 0 0 100 0 40 100 0 100 100 0 0 100 0 0 25 25 60 25 25 80 25 0 0 100 0 20 100 0 0 25 0 0 100 0 0 0 0 2 5 0 0 25 0 0 25 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 50 100 0 0 0 0 20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100 0 73 0 0 60 0 0100 0 0 0 0 0 0 0 100 40 9 17 0 60 0 75 100 100 82 50 50 60 0 0 0 0 0 0 0 0 0 0100 0 0 0 0 0 0 50 0 2 0 0 0 0 6 0 0 75 100 0 0 0 0 0 25 0100 0 0 0 0 0 0 0100 0 9 0 0 0 0 0 8 6 0 0 0 0 0 0 0100 0 0 0 0 0 0 0100 0 0 0 0 0 0 50100 0 0 0 0 0 2 5 50 100 0 0 0 0 20 0 0 0 0 0 0 0 0 0 17 25 50 100 0 0 0 25 0 25 0 0 50 100 11 11 11 0 11 0 0 100 80 100 100 100 0 100 100 100 100 100 100 0 100 0 100 100 100 100 0 100 0 100 100 100 100 0 100 100 100 100 100 100 40 100 0 75 100 100 100 0 100 17 100 100 100 100 0 83 35 7 0 40 0 0 0 0 0 0 0 60 0 2 0 0 0 82 45 45 0 0 0 45 0 75 33 0 0 75 0 0 33 25 0 0 17 100 44 8 46 5 48 4 49 5 0 0 0 100 0 0 0 0 0 0 43 9 0 22 11 33 89 89 33 33 78 89 100 100 4 100 11 33 100 100 67 100 0 0 25 0 0 0 0 0 0 0 0 100 0 50 20 0 0 0 0 0 0 0 0 0 0 0 0 60 100 100 0 0 20 0 50 0 100 100 100 100 0 25 80 33 12 0 0 100 0 0 0 0 0 0 0 0 37 60 100 100 0 75 100 0 88 0 24 20 75 100 0 75 20 0 0 0 0 0 0 0 0 25 40 0 50 0 0 0 0 0 0 50 60 100 20 0 12 60 100 100 0 100 0 37 20 100 100 100 33 100 100 0 5 0 100 78 0 2 5 0 100 100 0 0 0 0 0 0 0 0 0 0 0 0 100 100 100 100 100 60 100 100 100 100 100 100 100 100 100 100 100 100 100 0 100 33 100 100 0 0 0 0 0 8 0 100 20 64 33 100 100 0 100 100 100 100 0 100 25 0 0 0 0 0 0 37 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100 20 0 0 100 20 0 0 0 0 0 0 0 25 100 100 100 100 100 100 100 100 4 0 20 0 0 0 20 0 0 0 0 0 0 0 0 0 0 100 0 0 0 100 0 0 0 0 0 0 0 0 0 0 0 0 55 100 100 100 0 100 50 12 12 12 0 0 12 0 0 0 0 80 0 0 50 0 50 100 0 40 0 0 0 20 0 20 50 60 12 0 25 100 100 75 100 100 100 0 50 100 0 0 100 0 0 0 100 0 0 0 100 100 80 0 0 0 1872 F . G . PRIEST, M . GOODFELLOW A N D C . T O D D Table 4 (continued) Cluster number. . . Number of strains.. . 101. 37°C 102. 50°C 103. 65°C Growth in (%, wlv): 104. NaCl (2) 105. NaCl (5) 106. NaCl (10) Miscellaneous tests : 107. Anaerobic growth 108. Gas from glucose 109. Dihydroxyacetone production 110. Indole production 11 1. Growth on MacConkey agar 112. Methyl red test 113. Nitrate reduction 114. ONPG 115. Oxidase 1 16. Phenylalanine deamination 117. Phosphatase 1 18. Voges-Proskauer reaction 5 10 6 5 8 11 11 56 13 9 14 9 15 15 19 20 20 19 100 100 17 0 100 0 100 60 0 100 0 0 100 0 0 100 0 0 100 89 0 100 87 0 100 60 0 100 95 0 100 100 0 30 0 0 100 100 0 73 9 9 100 100 40 00 00 0 100 100 89 00 00 80 100 100 100 100 100 5 100 00 100 0 0 0 0 0 100 92 100 0 39 25 100 69 100 67 8 100 17 100 100 100 100 100 100 0 0 0 100 100 17 77 0 0 00 00 0 0 20 00 00 00 00 0 0 0 100 0 0 0 100 100 20 100 0 0 20 0 00 00 00 0 73 62 00 00 0 0 9 100 100 0 2 0 98 100 97 12 69 15 100 91 00 0 11 33 .O 67 100 0 11 0 0 0 0 0 100 0 67 11 78 89 0 89 100 100 0 0 0 0 60 100 0 0 0 95 33 95 00 0 00 100 0 10 0 0 67 15 100 100 100 0 95 0 0 95 100 100 10 95 100 100 3 4 4 6 100 100 0 0 0 0 00 50 0 1 13 100 92 0 analysis of phenotypic features (Logan et al., 1979), although there are no clear diagnostic features. The results of the present study indicate that strains associated with incidents of food poisoning cannot be separated easily from other strains using phenotypic tests. Strains of ‘ B . cereus var. mycoides’ were recovered in a separate subcluster (1 1I), and were distinguished by their characteristic colonial morphology. B. psychrosaccharolyticus was recovered as a well-defined cluster in this study, a result in line with earlier work (Laine, 1970; Gyllenberg & Laine, 1971).This species was not included in the Approved Lists of Bacterial Names (Skerman et al., 1980), but is listed as species incertae cedis in Bergey’s Manual of Systematic Bacteriology (Claus & Berkeley, 1986). It is evident from the present and earlier studies that the epithet B . psychrosaccharolyticus should be reintroduced (see below). The ‘ B . subtilis group’, defined at 78% SSM,contained clusters identified as B. amyloliquefaciens, B. licheniformis, B. pumilus and B . subtilis. B. amyloliquefaciens and B. subtilis strains share little DNA sequence homology (Welker & Campbell, 1967; Seki et al., 1975; Priest, 198l), can be separated by pyrolysis gas-liquid chromatography (O’Donnell et al., 1980) and can be distinguished by a few phenotypic properties (Priest et al., 1987). Our results support the recent demonstration that strains of B. amyloliquejaciens, unlike those of B . subtilis, produce acid from lactose (Nakamura, 1987), which is a useful distinguishing character. The clear separation of B. lichenformis, B. pumilis and B. subtilis has been noted in other taxometric studies (Bonde, 1975; Durand et al., 1979;O’Donnell et al., 1980; Logan & Berkeley, 1981). These species comprise discrete DNA homology groups (reviewed by Priest, 1981) and can be readily distinguished by a number of presumptively diagnostic features (Table 4). It was also encouraging that strains labelled as ‘B.aterrimus’ and ‘B. vulgatus’ fell within the B . subtilis cluster, as such strains were recovered in the B. subtilis DNA homology group by Seki et al. (1975). Three strains of ‘B.subtilis var. niger’, were assigned to a separate cluster that was closely related to B. subtilis. However further studies are required to determine the taxonomic status of ‘B.subtilis var. niger’, as a strain of this taxon showed 95 % DNA homology with the type strain of B . subtilis (Seki et al., 1975). Strains labelled as B . megaterium were assigned to three DNA homology groups by Hunger & Claus (1 98 1). The homology group corresponding to B. megaterium sensu strict0 is represented by cluster 22. A second DNA homology group contains strains originally labelled as ‘B. simp/ex’ Taxonomy of bacilli k Table 4 (continued) Y 5 6 1873 22 11 25 8 27 6 29 4 30 9 31 5 33 4 35 7 36 5 37 11 38 6 39 4 101. 100 100 100 100 100 100 100 100 100 100 100 100 102. 0 0 100 100 0 50 0 0 33 0 100 40 103. 0 0 0 0 0 0 0 0 0 0 0 0 104. 100 100 100 100 100 100 100 100 100 100 105. 100 100 100 100 100 100 100 100 0 100 106. 0 100 83 100 100 100 0 100 0 0 107. 0 0 0 0100 0 0 0 0 108. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 109. 1 1 0 . 0 0 0 0 0 0 0 0 0 111. 100 63 100 75 100 100 100 14 0 0 17 0 0 0 112. 73 0 0 0 0 0 89 100 0 0 100 113. 0 100 114. 100 0 83 100 89 0 0 100 0 115. 0 0 17 0 22 0 0 0 60 116. 100 75 67 50 56 0 0 0 0 0 0 20 0 0 80 117. 54 0 17 0 0 0 0 0 0 118. 0 0 0 67 100 33 100 0 0 40 5 42 4 43 9 44 8 46 5 48 4 49 5 100 100 100 100 75 0 0 0 00 100 0 0 0 0 0 100 100 100 100 0 0 80 80 0 25 100 100 25 89 100 0 12 0 20 0 0 50 25 0 80 60 0 0 0 0 0 00 50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 60 36 33 100 100 100 100 100 20 25 0 0 0 0 0 1 0 0 75 0 0 0 91 100 50 80 0 44 37 100 100 100 20 0 100 80 25 18 0 0 80 0 0 0 0 0 83 25 20 50 89 100 0 0 0 0 11 0 0 25 0 25 0 0 0 0 50 33 0 80 25 100 0 0 0 0 0 0 0 0 0 6 0 and ‘B. teres’. These strains are phenetically different from B . rnegaterium senw stricto, and in the taxometric study of Priest et al. (198 1) they were recovered in a different aggregate cluster from B. rnegaterium. In the present study, ‘B. simplex’ S210 and ‘R. teres’ S213 formed cluster 32 (cluster-group C), thereby supporting the distinction between these organisms and strains belonging to B. megaterium sensu stricto. The third DNA homology group described by Hunger & Claus (1981) encompassed strains originally labelled as ‘B. agrestis’ and ‘B.Jlexus’. These organisms were recovered in a separate phenon (cluster 23) in both this and a previous study (Priest et al., 1981) and clearly represent a distinct species. Since the name ‘B. flexus’ (Batchelor, 1919) has priority over ‘B. agrestis’ (Werner, 1933)the former epithet is used for the reintroduced taxon (see below). Single-member clusters recovered in cluster-group B included strains of ‘B. longissimus’ (Mishustin & Tepper, 1948) and ‘B. maroccanus’ (Delaporte & Sasson, 1967). More strains of these taxa must be isolated and studied before their taxonomic status can be clarified. Cluster-group C. Two groups of strains labelled as ‘B. carotarum’ and a third group associated with this name were recovered in cluster-group C. Strains S51 to S55 (cluster 31) were isolated by Gibson (1935) and identified by him as ‘B. carotarum’ sensu Koch 1888. Two other strains originally labelled as ‘B. carotarum’ (NRS 608 and NRS 828) were donated by Gordon and thought to be original isolates from G. Bredemann and C. Stapp & N . H. Claussen, respectively (R. E. Gordon, personal communication). These were recovered in cluster 33. The third group comprised strains originally labelled as ‘B. simplex’ and ‘B. teres’. Such strains were assigned to DNA homology group B by Hunger & Claus (1981) and were considered to belong to ‘B. carofarum’by Gibson & Gordon (1974). These were recovered as cluster 32. The fact that strains assigned to clusters 31, 32, and 33 have many properties in common helps to explain the confusion that has arisen with respect to the taxonomy of ‘B. carotarum’. It is, however, clear from the present study that these taxa are distinct. The integrity of cluster 32 is supported by DNA base composition and reassociation data (Hunger & Claus, 1981) and merits species status. Since these strains were not original isolates of Koch (1888), and to avoid confusion, they cannot be given the name ‘B. carofarum’. The name ‘B. simplex’ (Gottheil, 1901) should be reintroduced for the taxon represented by cluster 32 as this epithet has priority over ‘B. teres’ (Neide, 1904). Further DNA studies are needed to confirm the taxonomic status of clusters 31 and 33. 1874 F . G . P R I E S T , M . G O O D F E L L O W AND C . TODD Table 5 . Distribution of positive characters to minor clusters defined at the 83% leuel (&) 2 2 2 9 9 9 .g .g .g 3 Y fi u; 4 6 cri cri cri cri 3 6 cri cri 2 2 7 2 9 3 10 12 16 17 18 21 23 24 26 28 32 34 41 45 47 3 2 3 2 2 2 2 2 2 2 2 3 2 2 3 Colonial morphology 1. Flat/raised 2. Smooth 3. Rhizoidal 4. Entire 5. Opaque 6. Pigmented 7. Motile colonies 2 2 0 2 0 2 0 2 2 0 0 2 0 0 1 3 0 3 3 0 0 2 3 0 2 3 0 0 2 2 0 1 2 0 0 2 2 0 1 3 0 0 0 0 0 1 2 0 0 2 1 0 1 1 1 0 1 2 0 1 2 0 0 2 2 0 0 2 0 0 2 2 0 2 2 1 0 2 2 0 0 2 0 0 1 2 0 2 2 1 0 2 2 0 2 2 0 0 0 3 0 3 3 0 0 2 2 0 2 2 0 0 2 2 2 2 0 0 0 3 0 0 2 1 0 0 Cellular morphology 8. Length > 3 pm 9. Diameter >Om9 pm 10. Ends round 11. Single 12. Vacuoles present 13. Gram-variable 14. Gram-positive 15. Spores oval 16. Spores round 17. Spores central 18. Spores terminal 19. Spores bulging 20. Sporulation 24 h 21. Sporulation 72 h 22. Sporulation 120 h 23. Sporulation SxA 2 0 0 2 0 0 0 2 0 2 0 2 0 1 2 2 3 0 2 2 0 0 0 2 0 1 1 2 1 2 2 2 3 0 3 3 0 0 0 3 0 0 3 3 0 2 3 3 3 0 3 3 0 0 0 3 0 1 2 3 1 3 3 3 0 2 2 2 0 2 0 2 0 2 0 2 0 1 2 2 0 0 3 0 0 3 1 3 0 3 2 0 2 3 3 3 1 0 2 2 0 1 0 2 0 2 0 1 2 2 2 2 0 0 2 2 0 2 1 2 0 2 0 0 0 0 2 2 0 0 2 2 0 2 2 1 1 1 1 2 1 2 2 2 1 1 2 0 1 2 0 2 0 2 0 0 2 2 2 2 0 0 2 2 0 2 0 2 0 2 0 0 1 2 2 2 2 0 2 0 0 2 2 2 0 2 0 0 0 2 2 2 2 1 2 0 0 2 2 2 0 2 0 0 0 2 2 2 0 0 2 0 0 1 0 2 0 2 0 0 0 2 2 2 0 0 2 0 0 2 0 3 0 3 0 3 0 1 3 3 1 0 2 2 0 2 0 2 0 0 2 2 2 2 2 2 1 0 2 0 0 0 0 2 0 2 0 2 0 0 0 2 3 0 3 2 0 2 0 3 0 2 1 1 3 3 3 3 Degradation of: 24. Adenine 25. Aesculin 26. Allantoin 27. Arbutin 28. Casein 29. Chitin 30. DNA 31. Elastin 32. Gelatin 33. Guanine 34. Hippurate 35. Lecithin 36. Pectin 37. Pullulan 38. Pustulan 39. RNA 40. Starch 41. Testosterone 42. Tween 20 43. Tween 80 44. Tyrosine 45. Urea 0 2 0 2 2 0 2 0 2 0 2 2 0 2 0 2 2 0 2 2 2 2 0 2 0 2 2 0 2 0 2 0 0 0 2 0 2 2 2 0 2 2 0 0 0 3 0 3 2 0 0 0 0 0 1 0 0 3 0 3 3 3 3 3 0 0 0 3 0 3 1 0 2 0 2 0 3 0 0 0 1 1 2 1 3 3 0 2 0 2 2 2 2 0 2 2 2 0 0 2 0 2 0 2 2 0 2 2 0 2 1 3 0 3 3 2 3 1 3 0 0 3 1 2 0 3 3 0 3 3 0 0 0 2 0 2 2 0 2 2 2 0 1 2 0 0 0 2 2 0 2 2 0 0 1 0 2 2 0 2 2 2 2 2 0 2 2 2 2 1 2 2 0 0 0 0 1 ' 2 2 2 1 0 0 0 2 2 2 2 0 0 2 2 1 2 0 0 0 2 0 0 0 2 2 0 2 2 2 0 0 0 0 2 0 2 2 0 2 1 0 2 0 2 0 2 2 0 2 2 2 0 0 1 0 1 0 2 1 1 0 1 0 0 0 0 0 1 2 0 2 0 2 1 1 2 0 2 0 2 2 0 2 2 0 0 0 2 0 2 2 0 2 0 2 0 0 1 0 2 0 2 1 0 2 1 0 0 0 0 0 2 2 0 1 0 2 0 2 0 0 0 0 1 2 0 2 1 2 0 0 2 0 0 0 2 0 0 3 2 0 0 0 2 0 2 3 2 0 0 1 0 0 0 2 0 1 0 0 0 3 2 0 0 0 2 3 2 2 2 0 0 0 2 0 2 0 2 0 0 0 0 0 0 0 0 0 2 0 0 2 0 0 0 0 0 3 2 0 3 3 0 3 0 3 0 3 0 0 0 0 3 3 0 3 3 0 0 Cluster number.. . Number of strains.. . 1875 Taxonomy of bacilli Table 5 (continued) Cluster number.. Number of strains.. . . 2 2 7 2 9 3 10 12 16 17 18 21 23 24 26 28 32 34 41 45 47 3 2 3 2 2 2 2 2 2 2 2 3 2 2 3 0 0 0 0 0 2 0 0 2 2 0 0 2 2 0 0 2 2 0 2 0 0 2 2 2 2 2 2 0 0 0 0 2 2 1 1 2 2 0 0 0 0 0 1 3 3 0 0 0 0 3 3 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 1 1 1 1 1 1 0 0 1 2 0 0 0 0 0 1 0 1 1 1 3 3 1 2 2 3 0 0 3 3 0 0 3 3 0 1 1 2 0 3 0 1 0 0 1 2 0 0 2 2 0 0 2 2 2 2 0 0 2 2 0 0 0 0 0 2 0 0 2 2 0 0 2 2 1 2 2 2 2 2 0 0 2 2 0 0 2 2 2 2 2 2 2 2 0 2 2 2 0 2 0 0 0 0 0 0 1 2 1 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 2 2 0 0 0 0 0 2 0 0 0 1 2 2 2 2 0 1 2 2 2 2 0 0 0 0 2 2 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 1 0 0 0 0 2 2 0 1 1 1 0 0 0 0 0 0 0 0 1 3 0 0 1 1 0 3 3 3 0 0 0 2 0 0 0 0 2 2 2 2 2 2 2 2 2 2 2 2 1 1 2 2 0 0 0 0 0 0 2 2 0 0 0 0 1 2 0 1 0 0 2 2 0 0 0 0 0 0 0 0 0 0 0 0 3 3 0 0 0 0 0 0 0 0 0 0 2 0 0 0 2 2 2 2 0 2 0 0 2 0 2 0 2 2 0 0 0 2 0 0 2 1 2 2 1 2 2 2 2 2 0 2 1 2 2 2 0 0 3 0 0 3 3 3 2 0 3 3 3 1 3 0 3 0 3 3 3 1 0 2 0 0 3 3 3 1 0 3 3 0 1 3 0 2 0 3 3 1 0 0 1 0 0 2 0 2 2 0 0 2 1 2 0 0 1 1 1 2 2 0 0 3 0 0 3 3 3 3 3 1 3 3 3 1 0 3 3 3 3 3 0 1 2 0 0 2 0 2 2 2 1 1 2 1 2 0 2 2 2 2 1 0 0 0 0 0 2 0 2 0 0 0 2 1 0 0 0 0 1 2 2 0 0 0 2 0 0 2 2 2 2 2 0 2 2 2 2 1 2 2 2 2 2 0 0 0 0 0 2 2 2 2 1 2 2 2 0 2 0 1 0 2 2 0 0 0 1 0 2 2 2 2 2 0 0 2 2 2 2 0 0 0 2 2 0 0 0 0 0 1 0 1 1 2 0 0 1 0 0 0 0 0 0 1 2 0 0 0 0 0 0 0 0 0 2 0 0 1 0 0 0 0 0 0 0 1 0 0 0 2 0 2 0 2 2 2 1 0 2 1 1 0 0 0 0 2 2 0 0 0 0 0 3 0 3 3 2 0 3 3 3 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 2 2 2 0 1 0 1 2 0 2 0 0 0 1 0 2 0 0 0 0 3 0 3 3 2 0 0 3 3 3 0 0 0 0 3 3 0 Utilization of: 87. Acetate 88. Citrate 89. Formate 90. Gluconate 91. Lactate 92. Malonate 93. Succinate 1 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 1 0 0 3 0 0 1 3 3 0 2 0 0 1 0 1 0 0 3 3 3 0 3 0 3 2 1 2 0 0 0 1 0 0 1 0 0 0 2 2 2 0 1 0 0 2 2 2 2 0 0 0 2 1 1 2 0 0 0 2 1 2 2 3 0 2 2 0 0 0 1 0 1 0 2 2 2 1 1 0 2 2 3 1 0 0 0 3 2 0 2 0 0 0 1 0 0 1 2 2 0 0 0 3 1 1 1 0 0 Growth at: 94. pH 4.5 95. pH 6.0 96. pH 7.2 97. pH 8.0 98. pH9.5 99. 5°C 0 2 2 2 0 0 0 2 2 2 2 2 0 3 3 3 3 1 0 3 3 3 3 2 0 1 2 2 2 2 0 3 3 3 3 0 0 2 2 2 2 1 1 2 2 2 2 2 0 2 2 2 2 0 2 2 2 2 2 0 0 2 2 2 2 0 0 2 2 2 2 0 0 2 2 2 2 0 0 2 2 2 2 0 0 3 3 3 3 0 0 2 2 2 2 0 0 2 2 2 2 2 1 3 0 0 0 0 Resistance to (pg ml - ) : 46. Benzylpenicillin (8) 47. Benzylpenicillin (4) 48. Chloramphenicol (8) 49. Chloramphenicol (4) 50. Cycloserine (128) 5 1. Cycloserine (64) 52. Erythromycin (1) 53. Erythromycin (0.5) 54. Gramicidin (64) 55. Gramicidin (32) 56. Nalidixic acid (32) 57. Nalidixic acid (16) 58. Polymyxin (16) 59. Polymyxin (8) 60. Rifampicin (0.25) 61. Rifampicin (0.125) 62. Streptomycin (16) 63. Streptomycin (8) 64. Tetracycline (2) 65. Tetracycline (1) Acid from: 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. Adonitol Arabinose Cellobiose Dulcitol Erythritol Fructose Galactose Glucose Glycerol meso-Inositol Lactose Maltose Mannitol Mannose Raffinose Rhamnose Salicin Sorbitol Sucrose Trehalose Xylose 1876 F . G . P R I E S T , M. G O O D F E L L O W A N D C. T O D D Table 5 (continued) Cluster number. . . Number of strains. . . 100. 17°C 101. 37°C 102. 50 “C 103. 65°C Growth in (%, w/v): 104. NaCl (2) 105. NaCl (5) 106. NaCl (10) Miscellaneous tests : 107. Anaerobic growth 108. Gas from glucose 109. Di hydroxyacetone product ion 110. Indole production 1 1 1 . Growth on MacConkey agar 112. Methyl red test 1 1 3. Nitrate reduction 114. ONPG 115. Oxidase 116. Phenylalanine deamination 117. Phosphatase 118. Voges-Proskauer reaction 2 2 7 2 9 3 10 12 16 17 18 21 23 24 26 28 32 34 41 45 47 3 2 3 2 2 2 2 2 2 2 2 3 2 2 3 0 2 0 0 2 2 1 0 0 3 1 0 3 3 0 0 2 0 0 0 3 3 2 0 2 2 1 0 2 2 0 0 0 2 0 0 2 2 0 0 2 2 0 0 2 2 0 0 2 2 0 0 2 2 0 0 3 3 0 0 2 2 2 0 2 1 0 0 1 3 3 0 2 0 0 2 2 0 3 3 0 3 3 0 2 1 0 3 3 2 2 2 0 2 2 2 2 2 0 2 2 2 2 2 2 2 2 1 2 2 2 2 2 0 3 1 0 2 2 0 2 0 0 1 1 1 2 0 0 0 0 0 2 2 2 0 2 0 2 0 0 0 2 2 0 2 2 0 0 0 2 0 0 0 2 3 3 3 3 0 0 0 2 0 0 0 0 2 3 3 2 0 0 0 0 0 0 0 2 1 2 0 1 0 1 1 2 0 3 0 3 0 3 2 0 0 1 3 2 0 1 0 2 0 2 2 0 0 0 2 0 0 0 2 0 2 0 1 2 2 2 0 0 2 2 0 0 0 0 2 0 0 2 0 0 2 0 0 0 0 0 2 0 0 2 0 0 1 0 0 0 0 0 2 0 2 0 0 1 0 0 0 0 0 0 2 0 0 1 0 0 1 0 0 3 0 0 0 0 0 0 2 1 0 2 2 2 0 0 0 0 0 0 2 2 0 0 0 2 0 0 1 0 0 0 1 2 0 0 0 0 0 2 0 0 0 0 0 2 0 0 3 0 0 0 1 0 3 2 0 0 1 0 .o 2 0 2 2 0 0 1 2 Cluster-group C also contained isolates from marine or saline environments. In a numerical taxonomic study of 138 bacilli isolated from the North Sea, Boeyk & Aerts (1976) recognized two major clusters representing B. firmus and the B . subtilis group. Similarly, Bonde (1975, 1976) examined several hundred bacilli from marine sources and found that B.firmus, species of the ‘B. subtilis group’, and B. sphaericus were common. In the present investigation, most of the representatives from the studies of Bonde (1975, 1976) and Boeyk & Aerts (1976) were assigned to cluster-groups B and C. B.Jirmus has been a problematical taxon (Gordon et al., 1977). In the present study, the B. Jirrnus strains formed a compact phenon, a result in agreement with earlier work (Priest et a f . , 1981; Logan & Berkeley, 1981). B.Jirmus was also distinguished from B . lentus (cluster 44) in DNA pairing experiments (Priest, 1981; Seki et al., 1983) which showed that the sequence homology between representatives of these species was very low. Many bacilli isolated from saline environments have been described as intermediate between B.firmus and B . lentus and, as a result, strains in these taxa have been considered to form a ‘spectrum’ (Gordon et al., 1977). In the present numerical classification, however, most of the B.firmus/B.lentus intermediates were recovered in three related but distinct phena, clusters 27, 28 and 29, within cluster-group C. These findings are supported by DNA reassociation data which indicate that very little homology exists between ‘intermediate strains’ and B. firmus (Priest, 1981; Seki et al., 1983). Strains NRS 1575 and NRS 1570, for example, not only belong to different clusters but have been assigned to distinct DNA homology groups (Seki et al., 1983). Similarly, strain NRS 1151 was assigned to an individual homology group and was recovered in cluster 26. Further nucleic acid reassociation data are needed to resolve the taxonomic status of clusters 27, 28 and 29. Other phena that comprised distinct taxa within cluster-group C include B. pantothenticus and B. puluifaciens. Strains received as ‘Kruseflacascainensis’ (Castellani, 1954) produce ellipsoidal spores (Castellani, 1955; Gordon et a f . ,1973) and have been transferred to the genus Bacillus as ‘B. cascainensis’ (Castellani, 1955). The present study indicates that this epithet should be reintroduced, but DNA base composition data on representative strains are needed to complement the present description of this taxon. A single strain of ‘B.epiphytus’ was recovered on the periphery of the B.firrnus cluster; this relationship has been noted by others (Gibson & Taxonomy of bacilli 1877 Gordon, 1974; Bonde, 1976; Logan & Berkeley, 1981). DNA data are required to clarify the status of ‘B. epiphytus’. Similarly, ‘B. loehnisii’, ‘B.paciJicus’ and ‘B. macroides’ formed singlemember clusters in cluster-group C. ‘Bacillus loehnisii’ is generally regarded to be similar to B. pasteurii and ‘B.freudenreichii’ (Gibson, 1934) but in the present study, B . pasteurii strains were not included and ‘B.freundenreichii’ was recovered in cluster group D. ‘Bacillus macroides’, on the other hand, was assigned to the B.Jirmus aggregate group by Logan & Berkeley (1981); its placement in cluster-group C is consistent with this. Cluster-groupD.Most of the B . brevis strains were recovered in cluster 37, but the allocation of two strains to cluster 41 was in good agreement with an earlier taxometric study where B. brevis was shown to be heterogeneous (Priest et a/., 1981). Five strains of ‘B.aneurinolyticus’ formed a homogeneous phenon closely related to B . brevis. Previously, casein hydrolysis was considered the only feature available to distinguish between these taxa (Claus & Berkeley, 1986), but additional differential characteristics have been highlighted in this study. The name ‘B. aneurinolyticus’ should be reintroduced when confirmatory DNA base composition data become available. B. sphaericus strains have been assigned to at least five DNA homology groups (Seki et al., 1978; Krych et al., 1980), but still appear to be phenotypically uniform. Previous taxometric studies have placed B. sphaericus in a single phenon (Logan & Berkeley, 1981; Priest et al., 1981) but in the present analysis four strains labelled ‘B. sphaericus var. fusiformis’ were assigned to a separate cluster. This cluster corresponds to DNA homology group IIB of Krych et al. (1980). It is evident that the strains of cluster 41 merit species status given the good congruence between the DNA homology and numerical phenetic data. The name B .fusiformis has been proposed for this taxon (see below). A strain of ‘B.rotans’ was assigned to DNA homology group 111by Krych et al. (1980). The recovery of ‘B.sphaericus var. rotans’ (NCIB 8867) as a single-member cluster is in support with the view that this organism may also represent a new taxospecies. The integrity of B . azotoformans (Pichinoty et al., 1983) was supported by the assignment of six representatives of this species to cluster 38. A few psychrophilic strains were also recovered in cluster-group D. The numerical phenetic data support the current taxonomic status of B. globisporus, B. insolitus and B . psychrophilus (Larkin & Stokes, 1967; Ruger, 1983; Nakamura, 1984b). Cluster-groupE. The clear separation of B . lentus (cluster 44) from B.Jirmus (cluster 25, clustergroup C) confirms the independent status of these species. The recovery of two strains of B. macquariensis in cluster-group E casts doubt on the reported affinity between B . circulans and B. macquariensis (Gibson & Gordon, 1974; Logan & Berkeley, 1981). Cluster-group I;. The recovery of the B. coagulans and B . stearothermophilus strains in a single aggregate group is in good agreement with the earlier study of Logan & Berkeley (1981) which showed that these bacteria have many features in common beyond their ability to grow at high temperature. B . coagulans comprises at least two phenetic groups (Wolf & Barker, 1968), and although limited DNA reassociation studies indicated genetic homology (Seki et al., 1978), two DNA homology groups have subsequently been revealed (I. Blumenstock, personal communication: quoted by Claus & Berkeley, 1986). In the present study, strains of B . coagulans were similarly assigned to two clusters, cluster 46 representing B. coagulans sensu stricto. The recovery of the B . stearothermophilus strains in two major clusters and one single-member cluster provides yet further evidence for the heterogeneity of this taxon. It is generally accepted that B. stearothermophilus encompasses at least three distinct taxa (Baillie & Walker, 1968; Klaushofer & Hollaus, 1970; Walker & Wolf, 1971; Sharp et al., 1980). Cluster 48 contained strains of B . stearothermophilus sensu stricto (Walker & Wolf, 1971; group 3) although it also encompassed strains assigned by these workers to their group 2. Cluster 49, which was particularly well defined, corresponds to group 1 (‘B. kaustophilus’) of Walker & Wolf (1971). This taxon is phenetically and genotypically distinct from B. stearothermophilus (Sharp et al., 1980) and merits species status (see below). 1878 F . G . PRIEST, M . GOODFELLOW A N D C . T O D D The genus Bacillus, the emerging taxonomy It is appropriate in a wide-ranging study such as the present one, to draw some general conclusions and suggest priorities for the future. It is now evident that the genus Bacillus encompasses some 80 taxa of approximate species rank that can be assigned to five or more cluster-groups. The latter should be used as a framework for redefining the current genus and splitting it into several genera. An indication of how this might best be achieved has been revealed by Stackebrandt et al. (1987), who have shown that B. sphaericus and other species containing round-spored organisms can be distinguished from other bacilli on the basis of rRNA oligonucleotide sequencing, spore morphology and cell-wall composition studies. However, we agree with these authors that many more strains need to be studied by similar techniques before ‘a formal dissection of the genus Bacillus with consequent description of new genera is proposed’. It is also evident from the present study that several clusters merit species status given the appropriate supporting data from the literature, and formal proposals are given below. It is also highly likely that taxa such as ‘B. aneurinolyticus’, ‘B. apiarius’, ‘B. cascainensis’, ‘B. thiaminolyticus’ and the various halotolerant isolates described as ‘B. firmus-B. lentus intermediates’ should be raised to valid species status. Supporting DNA base composition and reassociation data are required before this can be recommended. Further comparative studies are needed to revise and clarify the classification of heterogeneous species such as B. brevis, B. circulans, B. coagulans, B. sphaericus and B . stearothermophilus.It is also possible that strains carrying names such as ‘B. cirroflagellosus’,‘B. epiphytus’, ‘B.filicolonicus’, ‘B. freudenreichii’, ‘B. globigii’, ‘B. loehnisii’, ‘B. longissimus’, ‘B. macroides’, ‘B.maroccanus’, ‘B.pacgcus’ and ‘B. repens’ represent new centres of variation, but additional representatives of these taxa need to be examined to determine their taxonomic status. NOMENCLATURE Description of Bacillus flexus (Batchelor, 1919) nom. rev. flex’us. L. adj. flexus, flexible. The description given below is taken from the present and earlier studies (Hunger & Claus, 1981; Claus & Berkeley, 1986). Strains in this species have similar properties to B. megaterium but differ from typical members of that species as cells are smaller (mean cell width 0.9 pm), poly-P-hydroxybutyrate is not formed, phenylalanine is not deaminated, neither is aesculin hydrolysed nor acid formed from pentoses. Strains of this species degrade casein, elastin, gelatin, pullulan and starch, are urease positive, but give a negative Voges-Proskauer reaction and do not reduce nitrate to nitrite. Additional properties are given in Table 5. The mol% G C content of the DNA of the two strains examined lies between 37 and 39 (T,). The type strain has little in common with either ‘B. carotarum’ or B. megaterium. Source: Faeces and soil. Type strain: DSM 1320 (= NRS 665). + Description of Bacillus fusiformis (Smith et al., 1946) comb. nov. (Bacillus sphaericus var. fusiformis Smith, Gordon & Clark, 1946, 97) fus.i.form’is. L. n. fusus spindle; L. n. forma shape, form; M.L. adj. fusiformis spindle-shaped. The description is taken from the present study and from that of Krych et al. (1980). Strains in this species have similar properties to B. sphaericus but differ from typical members of that species as they are urease positive, grow in the presence of NaCl(7%, w/v) and are sensitive to tetracycline (1 pg ml- ). They are oxidase positive, degrade gelatin and testosterone, but give a negative Voges-Proskauer reaction, and do not degrade starch or reduce nitrate to nitrite. Additional properties are given in Table 4. The mol% G C of the DNA falls within the range 35 to 36 (T,) for the eleven strains examined. These strains form a distinct DNA homology group that is related to a second homology group which accommodates strains pathogenic for mosquitoes (Krych et al., 1980). Source : Soil. Type strain: ATCC 7055. + Taxonomy of bacilli 1879 Description of Bacillus kaustophilus (Prickett, 1928) nom. rev. kau.sto.ph'il.us. Gr. n. kaustos, heat; Gr, adj.philus loving; M.L. adj. kaustophilus heat loving. The description is taken from the present and several other studies (Prickett, 1928; Walker & Wolf, 1971; Sharp et al., 1980). Strains in this species have similar properties to B . stearothermophilus but differ from members of this species by their ability to produce acid from cellobiose, meso-inositol and xylose, to degrade testosterone and to reduce nitrate to gas, and by their relative sensitivity to NaCl and failure to grow anaerobically. They produce oval to cylindrical spores that distend the sporangium to a greater or less extent, liquefy gelatin, degrade aesculin, arbutin, pullulan and starch (weakly), and grow optimally between 60 and 65 "C. Additional properties are given in Table 4. The mol% G C of the DNA of the five strains studied falls within the range 51 to 55 (T,). There is evidence that these strains form a distinct DNA homology group (Sharp et al., 1980). Source : Pasteurized milk, deteriorated canned food and probably soil. Type strain: ATCC 8005 (= N. R. Smith T281). + Description of Bacillus psychrosaccharolyticus (Larkin & Stokes, 1967) nom. rev. psy.chro.sac.char.o.lyt'i.cus. Gr. adj. psychros cold; Gr. n. saccharon sugar; Gr. adj. lytos dissolvable ; M .L. adj. psychrosaccharolyticus cold (adapted), sugar-fermenting. The description is taken from the present and two other studies (Larkins & Stokes, 1967; Claus & Berkeley, 1986). Cells are distinctly pleomorphic, varying from coccoid to elongate. On glucose media they may contain globules that are unstainable with fuchsin. Growth and sporulation occur at 0 "C. If sporulation does not occur, the organism may swell and become faintly stainable, often forming pear-shaped bodies up to 2 pm in diameter. The spore frequently fills most of the sporangium; it may occur in a lateral position. Relatively thick opaque growth without spreading or outgrowths occurs on agar media. Overgrowth of laboratory cultures by asporogenous mutants appears to occur frequently. Glucose promotes anaerobic growth only slightly. Aesculin, allantoin and arbutin are hydrolysed, and elastin, gelatin, lecithin, pullulan and starch are degraded. The mol% G C of the DNA lies within the range 43 to 44 ( T m ;F.G. Priest, unpublished data). Source: Soil and marshes. Type strain : NCIB 11729 ( = ATCC 23296 = DSM 6). Direct plating of soil frequently yields organisms which have the characteristics of B . psychrosaccharolyticus except that some of them may diverge from that species in their action on nitrate (none or denitrification), proteins, starch, particular sugars, or in utilization of glucose for anaerobic growth. These organisms, which do not appear to have been named, have yet to be the subject of comparative studies to determine their possible relationship to B . psychrosaccharoly ticus. + Description of Bacillus simplex (Gottheil, 1901) nom. rev. sim'plex. L. adj. simplex simple. The description is taken from the present study and that of Hunger & Claus (198 1). Strains in this species have properties in common with B. megateriurn but differ from typical members of that species as they reduce nitrate to nitrite, produce brownish colonies on tyrosine agar, fail to hydrolyse aesculin and urea, do not deaminate phenylalanine or form hydroxybutyrate and have cells that measure only 0.8 to 1.0 pm in diameter (a few broader cells are occasionally observed). They degrade arbutin, gelatin, starch and tyrosine but not chitin. They are negative for the Voges-Proskauer and egg-yolk tests and do not grow in the presence of lysozyme. Additional properties are given in Table 5. The mol% G + C content of the DNA of the six strains examined lies between 40 and 41 (T,). These strains form a distinct DNA homology group (Hunger & Claus, 1981). Source : Soil. Type strain: DSM 1321 (= NRS 960). 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