Introduction

Drug resistance is a major problem in almost all the drug-related fields. Among bacterial resistance, β-lactam-antibiotic resistance is the most prevailing and threatening area in public health, because β-lactam antibiotics have been most widely used for chemotherapy of infectious diseases even in 80 years since penicillin’s discovery.1 β-Lactam antibiotic resistance is caused mainly by two mechanisms: antibiotic-degrading enzymes, β-lactamases2 and modification of target sites, penicillin-binding proteins (PBPs).3

The phylum Actinobacteria constitute one of the largest phyla within the Bacteria.4, 5 Streptomyces species, which belong to Actinobacteria, are filamentous, soil-dwelling, high guanine+cytosine (G+C)-content Gram-positive bacteria and are characterized by their ability to undergo complex cellular differentiation like filamentous fungi.6 In addition, Streptomyces species produce a wide variety of secondary metabolites including β-lactam antibiotics.7, 8, 9 However, Streptomyces species are prokaryotic microorganisms and unlike penicillin- and cephalosporin-producing fungi they must protect themselves from the attack of antibiotics, thus they have to have self-resistant mechanisms.10, 11, 12, 13 In addition, Streptomyces species are known to be highly resistant to benzylpenicillin, although they are Gram-positive bacteria. This review discusses the role of PBPs in resistance to benzylpenicillin in Streptomyces belonging to Actinobacteria.

PBPs

The bacterial cell wall peptidoglycan is a three-dimensional, net-like mesh called sacculus in which glycan strands are cross-linked by peptide chains. It maintains cell shape and provides mechanical strength to resist osmotic pressure.14, 15 The peptidoglycan biosynthesis is catalyzed by glycosyltransferases to polymerize the glycan chains and by transpeptidases to catalyze peptide crosslinking between two adjacent glycan chains. The transpeptidases, also called PBPs, were initially identified as their ability to bind penicillins.16, 17 Depending on the structure and the catalytic activity of their N-terminal domain, they are classified into class A, B and C PBPs.14, 18, 19, 20 The C-terminal domains of both class A and class B PBPs have the transpeptidase activity. In class A PBPs, the N-terminal domain is responsible for their glycosyltransferase activity, whereas in class B PBPs, the glycosyltransferase domain is lacking. Class C PBPs are also called low-MW PBPs, having the carboxypeptidase activity, and are responsible for the maturation and recycling of the peptidoglycan.19 They are not essential and are excluded from further study.

Table 1 summarizes the genome sizes, G+C contents, numbers of PBPs, types of PBPs, class A and class B PBPs and some characters of 113 Actinobacterial species including 30 Streptomyces species. Most species have both class A and class B PBPs. However, the phylogenetic distribution of PBPs among taxa is uneven; Actinomyces odontolyticus ATCC 17982 encodes two PBPs per genome, whereas S. avermitilis MA-4680 and S. coelicolor A3(2) have 13 PBPs. In general, Streptomyces species possess >10 PBPs including class A and class B PBPs.21

Table 1 The numbers and types of putative PBP genes
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Class B PBPs

A phylogenetic tree constructed on the basis of their amino-acid sequences of 446 class B PBPs from 113 Actinobacterial species is classified into 10 clusters and is shown in Figure 1. In general, the PBPs from taxonomically related species go into the same clusters. PBPs of suborder Propionibacterineae belong to subcluster I. PBPs of the members of order Actinomycetales, such as Thermobispora, Streptosporangium, Actinomyces and Mobiluncus, form cluster II. PBPs of other members of Actinomycetales, such as Kytococcus, Kineococcus, Isoptericola, Cellulomonas, Sanguibacter, Beutenbergia, Janibacter, Catenulispora, Renibacterium, Arthrobacter, Saccharomonospora and Micromonospora, constitute cluster IV. Some PBPs of Actinomyces and Mobiluncus fall into subcluster IV-2. PBP members that from suborder Micrococcineae are partly overlapped and distributed in the clusters IV and V. Kytococcus, Isoptericola, Cellulomonas, Sanguibacter, Beutenbergia, Janibacter, Renibacterium and Arthrobacter are members of suborder Micrococcineae. PBPs of order Bifidobacteriales form subcluster III. The cluster V includes PBPs of suborder Micrococcineae members such as Clavibacter, Leifsonia, Tropheryma, Micrococcus, Rothia, Kocuria, Renibacterium and Arthorobacter. However, PBPs of suborder Micrococcineae are also distributed in the cluster IV, as described above. No PBPs of Micrococcineae make a phylogenetically distinct, coherent cluster and are dispersed in clusters IV and V.4 The subclusters VI-1, VI-2 and VI-3 consist of PBPs of suborder Corynebacterineae (Corynebacterium, Tsukamurella, Gordonia, Mycobacterium, Rhodococcus, Nocardia and Amycolicicoccus), suborder Pseudonocardineae (Saccharopolyspora, Saccharomonospora and Actinosynnema) and suborder Frankineae (Nakamurella). However, PBPs of Corynebacterineae and Pseudonocardineae compose different branches in the subclusters. The suborders Corynebacterineae and Pseudonocardineae are closely related taxonomically4, 22 and, in addition, Nakamurella multipartite, which is currently a member of suborder Frankineae, is proposed to be closely related to Pseudonocardineae.4 The PBPs of most Frankia species belong to subclusters VII-1, VII-2, VII-4 and VII-5 which contain also PBPs of suborder Frankineae (Frankia and Acidothermus), suborder Micromonosporineae (Salinispora, Micromonospora and Actinoplanes) and suborder Glycomycineae (Stackebrandtia).

Figure 1
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Phylogenetic tree of 446 class B PBPs listed in Table 1 from Actinobacteria. The tree was constructed by using ClustalX 240 as SCO4049 (penicillin acylase) as outgroup. A full color version of this figure is available at The Journal of Antibiotics journal online.

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As described above, Streptomyces species carry more PBPs than other species. Reflecting this fact, PBPs of Streptomyces species form seven large subclusters of VIII-1, VIII-2, VIII-3, VIII-4, VIII-5, VIII-6 and VIII-7. SCAB_10101 possesses neither essential serine nor lysine residues which are involved in the enzymatic catalysis (the amino-acid sequence in this region is Thr-Thr-Phe-Ser), so that it is excluded from further analysis.

Intriguingly, more than half of Streptomyces species hold two successive class B PBPs (Figure 1 and Table 1). For example, SSHG_03834 and SSHG_03835 (a similarity value E is 1.2e−62, the same hereafter), SAV_3603 and SAV_3604 (3e−68), SCLAV_4179 and SCLAV_4180 (1.5e−11), SMCF_7795 and SMCF_7796 (1.1e−48), SCO3156 and SCO3157 (2.8e−47), BN159_5121 and BN159_5122 (3.4e−65), SSFG_04216 and SSFG_04217 (1.3e−56), SGM_5988 and SGM_5989 (1.3e−58), SSRG_03705 and SSRG_03706 (3.2e−62), SHJG_4627 and SHJG_4628 (8.6e−62), SSPG_04382 and SSPG_04383 (2.7e−59), SSDG_07138 (125aa) and SSDG_07139 (too short to compare), SCAB_53611 and SCAB_53621 (3.2e−59), SSEG_00010 and SSEG_00011 (9.3e−61), and SSQG_03242 and SSQG_03243 (3.5e−61). The amino-acid sequences of these pairs of PBPs are not only very similar to each other, but also all the sequences are closely related and pertain to the subcluster VIII-7 in the phylogenetic tree (Figure 1). That is, the amino-acid sequence identity and similarity of PBPs in subcluster VIII-7 are in the range of 49.2–51.8% and 71.8–77.8%, respectively. Furthermore, the nucleotide sequences of each pair are arrayed in the same direction, indicating that they were duplicated and transferred to each other very recently. The pair of S. clavuligerus SCLAV_4179 and SCLAV_4180 is an exception. S. clavuligerus is a cephamycin and clavulanic acid producer. These PBPs of S. clavuligerus belong to the different subclusters (VIII-6 and VIII-2) and the similarity of the amino-acid sequences is very low (the E-value is 1.5e−11). Although S. clavuligerus possesses two PBPs in subcluster VIII-7, SCLAV_2276 and SCLAV_4198, their similarity value E is not so low (3.1e−28). In addition, S. cattleya, a cephamycin and thienamycin producer, carries only one PBP (SCAT_0768) in this subcluster. This peculiar behavior may be related to β-lactam production. The two PBPs of S. clavuligerus SCLAV_4179 and SCLAV_4180 are located at the end of cephamycin-clavulanic acid biosynthetic gene cluster, but arrayed in the reverse direction. Moreover, PBP SCLAV_4179 in S. clavuligerus is reported to have a low affinity to β-lactam antibiotics and is essential to the growth,23 consequently it is presumed to be involved in the self-resistance. Interestingly, the amino-acid sequence of SCLAV_4179 is highly similar to that of SCAT_5676 (the similarity value E is 8.4e−186) of S. cattleya, indicating that the PBP genes were interchanged between the two species as a whole-cephamycin biosynthetic gene cluster, and the clavulanic acid gene cluster was inserted in this region later. In S. cattleya, the protein of the similar amino-acid sequence to SCLAV_4180 is located not in the next to SCAT_5676 but in the completely different position as SCAT_3088, where no β-lactam biosynthetic gene is present. On the other hand, two proteins having highly similar amino-acid sequences to SCLAV_4179 (SCAT_5676 and SCAT_1730, the similarity E-values are 8.4e−186 and 1e−144, respectively) exist in S. cattleya. In S. clavuligerus, a similar PBP to SCLAV_4179 is present as SCLAV_1774 (E-value is 2.9e−159). Furthermore, a similar protein to SCLAV_4179 is also found in S. coelicolor (SCO2608, an E-value is 1.1e−160). Comparison of the genomic arrangements in these three species reveals similar arrangements of the genes, at least in the downstream of PBPs (Table 2). Moreover, similar proteins to SCLAV_4179 are present not only in Streptomyces species such as S. avermitilis MA-4680 (SAV_5458, an E-value is 2.5e−158, the same hereafter), S. lividans TK24 (SSPG_04919, 3.7e−169), S. viridochromogenes DSM 40736 (SSQG_02628, 5.6e−161), S. scabiei 87.22 (SCAB_60051, 1.7e−174), S. griseus (SGR_4934, 4.5e−165) and S. hygroscopicus (SHJG_4100, 2.9e−163), but also in Catenulispora acidiphila DSM 44928 (Caci_7282, 1.2e−101), Kitasatospora setae KM-6054 (KSE_26130, 5.9e−137), Kribbella flavida DSM 17836 (Kfla_2302, 4.3e−105) and Nocardioides sp. JS614 (Noca_3462, 2.0e -108).24 That is, the amino-acid sequence identity and similarity are in the range of 74.3–75.5% and 90.8–92.5% in Streptomyces, respectively, and 45.7–62.7% and 72.6–83.2% in other species, respectively. None of these species is reported to produce β-lactam antibiotics, suggesting that SCLAV_4179 and its analogs in Streptomyces at least are not related to β-lactam biosynthesis but associated only with β-lactam resistance. This is supported by the fact that similar proteins to SCLAV_4179 are also found in other Actinobacteria, such as Thermomonospora curvata DSM 43183 (Tcur_1542, 6.3e−99), Frankia sp. EuI1c (FraEuI1c_1960, 4.9e−76), Nocardiopsis dassonvillei (Ndas_3385, 5.2e−108) and Janibacter sp. HTCC2649 (JNB_05649, 4.3e−72), β-proteobacteria, such as Methylobacillus flagellatus KT (YP_546600, 5.2e−40) and Janthinobacterium lividum (WP_010393193, 3.9e−49), and γ-proteobacteria, such as Plesiomonas shigelloides (WP_010864271, 9.8e−36) and Pseudomonas putida (WP_009397921, 4.3e−38). The PBP of S. clavuligerus, SCLAV_4179, is reported to have a low affinity to β-lactam antibiotics.23 Therefore, the low-affinity PBP gene of S. clavuligerus (SCLAV_4179) is supposed to overspread to most Actinobacteria, especially to Streptomyces species, to have a major role in β-lactam resistance and to reflect on the fact that most Streptomyces species, in particular, are highly resistance to benzylpenicillin, although they are Gram-positive bacteria.25 In addition, two low-affinity type PBPs, SCLAV_1774 and SCLAV_4179, and SCAT_1730 and SCAT_5676, greedily present in S. clavuligerus and S. cattleya, β-lactam-antibiotic producers, reinforces their self-resistance to their own β-lactams.

Table 2 Comparison of genomic arrangements adjacent to PBPs in three Streptomyces species
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Another PBP proposed to be involved in the self-resistance in S. clavuligerus is SCLAV_4198.26, 27 SCLAV_4198 belongs to subcluster VIII-7 together with SCLAV_2276. Most Streptomyces species occupy two or three PBPs in this subcluster. These PBPs are supposed to strengthen further the self-resistance against β-lactam antibiotics in these Streptomyces species. Furthermore, most of these PBPs are adjacent to each other, as described above. S. clavuligerus is again an exception.

Goffin and Ghuysen18, 26 showed that class B PBPs from Gram-positive bacteria were classified into three distinct subclasses, B1 (whose prototype is Enterococcus faecium PBP5, X84859), B4 (whose prototype is S. pneumoniae PBP2x, P14677) and B5 (whose prototype is S. pneumoniae PBP2b, P10524). Phylogenetic and similarity analyses indicate that all the PBPs from Streptomyces analyzed in this paper form disparate clusters from these subclass members, and SCLAV_4179 is only distantly related to class B1/B2 PBPs, low-affinity class PBPs, rather than class B4 or B5 PBPs, whether they are analyzed in whole sequence or penicillin-binding core sequences26 (see Supplementary Figure 1).

Protein kinases are classified into two families based on their biochemical similarities and enzymatic specifications as following: the histidine kinase superfamily belonging to the two component systems28 and the serine/threonine and tyrosine protein kinase superfamily.29, 30 Recently, these serine/threonine and tyrosine protein kinases were shown to be involved in the regulation of cell morphogenesis of Streptococcus pneumonia,31 Corynebacterium glutamicum32 and Mycobacterium tuberculosis,33 germination of Bacillus subtilis spores34 and polar growth and hyphal branching in S. coelicolor.35 Although investigating the S. coelicolor homolog of PknB, a serine/threonine protein kinase of M. tuberculosis, Yeats et al.36 identified a novel domain called PASTA domain that is found in the C-termini of eukaryotic-like serine/threonine kinases and PBPs. This domain binds β-lactam antibiotics and their peptidoglycan analogs. It is intriguing in this connection that serine/threonine protein kinases are present next to PBPs in Streptomyces species. Furthermore, these protein kinases carry four PASTA domains in tandem in these molecules. Such protein kinases are K530_23511, SSHG_02907, SAV_4338, SBI_05406, SCAT_3089, SCATT_30800, SCLAV_2946, SMCF_8885, SCO3848, B446_19315, BN159_4479, SFLA_3201, SSFG_03588, SGM_3503, SSRG_03159, SGR_3725, SHJG_5218, SSPG_03807, SSDG_03054, SRIM_00070, SrosN1_010100017505, SCAB_45561, F750_3547, SACTE_3284, SSEG_02705, STSU_17414, SVEN_3632, STRV1_0274, SSQG_03956 and SZN_17937. However, although these PBPs adjacent to the protein kinases belong to the same subcluster VIII-3, they have no PASTA domain in their molecules in contrast to PBPs in other bacteria. The PBPs in Actinobacteria such as M. tuberculosis class A PBP (accession number is YP_178005, the same hereafter), Rhodococcus sp. DK17 (WP_016884523) and Nocardia sp. BMG111209 (WP_019931711) possess one PASTA domain each in their C-terminal region. Intriguingly, the amino-acid sequences of the protein kinases in this group are almost the same with each other, especially in N-terminal regions containing the protein kinase domains. Protein kinases located adjacent to PBPs are also seen in other Actinobacteria such as Cfla_0025 and Cfla_0026, AMIR_0021 and AMIR_0022, TCUR_0063 and TCUR_0064, Snas_6471 and Snas_6472, and Afer_0087 and Afer_0088. Two protein kinases arrange in tandem, and then comes PBP. Although the function of these protein kinases and the relationship to PBPs are not known yet, they might involve in peptidoglycan biosynthesis in concert with PBPs.

Class A PBPs

A phylogenetic tree constructed on the basis of their amino-acid sequences of 292 class A PBPs from Actinobacteria is classified into 10 clusters and is shown in Figure 2. Like the class B PBPs, the PBPs from taxonomically related species form the same clusters. Accordingly, cluster I consists of PBPs of subclass Rubrobacteridae (Rubrobacter and Conexibacter) and Coriobacteriae (Atopobium, Cryptobacterium and Slackia). All the PBPs of Trophyryma, Clavibacter and Leifsonia compose cluster II. The similarity of the amino-acid sequences between TW_0722 and TWT_0705 is 100% except the C-terminal amino acid, where glutamic acid is replaced by aspartic acid. Amino-acid sequence similarity values (E-values) are 1.3e−68 between TW_0722 and CMM_0915, 1.2e−86 between TW_0722 and LXX_03600, and 2.5e−70 between TW_0722 and LXX_02090. Among Micrococcineae PBPs, PBPs of three genera (Trophyryma, Clavibacter and Leifsonia) behave as a group like the class B PBPs. Cluster III is made up of PBPs of order Bifidobacteriales (Bifidobacterium and Gardnerella). These PBPs are divided into two subclusters, III-1 and III-2. PBPs of suborder Actinomycineae (Actinomyces and Mobiluncus) and Micrococcineae (Isoptericola, Beutenbergia, Brevibacterium, Micrococcus, Rothia, Kocuria, Janibacter, Renibacterium and Arthrobacter), together with two PBPs of genus Kineococcus (KRAD_0429 and KRAD_4341), form subclusters IV-1, IV-2 and IV-3. The class A PBPs of Kineococcus radiotolerans behave with those of suborder Micrococcineae ,such as Brevibacterium, but other PBPs of suborder Micrococcineae, such as Isoptericola and Beutenbergia, comport themselves with those of suborder Actinomycineae, such as Actinomyces and Mobiluncus (subclusters IV-1 and IV-3). PBPs of suborder Corynebacterineae (Corynebacterium, Tsukamurella, Gordonia, Mycobacterium, Rhodococcus, Nocardia and Amycolicicoccus) and Pseudonocardineae (Saccharopolyspora, Saccharomonospora and Actinosynnema) form subclusters V-1 and V-2 but different branches, although the amino acid similarity is not so different between PBPs of these branches. PBPs of genus Nakamurella move with those of suborder Pseudonocardineae like the class B PBPs, although genus Nakamurella is classified as suborder Frankineae. Subclusters VI-1 and VI-2 consist of PBPs of suborder Micromonosporineae (Salinispora, Micromonospora and Actinoplanes). PBPs of genus Stackebrandtia form outgroups in the phylogenetic tree as suggested by the taxonomic position. PBPs of genus Frankia form distinct subclusters VII-1 and VII-2, and those of genus Propionibacterium construct other discrete subclusters IX-1 and IX-2.

Figure 2
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Phylogenetic tree of 292 class A PBPs listed in Table 1 from Actinobacteria. The tree was constructed by using ClustalX 240 as SCO4049 (penicillin acylase) as outgroup. A full color version of this figure is available at The Journal of Antibiotics journal online.

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Like the class B PBPs, class A PBPs of Streptomyces species form large five subclusters, VIII-1, VIII-2, VIII-3, VIII-4 and VIII-5. Similarity analyses of class A PBPs from Streptomyces indicate that those inherent in the same subcluster have very low E-values irrespective of different species, especially PBPs in subcluster VIII-1 (Table 3). In addition, E-values between PBPs belonging to different subclusters but from the same species are not so different from those from different species. The amino-acid sequence similarity among class A PBPs are generally higher than those among class B PBPs. Another interesting fact clarified by amino-acid alignment analysis is that among 13 PBPs in subcluster VIII-3, 4 PBPs (SGM_0550, SHJG_3853, STRVI_2314 and SBI_03076) do not have essential serine residues in the motif SXXK. In addition, except two PBPs (SRIM_22689 and KSE_38960), they do not possess essential lysine residues, although other features18, 26 requisite for PBPs are conserved (Figure 3), suggesting that it is doubtful whether these PBPs function as transpeptidases or the transpeptidase activity is very low even though they retain penicillin-binding properties.

Table 3 Similarity of amino-acid sequences of class A PBPs in Streptomyces (E-values)
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Figure 3
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Amino-acid alignment of 13 PBPs in subcluster VIII-3 of the phylogenetic tree (Figure 2). The amino-acid sequences are aligned by using MUSCLE.41 The conserved motifs18 are boxed, and the essential SXXK sequences are marked with red, bold letters. A full color version of this figure is available at The Journal of Antibiotics journal online.

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Class A PBPs from Gram-positive bacteria are classified into five subclasses,26 A1 (whose prototype is Escherichia coli PBP1A), A2 (whose prototype is E. coli PBP1B), A3 (whose prototype is Streptococcus pneumoniae 1A), A4 (whose prototype is S. pneumoniae 2A) and A5 (whose prototype is S. pneumoniae 1B). Phylogenetic and similarity analyses indicate that all the class A PBPs from Streptomyces analyzed in this paper form a completely different cluster in a phylogenetic tree from these five clusters, where E-values are in the range of 3.4e−16 to 4.9e−31, indicating very low similarities (see Supplementary Figure 2). These results, together with the results in class B PBPs where E-values range from 9.3 to 7.7e−39, suggests strongly that the gene transfer and/or gene conversion occurred very rarely between PBPs in Streptomyces and those in Gram-positive and Gram-negative bacteria.

PBPs with low affinity to penicillins

Ogawara and Horikawa37 reported over 30 years ago that β-lactam-producing Streptomyces species possessed PBPs of very low affinity to benzylpenicillin. Later, two PBPs, that is, SCLAV_4179 and SCLAV_4198 were reported to have low affinity to penicillins.23, 27 A mutant disrupted in SCLAV_4198 gene exhibited a significant decrease in its resistance to benzylpenicillin and cephalosporins.27 Moreover, a probe containing SCLAV_4198 hybridized to genomic DNAs from β-lactam producers, S. jumonjinensis NRRL 5741, S. griseus NRRL 3851 and S. lipmanii NRRL 3584, suggesting that SCLAV_4198-like sequences and SCLAV_4198-mediated resistance mechanisms are likely to be present in these β-lactam-producing species. Table 4 lists low-affinity PBPs and some of the closely related PBPs in Streptomyces. The PBPs belonging to subclusters VIII-6 and VIII-7 in Figure 1 are assumed to have low affinity to penicillins.

Table 4 Low-affinity and some closely related PBPs in Streptomyces
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Conclusion

The work on self-resistance to β-lactam antibiotics in Actinobacteria in my research career started by the findings that most of the Streptomyces species constitutively produced β-lactamase independent of their resistance to β-lactam antibiotics and β-lactam production,25, 38 and the detection of PBPs in Streptomyces species by autoradiography took over 6 months instead of a few days in E. coli16 and B. subtilis.37 When I visited Dr Hamao Umezawa, my boss at that time, for the proofreading of the paper, he immediately said that ‘The avoidance of the contamination of Streptomyces species was the most important and absolute necessity in the fermentation of Penicillium for the production of benzylpenicillin. It caused the complete destruction of benzylpenicillin because of their production of β-lactamases.’ He knew by experience that most Streptomyces species produced β-lactamases. On the basis of these two findings, I proposed about 35 years ago in Antimicrobial Agents and Chemotherapy37 and Microbiological Reviews39 that low-affinity PBPs were the main cause of self-resistance to β-lactam antibiotics in Streptomyces. Since then, supporting evidence is gradually accumulating. This review offers some substantiating evidence from the points of PBPs for self-resistance and resistance in general to β-lactam antibiotics in Streptomyces even though they are Gram-positive bacteria.