Pseudomonas aeruginosa (PsA) is an opportunistic bacterial pathogen that causes life-threatening infections in individuals with compromised immune systems and exacerbates health concerns for those with cystic fibrosis (CF). PsA rapidly... more
Pseudomonas aeruginosa (PsA) is an opportunistic bacterial pathogen that causes life-threatening infections in individuals with compromised immune systems and exacerbates health concerns for those with cystic fibrosis (CF). PsA rapidly develops antibiotic resistance; thus, novel therapeutics are urgently needed to effectively combat this pathogen. Previously, we have shown that a novel cationic Zinc (II) porphyrin (ZnPor) has potent bactericidal activity against planktonic and biofilm-associated PsA cells, and disassembles the biofilm matrix via interactions with eDNA In the present study, we report that ZnPor caused a significant decrease in PsA populations in mouse lungs within an in vivo model of PsA pulmonary infection. Additionally, when combined with an obligately lytic phage PEV2, ZnPor at its minimum inhibitory concentration (MIC) displayed synergy against PsA in an established in vitro lung model resulting in greater protection of H441 lung cells versus either treatment alo...
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The purpose of this study was to determine whether there was an association between kinetics of atrazine mineralization, the number and type of atrazine-degrading microorganisms, and the presence of three genes representing different... more
The purpose of this study was to determine whether there was an association between kinetics of atrazine mineralization, the number and type of atrazine-degrading microorganisms, and the presence of three genes representing different steps in the degradative pathway of atrazine in three soils with different histories of atrazine application. Composite soil samples were collected from two agricultural fields and one
Research Interests: Chemistry, Kinetics, Biology, Hybridization, Biological Sciences, and 15 moreBiodegradation, Environmental Sciences, Pesticide, Carbon Isotopes, Pesticides, DNA Extraction, Bacteria, DNA hybridization, Mineralization, Soil Biology, Atrazine, Microbial community, Most Probable Number, Fungicide, and HPLC Chromatography
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The N-acyl homoserine lactone (AHL) quorum-sensing signals produced by Sinorhizobium meliloti strains AK631 and 1021 when cultured in a defined glucose-nitrate medium were identified by gas chromatography/mass spectrometry (GC/MS) and... more
The N-acyl homoserine lactone (AHL) quorum-sensing signals produced by Sinorhizobium meliloti strains AK631 and 1021 when cultured in a defined glucose-nitrate medium were identified by gas chromatography/mass spectrometry (GC/MS) and electrospray ionization tandem mass spectrometry (ESI MS/MS). Both strains synthesized several long-chain AHLs. Defined medium cultures of strain AK631 synthesized a complex mixture of AHLs with short acyl side chains. Strain 1021 produced no short-chain AHLs when grown on defined medium and made a somewhat different set of long-chain AHLs than previously reported for cultures in rich medium. While the two strains produced several AHLs in common, the differences in AHLs produced suggest that there may be significant differences in their patterns of quorum-sensing regulation.
Research Interests: Biochemistry, Microbiology, Chemistry, Mass Spectrometry, Medical Microbiology, and 10 moreMedicine, Quorum Sensing, Tandem Mass Spectrometry, High Pressure Liquid Chromatography, Liquid Chromatography / Electrospray Ionization Mass Spectrometry, Gas Chromatography/mass Spectrometry, Sinorhizobium meliloti, Culture Media, Butyrolactone, and Electrospray Ionization
Abstract In the present study, the use of bacteriophages to prevent growth and/or biofouling by Pseudomonas aeruginosa PAO1 was investigated in microcosms containing Jet A aviation fuel as the carbon source. Bacteriophages were found to... more
Abstract In the present study, the use of bacteriophages to prevent growth and/or biofouling by Pseudomonas aeruginosa PAO1 was investigated in microcosms containing Jet A aviation fuel as the carbon source. Bacteriophages were found to be effective at preventing biofilm formation but did not always prevent planktonic growth in the microcosms. This result was at odds with experiments conducted in nutrient-rich medium, demonstrating the necessity to test antimicrobial and antifouling strategies under conditions as near as possible to the ‘real world’. The success of the bacteriophages at preventing biofilm formation makes them potential candidates as antifouling agents for fuel systems.
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One of the greatest threats to human health is the rise in antibiotic-resistant bacterial infections. Pseudomonas aeruginosa (PsA) is an “opportunistic” pathogen known to cause life-threatening infections in immunocompromised individuals... more
One of the greatest threats to human health is the rise in antibiotic-resistant bacterial infections. Pseudomonas aeruginosa (PsA) is an “opportunistic” pathogen known to cause life-threatening infections in immunocompromised individuals and is the most common pathogen in adults with cystic fibrosis (CF). We report here a cationic zinc (II) porphyrin, ZnPor, that effectively kills planktonic and biofilm-associated cells of PsA. In standard tests against 16–18 h-old biofilms, concentrations as low as 16 µg/mL resulted in the extensive disruption and detachment of the matrix. The pre-treatment of biofilms for 30 min with ZnPor at minimum inhibitory concentration (MIC) levels (4 µg/mL) substantially enhanced the ability of tobramycin (Tobra) to kill biofilm-associated cells. We demonstrate the rapid uptake and accumulation of ZnPor in planktonic cells even in dedicated heme-uptake system mutants (ΔPhu, ΔHas, and the double mutant). Furthermore, uptake was unaffected by the ionophore ca...
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Motile swarmer cells of Hyphomicrobium strain W1-1B displayed positive chemotactic responses toward methylamine, dimethylamine, and trimethylamine but did not display significant chemotactic responses towards methanol and arginine.... more
Motile swarmer cells of Hyphomicrobium strain W1-1B displayed positive chemotactic responses toward methylamine, dimethylamine, and trimethylamine but did not display significant chemotactic responses towards methanol and arginine. Electron micrographs of negatively stained intact flagellar filaments indicated a novel striated surface pattern. The flagella were composed of two proteins of 39 and 41 kDa. Neither protein was a glycoprotein as determined by Schiff’s staining and by enzyme immunoassay. Protein fingerprints visualized from silver-stained polyacrylamide gels and Western blots of protease-digested samples indicated that the two proteins were similar but not identical. Monoclonal antibodies prepared to the complex flagella of Rhizobium meliloti cross-reacted with the striated flagella of Hyphomicrobium strain W1-1B; however, these antibodies did not cross-react with smooth-surface flagella. These results suggest that complex and striated flagella possess homologous epitope ...
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Two quorum-sensing systems (las and rhl) regulate virulence gene expression in Pseudomonas aeruginosa. The las system consists of a transcriptional activator, LasR, and LasI, which directs the synthesis of the autoinducer... more
Two quorum-sensing systems (las and rhl) regulate virulence gene expression in Pseudomonas aeruginosa. The las system consists of a transcriptional activator, LasR, and LasI, which directs the synthesis of the autoinducer N-(3-oxododecanoyl) homoserine lactone (PAI-1). Induction of lasB (encoding elastase) and other virulence genes requires LasR and PAI-1. The rhl system consists of a putative transcriptional activator, RhlR, and RhlI, which directs the synthesis of N-butyryl homoserine lactone (PAI-2). Rhamnolipid production in P. aeruginosa has been reported to require both the rhl system and rhlAB (encoding a rhamnosyltransferase). Here we report the generation of a delta lasI mutant and both delta lasI delta rhlI and delta lasR rhlR::Tn501 double mutants of strain PAO1. Rhamnolipid production and elastolysis were reduced in the delta lasI single mutant and abolished in the double-mutant strains. rhlAB mRNA was not detected in these strains at mid-logarithmic phase but was abunda...
Research Interests: Bacteriology, Biology, Biological Sciences, Humans, Movement, and 15 moreQuorum Sensing, Rhamnolipid, Pseudomonas aeruginosa, Genes, Epithelial cells, Glycolipids, Pseudomonas Aeruginosa, Bacterial Adhesion, DNA binding proteins, Lactones, Rhamnose, Plasminogen Activator Inhibitor, Cell Membrane, binding sites, and Medical and Health Sciences
A combination therapy for treating a bacterial biofilm comprises a therapeutically effective amount of an antibiotic comprising an aminoglycoside or tetracycline, or a combination of two or more thereof, and a cationic porphyrin in an... more
A combination therapy for treating a bacterial biofilm comprises a therapeutically effective amount of an antibiotic comprising an aminoglycoside or tetracycline, or a combination of two or more thereof, and a cationic porphyrin in an amount effective for enhancing the effectiveness of the antibiotic in treating the bacterial biofilm. A method of treating a bacterial biofilm comprising Pseudomonas aeruginosa comprises contacting the bacterial biofilm with a combination therapy comprising a therapeutically effective amount of an antibiotic, and a cationic porphyrin in an amount effective for enhancing the effectiveness of the antibiotic in treating the bacterial biofilm. A method of treating a bacterial biofilm comprising Pseudomonas aeruginosa in a patient comprises administering to the patient a combination therapy comprising a therapeutically effective amount of an antibiotic, and a cationic porphyrin in an amount effective for enhancing the effectiveness of the antibiotic in treating the bacterial biofilm
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Earlier work showed that higher plants produce unidentified compounds that specifically stimulate or inhibit quorum sensing (QS) regulated responses in bacteria. The ability of plants to produce substances that affect QS regulation may... more
Earlier work showed that higher plants produce unidentified compounds that specifically stimulate or inhibit quorum sensing (QS) regulated responses in bacteria. The ability of plants to produce substances that affect QS regulation may provide plants with important tools to manipulate gene expression and behavior in the bacteria they encounter. In order to examine the kinds of QS active substances produced by the model legume M. truncatula, young seedlings and seedling exudates were systematically extracted with various organic solvents, and the extracts were fractionated by reverse phase C18 high-performance liquid chromatography. M. truncatula appears to produce at least 15 to 20 separable substances capable of specifically stimulating or inhibiting responses in QS reporter bacteria, primarily substances that affect QS regulation dependent on N-acyl homoserine lactone (AHL) signals. The secretion of AHL QS mimic activities by germinating seeds and seedlings was found to change sub...
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Soil bacteria employ multitiered signaling mechanisms to structure multicellular communities, coordinate behaviors within these communities, and to interact with their eukaryotic hosts. Bacteria deploy signals to estimate numbers of... more
Soil bacteria employ multitiered signaling mechanisms to structure multicellular communities, coordinate behaviors within these communities, and to interact with their eukaryotic hosts. Bacteria deploy signals to estimate numbers of bacterial cells within diffusion-limited environments and then modulate gene expression in response to changes in population densities. This behavior is known as “quorum sensing” (QS). Many Gram-negative bacteria use N-acyl homoserine
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The unicellular soil-freshwater alga Chlamydomonas reinhardtii was found to secrete substances that mimic the activity of the N-acyl-l-homoserine lactone (AHL) signal molecules used by many bacteria for quorum sensing regulation of gene... more
The unicellular soil-freshwater alga Chlamydomonas reinhardtii was found to secrete substances that mimic the activity of the N-acyl-l-homoserine lactone (AHL) signal molecules used by many bacteria for quorum sensing regulation of gene expression. More than a dozen chemically separable but unidentified substances capable of specifically stimulating the LasR or CepR but not the LuxR, AhyR, or CviR AHL bacterial quorum sensing reporter strains were detected in ethyl acetate extracts of C. reinhardtii culture filtrates. Colonies of C. reinhardtii and Chlorella spp. stimulated quorum sensing-dependent luminescence in Vibrio harveyi, indicating that these algae may produce compounds that affect the AI-2 furanosyl borate diester-mediated quorum sensing system of Vibrio spp. Treatment of the soil bacterium Sinorhizobium meliloti with a partially purified LasR mimic from C. reinhardtii affected the accumulation of 16 of the 25 proteins that were altered in response to the bacterium's o...
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Legume-nodulating bacteria (rhizobia) usually produce N -acyl homoserine lactones, which regulate the induction of gene expression in a quorum-sensing (or population-density)-dependent manner. There is significant diversity in the types... more
Legume-nodulating bacteria (rhizobia) usually produce N -acyl homoserine lactones, which regulate the induction of gene expression in a quorum-sensing (or population-density)-dependent manner. There is significant diversity in the types of quorum-sensing regulatory systems that are present in different rhizobia and no two independent isolates worked on in detail have the same complement of quorum-sensing genes. The genes regulated by quorum sensing appear to be rather diverse and many are associated with adaptive aspects of physiology that are probably important in the rhizosphere. It is evident that some aspects of rhizobial physiology related to the interaction between rhizobia and legumes are influenced by quorum sensing. However, it also appears that the legumes play an active role, both in terms of interfering with the rhizobial quorum-sensing systems and responding to the signalling molecules made by the bacteria. In this article, we review the diversity of quorum-sensing regu...
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Many behaviors in bacteria, including behaviors important to pathogenic and symbiotic interactions with eukaryotic hosts, are regulated by a mechanism called quorum sensing (QS). A “quorum-quenching” approach was used here to identify... more
Many behaviors in bacteria, including behaviors important to pathogenic and symbiotic interactions with eukaryotic hosts, are regulated by a mechanism called quorum sensing (QS). A “quorum-quenching” approach was used here to identify QS-regulated behaviors in the N-fixing bacterial symbiont Sinorhizobium meliloti. The AiiA lactonase from Bacillus produced in S. meliloti was shown to enzymatically inactivate S. meliloti's N-acyl homoserine lactone (AHL) QS signals, thereby disrupting normal QS regulation. Sixty proteins were differentially accumulated in the AiiA-producing strain versus the control in early log or early stationary phase cultures. Fifty-two of these QS-regulated proteins, with putative functions that include cell division, protein processing and translation, metabolite transport, oxidative stress, and amino acid metabolism, were identified by peptide mass fingerprinting. Transcription of representative genes was reduced significantly in the AiiA-producing strain,...
Research Interests: Genetics, Microbiology, Plant Biology, Symbiosis, Biology, and 14 moreMedicine, Cell Division, Quorum Sensing, Molecular Plant Microbe Interactions, Medicago, Amino Acid Profile, Sinorhizobium meliloti, Quntitative Thin Layer Chromatography, Proteome, Host Plant, Butyrolactone, Quorum Quenching, Amino Acid Metabolism, and Structural similarity
In gram-negative bacteria, many important changes in gene expression and behavior are regulated in a popula tion density-dependent fashion by N-acyl homoserine lac tone (AHL) signal molecules. Exudates from pea (Pisum sativum) seedlings... more
In gram-negative bacteria, many important changes in gene expression and behavior are regulated in a popula tion density-dependent fashion by N-acyl homoserine lac tone (AHL) signal molecules. Exudates from pea (Pisum sativum) seedlings were found to contain several separable activities that mimicked AHL signals in well-characterized bacterial reporter strains, stimulating AHL-regulated be haviors in some strains while inhibiting such behaviors in others. The chemical nature of the active mimic com pounds is currently unknown, but all extracted differently into organic solvents than common bacterial AHLs. Various species of higher plants in addition to pea were found to secrete AHL mimic activities. The AHL signal-mimic compounds could prove to be important in determining the outcome of interactions between higher plants and a diver sity of pathogenic, symbiotic, and saprophytic bacteria.
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Proteome analysis revealed that two long-chain N -acyl homoserine lactones (AHLs) produced by Sinorhizobium meliloti 1021 induced significant differences in the accumulation of more than 100 polypeptides in early-log-phase cultures of the... more
Proteome analysis revealed that two long-chain N -acyl homoserine lactones (AHLs) produced by Sinorhizobium meliloti 1021 induced significant differences in the accumulation of more than 100 polypeptides in early-log-phase cultures of the wild type. Fifty-six of the corresponding proteins have been identified by peptide mass fingerprinting. The proteins affected by addition of these two AHLs had diverse functions in carbon and nitrogen metabolism, energy cycles, metabolite transport, DNA synthesis, and protein turnover. Two hours of exposure to 3-oxo-C 16:1 -homoserine lactone (3-oxo-C 16:1 -HL) affected the accumulation of 40 of the 56 identified proteins, whereas comparable exposure to C 14 -HL affected 13 of the 56 proteins. Levels of four proteins were affected by both AHLs. Exposure to 3-oxo-C 16:1 -HL for 8 h affected the accumulation of 17 proteins, 12 of which had reduced accumulation. Of the 80 proteins identified as differing in accumulation between early-log- and early-st...
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Current studies have indicated the utility of photodynamic therapy using porphyrins in the treatment of bacterial infections. Photoactivation of porphyrins results in the production of singlet oxygen ((1)O(2)) that damages biomolecules... more
Current studies have indicated the utility of photodynamic therapy using porphyrins in the treatment of bacterial infections. Photoactivation of porphyrins results in the production of singlet oxygen ((1)O(2)) that damages biomolecules associated with cells and biofilms, e.g., proteins, polysaccharides, and DNA. The effect of a cationic porphryin on P. aeruginosa PAO1 biofilms was assessed by exposing static biofilms to 5,10,15,20-tetrakis(1-methyl-pyridino)-21H,23H-porphine, tetra-p-tosylate salt (TMP) followed by irradiation. Biofilms were visualized using confocal laser scanning microscopy (CLSM) and cell viability determined using the LIVE/DEAD BacLight viability assay and standard plate counts. At a concentration of 100 μM TMP, there was substantial killing of P. aeruginosa PAO1 wild-type and pqsA mutant biofilms with little disruption of the biofilm matrix or structure. Exposure to 225 μM TMP resulted in almost complete killing as well as the detachment of wild-type PAO1 biofilms. In contrast, pqsA mutant biofilms that contain less extracellular DNA remained intact. Standard plate counts of cells recovered from attached biofilms revealed a 4.1-log(10) and a 3.9-log(10) reduction in viable cells of wild-type PAO1 and pqsA mutant strains, respectively. Our results suggest that the action of photoactivated TMP on P. aeruginosa biofilms is two-fold: direct killing of individual cells within biofilms and detachment of the biofilm from the substratum. There was no evidence of porphyrin toxicity in the absence of light; however, biofilms pretreated with TMP without photoactivation were substantially more sensitive to tobramycin than untreated biofilms.
Research Interests: Microbiology, Chemistry, Biofilms, Medical Microbiology, Life Sciences, and 13 moreConfocal Microscopy, Porphyrins, Photodynamic Therapy, Biofilm, Cell Viability, Pseudomonas aeruginosa, Porphyrin, Singlet Oxygen, Confocal Laser Scanning Microscopy, Pseudomonas Aeruginosa, Microbial Viability, Cations, and Tobramycin
Twitching motility allows Pseudomonas aeruginosa to respond to stimuli by extending and retracting its type IV pili (TFP). PilJ is a protein necessary for this surface-associated twitching motility and bears high sequence identity with... more
Twitching motility allows Pseudomonas aeruginosa to respond to stimuli by extending and retracting its type IV pili (TFP). PilJ is a protein necessary for this surface-associated twitching motility and bears high sequence identity with Escherichia coli methyl-accepting chemotaxis proteins (MCP). Here, we report that whereas wild-type P. aeruginosa PAO1 cells have extended pili at a single pole, pilJ mutant cells have shortened pili often at both poles despite normal levels of pilin accumulation, suggesting that PilJ is required for full TFP assembly/extension. Using yellow fluorescent protein fusions (pilJ-yfp), both plasmid born and in-frame chromosomal constructs, we determined that PilJ localizes to both poles of the cell. Overexpression of pilJ-yfp resulted in the protein accumulating between the poles.
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Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance... more
Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance as well as their ability to affect lateral gene transfer and to harbor bacterial 16S rRNA gene sequences. Here, we demonstrate the ability of broad-host-range, generalized transducing phages to acquire 16S rRNA genes and gene sequences. Using PCR and primers specific to conserved regions of the 16S rRNA gene, we have found that generalized transducing phages (D3112, UT1, and SN-T), but not specialized transducing phages (D3), acquired entire bacterial 16S rRNA genes. Furthermore, we show that the broad-host-range, generalized transducing phage SN-T is capable of acquiring the 16S rRNA gene from two different genera: Sphaerotilus natans , the host from which SN-T was originally isolated, and Pseudomonas aeruginosa . In sequential infections, SN-T harb...
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Many bacteria use quorum sensing (QS) as an intercellular signaling mechanism to regulate gene expression in local populations. Plant and algal hosts, in turn, secrete compounds that mimic bacterial QS signals, allowing these hosts to... more
Many bacteria use quorum sensing (QS) as an intercellular signaling mechanism to regulate gene expression in local populations. Plant and algal hosts, in turn, secrete compounds that mimic bacterial QS signals, allowing these hosts to manipulate QS-regulated gene expression in bacteria. Lumichrome, a derivative of the vitamin riboflavin, was purified and chemically identified from culture filtrates of the alga Chlamydomonas as a QS signal-mimic compound capable of stimulating the Pseudomonas aeruginosa LasR QS receptor. LasR normally recognizes the N-acyl homoserine lactone (AHL) signal, N-3-oxo-dodecanoyl homoserine lactone. Authentic lumichrome and riboflavin stimulated the LasR receptor in bioassays and lumichrome activated LasR in gel shift experiments. Amino acid substitutions in LasR residues required for AHL binding altered responses to both AHLs and lumichrome or riboflavin. These results and docking studies indicate that the AHL binding pocket of LasR recognizes both AHLs a...
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Pseudomonas aeruginosa translocates over solid surfaces by a type IV pilus-dependent form of multicellular motility known as twitching. We wondered whether cells utilize endogenous factors to organize twitching, and we purified from... more
Pseudomonas aeruginosa translocates over solid surfaces by a type IV pilus-dependent form of multicellular motility known as twitching. We wondered whether cells utilize endogenous factors to organize twitching, and we purified from wild-type cells a lipid that caused directed movement. Wild-type P. aeruginosa, but not a pilJ pilus-deficient mutant, showed biased movement up gradients of phosphatidylethanolamine (PE) established in agar. Activity was related to the fatty acid composition of the lipid, as two synthetic PE species, dilauroyl and dioleoyl PE, were capable of directing P. aeruginosa motility while many other species were inactive. P. aeruginosa PE did not contain either laurate or oleate, implying that the native attractant species contains different fatty acids. Uniform concentrations of PE increased cell velocity, suggesting that chemokinesis may be at least partly responsible for directed movement. We speculate that PE-directed twitching motility may be involved in b...