Brian J Wilkinson

Illinois State University, Maywood, Illinois, United States

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Publications (100)274.87 Total impact

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    ABSTRACT: Listeria monocytogenes is a psychrotolerant food borne pathogen, responsible for the high fatality disease listeriosis, and expensive food product recalls. Branched-chain fatty acids (BCFAs) of the membrane play a critical role in providing appropriate membrane fluidity and optimum membrane biophysics. The fatty acid composition of a BCFA-deficient mutant is characterized by high amounts of straight-chain fatty acids and even-numbered iso fatty acids, in contrast to the parent strain where odd-numbered anteiso fatty acids predominate. The presence of 2-methylbutyrate (C5) stimulated growth of the mutant at 37°C and restored growth at 10°C along with the content of odd-numbered anteiso fatty acids. The C6 branched-chain carboxylic acids 2-ethylbutyrate and 2-methylpentanoate also stimulated growth to a similar extent as 2-methylbutyrate. However, 3-methylpentanoate was ineffective in rescuing growth. 2-Ethylbutyrate and 2-methylpentanoate led to novel major fatty acids in the lipid profile of the membrane that were identified as 12-ethyltetradecanoic acid and 12-methylpentadecanoic acid respectively. Membrane anisotropy studies indicated that growth of strain MOR401 in the presence of these precursors increased its membrane fluidity to levels of the wild type. Cells supplemented with 2-methylpentanoate or 2-ethylbutyrate at 10°C shortened the chain length of novel fatty acids, thus showing homeoviscous adaptation. These experiments use the mutant as a tool to modulate the membrane fatty acid compositions through synthetic precursor supplementation, and show how existing enzymes in L. monocytogenes adapt to exhibit non-native activity yielding unique 'unnatural' fatty acid molecules, which nevertheless possess the correct biophysical properties for proper membrane function in the BCFA-deficient mutant. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 07/2015; 1851(10). DOI:10.1016/j.bbalip.2015.07.006 · 4.66 Impact Factor
  • 03/2015; 4(1):76-112. DOI:10.3390/antibiotics4010076
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    ABSTRACT: The Twin-arginine translocase (Tat) complex is a unique system that translocates folded proteins across the cytoplasmic membrane. In this study, the Tat-transporter system in Listeria monocytogenes was characterized to determine the role of Tat in the iron uptake pathway. A putative tatAC operon, containing conserved Fur-binding sequences in the promoter region, has been predicted to encode Tat-translocase components. Another operon, fepCAB, with a putative Fur- binding sequence in the promoter, close to TatAC, was identified in the complementary strands of L. monocytogenes. Electrophoretic mobility shift assay (EMSA) showed that the listerial Fur-repressor binds to the promoter of the tatAC-operon, suggesting that tat is under Fur regulation. Using a heterologous system in a reporter assay, FepB was shown to be translocated across the membrane. Mutations in tatC and fepB were constructed to determine the roles of Tat and FepB. In a whole-cell ferric reductase assay, the fepB and tatC mutants were found to have reduced levels of ferric reductase activities compared to that of the isogenic parent strain. Although ferric reductase activity has been demonstrated in Listeria, a conventional ferric reductase encoding sequence does not appear to be present in its genome. Hence, we propose that fepB encodes a ferric reductase enzyme which is translocated by the Tat-translocase system onto the bacteria cell surface, and plays an important role in the reductive iron uptake process in L. monocytogenes. Copyright © 2014, the Society for General Microbiology.
    Microbiology 11/2014; 161(Pt_2). DOI:10.1099/mic.0.083642-0 · 2.56 Impact Factor
  • Yang Song · Christopher S Lunde · Bret M Benton · Brian J Wilkinson
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    ABSTRACT: Telavancin is a novel semisynthetic lipoglycopeptide derivative of vancomycin with a dual mode of action. This study sought to understand the mechanisms of decreased telavancin susceptibility in a laboratory-derived Staphlococcus aureus mutant Tlv(DS)MED1952. There were extensive changes in the transcriptome of Tlv(DS)MED1952 compared to the susceptible parent strain MED1951. Genes upregulated included cofactor biosynthesis genes, cell wall-related genes, fatty acid biosynthesis genes, and stress genes. Downregulated genes included lysine operon biosynthesis genes and lrgB, which are induced by telavancin in susceptible strains, agr and kdpDE genes, various cell surface protein genes, phenol-soluble modulin genes, several protease genes, and genes involved in anaerobic metabolism. The decreased susceptibility mutant had somewhat thicker cell walls and a decreased autolytic activity that may be related to decreased proteolytic peptidoglycan hydrolase processing. Membrane fatty acid changes correlated with increased membrane fluidity were observed. It seems likely that there are multiple genetic changes associated with the development of decreased telavancin susceptibility. The Tlv(DS) mutant showed some similar features to vancomycin-intermediate S. aureus and decreased daptomycin susceptibility strains, but also exhibited its own unique features.
    Microbial drug resistance (Larchmont, N.Y.) 04/2013; 19(4). DOI:10.1089/mdr.2012.0195 · 2.49 Impact Factor
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    ABSTRACT: Daptomycin is an extensively used anti-staphylococcal agent due to the rise in methicillin-resistant Staphylococcus aureus, but the mechanism(s) of resistance is poorly understood. Comparative genome sequencing, transcriptomics, ultrastructure, and cell envelope studies were carried out on two relatively higher level (4 and 8 µg/ml(-1)) laboratory-derived daptomycin-resistant strains (strains CB1541 and CB1540 respectively) compared to their parent strain (CB1118; MW2). Several mutations were found in the strains. Both strains had the same mutations in the two-component system genes walK and agrA. In strain CB1540 mutations were also detected in the ribose phosphate pyrophosphokinase (prs) and polyribonucleotide nucleotidyltransferase genes (pnpA), a hypothetical protein gene, and in an intergenic region. In strain CB1541 there were mutations in clpP, an ATP-dependent protease, and two different hypothetical protein genes. The strain CB1540 transcriptome was characterized by upregulation of cap (capsule) operon genes, genes involved in the accumulation of the compatible solute glycine betaine, ure genes of the urease operon, and mscL encoding a mechanosensitive chanel. Downregulated genes included smpB, femAB and femH involved in the formation of the pentaglycine interpeptide bridge, genes involved in protein synthesis and fermentation, and spa encoding protein A. Genes altered in their expression common to both transcriptomes included some involved in glycine betaine accumulation, mscL, ure genes, femH, spa and smpB. However, the CB1541 transcriptome was further characterized by upregulation of various heat shock chaperone and protease genes, consistent with a mutation in clpP, and lytM and sceD. Both strains showed slow growth, and strongly decreased autolytic activity that appeared to be mainly due to decreased autolysin production. In contrast to previous common findings, we did not find any mutations in phospholipid biosynthesis genes, and it appears there are multiple pathways to and factors in daptomycin resistance.
    PLoS ONE 03/2013; 8(3):e58469. DOI:10.1371/journal.pone.0058469 · 3.23 Impact Factor
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    ABSTRACT: Tea tree oil (TTO) is a steam distillate of Melaleuca alternifolia that demonstrates broad-spectrum antibacterial activity. This study was designed to document how TTO challenge influences the Staphylococcus aureus transcriptome. Overall, bioinformatic analyses (S. aureus microarray meta-database) revealed that both ethanol and TTO induce related transcriptional alterations. TTO challenge led to the down-regulation of genes involved with energy-intensive transcription and translation, and altered the regulation of genes involved with heat shock (e.g. clpC, clpL, ctsR, dnaK, groES, groEL, grpE and hrcA) and cell wall metabolism (e.g. cwrA, isaA, sle1, vraSR and vraX). Inactivation of the heat shock gene dnaK or vraSR which encodes a two-component regulatory system that responds to peptidoglycan biosynthesis inhibition led to an increase in TTO susceptibility which demonstrates a protective role for these genes in the S. aureus TTO response. A gene (mmpL) encoding a putative resistance, nodulation and cell division efflux pump was also highly induced by TTO. The principal antimicrobial TTO terpene, terpinen-4-ol, altered ten genes in a transcriptional direction analogous to TTO. Collectively, this study provides additional insight into the response of a bacterial pathogen to the antimicrobial terpene mixture TTO. Copyright © 2012 John Wiley & Sons, Ltd.
    Phytotherapy Research 03/2013; 27(3). DOI:10.1002/ptr.4738 · 2.66 Impact Factor
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    ABSTRACT: Background Staphylococcus aureus is the pathogen most often and prevalently involved in skin and soft tissue infections. In recent decades outbreaks of methicillin-resistant S. aureus (MRSA) have created major problems for skin therapy, and burn and wound care units. Topical antimicrobials are most important component of wound infection therapy. Alternative therapies are being sought for treatment of MRSA and one area of interest is the use of essential oils. With the increasing interest in the use and application of natural products, we screened the potential application of terpeneless cold pressed Valencia orange oil (CPV) for topical therapy against MRSA using an in vitro dressing model and skin keratinocyte cell culture model. Methods The inhibitory effect of CPV was determined by disc diffusion vapor assay for MRSA and vancomycin intermediate-resistant S. aureus (VISA) strains. Antistaphylococcal effect of CPV in an in vitro dressing model was tested on S. aureus inoculated tryptic soya agar plate. Bactericidal effect of CPV on MRSA and VISA infected keratinocyte cells was examined by enumeration of extra- and intra-cellular bacterial cells at different treatment time points. Cytotoxic effects on human skin cells was tested by adding CPV to the keratinocyte (HEK001) cells grown in serum free KSFM media, and observed by phase-contrast microscope. Results CPV vapour effectively inhibited the MRSA and VISA strains in both disc diffusion vapour assay and in vitro dressing model. Compared to untreated control addition of 0.1% CPV to MRSA infected keratinocyte decreased the viable MRSA cells by 2 log CFU/mL in 1 h and in VISA strain 3 log CFU/mL reduction was observed in 1 h. After 3 h viable S. aureus cells were not detected in the 0.2% CPV treatment. Bactericidal concentration of CPV did not show any cytotoxic effect on the human skin keratinocyte cells in vitro. Conclusions At lower concentration addition of CPV to keratinocytes infected with MRSA and VISA rapidly killed the bacterial cells without causing any toxic effect to the keratinocytes. Therefore, the results of this study warrant further in vivo study to evaluate the potential of CPV as a topical antistaphylococcal agent.
    BMC Complementary and Alternative Medicine 08/2012; 12(1):125. DOI:10.1186/1472-6882-12-125 · 2.02 Impact Factor
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    ABSTRACT: Fatty acids (FAs) are the major structural component of cellular membranes, which provide a physical and chemical barrier that insulates intracellular reactions from environmental fluctuations. The native composition of membrane FAs establishes the topological and chemical parameters for membrane-associated functions and is therefore modulated diligently by microorganisms especially in response to environmental stresses. However, the consequences of altered FA composition during host-pathogen interactions are poorly understood. The food-borne pathogen Listeria monocytogenes contains mostly saturated branched-chain FAs (BCFAs), which support growth at low pH and low temperature. In this study, we show that anteiso-BCFAs enhance bacterial resistance against phagosomal killing in macrophages. Specifically, BCFAs protect against antimicrobial peptides and peptidoglycan hydrolases, two classes of phagosome antimicrobial defense mechanisms. In addition, the production of the critical virulence factor, listeriolysin O, was compromised by FA modulation, suggesting that FAs play a key role in virulence regulation. In summary, our results emphasize the significance of FA metabolism, not only in bacterial virulence regulation but also in membrane barrier function by providing resistance against host antimicrobial stress.
    Journal of bacteriology 07/2012; 194(19):5274-84. DOI:10.1128/JB.00045-12 · 2.81 Impact Factor
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    ABSTRACT: Transcriptional profiling of Staphylococcus aureus treated with cell wall-active antibiotics identified the 2-component system, VraSR, as one of the key players in response to antibiotic stress. Although it has been shown that a number of genes are regulated by the VraSR system, it has not been shown which genes are under direct VraSR regulation and which genes are not. In this study, chromatin immunoprecipitation techniques were used to identify the genes which are regulated by the direct interaction of VraR with their promoter regions. The results showed for the first time, that the VraSR mediated regulation of cell wall biosynthesis-associated genes, pbp2, murZ, and sgtB are facilitated by the direct binding of VraR to their respective promoters. Conversely, fmtA, indicated previously to be under VraSR regulation did not exhibit direct regulation by the binding of VraR to its promoter. The VraSR system plays a very important role in antibiotic resistance against cell wall-active antibiotics, and hence, it is essential to understand its complete regulatory mechanism.
    Canadian Journal of Microbiology 05/2012; 58(6):703-8. DOI:10.1139/w2012-043 · 1.22 Impact Factor
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    Yang Song · Christopher S Lunde · Bret M Benton · Brian J Wilkinson
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    ABSTRACT: Telavancin is a novel semisynthetic lipoglycopeptide derivative of vancomycin with a decylaminoethyl side chain that is active against Gram-positive bacteria, including Staphylococcus aureus strains resistant to methicillin or vancomycin. A dual mechanism of action has been proposed for telavancin involving inhibition of peptidoglycan biosynthesis and membrane depolarization. Here we report the results of genome-wide transcriptional profiling of the response of S. aureus to telavancin using microarrays. Short (15-min) challenge of S. aureus with telavancin revealed strong expression of the cell wall stress stimulon, a characteristic response to inhibition of cell wall biosynthesis. In the transcriptome obtained after 60-min telavancin challenge, in addition to induction of the cell wall stress stimulon, there was induction of various genes, including lrgA and lrgB, lysine biosynthesis operon (dap) genes, vraD and vraE, and hlgC, that have been reported to be induced by known membrane-depolarizing and active agents, including carbonyl cyanide m-chlorophenylhydrazone, daptomycin, bacitracin, and other antimicrobial peptides These genes were either not induced or only weakly induced by the parent molecule vancomycin. We suggest that expression of these genes is a response of the cell to mitigate and detoxify such molecules and is diagnostic of a membrane-depolarizing or membrane-active molecule. The results indicate that telavancin causes early and significant induction of the cell wall stress stimulon due to strong inhibition of peptidoglycan biosynthesis, with evidence in support of membrane depolarization and membrane activity that is expressed after a longer duration of drug treatment.
    Antimicrobial Agents and Chemotherapy 03/2012; 56(6):3157-64. DOI:10.1128/AAC.05403-11 · 4.48 Impact Factor
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    A Muthaiyan · E.M. Martin · S Natesan · P.G. Crandall · B.J. Wilkinson · S.C. Ricke
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    ABSTRACT: The objectives of this study were to evaluate the antistaphylococcal effect and elucidate the mechanism of action of orange essential oil against antibiotic-resistant Staphylococcus aureus strains. The inhibitory effect of commercial orange essential oil (EO) against six Staph. aureus strains was tested using disc diffusion and agar dilution methods. The mechanism of EO action on MRSA was analysed by transcriptional profiling. Morphological changes of EO-treated Staph. aureus were examined using transmission electron microscopy. Results showed that 0·1% of terpeneless cold-pressed Valencia orange oil (CPV) induced the cell wall stress stimulon consistent with the inhibition of cell wall synthesis. Transmission electron microscopic observation revealed cell lysis and suggested a cell wall lysis-related mechanism of CPV. CPV inhibits the growth of Staph. aureus, causes gene expression changes consistent with the inhibition of cell wall synthesis, and triggers cell lysis. Multiple antibiotics resistance is becoming a serious problem in the management of Staph. aureus infections. In this study, the altered expression of cell wall-associated genes and subsequent cell lysis in MRSA caused by CPV suggest that it may be a potential antimicrobial agent to control antibiotic-resistant Staph. aureus.
    Journal of Applied Microbiology 02/2012; 112(5):1020-33. DOI:10.1111/j.1365-2672.2012.05270.x · 2.48 Impact Factor
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    ABSTRACT: Wall teichoic acids (WTAs) are phosphate-rich, sugar-based polymers attached to the cell walls of most Gram-positive bacteria. In Staphylococcus aureus, these anionic polymers regulate cell division, protect cells from osmotic stress, mediate host colonization, and mask enzymatically susceptible peptidoglycan bonds. Although WTAs are not required for survival in vitro, blocking the pathway at a late stage of synthesis is lethal. We recently discovered a novel antibiotic, targocil, that inhibits a late acting step in the WTA pathway. Its target is TarG, the transmembrane component of the ABC transporter (TarGH) that exports WTAs to the cell surface. We examined here the effects of targocil on S. aureus using transmission electron microscopy and gene expression profiling. We report that targocil treatment leads to multicellular clusters containing swollen cells displaying evidence of osmotic stress, strongly induces the cell wall stress stimulon, and reduces the expression of key virulence genes, including dltABCD and capsule genes. We conclude that WTA inhibitors that act at a late stage of the biosynthetic pathway may be useful as antibiotics, and we present evidence that they could be particularly useful in combination with beta-lactams.
    Antimicrobial Agents and Chemotherapy 01/2012; 56(4):1810-20. DOI:10.1128/AAC.05938-11 · 4.48 Impact Factor
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    ABSTRACT: Listeria monocytogenes is a food-borne pathogen with the ability to grow at refrigeration temperatures. Knowledge of the mechanisms involved in low temperature growth is incomplete and here we report the results of a metabolomics investigation of this. The small molecule contents of L. monocytogenes 10403S grown at 37 °C and 8 °C were compared by gas chromatography/mass spectrometry (GC/MS). Over 500 peaks were detected in both 37 °C and 8 °C-grown cells, and 103 were identified. Of the identified metabolites, the concentrations of 56 metabolites were increased (P<0.05), while the concentrations of 8 metabolites were decreased at low temperature. Metabolites increasing in concentration at 8 °C included amino acids, sugars, organic acids, urea cycle intermediates, polyamines, and different compatible solutes. A principal component analysis (PCA) was used to visualize and compare the matrix containing the data in 6 samples, and this clearly identified the 37 °C and 8 °C metabolomes as different. The results indicated that an increase in solute concentrations in the cytoplasm was associated with low temperature adaptation, which may be a response to chill stress with the effect of lowering the freezing point of intracellular water and decreasing ice crystal formation.
    International journal of food microbiology 08/2011; 148(2):107-14. DOI:10.1016/j.ijfoodmicro.2011.05.008 · 3.08 Impact Factor
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    ABSTRACT: Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) which has been shown to increase the susceptibility of various bacteria to antimicrobials and demonstrated to have broad antimicrobial activity. This study describes transcriptome alterations in S. aureus strain COL grown with diclofenac and characterizes the effects of this NSAID on antibiotic susceptibility in laboratory, clinical and diclofenac reduced-susceptibility (DcRS) S. aureus strains. Transcriptional alterations in response to growth with diclofenac were measured using S. aureus gene expression microarrays and quantitative real-time PCR. Antimicrobial susceptibility was determined by agar diffusion MICs and gradient plate analysis. Ciprofloxacin accumulation was measured by fluorescence spectrophotometry. Growth of S. aureus strain COL with 80 μg/ml (0.2 × MIC) of diclofenac resulted in the significant alteration by ≥2-fold of 458 genes. These represented genes encoding proteins for transport and binding, protein and DNA synthesis, and the cell envelope. Notable alterations included the strong down-regulation of antimicrobial efflux pumps including mepRAB and a putative emrAB/qacA-family pump. Diclofenac up-regulated sigB (σB), encoding an alternative sigma factor which has been shown to be important for antimicrobial resistance. Staphylococcus aureus microarray metadatabase (SAMMD) analysis further revealed that 46% of genes differentially-expressed with diclofenac are also σB-regulated. Diclofenac altered S. aureus susceptibility to multiple antibiotics in a strain-dependent manner. Susceptibility increased for ciprofloxacin, ofloxacin and norfloxacin, decreased for oxacillin and vancomycin, and did not change for tetracycline or chloramphenicol. Mutation to DcRS did not affect susceptibility to the above antibiotics. Reduced ciprofloxacin MICs with diclofenac in strain BB255, were not associated with increased drug accumulation. The results of this study suggest that diclofenac influences antibiotic susceptibility in S. aureus, in part, by altering the expression of regulatory and structural genes associated with cell wall biosynthesis/turnover and transport.
    Annals of Clinical Microbiology and Antimicrobials 07/2011; 10(1):30. DOI:10.1186/1476-0711-10-30 · 2.19 Impact Factor
  • Sechan Wee · Brian J. Wilkinson
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    ABSTRACT: Exponential-phase Paracoccus denitrificans cells grown in a complex medium are known to be lysozyme susceptible without pretreatment with outer membrane perturbing agents. Stationary-phase cells are more lysozyme resistant, but simple washing of these cells with moderate concentrations (154 mM) of monovalent inorganic cations (NaCl, KCl, or LiCl), or 100 mM Tris(hydroxymethyl)aminomethane-HCl (Tris–HCl) rendered them lysozyme susceptible. Subsequent washing with divalent cations reversed the enhanced lysozyme susceptibility. Cells grown in succinate–salts medium were not rendered lysozyme susceptible by NaCl washing. Supplementation of complex medium with various salts used in succinate–salts medium showed that Mg2+ and Ca2+ supplementation resulted in increased growth yields and in cells that were not rendered lysozyme susceptible by NaCl washing. Measurement of the Mg2+ and Ca2+ content of peptone and yeast extract revealed Mg2+ and Ca2+ were present at 50–80 and 15–30 μM concentrations, respectively, in complex medium. Omission of Ca2+ or reduction of Mg2+ from 810 to 50 μM in succinate–salts medium resulted in cells that became lysozyme susceptible after NaCl washing. Incubation of cells grown in succinate–salts medium with Tris–HCl – ethylenediaminetetraacetate – sucrose caused them to become lysozyme susceptible, indicating that their lack of lysozyme susceptibility was due to a more effective outer membrane barrier than that of complex medium grown cells, rather than a lysozyme-resistant peptidoglycan. The protein composition of outer membranes from complex and complex + Mg2+ + Ca2+ grown cells was similar, suggesting that divalent cations did not have a major influence on protein production related to outer membrane stability. The results indicate that certain complex bacteriological media may be deficient in divalent cations for maximum cell envelope stability and growth yield. This might be particularly significant for soil bacteria such as P. denitrificans, which may have evolved to have outer membranes more dependent on divalent cations for structural integrity than animal host adapted bacteria such as enteric bacteria. The results also draw attention to NaCl as an outer membrane perturbing agent.
    Canadian Journal of Microbiology 02/2011; 34(8):952-959. DOI:10.1139/m88-168 · 1.22 Impact Factor
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    ABSTRACT: Our previous studies showed that both Sae and Fur are required for the induction of eap and emp expression in low iron. In this study, we show that expression of sae is also iron-regulated, as sae expression is activated by Fur in low iron. We also demonstrate that both Fur and Sae are required for full induction of the oxidative stress response and expression of non-covalently bound surface proteins in low-iron growth conditions. In addition, Sae is required for the induced expression of the important virulence factors isdA and isdB in low iron. Our studies also indicate that Fur is required for the induced expression of the global regulators Agr and Rot in low iron and a number of extracellular virulence factors such as the haemolysins which are also Sae- and Agr-regulated. Hence, we show that Fur is central to a complex regulatory network that is required for the induced expression of a number of important S. aureus virulence determinants in low iron.
    International journal of medical microbiology: IJMM 01/2011; 301(1):44-52. DOI:10.1016/j.ijmm.2010.05.003 · 3.61 Impact Factor
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    ABSTRACT: Alkali stress is an important means of inactivating undesirable pathogens in a wide range of situations. Unfortunately, Listeria monocytogenes can launch an alkaline tolerance response, significantly increasing persistence of the pathogen in such environments. This study compared transcriptome patterns of alkali and non-alkali-stressed L. monocytogenes 10403S cells, to elucidate the mechanisms by which Listeria adapts and/or grows during short- or long-term alkali stress. Transcription profiles associated with alkali shock (AS) were obtained by DNA microarray analysis of midexponential cells suspended in pH 9 media for 15, 30, or 60 min. Transcription profiles associated with alkali adaptation (AA) were obtained similarly from cells grown to midexponential phase at pH 9. Comparison of AS and AA transcription profiles with control cell profiles identified a high number of differentially regulated open-reading frames in all tested conditions. Rapid (15 min) changes in expression included upregulation of genes encoding for multiple metabolic pathways (including those associated with Na+/H+ antiporters), ATP-binding cassette transporters of functional compatible solutes, motility, and virulence-associated genes as well as the σ(B) controlled stress resistance network. Slower (30 min and more) responses to AS and adaptation during growth in alkaline conditions (AA) involved a different pattern of changes in mRNA concentrations, and genes involved in proton export.
    Foodborne Pathogens and Disease 10/2010; 7(10):1147-57. DOI:10.1089/fpd.2009.0501 · 1.91 Impact Factor
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    ABSTRACT: Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase (TP) with reduced susceptibility to β-lactam antibiotics. PBP2A cross-links nascent peptidoglycan when the native TPs are inhibited by β-lactams. Although mecA expression is essential for β-lactam resistance, it is not sufficient. Here we show that blocking the expression of wall teichoic acids (WTAs) by inhibiting the first enzyme in the pathway, TarO, sensitizes methicillin-resistant S. aureus (MRSA) strains to β-lactams even though the β-lactam-resistant transpeptidase, PBP2A, is still expressed. The dramatic synergy between TarO inhibitors and β-lactams is noteworthy not simply because strategies to overcome MRSA are desperately needed but because neither TarO nor the activities of the native TPs are essential in MRSA strains. The "synthetic lethality" of inhibiting TarO and the native TPs suggests a functional connection between ongoing WTA expression and peptidoglycan assembly in S. aureus. Indeed, transmission electron microscopy shows that S. aureus cells blocked in WTA synthesis have extensive defects in septation and cell separation, indicating dysregulated cell wall assembly and degradation. Our studies imply that WTAs play a fundamental role in S. aureus cell division and raise the possibility that synthetic lethal compound combinations may have therapeutic utility for overcoming antibiotic-resistant bacterial infections.
    ACS Chemical Biology 10/2010; 6(1):106-16. DOI:10.1021/cb100269f · 5.33 Impact Factor
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    Mudcharee Julotok · Atul K Singh · Craig Gatto · Brian J Wilkinson
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    ABSTRACT: Listeria monocytogenes is a food-borne pathogen that grows at refrigeration temperatures and increases its content of anteiso-C15:0 fatty acid, which is believed to be a homeoviscous adaptation to ensure membrane fluidity, at these temperatures. As a possible novel approach for control of the growth of the organism, the influences of various fatty acid precursors, including branched-chain amino acids and branched- and straight-chain carboxylic acids, some of which are also well-established food preservatives, on the growth and fatty acid composition of the organism at 37°C and 10°C were studied in order to investigate whether the organism could be made to synthesize fatty acids that would result in impaired growth at low temperatures. The results indicate that the fatty acid composition of L. monocytogenes could be modulated by the feeding of branched-chain amino acid, C4, C5, and C6 branched-chain carboxylic acid, and C3 and C4 straight-chain carboxylic acid fatty acid precursors, but the growth-inhibitory effects of several preservatives were independent of effects on fatty acid composition, which were minor in the case of preservatives metabolized via acetyl coenzyme A. The ability of a precursor to modify fatty acid composition was probably a reflection of the substrate specificities of the first enzyme, FabH, in the condensation of primers of fatty acid biosynthesis with malonyl acyl carrier protein.
    Applied and Environmental Microbiology 03/2010; 76(5):1423-32. DOI:10.1128/AEM.01592-09 · 3.67 Impact Factor
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    ABSTRACT: Iron is required by almost all bacteria, but concentrations above physiological levels are toxic. In bacteria, intracellular iron is regulated mostly by the ferric uptake regulator, Fur, or a similar functional protein. Iron limitation results in the regulation of a number of genes, especially those involved in iron uptake. A subset of these genes is the Fur regulon under the control of Fur. In the present study, we have identified Fur- and iron-regulated genes in Listeria monocytogenes by DNA microarray analysis using a fur mutant and its isogenic parent. To identify genes regulated exclusively in response to iron limitation, the whole-genome transcriptional responses to the iron limitation of a fur mutant and its isogenic parent were compared. Fur-regulated genes were identified by comparing the transcriptional profile of the parent with the transcriptional profile of the isogenic fur mutant. Our studies have identified genes regulated exclusively in response to iron and those that are negatively regulated by Fur. We have identified at least 14 genes that were negatively regulated directly by Fur. Under iron-limited conditions, these genes were upregulated, while the expression of fur was found to be downregulated. To further investigate the regulation of fur in response to iron, an ectopic fur promoter-lacZ transcriptional fusion strain was constructed, and its isogenic fur and perR mutant derivatives were generated in L. monocytogenes 10403S. Analysis of the iron limitation of the perR mutant indicated that the regulation of genes under the negative control of Fur was significantly inhibited. Our results indicate that Fur and PerR proteins negatively regulate fur and that under iron-limited conditions, PerR is required for the negative regulation of genes controlled by Fur.
    Applied and Environmental Microbiology 11/2009; 76(2):406-16. DOI:10.1128/AEM.01389-09 · 3.67 Impact Factor

Publication Stats

3k Citations
274.87 Total Impact Points


  • 1982–2014
    • Illinois State University
      • School of Biological Sciences
      Maywood, Illinois, United States
  • 2007–2008
    • A.T. Still University of Health Sciences
      • Department of Microbiology
      Kirksville, Missouri, United States
    • University of Ulster
      • Food Microbiology Research Group
      Belfast, NIR, United Kingdom
    • University of Louisville
      • Department of Microbiology and Immunology
      Louisville, Kentucky, United States
  • 2006
    • University of Minnesota Duluth
      Duluth, Minnesota, United States
  • 2003
    • New Mexico State University
      • Department of Biology
      Las Cruces, NM, United States
  • 2002
    • Midwestern University
      • Chicago College of Osteopathic Medicine
      Downers Grove, IL, United States
  • 1991
    • University of Alberta
      • Faculty of Pharmacy and Pharmaceutical Sciences
      Edmonton, Alberta, Canada