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doi: 10.1128/JB.00167-13pmid: 23687271
Gene Expression and Physiological Role of Pseudomonas aeruginosa Methionine Sulfoxide Reductases during Oxidative Stress Adisak Romsang a , Sopapan Atichartpongkul d , Wachareeporn Trinachartvanit b , Paiboon Vattanaviboon d , e and Skorn Mongkolsuk a , c , d , e Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand a Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand b Center of Excellence on Emerging Bacterial Infections, Faculty of Science, Mahidol University, Bangkok, Thailand c Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok, Thailand d Center of Excellence on Environmental Health and Toxicology, Bangkok, Thailand e ABSTRACT Pseudomonas aeruginosa PAO1 has two differentially expressed methionine sulfoxide reductase genes: msrA (PA5018) and msrB (PA2827). The msrA gene is expressed constitutively at a high level throughout all growth phases, whereas msrB expression is highly induced by oxidative stress, such as sodium hypochlorite (NaOCl) treatment. Inactivation of either msrA or msrB or both genes ( msrA msrB mutant) rendered the mutants less resistant than the parental PAO1 strain to oxidants such as NaOCl and H 2 O 2 . Unexpectedly, msr mutants have disparate resistance patterns when exposed to paraquat, a superoxide generator. The msrA mutant had a higher paraquat resistance level than the msrB mutant, which had a lower paraquat resistance level than the PAO1 strain. The expression levels of msrA showed an inverse correlation with the paraquat resistance level, and this atypical paraquat resistance pattern was not observed with msrB . Virulence testing using a Drosophila melanogaster model revealed that the msrA , msrB , and, to a greater extent, msrA msrB double mutants had an attenuated virulence phenotype. The data indicate that msrA and msrB are essential genes for oxidative stress protection and bacterial virulence. The pattern of expression and mutant phenotypes of P. aeruginosa msrA and msrB differ from previously characterized msr genes from other bacteria. Thus, as highly conserved genes, the msrA and msrB have diverse expression patterns and physiological roles that depend on the environmental niche where the bacteria thrive.
Maria R. Gomez-Garcia , Fariba Fazeli , Alexandra Grote , Arthur R. Grossman , and Devaki Bhaya
doi: 10.1128/JB.00207-13pmid: 23687278
Role of Polyphosphate in Thermophilic Synechococcus sp. from Microbial Mats Maria R. Gomez-Garcia a , Fariba Fazeli b , Alexandra Grote a , Arthur R. Grossman a and Devaki Bhaya a Carnegie Institution for Science, Department of Plant Biology, Stanford University, Stanford, California, USA a Aurora Biofuels, Alameda, California, USA b ABSTRACT Synechococcus OS-B′, a thermophilic unicellular cyanobacterium, recently isolated from the microbial mats in Octopus Spring (Yellowstone National Park), induces a suite of genes, including phosphatases and transporters, in response to phosphorus (P) starvation. Here we describe two different approaches to examine the ability of Synechococcus OS-B′ to synthesize and break down polyphosphate (poly P), a key storage compound in many prokaryotes. First, we developed a transformation protocol to create mutants in the polyphosphate kinase ( ppk ), the major enzyme responsible for the synthesis of poly P. The ppk mutant exhibited a pleiotropic phenotype with defects in poly P accumulation, aberrant levels of Pho regulon transcripts, growth defects, and changes in cell size and exopolysaccharide levels, among others. Second, we measured transcripts of ppk and ppx (encoding the polyphosphatase) directly from mat samples and found that the levels varied dramatically over a diel cycle. We also used Western blot analysis to quantify levels of PPK and PPX and found that these enzymes differentially accumulated during the diel cycle. Levels of polyphosphate kinase peaked at night, while polyphosphatase levels were highest during the early morning hours. We hypothesize that the opposing activities of these two enzymes allow cells to store and utilize poly P to optimize growth over a diel cycle.
Elaine Wood , Silvia Tamborero , Ismael Mingarro , and Maria D. Esteve-Gassent
doi: 10.1128/JB.00187-13pmid: 23687274
BB0172, a Borrelia burgdorferi Outer Membrane Protein That Binds Integrin α 3 β 1 Elaine Wood a , Silvia Tamborero b , Ismael Mingarro b and Maria D. Esteve-Gassent a Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA a Department of Biochemistry and Molecular Biology, Faculty of Biology, Burjassot, Valencia, Spain b ABSTRACT Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi infection. Upon infection, some B. burgdorferi genes are upregulated, including members of the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) protein family, which facilitate B. burgdorferi adherence to extracellular matrix components of the host. Comparative genome analysis has revealed a new family of B. burgdorferi proteins containing the von Willebrand factor A (vWFA) domain. In the present study, we characterized the expression and membrane association of the vWFA domain-containing protein BB0172 by using in vitro transcription/translation systems in the presence of microsomal membranes and with detergent phase separation assays. Our results showed evidence of BB0172 localization in the outer membrane, the orientation of the vWFA domain to the extracellular environment, and its function as a metal ion-dependent integrin-binding protein. This is the first report of a borrelial adhesin with a metal ion-dependent adhesion site (MIDAS) motif that is similar to those observed in eukaryotic integrins and has a similar function.
Kyle J. Perry and Darren E. Higgins
doi: 10.1128/JB.00210-13pmid: 23687268
A Differential Fluorescence-Based Genetic Screen Identifies Listeria monocytogenes Determinants Required for Intracellular Replication Kyle J. Perry and Darren E. Higgins Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA ABSTRACT Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen capable of causing severe invasive disease with high mortality rates in humans. While previous studies have largely elucidated the bacterial and host cell mechanisms necessary for invasion, vacuolar escape, and subsequent cell-to-cell spread, the L. monocytogenes factors required for rapid replication within the restrictive environment of the host cell cytosol are poorly understood. In this report, we describe a differential fluorescence-based genetic screen utilizing fluorescence-activated cell sorting (FACS) and high-throughput microscopy to identify L. monocytogenes mutants defective in optimal intracellular replication. Bacteria harboring deletions within the identified gene menD or pepP were defective for growth in primary murine macrophages and plaque formation in monolayers of L2 fibroblasts, thus validating the ability of the screening method to identify intracellular replication-defective mutants. Genetic complementation of the menD and pepP deletion strains rescued the in vitro intracellular infection defects. Furthermore, the menD deletion strain displayed a general extracellular replication defect that could be complemented by growth under anaerobic conditions, while the intracellular growth defect of this strain could be complemented by the addition of exogenous menaquinone. As prior studies have indicated the importance of aerobic metabolism for L. monocytogenes infection, these findings provide further evidence for the importance of menaquinone and aerobic metabolism for L. monocytogenes pathogenesis. Lastly, both the menD and pepP deletion strains were attenuated during in vivo infection of mice. These findings demonstrate that the differential fluorescence-based screening approach provides a powerful tool for the identification of intracellular replication determinants in multiple bacterial systems.
Valérie Duval , Laura M. McMurry , Kimberly Foster , James F. Head , and Stuart B. Levy
doi: 10.1128/JB.02224-12pmid: 23687277
Mutational Analysis of the Multiple-Antibiotic Resistance Regulator MarR Reveals a Ligand Binding Pocket at the Interface between the Dimerization and DNA Binding Domains Valérie Duval a , b , Laura M. McMurry a , b , Kimberly Foster a , b , James F. Head c and Stuart B. Levy a , b Center for Adaptation Genetics and Drug Resistance a Department of Molecular Biology and Microbiology, b Tufts University School of Medicine, Boston, Massachusetts, USA Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts, USA c ABSTRACT The Escherichia coli regulator MarR represses the multiple-antibiotic resistance operon marRAB and responds to phenolic compounds, including sodium salicylate, which inhibit its activity. Crystals obtained in the presence of a high concentration of salicylate indicated two possible salicylate sites, SAL-A and SAL-B. However, it was unclear whether these sites were physiologically significant or were simply a result of the crystallization conditions. A study carried out on MarR homologue MTH313 suggested the presence of a salicylate binding site buried at the interface between the dimerization and the DNA-binding domains. Interestingly, the authors of the study indicated a similar pocket conserved in the MarR structure. Since no mutagenesis analysis had been performed to test which amino acids were essential in salicylate binding, we examined the role of residues that could potentially interact with salicylate. We demonstrated that mutations in residues shown as interacting with salicylate at SAL-A and SAL-B in the MarR-salicylate structure had no effect on salicylate binding, indicating that these sites were not the physiological regulatory sites. However, some of these residues (P57, R86, M74, and R77) were important for DNA binding. Furthermore, mutations in residues R16, D26, and K44 significantly reduced binding to both salicylate and 2,4-dinitrophenol, while a mutation in residue H19 impaired the binding to 2,4-dinitrophenol only. These findings indicate, as for MTH313, the presence of a ligand binding pocket located between the dimerization and DNA binding domains.
Julie Liao , Michael J. Schurr , and Karin Sauer
doi: 10.1128/JB.00318-13pmid: 23687276
The MerR-Like Regulator BrlR Confers Biofilm Tolerance by Activating Multidrug Efflux Pumps in Pseudomonas aeruginosa Biofilms Julie Liao a , Michael J. Schurr b and Karin Sauer a Department of Biological Sciences, Binghamton University, Binghamton, New York, USA a Department of Microbiology, University of Colorado, School of Medicine, Aurora, Colorado, USA b ABSTRACT A defining characteristic of biofilms is antibiotic tolerance that can be up to 1,000-fold greater than that of planktonic cells. In Pseudomonas aeruginosa , biofilm tolerance to antimicrobial agents requires the biofilm-specific MerR-type transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm tolerance has not been elucidated. Genome-wide transcriptional profiling indicated that brlR was required for maximal expression of genes associated with antibiotic resistance, in particular those encoding the multidrug efflux pumps MexAB-OprM and MexEF-OprN. Chromatin immunoprecipitation (ChIP) analysis revealed a direct regulation of these genes by BrlR, with DNA binding assays confirming BrlR binding to the promoter regions of the mexAB-oprM and mexEF-oprN operons. Quantitative reverse transcriptase PCR (qRT-PCR) analysis further indicated BrlR to be an activator of mexAB-oprM and mexEF-oprN gene expression. Moreover, immunoblot analysis confirmed increased MexA abundance in cells overexpressing brlR . Inactivation of both efflux pumps rendered biofilms significantly more susceptible to five different classes of antibiotics by affecting MIC but not the recalcitrance of biofilms to killing by bactericidal agents. Overexpression of either efflux pump in a Δ brlR strain partly restored tolerance of Δ brlR biofilms to antibiotics. Expression of brlR in mutant biofilms lacking both efflux pumps partly restored antimicrobial tolerance of biofilms to wild-type levels. Our results indicate that BrlR acts as an activator of multidrug efflux pumps to confer tolerance to P. aeruginosa biofilms and to resist the action of antimicrobial agents.
Hyunkeun Kim , Hwiseop Lee , and Dongwoo Shin
doi: 10.1128/JB.00343-13pmid: 23708131
The FeoC Protein Leads to High Cellular Levels of the Fe(II) Transporter FeoB by Preventing FtsH Protease Regulation of FeoB in Salmonella enterica Hyunkeun Kim , Hwiseop Lee and Dongwoo Shin Division of Microbiology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, South Korea ABSTRACT In the gammaproteobacteria, the FeoA, FeoB, and FeoC proteins constitute the Feo system, which mediates ferrous iron (Fe(II)) import. Of these Feo proteins, FeoB is an inner membrane Fe(II) transporter that is aided by the small protein FeoA. However, the role of another small protein, FeoC, has remained unknown. Here we report that the FeoC protein is necessary for FeoB protein-mediated Fe(II) uptake in Salmonella experiencing low levels of oxygen and iron. The FeoC protein was found to directly bind to the FeoB transporter, leading to high cellular levels of FeoB. Depletion of the FtsH protease enabled high levels of FeoB in the absence of FeoC, suggesting that the FeoC protein protects the FeoB transporter from FtsH-mediated proteolysis. Our present study provides a singular example of bacteria that can control expression of iron uptake systems posttranslationally by employing a small iron transporter-binding protein.
Johannes Holert , Nina Jagmann , and Bodo Philipp
doi: 10.1128/JB.00410-13pmid: 23708132
The Essential Function of Genes for a Hydratase and an Aldehyde Dehydrogenase for Growth of Pseudomonas sp. Strain Chol1 with the Steroid Compound Cholate Indicates an Aldolytic Reaction Step for Deacetylation of the Side Chain Johannes Holert , Nina Jagmann and Bodo Philipp University of Münster, Institute of Molecular Microbiology and Biotechnology, Münster, Germany ABSTRACT In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C 5 acyl side chain of the C 24 steroid compound cholate involves the C 22 intermediate 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20 S -carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C 24 steroid (22 E )-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt , resulted in the accumulation of 7α,12α,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A- and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD + as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage.
Derek J. Fisher , Reinaldo E. Fernández , and Anthony T. Maurelli
doi: 10.1128/JB.00433-13pmid: 23708130
Chlamydia trachomatis Transports NAD via the Npt1 ATP/ADP Translocase Derek J. Fisher * , Reinaldo E. Fernández and Anthony T. Maurelli Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA ABSTRACT Obligate intracellular bacteria comprising the order Chlamydiales lack the ability to synthesize nucleotides de novo and must acquire these essential compounds from the cytosol of the host cell. The environmental protozoan endosymbiont Protochlamydia amoebophila UWE25 encodes five nucleotide transporters with specificities for different nucleotide substrates, including ATP, GTP, CTP, UTP, and NAD. In contrast, the human pathogen Chlamydia trachomatis encodes only two nucleotide transporters, the ATP/ADP translocase C. trachomatis Npt1 (Npt1 Ct ) and the nucleotide uniporter Npt2 Ct , which transports GTP, UTP, CTP, and ATP. The notable absence of a NAD transporter, coupled with the lack of alternative nucleotide transporters on the basis of bioinformatic analysis of multiple C. trachomatis genomes, led us to re-evaluate the previously characterized transport properties of Npt1 Ct . Using (adenylate- 32 P)NAD, we demonstrate that Npt1 Ct expressed in Escherichia coli enables the transport of NAD with an apparent K m and V max of 1.7 μM and 5.8 nM mg −1 h −1 , respectively. The K m for NAD transport is comparable to the K m for ATP transport of 2.2 μM, as evaluated in this study. Efflux and substrate competition assays demonstrate that NAD is a preferred substrate of Npt1 Ct compared to ATP. These results suggest that during reductive evolution, the pathogenic chlamydiae lost individual nucleotide transporters, in contrast to their environmental endosymbiont relatives, without compromising their ability to obtain nucleotides from the host cytosol through relaxation of transport specificity. The novel properties of Npt1 Ct and its conservation in chlamydiae make it a potential target for the development of antimicrobial compounds and a model for studying the evolution of transport specificity.
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