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4.  Transposon Mutagenesis Identified Chromosomal and Plasmid Genes Essential for Adaptation of the Marine Bacterium Dinoroseobacter shibae to Anaerobic Conditions 
Journal of Bacteriology  2013;195(20):4769-4777.
Anaerobic growth and survival are integral parts of the life cycle of many marine bacteria. To identify genes essential for the anoxic life of Dinoroseobacter shibae, a transposon library was screened for strains impaired in anaerobic denitrifying growth. Transposon insertions in 35 chromosomal and 18 plasmid genes were detected. The essential contribution of plasmid genes to anaerobic growth was confirmed with plasmid-cured D. shibae strains. A combined transcriptome and proteome approach identified oxygen tension-regulated genes. Transposon insertion sites of a total of 1,527 mutants without an anaerobic growth phenotype were determined to identify anaerobically induced but not essential genes. A surprisingly small overlap of only three genes (napA, phaA, and the Na+/Pi antiporter gene Dshi_0543) between anaerobically essential and induced genes was found. Interestingly, transposon mutations in genes involved in dissimilatory and assimilatory nitrate reduction (napA, nasA) and corresponding cofactor biosynthesis (genomic moaB, moeB, and dsbC and plasmid-carried dsbD and ccmH) were found to cause anaerobic growth defects. In contrast, mutation of anaerobically induced genes encoding proteins required for the later denitrification steps (nirS, nirJ, nosD), dimethyl sulfoxide reduction (dmsA1), and fermentation (pdhB1, arcA, aceE, pta, acs) did not result in decreased anaerobic growth under the conditions tested. Additional essential components (ferredoxin, cccA) of the anaerobic electron transfer chain and central metabolism (pdhB) were identified. Another surprise was the importance of sodium gradient-dependent membrane processes and genomic rearrangements via viruses, transposons, and insertion sequence elements for anaerobic growth. These processes and the observed contributions of cell envelope restructuring (lysM, mipA, fadK), C4-dicarboxylate transport (dctM1, dctM3), and protease functions to anaerobic growth require further investigation to unravel the novel underlying adaptation strategies.
doi:10.1128/JB.00860-13
PMCID: PMC3807425  PMID: 23974024
5.  FeoC from Klebsiella pneumoniae Contains a [4Fe-4S] Cluster 
Journal of Bacteriology  2013;195(20):4726-4734.
Iron is essential for pathogen survival, virulence, and colonization. Feo is suggested to function as the ferrous iron (Fe2+) transporter. The enterobacterial Feo system is composed of 3 proteins: FeoB is the indispensable component and is a large membrane protein likely to function as a permease; FeoA is a small Src homology 3 (SH3) domain protein that interacts with FeoB; FeoC is a winged-helix protein containing 4 conserved Cys residues in a sequence suitable for harboring a putative iron-sulfur (Fe-S) cluster. The presence of an iron-sulfur cluster on FeoC has never been shown experimentally. We report that under anaerobic conditions, the recombinant Klebsiella pneumoniae FeoC (KpFeoC) exhibited hyperfine-shifted nuclear magnetic resonance (NMR) and a UV-visible (UV-Vis) absorbance spectrum characteristic of a paramagnetic center. The electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) results were consistent only with the [4Fe-4S] clusters. Substituting the cysteinyl sulfur with oxygen resulted in significantly reduced cluster stability, establishing the roles of these cysteines as the ligands for the Fe-S cluster. When exposed to oxygen, the [4Fe-4S] cluster degraded to [3Fe-4S] and eventually disappeared. We propose that KpFeoC may regulate the function of the Feo transporter through the oxygen- or iron-sensitive coordination of the Fe-S cluster.
doi:10.1128/JB.00687-13
PMCID: PMC3807426  PMID: 23955005
6.  Editorial Board 
Journal of Bacteriology  2013;195(20):A1.
doi:10.1128/JB.masthead.195-20
PMCID: PMC3807427
7.  The MerR-Like Regulator BrlR Impairs Pseudomonas aeruginosa Biofilm Tolerance to Colistin by Repressing PhoPQ 
Journal of Bacteriology  2013;195(20):4678-4688.
While the MerR-like transcriptional regulator BrlR has been demonstrated to contribute to Pseudomonas aeruginosa biofilm tolerance to antimicrobial agents known as multidrug efflux pump substrates, the role of BrlR in resistance to cationic antimicrobial peptides (CAP), which is based on reduced outer membrane susceptibility, is not known. Here, we demonstrate that inactivation of brlR coincided with increased resistance of P. aeruginosa to colistin, while overexpression of brlR resulted in increased susceptibility. brlR expression correlated with reduced transcript abundances of phoP, phoQ, pmrA, pmrB, and arnC. Inactivation of pmrA and pmrB had no effect on the susceptibility of P. aeruginosa biofilms to colistin, while inactivation of phoP and phoQ rendered biofilms more susceptible than the wild type. The susceptibility phenotype of ΔphoP biofilms to colistin was comparable to that of P. aeruginosa biofilms overexpressing brlR. BrlR was found to directly bind to oprH promoter DNA of the oprH-phoPQ operon. BrlR reciprocally contributed to colistin and tobramycin resistance in P. aeruginosa PAO1 and CF clinical isolates, with overexpression of brlR resulting in increased tobramycin MICs and increased tobramycin resistance but decreased colistin MICs and increased colistin susceptibility. The opposite trend was observed upon brlR inactivation. The difference in susceptibility to colistin and tobramycin was eliminated by combination treatment of biofilms with both antibiotics. Our findings establish BrlR as an unusual member of the MerR family, as it not only functions as a multidrug transport activator, but also acts as a repressor of phoPQ expression, thus suppressing colistin resistance.
doi:10.1128/JB.00834-13
PMCID: PMC3807428  PMID: 23935054
8.  Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli 
Journal of Bacteriology  2013;195(20):4639-4649.
Gram-negative bacteria such as Escherichia coli build a peptidoglycan (PG) cell wall in their periplasm using the precursor known as lipid II. Lipid II is a large amphipathic molecule composed of undecaprenyl diphosphate and a disaccharide-pentapeptide that PG-synthesizing enzymes use to build the PG sacculus. During PG biosynthesis, lipid II is synthesized at the cytoplasmic face of the inner membrane and then flipped across the membrane. This translocation of lipid II must be assisted by flippases thought to shield the disaccharide-pentapeptide as it crosses the hydrophobic core of the membrane. The inner membrane protein MurJ is essential for PG biogenesis and homologous to known and putative flippases of the MOP (multidrug/oligo-saccharidyl-lipid/polysaccharide) exporter superfamily, which includes flippases that translocate undecaprenyl diphosphate-linked oligosaccharides across the cytoplasmic membranes of bacteria. Consequently, MurJ has been proposed to function as the lipid II flippase in E. coli. Here, we present a three-dimensional structural model of MurJ generated by the I-TASSER server that suggests that MurJ contains a solvent-exposed cavity within the plane of the membrane. Using in vivo topological studies, we demonstrate that MurJ has 14 transmembrane domains and validate features of the MurJ structural model, including the presence of a solvent-exposed cavity within its transmembrane region. Furthermore, we present functional studies demonstrating that specific charged residues localized in the central cavity are essential for function. Together, our studies support the structural homology of MurJ to MOP exporter proteins, suggesting that MurJ might function as an essential transporter in PG biosynthesis.
doi:10.1128/JB.00731-13
PMCID: PMC3807429  PMID: 23935042
9.  Copper Efflux Is Induced during Anaerobic Amino Acid Limitation in Escherichia coli To Protect Iron-Sulfur Cluster Enzymes and Biogenesis 
Journal of Bacteriology  2013;195(20):4556-4568.
Adaptation to changing environments is essential to bacterial physiology. Here we report a unique role of the copper homeostasis system in adapting Escherichia coli to its host-relevant environment of anaerobiosis coupled with amino acid limitation. We found that expression of the copper/silver efflux pump CusCFBA was significantly upregulated during anaerobic amino acid limitation in E. coli without the supplement of exogenous copper. Inductively coupled plasma mass spectrometry analysis of the total intracellular copper content combined with transcriptional assay of the PcusC-lacZ reporter in the presence of specific Cu(I) chelators indicated that anaerobic amino acid limitation led to the accumulation of free Cu(I) in the periplasmic space of E. coli, resulting in Cu(I) toxicity. Cells lacking cusCFBA and another copper transporter, copA, under this condition displayed growth defects and reduced ATP production during fumarate respiration. Ectopic expression of the Fe-S cluster enzyme fumarate reductase (Frd), or supplementation with amino acids whose biosynthesis involves Fe-S cluster enzymes, rescued the poor growth of ΔcusC cells. Yet, Cu(I) treatment did not impair the Frd activity in vitro. Further studies revealed that the alternative Fe-S cluster biogenesis system Suf was induced during the anaerobic amino acid limitation, and ΔcusC enhanced this upregulation, indicating the impairment of the Fe-S cluster assembly machinery and the increased Fe-S cluster demands under this condition. Taken together, we conclude that the copper efflux system CusCFBA is induced during anaerobic amino acid limitation to protect Fe-S cluster enzymes and biogenesis from the endogenously originated Cu(I) toxicity, thus facilitating the physiological adaptation of E. coli.
doi:10.1128/JB.00543-13
PMCID: PMC3807430  PMID: 23893112
10.  Deciphering the Interplay between Two Independent Functions of the Small RNA Regulator SgrS in Salmonella 
Journal of Bacteriology  2013;195(20):4620-4630.
Bacterial dual-function small RNAs regulate gene expression by RNA-RNA base pairing and also code for small proteins. SgrS is a dual-function small RNA in Escherichia coli and Salmonella that is expressed under stress conditions associated with accumulation of sugar-phosphates, and its activity is crucial for growth during stress. The base-pairing function of SgrS regulates a number of mRNA targets, resulting in reduced uptake and enhanced efflux of sugars. SgrS also encodes the SgrT protein, which reduces sugar uptake by a mechanism that is independent of base pairing. While SgrS base-pairing activity has been characterized in detail, little is known about how base pairing and translation of sgrT are coordinated. In the current study, we utilized a series of mutants to determine how translation of sgrT affected the efficiency of base pairing-dependent regulation and vice versa. Mutations that abrogated sgrT translation had minimal effects on base-pairing activity. Conversely, mutations that impaired base-pairing interactions resulted in increased SgrT production. Furthermore, while ectopic overexpression of sgrS mutant alleles lacking only one of the two functions rescued cell growth under stress conditions, the SgrS base-pairing function alone was indispensable for growth rescue when alleles were expressed from the native locus. Collectively, the results suggest that during stress, repression of sugar transporter synthesis via base pairing with sugar transporter mRNAs is the first priority of SgrS. Subsequently, SgrT is made and acts on preexisting transporters. The combined action of these two functions produces an effective stress response.
doi:10.1128/JB.00586-13
PMCID: PMC3807431  PMID: 23935052
11.  How Escherichia coli Tolerates Profuse Hydrogen Peroxide Formation by a Catabolic Pathway 
Journal of Bacteriology  2013;195(20):4569-4579.
When Escherichia coli grows on conventional substrates, it continuously generates 10 to 15 μM/s intracellular H2O2 through the accidental autoxidation of redox enzymes. Dosimetric analyses indicate that scavenging enzymes barely keep this H2O2 below toxic levels. Therefore, it seemed potentially problematic that E. coli can synthesize a catabolic phenylethylamine oxidase that stoichiometrically generates H2O2. This study was undertaken to understand how E. coli tolerates the oxidative stress that must ensue. Measurements indicated that phenylethylamine-fed cells generate H2O2 at 30 times the rate of glucose-fed cells. Two tolerance mechanisms were identified. First, in enclosed laboratory cultures, growth on phenylethylamine triggered induction of the OxyR H2O2 stress response. Null mutants (ΔoxyR) that could not induce that response were unable to grow. This is the first demonstration that OxyR plays a role in protecting cells against endogenous H2O2. The critical element of the OxyR response was the induction of H2O2 scavenging enzymes, since mutants that lacked NADH peroxidase (Ahp) grew poorly, and those that additionally lacked catalase did not grow at all. Other OxyR-controlled genes were expendable. Second, phenylethylamine oxidase is an unusual catabolic enzyme in that it is localized in the periplasm. Calculations showed that when cells grow in an open environment, virtually all of the oxidase-generated H2O2 will diffuse across the outer membrane and be lost to the external world, rather than enter the cytoplasm where H2O2-sensitive enzymes are located. In this respect, the periplasmic compartmentalization of phenylethylamine oxidase serves the same purpose as the peroxisomal compartmentalization of oxidases in eukaryotic cells.
doi:10.1128/JB.00737-13
PMCID: PMC3807432  PMID: 23913322
12.  Physiological and Transcriptional Responses to Osmotic Stress of Two Pseudomonas syringae Strains That Differ in Epiphytic Fitness and Osmotolerance 
Journal of Bacteriology  2013;195(20):4742-4752.
The foliar pathogen Pseudomonas syringae is a useful model for understanding the role of stress adaptation in leaf colonization. We investigated the mechanistic basis of differences in the osmotolerance of two P. syringae strains, B728a and DC3000. Consistent with its higher survival rates following inoculation onto leaves, B728a exhibited superior osmotolerance over DC3000 and higher rates of uptake of plant-derived osmoprotective compounds. A global transcriptome analysis of B728a and DC3000 following an osmotic upshift demonstrated markedly distinct responses between the strains; B728a showed primarily upregulation of genes, including components of the type VI secretion system (T6SS) and alginate biosynthetic pathways, whereas DC3000 showed no change or repression of orthologous genes, including downregulation of the T3SS. DC3000 uniquely exhibited improved growth upon deletion of the biosynthetic genes for the compatible solute N-acetylglutaminylglutamine amide (NAGGN) in a minimal medium, due possibly to NAGGN synthesis depleting the cellular glutamine pool. Both strains showed osmoreduction of glnA1 expression, suggesting that decreased glutamine synthetase activity contributes to glutamate accumulation as a compatible solute, and both strains showed osmoinduction of 5 of 12 predicted hydrophilins. Collectively, our results demonstrate that the superior epiphytic competence of B728a is consistent with its strong osmotolerance, a proactive response to an osmotic upshift, osmoinduction of alginate synthesis and the T6SS, and resiliency of the T3SS to water limitation, suggesting sustained T3SS expression under the water-limited conditions encountered during leaf colonization.
doi:10.1128/JB.00787-13
PMCID: PMC3807433  PMID: 23955010
13.  The Putative Enoyl-Coenzyme A Hydratase DspI Is Required for Production of the Pseudomonas aeruginosa Biofilm Dispersion Autoinducer cis-2-Decenoic Acid 
Journal of Bacteriology  2013;195(20):4600-4610.
In the present study, we report the identification of a putative enoyl-coenzyme A (CoA) hydratase/isomerase that is required for synthesis of the biofilm dispersion autoinducer cis-2-decenoic acid in the human pathogen Pseudomonas aeruginosa. The protein is encoded by PA14_54640 (PA0745), named dspI for dispersion inducer. The gene sequence for this protein shows significant homology to RpfF in Xanthomonas campestris. Inactivation of dspI was shown to abolish biofilm dispersion autoinduction in continuous cultures of P. aeruginosa and resulted in biofilms that were significantly greater in thickness and biomass than those of the parental wild-type strain. Dispersion was shown to be inducible in dspI mutants by the exogenous addition of synthetic cis-2-decenoic acid or by complementation of ΔdspI in trans under the control of an arabinose-inducible promoter. Mutation of dspI was also shown to abolish cis-2-decenoic acid production, as revealed by gas chromatography-mass spectrometry (GC-MS) analysis of cell-free spent culture medium. The transcript abundance of dspI correlated with cell density, as determined by quantitative reverse transcriptase (RT) PCR. This regulation is consistent with the characterization of cis-2-decenoic acid as a cell-to-cell communication molecule that regulates biofilm dispersion in a cell density-dependent manner.
doi:10.1128/JB.00707-13
PMCID: PMC3807434  PMID: 23935049
14.  A Novel Protein Protects Bacterial Iron-Dependent Metabolism from Nitric Oxide 
Journal of Bacteriology  2013;195(20):4702-4708.
Reactive nitrogen species (RNS), in particular nitric oxide (NO), are toxic to bacteria, and bacteria have mechanisms to allow growth despite this stress. Understanding how bacteria interact with NO is essential to understanding bacterial physiology in many habitats, including pathogenesis; however, many targets of NO and enzymes involved in NO resistance remain uncharacterized. We performed for the first time a metabolomic screen on NO-treated and -untreated bacteria to define broadly the effects of NO on bacterial physiology, as well as to identify the function of NnrS, a previously uncharacterized enzyme involved in defense against NO. We found many known and novel targets of NO. We also found that iron-sulfur cluster enzymes were preferentially inhibited in a strain lacking NnrS due to the formation of iron-NO complexes. We then demonstrated that NnrS is particularly important for resistance to nitrosative stress under anaerobic conditions. Our data thus reveal the breadth of the toxic effects of NO on metabolism and identify the function of an important enzyme in alleviating this stress.
doi:10.1128/JB.00836-13
PMCID: PMC3807435  PMID: 23935055
15.  Lability and Liability of Endogenous Copper Pools 
Journal of Bacteriology  2013;195(20):4553-4555.
doi:10.1128/JB.00891-13
PMCID: PMC3807436  PMID: 23913325
16.  The D3 Bacteriophage α-Polymerase Inhibitor (Iap) Peptide Disrupts O-Antigen Biosynthesis through Mimicry of the Chain Length Regulator Wzz in Pseudomonas aeruginosa 
Journal of Bacteriology  2013;195(20):4735-4741.
Lysogenic bacteriophage D3 causes seroconversion of Pseudomonas aeruginosa PAO1 from serotype O5 to O16 by inverting the linkage between O-specific antigen (OSA) repeat units from α to β. The OSA units are polymerized by Wzy to modal lengths regulated by Wzz1 and Wzz2. A key component of the D3 seroconversion machinery is the inhibitor of α-polymerase (Iap) peptide, which is able to solely suppress α-linked long-chain OSA production in P. aeruginosa PAO1. To establish the target specificity of Iap for Wzyα, changes in OSA phenotypes were examined via Western immunoblotting for wzz1 and wzz2 single-knockout strains, as well as a wzz1 wzz2 double knockout, following the expression of iap from a tuneable vector. Increased induction of Iap expression completely abrogated OSA production in the wzz1 wzz2 double mutant, while background levels of OSA production were still observed in either of the single mutants. Therefore, Iap inhibition of OSA biosynthesis was most effective in the absence of both Wzz proteins. Sequence alignment analyses revealed a high degree of similarity between Iap and the first transmembrane segment (TMS) of either Wzz1 or Wzz2. Various topology prediction analyses of the Iap sequence consistently predicted the presence of a single TMS, suggesting a propensity for Iap to insert itself into the inner membrane (IM). The compromised ability of Iap to abrogate Wzyα function in the presence of Wzz1 or Wzz2 provides compelling evidence that inhibition occurs after Wzyα inserts itself into the IM and is achieved through mimicry of the first TMS from the Wzz proteins of P. aeruginosa PAO1.
doi:10.1128/JB.00903-13
PMCID: PMC3807437  PMID: 23955007
17.  Roles of HynAB and Ech, the Only Two Hydrogenases Found in the Model Sulfate Reducer Desulfovibrio gigas 
Journal of Bacteriology  2013;195(20):4753-4760.
Sulfate-reducing bacteria are characterized by a high number of hydrogenases, which have been proposed to contribute to the overall energy metabolism of the cell, but exactly in what role is not clear. Desulfovibrio spp. can produce or consume H2 when growing on organic or inorganic substrates in the presence or absence of sulfate. Because of the presence of only two hydrogenases encoded in its genome, the periplasmic HynAB and cytoplasmic Ech hydrogenases, Desulfovibrio gigas is an excellent model organism for investigation of the specific function of each of these enzymes during growth. In this study, we analyzed the physiological response to the deletion of the genes that encode the two hydrogenases in D. gigas, through the generation of ΔechBC and ΔhynAB single mutant strains. These strains were analyzed for the ability to grow on different substrates, such as lactate, pyruvate, and hydrogen, under respiratory and fermentative conditions. Furthermore, the expression of both hydrogenase genes in the three strains studied was assessed through quantitative reverse transcription-PCR. The results demonstrate that neither hydrogenase is essential for growth on lactate-sulfate, indicating that hydrogen cycling is not indispensable. In addition, the periplasmic HynAB enzyme has a bifunctional activity and is required for growth on H2 or by fermentation of pyruvate. Therefore, this enzyme seems to play a dominant role in D. gigas hydrogen metabolism.
doi:10.1128/JB.00411-13
PMCID: PMC3807438  PMID: 23974026
18.  Characterization of IntA, a Bidirectional Site-Specific Recombinase Required for Conjugative Transfer of the Symbiotic Plasmid of Rhizobium etli CFN42 
Journal of Bacteriology  2013;195(20):4668-4677.
Site-specific recombination occurs at short specific sequences, mediated by the cognate recombinases. IntA is a recombinase from Rhizobium etli CFN42 and belongs to the tyrosine recombinase family. It allows cointegration of plasmid p42a and the symbiotic plasmid via site-specific recombination between attachment regions (attA and attD) located in each replicon. Cointegration is needed for conjugative transfer of the symbiotic plasmid. To characterize this system, two plasmids harboring the corresponding attachment sites and intA were constructed. Introduction of these plasmids into R. etli revealed IntA-dependent recombination events occurring at high frequency. Interestingly, IntA promotes not only integration, but also excision events, albeit at a lower frequency. Thus, R. etli IntA appears to be a bidirectional recombinase. IntA was purified and used to set up electrophoretic mobility shift assays with linear fragments containing attA and attD. IntA-dependent retarded complexes were observed only with fragments containing either attA or attD. Specific retarded complexes, as well as normal in vivo recombination abilities, were seen even in derivatives harboring only a minimal attachment region (comprising the 5-bp central region flanked by 9- to 11-bp inverted repeats). DNase I-footprinting assays with IntA revealed specific protection of these zones. Mutations that disrupt the integrity of the 9- to 11-bp inverted repeats abolish both specific binding and recombination ability, while mutations in the 5-bp central region severely reduce both binding and recombination. These results show that IntA is a bidirectional recombinase that binds to att regions without requiring neighboring sequences as enhancers of recombination.
doi:10.1128/JB.00714-13
PMCID: PMC3807439  PMID: 23935046
19.  Acrylyl-Coenzyme A Reductase, an Enzyme Involved in the Assimilation of 3-Hydroxypropionate by Rhodobacter sphaeroides 
Journal of Bacteriology  2013;195(20):4716-4725.
The anoxygenic phototroph Rhodobacter sphaeroides uses 3-hydroxypropionate as a sole carbon source for growth. Previously, we showed that the gene (RSP_1434) known as acuI, which encodes a protein of the medium-chain dehydrogenase/reductase (MDR) superfamily, was involved in 3-hydroxypropionate assimilation via the reductive conversion to propionyl-coenzyme A (CoA). Based on these results, we speculated that acuI encoded acrylyl-CoA reductase. In this work, we characterize the in vitro enzyme activity of purified, recombinant AcuI using a coupled spectrophotometric assay. AcuI from R. sphaeroides catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA. Two other members of the MDR012 family within the MDR superfamily, the products of SPO_1914 from Ruegeria pomeroyi and yhdH from Escherichia coli, were shown to also be part of this new class of NADPH-dependent acrylyl-CoA reductases. The activities of the three enzymes were characterized by an extremely low Km for acrylyl-CoA (<3 μM) and turnover numbers of 45 to 80 s−1. These homodimeric enzymes were highly specific for NADPH (Km = 18 to 33 μM), with catalytic efficiencies of more than 10-fold higher for NADPH than for NADH. The introduction of codon-optimized SPO_1914 or yhdH into a ΔacuI::kan mutant of R. sphaeroides on a plasmid complemented 3-hydroxypropionate-dependent growth. However, in their native hosts, SPO_1914 and yhdH are believed to function in the metabolism of substrates other than 3-hydroxypropionate, where acrylyl-CoA is an intermediate. Complementation of the ΔacuI::kan mutant phenotype by crotonyl-CoA carboxylase/reductase from R. sphaeroides was attributed to the fact that the enzyme also uses acrylyl-CoA as a substrate.
doi:10.1128/JB.00685-13
PMCID: PMC3807440  PMID: 23955006
20.  Library Screen Identifies Enterococcus faecalis CcpA, the Catabolite Control Protein A, as an Effector of Ace, a Collagen Adhesion Protein Linked to Virulence 
Journal of Bacteriology  2013;195(20):4761-4768.
The Enterococcus faecalis cell wall-anchored protein Ace is an important virulence factor involved in cell adhesion and infection. Expression of Ace on the cell surface is affected by many factors, including stage of growth, culture temperature, and environmental components, such as serum, urine, and collagen. However, the mechanisms that regulate or modulate Ace display are not well understood. With interest in identifying genes associated with Ace expression, we utilized a whole-cell enzyme-linked immunosorbent assay (ELISA)-based screening method to identify mutants from a transposon insertion mutant library which exhibited distinct Ace surface expression profiles. We identified a ccpA insertion mutant which showed significantly decreased levels of Ace surface expression at early growth phase versus those of wild-type OG1RF. Confirmation of the observation was achieved through flow cytometry and complementation analysis. Compared to the wild type, the E. faecalis ccpA mutant had an impaired ability to adhere to collagen when grown to early exponential phase, consistent with the lack of Ace expression in the early growth phase. As a key component of carbon catabolite regulation, CcpA has been previously reported to play a critical role in regulating expression of proteins involved in E. faecalis carbohydrate uptake and utilization. Our discovery is the first to associate CcpA with the production of a major E. faecalis virulence factor, providing new insights into the regulation of E. faecalis pathogenesis.
doi:10.1128/JB.00706-13
PMCID: PMC3807442  PMID: 23974022
21.  ExbB Cytoplasmic Loop Deletions Cause Immediate, Proton Motive Force-Independent Growth Arrest 
Journal of Bacteriology  2013;195(20):4580-4591.
The Escherichia coli TonB system consists of the cytoplasmic membrane proteins TonB, ExbB, and ExbD and multiple outer membrane active transporters for diverse iron siderophores and vitamin B12. The cytoplasmic membrane proteins harvest and transmit the proton motive force (PMF) to outer membrane transporters. This system, which spans the cell envelope, has only one component with a significant cytoplasmic presence, ExbB. Characterization of sequential 10-residue deletions in the ExbB cytoplasmic loop (residues 40 to 129; referred to as Δ10 proteins) revealed that it was required for all TonB-dependent activities, including interaction between the periplasmic domains of TonB and ExbD. Expression of eight out of nine of the Δ10 proteins at chromosomal levels led to immediate, but reversible, growth arrest. Arrest was not due to collapse of the PMF and did not require the presence of ExbD or TonB. All Δ10 proteins that caused growth arrest were dominant for that phenotype. However, several were not dominant for iron transport, indicating that growth arrest was an intrinsic property of the Δ10 variants, whether or not they could associate with wild-type ExbB proteins. The lack of dominance in iron transport also ruled out trivial explanations for growth arrest, such as high-level induction. Taken together, the data suggest that growth arrest reflected a changed interaction between the ExbB cytoplasmic loop and one or more unknown growth-regulatory proteins. Consistent with that, a large proportion of the ExbB cytoplasmic loop between transmembrane domain 1 (TMD1) and TMD2 is predicted to be disordered, suggesting the need for interaction with one or more cytoplasmic proteins to induce a final structure.
doi:10.1128/JB.00334-13
PMCID: PMC3807443  PMID: 23913327
22.  Staphylococcus aureus Mutants Lacking the LytR-CpsA-Psr Family of Enzymes Release Cell Wall Teichoic Acids into the Extracellular Medium 
Journal of Bacteriology  2013;195(20):4650-4659.
The LytR-CpsA-Psr (LCP) proteins are thought to transfer bactoprenol-linked biosynthetic intermediates of wall teichoic acid (WTA) to the peptidoglycan of Gram-positive bacteria. In Bacillus subtilis, mutants lacking all three LCP enzymes do not deposit WTA in the envelope, while Staphylococcus aureus Δlcp mutants display impaired growth and reduced levels of envelope phosphate. We show here that the S. aureus Δlcp mutant synthesized WTA yet released ribitol phosphate polymers into the extracellular medium. Further, Δlcp mutant staphylococci no longer restricted the deposition of LysM-type murein hydrolases to cell division sites, which was associated with defects in cell shape and increased autolysis. Mutations in S. aureus WTA synthesis genes (tagB, tarF, or tarJ2) inhibit growth, which is attributed to the depletion of bactoprenol, an essential component of peptidoglycan synthesis (lipid II). The growth defect of S. aureus tagB and tarFJ mutants was alleviated by inhibition of WTA synthesis with tunicamycin, whereas the growth defect of the Δlcp mutant was not relieved by tunicamycin treatment or by mutation of tagO, whose product catalyzes the first committed step of WTA synthesis. Further, sortase A-mediated anchoring of proteins to peptidoglycan, which also involves bactoprenol and lipid II, was not impaired in the Δlcp mutant. We propose a model whereby the S. aureus Δlcp mutant, defective in tethering WTA to the cell wall, cleaves WTA synthesis intermediates, releasing ribitol phosphate into the medium and recycling bactoprenol for peptidoglycan synthesis.
doi:10.1128/JB.00544-13
PMCID: PMC3807444  PMID: 23935043
23.  Nitrite Reductase NirBD Is Induced and Plays an Important Role during In Vitro Dormancy of Mycobacterium tuberculosis 
Journal of Bacteriology  2013;195(20):4592-4599.
Mycobacterium tuberculosis is one of the strongest reducers of nitrate among all mycobacteria. Reduction of nitrate to nitrite, mediated by nitrate reductase (NarGHJI) of M. tuberculosis, is induced during the dormant stage, and the enzyme has a respiratory function in the absence of oxygen. Nitrite reductase (NirBD) is also functional during aerobic growth when nitrite is the sole nitrogen source. However, the role of NirBD-mediated nitrite reduction during the dormancy is not yet characterized. Here, we analyzed nitrite reduction during aerobic growth as well as in a hypoxic dormancy model of M. tuberculosis in vitro. When nitrite was used as the sole nitrogen source in the medium, the organism grew and the reduction of nitrite was evident in both hypoxic and aerobic cultures of M. tuberculosis. Remarkably, the hypoxic culture of M. tuberculosis, compared to the aerobic culture, showed 32- and 4-fold-increased expression of nitrite reductase (NirBD) at the transcription and protein levels, respectively. More importantly, a nirBD mutant of M. tuberculosis was unable to reduce nitrite and compared to the wild-type (WT) strain had a >2-log reduction in viability after 240 h in the Wayne model of hypoxic dormancy. Dependence of M. tuberculosis on nitrite reductase (NirBD) was also seen in a human macrophage-based dormancy model where the nirBD mutant was impaired for survival compared to the WT strain. Overall, the increased expression and essentiality of nitrite reductase in the in vitro dormancy models suggested that NirBD-mediated nitrite reduction could be critical during the persistent stage of M. tuberculosis.
doi:10.1128/JB.00698-13
PMCID: PMC3807446  PMID: 23935045
24.  Control of the Escherichia coli Sialoregulon by Transcriptional Repressor NanR 
Journal of Bacteriology  2013;195(20):4689-4701.
NanR, one of >8,500 GntR superfamily helix-turn-helix transcriptional regulators, controls expression of the genes required for catabolism of sialic acids in Escherichia coli. It is predicted to do the same in related bacteria harboring orthologs of nanR. The sialic acids are a family of over 40 naturally occurring nine-carbon keto-sugar acids found mainly in the animal lineage, which includes starfish to humans in the deuterostome lineage. Sialic acids function in development, immunity, protein localization and stability, and homeostasis. They also serve as microbial carbon and nitrogen sources and ligands for cell recognition during host colonization. The importance of microbial sialic acid metabolism for host-microbe interactions has made it a target for therapeutic development. Exploiting this target depends on understanding sialometabolic pathways in a wide range of evolutionarily distinct bacteria. Here, we show by transcriptome, genetic, and biochemical analyses that the most common sialic acid, N-acetylneuraminate, induces the nanATEK-yhcH, yjhATS (nanCMS), and yjhBC operons by directly inactivating NanR, converting the predominantly dimeric form of the repressor to an inactive monomer of approximately 30-kDa. Additionally, other results identify critical amino acid residues and nucleotides in the regulator and operator, respectively. The combined results better define how sialic acids, acting through NanR, affect the metabolic flux of an important group of host-derived metabolites. Thus, E. coli serves as a valuable model for understanding sialocatabolic pathways in bacteria.
doi:10.1128/JB.00692-13
PMCID: PMC3807447  PMID: 23935044
25.  Evidence for Benzylsuccinate Synthase Subtypes Obtained by Using Stable Isotope Tools 
Journal of Bacteriology  2013;195(20):4660-4667.
We studied the benzylsuccinate synthase (Bss) reaction mechanism with respect to the hydrogen-carbon bond cleavage at the methyl group of toluene by using different stable isotope tools. Λ values (slopes of linear regression curves for carbon and hydrogen discrimination) for two-dimensional compound-specific stable isotope analysis (2D-CSIA) of toluene activation by Bss-containing cell extracts (in vitro studies) were found to be similar to previously reported data from analogous experiments with whole cells (in vivo studies), proving that Λ values generated by whole cells are caused by Bss catalysis. The Bss enzymes of facultative anaerobic bacteria produced smaller Λ values than those of obligate anaerobes. In addition, a partial exchange of a single deuterium atom in benzylsuccinate with hydrogen was observed in experiments with deuterium-labeled toluene. In this study, the Bss enzymes of the tested facultative anaerobes showed 3- to 8-fold higher exchange probabilities than those for the enzymes of the tested obligate anaerobic bacteria. The phylogeny of the Bss variants, determined by sequence analyses of BssA, the gene product corresponding to the α subunit of Bss, correlated with the observed differences in Λ values and hydrogen exchange probabilities. In conclusion, our results suggest subtle differences in the reaction mechanisms of Bss isoenzymes of facultative and obligate anaerobes and show that the putative isoenzymes can be differentiated by 2D-CSIA.
doi:10.1128/JB.00477-13
PMCID: PMC3807448  PMID: 23935041

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