Nonpolar Deletion Research Articles

Overexpression of the Escherichia coli TolQ protein leads to a null‐FtsN‐like division phenotype

Mutations involving the Tol-Pal complex of Escherichia coli result in a subtle phenotype in which cells chain when grown under low-salt conditions. Here, the nonpolar deletion of individual genes encoding the cytoplasmic membrane-associated components of the complex (TolQ, TolR, TolA) produced a similar phenotype. Surprisingly, the overexpression of one of these proteins, TolQ, resulted in a much more overt phenotype in which cells occurred as elongated rods coupled in long chains when grown under normal salt conditions. Neither TolR nor TolA overexpression produced a phenotype, nor was the presence of either protein required for the TolQ-dependent phenotype. Consistent with their native membrane topology, the amino-terminal domain of TolQ specifically associated in vivo with the periplasmic domain of FtsN in a cytoplasm-based two-hybrid analysis. Further, the concomitant overexpression of FtsN rescued the TolQ-dependent phenotype, suggesting a model wherein the overexpression of TolQ sequesters FtsN, depleting this essential protein from the divisome during Gram-negative cell division. The role of the Tol-Pal system in division is discussed.Over-expression of the cytoplasmic membrane protein TolQ resulted in a division phenotype similar to that seen in cells depleted for FtsN. Two hybrid analysis suggested that TolQ and FtsN physically interact through domains that localize in the periplasmic space; while the concurrent over-expression of FtsN alleviated the TolQ over-expression phenotype. Together these results suggest a model wherein over-expressed TolQ sequesters FtsN, disrupting normal cell division.

Flavobacterium johnsoniae GldK, GldL, GldM, and SprA Are Required for Secretion of the Cell Surface Gliding Motility Adhesins SprB and RemA

Flavobacterium johnsoniae cells move rapidly over surfaces by gliding motility. Gliding results from the movement of adhesins such as SprB and RemA along the cell surface. These adhesins are delivered to the cell surface by a Bacteroidetes-specific secretion system referred to as the type IX secretion system (T9SS). GldN, SprE, SprF, and SprT are involved in secretion by this system. Here we demonstrate that GldK, GldL, GldM, and SprA are each also involved in secretion. Nonpolar deletions of gldK, gldL, or gldM resulted in the absence of gliding motility and in T9SS defects. The mutant cells produced SprB and RemA proteins but failed to secrete them to the cell surface. The mutants were resistant to phages that use SprB or RemA as a receptor, and they failed to attach to glass, presumably because of the absence of cell surface adhesins. Deletion of sprA resulted in similar but slightly less dramatic phenotypes. sprA mutant cells failed to secrete SprB and RemA, but cells remained susceptible to some phages and retained some limited ability to glide. The phenotype of the sprA mutant was similar to those previously described for sprE and sprT mutants. SprA, SprE, and SprT are needed for secretion of SprB and RemA but may not be needed for secretion of other proteins targeted to the T9SS. Genetic and molecular experiments demonstrate that gldK, gldL, gldM, and gldN form an operon and suggest that the proteins encoded by these genes may interact to form part of the F. johnsoniae T9SS.

YgiW and qseBC are co-expressed in Aggregatibacter actinomycetemcomitans and regulate biofilm growth

The quorum-sensing Escherichia coli regulators B and C (QseBC) two-component system were previously shown to regulate biofilm growth of the oral pathogen Aggregatibacter actinomycetemcomitans and to be essential for virulence. In this study, we use RT-PCR to show that an open reading frame, ygiW, residing upstream of qseBC and encoding a hypothetical protein is co-expressed with qseBC. In addition, using a series of lacZ transcriptional fusion constructs and 5'-rapid amplification of cDNA Ends (RACE), the promoter that drives expression of the ygiW-qseBC operon and the transcriptional start site was mapped to the 372 bp intergenic region upstream from ygiW. No internal promoters drive qseBC expression independently from ygiW. However, qseBC expression is attenuated by approximately ninefold by a putative attenuator stem-loop (ΔG = -77.0 KJ/mol) that resides in the 137 bp intergenic region between ygiW and qseB. The QseB response regulator activates expression of the ygiW-qseBC operon and transcription from the ygiW promoter is drastically reduced in ΔqseB and ΔqseBC mutants of A. actinomycetemcomitans. In addition, transcriptional activity of the ygiW promoter is significantly reduced in a mutant expressing an in-frame deletion of qseC that lacks the sensor domain of QseC, suggesting that a periplasmic signal is required for QseB activation. Finally, a non-polar in-frame deletion in ygiW had little effect on biofilm depth but caused a significant increase in surface coverage relative to wild-type. Complementation of the mutant with a plasmid-borne copy of ygiW reduced surface coverage back to wild-type levels. Interestingly, deletion of the sensor domain of QseC or of the entire qseC open reading frame resulted in significant reductions in biofilm depth, biomass and surface coverage, indicating that the sensor domain is essential for optimal biofilm formation by A. actinomycetemcomitans. Thus, although ygiW and qseBC are co-expressed, they regulate biofilm growth by distinct mechanisms.

VraT/YvqF Is Required for Methicillin Resistance and Activation of the VraSR Regulon in Staphylococcus aureus

Staphylococcus aureus infections caused by strains that are resistant to all forms of penicillin, so-called methicillin-resistant S. aureus (MRSA) strains, have become common. One strategy to counter MRSA infections is to use compounds that resensitize MRSA to methicillin. S. aureus responds to diverse classes of cell wall-inhibitory antibiotics, like methicillin, using the two-component regulatory system VraSR (vra) to up- or downregulate a set of genes (the cell wall stimulon) that presumably facilitates resistance to these antibiotics. Accordingly, VraS and VraR mutations decrease resistance to methicillin, vancomycin, and daptomycin cell wall antimicrobials. vraS and vraR are encoded together on a transcript downstream of two other genes, which we call vraU and vraT (previously called yvqF). By producing nonpolar deletions in vraU and vraT in a USA300 MRSA clinical isolate, we demonstrate that vraT is essential for optimal expression of methicillin resistance in vitro, whereas vraU is not required for this phenotype. The deletion of vraT also improved the outcomes of oxacillin therapy in mouse models of lung and skin infection. Since vraT expressed in trans did not complement a vra operon deletion, we conclude that VraT does not inactivate the antimicrobial. Genome-wide transcriptional microarray experiments reveal that VraT facilitates resistance by playing a necessary regulatory role in the VraSR-mediated cell wall stimulon. Our data prove that VraTSR comprise a novel three-component regulatory system required to facilitate resistance to cell wall agents in S. aureus. We also provide the first in vivo proof of principle for using VraT as a sole target to resensitize MRSA to β-lactams.

Roles of the Structural Symbiosis Polysaccharide (syp) Genes in Host Colonization, Biofilm Formation, and Polysaccharide Biosynthesis in Vibrio fischeri

The symbiosis polysaccharide locus, syp, is required for Vibrio fischeri to form a symbiotic association with the squid Euprymna scolopes. It is also required for biofilm formation induced by the unlinked regulator RscS. The syp locus includes 18 genes that can be classified into four groups based on putative function: 4 genes encode putative regulators, 6 encode glycosyltransferases, 2 encode export proteins, and the remaining 6 encode proteins with other functions, including polysaccharide modification. To understand the roles of each of the 14 structural syp genes in colonization and biofilm formation, we generated nonpolar in-frame deletions of each gene. All of the deletion mutants exhibited defects in their ability to colonize juvenile squid, although the impact of the loss of SypB or SypI was modest. Consistent with their requirement for colonization, most of the structural genes were also required for RscS-induced biofilm formation. In particular, the production of wrinkled colonies, pellicles, and the matrix on the colony surface was eliminated or severely decreased in all mutants except for the sypB and sypI mutants; in contrast, only a subset of genes appeared to play a role in attachment to glass. Finally, immunoblotting data suggested that the structural Syp proteins are involved in polysaccharide production and/or export. These results provide important insights into the requirements for the syp genes under different environmental conditions and thus lay the groundwork for a more complete understanding of the matrix produced by V. fischeri to enhance cell-cell interactions and promote symbiotic colonization.

Is PhoR–PhoP partner fidelity strict? PhoR is required for the activation of the pho regulon in Streptomyces coelicolor

Two-component regulatory systems play a key role in the cell metabolism adaptation to changing nutritional and environmental conditions. The fidelity between the two cognate proteins of a two-component system is important since it determines whether a specific response regulator integrates the signals transmitted by different sensor kinases. Phosphate regulation in Streptomyces coelicolor is mostly mediated by the PhoR-PhoP two-component system. Previous studies elucidated the mechanisms that control phosphate regulation as well as the genes directly regulated by the response regulator PhoP (pho regulon) in this organism. However, the role of the histidine kinase PhoR in Streptomyces coelicolor had not been unveiled so far. In this work, we report the characterization of a non-polar ΔphoR deletion mutant in S. coelicolor that keeps its native promoter. Induction of the phoRP operon was dependent upon phosphorylation of PhoP, but the ΔphoR mutant expressed phoP at a basal level. RT-PCR and reporter luciferase assays demonstrated that PhoR plays a key role in the activation of the pho regulon in this organism. Our results point towards a strict cognate partner specificity in terms of the phosphorylation of PhoP by PhoR thus corroborating the tight interaction between the two-components of this system.

Biological Roles of Nontypeable Haemophilus influenzae Type IV Pilus Proteins Encoded by the pil and com Operons

We previously demonstrated that one or more products of the genes in the pil and com gene clusters of the opportunistic human respiratory pathogen nontypeable Haemophilus influenzae (NTHI) are required for type IV pilus (Tfp) biogenesis and function. Here, we have now demonstrated that the pilABCD and comABCDEF gene clusters are operons and that the product of each gene is essential for normal pilus function. Mutants with nonpolar deletions in each of the 10 pil and com genes had an adherence defect when primary human airway cells were used as the target. These mutants were also diminished in their ability to form a biofilm in vitro and, additionally, were deficient in natural transformation. Collectively, our data demonstrate that the product of each gene within these operons is required for the normal biogenesis and/or function of NTHI Tfp. Based on the similarity of PilA to other type IV pilins, we further predicted that the product of the pilA gene would be the major pilin subunit. Toward that end, we also demonstrated by immunogold labeling and mass spectrometry that PilA is indeed the majority type IV pilin protein expressed by NTHI. These new observations set the stage for experiments designed to dissect the function of each of the proteins encoded by genes within the pil and com gene clusters. The ability to characterize individual proteins with vital roles in NTHI colonization or pathogenesis has the potential to reduce the burden of NTHI-induced diseases through development of a Tfp-derived vaccine or a pilus-directed therapeutic.

Assessing the contributions of the LiaS histidine kinase to the innate resistance of Listeria monocytogenes to nisin, cephalosporins, and disinfectants.

The Listeria monocytogenes LiaSR two-component system (2CS) encoded by lmo1021 and lmo1022 plays an important role in resistance to the food preservative nisin. A nonpolar deletion in the histidine kinase-encoding component (ΔliaS) resulted in a 4-fold increase in nisin resistance. In contrast, the ΔliaS strain exhibited increased sensitivity to a number of cephalosporin antibiotics (and was also altered with respect to its response to a variety of other antimicrobials, including the active agents of a number of disinfectants). This pattern of increased nisin resistance and reduced cephalosporin resistance in L. monocytogenes has previously been associated with mutation of a second histidine kinase, LisK, which is a predicted regulator of liaS and a penicillin binding protein encoded by lmo2229. We noted that lmo2229 transcription is increased in the ΔliaS mutant and in a ΔliaS ΔlisK double mutant and that disruption of lmo2229 in the ΔliaS ΔlisK mutant resulted in a dramatic sensitization to nisin but had a relatively minor impact on cephalosporin resistance. We anticipate that further efforts to unravel the complex mechanisms by which LiaSR impacts on the antimicrobial resistance of L. monocytogenes could facilitate the development of strategies to increase the susceptibility of the pathogen to these agents.

Identification of a ferritin-like protein of Listeria monocytogenes as a mediator of β-lactam tolerance and innate resistance to cephalosporins

BackgroundThe food-borne pathogen Listeria monocytogenes is the causative agent of listeriosis. The β-lactam antibiotics penicillin G and ampicillin are the current drugs of choice for the treatment of listerial infections. While isolates of L. monocytogenes are susceptible to these antibiotics, their action is only bacteriostatic and consequently, this bacterium is regarded as tolerant to β-lactams. In addition, L. monocytogenes has a high level of innate resistance to the cephalosporin family of β-lactams frequently used to treat sepsis of unknown etiology. Given the high mortality rate of listeriosis despite rational antibiotic therapy, it is important to identify genes that play a role in the susceptibility and tolerance of L. monocytogenes to β-lactams.ResultsThe hly-based promoter trap system was applied to identify penicillin G-inducible genes of L. monocytogenes. The results of reporter system studies, verified by transcriptional analysis, identified ten penicillin G-inducible genes. The contribution of three of these genes, encoding a ferritin-like protein (fri), a two-component phosphate-response regulator (phoP) and an AraC/XylS family transcriptional regulator (axyR), to the susceptibility and tolerance of L. monocytogenes to β-lactams was examined by analysis of nonpolar deletion mutants. The absence of PhoP or AxyR resulted in more rapid growth of the strains in the presence of sublethal concentration of β-lactams, but had no effect on the MIC values or the ability to survive a lethal dose of these antibiotics. However, the Δfri strain showed impaired growth in the presence of sublethal concentrations of penicillin G and ampicillin and a significantly reduced ability to survive lethal concentrations of these β-lactams. A lack of Fri also caused a 2-fold increase in the sensitivity of L. monocytogenes to cefalotin and cephradine.ConclusionsThe present study has identified Fri as an important mediator of β-lactam tolerance and innate resistance to cephalosporins in L. monocytogenes. PhoP and AxyR are probably involved in transmitting signals to adjust the rate of growth of L. monocytogenes under β-lactam pressure, but these regulators do not play a significant role in susceptibility and tolerance to this class of antibiotics.

Structure-Function Analysis of the HrpB2-HrcU Interaction in the Xanthomonas citri Type III Secretion System

Bacterial type III secretion systems deliver protein virulence factors to host cells. Here we characterize the interaction between HrpB2, a small protein secreted by the Xanthomonas citri subsp. citri type III secretion system, and the cytosolic domain of the inner membrane protein HrcU, a paralog of the flagellar protein FlhB. We show that a recombinant fragment corresponding to the C-terminal cytosolic domain of HrcU produced in E. coli suffers cleavage within a conserved Asn264-Pro265-Thr266-His267 (NPTH) sequence. A recombinant HrcU cytosolic domain with N264A, P265A, T266A mutations at the cleavage site (HrcUAAAH) was not cleaved and interacted with HrpB2. Furthermore, a polypeptide corresponding to the sequence following the NPTH cleavage site also interacted with HrpB2 indicating that the site for interaction is located after the NPTH site. Non-polar deletion mutants of the hrcU and hrpB2 genes resulted in a total loss of pathogenicity in susceptible citrus plants and disease symptoms could be recovered by expression of HrpB2 and HrcU from extrachromossomal plasmids. Complementation of the ΔhrcU mutant with HrcUAAAH produced canker lesions similar to those observed when complemented with wild-type HrcU. HrpB2 secretion however, was significantly reduced in the ΔhrcU mutant complemented with HrcUAAAH, suggesting that an intact and cleavable NPTH site in HrcU is necessary for total functionally of T3SS in X. citri subsp. citri. Complementation of the ΔhrpB2 X. citri subsp. citri strain with a series of hrpB2 gene mutants revealed that the highly conserved HrpB2 C-terminus is essential for T3SS-dependent development of citrus canker symptoms in planta.

Ethanolamine Utilization Contributes to Proliferation of Salmonella enterica Serovar Typhimurium in Food and in Nematodes

Only three pathogenic bacterial species, Salmonella enterica, Clostridium perfringens, and Listeria monocytogenes, are able to utilize both ethanolamine and 1,2-propanediol as a sole carbon source. Degradation of these substrates, abundant in food and the gut, depends on cobalamin, which is synthesized de novo only under anaerobic conditions. Although the eut, pdu, and cob-cbi gene clusters comprise 40 kb, the conditions under which they confer a selection advantage on these food-borne pathogens remain largely unknown. Here we used the luciferase reporter system to determine the response of the Salmonella enterica serovar Typhimurium promoters P(eutS), P(pocR), P(pduF), and P(pduA) to a set of carbon sources, to egg yolk, to whole milk, and to milk protein or fat fractions. Depending on the supplements, specific inductions up to 3 orders of magnitude were observed for P(eutS) and P(pduA), which drive the expression of most eut and pdu genes. To correlate these significant expression data with growth properties, nonpolar deletions of pocR, regulating the pdu and cob-cbi genes, and of eutR, involved in eut gene activation, were constructed in S. Typhimurium strain 14028. During exponential growth of the mutants 14028ΔpocR and 14028ΔeutR, 2- to 3-fold-reduced proliferation in milk and egg yolk was observed. Using the Caenorhabditis elegans infection model, we could also demonstrate that the proliferation of S. Typhimurium in the nematode is supported by an active ethanolamine degradation pathway. Taking these findings together, this study quantifies the differential expression of eut and pdu genes under distinct conditions and provides experimental evidence that the ethanolamine utilization pathway allows salmonellae to occupy specific metabolic niches within food environments and within their host organisms.

Discovery of Novel Secreted Virulence Factors from Salmonella enterica Serovar Typhimurium by Proteomic Analysis of Culture Supernatants

Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis throughout the world. This pathogen has two type III secretion systems (TTSS) encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) that deliver virulence factors (effectors) to the host cell cytoplasm and are required for virulence. While many effectors have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this proteomic study, we identified effector proteins secreted into defined minimal medium designed to induce expression of the SPI-2 TTSS and its effectors. We compared the secretomes of the parent strain to those of strains missing essential (ssaK::cat) or regulatory (ΔssaL) components of the SPI-2 TTSS. We identified 20 known SPI-2 effectors. Excluding the translocon components SseBCD, all SPI-2 effectors were biased for identification in the ΔssaL mutant, substantiating the regulatory role of SsaL in TTS. To identify novel effector proteins, we coupled our secretome data with a machine learning algorithm (SIEVE, SVM-based identification and evaluation of virulence effectors) and selected 12 candidate proteins for further characterization. Using CyaA' reporter fusions, we identified six novel type III effectors and two additional proteins that were secreted into J774 macrophages independently of a TTSS. To assess their roles in virulence, we constructed nonpolar deletions and performed a competitive index analysis from intraperitoneally infected 129/SvJ mice. Six mutants were significantly attenuated for spleen colonization. Our results also suggest that non-type III secretion mechanisms are required for full Salmonella virulence.

Characterization of the O-antigen Polymerase (Wzy) of Francisella tularensis

The O-antigen polymerase of gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly(176), Asp(177), Gly(323), and Tyr(324)) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface.

TelA Contributes to the Innate Resistance of Listeria monocytogenes to Nisin and Other Cell Wall-Acting Antibiotics

Nisin is a class I bacteriocin (lantibiotic), which is employed by the food and veterinary industries and exhibits potent activity against numerous pathogens. However, this activity could be further improved through the targeting and inhibition of factors that contribute to innate nisin resistance. Here we describe a novel locus, lmo1967, which is required for optimal nisin resistance in Listeria monocytogenes. The importance of this locus, which is a homologue of the tellurite resistance gene telA, was revealed after the screening of a mariner random mutant bank of L. monocytogenes for nisin-susceptible mutants. The involvement of telA in nisin resistance was confirmed through an analysis of a nonpolar deletion mutant. In addition to being 4-fold-more susceptible to nisin, the ΔtelA strain was also 8-fold-more susceptible to gallidermin and 2-fold-more susceptible to cefuroxime, cefotaxime, bacitracin, and tellurite. This is the first occasion upon which telA has been investigated in a Gram-positive organism and also represents the first example of a link being established between a telA gene and resistance to cell envelope-acting antimicrobials.

Contribution of the Type VI Secretion System Encoded in SPI-19 to Chicken Colonization by Salmonella enterica Serotypes Gallinarum and Enteritidis

Salmonella Gallinarum is a pathogen with a host range specific to poultry, while Salmonella Enteritidis is a broad host range pathogen that colonizes poultry sub-clinically but is a leading cause of gastrointestinal salmonellosis in humans and many other species. Despite recent advances in our understanding of the complex interplay between Salmonella and their hosts, the molecular basis of host range restriction and unique pathobiology of Gallinarum remain largely unknown. Type VI Secretion System (T6SS) represents a new paradigm of protein secretion that is critical for the pathogenesis of many Gram-negative bacteria. We recently identified a putative T6SS in the Salmonella Pathogenicity Island 19 (SPI-19) of Gallinarum. In Enteritidis, SPI-19 is a degenerate element that has lost most of the T6SS functions encoded in the island. In this work, we studied the contribution of SPI-19 to the colonization of Salmonella Gallinarum strain 287/91 in chickens. Non-polar deletion mutants of SPI-19 and the clpV gene, an essential T6SS component, colonized the ileum, ceca, liver and spleen of White Leghorn chicks poorly compared to the wild-type strain after oral inoculation. Return of SPI-19 to the ΔSPI-19 mutant, using VEX-Capture, complemented this colonization defect. In contrast, transfer of SPI-19 from Gallinarum to Enteritidis resulted in transient increase in the colonization of the ileum, liver and spleen at day 1 post-infection, but at days 3 and 5 post-infection a strong colonization defect of the gut and internal organs of the experimentally infected chickens was observed. Our data indicate that SPI-19 and the T6SS encoded in this region contribute to the colonization of the gastrointestinal tract and internal organs of chickens by Salmonella Gallinarum and suggest that degradation of SPI-19 T6SS in Salmonella Enteritidis conferred an advantage in colonization of the avian host.

The ABC transporter AnrAB contributes to the innate resistance of Listeria monocytogenes to nisin, bacitracin, and various beta-lactam antibiotics.

A mariner transposon bank was used to identify loci that contribute to the innate resistance of Listeria monocytogenes to the lantibiotic nisin. In addition to highlighting the importance of a number of loci previously associated with nisin resistance (mprF, virRS, and telA), a nisin-sensitive phenotype was associated with the disruption of anrB (lmo2115), a gene encoding the permease component of an ABC transporter. The contribution of anrB to nisin resistance was confirmed by the creation of nonpolar deletion mutants. The loss of this putative multidrug resistance transporter also greatly enhanced sensitivity to bacitracin, gallidermin, and a selection of β-lactam antibiotics. A comparison of the relative antimicrobial sensitivities of a number of mutants established the ΔanrB strain as being one of the most bacitracin-sensitive L. monocytogenes strains identified to date.

Functional and molecular analysis of Escherichia coli strains lacking multiple DEAD-box helicases

DEAD-box RNA helicases are enzymes that unwind RNA duplexes and are found in virtually all organisms. Most organisms harbor multiple DEAD-box helicases, suggesting that these factors participate in distinct aspects of RNA metabolism. To define the individual and collective contribution of the five DEAD-box helicases in the bacterium Escherichia coli (E. coli), nonpolar deletion mutants lacking single or multiple DEAD-box genes were constructed. An analysis of the single-deletion strains indicated that the absence of either the DeaD or SrmB RNA helicase causes growth and/or ribosomal defects under typical laboratory growth conditions. The analysis of strains lacking multiple DEAD-box genes showed cumulative growth defects at low temperatures. A strain deleted for all five DEAD-box genes was also constructed for these studies, representing the first time all DEAD-box genes have been removed in any organism. Additional investigations revealed that the growth and ribosomal defects of such a DEAD-box deficient strain can be sharply attenuated under alternative conditions, indicating that the defects caused by a lack of DEAD-box genes are modulated by growth context.

Treponema denticola PrcB Is Required for Expression and Activity of the PrcA-PrtP (Dentilisin) Complex

The Treponema denticola surface protease complex, consisting of PrtP protease (dentilisin) and two auxiliary polypeptides (PrcA1 and PrcA2), is believed to contribute to periodontal disease by degrading extracellular matrix components and disrupting host intercellular signaling. Previously, we showed that transcription of the protease operon initiates upstream of TDE0760 (herein designated prcB), the open reading frame immediately 5' of prcA-prtP. The prcB gene is conserved in T. denticola strains. PrcB localizes to the detergent phase of Triton X-114 cell surface extracts and migrates as a 22-kDa polypeptide, in contrast to the predicted 17-kDa cytoplasmic protein encoded in the annotated T. denticola genome. Consistent with this observation, the PrcB N terminus is unavailable for Edman sequencing, suggesting that it is acylated. Nonpolar deletion of prcB in T. denticola showed that PrcB is required for production of PrtP protease activity, including native PrtP cleavage of PrcA to PrcA1 and PrcA2. A 6xHis-tagged PrcB protein coimmunoprecipitates with native PrtP, using either anti-PrtP or anti-His-tag antibodies, and recombinant PrtP copurifies with PrcB-6xHis in nickel affinity chromatography. Taken together, these data are consistent with identification of PrcB as a PrtP-binding lipoprotein that likely stabilizes the PrtP polypeptide during localization to the outer membrane.

Omics-based analysis of the luxS mutation in a clinical isolate of Escherichia coli O157:H7 in Korea

The purpose of this study was to investigate the relationship between the global regulatory mechanism known as quorum sensing and expression of virulence factors in Escherichia coli O157:H7. A non-polar luxS deletion was introduced into the chromosome of strain CI03J, a human clinical isolate from South Korea, to create the DeltaluxS mutant strain ML03J. Phenotypic characterization of wild-type and mutant strains demonstrated that ML03J had no obvious growth or metabolic defects on 0.2% glucose LB medium, produced a functionally-defective flagellum, and could not utilize sorbose; the biological significance of sorbose utilization is unknown. Omics-based analysis revealed the involvement of LuxS in the transcriptional activation of several flagella/chemotaxis-related genes (flhD; fliA, C, D, S, Z; cheA, Y, and Z), repression of glutamate-dependent acid resistance genes (gadAB), and expression of virulence factors including Shiga toxin, hemolysin, and SepD within the LEE pathogenicity island.

Surface Protein EF3314 Contributes to Virulence Properties of Enterococcus faecalis

Identification of putative new virulence factors as additional targets for therapeutic approaches alternative to antibiotic treatment of multi-resistant enterococcal infections. The EF3314 gene, coding for a putative surface-exposed antigen, was identified by the analysis of the Enterococcus faecalis V583 genome for LPXTG-motif cell wall anchor surface protein genes. A non-polar EF3314 gene deletion mutant in the E. faecalis 12030 human clinical isolate was obtained. The wild type and the isogenic mutant strain were investigated for biofilm formation, adherence to Hela cells, survival in human macrophages and a Caenorhabditis elegans infection model. The aminoterminal portion of the EF3314 protein was overexpressed in E. coli to obtain mouse polyclonal antibodies for use in Western blotting and immunolocalization experiments. The EF3314 gene has an unusually high GC content (46.88% vs. an average of 37.5% in the E. faecalis chromosome) and encodes a protein of 1744 amino acids that presents a series of 14 imperfect repeats of 90 amino acids covering almost the entire length of the protein. Its global organization is similar to the alpha-like protein family of group B streptococci, enterococcal surface protein Esp and biofilm associated protein Bap from S. aureus. The EF3314 gene was always present and specific for E. faecalis strains of human, food and animal origin. Differences in size depended on variable numbers of repeats in the repetitive region. EF3314 is a newly described, surface exposed protein that contributes to the virulence properties of E. faecalis.