Transcription Factor Sigma Research Articles

Temperature-Dependent Influence of FliA Overexpression on PHL628 E. coli Biofilm Growth and Composition.

Biofilm growth and survival pose a problem in both medical and industrial fields. Bacteria in biofilms are more tolerant to antibiotic treatment due to the inability of antibiotics to permeate to the bottom layers of cells in a biofilm and the creation of altered microenvironments of bacteria deep within the biofilm. Despite the abundance of information we have about E. coli biofilm growth and maturation, we are still learning how manipulating different signaling pathways influences the formation and fitness of biofilm. Understanding the impact of signaling pathways on biofilm formation may narrow the search for novel small molecule inhibitors or activators that affect biofilm production and stability. Here, we study the influence of the minor sigma transcription factor FliA (RpoF, sigma-28), which controls late-stage flagellar assembly and chemotaxis, on biofilm production and composition at various temperatures in the E. coli strain PHL628, which abundantly produces the extracellular structural protein curli. We examined FliA’s influence on external cellular structures like curli and flagella and the biomolecular composition of the biofilm’s extracellular polymeric substance (EPS) using biochemical assays, immunoblotting, and confocal laser scanning microscopy (CLSM). At 37°C, FliA overexpression results in the dramatic growth of biofilm in polystyrene plates and more modest yet significant biofilm growth on silica slides. We observed no significant differences in curli concentration and carbohydrate concentration in the EPS with FliA overexpression. Still, we did see significant changes in the abundance of EPS protein using CLSM at higher growth temperatures. We also noticed increased flagellin concentration, a major structural protein in flagella, occurred with FliA overexpression, specifically in planktonic cultures. These experiments have aided in narrowing our focus to FliA’s role in changing the protein composition of the EPS, which we will examine in future endeavors.

Listeria monocytogenes Biofilm Adaptation to Different Temperatures Seen Through Shotgun Proteomics.

Listeria monocytogenes is a foodborne pathogen that can cause invasive severe human illness (listeriosis) in susceptible patients. Most human listeriosis cases appear to be caused by consumption of refrigerated ready-to-eat foods. Although initial contamination levels in foods are usually low, the ability of these bacteria to survive and multiply at low temperatures allows it to reach levels high enough to cause disease. This study explores the set of proteins that might have an association with L. monocytogenes adaptation to different temperatures. Cultures were grown in biofilm, the most widespread mode of growth in natural and industrial realms. Protein extractions were performed from three different growth temperatures (10, 25, and 37°C) and two growth phases (early stage and mature biofilm). L. monocytogenes subproteomes were targeted using three extraction methods: trypsin-enzymatic shaving, biotin-labeling and cell fractionation. The different subproteomes obtained were separated and analyzed by shotgun proteomics using high-performance liquid chromatography combined with tandem mass spectrometry (LC-OrbiTrap LTQVelos, ThermoFisher Scientific). A total of 141 (biotinylation), 98 (shaving) and 910 (fractionation) proteins were identified. Throughout the 920 unique proteins identified, many are connected to basic cell functions, but some are linked with thermoregulation. We observed some noteworthy protein abundance shifts associated with the major adaptation to cold mechanisms present in L. monocytogenes, namely: the role of ribosomes and the stressosome with a higher abundance of the general stress protein Ctc (Rl25) and the general stress transcription factor sigma B (σB), changes in cell fluidity and motility seen by higher levels of foldase protein PrsA2 and flagellin (FlaA), the uptake of osmolytes with a higher abundance of glycine betaine (GbuB) and carnitine transporters (OpucA), and the relevance of the overexpression of chaperone proteins such as cold shock proteins (CspLA and Dps). As for 37°C, we observed a significantly higher percentage of proteins associated with transcriptional or translational activity present in higher abundance upon comparison with the colder settings. These contrasts of protein expression throughout several conditions will enrich databases and help to model the regulatory circuitry that drives adaptation of L. monocytogenes to environments.

Transcription is a major driving force for plastid genome instability in Arabidopsis.

Though it is an essential process, transcription can be a source of genomic instability. For instance, it may generate RNA:DNA hybrids as the nascent transcript hybridizes with the complementary DNA template. These hybrids, called R-loops, act as a major cause of replication fork stalling and DNA breaks. In this study, we show that lowering transcription and R-loop levels in plastids of Arabidopsis thaliana reduces DNA rearrangements and mitigates plastid genome instability phenotypes. This effect can be observed on a genome-wide scale, as the loss of the plastid sigma transcription factor SIG6 prevents DNA rearrangements by favoring conservative repair in the presence of ciprofloxacin-induced DNA damage or in the absence of plastid genome maintenance actors such as WHY1/WHY3, RECA1 and POLIB. Additionally, resolving R-loops by the expression of a plastid-targeted exogenous RNAse H1 produces similar results. We also show that highly-transcribed genes are more susceptible to DNA rearrangements, as increased transcription of the psbD operon by SIG5 correlates with more locus-specific rearrangements. The effect of transcription is not specific to Sigma factors, as decreased global transcription levels by mutation of heat-stress-induced factor HSP21, mutation of nuclear-encoded polymerase RPOTp, or treatment with transcription-inhibitor rifampicin all prevent the formation of plastid genome rearrangements, especially under induced DNA damage conditions.

Salt stress affects global protein expression profiles of extracellular membrane-derived vesicles of Listeria monocytogenes

Our previous study has suggested that Listeria monocytogenes produces extracellular membrane vesicles (MVs) and its general stress transcription factor sigma B (σB) affects the production of MVs under energy stress. The objective of this study was to evaluate the production of MVs and perform global protein profiling for MVs with or without salt stress to understand the function of MVs in the pathogenesis of L. monocytogenes. When cells of L. monocytogenes were grown under 0.5 M salt stress, protein concentrations of MVs derived from wild-type strain and its isogenic ΔsigB mutant were approximately doubled compared to those of MVs derived from cells without salt stress. Proteomic analyses showed that the number of MV proteins expressed in wild-type strain was similar to that in ΔsigB mutant under salt stress. However, global protein expression profiles were dramatically changed under salt stress compared to those without salt stress. Fifteen σB dependent proteins were expressed in MVs of wild-type strain under salt stress, including osmolyte transporter OpuCABCD. In addition, MVs produced by salt stressed wild-type and ΔsigB mutant inhibited biofilm formation abilities of both strains. Taken together, our results suggest that salt stress can promote the production of MVs involved in carnitine transporter proteins, with σB playing a pivotal role in biological event.

Coupling of Flagellar Gene Expression with Assembly in Salmonella enterica.

There are more than 70 genes in the flagellar and chemosensory regulon of Salmonella enterica. These genes are organized into a transcriptional hierarchy of three promoter classes. At the top of the transcriptional hierarchy is the flhDC operon, also called the flagellar master operon, which is transcribed from the flagellar class 1 promoter region. The protein products of the flhDC operon form a hetero-multimeric complex, FlhD4C2, which directs σ70 RNA polymerase to transcribe from class 2 flagellar promoters. Products of flagellar class 2 transcription are required for the structure and assembly of the hook-basal body (HBB) complex. One of the class 2 flagellar genes, fliA, encodes an alternative sigma transcription factor, σ28, which directs transcription from flagellar class 3 promoters. The class 3 promoters direct transcription of gene products needed after HBB completion including the motor force generators, the filament, and the chemosensory genes. Flagellar gene transcription is coupled to assembly at the level of hook-basal body completion. Two key proteins, σ28 and FliT, play assembly roles prior to HBB completion and upon HBB completion act as positive and negative regulators, respectively. HBB completion signals a secretion-specificity switch in the flagellar type III secretion system, which results in the secretion of σ28 and FliT antigonists allowing these proteins to perform their roles in transcriptional regulation of flagellar genes. Genetic methods have provided the principle driving forces in our understanding of how flagellar gene expression is controlled and coupled to the assembly process.

Mycobacterium tuberculosis gene expression at different stages of hypoxia-induced dormancy and upon resuscitation.

The physiology of dormant Mycobacterium tuberculosis was studied in detail by examining the gene expression of 51 genes using quantitative Reverse-Transcription Polymerase Chain Reaction. A forty-day period of dormancy in the Wayne culture model depicted four major transcription patterns. Some sigma factors and many metabolic genes were constant, whereas genes belonging to the dormancy regulon were activated on day 9. In particular, alpha-crystallin mRNA showed more than a 1,000-fold increase compared to replicating bacilli. Genes belonging to the enduring hypoxic response were up-regulated at day 16, notably, transcription factors sigma B and E. Early genes typical of log-phase bacilli, esat-6 and fbpB, were uniformly down-regulated during dormancy. Late stages of dormancy showed a drop in gene expression likely due to a lack of substrates in anaerobic respiration as demonstrated by the transcriptional activation observed following nitrates addition. Among genes involved in nitrate metabolism, narG was strongly up-regulated by nitrates addition. Dormant bacilli responded very rapidly when exposed to oxygen and fresh medium, showing a transcriptional activation of many genes, including resuscitation-promoting factors, within one hour. Our observations extend the current knowledge on dormant M. tuberculosis gene expression and its response to nutrients and to aerobic and anaerobic respiration.

Transfer of plasmid DNA to clinical coagulase-negative staphylococcal pathogens by using a unique bacteriophage.

Genetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a unique Staphylococcus aureus strain via a specific S. aureus bacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinical Staphylococcus epidermidis isolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

Phylogenetic and functional features of the plastid transcription kinase cpCK2 from Arabidopsis signify a role of cysteinyl SH‐groups in regulatory phosphorylation of plastid sigma factors

A plastidic serine/threonine protein kinase, initially named plastid transcription kinase (PTK) has been implicated in phosphorylation and redox control of chloroplast transcription. This kinase was later renamed as chloroplast casein kinase 2 (cpCK2) because of its physical and functional similarity to nucleocytosolic casein kinase 2 (ncCK2). It shares all four of its cysteine residues with ncCK2 from land plants, while only three of these residues are conserved in algal CK2-type sequences, and just two in animals. Using bacterial overexpression of cpCK2 from Arabidopsis thaliana, here we show the principal features of this enzyme and assign functional determinants of its role as a transcriptional regulator in vitro. The recombinant protein is capable of using various plant sigma transcription factors as phosphorylation substrates. Electrophoretic mobility shift DNA-binding assays reveal differential effects of sigma phosphorylation, depending on the factor and the promoter used. Treatment of the kinase with redox-active reagents indicate a critical involvement of thiol groups in both its enzymatic activity and interaction capabilities. Mutational exchanges of cysteine to serine residues, in combination with in vitro assays, have provided clues to the possible role of individual cysteines. For instance, while Cys4 but not Cys2 is essential for activity, the latter seems to be involved in the formation of intermolecular (regulatory) disulfide bonds.

Structural Characterization of the Multidomain Regulatory Protein Rv1364c from Mycobacterium tuberculosis

The open reading frame rv1364c of Mycobacterium tuberculosis, which regulates the stress-dependent σ factor, σ(F), has been analyzed structurally and functionally. Rv1364c contains domains with sequence similarity to the RsbP/RsbW/RsbV regulatory system of the stress-response σ factor of Bacillus subtilis. Rv1364c contains, sequentially, a PAS domain (which shows sequence similarity to the PAS domain of the B. subtilis RsbP protein), an active phosphatase domain, a kinase (anti-σ(F) like) domain anda C-terminal anti-σ(F) antagonist like domain. The crystal structures of two PAS domain constructs (at 2.3 and 1.6Å) and a phosphatase/kinase dual domain construct (at 2.6Å) are described. The PAS domain is shown to bind palmitic acid but to have 100 times greater affinity for palmitoleic acid. The full-length protein can exist in solution as both monomer and dimer. We speculate that a switch between monomer and dimer, possibly resulting from fatty acid binding,affects the accessibility of the serine of the C-terminal, anti-σ(F) antagonist domain for dephosphorylation by the phosphatase domain thus indirectly altering the availability of σ(F).

Determining the the Effects of Disorder on Binding Affinity

It has long been believed that proteins require a defined three-dimensional structure to perform their specific functions. However, a class of proteins called intrinsically disordered proteins has been identified that do not require a stable structure to perform their functions. These proteins play important roles in many diverse biological processes including signal transduction, transcription, and cell division. Therefore, understanding how these proteins recognize and bind to other proteins to perform their functions is an important question. FlgM is an 88-residue intrinsically disordered protein from bacteria that regulates flagella synthesis by binding the RNA transcription factor Sigma 28. When FlgM is bound to Sigma 28, it inhibits transcription of the genes encoding the late flagella proteins. The FlgM protein is an interesting IDP since FlgM genes from different bacteria exhibit different degree of disorder region. Specifically, our lab has shown that the FlgM gene from A. aeolicus is significantly more ordered than the S. typhimurium FlgM. It is predicted that the more ordered the protein, the higher the affinity of the FlgM for Sigma 28. We are using a combination of Isothermal Titration Calorimetry (ITC) and fluorescence to determine the equilibrium binding constant and the binding kinetics for FlgM binding to Sigma 28 using proteins from a series of different bacteria, including A. aeolicus, S. typhimurium, E. coli, P. aeruginosa, and B. subtilis.

Novel Secretion Apparatus Maintains Spore Integrity and Developmental Gene Expression in Bacillus subtilis

Sporulation in Bacillus subtilis involves two cells that follow separate but coordinately regulated developmental programs. Late in sporulation, the developing spore (the forespore) resides within a mother cell. The regulation of the forespore transcription factor σG that acts at this stage has remained enigmatic. σG activity requires eight mother-cell proteins encoded in the spoIIIA operon and the forespore protein SpoIIQ. Several of the SpoIIIA proteins share similarity with components of specialized secretion systems. One of them resembles a secretion ATPase and we demonstrate that the ATPase motifs are required for σG activity. We further show that the SpoIIIA proteins and SpoIIQ reside in a multimeric complex that spans the two membranes surrounding the forespore. Finally, we have discovered that these proteins are all required to maintain forespore integrity. In their absence, the forespore develops large invaginations and collapses. Importantly, maintenance of forespore integrity does not require σG. These results support a model in which the SpoIIIA-SpoIIQ proteins form a novel secretion apparatus that allows the mother cell to nurture the forespore, thereby maintaining forespore physiology and σG activity during spore maturation.

Polar Localization and Compartmentalization of ClpP Proteases during Growth and Sporulation inBacillus subtilis

Spatial control of proteolysis is emerging as a common feature of regulatory networks in bacteria. In the spore-forming bacterium Bacillus subtilis, the peptidase ClpP can associate with any of three ATPases: ClpC, ClpE, and ClpX. Here, we report that ClpCP, ClpEP, and ClpXP localize in foci often near the poles of growing cells and that ClpP and the ATPase are each capable of polar localization independently of the other component. A region of ClpC containing an AAA domain was necessary and sufficient for polar localization. We also report that ClpCP and ClpXP proteases differentially localize to the forespore and mother cell compartments of the sporangium during spore formation. Moreover, model substrates for each protease created by appending recognition sequences for ClpCP or ClpXP to the green fluorescent protein were preferentially eliminated from the forespore or the mother cell, respectively. Biased accumulation of ClpCP in the forespore may contribute to the cell-specific activation of the transcription factor sigma(F) by preferential ClpCP-dependent degradation of the anti-sigma(F) factor SpoIIAB.

Peptide inhibitor of cytokinesis during sporulation in Bacillus subtilis

Cytokinesis in bacteria is mediated by the tubulin-like protein FtsZ, which forms a Z-ring at the division site. Using FtsZ as bait in a two-hybrid screen, we discovered a 40-amino-acid peptide, termed MciZ, from Bacillus subtilis that appeared to interact with FtsZ. Cells engineered to produce MciZ during growth formed aseptate filaments that lacked Z-rings. A mutant resistant to the toxic effects of MciZ during growth harboured an amino acid substitution near the GTP binding pocket of FtsZ. Synthetic MciZ inhibited the GTPase activity of FtsZ and its ability to polymerize. MciZ was produced during sporulation under the control of the transcription factor sigma(E). In the absence of MciZ, the mother-cell compartment of the sporangium aberrantly formed a Z-ring at a time in development when cytokinetic events normally have ceased. We conclude that MciZ is a previously unrecognized inhibitor of FtsZ that prevents inappropriate Z-ring formation during sporulation. MciZ showed little sequence similarity to other peptides in the databases, except the mouse antimicrobial peptide CRAMP, which we speculate works in part by inhibiting cytokinesis.

SKPDT is a signaling peptide that stimulates sporulation and cry1Aa expression in Bacillus thuringiensis but not in Bacillus subtilis

We have identified and characterized in the supernatant of the transition phase of Bacillus thuringiensis var. kurstaki the peptide SKPDT. This peptide was previously identified by in silico analysis by Pottathil and Lazazzera (Front Biosci 8:32-45 2003) as a putative signaling peptide (NprRB) of the Phr family in B. thuringiensis. The chemically synthesized NprRB did not affect the growth kinetics of B. thuringiensis var. kurstaki but stimulated the sporulation, spore release, and transcription of cry1Aa when added to cultures during the transition phase. In fact, when the peptide (100 nM) was added to a culture in transition phase, the transcription of cry1Aa was stimulated almost threefold, mainly from the late promoter BtII, which requires the late-stage sporulation-specific transcription factor sigma (K). On the other hand, NprRB did not have any effect on B. subtilis. Thus, SKPDT seems to be a signaling peptide specific for B. thuringiensis.

Holding Back the Tide of Antibiotic Resistance

Another novel approach is being pursued by Wisconsin-based ConjuGon (http://www.conjugon.com/). This eight-person startup is using bacterial sexual reproduction to spread lethal genes in pathogen communities. When the lethal genes are expressed, the bacterial population dies out.“I'm a plasmid biologist,” says Marcin Filutowicz, professor of bacteriology at the University of Wisconsin-Madison, and one of ConjuGon's founders. “Bacterial plasmids are extra chromosomal elements that bacteria don't need to live. They transfer DNA molecules among bacteria. They carry genes which frequently encode for antibiotic resistance. Most of the DNA that is transferred between bacteria is done through plasmid exchange.” ConjuGon engineers specific plasmids with DNA that encodes toxins as well as benign bacteria that are immune to the toxins encoded by the plasmids. When immune bacteria transfer the plasmid that encodes for the toxins to the recipient bacteria, such as a pathogen like Pseudomonas aeruginosa, during conjugation the toxin is expressed, killing the pathogen.“It is a Trojan-horse-like approach where we introduce a DNA molecule from donor to recipient and then express in the recipients multiple toxins or single toxins,” says Filutowicz. “When we express multiple toxins, we reduce the probability for the recipient bacteria to acquire immunity.”ConjuGon is targeting genes that encode for antibiotics that block a certain RNA polymerase at several different locations in bacteria. The company has a patent on “Sigma Binding of RNA Polymerase and Uses Therof” for its method of high-throughput screening for small-molecule drugs that interfere with the binding of transcription factor sigma to the core RNA polymerase. ConjuGon chose this target because it is unlikely that bacteria would mutate in enough multiple ways to evade a compound that interferes with this interaction.Conjugon recently received a phase I SBIR grant from the Army to do research on a treatment for antibiotic-resistant Acineobacter baumannii infections contracted by wounded soldiers in Iraq and Afghanistan. Additionally, Ravi Shankar, associate professor, Department of Surgery and Cell Biology, Neurobiology & Anatomy Burn and Shock Trauma Unit at Loyola University Medical Center, is conducting the proof-of-concept studies on ConjuGon's approach on mice. Shankar is interested in ConjuGon's approach as a specialized or adjunct therapy for patients with burn or trauma wounds who have a long healing process. “Much of the time, especially with burn-wound cases, it is not about life or death. It is about achieving adequate wound coverage with grafts or synthetic material so the person can heal faster,” Shankar says. “But unfortunately, if you have an infection, many of the grafts fail. They lift off.” Conventional antibiotic therapy can be problematic as the antibiotic has to be delivered to the actual wound area of the patient. Thus, a systemic antibiotic is not enough. A majority of time, wound care is delivered via topical antibiotics and antimicrobials, such as topical silver emulsions.Shankar says that in the mouse studies that pathogens were totally eliminated within 30 min of administering ConjuGon treatment. “It actually works…. We have to see if it works on human beings,” Shankar says.

Mutational analysis of Escherichia coli heat shock transcription factor sigma 32 reveals similarities with sigma 70 in recognition of the -35 promoter element and differences in promoter DNA melting and -10 recognition.

Upon the exposure of Escherichia coli to high temperature (heat shock), cellular levels of the transcription factor sigma32 rise greatly, resulting in the increased formation of the sigma32 holoenzyme, which is capable of transcription initiation at heat shock promoters. Higher levels of heat shock proteins render the cell better able to cope with the effects of higher temperatures. To conduct structure-function studies on sigma32 in vivo, we have carried out site-directed mutagenesis and employed a previously developed system involving sigma32 expression from one plasmid and a beta-galactosidase reporter gene driven by the sigma32-dependent groE promoter on another in order to monitor the effects of single amino acid substitutions on sigma32 activity. It was found that the recognition of the -35 region involves similar amino acid residues in regions 4.2 of E. coli sigma32 and sigma70. Three conserved amino acids in region 2.3 of sigma32 were found to be only marginally important in determining activity in vivo. Differences between sigma32 and sigma70 in the effects of mutation in region 2.4 on the activities of the two sigma factors are consistent with the pronounced differences between both the amino acid sequences in this region and the recognized promoter DNA sequences.

Induction of Fibronectin Adhesins in Quinolone-Resistant Staphylococcus aureus by Subinhibitory Levels of Ciprofloxacin or by Sigma B Transcription Factor Activity Is Mediated by Two Separate Pathways

We recently reported on the involvement of a RecA-LexA-dependent pathway in the ciprofloxacin-triggered upregulation of fibronectin-binding proteins (FnBPs) by fluoroquinolone-resistant Staphylococcus aureus. The potential additional contribution of the transcription factor sigma B (SigB) to the ciprofloxacin-triggered upregulation of FnBPs was studied in isogenic mutants of fluoroquinolone-resistant strain RA1 (a topoisomerase IV gyrase double mutant of S. aureus NCTC strain 8325), which exhibited widely different levels of SigB activity, as assessed by quantitative reverse transcription-PCR of their respective sigB and SigB-dependent asp23 transcript levels. These mutants were Tn551 insertion sigB strain TE1 and rsbU(+) complemented strain TE2, which exhibited a wild-type SigB operon. Levels of FnBP surface display and fibronectin-mediated adhesion were lower in sigB mutant TE1 or higher in the rsbU(+)-restored strain TE2 compared to their sigB(+) but rsbU parent, strain RA1, exhibiting low levels of SigB activity. Steady-state fnbA and fnbB transcripts levels were similar in strains TE1 and RA1 but increased by 4- and 12-fold, respectively, in strain TE2 compared to those in strain RA1. In contrast, fibronectin-mediated adhesion of strains TE1, RA1, and TE2 was similarly enhanced by growth in the presence of one-eighth the MIC of ciprofloxacin, which led to a significantly higher increase in their fnbB transcript levels compared to the increase in their fnbA transcript levels. Increased SigB levels led to a significant reduction in agr RNAIII; in contrast, it led to a slight increase in sarA transcript levels. In conclusion, upregulation of FnBPs by increased SigB levels and ciprofloxacin exposure in fluoroquinolone-resistant S. aureus occurs via independent pathways whose concerted actions may significantly promote bacterial adhesion and colonization.

Alternative Sigma Factor σB Is Not Essential for Listeria monocytogenes Surface Attachment

Listeria monocytogenes is a foodborne pathogen frequently isolated from the food processing environment. Multiple lines of evidence suggested a possible role for the L. monocytogenes alternative transcription factor sigma B (σB) in surface attachment and biofilm formation. Therefore, through plate count and microscopic techniques, the L. monocytogenes 10403S strain and an otherwise isogenic ΔsigB strain were tested for attachment to stainless steel. Analysis of microscopic images revealed that after 72 h at 24°C under static conditions the tested L. monocytogenes strains attached uniformly to surfaces as single cells. Both strains were capable of rapid attachment (i.e., numbers of attached cells were essentially the same after either 5 min or 24 h of incubation). Numbers of attached ΔsigB cells were significantly lower than those of the wild-type strain after 48 and 72 h of incubation at 24°C (P = 0.001). Similar numbers of the ΔsigB strain attached to stainless steel regardless of temperature (24 or 37°C); however, ΔsigB cells attached at higher relative numbers in the presence of 6% NaCl after 48 and 72 h. Furthermore, in the presence of Pseudomonas fluorescens, similarly high numbers of wild-type and ΔsigB cells attached to the surfaces, forming mixed biofilms. Our data suggest that σB is not required for initial surface attachment of L. monocytogenes.

Modulation of fibronectin adhesins and other virulence factors in a teicoplanin-resistant derivative of methicillin-resistant Staphylococcus aureus.

The impact of glycopeptide resistance on the molecular regulation of Staphylococcus aureus virulence and attachment to host tissues is poorly documented. We compared stable teicoplanin-resistant methicillin-resistant S. aureus (MRSA) strain 14-4 with its teicoplanin-susceptible MRSA parent, strain MRGR3, which exhibits a high degree of virulence in a rat model of chronic foreign body MRSA infection. The levels of fibronectin-mediated adhesion and surface display of fibronectin-binding proteins were higher in teicoplanin-resistant strain 14-4 than in its teicoplanin-susceptible parent or a teicoplanin-susceptible revertant (strain 14-4rev) that spontaneously emerged during tissue cage infection. Quantitative reverse transcription-PCR (qRT-PCR) showed four- and twofold higher steady-state levels of fnbA and fnbB transcripts, respectively, in strain 14-4 than in its teicoplanin-susceptible counterparts. Analysis of global regulatory activities by qRT-PCR revealed a strong reduction in the steady-state levels of RNAIII and RNAII in the teicoplanin-resistant strain compared to in its teicoplanin-susceptible counterparts. In contrast, sarA mRNA levels were more than fivefold higher in strain 14-4 than in MRGR3 and 14-4rev. Furthermore, the alternative transcription factor sigma B had a higher level of functional activity in the teicoplanin-resistant strain than in its teicoplanin-susceptible counterparts, as evidenced by significant increases in both the sigma B-dependent asp23 mRNA levels and the sarA P3 promoter-derived transcript levels, as assayed by qRT-PCR and Northern blotting, respectively. These data provide further evidence that the emergence of glycopeptide resistance is linked by still poorly understood molecular pathways with significant pleiotropic changes in the expression and regulation of some major virulence genes. These molecular and phenotypic changes may have a profound impact on the bacterial adhesion and colonization properties of such multiresistant organisms.

A multicomponent protein complex mediates environmental stress signaling in Bacillus subtilis.

Activity of the general stress transcription factor sigma(B) of Bacillus subtilis is regulated directly by a partner-switching mechanism in which key protein interactions are governed by serine phosphorylation. Signals of energy or environmental stress are conveyed to sigma(B) by independent pathways, each terminating with a differentially regulated serine phosphatase, whose activity is required to control the partner-switching regulators. We present genetic and biochemical evidence that activation of the RsbU environmental signaling phosphatase is modulated by a second, atypical partner switch that comprises redundant negative regulatory proteins in a large, multicomponent signaling complex. In the current model, negative regulation of the RsbU phosphatase depends solely on the RsbS antagonist protein. Here, we perform a critical genetic test that invalidates this model and demonstrates that the RsbS antagonist alone is insufficient to prevent environmental signaling. Also required is one of a family of four co-antagonist proteins, here renamed RsbRA, RsbRB, RsbRC, and RsbRD, each with a carboxyl-terminal domain closely resembling the entire RsbS protein. Because any single member of the RsbR family, together with RsbS, was sufficient for environmental signaling, we conclude that the RsbR proteins serve as redundant co-antagonists necessary for RsbS antagonist function. Moreover, purification of RsbRA from cell extracts by nickel affinity and gel-filtration chromatography found a multicomponent complex containing the RsbRA and RsbRB co-antagonists together with the RsbS antagonist. We propose that this complex serves as a machine to transmit stress signals to sigma(B), and that the properties of the complex may contribute to environmental stress sensing.