SigB Activity Research Articles

The Virulence and Infectivity of Listeria monocytogenes Are Not Substantially Altered by Elevated SigB Activity.

Listeria monocytogenes is a bacterial pathogen capable of causing severe infections but also thriving outside the host. To respond to different stress conditions, L. monocytogenes mainly utilizes the general stress response regulon, which largely is controlled by the alternative sigma factor Sigma B (SigB). In addition, SigB is important for virulence gene expression and infectivity. Upon encountering stress, a large multicomponent protein complex known as the stressosome becomes activated, ultimately leading to SigB activation. RsbX is a protein needed to reset a "stressed" stressosome and prevent unnecessary SigB activation in nonstressed conditions. Consequently, absence of RsbX leads to constitutive activation of SigB even without prevailing stress stimulus. To further examine the involvement of SigB in the virulence of this pathogen, we investigated whether a strain with constitutively active SigB would be affected in virulence factor expression and/or infectivity in cultured cells and in a chicken embryo infection model. Our results suggest that increased SigB activity does not substantially alter virulence gene expression compared with the wild-type (WT) strain at transcript and protein levels. Bacteria lacking RsbX were taken up by phagocytic and nonphagocytic cells at a similar frequency to WT bacteria, both in stressed and nonstressed conditions. Finally, the absence of RsbX only marginally affected the ability of bacteria to infect chicken embryos. Our results suggest only a minor role of RsbX in controlling virulence factor expression and infectivity under these conditions.

Prevalence of the SigB-Deficient Phenotype among Clinical Staphylococcus aureus Isolates Linked to Bovine Mastitis.

Phenotypic adaptation has been associated with persistent, therapy-resistant Staphylococcus aureus infections. Recently, we described within-host evolution towards a Sigma factor B (SigB)-deficient phenotype in a non-human host, a naturally infected dairy cow with chronic, persistent mastitis. However, to our knowledge, the prevalence of SigB deficiency among clinical S. aureus isolates remains unknown. In this study, we screened a collection of bovine mastitis isolates for phenotypic traits typical for SigB deficiency: decreased carotenoid pigmentation, increased proteolysis, secretion of α-hemolysin and exoproteins. Overall, 8 out of 77 (10.4%) isolates of our bovine mastitis collection exhibited the SigB-deficient phenotype. These isolates were assigned to various clonal complexes (CC8, CC9, CC97, CC151, CC3666). We further demonstrated a strong positive correlation between asp23-expression (a marker of SigB activity) and carotenoid pigmentation (r = 0.6359, p = 0.0008), underlining the role of pigmentation as a valuable predictor of the functional status of SigB. Sequencing of the sigB operon (mazEF-rsbUVW-sigB) indicated the phosphatase domain of the RsbU protein as a primary target of mutations leading to SigB deficiency. Indeed, by exchanging single nucleotides in rsbU, we could either induce SigB deficiency or restore the SigB phenotype, demonstrating the pivotal role of RsbU for SigB functionality. The data presented highlight the clinical relevance of SigB deficiency, and future studies are needed to exploit its role in staphylococcal infections.

Phylogenetic and Phenotypic Analyses of a Collection of Food and Clinical Listeria monocytogenes Isolates Reveal Loss of Function of Sigma B from Several Clonal Complexes.

To understand the molecular mechanisms that contribute to the stress responses of the important foodborne pathogen Listeria monocytogenes, we collected 139 strains (meat, n = 25; dairy, n = 10; vegetable, n = 8; seafood, n = 14; mixed food, n = 4; and food processing environments, n = 78), mostly isolated in Ireland, and subjected them to whole-genome sequencing. These strains were compared to 25 Irish clinical isolates and 4 well-studied reference strains. Core genome and pan-genome analysis confirmed a highly clonal and deeply branched population structure. Multilocus sequence typing showed that this collection contained a diverse range of strains from L. monocytogenes lineages I and II. Several groups of isolates with highly similar genome content were traced to single or multiple food business operators, providing evidence of strain persistence or prevalence, respectively. Phenotypic screening assays for tolerance to salt stress and resistance to acid stress revealed variants within several clonal complexes that were phenotypically distinct. Five of these phenotypic outliers were found to carry mutations in the sigB operon, which encodes the stress-inducible sigma factor sigma B. Transcriptional analysis confirmed that three of the strains that carried mutations in sigB, rsbV, or rsbU had reduced SigB activity, as predicted. These strains exhibited increased tolerance to salt stress and displayed decreased resistance to low pH stress. Overall, this study shows that loss-of-function mutations in the sigB operon are comparatively common in field isolates, probably reflecting the cost of the general stress response to reproductive fitness in this pathogen. IMPORTANCE The bacterial foodborne pathogen Listeria monocytogenes frequently contaminates various categories of food products and is able to cause life-threatening infections when ingested by humans. Thus, it is important to control the growth of this bacterium in food by understanding the mechanisms that allow its proliferation under suboptimal conditions. In this study, intraspecies heterogeneity in stress response was observed across a collection consisting of mainly Irish L. monocytogenes isolates. Through comparisons of genome sequence and phenotypes observed, we identified three strains with impairment of the general stress response regulator SigB. Two of these strains are used widely in food challenge studies for evaluating the growth potential of L. monocytogenes. Given that loss of SigB function is associated with atypical phenotypic properties, the use of these strains in food challenge studies should be re-evaluated.

Impact of osmotic stress on the phosphorylation and subcellular location of Listeria monocytogenes stressosome proteins

Listeria monocytogenes responds to environmental stress using a supra-macromolecular complex, the stressosome, to activate the stress sigma factor SigB. The stressosome structure, inferred from in vitro-assembled complexes, consists of the core proteins RsbR (here renamed RsbR1) and RsbS and, the kinase RsbT. The active complex is proposed to be tethered to the membrane and to support RsbR1/RsbS phosphorylation by RsbT and the subsequent release of RsbT following signal perception. Here, we show in actively-growing cells that L. monocytogenes RsbR1 and RsbS localize mostly in the cytosol in a fully phosphorylated state regardless of osmotic stress. RsbT however distributes between cytosolic and membrane-associated pools. The kinase activity of RsbT on RsbR1/RsbS and its requirement for maximal SigB activation in response to osmotic stress were demonstrated in vivo. Cytosolic RsbR1 interacts with RsbT, while this interaction diminishes at the membrane when RsbR1 paralogues (RsbR2, RsbR3 and RsbL) are present. Altogether, the data support a model in which phosphorylated RsbR1/RsbS may sustain basal SigB activity in unstressed cells, probably assuring a rapid increase in such activity in response to stress. Our findings also suggest that in vivo the active RsbR1-RsbS-RsbT complex forms only transiently and that membrane-associated RsbR1 paralogues could modulate its assembly.

Structural insights into the regulation of SigB activity by RsbV and RsbW.

Bacillus subtilis SigB is an alternative sigma factor that initiates the transcription of stress-responsive genes. The anti-sigma factor RsbW tightly binds SigB to suppress its activity under normal growth conditions and releases it when nonphosphorylated RsbV binds to RsbW in response to stress signals. To understand the regulation of SigB activity by RsbV and RsbW based on structural features, crystal structures and a small-angle X-ray scattering (SAXS) envelope structure of the RsbV-RsbW complex were determined. The crystal structures showed that RsbV and RsbW form a heterotetramer in a similar manner to a SpoIIAA-SpoIIAB tetramer. Multi-angle light scattering and SAXS revealed that the RsbV-RsbW complex is an octamer in solution. Superimposition of the crystal structure on the SAXS envelope structure showed that the unique dimeric interface of RsbW mediates the formation of an RsbV-RsbW octamer and does not prevent RsbV and SigB from binding to RsbW. These results provide structural insights into the molecular assembly of the RsbV-RsbW complex and the regulation of SigB activity.

Revisiting the regulation of the capsular polysaccharide biosynthesis gene cluster in Staphylococcus aureus.

Capsular polysaccharide (CP) biosynthesis in Staphylococcus aureus is tightly controlled resulting in a heterogeneous phenotype within a population and CP being mainly detectable in nongrowing cells. Expression of the corresponding biosynthesis gene cluster is driven by one promoter element (Pcap ). Here, we demonstrate that Pcap contains a main SigB-dependent promoter. The SigB consensus motif overlaps with a previously described inverted repeat (IR) that is crucial for cap expression. The essentiality of the IR is derived from this region acting as a SigB binding site rather than as an operator site for the proposed cap activators RbsR and MsaB. Furthermore, Pcap contains an extensive upstream region harboring a weak SigA-dependent promoter and binding sites for cap repressors such as SaeR, CodY and Rot. Heterogeneous CP synthesis is determined by SigB activity and repressor binding to the upstream region. SigB dependency and regulation by the upstream repressors are also sufficient to explain the temporal gene expression pattern at the transcriptional level. However, CP synthesis remains growth phase-dependent even when transcription is rendered constitutive, suggesting additional posttranscriptional regulatory circuits. Thus, the interference of multiple repressors with SigB-dependent promoter activity as well as post-transcriptional mechanisms ensure the appropriate regulation of CP synthesis.

Stress-Responsive Alternative Sigma Factor SigB Plays a Positive Role in the Antifungal Proficiency of Bacillus subtilis.

Different Bacillus species with PGPR (plant growth-promoting rhizobacterium) activity produce potent biofungicides and stimulate plant defense responses against phytopathogenic fungi. However, very little is known about how these PGPRs recognize phytopathogens and exhibit the antifungal response. Here, we report the antagonistic interaction between Bacillus subtilis and the phytopathogenic fungus Fusarium verticillioides We demonstrate that this bacterial-fungal interaction triggers the induction of the SigB transcription factor, the master regulator of B. subtilis stress adaptation. Dual-growth experiments performed with live or dead mycelia or culture supernatants of F. verticillioides showed that SigB was activated and required for the biocontrol of fungal growth. Mutations in the different regulatory pathways of SigB activation in the isogenic background revealed that only the energy-related RsbP-dependent arm of SigB activation was responsible for specific fungal detection and triggering the antagonistic response. The activation of SigB increased the expression of the operon responsible for the production of the antimicrobial cyclic lipopeptide surfactin (the srfA operon). SigB-deficient B. subtilis cultures produced decreased amounts of surfactin, and B. subtilis cultures defective in surfactin production (ΔsrfA) were unable to control the growth of F. verticillioidesIn vivo experiments of seed germination efficiency and early plant growth inhibition in the presence of F. verticillioides confirmed the physiological importance of SigB activity for plant bioprotection.IMPORTANCE Biological control using beneficial bacteria (PGPRs) represents an attractive and environment-friendly alternative to pesticides for controlling plant diseases. Different PGPR Bacillus species produce potent biofungicides and stimulate plant defense responses against phytopathogenic fungi. However, very little is known about how PGPRs recognize phytopathogens and process the antifungal response. Here, we report how B. subtilis triggers the induction of the stress-responsive sigma B transcription factor and the synthesis of the lipopeptide surfactin to fight the phytopathogen. Our findings show the participation of the stress-responsive regulon of PGPR Bacillus in the detection and biocontrol of a phytopathogenic fungus of agronomic impact.

Rhodomyrtone decreases Staphylococcus aureus SigB activity during exponentially growing phase and inhibits haemolytic activity within membrane vesicles

Sigma factor B (SigB) controls the expression of Staphylococcus aureus genes including virulence factors and plays a role in the bacterial secretion system through membrane vesicle production. Inhibition of SigB could attenuate SigB dependent virulence and secretion system. The objective of this study was to determine the effects of rhodomyrtone on SigB and virulence factors related to SigB. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of rhodomyrtone against 67 clinical methicillin-resistant S. aureus isolates were 0.25–8 μg/ml, which were similar to those of vancomycin. Using luciferase gene fused to SigB dependent promoters of asp23, five time reduction in SigB activity was observed when the bacteria were treated with rhodomyrtone for 3 h. Rhodomyrtone significantly reduced SigB activity in a concentration dependent manner in exponentially growing cells (P < 0.05). In addition, sigB mutant was more sensitive towards increasing concentrations of rhodomyrtone than the wild type and yabJ-spoVG mutant. Rhodomyrtone at 0.625 μg/ml reduced the growth of sigB mutant by approximately 99%, compared with the yabJ-spoVG mutant and the wild type. Membrane vesicles were significantly reduced in the bacterial cells when treated with 0.5 × MIC rhodomyrtone (P < 0.05). Decreased haemolytic activity was detected within rhodomyrtone-treated membrane vesicles. The results indicated that rhodomyrtone inhibited S. aureus SigB activity during exponentially growing phase and inhibited haemolytic activity within membrane vesicles.

Comparative Secretome Analyses of Human and Zoonotic Staphylococcus aureus Isolates CC8, CC22, and CC398

The spread of methicillin-resistant Staphylococcus aureus (MRSA) in the community, hospitals and in livestock is mediated by highly diverse virulence factors that include secreted toxins, superantigens, enzymes and surface-associated adhesins allowing host adaptation and colonization. Here, we combined proteogenomics, secretome and phenotype analyses to compare the secreted virulence factors in selected S. aureus isolates of the dominant human- and livestock-associated genetic lineages CC8, CC22, and CC398. The proteogenomic comparison revealed 2181 core genes and 1306 accessory genes in 18 S. aureus isolates reflecting the high genome diversity. Using secretome analysis, we identified 869 secreted proteins with 538 commons in eight isolates of CC8, CC22, and CC398. These include 64 predicted extracellular and 37 cell surface proteins that account for 82.4% of total secretome abundance. Among the top 10 most abundantly secreted virulence factors are the major autolysins (Atl, IsaA, Sle1, SAUPAN006375000), lipases and lipoteichoic acid hydrolases (Lip, Geh, LtaS), cytolytic toxins (Hla, Hlb, PSMβ1) and proteases (SspB). The CC398 isolates showed lower secretion of cell wall proteins, but higher secretion of α- and β-hemolysins (Hla, Hlb) which correlated with an increased Agr activity and strong hemolysis. CC398 strains were further characterized by lower biofilm formation and staphyloxanthin levels because of decreased SigB activity. Overall, comparative secretome analyses revealed CC8- or CC22-specific enterotoxin and Spl protease secretion as well as Agr- and SigB-controlled differences in exotoxin and surface protein secretion between human-specific and zoonotic lineages of S. aureus.

SigB Is a Dominant Regulator of Virulence in Staphylococcus aureus Small-Colony Variants

Staphylococcus aureus small-colony variants (SCVs) are persistent pathogenic bacteria characterized by slow growth and, for many of these strains, an increased ability to form biofilms and to persist within host cells. The virulence-associated gene expression profile of SCVs clearly differs from that of prototypical strains and is often influenced by SigB rather than by the agr system. One objective of this work was to confirm the role of SigB in the control of the expression of virulence factors involved in biofilm formation and intracellular persistence of SCVs. This study shows that extracellular proteins are involved in the formation of biofilm by three SCV strains, which, additionally, have a low biofilm-dispersing activity. It was determined that SigB activity modulates biofilm formation by strain SCV CF07-S and is dominant over that of the agr system without being solely responsible for the repression of proteolytic activity. On the other hand, the expression of fnbA and the control of nuclease activity contributed to the SigB-dependent formation of biofilm of this SCV strain. SigB was also required for the replication of CF07-S within epithelial cells and may be involved in the colonization of lungs by SCVs in a mouse infection model. This study methodically investigated SigB activity and associated mechanisms in the various aspects of SCV pathogenesis. Results confirm that SigB activity importantly influences the production of virulence factors, biofilm formation and intracellular persistence for some clinical SCV strains.

Occurrence of mutations impairing sigma factor B (SigB) function upon inactivation of Listeria monocytogenes genes encoding surface proteins

Bacteria of the genus Listeria contain the largest family of LPXTG surface proteins covalently anchored to the peptidoglycan. The extent to which these proteins may function or be regulated cooperatively is at present unknown. Because of their unique cellular location, we reasoned that distinct LPXTG proteins could act as elements contributing to cell wall homeostasis or influencing the stability of other surface proteins bound to peptidoglycan. To test this hypothesis, we used proteomics to analyse mutants of the intracellular pathogen Listeria monocytogenes lacking distinct LPXTG proteins implicated in pathogen-host interactions, such as InlA, InlF, InlG, InlH, InlJ, LapB and Vip. Changes in the cell wall proteome were found in inlG and vip mutants, which exhibited reduced levels of the LPXTG proteins InlH, Lmo0610, Lmo0880 and Lmo2085, all regulated by the stress-related sigma factor SigB. The ultimate basis of this alteration was uncovered by genome sequencing, which revealed that these inlG and vip mutants carried loss-of-function mutations in the rsbS, rsbU and rsbV genes encoding regulatory proteins that control SigB activity. Attempts to recapitulate this negative selection of SigB in a large series of new inlG or vip mutants constructed for this purpose were, however, unsuccessful. These results indicate that inadvertent secondary mutations affecting SigB functionality can randomly arise in L. monocytogenes when using common genetic procedures or during subculturing. Testing of SigB activity could be therefore valuable when manipulating genetically L. monocytogenes prior to any subsequent phenotypic analysis. This test may be even more justified when generating deletions affecting cell envelope components.

An insight into the significance of the DnaK heat shock system in Staphylococcus aureus

The bacterium Staphylococcus aureus utilizes a variety of different mechanisms to survive unfavorable stress conditions that are critical for its persistence in the environment and for pathogenicity. The staphylococcal DnaK heat shock system functions as a major protein folding machine under stress conditions that cause aggregation and un-folding of proteins. In prior studies, S. aureus cells with a non-functional DnaK system showed reduced tolerance to heat, oxidative and antibiotic stresses, a lowered carotenoid production, and decreased survival in a murine host. This study provides insights that the altered phenotypes of the dnaK mutant cells are not due to decreased SigB activity in the mutant cell. Transcriptomic profiling studies provide evidence that a large number of genes encoding proteins involved in cell wall biogenesis, virulence and general stress tolerance, and genes encoding proteins involved in metabolic processes are differentially regulated in dnaK mutant cells relative to wild-type S. aureus. It was also determined that loss of functional DnaK caused a reduction in the ability of S. aureus to make biofilms and its adherence to eukaryotic cells. This study provides evidence of a global significance of DnaK heat shock system in S. aureus.

Intrahost passage alters SigB-dependent acid resistance and host cell-associated kinetics of Listeria monocytogenes

Listeria monocytogenes is a foodborne pathogen that causes gastroenteritis, maternofetal infections and meningoencephalitis in humans. Here we report that an intrahost genome mutation alters bacterial acid resistance and the abilities for replication/invasion in tissue cell culture. Among the L. monocytogenes isolates from the recent outbreak in Japan, we found that one food strain, 668, exhibited the greatest acid resistance, whereas one human clinical strain, 690, sharing identical pulsed-field gel electrophoresis (PFGE) and ribotyping patterns, exhibited an acid-sensitive phenotype. Passage of the 668 food strain through the mouse intestine increased its acid sensitivity without altering the macrogenotypes, indicating intrahost alteration of the bacterial acid-resistant phenotype. Genetic and proteomic analyses revealed a link between acid resistance and SigB (RNA polymerase SigmaB subunit) activity. Compared with the strain 668, the clinical and 4 of 5 mice-passaged strains showed a mutation in the rsbW locus, whose product controls the regulation of SigB activity. Corresponding to the SigB activity, the host-passaged strains had reduced abilities to survive inside macrophages and to invade Caco-2 cells, compared with the food strain 668. Overall, we have demonstrated the first example of a host environment promoting the alteration of SigB-dependent acid resistance and host cell-associated actions of L. monocytogenes. Our study provides new insight into the potential role of intrahost environment in the process of bacterial evolution.

The alternative sigma factor B modulates virulence gene expression in a murine Staphylococcus aureus infection model but does not influence kidney gene expression pattern of the host

Infections caused by Staphylococcus aureus are associated with significant morbidity and mortality and are an increasing threat not only in hospital settings. The expression of the staphylococcal virulence factor repertoire is known to be affected by the alternative sigma factor B (SigB). However, its impact during infection still is a matter of debate. Kidney tissues of controls or mice infected with S. aureus HG001 or its isogenic sigB mutant were analyzed by transcriptome profiling to monitor the host response, and additionally expression of selected S. aureus genes was monitored by RT-qPCR. Direct transcript analysis by RT-qPCR revealed significant SigB activity in all mice infected with the wild-type strain, but not in its isogenic sigB mutant ( p < 0.0001). Despite a clear-cut difference in the SigB-dependent transcription pattern of virulence genes ( clfA, aur, and hla), the host reaction to infection (either wild type or sigB mutant) was almost identical. Despite its significant activity in vivo, loss of SigB did neither have an effect on the outcome of infection nor on murine kidney gene expression pattern. Thus, these data support the role of SigB as virulence modulator rather than being a virulence determinant by itself.

A role for sigma factor B in the emergence of Staphylococcus aureus small-colony variants and elevated biofilm production resulting from an exposure to aminoglycosides

Staphylococcus aureus small-colony variants (SCVs) and biofilms are linked to chronic infections. It is known that the presence of aminoglycoside antibiotics may contribute to the emergence of SCVs and it is thought that molecular mechanisms are involved in the ability of S. aureus to adopt this phenotype. No study has addressed the possible role of the stress- and colonization-related alternative sigma factor B (SigB) in the emergence of SCVs, although a sustained SigB activity was reported in these variants. Here, we demonstrate that SigB is involved in the emergence of SCVs resulting from an exposure to a sub-inhibitory concentration of aminoglycosides. Monitoring of gene expression in an aminoglycoside-treated prototypical strain or in clinical SCVs showed the activation of SigB, whereas the accessory gene regulator ( agr) system was not. Furthermore, gentamicin-treated prototypical bacteria and SCVs had an increased ability to form biofilm only in a SigB functional background. The administration of a sub-inhibitory concentration of gentamicin significantly increased the formation of SCVs for a prototypical strain but not for the sigB mutant in a mouse model of S. aureus-induced mastitis. Collectively, our results show that SigB may positively influence the appearance of S. aureus SCVs and the production of biofilm upon aminoglycoside exposure.

Role of RsbU in controlling SigB activity in Staphylococcus aureus following alkaline stress.

SigB is an alternative sigma factor that controls a large regulon in Staphylococcus aureus. Activation of SigB requires RsbU, a protein phosphatase 2C (PP2C)-type phosphatase. In a closely related organism, Bacillus subtilis, RsbU activity is stimulated upon interaction with RsbT, a kinase, which following an activating stimulus switches from a 25S high-molecular-weight complex, the stressosome, to the N-terminal domain of RsbU. Active RsbU dephosporylates RsbV and thereby triggers the release of SigB from its inhibitory complex with RsbW. While RsbU, RsbV, RsbW, and SigB are conserved in S. aureus, proteins similar to RsbT and the components of the stressosome are not, raising the question of how RsbU activity and hence SigB activity are controlled in S. aureus. We found that in contrast to the case in B. subtilis, the induced expression of RsbU was sufficient to stimulate SigB-dependent transcription in S. aureus. However, activation of SigB-dependent transcription following alkaline stress did not lead to a clear accumulation of SigB and its regulators RsbV and RsbW or to a change in the RsbV/RsbV-P ratio in S. aureus. When expressed in B. subtilis, the S. aureus RsbU displayed a high activity even in the absence of an inducing stimulus. This high activity could be transferred to the PP2C domain of the B. subtilis RsbU protein by a fusion to the N-terminal domain of the S. aureus RsbU. Collectively, the data suggest that the activity of the S. aureus RsbU and hence SigB may be subjected to different regulation in comparison to that in B. subtilis.

Interconnections between Sigma B, agr , and Proteolytic Activity in Staphylococcus aureus Biofilm Maturation

Staphylococcus aureus is a proficient biofilm former on host tissues and medical implants. We mutagenized S. aureus strain SH1000 to identify loci essential for ica-independent mechanisms of biofilm maturation and identified multiple insertions in the rsbUVW-sigB operon. Following construction and characterization of a sigB deletion, we determined that the biofilm phenotype was due to a lack of sigma factor B (SigB) activity. The phenotype was conserved in a sigB mutant of USA300 strain LAC, a well-studied community-associated methicillin-resistant S. aureus isolate. We determined that agr RNAIII levels were elevated in the sigB mutants, and high levels of RNAIII expression are known to have antibiofilm effects. By introducing an agr mutation into the SH1000 or LAC sigB deletion strain, S. aureus regained biofilm capacity, indicating that the biofilm phenotype was agr dependent. Protease activity is linked to agr activity and ica-independent biofilm formation, and we observed that the protease inhibitors phenylmethylsulfonyl fluoride and alpha-macroglobulin could reverse the sigB biofilm defect. Similarly, inactivating genes encoding both the aureolysin and Spl extracellular proteases in the sigB mutant restored biofilm capacity. Due to the growing link between murein hydrolase activity and biofilm maturation, autolysin zymography was performed, which revealed an altered profile in the sigB mutant; again, the phenotype could be repaired through protease inactivation. These findings indicate that the lack of SigB activity results in increased RNAIII expression, thus elevating extracellular protease levels and altering the murein hydrolase activity profile. Altogether, our observations demonstrate that SigB is an essential regulator of S. aureus biofilm maturation.

Staphylococcus aureus SigB activity promotes a strong fibronectin–bacterium interaction which may sustain host tissue colonization by small‐colony variants isolated from cystic fibrosis patients

Genes encoding cell-surface proteins regulated by SigB are stably expressed in Staphylococcus aureus small-colony variants (SCVs) isolated from cystic fibrosis (CF) patients. Our hypothesis is that CF-isolated SCVs are locked into a colonization state by sustaining the expression of adhesins such as fibronectin-binding proteins (FnBPs) throughout growth. Force spectroscopy was used to study the fibronectin-FnBPs interaction among strains varying for their SigB activity. The fibronectin-FnBPs interaction was described by a strength of 1000+/-400 pN (pulling rate of 2 microm s(-1)), an energetic barrier width of 0.6+/-0.1 A and an off-rate below 2 x 10(-4) s(-1). A CF-isolated SCV highly expressed fnbA throughout growth and showed a sustained capacity to bind fibronectin, whereas a prototypic strain showed a reduced frequency of fibronectin-binding during the stationary growth phase when its fnbA gene was down-regulated. Reduced expression of fnbA was observed in sigB mutants, which was associated with an overall decrease adhesion to fibronectin. These results suggest that the fibronectin-FnBPs interaction plays a role in the formation of a mechanically resistant adhesion of S. aureus to host tissues and supports the hypothesis that CF-isolated SCVs are locked into a colonization state as a result of a sustained SigB activity.

Genetic characterization of the natural SigB variants found in clinical isolates of Staphylococcus aureus

The SigB concentrations in clinical isolates of Staphylococcus aureus were measured to examine their correlation with the antibiotic resistance. The SigB concentrations in methicillin-resistant S. aureus (MRSA) were higher than in the control strain, N315, and many of methicillin-susceptible S. aureus (MSSA). Sequencing analyses of the sigB genes revealed that the strains exhibiting the high SigB concentrations have three amino acid substitutions in SigB: I11V, D141N, and Q256K. Further analysis using isogenic mutants demonstrated that D141N (or both D141N and Q256K) is essential to maintain the high SigB concentration. These substitutions affected the UV tolerance, but had no effect on the antibiotic resistance. The SigB activity was affected by these substitutions toward the stationary phase, but not during the transient heat shock response.

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.