Oxidative Disinfectants Activate Different Responses in Vibrio parahaemolyticus

ABSTRACT Vibrio parahaemolyticus is a prevalent seafoodborne enteropathogen that has become a global concern since the spread of its pandemic strain in 1996. This study investigates the responses of this pathogen to the oxidative disinfectants hydrogen peroxide, chlorine dioxide, and peracetic acid. Expression of the regulator genes oxyR and rpoS, determined by reverse transcription PCR, in V. parahaemolyticus wild-type, oxyR mutant, and rpoS mutant strains exhibited similar patterns in response to the tested oxidative disinfectants. The transcription of the rpoS gene was markedly enhanced in the oxyR mutant strain in the exponential phase. The expression of catalase KatE1 was tracked by using a LacZ fusion reporter in these strains. The experimental results revealed that KatE1 was a significant scavenger of hydrogen peroxide and peracetic acid in V. parahaemolyticus, and RpoS may partially compensate for the regulatory role of OxyR in the oxyR mutant strain. In contrast to its responses to hydrogen peroxide and paracetic acid, KatE1 was not the primary scavenger of chlorine dioxide in these V. parahaemolyticus strains. This study shows that these disinfectants activated a basic oxidative response in this pathogen with different features.

Two Distinct Mechanisms Govern RpoS-Mediated Repression of Tick-Phase Genes during Mammalian Host Adaptation byBorrelia burgdorferi, the Lyme Disease Spirochete

ABSTRACTThe alternative sigma factor RpoS plays a key role modulating gene expression inBorrelia burgdorferi, the Lyme disease spirochete, by transcribing mammalian host-phase genes and repressing σ70-dependent genes required within the arthropod vector. To identifycisregulatory elements involved in RpoS-dependent repression, we analyzed green fluorescent protein (GFP) transcriptional reporters containing portions of the upstream regions of the prototypical tick-phase genesospAB, theglpoperon, andbba74. As RpoS-mediated repression occurs only following mammalian host adaptation, strains containing the reporters were grown in dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats. Wild-type spirochetes harboringospAB- andglp-gfpconstructs containing only the minimal (−35/−10) σ70promoter elements had significantly lower expression in DMCs relative to growthin vitroat 37°C; no reduction in expression occurred in a DMC-cultivated RpoS mutant harboring these constructs. In contrast, RpoS-mediated repression ofbba74required a stretch of DNA located between −165 and −82 relative to its transcriptional start site. Electrophoretic mobility shift assays employing extracts of DMC-cultivatedB. burgdorferiproduced a gel shift, whereas extracts from RpoS mutant spirochetes did not. Collectively, these data demonstrate that RpoS-mediated repression of tick-phase borrelial genes occurs by at least two distinct mechanisms. One (e.g.,ospABand theglpoperon) involves primarily sequence elements near the core promoter, while the other (e.g.,bba74) involves an RpoS-induced transacting repressor. Our results provide a genetic framework for further dissection of the essential “gatekeeper” role of RpoS throughout theB. burgdorferienzootic cycle.IMPORTANCEBorrelia burgdorferi, the Lyme disease spirochete, modulates gene expression to adapt to the distinctive environments of its mammalian host and arthropod vector during its enzootic cycle. The alternative sigma factor RpoS has been referred to as a “gatekeeper” due to its central role in regulating the reciprocal expression of mammalian host- and tick-phase genes. While RpoS-dependent transcription has been studied extensively, little is known regarding the mechanism(s) of RpoS-mediated repression. We employed a combination of green fluorescent protein transcriptional reporters along with anin vivomodel to definecisregulatory sequences responsible for RpoS-mediated repression of prototypical tick-phase genes. Repression ofospABand theglpoperon requires only sequences near their core promoters, whereas modulation ofbba74expression involves a putative RpoS-dependent repressor that binds upstream of the core promoter. Thus, Lyme disease spirochetes employ at least two different RpoS-dependent mechanisms to repress tick-phase genes within the mammal.

The Polymorphic Aggregative Phenotype of Shiga Toxin-Producing Escherichia coli O111 Depends on RpoS and Curli

ABSTRACTEscherichia coliO111 is an emerging non-O157:H7 serotype of Shiga toxin-producingE. coli(STEC). We previously reported that outbreak and environmental, but not sporadic-case, strains of STEC O111 share a distinct aggregation phenotype (M. E. Diodati, A. H. Bates, M. B. Cooley, S. Walker, R. E. Mandrell, and M. T. Brandl, Foodborne Pathog Dis 12:235−243, 2015,http://dx.doi.org/10.1089/fpd.2014.1887). We show here the natural occurrence of nonaggregative variants in single STEC O111 strains. These variants do not produce curli fimbriae and lack RpoS function but synthesize cellulose. The deletion ofcsgBACorrpoSin an aggregative outbreak strain abolished aggregate formation, which was rescued when curli biogenesis or RpoS function, respectively, was restored. Complementation of a nonaggregative variant with RpoS also conferred curli production and aggregation. These observations were supported by Western blotting with an anti-CsgA antibody. Immunomicroscopy revealed that curli were undetectable on the cells of the nonaggregative variant and the RpoS mutant but were present in large quantities in the intercellular matrix of the assemblages formed by aggregative strains. Sequence analysis ofrpoSin the aggregative strain and its variant showed a single substitution of threonine for asparagine at amino acid 124. Our results indicate that the multicellular behavior of STEC O111 is RpoS dependent via positive regulation of curli production. Aggregation may confer a fitness advantage in O111 outbreak strains under stressful conditions in hydrodynamic environments along the food production chain and in the host, while the occurrence of nonaggregative variants may allow the cell population to adapt to conditions benefiting a planktonic lifestyle.

Filament formation of Salmonella Paratyphi A accompanied by FtsZ assembly impairment and low level ppGpp

Previously, we reported that Salmonella enterica serovar Paratyphi A strain S602 grew into multinuclear, nonseptate, and nonlethal filaments on agar plates containing nitrogenous salts. Strain S602 was more sensitive to osmotic and oxidative stress than the reference strain 3P243 of nonfilamentous Salmonella Paratyphi A. Strain S602 had an amber mutation (C154T) in rpoS. The revertant of this mutant, SR603, was repressed to form filaments under conditions with abundant nitrogenous salts. However, 3PR244, an rpoS mutant of 3P243 (C154T), did not form filaments, which implies that the rpoS mutation is not the sole cause of filamentation in strain S602. Next, we examined whether the level of guanosine 5′-diphosphate 3′-diphosphate (ppGpp) in S602 strain is involved in filament formation. The intracellular ppGpp level in filamentous cells was lower than that in nonfilamentous cells. Furthermore, cells belonging to strain RE606, a derivative of S602 where the intracellular concentration of ppGpp was increased by overexpression of the relA gene, exhibited normal Z-ring formation and cell division. In the S602 strain, the decrease in the ppGpp level induced by the presence of nitrogenous salt and the rpoS mutation led to the inhibition of Z-ring formation and the subsequent filamentation of cells.

The GacS-regulated sigma factor RpoS governs production of several factors involved in biocontrol activity of the rhizobacterium Pseudomonas chlororaphis O6

Pseudomonas chlororaphis O6 possesses many beneficial traits involved in biocontrol of plant diseases. In this paper, we examined the effect of a mutation in rpoS encoding a stress-related alternative sigma factor to better understand the regulation of these traits. Biochemical studies indicated that production of acyl homoserine lactones was altered and phenazine was increased in the P. chlororaphis O6 rpoS mutant. The rpoS mutation reduced hydrogen cyanide levels, but the rpoS mutant still displayed a level of in vitro antifungal activity against Fusarium graminearum and Alternaria alternata. Tomato root colonization by the rpoS mutant was lower than that by the wild type at 5, 7, and 13 days after inoculation. The rpoS mutant was less effective than the wild type in induction of systemic resistance to two foliar pathogens after root inoculation of the tomato plants. Our findings demonstrate that the stationary-phase sigma factor RpoS regulates production of several key factors involved in the biocontrol potential of P. chlororaphis O6, some independently of the global regulator GacS.

Crl Binds to Domain 2 of σS and Confers a Competitive Advantage on a Natural rpoS Mutant of Salmonella enterica Serovar Typhi

ABSTRACTThe RpoS sigma factor (σS) is the master regulator of the bacterial response to a variety of stresses. Mutants inrpoSarise in bacterial populations in the absence of stress, probably as a consequence of a subtle balance between self-preservation and nutritional competence. We characterized here one naturalrpoSmutant ofSalmonella entericaserovar Typhi (Ty19). We show that therpoSallele of Ty19 (rpoSTy19) led to the synthesis of a σSTy19protein carrying a single glycine-to-valine substitution at position 282 in σSdomain 4, which was much more dependent than the wild-type σSprotein on activation by Crl, a chaperone-like protein that increases the affinity of σSfor the RNA polymerase core enzyme (E). We used the bacterial adenylate cyclase two-hybrid system to demonstrate that Crl bound to residues 72 to 167 of σSdomain 2 and that G282V substitution did not directly affect Crl binding. However, this substitution drastically reduced the ability of σSTy19to bind E in a surface plasmon resonance assay, a defect partially rescued by Crl. The modeled structure of the EσSholoenzyme suggested that substitution G282V could directly disrupt a favorable interaction between σSand E. TherpoSTy19allele conferred a competitive fitness when the bacterial population was wild type forcrlbut was outcompeted in Δcrlpopulations. Thus, these results indicate that the competitive advantage of therpoSTy19mutant is dependent on Crl and suggest thatcrlplays a role in the appearance ofrpoSmutants in bacterial populations.