β-Lactams disrupt bacterial cell wall synthesis, and these agents are the most widely used antibiotics. One of the principle mechanisms by which bacteria resist the action of β-lactams is by producing β-lactamases, enzymes that degrade β-lactams. In Gram-negative bacteria, production of β-lactamases is often induced in response to the antibiotic-associated damage to the cell wall. Here, we have identified a previously unidentified mechanism that governs β-lactamase production. In the Gram-negative enteric pathogen Vibrio parahaemolyticus, we found a histidine kinase/response regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam antibiotics, but not by other lactams. However, single amino acid substitutions in the putative periplasmic binding pocket of VbrK leads its phosphorylation in response to both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that can directly detect β-lactam antibiotics instead of detecting the damage to cell wall resulting from β-lactams. In strong support of this idea, we found that purified periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for VbrK autophosphorylation and β-lactamase production. Direct recognition of β-lactam antibiotics by a histidine kinase receptor may represent an evolutionarily favorable mechanism to defend against β-lactam antibiotics.

In vibrios, the expression of virulence factors is often controlled by LuxR, the master quorum-sensing regulator. Here, we investigate the interplay between LuxR and σ^E, an alternative sigma factor, during the control of virulence-related gene expression and adaptations to temperature elevations in the zoonotic pathogen Vibrio alginolyticus. An rpoE null V. alginolyticus mutant was unable to adapt to various stresses and was survival-deficient in fish. In wild type V. alginolyticus, the expression of LuxR-regulated virulence factors increased as the temperature was increased from 22°C to 37°C, but mutants lacking σ^E did not respond to temperature, indicating that σ^E is critical for the temperature-dependent upregulation of virulence genes. Further analyses revealed that σ^E binds directly to -10 and -35 elements in the luxR promoter that drive its transcription. ChIP assays showed that σ^E binds to the promoter regions of luxR, rpoH and rpoE at high temperatures (e.g., 30°C and 37°C). However, at higher temperatures (42°C) that induce thermal stress, σ^E binding to the luxR promoter decreased, while its binding to the rpoH and rpoE promoters was unchanged. Thus, the temperature-dependent binding of σ^E to distinct promoters appears to underlie a σ^E-controlled switch between the expression of virulence genes and adaptation to thermal stress. This study illustrates how a conserved temperature response mechanism integrates into quorum-sensing circuits to regulate both virulence and stress adaptation. Zoonotic Vibrio outbreaks are believed to be closely associated with increases in environmental temperature. The mechanisms underlying this phenomenon have not been defined. Here, we show that the expression of the V. alginolyticus exotoxin Asp and other quorum-sensing (QS)-regulated virulence factors are induced by increasing temperatures, with the maximum expression observed at approximately 37°C. σ^E plays an essential role in regulating the QS master regulator LuxR in response to temperature shifts by binding directly to the -10 and -35 regions of the luxR promoter to drive its transcription. However, at higher thermal stress temperatures, σ^E binding to the luxR promoter decreased, resulting in a reduction in luxR transcription. This change underlies a binomial switch mechanism that regulates σ^E-controlled virulence gene expression patterns. Furthermore, we found that anti-σ^E signaling was involved in this stress and virulence reciprocal switch. This study suggests that a common temperature response mechanism is integrated into QS circuits to regulate both virulence and adaptation in related Vibrio taxa.

Vibrio parahaemolyticus is the leading cause of seafood-borne diarrheal diseases. Experimental overproduction of a type 3 secretion system (T3SS1) in this pathogen leads to decreased intestinal colonization, which suggests that T3SS1 repression is required for maximal virulence. However, the mechanisms by which T3SS1 is repressed in vivo are unclear. Here, we show that host-derived nitrite modifies the activity of a bacterial histidine kinase and mediates T3SS1 repression. More specifically, nitrite activates histidine kinase sensor VbrK through S-nitrosylation on cysteine 86, which results in downregulation of the entire T3SS1 operon through repression of its positive regulator exsC. Replacement of cysteine 86 with a serine (VbrK C86S mutant) leads to increased expression of inflammatory cytokines in infected Caco-2 cells. In an infant rabbit model of infection, the VbrK C86S mutant induces a stronger inflammatory response at the early stage of infection, and displays reduced intestinal colonization and virulence at the later stage of infection, in comparison with the parent strain. Our results indicate that the pathogen V. parahaemolyticus perceives nitrite as a host-derived signal and responds by downregulating a proinflammatory factor (T3SS1), thus enhancing intestinal colonization and virulence.