A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR

Abstract

The toxR gene of Vibrio cholerae encodes a transmembrane, DNA-binding protein that activates transcription of the cholera toxin operon and a gene (tcpA) for the major subunit of a pilus colonization factor. We constructed site-directed insertion mutations in the toxR gene by a novel method employing the chromosomal integration of a mobilizable suicide plasmid containing a portion of the toxR coding sequence. Mutants containing these new toxR alleles had an altered outer membrane protein profile, suggesting that two major outer membrane proteins (OmpT and OmpU) might be under the control of toxR. Physiological studies indicated that varying the concentration of the amino acids asparagine, arginine, glutamate, and serine caused coordinate changes in the expression of cholera toxin, TcpA, OmpT, and OmpU. Changes in the osmolarity of a tryptone-based medium also produced coordinate changes in the expression of these proteins. Other environmental signals (temperature and pH) had a more pronounced effect on the expression of cholera toxin and TcpA than they did on the outer membrane proteins. These results suggest that certain environmental signals (i.e., osmolarity and the presence of amino acids) are tightly coupled to the expression of toxR-regulated proteins and therefore may be signals that are directly sensed by the ToxR protein.Little is known about the in vivo environmental signals that control expression of bacterial virulence determinants. The effects of nutritional and physical parameters on the production of virulence factors in laboratory media reflect the existence of regulatory mechanisms that may help the microbe determine when it is appropriate to express these rather specialized properties. This regulation presumably allows the organism to avoid the metabolic drain of producing toxins, colonization factors, capsules, and other virulence-enhancing proteins in environments where their action is not needed. A wide spectrum of compounds and growth conditions have been implicated in the regulation of virulence properties, including iron (2, 23), divalent cations (24,25,29,33), atmospheric gases (26), temperature (14), and even complex organic molecules like nicotinic acid (33) and phenolic compounds (28). Understanding the signals and mechanisms that are involved in the control of virulence gene expression might someday lead to applications in vaccine development and chemotherapy of bacterial infections.Vibrio cholerae is a gram-negative bacterium that causes a severe diarrheal disease by colonizing the upper intestine of humans and elaborating a protein exotoxin (1). Cholera toxin is a multimeric protein composed of two types of subunits, A and B, that are encoded by the genes ctxA and ctxB, respectively (18). The ctxA and ctxB genes form an operon that is positively regulated at the transcriptional level by the product of the toxR gene (20)(21)(22). Recently, we have shown that the toxR gene regulates not only the ctx operon but also the gene (tcpA) for the major subunit of a toxin-coregulated pilus col...

Complete genome sequence of the entomopathogenic and metabolically versatile soil bacterium Pseudomonas entomophila

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