Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of an extracellular signal molecule (autoinducer) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode functions necessary for the synthesis of, and response to, a signal molecule. Sequence analysis of the region encoding these functions revealed three open reading frames, two (luxL and luxM) that are required for production of an autoinducer substance and a third (luxN) that is required for response to this signal substance. The LuxL and LuxM proteins are not similar in amino acid sequence to other proteins in the database, but the LuxN protein contains regions of sequence resembling both the histidine protein kinase and the response regulator domains of the family of two-component, signal transduction proteins. The phenotypes of mutants with luxL, luxM and luxN defects indicated that an additional signal-response system controlling density-dependent expression of luminescence remains to be identified.

Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium. One signal-response system is encoded by the luxL,M,N locus. The luxL and luxM genes are required for the production of an autoinducer (probably beta-hydroxybutyl homoserine lactone), and the luxN gene is required for the response to that autoinducer. Analysis of the phenotypes of LuxL,M and N mutants indicated that an additional signal-response system also controls density sensing. We report here the identification, cloning and analysis of luxP and luxQ, which encode functions required for a second density-sensing system. Mutants with defects in luxP and luxQ are defective in response to a second autoinducer substance. LuxQ, like LuxN, is similar to members of the family of two-component, signal transduction proteins and contains both a histidine protein kinase and a response regulator domain. Analysis of signalling mutant phenotypes indicates that there are at least two separate signal-response pathways which converge to regulate expression of luminescence in V. harveyi.

Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode a function required for the density-dependent response. Transposon Tn5 insertions in the recombinant clone were isolated, and the mutations were transferred to the genome of V. harveyi for examination of mutant phenotypes. Expression of luminescence in V. harveyi strains with transposon insertions in one locus, luxO, was independent of the density of the culture and was similar in intensity to the maximal level observed in wild-type bacteria. Sequence analysis of luxO revealed one open reading frame that encoded a protein, LuxO, similar in amino acid sequence to the response regulator domain of the family of two-component, signal transduction proteins. The constitutive phenotype of LuxO- mutants indicates that LuxO acts negatively to control expression of luminescence, and relief of repression by LuxO in the wild type could result from interactions with other components in the Lux signalling system.

Vibrio parahaemolyticus possesses two distinct motility systems, the polar system used for swimming in liquid environments and the lateral system used for swarming over surfaces. Growth on surfaces induces swarmer cell differentiation and expression of the lateral motility system. Mutants, created by transposon mutagenesis of a clone expressing lateral flagellin and gene disruption in V. parahaemolyticus, were unable to swarm and failed to make lateral flagellin; therefore, unlike the case for the polar system, there is one gene (lafA) encoding lateral flagellin. In addition to lafA, other genes required for swarming but not for swimming were identified by gene replacement mutagenesis. The nucleotide sequence of the clone determined open reading frames (ORFs) and deduced amino acid sequences showed similarities to flagellar components of other bacteria: flagellin, hook-associated protein (HAP2), motor components, and flagellar sigma factor (&2s). Many &28 factors have been shown to recognize cognate promoters; however, expression of lafA in Escherichia coli required LafS, and E. coli r2' did not substitute. Also, there were no sequences preceding genes encoding flagellin or HIAP2 resembling the &8 consensus promoter. The product of the sigma-like gene seems to be a unique member of the or"' cluster. It appears the result of requiring expression for immunodetection of flagellin clones was that the sigma locus was fortuitously cloned, since the sigma and lafA loci were not contiguous in the chromosome.This work initiates identification and placement of genes in a scheme of control for swarmer cell differentiation; three levels have been identified in the transcriptional hierarchy.The marine bacterium and human pathogen Vibrio parahaemolyticus possesses two distinct motility systems, each appropriate for movement under different circumstances (2, 48). Translocation over surfaces, or swarming, results in radially spreading colonies and colonization of surfaces, while movement in liquid is called swimming (20,52). The polar flagellar system (Fla) propels the bacterium in liquid medium. Growth on surfaces induces the developmental program called swarmer cell differentiation, in which septation ceases, the cells elongate, and a new flagellar system, the lateral system (Laf), is synthesized. The polar system is expressed essentially constitutively, while the lateral flagellar system is expressed only when the bacterium is in contact with surfaces or in a viscous environment, i.e., under conditions in which the polar flagellum is not functional (38). Thus, the two gene systems interact: performance of the polar system is in some way coupled to transcription of the swarmer cell gene system. To elucidate the signal transduction event by which physical information is processed and converted to a signal that leads to swarmer cell differentiation, we are defining some of the key genes and the regulatory hierarchies of the two flagellar systems.The gene systems that encode the two motility systems are large, each composed of 4...

Barophilic bacteria inhabit the deep oceans, and the specific functional modifications and regulatory mechanisms which govern adaptation to hydrostatic pressure are beginning to be understood. For example, the rate of production of several proteins by some hydrothermal vent archaebacteria and the degree of saturation of membrane lipids in other deep-sea bacteria have been found to change as a result of cultivation at high pressure. We report here the cloning of gene, ompH, which encodes a major pressure-inducible protein of strain SS9, a gram-negative eubacterium isolated from a depth of 2.5 kilometres in the Sulu Sea. Messenger RNA encoded by ompH is expressed when cells are grown at 280 atm but not at 1 atm, indicating that transcription of the ompH gene is controlled by hydrostatic pressure. The function of the OmpH protein in adaptation to high pressure and the use of the ompH gene in studying how bacteria sense and respond to pressure is discussed.