Michael Silverman

Bacterial bioluminescence: Isolation and genetic analysis of functions from Vibrio fischeri

Recombinant E. coli that produce light were found in a clone library of hybrid plasmids containing DNA from the marine bacterium Vibrio fischeri. All luminescent clones had a 16 kb insert that encoded enzymatic activities for the light reaction as well as regulatory functions necessary for expression of the luminescence phenotype (Lux). Mutants generated by transposons Tn5 and mini-Mu were used to define Lux functions and to determine the genetic organization of the lux region. Regulatory and enzymatic functions were assigned to regions of two lux operons. With transcriptional fusions between the lacZ gene or transposon mini-Mu and the target gene, expression of lux operons could be measured in the absence of light production. The direction of transcription of lux operons was deduced from the orientation of mini-Mu insertions in the fusion plasmids. Induction of transcription of one lux operon required a function encoded by that operon (autoregulation). From these and other regulatory relationships, we propose a model for genetic control of light production.

Flagellar dynamometer controls swarmer cell differentiation of V. parahaemolyticus

Swarmer cell genes, laf, are induced when V. parahaemolyticus is grown on the surface of solidified media, embedded in solidified media, suspended in viscous media, or agglutinated with antibody in liquid media. These conditions have in common the constraint of the movement of the polar flagellum. To test the hypothesis that the polar flagellum functions as a sensor necessary for control of swarmer cell formation, we have constructed a variety of mutations in genes encoding components of the polar flagellum, fla. The consequence of such mutations is the constitutive expression of laf genes. So, the performance of the polar flagellum is coupled to the transcription of laf genes such that when function is perturbed, either physically or genetically, swarmer cell genes are induced. Because the polar flagellum appears to be capable of sensing external forces influencing its motion, we suggest it is acting as a dynamometer.

[24] Genetic analysis in Vibrio

Bacteria of the genus Vibrio are remarkably diverse, and until recently the methodology for genetic analysis consisted of a patchwork of different approaches, many of which were narrowly applicable to a single species. The invention of the recombinant DNA technology and the subsequent innovations in transposon mutagenesis and in transductive and conjugative gene transfer techniques have led to the development of very powerful and general strategies for genetic analysis of species of Vibrio. The striking synergy of combining recombinant DNA, transposon, and gene transfer methods is particularly evident in the construction of transposons which generate gene fusions and of broad host range plasmids which deliver transposons and mutated genes and which mobilize chromosomes. With such tools it should be possible to perform advanced genetic analysis on the many undomesticated species of Vibrio still to be explored.

Genetic analysis of surface sensing in Vibrio parahaemolyticus

Growth of Vibrio parahaemolyticus on the surfaces of solid media induces formation of a specialized bacterium called the swarmer cell. Differentiation to this cell is controlled by an information transduction mechanism which couples perception of signals specific for life on a surface to expression of genes encoding the swarmer cell phenotype. A variety of genetic tools used to analyze surface sensing in V. parahaemolyticus are described. A summary is given of what has been learned about the signals and sensors controlling differentiation and other possible genetic mechanisms for adaptation to growth on a surface or biofilm habitat are discussed.

[8] Techniques for cloning and analyzing bioluminescence genes from marine bacteria

Publisher Summary This chapter discusses the techniques for cloning and analyzing bioluminescence genes from marine bacteria. The isolation by recombinant DNA techniques of genes for bioluminescence (lux) from marine bacteria has resulted in a rapid expansion of knowledge of the biochemical activities necessary for light production and of the regulatory mechanisms, which govern the expression of these functions. The chapter describes a variety of genetic methodologies for cloning DNA fragments encoding luminescence functions, eliciting expression of luminescence genes, defining individual lux genes and transcriptional units containing lux genes, identifying the products of cloned lux genes, exploring the regulatory control of lux genes, and using lux gene fusions to measure transcriptional control of other gene systems. Most genetic analysis has been performed with luminescent Vibrio, and the attention is confined to bacteria of this genus.