Cloning, Sequencing, and Phenotypic Characterization of the rpoS Gene from Pseudomonas putida KT2440

Abstract

A gene homologous to the rpoS gene of Escherichia coli was cloned from a Pseudomonas putida KT2440 gene bank by complementation of the rpoS-deficient strainE. coli ZK918. The rpoS gene of P. putida complemented the acid sensitivity and catalase deficiency of the rpoS mutant of E. coli and stimulated expression of the RpoS-controlled promoter,bolAp 1. The gene was sequenced and found to be highly similar to the rpoS genes of other gram-negative bacteria. Like in other gram-negative bacteria, a homolog of thenlpD gene was found upstream to the rpoS gene. A transcriptional fusion of the promoter of the P. putida rpoS gene to the luxAB genes from Vibrio harveyi was constructed and used as an inactivated allele ofrpoS for gene replacement of the wild-type copy in the chromosome of P. putida. The resultantrpoS mutant of P. putida, C1R1, showed reduced survival of carbon starvation and reduced cross-protection against other types of stress in cells starved for carbon, in particular after a challenge with ethanol. Survival in soil amended with m-methylbenzoate was also reduced in the mutant strain P. putida C1R1. The RpoS protein ofP. putida controls the expression of more than 50 peptides, which are normally expressed in cells after a short period of carbon starvation.

Mutation ofrpoS gene decreased resistance to environmental stresses, synthesis of extracellular products and virulence ofVibrio anguillarum

“…Ã P o 0.05 was taken to indicate statistical significance. (Kim et al, 1995;Ramos-González & Molin, 1998); the latter is a s factor regulating nitrogen metabolism in several bacteria. Our result in V. anguillarum suggested that alternative systems may be available to protect against carbon starvation in V. anguillarum.…”

“…Virulence to zebra fish of the mutant and the wild-type cells of Vibrio anguillarum -4 weeks, but the viable count of an rpoSdeficient strain was reduced about 100-fold after 2 weeks of carbon starvation(Ramos-González & Molin, 1998). Most bacterial genomes encode multiple s factors.…”

Regulation of the cyclopropane synthase cfaB gene in Pseudomonas putida KT2440

“…The PCR fragments were cut with EcoRI and PstI and cloned into pMP220 (Spaink et al, 1987), previously cut with the same enzymes, to construct the plasmid pMPcfaBKT2440. This plasmid was electroporated into P. putida KT2440 and into P. putida C1R1, a P. putida KT2440 RpoS mutant (Ramos-González & Molin, 1998).…”

“…In all the different environments these bacteria can inhabit, they are threatened by diverse biotic and abiotic factors; however, bacterial cells have developed mechanisms to cope with these threats Daniels et al, 2010). The ability to colonize multiple habitats reflects a high adaptability and this trait correlates with the comparative high number of sigma factors present in bacteria (Ramos-González & Molin, 1998;Martinez-Bueno et al, 2002;Venturi, 2003;Potvin et al, 2008). One extensively studied alternative sigma factor is RpoS (s S or s 38 ), which controls the expression of genes involved in survival to starvation and other stresses that lead to growth reduction (stationary phase).…”

Transcription analysis of rpoH in Pseudomonas putida

“…The major c gene, rpoD, and groEL are transcribed with RNA polymerase containing heat-shock c, c H [4^6]. The amino acid sequences of RpoD (c 70 protein), RpoH (c H protein) and RpoS (c S protein) show 75%, 75% and 88% identity between Pseudomonas putida and Pseudomonas aeruginosa, respectively, and each c protein has a characteristic region which is highly conserved in these species [4,7,8]. The P. putida rpoE gene, encoding c E protein, has not been cloned yet.…”