No abstract

The alternative sigma factor RpoS controls the expression of many stationary-phase genes in Escherichia coli and other bacteria. Though the RpoS regulon is a large, conserved system that is critical for adaptation to nutrient deprivation and other stresses, it remains incompletely characterized. In this study, we have used oligonucleotide arrays to delineate the transcriptome that is controlled by RpoS during entry into stationary phase of cultures growing in rich medium. The expression of known RpoS-dependent genes was confirmed to be regulated by RpoS, thus validating the use of microarrays for expression analysis. The total number of positively regulated stationary-phase genes was found to be greater than 100. More than 45 new genes were identified as positively controlled by RpoS. Surprisingly, a similar number of genes were found to be negatively regulated by RpoS, and these included almost all genes required for flagellum biosynthesis, genes encoding enzymes of the TCA cycle, and a physically contiguous group of genes located in the Rac prophage region. Negative regulation by RpoS is thus much more extensive than has previously been recognized, and is likely to be an important contributing factor to the competitive growth advantage of rpoS mutants reported in previous studies.

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Horizontal gene transfer is now recognized as an important mechanism of evolution. Several methods to detect horizontally transferred genes have been suggested. These methods are based on either nucleotide composition or the failure to find a similar gene in closely related species. Genes that evolve vertically between closely related species can be divided into those that retain homologous chromosomal positions (positional orthologs) and those that do not. By comparing open reading frames in the Escherichia coli and Salmonella typhi genomes, we identified 2,728 positional orthologs since these species split 100 MYA. A group of 1,144 novel E. coli genes were unusually diverged from their S. typhi counterparts. These novel genes included those that had been horizontally transferred into E. coli, as well as members of gene pairs that had been rearranged or deleted. Positional orthologs were used to investigate compositional methods of identifying horizontally transferred genes. A large number of E. coli genes with normal nucleotide composition have no apparent ortholog in S. typhi, and many genes of atypical composition do, in fact, have positional orthologs. A phylogenetic approach was employed to confirm selected examples of horizontal transmission among the novel groups of genes. Our analysis of 80 E. coli genes determined that a number of genes previously classified as horizontally transferred based on base composition and codon bias were native, and genes previously classified as native appeared to be horizontally transferred. Hence, atypical nucleotide composition alone is not a reliable indicator of horizontal transmission.

We report on the biochemical, phylogenetic and genetic regulation of PhoX, the major alkaline phosphatase protein from the soil bacterium Sinorhizobium meliloti. The protein is shown to be a member of a recently identified family of PhoX alkaline phosphatase proteins that is distinct from the well-characterized PhoA family. The mature S. meliloti PhoX protein is located in the periplasm and lacks a 76-amino-acid N-terminal Tat signal peptide. Its phosphatase activity was stimulated by Ca(+2) and was optimal at pH 9-11. Except for phytic acid and phosphatidic acid, the enzyme was active against a wide range of phosphorylated substrates (77 nucleotides, phosphorylated carbohydrates and amino acids) and thus exhibited low substrate specificity for C-O-P bonds. No C-P bond substrate was dephosphorylated while the protein was active with two of six phosphoramidate substrates (N-P bond) tested. Sinorhizobium meliloti phoX was induced when cells were starved for phosphorous and the induction was dependent on the PhoB-regulatory protein. We demonstrate by in vitro analysis that PhoB protein binds to two tandem 22 nt PhoB binding sites located 64-21 nt upstream from the phoX transcription start site. Analysis of 95 PhoX orthologues from diverse bacteria revealed two distinct phylogenetic groups of PhoX proteins. The two groups differed in having a conserved glycine (PhoX-I) or asparagine (PhoX-II) next to their putative catalytic Ca(+2) binding site. Analysis of the phoX promoter regions from many of these bacteria also revealed the presence of PhoB binding sites. Alkaline phosphatase proteins of either the PhoX or PhoA family (but rarely both) are found in many bacteria, thus it appears that these are functionally equivalent.