SummaryLarge extrachromosomal replicons in many members of the a a a a -proteobacteria encode genes that are required for plant or animal pathogenesis or symbiosis. Most of these replicons encode repABC genes that control their replication and faithful segregation during cell division. In addition to its chromosome, the plant endosymbiont Sinorhizobium meliloti also maintains the 1.4 Mb pSymA and 1.7 Mb pSymB symbiotic megaplasmids both of which are repABC -type replicons. In all repABC loci that have been characterized, an apparently untranslated intergenic region between the repB and repC genes encodes a strong incompatibility determinant (referred to as inc a a a a ). Here we report the isolation of mutations within the inc a regions of pSymA and pSymB that eliminate incompatibility. These mutations map to and inactivate a promoter in the intergenic region that drives the expression of an approximately 56 nucleotide untranslated RNA molecule that mediates incompatibility. This gene, that we have named incA , is transcribed antisense to the repABC genes. Our analysis suggests that the incA gene is conserved in repABC loci from a diverse spectrum of bacteria.

The ability to recognize and predict non-s 54 promoters in the alphaproteobacteria is not well developed. In this study, 25 experimentally verified Sinorhizobium meliloti promoter sequences were compiled and used to predict the location of other related promoters in the S. meliloti genome. Fourteen candidate predictions were targeted for verification and of these at least 12 proved to be genuine promoters. As a result, the experimental identification of 12 novel promoters linked to genes rpoD, topA, rpmJ, trpS, ropB1, metC, rpsT, secE, trkH and three tRNA genes is reported. In all, 99 predicted and verified promoters are reported, including those linked with 13 tRNA genes, eight ribosomal protein genes and a number of other physiologically important or essential genes. On the basis of sequence conservation and a mutational analysis of promoter activity, the "35 and "10 consensus for these promoters is 5-CTTGAC-N 17 -CTATAT. This promoter structure, which seems to be widely conserved amongst several other genera in the alphaproteobacteria, shares significant similarity with, but is skewed by a 1 nt step from, the canonical Escherichia coli s 70 promoter. Perhaps this difference is responsible for the observation that S. meliloti promoters are often poorly expressed in E. coli. In this regard, expression data from plasmid-borne gfp-reporter fusions to eight of the S. meliloti promoters verified in this work revealed that while these promoters were very active in S. meliloti and Agrobacterium tumefaciens only very low, near-background activity was detected in E. coli.

Osmoregulatory transporters Prop and ProU mediate the use of betaines as osmoprotectants by Escherichia coli. Glycine betaine and proline betaine are present in mammalian urines. Betaine uptake may therefore facilitate the growth of Em coli in the urinary tract, an environment of fluctuating osmolality. Prop transporter activity was approximately threefold higher in a pyelonephritis isolate, E. coli HU734, than in E. coli K-12. The growth rate of E. coli HU734 in aerated minimal salts medium was reduced twofold by 0 2 M NaCl in the absence and by 055 M NaCl in the presence of glycine betaine.Maximal growth rate stimulation was achieved when glycine betaine was added a t a concentration as low as 25 pM. Deletion of the prop locus impaired the growth rate of E. coli HU734 in human urine but not in minimal medium supplemented with NaCl (04 M), with or without glycine betaine (0-1 mM). The expression of pyelonephritis-associated (P) pili was reduced when E. coli HU734 was cultured in a rich culture medium (LB) of elevated salinity. The prop lesion had no influence on P pilus expression in witm or on the recovery of bacteria from the kidneys of inoculated mice. However, it did reduce their recovery from the bladders of inoculated mice 100-fold. These data provide the first direct evidence that osmoprotective betaine accumulation and transporter Prop are pertinent to both growth in human urine and colonization of the murine urinary tract by uropathogenic E. coli.

SummaryWe have investigated the function of a cell envelope stress-inducible gene, yvrI, which encodes a 22.5 kDa protein that includes a predicted s 70 region 4 domain, but lacks an apparent region 2 domain. YvrI interacts with RNA polymerase and overexpression of YvrI results in induction of OxdC, an oxalate decarboxylase maximally expressed under low-pH conditions. We have used microarray-based analyses to define the YvrI regulon. YvrI is required for the transcription of three operons (oxdC-yvrL, yvrJ and yvrI-yvrHa) each of which is preceded by a highly similar promoter sequence. Activation of these promoters requires both YvrI and the product of the second gene in the yvrI-yvrHa operon, YvrHa. YvrI and YvrHa together allow recognition of the oxdC promoter, stimulate DNA melting and activate transcription by core RNA polymerase. Together, these results suggest that YvrI is a previously unrecognized s factor in Bacillus subtilis and that the 9.5 kDa YvrHa protein acts as a required co-activator of transcription. A yvrL deletion results in the upregulation of YvrI activity suggesting that YvrL is a negative regulator of YvrI-dependent transcription, possibly functioning as an anti-s factor.

The basic replication unit of many plasmids and second chromosomes in the alpha-proteobacteria consists of a repABC locus that encodes the trans- and cis-acting components required for both semiautonomous replication and replicon maintenance in a cell population. In terms of physical genetic organization and at the nucleotide sequence level, repABC loci are well conserved across various genera. As with all repABC-type replicons that have been genetically characterized, the 1.4 Mb pSymA and 1.7 Mb pSymB megaplasmids from the plant endosymbiont Sinorhizobium meliloti encode strong incompatibility (inc) determinants. We have identified a novel inc sequence upstream of the repA2 gene in pSymA that is not present on pSymB and not reported in other repABC plasmids that have been characterized. This region, in concert with the repA and repB genes, stabilizes a test plasmid indicating that it constitutes a partitioning (par) system for the megaplasmid. Purified RepB binds to this sequence and binding may be enhanced by RepA. We have isolated 19 point mutations that eliminate incompatibility, reduce RepB binding or the stabilization phenotype associated with this sequence and all of these map to a 16-nucleotide palindromic sequence centred 330 bp upstream of the repA2 gene. An additional five near-perfect repeats of this palindrome are located further upstream of the repA2 gene and we show that they share some conservation with known RepB binding sites in different locations on other repABC plasmids and to two sequences found on the tumour inducing plasmid of Agrobacterium tumefaciens. These additional palindromes also bind RepB but one of them does not display obvious incompatibility effects. A heterogenic distribution of par sequences demonstrates unexpected diversity in the structural genetic organization of repABC loci, despite their obvious levels of similarity.