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Pseudomonas fluorescens SBW25 is capable of growing on histidine as a sole source of carbon and/or nitrogen. Previous work showed that the two-component regulatory system CbrAB is required for expression of the histidine utilization (hut) locus when histidine is the sole source of carbon and nitrogen. Here, using mutational analysis and transcriptional assays, we demonstrate involvement of a second two-component system, NtrBC. When histidine is the sole carbon source, transcription of the hutU operon is initiated from [[sigma].sup.54]-type promoter and requires CbrB (an enhancer binding protein for [[sigma].sup.54]-recruitment). However, when histidine is the sole nitrogen source, the hutU operon is transcribed from a [[sigma].sup.70]-type promoter and requires either CbrB or the nitrogen regulator, NtrC. No role was found for the SBW25 homolog of the nitrogen assimilation control protein (NAC). Biolog phenotypic microarray analysis of the ability of the three mutants ([DELTA]cbrB, [DELTA]ntrC, and [DELTA]cbrB [DELTA]ntrC) to utilize 190 carbon and 95 nitrogen substrates confirmed the central regulatory roles of CbrAB and NtrBC in cellular carbon and nitrogen catabolism: deletion of cbrB abolished growth on 20 carbon substrates; deletion of ntrC eliminated growth on 28 nitrogen substrates. A double cbrB-ntrC mutant was unable to utilize a further 14 nitrogen substrates (including histidine, proline, leucine, isoleucine, and valine). Our data show that CbrAB plays a role in regulation of both carbon and nitrogen catabolism and maintains activity of catabolic pathways under different C:N ratios.

The histidine utilization (hut) locus of Pseudomonas fluorescens SBW25 confers the ability to utilize histidine as a sole carbon and nitrogen source. Genetic analysis using a combination of site-directed mutagenesis and chromosomally integrated lacZ fusions showed the hut locus to be composed of 13 genes organized in 3 transcriptional units: hutF, hutCD, and 10 genes from hutU to hutG (which includes 2 copies of hutH, 1 of which is nonfunctional). Inactivation of hutF eliminated the ability to grow on histidine, indicating that SBW25 degrades histidine by the five-step enzymatic pathway. The 3 hut operons are negatively regulated by the HutC repressor with urocanate (the first intermediate of the histidine degradation pathway) as the physiological inducer. 5'-RACE analysis of transcriptional start sites revealed involvement of both cr54 (for the hutU-G operon) and [[sigma].sup.70] (for hutF); the involvement of [[sigma].sup.54] was experimentally demonstrated. CbrB (an enhancer binding protein for [[sigma].sup.54] recruitment) was required for bacterial growth on histidine, indicating positive control of hut gene expression by CbrB. Recognition that a gene (named hutl) encoding a widely distributed conserved hypothetical protein is transcribed along with hutC led to analysis of its role. Mutational and gene fusion studies showed that HutD functions independently of HutC. Growth and fitness assays in laboratory media and on sugar beet seedlings suggest that HutD acts as a governor that sets an upper bound to the level of hut activity.

Author(s): Tadashi Fukami (corresponding author) [1]; Hubertus J. E. Beaumont [2]; Xue-Xian Zhang [2]; Paul B. Rainey [2] Diversity in biological communities is a historical product of immigration, diversification and [...]

Abstract Stochastic phenotype switching – or bet hedging – is a pervasive feature of living systems and common in bacteria that experience fluctuating (unpredictable) environmental conditions. Under such conditions, the capacity to generate variable offspring spreads the risk of being maladapted in the present environment, against offspring likely to have some chance of survival in the future. While a rich subject for theoretical studies, little is known about the selective causes responsible for the evolutionary emergence of stochastic phenotype switching. Here we review recent work – both theoretical and experimental – that sheds light on ecological factors that favour switching types over non-switching types. Of particular relevance is an experiment that provided evidence for an adaptive origin of stochastic phenotype switching by subjecting bacterial populations to a selective regime that mimicked essential features of the host immune response. Central to the emergence of switching types was frequent imposition of ‘exclusion rules’ and ‘population bottlenecks’ – two complementary faces of frequency dependent selection. While features of the immune response, exclusion rules and bottlenecks are likely to operate in many natural environments. Together these factors define a set of selective conditions relevant to the evolution of stochastic switching, including antigenic variation and bacterial persistence.

The ability to monitor the spatial and temporal distribution of signals in complex environments is necessary for an understanding of the function of bacteria in the wild. To this end, an existing recombinase-based transcriptional reporter strategy (recombinase-based in vivo expression technology, RIVET) has been extended and applied to the plant-colonizing bacterium Pseudomonas fluorescens SBW25. Central to the project was a rhizosphere-inducible locus, rhi14, which functional analyses show is hutT, a histidine-inducible gene that is required for histidine utilization. A transcriptional fusion between hutT and a promoterless site-specific recombinase (tnp[R.sup.mut168]) results in excision of a chromosomally integrated tetracycline-resistance cassette in a histidine-dependent manner. The dose- and time-responsiveness of the promoterless recombinase to histidine closely mirrored the histidine responsiveness of an identical hutT fusion to promoterless lacZ. To demonstrate the effectiveness of the strategy, the activity of hutT was monitored on sugar beet seedlings. Low levels of transcriptional activity were detected in the phyllosphere, rhizosphere and in plant extract, but not in vermiculite devoid of seedlings. The histidine concentration in the rhizosphere was estimated to be 0.6 [micro]g [ml.sup.-1]. The ecological significance of the hut locus was examined by competing a hutT deletion mutant against the wild-type during colonization of sugar beet seedlings. No impact on competitive fitness was detected, suggesting that the ability to utilize plant-derived histidine is not essential for bacterial colonization.

Application of a promoter-trapping strategy to identify plant-inducible genes carried on an indigenous Pseudomonas plasmid, pQBR103, revealed the presence of a putative oligoribonuclease (orn) gene that encodes a highly conserved 3' to 5' exoribonuclease specific for small oligoribonucleotides. The deduced amino acid sequence of the plasmid-derived orn ([orn.sub.pl]) showed three conserved motifs characteristic of Orn from both prokaryotes and eukaryotes. Deletion of [orn.sub.pl] generated no observable phenotype, but inactivation of the chromosomal copy caused slow growth in Pseudomonas putida KT2440. This defect was fully restored by complementation with orn from Escherichia coli ([orn.sub.E.coli]). Plasmid-derived [orn.sub.pl] was capable of partially complementing the P. putida orn mutant, demonstrating functionality of [orn.sub.pl]. Phylogenetic analysis showed that plasmid-encoded Orn was distinct from Orn encoded by the chromosome of proteobacteria. A survey of [orn.sub.pl] from related Pseudomonas plasmids showed a sporadic distribution but no sequence diversity. These data suggest that the [orn.sub.pl] was acquired by pQBR103 in a single gene-transfer event: the donor is unknown, but is unlikely to be a member of the Proteobacteria.

Gene rearrangement between a symbiotic plasmid and a nonsymbiotic plasmid in Rhizobium leguminosarum bv. viciae VF39 results in relocation of most of the symbiotic genes. Data indicate that the plasmid rearrangement causes an unequal DNA exchange.

Summary: Pseudomonas aeruginosa rugose small‐colony variants (RSCVs) are frequently isolated from chronic infections, yet, they are rarely reported in environmental isolates. Here, during the comparative genomic analysis of two P. aeruginosa strains isolated from crude oil, we discovered a spontaneous in‐frame deletion, wspAΔ280–307, which led to hyper‐biofilm and RSCV phenotypes. WspA is a homologue of methyl‐accepting chemotaxis proteins (MCPs) that senses surfaces to regulate biofilm formation by stimulating cyclic‐di‐guanosine monophosphate (c‐di‐GMP) synthesis through the Wsp system. However, the methylation sites of WspA have never been identified. In this study, we identified E280 and E294 of WspA as methylation sites. The wspAΔ280–307 mutation enabled the Wsp system to lock into a constitutively active state that is independent of regulation by methylation. The result is an enhanced production of c‐di‐GMP. Sequence alignment revealed three conserved repeat sequences within the amino acid residues 280–313 (aa280–313) region of WspA homologues, suggesting that a spontaneous deletion within this DNA encoding region was likely a result of intragenic recombination and that similar mutations might occur in several related bacterial genera. Our results provide a plausible explanation for the selection of RSCVs and a mechanism to confer a competitive advantage for P. aeruginosa in a crude‐oil environment. [ABSTRACT FROM AUTHOR]