Pseudomonas stutzeri MJL19, a rhizosphere-colonizing bacterium that promotes plant growth under saline stress.

AIMS: The aim of this study was to find and use rhizobacteria able to confer plants advantages to deal with saline conditions. METHODS AND RESULTS: We isolated 24 different bacterial species from the rhizosphere of halophyte plants growing in Santiago del Estero, Argentina salt flat. Four strains were selected upon their ability to grow in salinity and their biochemical traits associated with plant growth promotion. Next, we tested the adhesion on soybean seeds surface and root colonization with the four selected isolates. Isolate 19 stood out from the rest and was selected for further experiments. This strain showed positive chemotaxis towards soybean root exudates and a remarkable ability to form biofilm both in vitro conditions and on soybean roots. Interestingly, this trait was enhanced in high saline conditions, indicating the extremely adapted nature of the bacterium to high salinity. In addition, this strain positively impacted on seed germination, plant growth and general plant health status also under saline stress. CONCLUSIONS: A bacterium isolate with outstanding ability to promote seed germination and plant growth under saline conditions was found. SIGNIFICANCE AND IMPACT OF THE STUDY: The experimental approach allowed us to find a suitable bacterial candidate for a biofertilizer intended to alleviate saline stress on crops. This would allow the use of soil now considered inadequate for agriculture and thus prevent further advancement of agriculture frontiers into areas of environmental value.

Expression of a Pseudomonas putida aminotransferase involved in lysine catabolism is induced in the rhizosphere.

Using a transposon carrying a promoterless lux operon to generate transcriptional fusions by insertional mutagenesis, we have identified a Pseudomonas putida gene with increased expression in the presence of corn root exudates. Expression of the transcriptional fusion, induced by the amino acid lysine, was detected in P. putida in the rhizosphere of plants as well as in response to seed exudates. The mutant was unable to grow on lysine or delta-aminovalerate as carbon sources, which indicates that the affected function is involved in the pathway for lysine catabolism. However, the mutant strain grew with glutaric acid, the product of delta-aminovalerate metabolism via glutaric acid semialdehyde, as a C source. The translated sequence of the interrupted gene showed high levels of similarity with aminotransferases. These sets of data suggest that the product of this gene has delta-aminovalerate aminotransferase activity. This is the first direct genetic evidence correlating a DNA sequence with such activity in Pseudomonadaceae.

Responses of Gram-negative bacteria to certain environmental stressors.

Bacteria in nature are exposed to variations in temperature, and are affected by the availability of nutrients and water and the presence of toxic molecules. Their reactions to these changes require a series of rapid adaptive responses. Although transcriptional regulation is of primary importance in these responses, translational regulation and even activation of 'silenced' enzymes are critical for survival in changing environments. Bacteria have developed a series of mechanisms at the membrane structure level to cope with high concentrations of solvents. In addition, solvent-tolerant strains express highly effective efflux pumps to remove solvents from the cytoplasm. Desiccation tolerance is based on the synthesis and accumulation of osmoprotectants together with changes in fatty acid composition to preserve membrane structure. Both cold shock and heat shock responses are mainly regulated at a post-transcriptional level, translation efficiency in the case of cold shock and mRNA half-life and sigma32 stability in the case of heat shock.

Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds.

Many agricultural uses of bacteria require the establishment of efficient bacterial populations in the rhizosphere, for which colonization of plant seeds often constitutes a critical first step. Pseudomonas putida KT2440 is a strain that colonizes the rhizosphere of a number of agronomically important plants at high population densities. To identify the functions involved in initial seed colonization by P. putida KT2440, we subjected this strain to transposon mutagenesis and screened for mutants defective in attachment to corn seeds. Eight different mutants were isolated and characterized. While all of them showed reduced attachment to seeds, only two had strong defects in their adhesion to abiotic surfaces (glass and different plastics). Sequences of the loci affected in all eight mutants were obtained. None of the isolated genes had previously been described in P. putida, although four of them showed clear similarities with genes of known functions in other organisms. They corresponded to putative surface and membrane proteins, including a calcium-binding protein, a hemolysin, a peptide transporter, and a potential multidrug efflux pump. One other showed limited similarities with surface proteins, while the remaining three presented no obvious similarities with known genes, indicating that this study has disclosed novel functions.

A novel system for efficient gene expression and monitoring of bacteria in aquatic environments.

In a previous study, we reported the identification of Escherichia coli genes with increased expression in an aquatic environment. Here, we describe the use of one of these genes, gapC, as an expression system in freshwater habitats. We have identified the transcriptional start site of gapC and analysed the synthesis of the GapC protein during incubation in aquatic medium. The promoter of gapC was used to construct fusions to the reporter genes lacZ and gfp. Analysis of these fusions indicates the potential of gapC as a valuable tool for the detection of E. coli in freshwater habitats, as well as for expressing other genes in aquatic environments.

Escherichia coli genes expressed preferentially in an aquatic environment.

Enteric bacteria are frequently found in aquatic environments, where they may pose a risk to human health. Although bacterial survival and persistence in such habitats has been studied extensively, there is almost no information about bacterial adaptation to these conditions at the level of changes in gene expression. As a first exploration of this field, we have carried out a screen designed to identify Escherichia coli genes that show increased expression in an aquatic environment. The screen was performed by subtractive hybridization on a genomic library and led to the identification of several RNA species more abundant in cells inoculated in this medium than in stationary-phase cultures after growth in rich medium. The genes identified include specific tRNA operons and a gene of unknown function, gapC, with similarities to glyceraldehyde-3-phosphate dehydrogenases. E. coli K-12 strains appear to have accumulated mutations in gapC, which may impede its translation, whereas natural isolates have an intact gapC gene. Sequence comparison of gapC with related genes suggests its acquisition by horizontal gene transfer from gram-positive bacteria.

Sigma s regulates pLS1 maintenance in stationary-phase Escherichia coli.

We have studied the influence of sigma s on the stability and number of copies of the promiscuous plasmid pLS1 in Escherichia coli. Our results indicate that pLS1 is less stable and has a lower number of copies in a rpoS mutant than in a wild-type strain during stationary phase. This behaviour does not seem to be due to differences in the expression of pLS1 replication regulators, but to be related to plasmid topology.

Sigma s-dependent promoters in Escherichia coli are located in DNA regions with intrinsic curvature.

Expression of a number of genes during stationary phase in Escherichia coli is controlled by the alternative sigma factor sigma s (KatF). Promoters recognized by sigma s do not present a well-defined consensus sequence in their -10 and -35 regions. By polyacrylamide gel electrophoresis of DNA fragments performed at different temperatures, and by computer prediction analyses, we have found that sigma s-regulated promoters are located in regions where DNA shows intrinsic curvatures. This feature does not appear in a stationary-phase-induced promoter which is not controlled by sigma s. We propose that DNA bending may help in recognition and/or binding of sigma s to stationary-phase-induced promoters.

Stationary-phase-inducible "gearbox" promoters: differential effects of katF mutations and role of sigma 70.

Many of the changes in gene expression observed when Escherichia coli cells enter stationary phase are regulated at the level of transcription initiation. A group of stationary-phase-inducible promoters, known as "gearbox" promoter, display a characteristic sequence in the -10 region which differs greatly from the consensus sequence for sigma 70-dependent promoters. Here we describe our studies on the gearbox promoters bolAp1 and mcbAp, responsible for the temporally regulated transcription of bolA and the genes involved in the synthesis of the peptide antibiotic microcin B17, respectively. Deletion analysis of mcbAp demonstrated that the stationary-phase-inducible properties of this promoter are found in a DNA fragment extending from -54 to +11 bp, surrounding the transcriptional start site, and are separable from DNA sequences responsible for the OmpR-dependent stimulation of transcription of mcbAp. In vitro transcription studies indicate that the RNA polymerase holoenzyme involved in the transcription of mcbAp contains sigma 70. In this and an accompanying paper (R. Lange and R. Hengge-Aronis, J. Bacteriol. 173: 4474-4481, 1991), experiments are described which show that the product of katF, a global regulator of stationary-phase gene expression and a putative sigma factor, is required for the expression of bolAp1 fused to the reporter gene lacZ. In contrast, mcbAp appears to be negatively regulated by katF. We discuss the implications of these results for postexponential gene expression and the role of gearbox sequences in the regulation of promoter activity.