Ribosomal RNA content in microcolony forming soil bacteria measured by quantitative 16S rRNA hybridization and image analysis

The correlation between the growth rate, rRNA concentration and number of rrna operons was studied in Alcaligenes sp. A2, Pseudomonas fluorescens R2f and Bacillus sp. B1 cells grown exponentially in liquid 1/10 strength tryptic soy broth (1/10 TSB) medium and grown to microcolony (mCFU) size on polycarbonate membrane filters floating on 1/10 TSB. The rRNA concentration was also determined in cells forming mCFUs after incubation on membranes of rhizosphere and bulk soil samples taken from a barley (Hordeum vulgare) microcosm experiment. A protocol for fluorescent in situ hybridization directly on polycarbonate membrane filters combined with confocal laser scanning microscopy and image analysis was used to measure the probing intensity from individualcells within the mCFUs. We found that cells forming mCFU on membranes had a rRNA concentration slightly higher than cells grown in fluid medium. Mean values of growth rates and rRNA concentration measured as a probing intensity per cell area were 0.31, 0.61 and 0.63 h-1 and 52, 63 and 146 units per cell area for Alcaligenes sp. A2, P. fluorescens R2f, and Bacillus sp. B1, respectively, when grown on membranes. The number of rrna operons carried by the three bacteria was determined to be between two to three, four and seven to nine. Alcaligenes sp. A2 which carried only two to three rrna operons had the lowest concentration of rRNA. Furthermore this organism had a lower growth rate compared to P. fluorescens R2f, and Bacillus sp. B1. Although P. fluorescens R2f and Bacillus sp. B1 had the same growth rate, Bacillus sp. B1 expressed a much higher rRNA concentration, which reflected well the high number of rrna operons carried by this bacterium. The mean probing intensity of mCFUs obtained after incubation of rhizosphere and bulk soil samples varied more than four-fold in both environments. The percentage of mCFU forming bacteria expressing a low concentration of rRNA was at least as high in the rhizosphere as in the bulk soil. The rationale behind carrying a high copy number of rrna operons and maintaining high concentrations of rRNA during growth is discussed

Chemical and isotopic switching within the subglacial environment of a high Arctic glacier.

Natural environmental isotopes of nitrate, sulphate and inorganic carbon are discussed in conjunction with major ion chemistry of subglacial runoff from a High Arctic glacier, Midre Lovénbreen, Svalbard. The chemical composition of meltwaters is observed to switch in accordance with subglacial hydrological evolution and redox status. Changing rapidly from reducing to oxidizing conditions, subglacial waters also depict that 15N/14N values show microbial denitrification is an active component of nutrient cycling beneath the glacier. 18O/16O ratios of sulphate are used to elucidate mechanisms of biological and abiological sulphide oxidation. Concentrations of bicarbonate appear to be governed largely by the degree of rock:water contact encountered in the subglacial system, rather than the switch in redox status, although the potential for microbiological activity to influence ambient bicarbonate concentrations is recognised. Glaciers are therefore highlighted as cryospheric ecosystems supporting microbial life which directly impacts upon the release of solute through biogeochemically mediated processes

Impact of biocontrol strain Pseudomonas fluorescens CHA0 on rhizosphere bacteria isolated from barley (Hordeum vulgare L.) with special reference to Cytophaga-like bacteria.

AIMS: To assess the impact of the biocontrol strain Pseudomonas fluorescens CHA0 on a collection of barley rhizosphere bacteria using an agar plate inhibition assay and a plant microcosm, focusing on a CHA0-sensitive member of the Cytophaga-like bacteria (CLB). METHODS AND RESULTS: The effect of strain CHA0 on a collection of barley rhizosphere bacteria, in particular CLB and fluorescent pseudomonads sampled during a growth season, was assessed by a growth inhibition assay. On average, 85% of the bacteria were sensitive in the May sample, while the effect was reduced to around 68% in the July and August samples. In the May sample, around 95% of the CLB and around 45% of the fluorescent pseudomonads were sensitive to strain CHA0. The proportion of CHA0-sensitive CLB and fluorescent pseudomonad isolates decreased during the plant growth season, i.e. in the July and August samples. A particularly sensitive CLB isolate, CLB23, was selected, exposed to strain CHA0 (wild type) and its genetically modified derivatives in the rhizosphere of barley grown in gnotobiotic soil microcosms. Two dry-stress periods were imposed during the experiment. Derivatives of strain CHA0 included antibiotic or exopolysaccharide (EPS) overproducing strains and a dry-stress-sensitive mutant. Despite their inhibitory activity against CLB23 in vitro, neither wild-type strain CHA0, nor any of its derivatives, had a major effect on culturable and total cell numbers of CLB23 during the 23-day microcosm experiment. Populations of all inoculants declined during the two dry-stress periods, with soil water contents below 5% and plants reaching the wilting point, but they recovered after re-wetting the soil. Survival of the dry-stress-sensitive mutant of CHA0 was most affected by the dry periods; however, this did not result in an increased population density of CLB23. CONCLUSIONS: CLB comprise a large fraction of barley rhizosphere bacteria that are sensitive to the biocontrol pseudomonad CHA0 in vitro. However, in plant microcosm experiments with varying soil humidity conditions, CHA0 or its derivatives had no major impact on the survival of the highly sensitive CLB strain, CLB23, during two dry-stress periods and a re-wetting period; all co-existed well in the rhizosphere of barley plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate a lack of interaction between the biocontrol pseudomonad CHA0 and a sensitive CLB when the complexity increases from agar plate assays to plant microcosm experiments. This suggests the occurrence of low levels of antibiotic production and/or that the two bacterial genera occupy different niches in the rhizosphere