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Bacillus amyloliquefaciens ssp. plantarum FZB42 represents the prototype of Gram-positive plant growth promoting and biocontrol bacteria. In this study, we applied transposon mutagenesis to generate a transposon library, which was screened for genes involved in multicellular behavior and biofilm formation on roots as a prerequisite of plant growth promoting activity. Transposon insertion sites were determined by rescue-cloning followed by DNA sequencing. As in B. subtilis, the global transcriptional regulator DegU was identified as an activator of genes necessary for swarming and biofilm formation, and the DegU-mutant of FZB42 was found impaired in efficient root colonization. Direct screening of 3,000 transposon insertion mutants for plant-growth-promotion revealed the gene products of nfrA and RBAM_017140 to be essential for beneficial effects exerted by FZB42 on plants. We analyzed the performance of GFP-labeled wild-type and transposon mutants in the colonization of lettuce roots using confocal laser scanning microscopy. While the wild-type strain heavily colonized root surfaces, the nfrA mutant did not colonize lettuce roots, although it was not impaired in growth in laboratory cultures, biofilm formation and swarming motility on agar plates. The RBAM17410 gene, occurring in only a few members of the B. subtilis species complex, was directly involved in plant growth promotion. None of the mutant strains were affected in producing the plant growth hormone auxin. We hypothesize that the nfrA gene product is essential for overcoming the stress caused by plant response towards bacterial root colonization. [ABSTRACT FROM AUTHOR]

The soil-borne pathogen Rhizoctonia solani is responsible for crop losses on a wide range of important crops worldwide. The lack of effective control strategies and the increasing demand for organically grown food has stimulated research on biological control. The aim of the present study was to evaluate the rhizosphere competence of the commercially available inoculant Bacillus amyloliquefaciens FZB42 on lettuce growth and health together with its impact on the indigenous rhizosphere bacterial community in field and pot experiments. Results of both experiments demonstrated that FZB42 is able to effectively colonize the rhizosphere (7.45 to 6.61 Log 10 CFU g−1 root dry mass) within the growth period of lettuce in the field. The disease severity (DS) of bottom rot on lettuce was significantly reduced from severe symptoms with DS category 5 to slight symptom expression with DS category 3 on average through treatment of young plants with FZB42 before and after planting. The 16S rRNA gene based fingerprinting method terminal restriction fragment length polymorphism (T-RFLP) showed that the treatment with FZB42 did not have a major impact on the indigenous rhizosphere bacterial community. However, the bacterial community showed a clear temporal shift. The results also indicated that the pathogen R. solani AG1-IB affects the rhizosphere microbial community after inoculation. Thus, we revealed that the inoculant FZB42 could establish itself successfully in the rhizosphere without showing any durable effect on the rhizosphere bacterial community. [ABSTRACT FROM AUTHOR]

Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H2 possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H2-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H2 oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 106 to 108 hhyL gene copies g (dry weight)-1. Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H2-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H2 oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H2 by soil, because high-affinity H2 oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur. [ABSTRACT FROM AUTHOR]

Abstract: Two complementary experimental approaches were utilized to examine the extent to which free soil hydrogenases and H2-oxidizing bacteria contribute to the soil uptake of atmospheric H2. First, high affinity hydrogenase activity and H2-oxidizing bacteria were fractionated in non-axenic soil and axenic soil colonized with the high affinity H2-oxidizing bacterium Streptomyces sp. PCB7. Non-axenic soil was fractionated by buoyant density centrifugation. High affinity H2 oxidation activity measured in individual fractions was proportional to the copy number of hhyL gene, specifying the large subunit of putative high affinity [NiFe]-hydrogenases. 2.5% of the hydrogenase activity was recovered in bacteria-free soil extract. Similarly, sequential centrifugation and wet filtrations of strain PCB7-colonized soil dispersed in solubilization buffer caused a loss of the activity, at a ratio proportional to the number of living cells removed. No abiontic hydrogenase activity was detected in bacteria-free fractions. The second experimental approach was designed to verify whether or not the [NiFe]-hydrogenase of strain PCB7 retains high affinity H2 oxidation activity in soil, under the abiontic state. H2 oxidation rates of crude enzyme extract of strain PCB7 measured under aerobic and anaerobic conditions were indistinguishable, indicating that the high affinity hydrogenase of strain PCB7 is oxygen-tolerant. The hydrogenase activity of sterile soil spiked with as much as 0.14mg(protein) g(soil-dw) −1 was equivalent to the H2-oxidation activity of only 106–107 CFU of strain PCB7 g(soil-dw) −1. Taken together, our results indicate that high affinity hydrogenase activity is proportional to the abundance of H2-oxidizing bacteria in soil and, that abiontic hydrogenases contribute only a few percent of the total high affinity H2 oxidation activity detected in soil. [Copyright &y& Elsevier]

Abstract: Soils are the most important sink for atmospheric hydrogen, which is assumed to be oxidized by abiontic soil hydrogenases or by putative high-affinity hydrogenases of microbial origin. The activity of soil hydrogenases has been found to change with soil temperature as it changes during the day and the season. However, it is unclear whether and to which extent the soil hydrogenases are deactivated by increased temperature. Therefore, we incubated soils from different climates and different ecosystems (forest, agricultural, arid, hyper-arid, paddy, peat) at elevated temperature and measured the residual activity at 25 °C after different incubation times. We found that at least part of the soil hydrogenase is deactivated irreversibly already at relatively low temperatures (>30 °C) and short exposure times (>10 min) and that the deactivation was more pronounced in soil from cold versus hot climate. The deactivation kinetics could be fitted to a biexponential model, but were complex with respect to soil type and deactivation temperature. The results show that new hydrogenase activity has to be generated in the soil to compensate for activity loss by diurnal and seasonally increased temperature. [Copyright &y& Elsevier]

Salinity stress inhibits the growth and nitrogen fixation ability of the plant growth-promoting rhizobacterium Azospirillum brasilense. Five strains of A. brasilense were isolated from the rhizosphere of Indian cereals and grasses and identified on the basis of their phenotypic features and 16S rRNA gene sequence. The five Indian isolates and two standard strains of A. brasilense, Sp7 and Cd, showed notable differences in growth, acetylene-reducing activity under salt stress, and ability to take up and use glycine betaine for the restoration of growth and acetylene-reducing activity under salt stress. Salt stress also enhanced the production of exopolysaccharides and cell aggregates, the extent of which varied in different strains of A. brasilense at different carbon to nitrogen ratios in the culture medium. It can be concluded that the production of exopolysaccharides and cell aggregates is a more consistent physiological response of A. brasilense to salt stress than is the uptake and osmoprotection by glycine betaine. [ABSTRACT FROM AUTHOR]

Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H(2) possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H(2)-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H(2) oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 10(6) to 10(8) hhyL gene copies g (dry weight)(-1). Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H(2)-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H(2) oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H(2) by soil, because high-affinity H(2) oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur.