Metagenomics-Guided Survey, Isolation, and Characterization of Uranium Resistant Microbiota from the Savannah River Site, USA.

Despite the recent advancements in culturomics, isolation of the majority of environmental microbiota performing critical ecosystem services, such as bioremediation of contaminants, remains elusive. Towards this end, we conducted a metagenomics-guided comparative assessment of soil microbial diversity and functions present in uraniferous soils relative to those that grew in diffusion chambers (DC) or microbial traps (MT), followed by isolation of uranium (U) resistant microbiota. Shotgun metagenomic analysis performed on the soils used to establish the DC/MT chambers revealed Proteobacterial phyla and Burkholderia genus to be the most abundant among bacteria. The chamber-associated growth conditions further increased their abundances relative to the soils. Ascomycota was the most abundant fungal phylum in the chambers relative to the soils, with Penicillium as the most dominant genus. Metagenomics-based taxonomic findings completely mirrored the taxonomic composition of the retrieved isolates such that the U-resistant bacteria and fungi mainly belonged to Burkholderia and Penicillium species, thus confirming that the chambers facilitated proliferation and subsequent isolation of specific microbiota with environmentally relevant functions. Furthermore, shotgun metagenomic analysis also revealed that the gene classes for carbohydrate metabolism, virulence, and respiration predominated with functions related to stress response, membrane transport, and metabolism of aromatic compounds were also identified, albeit at lower levels. Of major note was the successful isolation of a potentially novel Penicillium species using the MT approach, as evidenced by whole genome sequence analysis and comparative genomic analysis, thus enhancing our overall understanding on the uranium cycling microbiota within the tested uraniferous soils.

[Effect of soil microflora on 137Cs transition to plants].

The impact of certain types of microorganisms on 137Cs transfer from the substrate into the plant was analyzed in the experiment on artificial mediums. It was found that certain types of microorganisms could either reduce or increase the ratio of 137Cs transfer from the substrate to the plant. It is shown that this property is independent of the localization of the microorganism on the surface of the root, for all the analyzed bacteria belonging to the rhizospheric group. Azotobacter chroococcum UKM B-6003 stimulated the radionuclide transfer to plants up to 1.5 times, while the best bacteria for reducing its accumulation is Burkholderia sp IMER-B1 -53 - 1.3 times in comparison with the control. It was shown that the strain Bacillus megaterium UKM B-5724 from the collection of the Institute of Microbiology and Virology of NASU has a high ability to accumulate radionuclides.

The roles of the shikimate pathway genes, aroA and aroB, in virulence, growth and UV tolerance of Burkholderia glumae strain 411gr-6.

Burkholderia glumae is the major causal agent of bacterial panicle blight of rice, which is a growing disease problem for rice growers worldwide. In our previous study, some B. glumae strains showed pigmentation phenotypes producing at least two (yellow-green and purple) pigment compounds in casein-peptone-glucose agar medium. The B. glumae strains LSUPB114 and LSUPB116 are pigment-deficient mutant derivatives of the virulent and pigment-proficient strain 411gr-6, having mini-Tn5gus insertions in aroA encoding 3-phosphoshikimate 1-carboxyvinyltransferase and aroB encoding 3-dehydroquinate synthase, respectively. Both enzymes are known to be involved in the shikimate pathway, which leads to the synthesis of aromatic amino acids. Here, we demonstrate that aroA and aroB are required for normal virulence in rice and onion, growth in M9 minimal medium and tolerance to UV light, but are dispensable for the production of the phytotoxin toxoflavin. These results suggest that the shikimate pathway is involved in bacterial pathogenesis by B. glumae without a significant role in the production of toxoflavin, a major virulence factor of this pathogen.

Regulation of universal stress protein genes by quorum sensing and RpoS in Burkholderia glumae.

Burkholderia glumae possesses a quorum-sensing (QS) system mediated by N-octanoyl-homoserine lactone (C(8)-HSL) and its cognate receptor TofR. TofR/C(8)-HSL regulates the expression of a transcriptional regulator, qsmR. We identified one of the universal stress proteins (Usps), Usp2, from a genome-wide analysis of QS-dependent proteomes of B. glumae. In the whole genome of B. glumae BGR1, 11 usp genes (usp1 to usp11) were identified. Among the stress conditions tested, usp1 and usp2 mutants died 1 h after heat shock stress, whereas the other usp mutants and the wild-type strain survived for more than 3 h at 45°C. The expressions of all usp genes were positively regulated by QS, directly by QsmR. In addition, the expressions of usp1 and usp2 were dependent on RpoS in the stationary phase, as confirmed by the direct binding of RpoS-RNA holoenzyme to the promoter regions of the usp1 and usp2 genes. The expression of usp1 was upregulated upon a temperature shift from 37°C to either 28°C or 45°C, whereas the expression of usp2 was independent of temperature stress. This indicates that the regulation of usp1 and usp2 expression is different from what is known about Escherichia coli. Compared to the diverse roles of Usps in E. coli, Usps in B. glumae are dedicated to heat shock stress.

The quorum sensing-dependent gene katG of Burkholderia glumae is important for protection from visible light.

Quorum sensing (QS) plays important roles in the pathogenicity of Burkholderia glumae, the causative agent of bacterial rice grain rot. We determined how QS is involved in catalase expression in B. glumae. The QS-defective mutant of B. glumae exhibited less catalase activity than wild-type B. glumae. A beta-glucuronidase assay of a katG::Tn3-gusA78 reporter fusion protein revealed that katG expression is under the control of QS. Furthermore, katG expression was upregulated by QsmR, a transcriptional activator for flagellar-gene expression that is regulated by QS. A gel mobility shift assay confirmed that QsmR directly activates katG expression. The katG mutant produced toxoflavin but exhibited less severe disease than BGR1 on rice panicles. Under visible light conditions and a photon flux density of 61.6 micromol(-1) m(-2), the survival rate of the katG mutant was 10(5)-fold lower than that of BGR1. This suggests that KatG is a major catalase that protects bacterial cells from visible light, which probably results in less severe disease caused by the katG mutant.

Propionic acid metabolism and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P3HB-co-3HV) production by Burkholderia sp.

Mutants of Burkholderia sp. that are unable to grow on propionic acid (prp) but still accumulate P3HB-co-3HV from carbohydrate and propionic acid were studied. In shaken flask tests, yields of 3HV from propionic acid (Y(3HV/Prop)) increased from 0.10 g g(-1) in the wild type to c.a. 0.35 g g(-1) in mutants affected in alpha-oxidation pathway or to 0.80 g g(-1) in mutants not affected in that pathway. In bioreactor tests, mutant IPT 189 showed Y(3HV/Prop) = 1.20 g g(-1), a yield very close to the theoretical maximum of 1.35 g g(-1). Accumulation of 3HV units from unrelated carbon sources was undetectable in these mutants indicating that 3HV units are produced directly from propionic acid. Thus, the industrial use of those mutants to produce the copolymer from sucrose and propionic acid could significantly reduce the production costs. The results strongly suggest the existence of at least two pathways that are involved in the oxidation of propionic acid in Burkholderia sp. Their rates would be modulated by the availability of propionic acid.