SummaryIn this study we disrupted two Herbaspirillum seropedicae genes, rfbB and rfbC, responsible for rhamnose biosynthesis and its incoporation into LPS. GC-MS analysis of the H. seropedicae wild-type strain LPS oligosaccharide chain showed that rhamnose, glucose and N-acetyl glucosamine are the predominant monosaccharides, whereas rhamnose and N-acetyl glucosamine were not found in the rfbB and rfbC strains. The electrophoretic pattern of the mutants LPS was drastically altered when compared with the wild type. Knockout of rfbB or rfbC increased the sensitivity towards SDS, polymyxin B sulfate and salicylic acid. The mutants attachment capacity to maize root surface plantlets was 100-fold lower than the wild type. Interestingly, the wild-type capacity to attach to maize roots was reduced to a level similar to that of the mutants when the assay was performed in the presence of isolated wild-type LPS, glucosamine or N-acetyl glucosamine. The mutant strains were also significantly less efficient in endophytic colonization of maize. Expression analysis indicated that the rfbB gene is upregulated by naringenin, apigenin and CaCl 2. Together, the results suggest that intact LPS is required for H. seropedicae attachment to maize root and internal colonization of plant tissues.
Many naturally-occurring cellulolytic microorganisms are not readily cultivable, demanding a culture-independent approach in order to study their cellulolytic genes. Metagenomics involves the isolation of DNA from environmental sources and can be used to identify enzymes with biotechnological potential from uncultured microbes. In this study, a gene encoding an endoglucanase was cloned from red rice crop residues using a metagenomic strategy. The amino acid identity between this gene and its closest published counterparts is lower than 70%. The endoglucanase was named EglaRR01 and was biochemically characterized. This recombinant protein showed activity on carboxymethylcellulose, indicating that EglaRR01 is an endoactive lytic enzyme. The enzymatic activity was optimal at a pH of 6.8 and at a temperature of 30˝C. Ethanol production from this recombinant enzyme was also analyzed on EglaRR01 crop residues, and resulted in conversion of cellulose from red rice into simple sugars which were further fermented by Saccharomyces cerevisiae to produce ethanol after seven days. Ethanol yield in this study was approximately 8 g/L. The gene found herein shows strong potential for use in ethanol production from cellulosic biomass (second generation ethanol).
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