The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species.

BackgroundMetagenomics, the application of molecular genomics to consortia of non-cultivated microbes, has the potential to have a substantial impact on the search for novel industrial enzymes such as esterases (carboxyl ester hydrolases, EC 3.1.1.1) and lipases (triacylglycerol lipases, EC 3.1.1.3). In the current work, a novel lipase gene was identified from a fosmid metagenomic library constructed with the "prokaryotic-enriched" DNA from a fat-contaminated soil collected from a wastewater treatment plant.ResultsIn preliminary screening on agar containing 1% tributyrin, 2661 of the approximately 500,000 clones in the metagenomic library showed activity. Of these, 127 showed activity on agar containing 1% tricaprylin, while 32 were shown to be true lipase producers through screening on agar containing 1% triolein. The clone with the largest halo was further characterized. Its lipase gene showed 72% identity to a putative lipase of Yersinia enterocolitica subsp. palearctica Y11. The lipase, named LipC12, belongs to family I.1 of bacterial lipases, has a chaperone-independent folding, does not possess disulfide bridges and is calcium ion dependent. It is stable from pH 6 to 11 and has activity from pH 4.5 to 10, with higher activities at alkaline pH values. LipC12 is stable up to 3.7 M NaCl and from 20 to 50°C, with maximum activity at 30°C over a 1 h incubation. The pure enzyme has specific activities of 1722 U/mg and 1767 U/mg against olive oil and pig fat, respectively. Moreover, it is highly stable in organic solvents at 15% and 30% (v/v).ConclusionsThe combination of the use of a fat-contaminated soil, enrichment of prokaryotic DNA and a three-step screening strategy led to a high number of lipase-producing clones in the metagenomic library. The most notable properties of the new lipase that was isolated and characterized were a high specific activity against long chain triacylglycerols, activity and stability over a wide range of pH values, good thermal stability and stability in water-miscible organic solvents and at high salt concentrations. These characteristics suggest that this lipase has potential to perform well in biocatalytic processes, such as for hydrolysis and synthesis reactions involving long-chain triacylglycerols and fatty acid esters.

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The Brazilian Atlantic Forest is one of the 25 biodiversity hot spots in the world. Although the diversity of its fauna and flora has been studied fairly well, little is known of its microbial communities. In this work, we analyzed the Atlantic Forest ecosystem to determine its bacterial biodiversity, using 16S rRNA gene sequencing, and correlated changes in deduced taxonomic profiles with the physicochemical characteristics of the soil. DNAs were purified from soil samples, and the 16S rRNA gene was amplified to construct libraries. Comparison of 754 independent 16S rRNA gene sequences from 10 soil samples collected along a transect in an altitude gradient showed the prevalence of Acidobacteria (63%), followed by Proteobacteria (25.2%), Gemmatimonadetes (1.6%), Actinobacteria (1.2%), Bacteroidetes (1%), Chloroflexi (0.66%), Nitrospira (0.4%), Planctomycetes (0.4%), Firmicutes (0.26%), and OP10 (0.13%). Forty-eight sequences (6.5%) represented unidentified bacteria. The Shannon diversity indices of the samples varied from 4.12 to 3.57, indicating that the soils have a high level of diversity. Statistical analysis showed that the bacterial diversity is influenced by factors such as altitude, Ca 2؉ /Mg 2؉ ratio, and Al 3؉ and phosphorus content, which also affected the diversity within the same lineage. In the samples analyzed, pH had no significant impact on diversity.

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.

The fixation of atmospheric nitrogen by the prokaryotic enzyme nitrogenase is an energyexpensive process and consequently it is tightly regulated at a variety of levels. In many diazotrophs this includes post-translational regulation of the enzyme's activity, which has been reported in both bacteria and archaea. The best understood response is the short-term inactivation of nitrogenase in response to a transient rise in ammonium levels in the environment. A number of proteobacteria species effect this regulation through reversible ADP-ribosylation of the enzyme, but other prokaryotes have evolved different mechanisms. Here we review current knowledge of post-translational control of nitrogenase and show that, for the response to ammonium, the P II signal transduction proteins act as key players. IntroductionBiological nitrogen fixation, the reduction of atmospheric N 2 to NH 3 by nitrogen-fixing bacteria, is a key step in the nitrogen cycle. This process is catalysed by nitrogenase, the most common form of which is the molybdenum nitrogenase, composed of dinitrogenase (MoFe protein or NifDK), an a 2 b 2 tetramer encoded by the nifD and nifK genes, respectively, and dinitrogenase reductase (Fe protein or NifH), a c 2 homodimer encoded by the nifH gene. The NifH protein is responsible for ATP-hydrolysisdriven electron transport to the NifDK protein, which contains the site for the reduction of dinitrogen to ammonium (Seefeldt et al., 2009). The reduction of N 2 to two molecules of ammonium is an energy-expensive process requiring the hydrolysis of 16 ATPs.To avoid energy wastage, diazotrophs have evolved both transcriptional and post-translational mechanisms to shut-down nitrogen fixation when ammonium is available in the environment. Post-translational control of nitrogenase activity has been found in a range of diazotrophs and affords a rapid and reversible mechanism by which the organism can respond to transient changes in the environment. Here we review current knowledge of the different mechanisms of post-translational control of nitrogenase. We focus on the two best-described systems: ADP-ribosylation of NifH, which occurs in proteobacteria, and the interaction of NifI regulatory proteins with NifDK in archaea, which potentially also operates in some anaerobic diazotrophic bacteria. Regulation of nitrogenase activity by reversible ADP-ribosylation Historical perspectiveThe process of metabolic inactivation of nitrogenase in response to ammonium was first described in Azotobacter vinelandii (Burris & Wilson, 1946); this phenomenon was later identified in other prokaryotes and named nitrogenase 'switch-off' (Zumft & Castillo, 1978). Different mechanisms are used to regulate nitrogenase post-translationally depending on the organism. The best-studied mechanism operates through reversible ADP-ribosylation of NifH (reviewed by Nordlund, 2000). This system responds to not only the presence of ammonium but also a decrease in the availability of cellular energy, in response to either darkness in phototrophs such as ...

Molecular mechanisms of plant recognition and colonization by diazotrophic bacteria are barely understood. Herbaspirillum seropedicae is a Betaproteobacterium capable of colonizing epiphytically and endophytically commercial grasses, to promote plant growth. In this study, we utilized RNA-seq to compare the transcriptional profiles of planktonic and maize root-attached H. seropedicae SmR1 recovered 1 and 3 days after inoculation. The results indicated that nitrogen metabolism was strongly activated in the rhizosphere and polyhydroxybutyrate storage was mobilized in order to assist the survival of H. seropedicae during the early stages of colonization. Epiphytic cells showed altered transcription levels of several genes associated with polysaccharide biosynthesis, peptidoglycan turnover and outer membrane protein biosynthesis, suggesting reorganization of cell wall envelope components. Specific methyl-accepting chemotaxis proteins and two-component systems were differentially expressed between populations over time, suggesting deployment of an extensive bacterial sensory system for adaptation to the plant environment. An insertion mutation inactivating a methyl-accepting chemosensor induced in planktonic bacteria, decreased chemotaxis towards the plant and attachment to roots. In summary, analysis of mutant strains combined with transcript profiling revealed several molecular adaptations that enable H. seropedicae to sense the plant environment, attach to the root surface and survive during the early stages of maize colonization.

Several bacteria are able to degrade flavonoids either to use them as carbon sources or as a detoxification mechanism. Degradation pathways have been proposed for several bacteria, but the genes responsible are not known. We identified in the genome of the endophyte Herbaspirillum seropedicae SmR1 an operon potentially associated with the degradation of aromatic compounds. We show that this operon is involved in naringenin degradation and that its expression is induced by naringenin and chrysin, two closely related flavonoids. Mutation of fdeA, the first gene of the operon, and fdeR, its transcriptional activator, abolished the ability of H. seropedicae to degrade naringenin.