Project description:To address the question of how photosynthetic bacterium Rhodopseudomonas palustris differentially regulates gene expression of three nitrogenase isozymes (Mo, V, and Fe nitrogenases), we constructed Mo strain (Mo nitrogenase only strain), V strain (V nitrogenase only strain), and Fe strain (Fe nitrogenase only strain), and analyzed the whole genome transcriptome profiles of each mutant and wild-type cells grown under nitrogen-fixing conditions. Keywords: Genetic modification
Project description:L. helveticus is used to modulate cheese flavor and as a starter organism in certain cheese varieties. Our group has compiled a draft (4x) sequence for the 2.4 Mb genome of an industrial strain L. helveticus CNRZ32. The primary aim was to investigate expression of 168 completely sequenced genes during growth in milk and MRS medium using microarrays. Oligonucleotide probes against each of the completely sequenced genes were compiled on maskless photolithography-based DNA microarrays. Additionally, the entire draft genome sequence was used to produce tiled microarrays where the non-interrupted sequence contigs were covered by consecutive 24-mer probes. Keywords: growth conditions response
Project description:Mycorrhiza helper bacteria (MHB) promote the formation of ectomycorrhizae between tree roots and ectomycorrhizal fungi. Despite the high relevance of MHB for forestry and for sustainable tree production in tree nurseries, little is known about the properties of the bacteria that contribute to their helper abilities. The MHB strain Pseudomonas fluorescens BBc6R8 is used as a model to study the mechanisms of the helper effect. We took advantage of new technologies to obtain, for the first time, the whole genome sequence of an MHB. Analyses reveal an important plasticity of the genome with numerous functions acquired by horizontal gene tranfer. Genome mining was combined with transcriptomic and mutagenesis approaches to reveal molecular determinants of the helper effect. The data suggest that the production of helper molecules is likely to be constitutive in vitro. The helper effect appears to be pleiotropic and to rely, for a substantial part, on trophic interactions. Despite its helper abilities, the bacterium is also able in specific conditions to outcompete ectomycorrhizal fungi and inhibit their growth. We conclude that the helper bacterium possess a broad range of properties whose expression depending on the biotic and abiotic conditions can result in either a beneficial, neutral or antagonistic interaction between the plant, the ectomycorrhizal fungus and the bacterium.
Project description:Polyamines, such as putrescine and spermidine, are aliphatic organic compounds with multiple amino groups. They are found ubiquitously in marine systems. However, compared with the extensive studies on the concentration and fate of other dissolved organic nitrogen compounds in seawater, such as dissolved free amino acids (DFAA), investigations of bacterially-mediated polyamine transformations have been rare. Bioinformatic analysis identified genes encoding polyamine transporters in 74 of 109 marine bacterial genomes surveyed, a surprising frequency for a class of organic nitrogen compounds not generally recognized as an important source of carbon and nitrogen for marine bacterioplankton. The genome sequence of marine model bacterium Silicibacter pomeroyi DSS-3 contains a number of genes putatively involved in polyamine use, including six four-gene ATP-binding cassette transport systems. In the present study, polyamine uptake and metabolism by S. pomeroyi was examined to confirm the role of putative polyamine-related genes, and to investigate how well current gene annotations reflect function. A comparative whole-genome microarray approach (Bürgmann et al., 2007) allowed us to identify key genes for transport and metabolism of spermidine in this bacterium, and specify candidate genes for in situ monitoring of polyamine transformations in marine bacterioplankton communities.
Project description:Many bacteria, often associated with eukaryotic hosts and of relevance for biotechnological applications, harbor a multipartite genome composed of more than one replicon. Biotechnologically relevant phenotypes are often encoded by genes residing on the secondary replicons. A synthetic biology approach to developing enhanced strains for biotechnological purposes could therefore involve merging pieces or entire replicons from multiple strains into a single genome. Here we report the creation of a genomic hybrid strain in a model multipartite genome species, the plant-symbiotic bacterium Sinorhizobium meliloti. We term this strain as cis-hybrid, since it is produced by genomic material coming from the same species' pangenome. In particular, we moved the secondary replicon pSymA (accounting for nearly 20% of total genome content) from a donor S. meliloti strain to an acceptor strain. The cis-hybrid strain was screened for a panel of complex phenotypes (carbon/nitrogen utilization phenotypes, intra- and extracellular metabolomes, symbiosis, and various microbiological tests). Additionally, metabolic network reconstruction and constraint-based modeling were employed for in silico prediction of metabolic flux reorganization. Phenotypes of the cis-hybrid strain were in good agreement with those of both parental strains. Interestingly, the symbiotic phenotype showed a marked cultivar-specific improvement with the cis-hybrid strains compared to both parental strains. These results provide a proof-of-principle for the feasibility of genome-wide replicon-based remodelling of bacterial strains for improved biotechnological applications in precision agriculture.
Project description:Acetic acid bacteria are obligately aerobic alphaproteobacteria that have a unique ability to incompletely oxidize various alcohols and sugars to organic acids. The ability of these bacteria to incompletely oxidize ethanol to acetate has been historically utilized for vinegar production. The mechanism of switching between incomplete oxidation and assimilatory oxidation and the control of energy and carbon metabolism in acetic acid bacteria are not fully understood. To understand the physiology and molecular biology of acetic acid bacteria better, we determined the draft genome sequence of Acetobacter aceti NBRC 14818, which is the type strain of the genus. Based on this draft genome sequence, the transcriptome profiles in A. aceti cells grown on ethanol, acetate, glucose, or mix of ethanol and glucose was determined by using NimbleGen Prokaryotic Expression array (4x72K).
Project description:Desulfotomaculum reducens strain MI-1 is a Gram-positive, sulfate-reducing bacterium also capable of reducing Fe(III). Metal reduction in Gram-positive bacteria is poorly understood. Here, we investigated Fe(III) reduction with lactate, a non-fermentable substrate, as the electron donor. Lactate consumption is concomitant to Fe(III) reduction, but does not support significant growth, suggesting that little energy can be conserved from this process and that it may occur fortuitously. D. reducens can reduce both soluble (Fe(III)-citrate) and insoluble (hydrous ferric oxide, HFO) Fe(III). Because physically inaccessible HFO was not reduced, we concluded that reduction requires direct contact under these experimental conditions. This implies the presence of a surface exposed reductase capable of transferring electrons from the cell to the extracellular electron acceptor. With the goal of identifying candidate Fe(III) reductases, we carried out an investigation of the surface proteome (surfaceome) of D. reducens. Cell surface exposed proteins were extracted by trypsin cell shaving or by lysozyme treatment, and analyzed by liquid chromatography-tandem mass spectrometry.
Project description:Differences in genome size and gene content are among the most important signatures of microbial adaptation and genome evolution. Here, we investigated the patterns of genome variation among strains of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti. Using the sequenced strain Rm1021 as a reference, the genome size and gene content variations were analyzed among ten diverse natural strains, through pulse field gel electrophoresis (PFGE) and whole-genome microarray hybridizations. Our PFGE analysis showed a genome size range of 6.45-7.01Mbp, with the greatest variation arising from the pSymA replicon, followed by that of pSymB. No observable size difference was evident among the chromosomes. Consistent with this pattern of size differences, 41.2% of ORFs on pSymA were variably absent/present, followed by 12.7% on pSymB, and 3.7% on the chromosome. However, the percentages of ORFs that were variably duplicated were more evenly distributed among the three replicons, 11.0%, 16.5% and 15.3% respectively for ORFs on pSymA, pSymB and the chromosome. Among the 10 strains, the percentages of absent ORFs ranged from 1.51% to 6.35% and those of duplicated ORFs ranged from 0.27% to 8.56%. Our analyses showed that host plants, geographic origins, multilocus enzyme electrophoretic types, and replicon sizes had little influence on the distribution patterns of absent or duplicated ORFs. The proportions of ORFs that were either variably absent/present or variably duplicated differed greatly among the functional categories, for each of the three replicons as well as for the whole genome. Interestingly, we observed positive correlations among the three replicons in their numbers of absent ORFs as well as the numbers of duplicated ORFs, consistent with coordinated gene gains/losses in this important bacterium in nature. microarray:Sm6kOligo
Project description:Mycorrhiza helper bacteria (MHB) promote the formation of ectomycorrhizae between tree roots and ectomycorrhizal fungi. Despite the high relevance of MHB for forestry and for sustainable tree production in tree nurseries, little is known about the properties of the bacteria that contribute to their helper abilities. The MHB strain Pseudomonas fluorescens BBc6R8 is used as a model to study the mechanisms of the helper effect. We took advantage of new technologies to obtain, for the first time, the whole genome sequence of an MHB. Analyses reveal an important plasticity of the genome with numerous functions acquired by horizontal gene tranfer. Genome mining was combined with transcriptomic and mutagenesis approaches to reveal molecular determinants of the helper effect. The data suggest that the production of helper molecules is likely to be constitutive in vitro. The helper effect appears to be pleiotropic and to rely, for a substantial part, on trophic interactions. Despite its helper abilities, the bacterium is also able in specific conditions to outcompete ectomycorrhizal fungi and inhibit their growth. We conclude that the helper bacterium possess a broad range of properties whose expression depending on the biotic and abiotic conditions can result in either a beneficial, neutral or antagonistic interaction between the plant, the ectomycorrhizal fungus and the bacterium. The study was performed along a kinetic with three sampling times: before contact between the bacterial colonies and the fungus (14 days after inoculation), at the time of contact (16 days after inoculation) and after an extended period of contact (21 days). For each time, three independent replicates were performed. A control treatment without the fungus was performed in triplicate and sampled at 14 days.