132 research outputs found
Long term adaptation of a microbial population to a permanent metabolic constraint: overcoming thymineless death by experimental evolution of Escherichia coli
BACKGROUND: To maintain populations of microbial cells under controlled conditions of growth and environment for an indefinite duration is a prerequisite for experimentally evolving natural isolates of wild-type species or recombinant strains. This goal is beyond the scope of current continuous culture apparatus because these devices positively select mutants that evade dilution, primarily through attachment to vessel surfaces, resulting in persistent sub-populations of uncontrollable size and growth rate. RESULTS: To overcome this drawback, a device with two growth chambers periodically undergoing transient phases of sterilization was designed. The robustness of this device was assessed by propagating an E. coli strain under permanent thymine starvation for over 880 days, i.e. metabolic conditions notoriously known to lead to cell death and clogging of cultivation vessels. Ten thousand generations were required to obtain a descendant lineage that could resist thymine starvation and had recovered wild-type growth rate. CONCLUSIONS: This approach provides a technological framework for the diversification and improvement of microbial strains by long-term adaptation to inescapable metabolic constraints. An E. coli strain that is totally resistant to thymineless death was selected
RNomics and Modomics in the halophilic archaea Haloferax volcanii: identification of RNA modification genes
<p>Abstract</p> <p>Background</p> <p>Naturally occurring RNAs contain numerous enzymatically altered nucleosides. Differences in RNA populations (RNomics) and pattern of RNA modifications (Modomics) depends on the organism analyzed and are two of the criteria that distinguish the three kingdoms of life. If the genomic sequences of the RNA molecules can be derived from whole genome sequence information, the modification profile cannot and requires or direct sequencing of the RNAs or predictive methods base on the presence or absence of the modifications genes.</p> <p>Results</p> <p>By employing a comparative genomics approach, we predicted almost all of the genes coding for the t+rRNA modification enzymes in the mesophilic moderate halophile <it>Haloferax volcanii</it>. These encode both guide RNAs and enzymes. Some are orthologous to previously identified genes in Archaea, Bacteria or in <it>Saccharomyces cerevisiae</it>, but several are original predictions.</p> <p>Conclusion</p> <p>The number of modifications in t+rRNAs in the halophilic archaeon is surprisingly low when compared with other Archaea or Bacteria, particularly the hyperthermophilic organisms. This may result from the specific lifestyle of halophiles that require high intracellular salt concentration for survival. This salt content could allow RNA to maintain its functional structural integrity with fewer modifications. We predict that the few modifications present must be particularly important for decoding, accuracy of translation or are modifications that cannot be functionally replaced by the electrostatic interactions provided by the surrounding salt-ions. This analysis also guides future experimental validation work aiming to complete the understanding of the function of RNA modifications in Archaeal translation.</p
Comparative genomics of bacterial and plant folate synthesis and salvage: predictions and validations
<p>Abstract</p> <p>Background</p> <p>Folate synthesis and salvage pathways are relatively well known from classical biochemistry and genetics but they have not been subjected to comparative genomic analysis. The availability of genome sequences from hundreds of diverse bacteria, and from <it>Arabidopsis thaliana</it>, enabled such an analysis using the SEED database and its tools. This study reports the results of the analysis and integrates them with new and existing experimental data.</p> <p>Results</p> <p>Based on sequence similarity and the clustering, fusion, and phylogenetic distribution of genes, several functional predictions emerged from this analysis. For bacteria, these included the existence of novel GTP cyclohydrolase I and folylpolyglutamate synthase gene families, and of a trifunctional <it>p</it>-aminobenzoate synthesis gene. For plants and bacteria, the predictions comprised the identities of a 'missing' folate synthesis gene (<it>folQ</it>) and of a folate transporter, and the absence from plants of a folate salvage enzyme. Genetic and biochemical tests bore out these predictions.</p> <p>Conclusion</p> <p>For bacteria, these results demonstrate that much can be learnt from comparative genomics, even for well-explored primary metabolic pathways. For plants, the findings particularly illustrate the potential for rapid functional assignment of unknown genes that have prokaryotic homologs, by analyzing which genes are associated with the latter. More generally, our data indicate how combined genomic analysis of both plants and prokaryotes can be more powerful than isolated examination of either group alone.</p
Whole-Genome Sequence of Streptomyces kaniharaensis Shomura and Niida SF-557.
Streptomyces kaniharaensis is a Gram-positive bacterium that produces formycin A 5'-phosphate, a C nucleotide with antimicrobial and anticancer activity. Here, we report the sequencing, assembly, and annotation of the draft genome sequence of Streptomyces kaniharaensis Shomura and Niida
A subset of the diverse COG0523 family of putative metal chaperones is linked to zinc homeostasis in all kingdoms of life
<p>Abstract</p> <p>Background</p> <p>COG0523 proteins are, like the nickel chaperones of the UreG family, part of the G3E family of GTPases linking them to metallocenter biosynthesis. Even though the first COG0523-encoding gene, <it>cobW</it>, was identified almost 20 years ago, little is known concerning the function of other members belonging to this ubiquitous family.</p> <p>Results</p> <p>Based on a combination of comparative genomics, literature and phylogenetic analyses and experimental validations, the COG0523 family can be separated into at least fifteen subgroups. The CobW subgroup involved in cobalamin synthesis represents only one small sub-fraction of the family. Another, larger subgroup, is suggested to play a predominant role in the response to zinc limitation based on the presence of the corresponding COG0523-encoding genes downstream from putative Zur binding sites in many bacterial genomes. Zur binding sites in these genomes are also associated with candidate zinc-independent paralogs of zinc-dependent enzymes. Finally, the potential role of COG0523 in zinc homeostasis is not limited to Bacteria. We have predicted a link between COG0523 and regulation by zinc in Archaea and show that two COG0523 genes are induced upon zinc depletion in a eukaryotic reference organism, <it>Chlamydomonas reinhardtii</it>.</p> <p>Conclusion</p> <p>This work lays the foundation for the pursuit by experimental methods of the specific role of COG0523 members in metal trafficking. Based on phylogeny and comparative genomics, both the metal specificity and the protein target(s) might vary from one COG0523 subgroup to another. Additionally, Zur-dependent expression of <it>COG0523 </it>and putative paralogs of zinc-dependent proteins may represent a mechanism for hierarchal zinc distribution and zinc sparing in the face of inadequate zinc nutrition.</p
SOLiD sequencing of four Vibrio vulnificus genomes enables comparative genomic analysis and identification of candidate clade-specific virulence genes
<p>Abstract</p> <p>Background</p> <p><it>Vibrio vulnificus </it>is the leading cause of reported death from consumption of seafood in the United States. Despite several decades of research on molecular pathogenesis, much remains to be learned about the mechanisms of virulence of this opportunistic bacterial pathogen. The two complete and annotated genomic DNA sequences of <it>V. vulnificus </it>belong to strains of clade 2, which is the predominant clade among clinical strains. Clade 2 strains generally possess higher virulence potential in animal models of disease compared with clade 1, which predominates among environmental strains. SOLiD sequencing of four <it>V. vulnificus </it>strains representing different clades (1 and 2) and biotypes (1 and 2) was used for comparative genomic analysis.</p> <p>Results</p> <p>Greater than 4,100,000 bases were sequenced of each strain, yielding approximately 100-fold coverage for each of the four genomes. Although the read lengths of SOLiD genomic sequencing were only 35 nt, we were able to make significant conclusions about the unique and shared sequences among the genomes, including identification of single nucleotide polymorphisms. Comparative analysis of the newly sequenced genomes to the existing reference genomes enabled the identification of 3,459 core <it>V. vulnificus </it>genes shared among all six strains and 80 clade 2-specific genes. We identified 523,161 SNPs among the six genomes.</p> <p>Conclusions</p> <p>We were able to glean much information about the genomic content of each strain using next generation sequencing. Flp pili, GGDEF proteins, and genomic island XII were identified as possible virulence factors because of their presence in virulent sequenced strains. Genomic comparisons also point toward the involvement of sialic acid catabolism in pathogenesis.</p
Comparative genomic analysis of the DUF71/COG2102 family predicts roles in diphthamide biosynthesis and B12 salvage
Background: The availability of over 3000 published genome sequences has enabled the use of comparative genomic approaches to drive the biological function discovery process. Classically, one used to link gene with function by genetic or biochemical approaches, a lengthy process that often took years. Phylogenetic distribution profiles, physical clustering, gene fusion, co-expression profiles, structural information and other genomic or post-genomic derived associations can be now used to make very strong functional hypotheses. Here, we illustrate this shift with the analysis of the DUF71/COG2102 family, a subgroup of the PP-loop ATPase family. Results: The DUF71 family contains at least two subfamilies, one of which was predicted to be the missing diphthine-ammonia ligase (EC 6.3.1.14), Dph6. This enzyme catalyzes the last ATP-dependent step in the synthesis of diphthamide, a complex modification of Elongation Factor 2 that can be ADP-ribosylated by bacterial toxins. Dph6 orthologs are found in nearly all sequenced Archaea and Eucarya, as expected from the distribution of the diphthamide modification. The DUF71 family appears to have originated in the Archaea/Eucarya ancestor and to have been subsequently horizontally transferred to Bacteria. Bacterial DUF71 members likely acquired a different function because the diphthamide modification is absent in this Domain of Life. In-depth investigations suggest that some archaeal and bacterial DUF71 proteins participate in B12 salvage. Conclusions: This detailed analysis of the DUF71 family members provides an example of the power of integrated data-miming for solving important “missing genes” or “missing function” cases and illustrates the danger of functional annotation of protein families by homology alone. Reviewers’ names: This article was reviewed by Arcady Mushegian, Michael Galperin and L. Aravind
Erratum to “Towards a Systems Approach in the Genetic Analysis of Archaea: Accelerating Mutant Construction and Phenotypic Analysis in Haloferax volcanii”
A Gateway platform for functional genomics in Haloferax volcanii
In part due to the existence of simple methods for its cultivation and genetic manipulation, Haloferax volcanii is a major archaeal model organism. It is the only archaeon for which the whole set of post-transcriptionally modified tRNAs has been sequenced, allowing for an in silico prediction of all RNA modification genes present in the organism. One approach to check these predictions experimentally is via the construction of targeted gene deletion mutants. Toward this goal, an integrative “Gateway vector” that allows gene deletion in H. volcanii uracil auxotrophs was constructed. The vector was used to delete three predicted tRNA modification genes: HVO_2001 (encoding an archaeal transglycosyl tranferase or arcTGT), which is involved in archeosine biosynthesis; HVO_2348 (encoding a newly discovered GTP cyclohydrolase I), which catalyzes the first step common to archaeosine and folate biosynthesis; and HVO_2736 (encoding a member of the COG1444 family), which is involved in N4-acetylcytidine (ac4C) formation. Preliminary phenotypic analysis of the deletion mutants was conducted, and confirmed all three predictions
Towards a Systems Approach in the Genetic Analysis of Archaea: Accelerating Mutant Construction and Phenotypic Analysis in Haloferax volcanii
With the availability of a genome sequence and increasingly sophisticated genetic tools, Haloferax volcanii is becoming a model for both Archaea and halophiles. In order for H. volcanii to reach a status equivalent to Escherichia coli, Bacillus subtilis, or Saccharomyces cerevisiae, a gene knockout collection needs to be constructed in order to identify the archaeal essential gene set and enable systematic phenotype screens. A streamlined gene-deletion protocol adapted for potential automation was implemented and used to generate 22 H. volcanii deletion strains and identify several potentially essential genes. These gene deletion mutants, generated in this and previous studies, were then analyzed in a high-throughput fashion to measure growth rates in different media and temperature conditions. We conclude that these high-throughput methods are suitable for a rapid investigation of an H. volcanii mutant library and suggest that they should form the basis of a larger genome-wide experiment
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