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

The Escherichia coli COG1738 Member YhhQ Is Involved in 7-Cyanodeazaguanine (preQ0) Transport

Queuosine (Q) is a complex modification of the wobble base in tRNAs with GUN anticodons. The full Q biosynthesis pathway has been elucidated in Escherichia coli. FolE, QueD, QueE and QueC are involved in the conversion of guanosine triphosphate (GTP) to 7-cyano-7-deazaguanine (preQ0), an intermediate of increasing interest for its central role in tRNA and DNA modification and secondary metabolism. QueF then reduces preQ0 to 7-aminomethyl-7-deazaguanine (preQ1). PreQ1 is inserted into tRNAs by tRNA guanine(34) transglycosylase (TGT). The inserted base preQ1 is finally matured to Q by two additional steps involving QueA and QueG or QueH. Most Eubacteria harbor the full set of Q synthesis genes and are predicted to synthesize Q de novo. However, some bacteria only encode enzymes involved in the second half of the pathway downstream of preQ0 synthesis, including the signature enzyme TGT. Different patterns of distribution of the queF, tgt, queA and queG or queH genes are observed, suggesting preQ0, preQ1 or even the queuine base being salvaged in specific organisms. Such salvage pathways require the existence of specific 7-deazapurine transporters that have yet to be identified. The COG1738 family was identified as a candidate for a missing preQ0/preQ1 transporter in prokaryotes, by comparative genomics analyses. The existence of Q precursor salvage was confirmed for the first time in bacteria, in vivo, through an indirect assay. The involvement of the COG1738 in salvage of a Q precursor was experimentally validated in Escherichia coli, where it was shown that the COG1738 family member YhhQ is essential for preQ0 transport

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

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

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 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