Biomedical subjects
David Romero
Publications and source records attributed to David Romero.
Gene conversion and concerted evolution in bacterial genomes.
Gene conversion is defined as the non-reciprocal transfer of information between homologous sequences. Despite methodological problems to establish non-reciprocity, gene conversion has been demonstrated in a wide variety of bacteria. Besides examples of high-frequency reversion of mutations in repeated genes, gene conversion in bacterial genomes has been implicated in concerted evolution of multigene families. Gene conversion also has a prime importance in the generation of antigenic variation, an interesting mechanism whereby some bacterial pathogens are able to avoid the host immune system. In this review, we analyze examples of bacterial gene conversion (some of them spawned from the current genomic revolution), as well as the molecular models that explain gene conversion and its association with crossovers.
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Gene conversion tracts associated with crossovers in Rhizobium etli.
Gene conversion has been defined as the nonreciprocal transfer of information between homologous sequences. Despite its broad interest for genome evolution, the occurrence of this mechanism in bacteria has been difficult to ascertain due to the possible occurrence of multiple crossover events that would mimic gene conversion. In this work, we employ a novel system, based on cointegrate formation, to isolate gene conversion events associated with crossovers in the nitrogen-fixing bacterium Rhizobium etli. In this system, selection is applied only for cointegrate formation, with gene conversions being detected as unselected events. This minimizes the likelihood of multiple crossovers. To track the extent and architecture of gene conversions, evenly spaced nucleotide changes were made in one of the nitrogenase structural genes (nifH), introducing unique sites for different restriction endonucleases. Our results show that (i) crossover events were almost invariably accompanied by a gene conversion event occurring nearby; (ii) gene conversion events ranged in size from 150 bp to 800 bp; (iii) gene conversion events displayed a strong bias, favoring the preservation of incoming sequences; (iv) even small amounts of sequence divergence had a strong effect on recombination frequency; and (v) the MutS mismatch repair system plays an important role in determining the length of gene conversion segments. A detailed analysis of the architecture of the conversion events suggests that multiple crossovers are an unlikely alternative for their generation. Our results are better explained as the product of true gene conversions occurring under the double-strand break repair model for recombination.
Identification of the rctA gene, which is required for repression of conjugative transfer of rhizobial symbiotic megaplasmids.
An analysis of the conjugative transfer of pRetCFN42d, the symbiotic plasmid (pSym) of Rhizobium etli, has revealed a novel gene, rctA, as an essential element of a regulatory system for silencing the conjugative transfer of R. etli pSym by repressing the transcription of conjugal transfer genes in standard laboratory media. The rctA gene product lacks sequence conservation with other proteins of known function but may belong to the winged-helix DNA-binding subfamily of transcriptional regulators. Similar to that of many transcriptional repressors, rctA transcription seems to be positively autoregulated. rctA expression is greatly reduced upon overexpression of another gene, rctB, previously identified as a putative activator of R. etli pSym conjugal transfer. Thus, rctB seems to counteract the repressive action of rctA. rctA homologs are present in at least three other bacterial genomes within the order Rhizobiales, where they are invariably located adjacent to and divergently transcribed from putative virB-like operons. We show that similar to that of R. etli pSym, conjugative transfer of the 1.35-Mb symbiotic megaplasmid A of Sinorhizobium meliloti is also subjected to the inhibitory action of rctA. Our data provide strong evidence that the R. etli and S. meliloti pSym plasmids are indeed self-conjugative plasmids and that this property would only be expressed under optimal, as yet unknown conditions that entail inactivation of the rctA function. The rctA gene seems to represent novel but probably widespread regulatory systems controlling the transfer of conjugative elements within the order Rhizobiales.
The effects of ethanol and silymarin treatment during gestation on spatial working memory.
BACKGROUND: Using a rat model we have found that the bioflavonoid silymarin (SY) ameliorates some of the negative consequences of in utero exposure to ethanol (EtOH). In the current study our aim was to determine if spatial working memory (SWM) was impaired in offspring whose mothers were maintained on a liquid diet containing EtOH during different gestational weeks. We also determined if SWM was altered with a concomitant administration of SY with EtOH during specific gestational weeks. METHODS: We provided pregnant Fischer/344 rats with liquid diets containing 35% EtOH derived calories (EDC) during specific weeks of the gestational period. A silymarin/phospholipid compound containing 29.8% silybin co-administered with EtOH was also administered during specific weeks of the gestational period. We tested SWM of the offspring with a radial arm maze on postnatal day (PND) 60. After testing the rats were sacrificed and their brains perfused for later analysis. RESULTS: We observed SWM deficits, as well as a significantly lower brain weight in female offspring born of mothers treated with EtOH during the third week of gestation in comparison to mothers treated during either the first or second weeks of gestation. Rats from any group receiving EtOH in co-administration with SY showed no significant deficits in SWM. CONCLUSION: EtOH treatment during the last week of gestation had the greatest impact on SWM. The addition of SY to the EtOH liquid diet appeared to ameliorate the EtOH-induced learning deficits.
Transfer of the symbiotic plasmid of Rhizobium etli CFN42 requires cointegration with p42a, which may be mediated by site-specific recombination.
Plasmid p42a from Rhizobium etli CFN42 is self-transmissible and indispensable for conjugative transfer of the symbiotic plasmid (pSym). Most pSym transconjugants also inherit p42a. pSym transconjugants that lack p42a always contain recombinant pSyms, which we designated RpSyms*. RpSyms* do not contain some pSym segments and instead have p42a sequences, including the replication and transfer regions. These novel recombinant plasmids are compatible with wild-type pSym, incompatible with p42a, and self-transmissible. The symbiotic features of derivatives simultaneously containing a wild-type pSym and an RpSym* were analyzed. Structural analysis of 10 RpSyms* showed that 7 shared one of the two pSym-p42a junctions. Sequencing of this common junction revealed a 53-bp region that was 90% identical in pSym and p42a, including a 5-bp central region flanked by 9- to 11-bp inverted repeats reminiscent of bacterial and phage attachment sites. A gene encoding an integrase-like protein (intA) was localized downstream of the attachment site on p42a. Mutation or the absence of intA abolished pSym transfer from a recA mutant donor. Complementation with the wild-type intA gene restored transfer of pSym. We propose that pSym-p42a cointegration is required for pSym transfer; cointegration may be achieved either through homologous recombination among large reiterated sequences or through IntA-mediated site-specific recombination between the attachment sites. Cointegrates formed through the site-specific system but resolved through RecA-dependent recombination or vice versa generate RpSyms*. A site-specific recombination system for plasmid cointegration is a novel feature of these large plasmids and implies that there is unique regulation which affects the distribution of pSym in nature due to the role of the cointegrate in conjugative transfer.
The recombination genes addAB are not restricted to gram-positive bacteria: genetic analysis of the recombination initiation enzymes RecF and AddAB in Rhizobium etli.
Single-strand gaps (SSGs) and double-strand breaks (DSBs) are the major initiation sites for recombination. In bacteria, the SSGs are repaired by RecFOR, while the DSBs are processed by RecBCD in gram-negative bacteria and AddAB in gram-positive bacteria. Unexpectedly, instead of recBCD genes, the addAB genes were found in members of the alpha-proteobacteria group (gram negative). Taking Rhizobium etli as a model, the role of recF and addAB genes in homologous recombination and repair of damaged DNA was evaluated. Inactivation of either recF or addA provoked strong sensitivity to UV radiation and mitomycin C, while an additive effect was observed in the recF-addA mutant. The DSBs generated by nalidixic acid caused low viability only in the addA mutant. The recombination frequency of large and small plasmids was reduced in the recF mutant (24- and 36-fold, respectively), whereas a slight decrease (threefold) in the addA mutant was observed. Moreover, an additive effect (47- and 90-fold, respectively) was observed in the double mutant, but it was not as dramatic as that in a recA mutant. Interestingly, the frequency of deletion and Campbell-type recombination was slightly affected in either single or double mutants. These results suggest that another pathway exists that allows plasmid and Campbell-type recombination in the absence of recF and addA genes.
The mosaic structure of the symbiotic plasmid of Rhizobium etli CFN42 and its relation to other symbiotic genome compartments.
BACKGROUND: Symbiotic bacteria known as rhizobia interact with the roots of legumes and induce the formation of nitrogen-fixing nodules. In rhizobia, essential genes for symbiosis are compartmentalized either in symbiotic plasmids or in chromosomal symbiotic islands. To understand the structure and evolution of the symbiotic genome compartments (SGCs), it is necessary to analyze their common genetic content and organization as well as to study their differences. To date, five SGCs belonging to distinct species of rhizobia have been entirely sequenced. We report the complete sequence of the symbiotic plasmid of Rhizobium etli CFN42, a microsymbiont of beans, and a comparison with other SGC sequences available. RESULTS: The symbiotic plasmid is a circular molecule of 371,255 base-pairs containing 359 coding sequences. Nodulation and nitrogen-fixation genes common to other rhizobia are clustered in a region of 125 kilobases. Numerous sequences related to mobile elements are scattered throughout. In some cases the mobile elements flank blocks of functionally related sequences, thereby suggesting a role in transposition. The plasmid contains 12 reiterated DNA families that are likely to participate in genomic rearrangements. Comparisons between this plasmid and complete rhizobial genomes and symbiotic compartments already sequenced show a general lack of synteny and colinearity, with the exception of some transcriptional units. There are only 20 symbiotic genes that are shared by all SGCs. CONCLUSIONS: Our data support the notion that the symbiotic compartments of rhizobia genomes are mosaic structures that have been frequently tailored by recombination, horizontal transfer and transposition.
BacS: an abundant bacteroid protein in Rhizobium etli whose expression ex planta requires nifA.
Rhizobium etli CFN42 bacteroids from bean nodules possessed an abundant 16-kDa protein (BacS) that was found in the membrane pellet after cell disruption. This protein was not detected in bacteria cultured in tryptone-yeast extract. In minimal media, it was produced at low oxygen concentration but not in a mutant whose nifA was disrupted. N-terminal sequencing of the protein led to isolation of a bacS DNA fragment. DNA hybridization and nucleotide sequencing revealed three copies of the bacS gene, all residing on the main symbiotic plasmid of strain CFN42. A stretch of 304 nucleotides, exactly conserved upstream of all three bacS open reading frames, had very close matches with the NifA and sigma 54 consensus binding sequences. The only bacS homology in the genetic sequence databases was to three hypothetical proteins of unknown function, all from rhizobial species. Mutation and genetic complementation indicated that each of the bacS genes gives rise to a BacS polypeptide. Mutants disrupted or deleted in all three genes did not produce the BacS polypeptide but were Nod+ and Fix+ on Phaseolus vulgaris.
Effect of Naturally Occurring nif Reiterations on Symbiotic Effectiveness in Rhizobium phaseoli.
Most naturally occurring strains of Rhizobium phaseoli possess reiteration of the nif genes. Three regions contain nitrogenase structural genes in strain CFN42. Two of these regions (a and b) have copies of nifH, nifD, and nifK, whereas the third region (c) contains only nifH. Strains containing mutations in either nif region a or nif region b had significantly diminished symbiotic effectiveness compared with the wild-type strain on the basis of nodule mass, total nitrogenase activity per plant, nitrogenase specific activity, total nitrogen in the shoot, and percentage of nitrogen. A strain containing mutations in both nif region a and nif region b was totally ineffective. These data indicate that both nif region a and nif region b are needed for full symbiotic effectiveness in R. phaseoli.