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PubMed1997

Antisense RNA-regulated programmed cell death.

Eubacterial plasmids and chromosomes encode multiple killer genes belonging to the hok gene family. The plasmid-encoded killer genes mediate plasmid stabilization by killing plasmid-free cells. This review describes the genetics, molecular biology, and evolution of the hok gene family. The complicated antisense RNA-regulated control-loop that regulates posttranscriptional and postsegregational activation of killer mRNA translation in plasmid-free cells is described in detail. Nucleotide covariations in the mRNAs reveal metastable stem-loop structures that are formed at the mRNA 5' ends in the nascent transcripts. The metastable structures prevent translation and antisense RNA binding during transcription. Coupled nucleotide covariations provide evidence for a phylogenetically conserved mRNA folding pathway that involves sequential dynamic RNA rearrangements. Our analyses have elucidated an intricate mechanism by which translation of an antisense RNA-regulated mRNA can be conditionally activated. The complex phylogenetic relationships of the plasmid- and chromosome-encoded systems are also presented and discussed.

Apoptosis↗
PubMed2026

Genetic Tools in the Nakaseomyces clade for Evolutionary Comparisons of Signal Transduction Pathways.

The genus Nakaseomyces provides four species that are closely related but have different characteristics. For example, N. glabratus (formerly known as Candida glabrata) is a common human pathogen, whereas N. bracarensis and N. nivariensis have been isolated in clinical settings but are not common human pathogens. N. delphensis was isolated from fruit and there is no evidence it is pathogenic. Given the differences, we developed the clade as a molecular genetic system where we could introduce plasmids and assess transcriptional output from cloned promoters. We engineered a CRISPR/Cas9 plasmid that allows for rapid Gibson cloning of gRNAs, generated auxotrophic strains for amino acids and nucleotides, and introduced plasmids into each species. We used promoter-YFP plasmids to determine that while there are differences between the species, each species likely has intact thiamine and phosphate (THI and PHO) signal transduction pathways, and that gene expression in N. glabratus and N. bracarensis is more similar to one another than to the other two species. Finally, we determine that N. glabratus, N. bracarensis, and N. nivariensis persist in a murine macrophage for 24 h, whereas N. delphensis does not. This work describes new molecular tools for genetic manipulation in the Nakaseomyces clade and allows for evolutionary questions to be explored.

Signal Transduction↗
PubMed1994

Cryptic plasmids from Lactobacillus helveticus and their evolutionary relationship.

Three different cryptic plasmids from Lactobacillus helveticus have been identified and their DNA sequences determined. Analysis and comparisons of their primary structures revealed stretches of DNA with considerable homology. Thus, large portions of the plasmid non-coding sequences were conserved at 80-90% identity between the different plasmids identified so far in L. helveticus. Nevertheless, different plasmids found in a same host strain utilise different genes of replication, probably acquired during evolution from different replicons from Gram-positive bacterial origins. A remnant structure of such a possible genetic integration of a foreign replication gene into one of the plasmids of L. helveticus was identified.

Amino Acid Sequence↗
PubMedDate not supplied

A molecular strategy for the study of bacterial invasion.

Bacterial populations are often clonal, and even within a bacterial species, the frequency of gene exchange and recombination is quite low. Consequently, mobile genetic elements--plasmids, bacteriophages, and transposons--have been the central factors in the evolution of pathogenic traits. One central feature of pathogenicity, the capacity to enter epithelial cells, is encoded by the bacterial chromosome of Yersinia pseudotuberculosis but by plasmid genes in enteroinvasive Escherichia coli. A single Yersinia gene, inv, which encodes a single 107,000-dalton protein, can be cloned in E. coli K12, and its presence is sufficient to permit the bacteria to enter cultured human cells. In contrast, fully 70 kilobases of a virulence plasmid from enteroinvasive E. coli must be transferred to E. coli K12 to achieve the same result. While researchers are at the early stages of understanding microbial entry into host cells, they can now investigate the molecular basis of this event in greater detail than has previously been possible.

Animals↗
PubMed1975

RP1 properties and fertility inhibition among P, N, W, and X incompatibility group plasmids.

Incompatibility group P plasmids demonstrate strong entry exclusion properties. Stringent incompatibility is also observed in the absence of entry exclusion. These observations have been facilitated by the study of a nontransmissible plasmid, RP1-S2, derived from RP1 by transductional shortening. RP1-S2 retains carbenicillin and tetracycline resistances as well as loci that cause either the loss of P plasmids (incp) or a locus specifying susceptibility to curing (sinp) in the presence of a P plasmid. RP1-S2 can be mobilized by an incompatibility group W plasmid, R388, and also freely forms recombinants with R388. P, N, and W incompatibility group plasmids all encode information for the receptor of the cell wall-adsorbing phage PRD1. Based on the premise that the location of this receptor is analogous to entry exclusion factors for F-like plasmids and hence a regulated transfer region determinant, we tested fertility inhibition relationships among these plasmid groups. We detected both reciprocal and nonreciprocal fertility inhibition relationships for bacteria containing various combinations of W, N, and P group plasmids. The nonreciprocal nature of some combinations, we believe, reflects the identity of the point mutation reading to derepression of the plasmid in question. Reciprocal fertility inhibition, on the other hand, may reflect the reconstruction of a fertility inhibition system through complementation. An X incompatibility group plasmid, known to affect the fertility of an N group plasmid, was also shown to inhibit P plasmid fertility. These observations may indicate a possible evolutionary relationship(s) of plasmids unrelated by the criteria of incompatibility, pilus phage specificity, or plasmid host range.

Biological Evolution↗
PubMed1986

An inhibitor of SOS induction, specified by a plasmid locus in Escherichia coli.

Plasmid R6-5 contains a locus whose product inhibits induction of sfiA and prophage lambda in a recA441 mutant at 42 degrees C and in a recA+ host after treatment with nalidixic acid. This plasmidic SOS-inhibition locus (psi) is situated on an 8.1-kilobase DNA fragment near oriT, the origin of plasmid R6-5 conjugational transfer. Loss of the Psi function, resulting from the insertion of Tn3 into psi+, greatly reduced the synthesis of two proteins, designated PsiA (Mr 24,500) and PsiB (Mr 12,500). Using host cells in which there was an inactive LexA repressor, we found that Psi function does not act by interfering with the expression of the SOS pathway. The Psi function may affect the generation of an SOS signal. We postulate that during the course of evolution, the Psi function has been selected in some conjugative plasmids so as to permit them to transfer single-stranded DNA without generating an SOS signal.

Biological Evolution↗
PubMed1985

Genes specifying degradation of 3-chlorobenzoic acid in plasmids pAC27 and pJP4.

All of the structural genes for 3-chlorobenzoate degradation are clustered in a 4.2-kilobase (kb) region of plasmid pAC25 (or pAC27) in Pseudomonas putida. An approximate 10-kb DNA segment containing three structural genes for chlorocatechol metabolism present on plasmid pJP4 in Alcaligenes eutrophus shows homology with the above 4.2-kb region of pAC27. In spite of the detectable sequence homology in the structural genes present on both plasmids, the regulation of their expression seems quite different; unlike pAC27, structural rearrangements are prerequisite for efficient expression of the 3-chlorobenzoate genes on plasmid pJP4. Structural features such as stem-loop structures present on plasmid pJP4 are most likely the starting materials for such rearrangements.

Alcaligenes↗
PubMed1992

The chromosomal integration site for the Streptomyces plasmid SLP1 is a functional tRNA(Tyr) gene essential for cell viability.

The genetic element SLP1 exists in nature as a single DNA segment integrated into the genome of Streptomyces coelicolor. Upon mating with Streptomyces lividans, a closely related species, SLP1 undergoes precise excision from its chromosomal site and is transferred into the recipient where it integrates chromosomally. Previous work has shown that integration and excision involve site-specific recombination between a chromosomal site, attB, and a virtually identical sequence, attP, on SLP1. We demonstrate here by means of gene replacement that a tRNA(Tyr) sequence that overlaps part of the attB site of S. lividans is both biologically functional and essential for cell viability. The requirement for this tRNA gene has been used to stabilize the inheritance of a segrationally unstable plasmid in cells lacking a chromosomal attB site. The evolution of an essential DNA locus as an attachment site for a chromosomally integrating genetic element represents a novel mechanism of biological adaptation.

Base Sequence↗
PubMed2006

Conversion of hydroxyphenylpyruvate dioxygenases into hydroxymandelate synthases by directed evolution.

Hydroxymandelate synthase (HmaS) and hydroxyphenylpyruvate dioxygenase (HppD) are non-heme iron-dependent dioxygenases, which share a common substrate and first catalytic step. The catalytic pathways then diverge to yield hydroxymandelate for secondary metabolism, or homogentisate in tyrosine catabolism. To probe the differences between these related active sites that channel a common intermediate down alternative pathways, we attempted to interconvert their activities by directed evolution. HmaS activity was readily introduced to HppD by just two amino acid changes. A parallel attempt to engineer HppD activity in HmaS was unsuccessful, suggesting that homogentisate synthesis places greater chemical and steric demands on the active site.

Catalysis↗
PubMed1997

Mutation for survival.

Adaptive mutations appear in response to selection. In the best-studied system, the two most controversial issues were resolved this year. The mutations are neither Lamarckian nor a peculiarity of bacterial sex, as had been suggested. They occur genome-wide in a hypermutable subpopulation of stressed cells. Genomic 'hot' and 'cold' regions may explain previous failures to detect similar mutations in other systems and at other sites. Stationary phase specific limitation of mismatch repair has also been discovered.

Adaptation, Biological↗
PubMed1994

Nucleotide polymorphism in colicin E1 and Ia plasmids from natural isolates of Escherichia coli.

We examined DNA sequence polymorphism for the colicin gene clusters of seven ColE1 and six ColIa plasmids obtained from natural isolates of Escherichia coli. These gene clusters harbor levels of nucleotide diversity ranging from 0.006 (ColIa) to 0.054 (ColE1). This level of diversity is similar to that observed for chromosomally encoded E. coli genes. However, the variance associated with these estimates is severalfold higher for the plasmid-encoded genes. This increased variance may be due to the differing plasmid population sizes. The pattern of colicin gene cluster polymorphism suggests that the two colicins are evolving in different fashions. ColE1 accumulates polymorphism at an elevated rate in the central domain of the colicin protein, while ColIa polymorphism is distributed evenly along the gene cluster. Comparison of the patterns of divergence between colicin and related proteins of ColIa and Ib and patterns of polymorphism within ColIa suggest that this gene cluster is not evolving in a neutral fashion. These data lend support to the hypothesis that colicin gene clusters may evolve under the influence of diversifying selection.

Bacteriocin Plasmids↗
PubMed1980

The nucleotide sequence of the major beta-globin mRNA from Xenopus laevis.

The nucleotide sequence of a cloned fragment containing an almost complete copy of the mRNA encoding the major adult beta-globin polypeptide in Xenopus laevis, the South African Clawed Toad, is presented. A procedure for strand separation by hybridization to complementary mRNA was used to determine some of the sequence and this technique is described. The complete amino acid sequence of the polypeptide has been deduced and comparison with other vertebrate beta-globins reveals several highly conserved, and therefore potentially important, regions of the protein. The sequence of beta-globin mRNA has been determined in several mammals, and in the chicken. Thus we have searched for conserved regions in the non-coding portions of these mRNA sequences, which encode the same protein, but which have been evolving separately for several hundred million years.

Amino Acid Sequence↗
PubMed2005

A highly sensitive selection method for directed evolution of homing endonucleases.

Homing endonucleases are enzymes that catalyze DNA sequence specific double-strand breaks and can significantly stimulate homologous recombination at these breaks. These enzymes have great potential for applications such as gene correction in gene therapy or gene alteration in systems biology and metabolic engineering. However, homing endonucleases have a limited natural repertoire of target sequences, which severely hamper their applications. Here we report the development of a highly sensitive selection method for the directed evolution of homing endonucleases that couples enzymatic DNA cleavage with the survival of host cells. Using I-SceI as a model homing endonuclease, we have demonstrated that cells with wild-type I-SceI showed a high cell survival rate of 80-100% in the presence of the original I-SceI recognition site, whereas cells without I-SceI showed a survival rate <0.003%. This system should also be readily applicable for directed evolution of other DNA cleavage enzymes.

Deoxyribonucleases, Type II Site-Specific↗
PubMed1988

Frequency-dependent selection in bacterial populations.

There are many situations in which the direction and intensity of natural selection in bacterial populations will depend on the relative frequencies of genotypes. In some cases, this selection will favour rare genotypes and result in the maintenance of genetic variability; this is termed stabilizing frequency-dependent selection. In other cases, selection will only favour genotypes when they are common. Rare types cannot invade and genetic variability will not be maintained; this is known as disruptive frequency-dependent selection. Phage-mediated selection for bacteria with novel restriction-modification systems is frequency-dependent and stabilizing. In mass culture, selection for the production of toxins and allelopathic agents is likely to be frequency-dependent but disruptive. This also occurs in selection favouring genes and transposable elements that cause mutations. Here I review the results of theoretical and experimental studies of stabilizing and disruptive frequency-dependent selection in bacterial populations, and speculate on the importance of this kind of selection in the adaptation and evolution of these organisms and their accessory elements (plasmid, phage and transposons).

Bacteria↗
PubMed2006

Genetic characterization of the phenylacetyl-coenzyme A oxygenase from the aerobic phenylacetic acid degradation pathway of Escherichia coli.

We show here that the paaABCDE genes of the paa cluster responsible for phenylacetate degradation in Escherichia coli W encode a five-component oxygenase that hydroxylates phenylacetyl-coenzyme A (CoA), the first intermediate of the pathway. The primary structure of the subunits of bacterial phenylacetyl-CoA oxygenases revealed that these enzymes constitute the prototype of a new and distinct group of the large bacterial diiron multicomponent oxygenase family.

Acetyl Coenzyme A↗
PubMed1994

Comparative analysis of the replicon regions of eleven ColE2-related plasmids.

The incA gene product of ColE2-P9 and ColE3-CA38 plasmids is an antisense RNA that regulates the production of the plasmid-coded Rep protein essential for replication. The Rep protein specifically binds to the origin and synthesizes a unique primer RNA at the origin. The IncB incompatibility is due to competition for the Rep protein among the origins of the same binding specificity. We localized the regions sufficient for autonomous replication of 15 ColE plasmids related to ColE2-P9 and ColE3-CA38 (ColE2-related plasmids), analyzed their incompatibility properties, and determined the nucleotide sequences of the replicon regions of 9 representative plasmids. The results suggest that all of these plasmids share common mechanisms for initiation of DNA replication and its control. Five IncA specificity types, 4 IncB specificity types, and 9 of the 20 possible combinations of the IncA and IncB types were found. The specificity of interaction of the Rep proteins and the origins might be determined by insertion or deletion of single nucleotides and substitution of several nucleotides at specific sites in the origins and by apparently corresponding insertion or deletion and substitution of amino acid sequences at specific regions in the C-terminal portions of the Rep proteins. For plasmids of four IncA specificity types, the nine-nucleotide sequences at the loop regions of the stem-loop structures of antisense RNAs are identical, suggesting an evolutionary significance of the sequence. The mosaic structures of the replicon regions with homologous and nonhomologous segments suggest that some of them were generated by exchanging functional parts through homologous recombination.

Amino Acid Sequence↗
PubMed2003

Simple sequence repeats and compositional bias in the bipartite Ralstonia solanacearum GMI1000 genome.

BACKGROUND: Ralstonia solanacearum is an important plant pathogen. The genome of R. solananearum GMI1000 is organised into two replicons (a 3.7-Mb chromosome and a 2.1-Mb megaplasmid) and this bipartite genome structure is characteristic for most R. solanacearum strains. To determine whether the megaplasmid was acquired via recent horizontal gene transfer or is part of an ancestral single chromosome, we compared the abundance, distribution and composition of simple sequence repeats (SSRs) between both replicons and also compared the respective compositional biases. RESULTS: Our data show that both replicons are very similar in respect to distribution and composition of SSRs and presence of compositional biases. Minor variations in SSR and compositional biases observed may be attributable to minor differences in gene expression and regulation of gene expression or can be attributed to the small sample numbers observed. CONCLUSIONS: The observed similarities indicate that both replicons have shared a similar evolutionary history and thus suggest that the megaplasmid was not recently acquired from other organisms by lateral gene transfer but is a part of an ancestral R. solanacearum chromosome.

Base Composition↗