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PubMed1998

Plasmid (1952-1997).

The term "plasmid" was introduced 45 years ago (J. Lederberg, 1952, Physiol. Rev. 32, 403-430) as a generic term for any extrachromosomal genetic particle. It was intended to clarify the classification of agents that had been thought of disjunctively as parasites, symbionts, organelles, or genes. For a decade or more it was confused with "episome," although that was carefully crafted (F. Jacob and E. L. Wollman, 1958, C. R. Acad. Sci. 247, 154-156) to mean agents with traffic in and out of chromosomes. Starting about 1970, plasmids became important reagents in molecular genetic research and biotechnology. They also play a cardinal role in the evolution of microbial resistance and of pathogenicity. The usage of the term has then escalated to its current peak of about 3000 published articles per year. The bedrock of genetic mechanism is no longer mitosis and meiosis of chromosomes; it is template-directed DNA assembly. This is often more readily studied and managed with the use of plasmids, which replicate autonomously outside the chromosomes. Some plasmids are also episomes, namely, they interact with the chromosomal genome, and other mobile elements may be transposed from one chromosomal locus to another without replicating autonomously.

Animals↗
PubMed1998

Analysis of two Staphylococcus epidermidis plasmids coding for resistance to streptogramin A.

The two Staphylococcus epidermidis plasmids pIP1629 (7.5 kb) and pIP1630 (14.4 kb) contain the vga gene conferring resistance to streptogramin A. All the sequences of pIP1629, except two of the four 22-nt iterons preceding the replication gene, were found in pIP1630. The additional 6.9-kb fragment of pIP1630 is similar to the mobilizable S. epidermidis plasmid pSK639, carrying the dfrA-thyE-orf140 operon and thought to replicate by an iteron controlled theta-type replication mechanism. The replication-mobilization elements of pIP1629 and pSK639 are very similar despite having been isolated in France and in Australia, respectively, showing that they are geographically widely dispersed in S. epidermidis. The gene thyE encoding thymidylate synthetase carried by pSK639 is not present in pIP1630. pIP1630 probably arose by the recombination of two homologous plasmids carrying distinct resistance determinants.

Australia↗
PubMed1986

Microbial metabolism of chlorosalicylates: accelerated evolution by natural genetic exchange.

Methylsalicylate-grown cells of Pseudomonas sp. WR401 cometabolized 3-, 4- and 5-substituted halosalicylates to the corresponding halocatechols. Further degradation was unproductive due to the presence of high levels of catechol 2,3-dioxygenase. This strain acquired the ability to utilize 3-chlorobenzoate following acquisition of genes from Pseudomonas sp. B13 which are necessary for the assimilation of chlorocatechols. This derivative (WR4011) was unable to use 4- or 5-chlorosalicylates. Derivatives able to use these compounds were obtained by plating WR4011 on 5-chlorosalicylate minimal medium; one such derivative was designated WR4016. The acquisition of this property was accompanied by concomitant loss of the methylsalicylate phenotype. During growth on 4- or 5-chlorosalicylate the typical enzymes of chlorocatechol assimilation were detected in cell free extracts, whereas catechol 2,3-dioxygenase activity was not induced. Repeated subcultivation of WR4016 in the presence of 3-chlorosalicylate produced variants (WR4016-1) which grew on all three isomers.

Biodegradation, Environmental↗
PubMed1996

Identification of a partition and replication region in the Alcaligenes eutrophus megaplasmid pMOL28.

A 4.64 kb region of the 180 kb heavy metal resistance plasmid pMOL28 of Alcaligenes eutrophus CH34, previously shown to be able to replicate autonomously, was sequenced and analyzed. Three genes involved in plasmid maintenance were identified: parA28 and parB28 are involved in plasmid partitioning and stability, while repA28 encodes a protein required for replication. In addition to the par AB28 genes, a third locus, parS28, required in cis active partitioning was identified. The parABS28 locus of pMOL28 shows strong similarity in organization to the sop, par and rep regions, respectively, of the Escherichia coli F-factor, the E.coli P1 and P7 prophages and the Agrobacterium pTiB6S3 and pRiA4b plasmids. The ParAB28 proteins of pMOL28 also show similarity to the proteins encoded by two conserved open reading frames present in the replication regions of the Pseudomonas putida and Bacillus subtilis chromosomes. The functionality of the pMOL28 par region was examined by performing stability and incompatibility tests between pMOL28 and pMOL846 or pMOL850 which contain the 4.64 EcoRI replicon fragment of pMOL28, cloned in opposite orientations into pSUP202, which is itself unable to replicate in A. eutrophus. The RepA2 8 replication protein showed similarity to the RepL protein of P1, which is required for lytic replication of this E. coli phage. The replication origin of pMOL28, oriV28, seems to be located within the repA28 coding region, and pMOL28 replication may depend on transcriptional activation of oriV28.

Alcaligenes↗
PubMed1996

Relationships between bacterial drug resistance pumps and other transport proteins.

We have used three reference sequences representative of bacterial drug resistance pumps and sugar transport proteins to collect the 91 most closely related sequences from a composite, nonredundant protein sequence database. Having eliminated certain very close relatives, the remainder were subjected to analysis and alignment by using two different similarity matrices: one of these was a matrix based on structural conservation of amino acid residues in proteins of known conformation and the other was based on the more familiar mutational matrix. Unrooted similarity trees for these proteins were constructed for each matrix and compared. A systematic analysis of the differences between these trees was undertaken and the sequences were analyzed for the presence or absence of certain sequence motifs. The results show that the clades created by the two methods are broadly comparable but that there are some clusters of sequences that are significantly different. Further analysis confirmed that (1) the sequences collected by this objective method are all known or putative 12-helix (in some cases reported as 14-helix) transmembrane proteins, (2) there is evidence for few cases of an origin based on gene duplication, (3) the bacterial drug resistance pumps are distributed in more than one clade and cannot be regarded as a definitive subset of these proteins, and that (4) the diversity is such that there is no evidence of a single ancestral protein. The possible extension of the methods to other cases of divergent protein sequences is discussed.

Amino Acid Sequence↗
PubMed1999

Class 1 integrons, gene cassettes, mobility, and epidemiology.

Integrons are genetic elements that, although unable to move themselves, contain gene cassettes that can be mobilized to other integrons or to secondary sites in the bacterial genome. The majority of approximately 60 known gene cassettes encode resistance to antibiotics. Recently, a number of gene cassettes encoding extended-spectrum beta-lactamases or carbapenemases have been described. Up to at least five cassettes may be present in an integron, which leads to multiresistance. Frequently, more than one integron is observed within the same bacterial cell. Integrons are widespread in their species distribution. Although integrons are normally reported from Enterobacteriaceae and other gram-negative bacteria, an integron has been described in Corynebacterium glutamicum, a gram-positive species. The gene cassette in this integron showed even higher expression when compared to the expression in Escherichia coli. Integrons have been reported from all continents and are found frequently. The widespread occurrence of integrons is thought to be due to their association with transposon plasmids, conjugative plasmids, or both. Integrons form an important source for the spread of antibiotic resistance, at least in gram-negative bacteria but also potentially in gram-positive bacteria. The aim of this review is to describe the versatility of integrons, especially their mobility and their ability to collect resistance genes.

Animals↗
PubMed2000

Dynamics of dominance of a dipeptide insertion in reverse transcriptase of HIV-1 from patients subjected to prolonged therapy.

A small proportion (0.8%) of individuals of a cohort of HIV-1 infected patients subjected to prolonged therapy with nucleoside analogues included a recently recognised dipeptide insertion in their RT (Ser-Ser or Ser-Gly between RT codons 69 and 70). To study the dynamics of dominance of genomes with this genetic change, sequential HIV-1 isolates from two patients were analyzed with regard to consensus sequences and complexity of mutant spectra. The two patients displayed completely different, complex evolutionary patterns leading to temporary dominance of dipeptide insertions. In one patient, a virus very closely related to an ancestor virus from the same patient overtook the population at late times, displacing genomes encoding a Ser-Ser insertion. In another patient the sequential dominance of genomes with Ser-Ser insertion-->no insertion-->Ser-Gly insertion was observed. These three types of genomes coexisted in the mutant spectrum of one HIV-1 isolate. Complexity was also reflected in the shape of phylogenetic trees derived with genomes from the mutant spectrum at each time point. The results suggest that HIV-1 genomes encoding a dipeptide insertion between RT codons 69 and 70 do not show a clear selective advantage over other genomes lacking the insertion. Such an absence of a clear selective advantage will favor that such genomes encoding this RT insertion become dominant only in a transient fashion, and following disparate kinetics in different patients.

Adult↗
PubMed2000

Horizontal gene transfer and the origin of species: lessons from bacteria.

In bacteria, horizontal gene transfer (HGT) is widely recognized as the mechanism responsible for the widespread distribution of antibiotic resistance genes, gene clusters encoding biodegradative pathways and pathogenicity determinants. We propose that HGT is also responsible for speciation and sub-speciation in bacteria, and that HGT mechanisms exist in eukaryotes.

Bacteria↗
PubMed1998

A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications.

BACKGROUND: Glutathione S-transferases (GSTs) are a multifunctional group of enzymes, widely distributed in aerobic organisms, that have a critical role in the cellular detoxification process. Unlike their mammalian counterparts, bacterial GSTs often catalyze quite specific reactions, suggesting that their roles in bacteria might be different. The GST from Proteus mirabilis (PmGST B1-1) is known to bind certain antibiotics tightly and reduce the antimicrobial activity of beta-lactam drugs. Hence, bacterial GSTs may play a part in bacterial resistance towards antibiotics and are the subject of intense interest. RESULTS: Here we present the structure of a bacterial GST, PmGST B1-1, which has been determined from two different crystal forms. The enzyme adopts the canonical GST fold although it shares less than 20% sequence identity with GSTs from higher organisms. The most surprising aspect of the structure is the observation that the substrate, glutathione, is covalently bound to Cys 10 of the enzyme. In addition, the highly structurally conserved N-terminal domain is found to have an additional beta strand. CONCLUSIONS: The crystal structure of PmGST B1-1 has highlighted the importance of a cysteine residue in the catalytic cycle. Sequence analyses suggest that a number of other GSTs share this property, leading us to propose a new class of GSTs - the beta class. The data suggest that the in vivo role of the beta class GSTs could be as metabolic or redox enzymes rather than conjugating enzymes. Compelling evidence is presented that the theta class of GSTs evolved from an ancestral member of the thioredoxin superfamily.

Amino Acid Sequence↗
PubMed2000

Lateral gene transfer and the nature of bacterial innovation.

Unlike eukaryotes, which evolve principally through the modification of existing genetic information, bacteria have obtained a significant proportion of their genetic diversity through the acquisition of sequences from distantly related organisms. Horizontal gene transfer produces extremely dynamic genomes in which substantial amounts of DNA are introduced into and deleted from the chromosome. These lateral transfers have effectively changed the ecological and pathogenic character of bacterial species.

Bacteria↗
PubMed2000

Micronutrients and infection: interactions and implications with enteric and other infections and future priorities.

Symposium presentations have focused on the elegant molecular science and the biologic mechanisms by which micronutrients play critical roles in cellular and humoral immune responses, cellular signaling and function, and even in the evolution of microbial virulence. The concluding session examined the practical issues of how best to evaluate the nutritionally at-risk host, especially in the areas of greatest need-an analytical model of nutrient-immune interactions, implications of nutritional modulation of the immune response for disease, and the implications for international research and child health. This overview illustrated how malnutrition may be a major consequence of early childhood diarrhea and enteric infections, as enteric infections may critically impair intestinal absorptive function with potential long-term consequences for growth and development. The potentially huge, largely undefined DALY (disability-adjusted life years) impact of early childhood diarrheal illnesses demonstrates the importance of quantifying the long-term functional impact of largely preventable nutritional and infectious diseases, especially in children in developing areas.

Child↗
PubMed1999

Increased polymerase fidelity of lamivudine-resistant HIV-1 variants does not limit their evolutionary potential.

OBJECTIVE: Anti HIV-1 therapy with nucleoside reverse transcriptase inhibitors can select for drug-resistant reverse transcriptase variants with altered enzyme properties. Some of the mutations, e.g. Met184Val and Met184Ile, result in an increase in polymerase fidelity of the enzyme as measured in biochemical assays; however, the effect of such changes on the fidelity during viral replication is largely unknown. In this study, the codon 184 variants were used to investigate whether the mutation at codon 184 affects the mutation spectrum and mutation rate of the mutant viruses. DESIGN AND METHOD: In vitro selection experiments with either wild-type or lamivudine-resistant viruses (Met184Val and Met184Ile) were performed using a protease inhibitor as the selective drug. In addition, a novel selection approach was developed using a mixture of viruses, instead of individual viruses, during the selection process. RESULTS: Comparison of a total of 108 protease-resistant variants revealed no significant difference in the mutational spectrum of the wild-type and the lamivudine-resistant variants. In addition, the selection experiments with the viral mixtures demonstrated no delay in the kinetics of mutation generation in response to an antiviral drug. CONCLUSION: This study demonstrates that the Met184Val and Met184Ile mutations in the HIV-1 reverse transcriptase enzyme do not significantly affect the evolutionary potential of the corresponding viruses.

Codon↗
PubMed2025

Pseudoalteromonas is a novel symbiont of marine invertebrates that exhibits broad patterns of phylosymbiosis.

Despite growing insights into the composition of marine invertebrate microbiomes, our understanding of their ecological and evolutionary patterns remains poor, owing to limited sampling depth and low-resolution datasets. Previous studies have provided mixed results when evaluating patterns of phylosymbiosis between marine invertebrates and marine bacteria. Here, we investigated potential animal-microbe symbioses in Pseudoalteromonas, an overlooked bacterial genus consistently identified as a core microbiome taxon in diverse invertebrates. Using a pangenomic analysis of 236 free-living and invertebrate-associated bacterial strains (including two new nematode-associated isolates generated in this study), we confirm that Pseudoalteromonas is a novel symbiont with substantial evidence of phylosymbiosis across at least three marine invertebrate phyla (e.g., Nematoda, Mollusca, and Cnidaria). Patterns of symbiosis were consistent irrespective of geography (including in Antarctica), with FISH images from nematodes indicating that bacterial symbionts form biofilms in the mouth and esophagus. The evolutionary history of Pseudoalteromonas is marked by substantial host-switching and lifestyle transitions, and host-associated genomes suggest that these bacteria are facultative symbionts involved in nutritional mutualisms. In marine environments, we hypothesize that horizontally-acquired symbionts may have co-evolved with invertebrates, using host mucus as a physical niche and food source, while providing their animal hosts with Vitamin B, amino acids, and bioavailable carbon compounds in return.

Marine Invertebrates↗
PubMed1997

Mismatch repair protein MutL becomes limiting during stationary-phase mutation.

Postsynthesis mismatch repair is an important contributor to mutation avoidance and genomic stability in bacteria, yeast, and humans. Regulation of its activity would allow organisms to regulate their ability to evolve. That mismatch repair might be down-regulated in stationary-phase Escherichia coli was suggested by the sequence spectrum of some stationary-phase ("adaptive") mutations and by the observations that MutS and MutH levels decline during stationary phase. We report that overproduction of MutL inhibits mutation in stationary phase but not during growth. MutS overproduction has no such effect, and MutL overproduction does not prevent stationary-phase decline of either MutS or MutH. These results imply that MutS and MutH decline to levels appropriate for the decreased DNA synthesis in stationary phase, whereas functional MutL is limiting for mismatch repair specifically during stationary phase. Modulation of mutation rate and genetic stability in response to environmental or developmental cues, such as stationary phase and stress, could be important in evolution, development, microbial pathogenicity, and the origins of cancer.

Adenosine Triphosphatases↗
PubMed1997

Promoting changes in clinical practice. Treatment time and outcome studies in a Danish public child dental health clinic.

This paper examines the characteristics of the process of innovation as applied to provision of dental health to children. The data are from a demonstration project conducted in a Danish public child dental health clinic to evaluate the appropriateness and effectiveness of an individualized and alternative, mainly non-operative caries treatment strategy that was implemented in 1987. The aim of the strategy was to avoid operative intervention and sealants, and was based on three principles: caries is a localized disease; intensive training in home-based treatment, and individualized risk assessments and controls in conjunction with professional plaque removal. The major influence on the innovation process appeared to be personal education of the personnel, active participation in research, and feedback from clinical observations. In 1992 an additional feedback measure was introduced addressing the question: how much time does the individual child spend on treatment during one year? Mean total treatment time varied from 12 min 1- to 4-yr-olds to 44-58 min for 7- to 17-yr-olds. About 60% of the total treatment time was used on non-operative caries treatment (diagnosis, risk assessment, plaque removal, toothbrushing training) and about 25% on operative caries treatment for 3- to 17-yr-olds. The 1-2-yr-olds required only non-operative treatment. The new treatment strategy reduced operative procedures by 60%. The paper introduces an easily applicable measure of treatment outcome in terms of teeth that have not been restored or sealed as a result of regular professional treatment or care. The main conclusion drawn is that the dominant moving forces in the innovation process were professional responsibility and self-adjustment by a constant learning process involving clinical feedback measures in conjunction with modern understanding of caries as a localized disease caused by local evolution of microbial cariogenic biofilms.

Adolescent↗
PubMed1997

Identification and molecular genetic analysis of multiple loci contributing to high-level tellurite resistance in Rhodobacter sphaeroides 2.4.1.

The ability of the facultative photoheterotroph Rhodobacter sphaeroides to tolerate and reduce high levels of tellurite in addition to at least 10 other rare earth metal oxides and oxyanions has considerable potential for detoxification and bioremediation of contaminated environments. We report the identification and characterization of two loci involved in high-level tellurite resistance. The first locus contains four genes, two of which, trgAB, confer increased tellurite resistance when introduced into the related bacterium Paracoccus denitrificans. The trgAB-derived products display no significant homology to known proteins, but both are likely to be membrane-associated proteins. Immediately downstream of trgB, the cysK (cysteine synthase) and orf323 genes were identified. Disruption of the cysK gene resulted in decreased tellurite resistance in R. sphaeroides, confirming earlier observations on the importance of cysteine metabolism for high-level tellurite resistance. The second locus identified is represented by the telA gene, which is separated from trgAB by 115 kb. The telA gene product is 65% similar to the product of the klaB (telA) gene from the tellurite-resistance-encoding kilA operon from plasmid RK2. The genes immediately linked to the R. sphaeroides telA gene have no similarity to other components of the kilA operon. R. sphaeroides telA could not functionally substitute for the plasmid RK2 telA gene, indicating substantial functional divergence between the two gene products. However, inactivation of R. sphaeroides telA resulted in a significant decrease in tellurite resistance compared to the wild-type strain. Both cysK and telA null mutations readily gave rise to suppressors, suggesting that the phenomenon of high-level tellurite resistance in R. sphaeroides is complex and other, as yet uncharacterized, loci may be involved.

Amino Acid Sequence↗