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At least 289 records · Page 16Linked to original sources
PubMed2006

Design of an Escherichia coli system for whole cell mediated steroid synthesis and molecular evolution of steroid hydroxylases.

The 15beta-hydroxylase (CYP106A2) from Bacillus megaterium, one of the few bacterial steroid hydroxylases, which has been isolated and characterized so far, catalyses the 15beta-hydroxylation of a variety of steroids. The enzyme can be supported in its activity with adrenodoxin (Adx) and adrenodoxin reductase (AdR) from bovine adrenals, supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. This three-component electron transfer chain was implemented in Escherichia coli by coexpression of the corresponding coding sequences from two plasmids, containing different selection markers and compatible origins of replication. The cDNAs of AdR and Adx on the first plasmid were separated by a ribosome binding sequence, with the reductase preceding the ferredoxin. The second plasmid for CYP106A2 expression was constructed with all features necessary for a molecular evolution approach. The transformed bacteria show the inducible ability to efficiently convert 11-deoxycorticosterone (DOC) to 15beta-DOC at an average rate of 1 mM/d in culture volumes of 300 ml. The steroid conversion system was downscaled to the microtiter plate format and a robot set-up was developed for a fluorescence-based conversion assay as well as a CO difference spectroscopy assay, which enables the screening for enzyme variants with higher activity and stability.

Adrenodoxin↗
PubMed2002

Plasmids spread very fast in heterogeneous bacterial communities.

Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes.

Bacteria↗
PubMedDate not supplied

Primary structure of a chloramphenicol acetyltransferase specified by R plasmids.

Naturally occurring isolates of chloramphenicol-resistant bacteria commonly synthesise chloramphenicol acetyltransferase (EC 2.3.28; CAT) in amounts which are sufficient to account for the resistance phenotype and often harbour plasmids which carry the structural gene for CAT. The findings of CAT in such diverse prokaryotes as Proteus mirabilis, Agrobacterium tumefaciens, Streptomyces sp., and a soil Flavobacterium has led to speculation concerning the origin and evolution of the more commonly observed CAT variants specified by plasmids in clinically important bacteria. To provide a more solid basis for studying the evolution and spread of CAT within prokaryotes we chose to determine the complete amino acid sequence of a type I variant of CAT, the variant known to be associated with most F-like plasmids conferring chloramphenicol resistance. The sequence has been determined by combining the results obtained from manual and automated sequential degradation with those obtained by mass spectrometry of peptides generated by enzymatic digestion. The directly determined primary structure is identical with that predicted by the DNA sequence analysis of the chloramphenicol resistance transponson Tn9 known to specify a type I variant of chloramphenicol acetyltransferase.

Acetyltransferases↗
PubMed2000

Phylogeny of the replication regions of pPT23A-like plasmids from Pseudomonas syringae.

It was previously shown that most Pseudomonas syringae strains contain one or more plasmids with cross-hybridizing replication regions and other areas of homology, and these plasmids were designated the pPT23A-like family. The majority of these plasmids encode genes conferring epiphytic fitness or resistance to antibacterial compounds and those investigated in this study are essential for pathogenicity or increased virulence. The phylogeny of 14 pPT23A-like plasmids from five P. syringae pathovars was studied by comparing a fragment of the sequence of their repA genes (encoding a replicase essential for replication). In the phylogenetic tree obtained, four groups (< or =88.8% identity between their members) could be identified. The first group contained the plasmids from three P. syringae pv. tomato strains, a P. syringae pv. apii strain and five out of the seven P. syringae pv. syringae strains, with identity ranging between 88.8 and 100%. The clustering of the pv. syringae strains did not reflect host specialization or previously reported phylogenetic relationships. The second group contained the plasmids from two strains of pv. glycinea and pv. tomato (95.5% identity), and it also included the previously sequenced replicon of a pathogenicity plasmid from P. syringae pv. phaseolicola. The plasmids from the remaining two pv. syringae strains were distantly related to the other plasmid sequences. Hybridization experiments using different genes or transposable elements previously described as plasmid-borne in P. syringae, showed that the gene content of highly related plasmids could be dissimilar, suggesting the occurrence of major plasmid reorganizations. Additionally, the phylogeny of the different native plasmids did not always correlate with the phylogeny of their harbouring strains, as determined by the analysis of extragenic repetitive consensus (ERIC) and arbitrarily primed PCR (AP-PCR) products. Collectively, these results suggest that pPT23A-like plasmids were, in most cases, acquired early during evolution.

Base Sequence↗
PubMed2003

Characterization of blaCMY-10 a novel, plasmid-encoded AmpC-type beta-lactamase gene in a clinical isolate of Enterobacter aerogenes.

AIMS: We report the description of a novel plasmid-encoded AmpC beta-lactamase gene (blaCMY-10) from Enterobacter aerogenes K9911729 that was isolated from a patient suffering from pneumonia in South Korea. METHODS AND RESULTS: Using antibiotic susceptibility testing, plasmid analysis, transconjugation and Southern blot analysis, the cefoxitin resistance phenotype reflects the presence of a large plasmid [pYMG-1 (130 kb)] in Ent. aerogenes K9911729. One beta-lactamase with the pI of 8.0 from transconjugant of Ent. aerogenes K9911729 was identified by isoelectric focusing on a gel. A 1475 bp DNA fragment containing the blaCMY-10 gene, identified on pYMG-1 of Ent. aerogenes K9911729, was sequenced and an open reading frame coding for 382 amino acid, CMY-10, was found. The 37 class C beta-lactamases were subclassified into 1a to 1j and CMY-10 into 1a by phylogenetic analysis. A sequence identical to the common regions in In6, In7 and a novel integron from pSAL-1 was found upstream from blaCMY-10 gene at nucleotide 1-71. CONCLUSIONS: These results clearly show that blaCMY-10 gene belongs to the group of ampC-related bla genes. Homology analysis among AmpC enzymes or ampC genes implied that integration of the chromosomal ampC gene into a large resident plasmid, followed by transconjugation, was involved in the evolution of blaCMY-10 gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The first identification of the blaCMY-10 gene is of concern as chromosomal beta-lactamases may cause serious therapeutic problems if their genes are translocated onto plasmids.

Base Sequence↗
PubMed1984

Inhibition of polyoma gene expression in transformed mouse cells by hypermethylation.

The evolution of mouse cells transformed by a recombinant plasmid containing the genome of the tsA mutant of polyoma virus (Py) cloned at the BamHI site into the plasmid pML, whose sequences therefore interrupt the Py late region, has been studied. Clones of transformed cells were selected at 39 degrees (nonpermissive temperature for large T antigen). Under these conditions viral DNA integration is stable and the cells display a uniformed transformed phenotype. Also studied in detail was the evolution of one of these cell lines (A4) upon shift to a temperature permissive for large T-Ag function (33 degrees); immediately after shift, 90% of the population became intensely positive for T-Ag and a considerable amount of free-viral DNA was produced, accompanied by a clear cytopathic effect. Surviving cells proliferated actively after 4 weeks at 33 degrees and showed a decreased expression of large T-Ag (only 2-3% of the population was T-Ag positive by immunofluorescence), a drastic reduction in the amount of free-viral DNA produced, but no apparent change in the pattern of integration of Py DNA in the host chromosomes. Analysis of the high-molecular-weight DNA with the restriction enzymes HpaII and MspI revealed that the cytosines in the recognition sequences of these enzymes were methylated. Accordingly, treating the cells with 5-Azacytidine, a methylation inhibitor, results in the expression of viral T-Ags in more than 80% of the cell population. Analysis of DNA transcription revealed a dramatic reduction of virus-specific poly(A)+ mRNA in the methylated cells; in addition, the phenotype of the 33 degrees A4 populations was much less transformed than that of the original cultures. The block of Py expression by methylation is not complete; approximately 2% of the cells remain T-Ag positive and viral transcription is not completely suppressed. This could be explained by an incomplete methylation which randomly leaves unmethylated sequences essential for Py gene expression, or by the fact that methylation is not sufficient to block transcription completely. Possible mechanisms underlying this type of evolution are discussed.

Animals↗
PubMed1982

Sarcomeric myosin heavy chain is coded by a highly conserved multigene family.

pMHC25, a recombinant plasmid containing myosin heavy chain (MHC) cDNA sequences from differentiated myotubes of the L6E9 rat cell line, has been shown to hybridize to all sarcomeric MHC mRNAs so far tested but not to nonsarcomeric MHC mRNAs. In addition, pMHC25 hybridizes to multiple restriction endonuclease-digested fragments of rat genomic DNA corresponding to different MHC genomic sequences. Thus, the MHC gene represented by pMHC25 is a member of a sarcomeric MHC multigene family that has regions of sequence homology shared among its members. This sarcomeric MHC multigene family has been estimated to be composed of a minimum of seven genes, some of which are polymorphic in the rat. We have also determined that pMHC25 hybridizes to MHC gene sequences in genomic DNA of all species that have striated muscle, ranging from nematodes to man. Sarcomeric MHC genes, therefore, have been horizontally and vertically conserved in evolution. Additionally, we have used the pMHC25 plasmid to demonstrate that MHC genes do not undergo rearrangement or amplification during muscle cell differentiation.

Animals↗
PubMed1996

[Comparative genetics of representatives of the genus Yersinia and evolution of their pathogenicity].

This paper was prepared on the basis of a report presented at the first Congress of the All-Russian Society of Geneticists and Breeders (December 1994, Saratov, Russia). Analysis of published data and the authors' own investigations enabled them to propose a concept of evolution of pathogenicity within the Yersinia genus. They concluded that phenotypic features conferred by yersinia plasmids should be considered taxonomic traits.

Biological Evolution↗
PubMed2006

Plasmids in the aphid endosymbiont Buchnera aphidicola with the smallest genomes. A puzzling evolutionary story.

Buchnera aphidicola, the primary endosymbiont of aphids, has undergone important genomic and biochemical changes as an adaptation to intracellular life. The most important structural changes include a drastic genome reduction and the amplification of genes encoding key enzymes for the biosynthesis of amino acids by their translocation to plasmids. Molecular characterization through different aphid subfamilies has revealed that the genes involved in leucine and tryptophan biosynthesis show a variable fate, since they can be located on plasmids or on the chromosome in different lineages. This versatility contrasts with the genomic stasis found in three distantly related B. aphidicola strains already sequenced. We present the analysis of three B. aphidicola strains (BTg, BCt and BCc) belonging to aphids from different tribes of the subfamily Lachninae, that was estimated to harbour the bacteria with the smallest genomes. The presence of both leucine and tryptophan plasmids in BTg, a chimerical leucine-tryptophan plasmid in BCt, and only a leucine plasmid in BCc, indicates the existence of many recombination events in a recA minus bacterium. In addition, these B. aphidicola plasmids are the simplest described in this species, indicating that plasmids are also involved in the genome shrinkage process.

Amino Acids↗
PubMed1980

Plasmids.

Explore the source record for details and available documents.

Bacillus↗
PubMed2026

Genomic surveillance reveals escalating antimicrobial resistance and plasmid diversity in clinical Salmonella 1,4,[5],12:i:- ST34 isolates from Guizhou Province, China.

INTRODUCTION: Salmonella 1,4,[5],12:i:- ST34 has emerged as a significant public health issue due to its association with various antimicrobial resistance genes (ARGs) and transferable plasmids. However, its genomic characteristics and potential influence on public health in Guizhou have not been comprehensively assessed. METHODS: From 2019 to 2023, a 5-year surveillance was conducted in nine cities (prefectures) of Guizhou Province. We integrated phenotypic and genomic analyses of 281 clinical Salmonella 1,4,[5],12:i:- ST34 isolates to investigate the prevalence of ARGs and plasmids and to analyze the molecular epidemiology and evolution. RESULTS: The isolates exhibited resistance to first-line antibiotics, with 22.4% for ciprofloxacin, 11.4% for azithromycin, 18.5% for ceftazidime, and 39.1% for cefotaxime. ARGs showed substantial agreement with phenotypes for tetracycline, macrolides, third-generation cephalosporins (3GCs), carbapenems, and colistin (80.8-100.0% consistency; Kappa: 0.50-1.00). Plasmid analysis identified IncQ1 (84.3%) and IncHI2/IncHI2A (26.3%) as the main replicons, with the variety of plasmid replicons increasing from 7 to 21 over the 5 years. ARGs associated with resistance to critically important antibiotics (CIAs) were frequently predicted to be located on plasmid-associated contigs, with significant associations observed between IncHI2/IncHI2A plasmids and ARGs conferring resistance to fluoroquinolones, macrolides, and cephalosporins (P < 0.05). Molecular typing divided 281 isolates into 37 cgSTs, with cgST52428 being the most common. Molecular epidemiological analysis revealed that Guizhou isolates primarily clustered together, sharing close genetic ties with those from Sichuan and Guangdong, and exhibited the highest genetic similarity to pork-derived isolates. Phylogenetic analysis revealed clustering of CIA-resistant ARGs and plasmids in Clades 4 and 5, with a significant association between IncHI2/IncHI2A plasmids and CIA-resistant ARGs (&#x3c7;2 = 112.12, P < 0.001). Additionally, class 1 integron was associated with higher ARG burdens, while virulence-associated genes were conserved and predominantly chromosome-associated. Gene-content analysis revealed that isolates in Clades 4 and 5 harbored the largest mean gene complements, and cgST52428 isolates also harbored the largest among dominant cgSTs. DISCUSSION: This study presents a comprehensive genomic profile of Salmonella 1,4,[5],12:i:- ST34 in Guizhou, providing essential data for exploring the resistance characteristics and investigating the molecular epidemiology of Salmonella 1,4,[5],12:i:-.

ST34↗
PubMed2000

Paradigms of plasmid organization.

Plasmids are extrachromosomal elements built from a selection of generally quite well understood survival and propagation functions, including replication, partitioning, multimer resolution, post-segregational killing and conjugative transfer. Evolution has favoured clustering of these modules to form plasmid cores or backbones. Co-regulation of these core genes can also provide advantages that favour retention of the backbone organization. Tumour-inducing and symbiosis-determining plasmids appear to co-regulate replication and transfer in response to cell density, both being stimulated at high density. Broad-host-range plasmids of the IncP-1 group, on the other hand, have autogenous control circuits, which allow a burst of expression during establishment in a new host, but a minimum of expression during maintenance. The lessons that plasmids have for clustering and co-regulation may explain the logic and organization of many small bacterial genomes currently being investigated.

DNA Replication↗
PubMed2005

Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.

Prokaryotes contain short DN repeats known as CRISPR, recognizable by the regular spacing existing between the recurring units. They represent the most widely distributed family of repeats among prokaryotic genomes suggesting a biological function. The origin of the intervening sequences, at present unknown, could provide clues about their biological activities. Here we show that CRISPR spacers derive from preexisting sequences, either chromosomal or within transmissible genetic elements such as bacteriophages and conjugative plasmids. Remarkably, these extrachromosomal elements fail to infect the specific spacer-carrier strain, implying a relationship between CRISPR and immunity against targeted DNA. Bacteriophages and conjugative plasmids are involved in prokaryotic population control, evolution, and pathogenicity. All these biological traits could be influenced by the presence of specific spacers. CRISPR loci can be visualized as mosaics of a repeated unit, separated by sequences at some time present elsewhere in the cell.

Base Sequence↗
PubMed1987

Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains two genes which encode cyclic AMP (cAMP) phosphodiesterase. We previously isolated and characterized PDE2, which encodes a high-affinity cAMP phosphodiesterase. We have now isolated the PDE1 gene of S. cerevisiae, which encodes a low-affinity cAMP phosphodiesterase. These two genes represent highly divergent branches in the evolution of phosphodiesterases. High-copy-number plasmids containing either PDE1 or PDE2 can reverse the growth arrest defects of yeast cells carrying the RAS2(Val-19) mutation. PDE1 and PDE2 appear to account for the aggregate cAMP phosphodiesterase activity of S. cerevisiae. Disruption of both PDE genes results in a phenotype which resembles that induced by the RAS2(Val-19) mutation. pde1- pde2- ras1- ras2- cells are viable.

3',5'-Cyclic-AMP Phosphodiesterases↗