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PubMed1994

Variation of clonal, mesquite-associated rhizobial and bradyrhizobial populations from surface and deep soils by symbiotic gene region restriction fragment length polymorphism and plasmid profile analysis.

Genetic characteristics of 14 Rhizobium and 9 Bradyrhizobium mesquite (Prosopis glandulosa)-nodulating strains isolated from surface (0- to 0.5-m) and deep (4- to 6-m) rooting zones were determined in order to examine the hypothesis that surface- and deep-soil symbiont populations were related but had become genetically distinct during adaptation to contrasting soil conditions. To examine genetic diversity, Southern blots of PstI-digested genomic DNA were sequentially hybridized with the nodDABC region of Rhizobium meliloti, the Klebsiella pneumoniae nifHDK region encoding nitrogenase structural genes, and the chromosome-localized ndvB region of R. meliloti. Plasmid profile and host plant nodulation assays were also made. Isolates from mesquite nodulated beans and cowpeas but not alfalfa, clover, or soybeans. Mesquite was nodulated by diverse species of symbionts (R. meliloti, Rhizobium leguminosarum bv. phaseoli, and Parasponia bradyrhizobia). There were no differences within the groups of mesquite-associated rhizobia or bradyrhizobia in cross-inoculation response. The ndvB hybridization results showed the greatest genetic diversity among rhizobial strains. The pattern of ndvB-hybridizing fragments suggested that surface and deep strains were clonally related, but groups of related strains from each soil depth could be distinguished. Less variation was found with nifHDK and nodDABC probes. Large plasmids (>1,500 kb) were observed in all rhizobia and some bradyrhizobia. Profiles of plasmids of less than 1,000 kb were related to the soil depth and the genus of the symbiont. We suggest that interacting selection pressures for symbiotic competence and free-living survival, coupled with soil conditions that restrict genetic exchange between surface and deep-soil populations, led to the observed patterns of genetic diversity.

Journal Article↗
PubMed1992

Use of genomic fingerprinting in the characterization of Neisseria gonorrhoeae isolated in Valencia, Spain.

Ninety-five Neisseria gonorrhoeae organisms isolated in Valencia, Spain, were characterized by antibiotic sensitivity testing, auxotyping, serotyping, plasmid analysis and restriction endonuclease fingerprinting (HindIII digestion). Cluster analysis of the restriction patterns revealed that 31 isolates (32.6%) formed 12 clearly defined clusters. Penicillinase-producing Neisseria gonorrhoeae strains formed four of these groups. Eight groups of gonococcal strains were identified by auxotyping, although 83% of isolates belonged to two auxotypes (Proto, Pro-). Twenty-three different serovars were identified by serotyping. The serovar pattern IB/rop was found in 38% of isolates. A 60% coincidence was found between gonococcal groupings obtained by combination of auxotyping, serotyping and plasmid analysis and those obtained with the restriction enzyme fingerprinting technique. The specificity of enzyme restriction patterns of Neisseria gonorrhoeae is confirmed to be of practical importance in the epidemiologic study of gonorrhoea.

Bacterial Typing Techniques↗
PubMed1997

Salmonella serogroups identified from adult diarrhoeal out-patients in Addis Ababa, Ethiopia: antibiotic resistance and plasmid profile analysis.

Forty five Salmonella strains were isolated from 700 diarrhoeal samples collected from adult diarrhoeal out-patients in Addis Ababa. Among the isolates, serogroup C comprised 31.1%, B 24.4%, S.typhi 15.6%, D 13.3%, A 8.9% and E 6.7%. Among the isolates, 71.1% were resistant to tetracycline, 68.9% to ampicillin, 66.7% to cephalothin, 57.8% to trimethoprim-sulphamethoxazole, 53.9% to kanamycin, 46.7% to chloramphenicol and less than 31.8% of the isolates were resistant to other drugs. Among S.typhi isolates, 28.6% were resistant to chloramphenicol and this shows the emergence of chloramphenicol resistant S.typhi strains in Ethiopia. Gentamicin and polymyxin B were found to be the drugs of choice for cases of salmonellosis including S.typhi. All drug resistant isolates analysed for plasmids contained multiple plasmids ranging in sizes from 1.8 to greater than 21 Kilobases.

Adolescent↗
PubMedDate not supplied

Evaluation of coagulase-negative staphylococcal isolates from serial nasopharyngeal cultures of premature infants.

The present study was undertaken to determine whether very low birth weight infants in a neonatal intensive care unit became colonized with virulent strains of coagulase-negative staphylococci (C-S) over time (i.e., those characterized as Staphylococcus epidermidis, slime positive, and/or multiply antibiotic resistant), and if so, whether the initial colonizing strains developed these characteristics or whether the strains themselves changed. Nasopharyngeal (NP) cultures were obtained weekly on 28 very low birth weight (less than 1750 g) infants hospitalized for a mean of 8 wk (range 4-15 wk). There were 105 isolates of C-S recovered from 96 cultures that were characterized by species, biotype, antibiotic susceptibility pattern, and slime production (screening parameters). Isolates from the same infant with highly similar screening parameters then underwent phage typing and plasmid analysis to increase the likelihood of establishing strain identity. C-S colonization rose from 12% on admission to 75% by wk 2, then gradually declined to 30% by wk 6 and remained stable through wk 10. There were no significant differences among C-S isolates from wk 1 compared with wk 10 of hospital stay with respect to distribution of species, slime positive, or multiply antibiotic resistant strains. One biotype of S. epidermidis was recovered from 46% of study infants, but only one infant was colonized with a predominant biotype of S. epidermidis throughout hospitalization. Thirteen pairs of isolates recovered from 12 of the infants on two or more wk were found to be identical by phage typing and plasmid analysis. Only seven of these 13 pairs of isolates had concordant results for all the screening parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Typing Techniques↗
PubMed1994

A framework for IS200, 16S rRNA gene and plasmid-profile analysis in Salmonella serogroup D1.

Chromosomal fingerprinting of the type strains of serotypes of Salmonella O-serogroup D1 with the DNA insertion sequence IS200 generated patterns which were either serotype-specific (e.g., Typhi), or conserved among groups of related serotypes (e.g., Dublin, Rostock and certain phage types of Enteritidis). The number of IS200 copies varied considerably, and the IS200 patterns of type strains of serotypes associated with systemic infections in man were specific and suitable for identifying strains within those serotypes. Polymorphism at 16S rRNA gene loci was examined among type strains and 11 16S rRNA gene profiles were characterised. The most prevalent of these was conserved among type strains of 11 serotypes, and the next most prevalent among type strains of nine serotypes; together, they encompassed 15 unique IS200 profiles. The distribution and mol. wts of plasmids carrying spvBC (virulence) genes could be directly related to certain chromosomal genotypes defined by IS200 patterns. The presence of virulence plasmids in serotypes Lomalinda, Antarctica and Wangata is reported for the first time. Comparison of 16S rRNA gene profiles and IS200 patterns provides a definition of genotype that is applicable to epidemiological studies of various group D1 serotypes and should prove particularly useful for those lacking plasmid DNA.

Blotting, Southern↗
PubMed2001

Quantitative plasmid mixture analysis using the fluorogenic 5'-nuclease polymerase chain reaction assay.

The fluorogenic 5'-nuclease polymerase chain reaction (PCR) assay has been shown to be useful for quantifying a given DNA target in a sample. Here we show how an existing PCR protocol can be amended for quantification by incorporating distinctive dual-labeled, sequence-specific oligonucleotide probes and resulting in a two- to threefold broader and more reliable dynamic range than that of conventional end-point analysis of PCR products. Moreover, we show a multiplex situation in which two targets, one normal and one mutated, can be amplified and quantified simultaneously and in the same reaction tube. Use of this novel approach for quantitative PCR applications eliminates the need for post-PCR processing and has clinical- and research-based diagnostic applications, particularly for measuring levels of mutations in a mixture.

Base Sequence↗
PubMedDate not supplied

[Molecular epidemiology of Salmonella enterica poisoning: correlation of the serotype and protein profile and plasmid DNA analysis].

Food-borne diseases nosocomial outbreaks due to Salmonella enterica strains have a high incidence nowadays. We describe here an outbreak that occurred in July 1990 in the Malaga University Hospital, that only involves one shift of health care workers. Salmonella enterica was detected in stool samples from HCW and a first analysis revealed the presence of two different antibiotic susceptibility patterns (resistotypes) among isolated strains. Two months after the outbreak started, the CNVIS (Majadahonda, Spain) confirmed the presence of two different serotypes (bovismorbificans and enteritidis). The delayed availability of the results (due to the lack of specific sera needed in most of the clinical Laboratories) was responsible for finishing only a partial and restricted epidemiologic study in terms of source identification for one particular serotype as well as for the antimicrobial resistance studies. The molecular identification techniques, particularly the electrophoretic protein profile could overcome these problems.

Antigens, Bacterial↗
PubMed1995

Molecular epidemiology of enterococci with high-level resistance to aminoglycosides.

DNA-based methodologies are considerably more powerful than other phenotype-based typing systems, providing a finer level of epidemiological discrimination, differentiating both closely and distantly related independent isolates that otherwise may appear as identical. In this study, plasmid analysis and pulsed-field gel electrophoresis were used to compare 28 isolates of Enterococci (respectively 13 strains of Enterococcus faecalis and 15 strains of Enterococcus faecium) with high-level resistance to aminoglycosides, isolated in Catania (Italy). Plasmid profile analysis resolved 20 different patterns among 24 plasmid harboring strains; many isolates showed one or two plasmids of the same size, but different plasmid content. Analysis of the PFGE-based RFLP patterns after SmaI digestion of genomic DNA resolved 26 different clones from 28 isolates: particularly, it resolved two different clones from three isolates showing identical plasmid profiles, and it identified as a single clone two isolates exhibiting different plasmid profiles. Thus, on the basis of our PFGE-based RFLP analysis data, we concluded that all the strains included in the study were genetically unrelated with two exceptions.

Aminoglycosides↗
PubMed1999

Codon optimization effect on translational efficiency of DNA vaccine in mammalian cells: analysis of plasmid DNA encoding a CTL epitope derived from microorganisms.

Interspecific difference of codon usage is one of the major obstacles for effective induction of specific immune responses against bacteria and protozoa by DNA immunization. Using genes encoding major histocompatibility complex class I-restricted cytotoxic T-lymphocyte (CTL) epitopes, derived from an intracellular bacterium, Listeria monocytogenes and a mouse malaria parasite, Plasmodium yoelii, we report here that the codon optimization level of the genes is not precisely proportional to, but does correlate well with the translational efficiency in mammalian cells, which is concomitantly associated with the induction level of specific CTL response in the mouse. These results suggest that DNA immunization using the gene codon-optimized to mammals through the entire region is very effective.

Animals↗
PubMed1997

Sequence analysis of plasmid pCC5.2 from cyanobacterium Synechocystis PCC 6803 that replicates by a rolling circle mechanism.

The cyanobacterium Synechocystis sp. strain PCC 6803 contains several cryptic plasmids of 2.4, 5.2, and about 50 or 100 kbp. The complete nucleotide sequence of the 5.2-kbp plasmid, pCC5.2, has been analyzed and is reported here. This plasmid contains 5214 bp and 53.1% A+T. Six open reading frames, ORFs A-F (encoding peptides of larger than 90 amino acid residues), were located on both strands of pCC5.2. ORF B codes for a potential replication protein containing 971 amino acids, for which there are three homologous proteins encoded by other cyanobacterial plasmids. ORF C encodes a polypeptide of 93 amino acids which shares homologies with products of two ORFs found in the protein database. Counterparts of the products of ORF A, D, E, and F could not be found in the protein database. Detection of a single-stranded DNA intermediate during replication of pCC5.2 indicates that this plasmid may also replicate by a rolling circle mechanism, as has been reported for pCA2.4 and pCB2.4 from the same strain of Synechocystis (PCC 6803).

Amino Acid Sequence↗
PubMed1998

Sequence analysis of plasmid pTF5, a 19.8-kb geographically widespread member of the Thiobacillus ferrooxidans pTFI91-like plasmid family.

The most important features of plasmid pTF5, a 19,793-bp plasmid that was isolated from Thiobacillus ferrooxidans ATCC33020, are presented. The plasmid contained at least 14 complete open reading frames (ORFs), most of which had clear amino acid sequence similarity to previously identified proteins. The majority of ORFs were related to proteins commonly found on plasmids such as replication-, partition-, and stability-associated proteins or on transposons such as transposases, an invertase, and a resolvase. Products of three of the ORFs were related to redox-active proteins and possibly constitute an electron transport system. Plasmids with restriction endonuclease maps identical to that of pTF5 have been widely reported among T. ferrooxidans strains, and in this study, pTF5 was itself shown to be a member of the widely distributed pTFI91 T. ferrooxidans plasmid family. A comparison of restriction endonuclease maps indicated that a pTFI91-like plasmid (9.8 kb) appeared to be contained entirely within pTF5.

Amino Acid Sequence↗
PubMed1999

Structural analysis of plasmid pLQ510 from Moraxella catarrhalis E22.

The complete nucleotide sequence of plasmid pLQ510 from Moraxella catarrhalis strain E22 has been determined. This plasmid contained 12,082 bp with 38% GC content. Five open reading frames that encoded predicted proteins with homology to plasmid-encoded proteins from other bacteria were identified. A putative origin of replication that contained an AT-rich region followed by four direct repeats and an inverted repeat was identified.

Amino Acid Sequence↗
PubMed1994

Sequence of the cellular T-DNA in the untransformed genome of Nicotiana glauca that is homologous to ORFs 13 and 14 of the Ri plasmid and analysis of its expression in genetic tumours of N. glauca x N. langsdorffii.

A region homologous to the TL-DNA of Agrobacterium rhizogenes was previously detected in the genome of untransformed Nicotiana glauca and designated cellular T-DNA (cT-DNA). Subsequently, part of this region was sequenced and two genes, which corresponded to rolB and rolC and were named NgrolB and NgrolC, were found. We have now sequenced a region of the cT-DNA other than the region that includes NgrolB and C and we have found two other open reading frames (ORFs), NgORF13 and NgORF14. These ORFs correspond to ORFs 13 and 14 of the TL-DNA of A. rhizogenes and exhibit a high degree of homology to these ORFs, without having a nonsense codon. We have not found any sequence homologous to rolD (ORF15). The two genes, NgORF13 and 14, as well as the NgrolB and C genes, are expressed in genetic tumors of hybrids between N. glauca and N. langsdorffii but not in leaf tissues of the hybrid.

Amino Acid Sequence↗
PubMedDate not supplied

Osmophilic linear plasmids from the salt-tolerant yeast Debaryomyces hansenii.

Three novel linear plasmids, pDHL1 (8.4 kb), pDHL2 (9.2 kb) and pDHL3 (15.0 kb), were discovered in the halophilic (salt-tolerant) yeast Debaryomyces hansenii. Exonuclease treatment indicated that all three plasmids were blocked at their 5' ends, presumably, by analogy with most other eukaryotic linear plasmids which involved protein attachment. The Debaryomyces plasmids were entirely cured simply by growing cells in normal culture medium, but were stably maintained in culture medium containing salts, sorbitol or glycerol at suitable concentrations. This suggested that the pDHL plasmids required an osmotic pressure for stable replication and maintenance. The Debaryomyces yeast secreted a killer toxin against various yeasts species. Toxin activity was demonstrated only in the presence of salts such as NaCl or KCl, but this killer phenotype was not associated with the pDHL plasmids. Analysis of the plasmid-curing pattern suggested that pDHL3 may play a key role in the replication of the Debaryomyces plasmids. Southern hybridization showed that an extensive homology exists between specific regions of pDHL1 and pDHL2, whereas pDHL3 is unique.

Blotting, Southern↗
PubMed1991

Replication analysis of plasmid DNAs injected into Drosophila embryos.

From a "shotgun" collection of DNA fragments, isolated from Drosophila melanogaster, we selected sequences that function as autonomously replicating sequences (ARS) in the yeast Saccharomyces cerevisiae. To investigate the replicative potential of such sequences in Drosophila, five of these ARS elements and also the Adh gene of D. melanogaster, which has been described earlier to have ARS function in yeast, were microinjected into developing Drosophila eggs and analysed after reisolation from first instar larvae. As an assay for DNA replication, we determined the sensitivity of recovered plasmid DNA to restriction enzymes that discriminate between adenine methylation and non-methylation. Within the limits of detection our results show that none of the plasmids replicated two or more rounds. However, a fraction of all injected plasmid DNAs, including vector DNA, seems to replicate once. The same result was obtained for a DNA sequence from mouse that had been reported to have replication origin function in mouse tissue culture cells. We excluded the possibility that methylation of the plasmids is the reason for their inability to replicate. These results demonstrate that homologous and heterologous DNA sequences that drive replication of plasmids in cells of other species are not sufficient to fulfil this function in Drosophila embryos.

Animals↗
PubMed1991

Biochemical and biophysical analysis of plasmid pMJ600-encoded tellurite [TeO2(3-)] resistance.

Escherichia coli AB1157, and the transconjugant AB1157 (pMJ600), were used to study the mechanism of tellurite resistance conferred by pMJ600, which contains the tellurite resistance determinant cloned from the IncHI-2 conjugative plasmid pMER610. The transconjugant can tolerate a 100-fold higher concentration of potassium tellurite [K2TeO3] than the plasmid-free strain. Both strains were found to accumulate tellurite irreversibly at equivalent rates, with elemental tellurium being deposited intracellularly, direct efflux of tellurite was not found to contribute to the resistance mechanism. However, under these conditions, growth, protein synthesis and oxygen uptake ceased in AB1157, but-were unaffected in the transconjugant. No NADH- or NADPH-linked reduction of tellurite was detected in crude cell extracts of either strain; however, cell extracts of both reduced tellurite at alkaline pH in the absence of any co-factors.

Drug Resistance, Microbial↗
PubMed2007

Analysis of plasmids encoding extended-spectrum beta-lactamases (ESBLs) from Escherichia coli isolated from non-hospitalised patients in Seville.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli is an emerging pathogen in the community. We investigated the specific types of ESBLs in E. coli isolates from non-hospitalised patients and the plasmids harbouring these ESBLs. Forty-eight consecutive ESBL-producing E. coli were studied. All the isolates were clonally unrelated. ESBLs were characterised by sequencing. Mobility of the undigested plasmids was investigated both by conventional electrophoresis and by pulsed-field gel electrophoresis. Southern restriction fragment length polymorphism (RFLP) experiments were carried out by digesting plasmids with EcoRI and HindIII. CTX-M-14 was the most frequent ESBL (28 isolates), followed by SHV-12 (16 isolates). Plasmids encoding CTX-M-14 appeared homogeneous both in hybridisation profile and mobility. In contrast, plasmids expressing SHV-12 were more varied in size and hybridisation patterns. Our data suggest that a single type or very closely related plasmids harbouring CTX-M-14 are spreading in the community among unrelated E. coli isolates in our area.

Blotting, Southern↗