Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria.

The phenotypic diversity of about 200 bacterial strains isolated from soil was compared with the genotypic diversity of the same population. The strains were phenotypically characterized by the API 20B test system. The results of these tests were subjected to cluster analysis, which revealed 41 biotypes at 80% similarity. The five dominating biotypes contained 43% of the strains. The phenotypic diversity as determined by the Shannon index, equitability, rarefaction, and cumulative differences was high, but indicated some dominant biotypes. The genetic diversity was measured by reassociation of mixtures of denatured DNA isolated from the bacterial strains (C0t plots). The observed genetic diversity was high. Reassociation of DNA from all bacterial strains together revealed that the population contained heterologous DNA equivalent to 20 totally different bacterial genomes (i.e., genomes that have no homology). This study showed that reassociation of DNA isolated from a collection of bacteria gave a good estimate of the diversity of the collection and that there was good agreement with different phenotypic diversity measures. The Shannon index in particular has features in common with the genetic diversity measure presented here.     

Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria

The phenotypic diversity of about 200 bacterial strains isolated from soil was compared with the genotypic diversity of the same population. The strains were phenotypically characterized by the API 20B test system. The results of these tests were subjected to cluster analysis, which revealed 41 biotypes at 80% similarity. The five dominating biotypes contained 43% of the strains. The phenotypic diversity as determined by the Shannon index, equitability, rarefaction, and cumulative differences was high, but indicated some dominant biotypes. The genetic diversity was measured by reassociation of mixtures of denatured DNA isolated from the bacterial strains (C0t plots). The observed genetic diversity was high. Reassociation of DNA from all bacterial strains together revealed that the population contained heterologous DNA equivalent to 20 totally different bacterial genomes (i.e., genomes that have no homology). This study showed that reassociation of DNA isolated from a collection of bacteria gave a good estimate of the diversity of the collection and that there was good agreement with different phenotypic diversity measures. The Shannon index in particular has features in common with the genetic diversity measure presented here.     

Genetic heterogeneity in Streptococcus mutans

The genetic homogeneity among eight cariogenic strains of Streptococcus mutans was assessed by deoxyribonucleic acid (DNA)-DNA reassociation experiments. DNA species were extracted from strains GS5, Ingbritt, 10449, FAl, BHT, E49, SLl, and KlR. Labeled DNA ((14)C-DNA) was extracted from strains 10449, FAl, and SLl. Denatured (14)C-DNA fragments were allowed to reassociate, i.e., form hybrid duplexes, with denatured DNA immobilized on membrane filters incubated in 0.45 m NaCl-0.045 m sodium citrate at 67 or 75 C. At 67 C, 10449 (14)C-DNA reassociated extensively only with GS5 and Ingbritt DNA. FAl (14)C-DNA hybridized extensively only with BHT DNA, and SLl (14)C-DNA reassociated with KlR and E49 DNA. DNA which hybridized extensively at 67 C also reassociated to a high degree at 75 C. Thermal elution of (14)C-FAl-BHT duplexes showed that the hybrid duplexes were thermostable. The results indicate that S. mutans is a genetically heterogeneous species. The strains studied can be divided into three (possibly four) genetic groups, and these groups closely parallel antigenic groups.     

Decreased resistance to antibiotics and plasmid loss in plasmid-carrying strains of Staphylococcus aureus treated with ascorbic acid

The effect of ascorbic acid on plasmid-coded antibiotic resistance in Staphylococcus aureus was investigated. Several strains of S. aureus were cultured in the presence of 1 mM ascorbate for 6 h. This treatment induced an increased loss of resistance markers in 4 of 6 strains tested, and agarose gel electrophoresis showed this disappearance of plasmid DNA in ascorbate-induced susceptible colonies. The presence of ascorbate induced a 50-75% decrease in minimal inhibitory concentrations of different antibiotics for resistant strains. When ascorbate is added, formerly subinhibitory concentrations of penicillin or tetracycline have an increased inhibitory effect on resistant strains and even induced the death of 25-93% of the initial population. These results suggest that ascorbate can induce the loss of several plasmids of S. aureus, and that the levels of antibiotic resistance are also affected by the presence of this compound.     

Cloning and sequence determination of six Staphylococcus aureus beta-lactamases and their expression in Escherichia coli and Staphylococcus aureus

The plasmid-encoded beta-lactamase genes of six strains of Staphylococcus aureus were cloned and shown to be expressed in Escherichia coli. The cloned genes were re-introduced into S. aureus via a shuttle vector, and expressed beta-lactamase. However, clones containing only the small amount of DNA found necessary for expression of ampicillin resistance in E. coli did not express beta-lactamase in S. aureus. Much larger pieces of DNA from the original plasmid were necessary to obtain expression in S. aureus. Some of the six strains of S. aureus synthesized beta-lactamase constitutively and some released only a small proportion of the enzyme into the medium. Both these characteristics were maintained in the clones so it is concluded that they are features either of the gene itself or of the surrounding DNA. The cloned genes were sequenced and the putative amino acid sequences of the beta-lactamases were compared. There are several differences between the sequences and in particular one change in the N-terminal region, at a position believed to be especially important for export of proteins from the cell, is thought to have a key effect on whether or not the enzyme is found in the medium.     

Molecular analyses of conjugative, gentamicin-resistance plasmids from staphylococcal clinical isolates

Gentamicin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains which were isolated from infants with staphylococcal bacteremia were analyzed for the presence of self-transmissible gentamicin-resistance (Gmr) plasmids. Conjugative GMr plasmids of approximately 43.8-63 kilobases (kb) were found in all S. aureus strains. Inter- and intra-species transfer of Gmr plasmids by conjugation was observed from S. aureus to S. aureus and to S. epidermidis recipient strains. However, neither inter- nor intra-species transfer of gentamicin resistance by conjugation was observed with nine out of nine S. epidermidis donor strains which were mated with either S. epidermidis or S. aureus recipient strains. These conjugative Gmr plasmids were unable to comobilize a smaller (15-kb) plasmid present in all but two S. aureus clinical isolates. Many of the conjugative Gmr plasmids also carried genetic determinants for kanamycin, tobramycin, neomycin, and ethidium bromide resistance, and for beta-lactamase synthesis. EcoRI restriction endonuclease digests of the S. aureus Gmr conjugative plasmids revealed three different digestion patterns. Four EcoRI restriction endonuclease digestion fragments of 15, 11.4, 6.3, and 4.6 kb in size were common to all plasmids. These plasmids and conjugative Gmr staphylococcal plasmids from other geographical regions shared restriction digestion fragments of similar molecular weights. DNA hybridization with biotinylated S. aureus plasmid pIZ7814 DNA revealed a high degree of homology among these plasmids. A 50.9-kb plasmid from one of the nonconjugative S. epidermidis clinical isolates showed homology with the probe DNA but lacked a portion of a 6.3-kb fragment which was present in all conjugative plasmids and believed to carry much genetic information for conjugation.     

Absence of Circular Plasmid Deoxyribonucleic Acid Attributable to a Genetic Determinant for Methicillin Resistance in Staphylococcus aureus

Plasmid deoxyribonucleic acid was not detected by centrifugal analysis of lysates of penicillinase-negative strains of Staphylococcus aureus harboring a determinant of methicillin resistance derived from strain Villaluz. When these strains contained a penicillinase plasmid, the plasmid deoxyribonucleic acid of methicillin-resistant and methicillin-susceptible strains was indistinguishable by the methods employed. The results indicate that the genetic determinant for methicillin resistance in the strains examined was not associated with a circular plasmid comparable to those that have been shown to determine resistance to benzylpenicillin, tetracycline, and chloramphenicol in S. aureus.     

Genomic Relatedness of Xanthomonas campestris Strains Causing Diseases of Citrus

Xanthomonas campestris strains that cause disease in citrus were compared by restriction endonuclease analysis of DNA fragments separated by pulsed-field gel electrophoresis and by DNA reassociation. Strains of X. campestris pv. citrumelo, which cause citrus bacterial spot, were, on average, 88% related to each other by DNA reassociation, although these strains exhibited diverse restriction digest patterns. In contrast, strains of X. campestris pv. citri groups A and B, which cause canker A and canker B, respectively, had relatively homogeneous restriction digest patterns. The groups of strains causing these three different citrus diseases were examined by DNA reassociation and were found to be from 55 to 63% related to one another. Several pathovars of X. campestris, previously shown to cause weakly aggressive symptoms on citrus, ranged from 83 to 90% similar to X. campestris pv. citrumelo by DNA reassociation. The type strain of X. campestris pv. campestris ranged from 30 to 40% similar in DNA reassociation experiments to strains of X. campestris pv. citrumelo and X. campestris pv. citri groups A and B. Whereas DNA reassociation quantified the difference between relatively unrelated groups of bacterial strains, restriction endonuclease analysis distinguished between closely related strains.     

Rapid isolation and sequencing of purified plasmid DNA from Bacillus subtilis.

We report two methods for isolation of plasmid DNA from the gram-positive bacterium Bacillus subtilis. The protoplast alkaline lysis procedure was developed for general use, and the protoplast alkaline lysis magic procedure was developed for isolation of DNA for sequencing. Both procedures yielded large amounts of high-quality DNA in less than 1 h, while current protocols require 4 to 7 h to perform and give lower yields and quality. Plasmid DNA was obtained from strains containing either high- or low-copy-number plasmids. In addition, the procedures were easily adapted to yield large amounts of plasmid DNA suitable for sequencing from another gram-positive organism, Staphylococcus aureus. Further, we demonstrated that neither chloramphenicol, used for plasmid selection, nor the mutation recE4 reduced plasmid DNA yield from the strains we examined.     

Heterogeneity of deoxyribonucleic acid molecules isolated from Mycobacterium smegmatis.

Bulk chromosomal deoxyribonucleic acids (DNAs) of Mycobacterium smegmatis strains 607+ (wild type) and 607-1 (Strr) and orange-red pigmented variants (OR) were separated into two distinct bands (types 1 and 2) by cesium chloride density gradient centrifugation. Thermal denaturation analyses showed that type 1 and 2 DNA fragments of these strains possessed guanine plus cytosine contents averaging 69.2% and 60.8%, respectively. Type 1 and 2 DNAs from all strains tested were recovered in relatively equal quantities upon isolation and were found to have similar molecular weights (3.0 x 10(7)). Spectrophotometric assay of DNA reassociation showed that homology between any type 1 and 2 DNA fragments was always very low (29 to 33%), even within the same strain. Homologies among type 1 DNAs isolated from any strain were always high (92 to 98%), whereas homologies between type 2 DNA isolated from OR strains and that from their parental strain 607-1 were lower (51 to 55%). Transformation experiments revealed that methionine, leucine, folic acid, and streptomycin markers were found exclusively in type 1 DNA fragments. In addition to the two types of chromosomal DNA, plasmid DNA possessing a molecular weight of about 4 x 10(6) was found in strain 607-1.     

Plasmid required for virulence of Agrobacterium tumefaciens.

The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37 C is shown to be due to loss of a large plasmid (1.2 X 10-8 daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6 is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 X A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.     

Insertion of foreign DNA into plasmids from gram-positive bacteria induces formation of high-molecular-weight plasmid multimers.

Plasmids pUB110, pC194, pE194, and pT181 are commonly used as cloning vectors in both Bacillus subtilis and Staphylococcus aureus. We report that insertion of foreign DNA into any of these plasmids results in the generation of high-molecular-weight plasmid multimers (HMW) of the recombinant, present as tandem head-to-tail copies. HMW was detected in wild-type B. subtilis and S. aureus strains. The production of HMW depended on the nature of the DNA insertion. Inserts of Escherichia coli DNA, e.g., pBR322 or pUC18, resulted in large amounts of HMW, whereas some inserts of S. aureus DNA of the same size had no effect on plasmid profile. The generation of HMW depended on the mode of plasmid replication; plasmids which replicate via a single-stranded DNA intermediate produced HMW upon foreign DNA insertion, whereas plasmid pAM beta 1, which does not generate single-stranded DNA, did not generate HMW. We propose that HMW is a product of imparied termination of rolling-circle replication and that the impairment is due to the nature of the DNA insertion.     

Comparative effect of microwaves and boiling on the denaturation of DNA

The effect of heat and microwave denaturation of small volumes of double-stranded plasmid DNA has been compared. Samples of intact plasmid DNA had plasmid DNA linearized by digestion with EcoRI were conventionally denatured in a boiling water bath or denatured by 2450 MHz of microwave energy for 0-300 s. Heat denaturation for periods longer than 120 s caused breakdown of linearized plasmid DNA; however, microwave denaturation for 10-300 s caused no apparent degradation of linearized DNA. Breakdown of DNA forms II and III was noted in plasmid DNA subjected to 300 s of either heat or microwave denaturation but breakdown of forms II and III occurred more quickly with heat than with microwave treatment. Microwave treatment was also found to be better than heat to denature 32P-labeled DNA probes subsequently used to detect homologous DNA samples immobilized on nitrocellulose filters. A microwave-treated 32P-labeled DNA probe was able to hybridize to DNA samples 20 times more dilute than a heat-treated 32P-labeled DNA probe. Depending on the form of DNA to be analyzed, these results indicate that small volumes of DNA solutions and radiolabeled DNA probes can be effectively denatured in a conventional microwave oven.     

Co-Transduction of Plasmids Mediating Resistance to Tetracycline and Chloramphenicol in Staphylococcus aureus

Independent plasmids mediating resistance either to tetracycline (Tc) or chloramphenicol (Cm) were transduced successively into Staphylococcus aureus strain 8325. From this doubly resistant donor strain, Tc was co-transduced with a frequency of 40 to 50% when Cm was selected. Co-transduction of Cm was 5 to 10% with Tc selection. Plasmid elimination was infrequent and restricted to the Cm plasmid. A variant, doubly resistant strain gave 100% co-transduction of Tc and Cm and a high rate of joint elimination of both plasmid markers. Co-transduction of the plasmids from recombination-deficient donor strains was much reduced if the plasmids had been introduced separately into the donor strain, but occurred at the normal high rate if they had been introduced jointly. The plasmids were co-transformed at relatively low rates with closed circular deoxyribonucleic acid (DNA) from doubly resistant donors, but not with DNA from a mixed lysate of singly resistant strains. Our evidence favored a hypothesis of recombination-dependent, reversible linkage between the two plasmids as the basis for their co-transduction. Examination of plasmid DNA from the doubly resistant strains by ultracentrifugal and electron microscopic methods did not disclose any physical differences between singly and doubly resistant strains that might account for the observed co-transduction.     

Differentiation of metaxylem cell line in the root ofAllium cepa

Summary The first step of differentiation in the root segments ofAllium cepa containing metaxylem cells in different stages of differentiation were studied by DNA reassociation curves and compared to meristem cell extracted DNA. Upon sonication of DNA samples to about 400 base pairs, the reassociation profiles of the heat denatured DNA, were spectrophotometrically followed at two different concentrations. The kinetic complexities,i.e., the number of base pairs per haploid genome of a given sequence and its redundancy were calculated. Differences were found at the level of highly and medium repetitive sequences, thus demonstrating that some DNA reassociation classes may undergo amplification during root development.     

Plasmids of Staphylococcus aureus associated with live and processed poultry

A series of 23 strains of Staphylococcus aureus originally isolated from processed poultry was screened for the presence of plasmids. Plasmids were more common in strains of Staph. aureus characteristically associated with live poultry than with strains endemic in poultry plants and strains of human origin. Two plasmids with sizes of 1·65 and 18·2 kilobase pairs (kBp) were present in three strains considered typical of Staph. aureus forma specialis 'altilis' and two plasmids with sizes of 1·65 and 17 kBp were present in three of four strains of Staph. aureus var. gallinae. A 1·65 kBp plasmid was present in all seven strains of these poultry biotypes and in three of 14 'endemic' strains. All the 1·65 kBp plasmids were shown by blot hybridization to share sequence homology. There was also some sequence homology between the 18·2 kBp and 17 kBp plasmids. These results were supported by restriction enzyme digest analyses. A study of cured derivatives of strain PS221 f.sp. 'altilis' suggested that the 18·2 kBp plasmid encoded the genetic determinant(s) responsible for caseolysis. Both the 1·65 and the 18·2 kBp plasmids also exerted an effect on the production of acid from lactose. In no other characteristic did cured strains resemble the plasmid-free 'endemic' strains. This was therefore consistent with the notion that the genetic determinants associated with the cultural characteristics of endemic strains are chromosomally located.     

Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis

Evidence is presented that crown gall tumors are caused by the incorporation of part of a virulence plasmid carried by the inciting bacterium, Agrobacterium tumefaciens. The rate of reassociation of labeled plasmid DNA was slightly accelerated in the presence of tobacco crown gall tumor DNA, but not normal tobacco DNA. Treatment of tumor DNA with DNAase abolished the acceleration. To determine whether all plasmid sequences are represented in tumor DNA, the labeled plasmid DNA was separated into specific fragments after digestion with restriction endonuclease Sma I. Renaturation rates for DNA from bands 1, 2, 7, 8, 9, 12 and 14 were not affected by tumor DNA. DNA from band 3 showed a slight rate increase in the presence of tumor DNA, indicating 21–27 copies of 14–18% of the DNA sequences in this (doublet) band. The band 3 doublet was separated by electrophoresis into bands 3a and 3b. Tumor DNA had little effect on the rate of reassociation of labeled band 3a DNA. Band 3b DNA renatured rapidly in the presence of tumor DNA, and its rate increase indicated that approximately 18 copies of 40% of band 3b DNA sequences are present per diploid tumor cell. This amounts to 3.7 × 10⁶ daltons of foreign genetic information and represents a contribution of 0.0011% to the DNA content of the tumor cell. The relationship between this plant tumor and virally induced animal tumor systems is discussed.     

Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones.

The norA gene cloned from chromosomal DNA of quinolone-resistant Staphylococcus aureus TK2566 conferred relatively high resistance to hydrophilic quinolones such as norfloxacin, enoxacin, ofloxacin, and ciprofloxacin, but only low or no resistance at all to hydrophobic ones such as nalidixic acid, oxolinic acid, and sparfloxacin in S. aureus and Escherichia coli. The 2.7-kb DNA fragment containing the norA gene had a long open reading frame coding for 388 amino acid residues with a molecular weight of 42,265, which was consistent with the experimental value of about 49,000 obtained on DNA-directed translation. The deduced NorA polypeptide has 12 hydrophobic membrane-spanning regions and is partly homologous to tetracycline resistance protein and sugar transport proteins. The uptake of a hydrophilic quinolone, enoxacin, by S. aureus harboring a plasmid carrying the norA gene was about 50% that by the parent strain lacking the plasmid, but it increased to almost the same level as that by the latter strain with carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, the uptake of a hydrophobic quinolone, sparfloxacin, was similar in the two strains. These results suggest that the NorA polypeptide may constitute a membrane-associated active efflux pump of hydrophilic quinolones.     

Characteristics and distribution of plasmids in a clonally diverse set of methicillin-resistant Staphylococcus aureus strains

The aim of this study was to compare the plasmid contents of methicillin-resistant Staphylococcus aureus (MRSA) strains classified into different clonal clusters (CCs). The isolates were collected from 15 Czech hospitals in 2000-2008. Plasmid DNA was detected in 65 (89%) strains, and 33 of them harbored more than one plasmid type. Altogether 24 different types of plasmids were identified, ranging in size from 1.3 to 55 kb. Restriction endonuclease analysis, plasmid elimination, DNA hybridization, and sequencing were used for their further characterization. It has been found that the conjugative, erythromycin resistance and enterotoxin D encoding plasmids are harbored by strains from different CCs. On the other hand, chloramphenicol and tetracycline resistance plasmids, and most of the penicillinase and cryptic plasmids were only detected in certain CCs. Especially, the pUSA300-like plasmids were found exclusively in the USA300 clone strains. The high diversity in plasmid content detected in the study strains implies that plasmids play a major role in evolution of MRSA clonal lineages.