Evidence for a plasmid in a methanogenic bacterium.

Among 15 strains of methanogens, one plasmid, pMP1, was identified in the new coccoid isolate PL-12/M. It could not be detected in the cleared lysate, but it was detected in the viscous pellet. The plasmid had a molecular weight of ca. 4.6 x 10(6). A restriction enzyme cleavage map of the cloned plasmid was derived.     

Megaplasmids encode differing combinations of lantibiotics in Streptococcus salivarius

Streptococcus salivarius strains commonly produce bacteriocins as putative anticompetitor or signalling molecules. Here we report that bacteriocin production by the oral probiotic strain S. salivarius K12 is encoded by a large (ca. 190 kb) plasmid. Oral cavity transmission of the plasmid from strain K12 to a plasmid-negative variant of this bacterium was demonstrated in two subjects. Tests of additional S. salivarius strains showed large (up to ca. 220 kb) plasmids present in bacteriocin-producing isolates. Various combinations (up to 3 per plasmid) of loci encoding the known streptococcal lantibiotics salivaricin A, salivaricin B, streptin and SA-FF22 were localised to these plasmids. Since all bacteriocin-producing strains of S. salivarius tested to date appear to harbour plasmids, it appears that they may function as mobile repositories for bacteriocin loci, especially those of the lantibiotic class.     

E. coli phosphofructokinase synthesized in vitro from a ColE1 hybrid plasmid.

A hybrid plasmid, pLC 16-4, from the ColE1-DNA (E. coli) bank of Clarke and Carbon (1976) carrying pfkA was used to program an in vitro protein synthesis system from E. coli. Phosphofructokinase was the main product, as determined by enzyme assay, immunoprecipitation and gel electrophoresis. The enzyme was synthesized in vitro without added cAMP at a rate (enzyme/genome/h) ca. 30% the in vivo value, a higher efficiency than usually found in cell free systems. The plasmic molecular weight is ca. 16.10(6) daltons.     

Plasmid-determined bleomycin resistance in Staphylococcus aureus

A 1580-bp fragment of Staphylococcus aureus plasmid pUB110 encoding resistance to the DNA synthesis inhibitor bleomycin has been cloned and sequenced. A DNA sequence containing an open reading frame of 405 bp was subcloned into several expression vectors and bleomycin resistance was expressed at high level in Escherichia coli under the control of lambda PL promoter. On induction, a ca. 14,000-Da protein was detected by gel electrophoresis. The bleomycin resistance determinant of the gram-positive plasmid pUB110 was compared to that of the enterobacterial transposon Tn5; limited regions of close relatedness could be identified.     

Isolation and preliminary characterization of a plasmid mutant derepressed for conjugal transfer in Staphylococcus aureus

The plasmid pCRG1600 is a 52.9-kb self-transmissible plasmid coding for resistance to aminoglycoside and β-lactam antibiotics in Staphylococcus aureus. When transferred by transduction, plasmid deletion mutants affecting one or more antibiotic-resistance genes were readily obtained. Of these, one derivative (pCRG1690) was found to exhibit a conjugal transfer frequency ca. 100-fold higher than that of the wild-type plasmid. A preliminary physical-genetic map of pCRG1600 located tra in a 14.6-kb region within the 16.9-kb XbaI-A fragment. An 8.5-kb deletion to the left of tra in pCRG1690 was specifically associated with the increased conjugal transferability of the plasmid. Thus, pCRG1690 appears similar to plasmids derepressed for conjugal transfer (drd) in gram-negative bacterial species.     

Characterization of pER371-based Streptococcus thermophilus-Escherichia coli shuttle vectors

Native plasmid of Streptococcus thermophilus ST137, pER371 (2.7 kb) linearized at various unique restriction sites was individually subcloned into Escherichia coli plasmid pUC19 to generate the pUER-series recombinants. A selection cassette consisting of chloramphenicol- and erythromycin-resistance genes was spliced into each construct to generate the pMEU shuttle vectors. Electrotransformation of Streptococcus thermophilus with these vectors showed that a ca. 1.7 kb BstEII/ BanII fragment is essential for plasmid replication. A shuttle vector, pMEU149-1 (5.3 kb), was constructed using the minimally required fragment for replication. A chloramphenicol acetyltransferase ( cat) gene was successfully expressed in the ultimate S. thermophilus host by using pMEU149-1. Cloning vectors derived from pER371 should provide valuable alternative gene delivery vehicles for the genetic engineering of lactic acid bacteria.     

Enterotoxins and toxic shock syndrome toxin in Staphylococcus aureus recovered from human nasal carriers and manually handled foods: epidemiological and genetic findings

A set of 269 Staphylococcus aureus isolates recovered from nasal carriers and manually handled foods in a region of Spain was analyzed for pyrogenic toxin production and toxin genes. Fifty-seven isolates producing at least one of four enterotoxins (SEA, SEB, SEC, SED), 10 isolates producing only toxic shock syndrome toxin (TSST-1), and 10 isolates producing both toxin types were found. The 77 toxigenic isolates were discriminated into 36 SmaI genomic and 13 EcoRI plasmid profiles. A strong relationship between toxin profiles with both SmaI genomic and EcoRI plasmid profiles was revealed. SmaI genomic profiles showing six or less mismatching fragments and similarity coefficient > or =0.7 were included in a lineage. Eight lineages were differentiated; six of them grouped both human and food isolates and two of these also included outbreak-implicated isolates. Two lineages, represented by TSST-SEA and TSST-1, on the one hand, and SEC and SEC-SED isolates, on the other hand, were the most frequent, but only the second was outbreak-related. When SmaI genomic and EcoRI plasmid profiles were hybridized with tst, sea, seb, and sec toxin probes, it was observed that each probe mapped on a different SmaI fragment from isolates falling into the same lineage. All of the probes only mapped on genomic fragments, but sed also mapped on three plasmid fragments. When sej and ser probes were included, they mapped together with sed on the chromosome and on the plasmids. Two plasmids (ca. 33 and 36 kb) carried the expected sed-sej-ser genes, while the other (ca. 53.5 kb) carried sed-sej and ser-like genes. The latter plasmid and the chromosomal location of sed-sej-ser are new findings from this study.     

Plasmids in avirulent strains of Agrobacterium.

Twelve strains of Agrobacterium radiobacter isolated from naturally occurring crown galls or soil were found to be avirulent on sunflower, tomato, Kalanchoe, and carrot. Eleven strains contained plasmids of molecular weights 77 X 10(6) to 182 X 10(6) as determined by electron microscopy. One strain contained only a smaller plasmid (50 X 10(6) daltons). Several strains had both large and small (ca. 11 X 10(6) daltons) plasmids; one strain contained two large plasmids (112 X 10(6) and 136 X 10(6) daltons). Hybridization reactions of virulence plasmids from Agrobacterium tumefaciens strains C58 and A6 with plasmids from each of the A. radiobacter strains revealed that some A. radiobacter plasmids had less than 10% homology to either the C58 or A6 plasmids. Plasmids from some strains had approximately 50% homology with the C58 plasmid, but only one A. radiobacter plasmid contained more than 10% homology to the A6 plasmid. The presence of large plasmids in A. radiobacter strains did not correlate with sensitivity to agrocin 84; however, the utilization of the amino acid derivatives octopine and nopaline was generally correlated to partial base sequence homology to the C58 plasmid. We conclude that all large plasmids found in Agrobacterium strains are not virulence associated, although they may share base sequence homology with a virulence-associated plasmid. Further, plasmids from tumorigenic strains may be more closely related by base sequence homology to plasmids from nonpathogenic strains than to plasmids from other pathogenic strains.     

Transposon mutagenesis in Proteus mirabilis.

A technique of transposon mutagenesis involving the use of Tn5 on a suicide plasmid was developed for Proteus mirabilis. Analysis of the resulting exconjugants indicated that Tn5 transposed in P. mirabilis at a frequency of ca. 4.5 x 10(-6) per recipient cell. The resulting mutants were stable and retained the transposon-encoded antibiotic resistance when incubated for several generations under nonselective conditions. The frequency of auxotrophic mutants in the population, as well as DNA-DNA hybridizaiton to transposon sequences, confirmed that the insertion of the transposon was random and the Proteus chromosome did not contain significant insertional hot spots of transposition. Approximately 35% of the mutants analyzed possessed plasmid-acquired ampicillin resistance, although no extrachromosomal plasmid DNA was found. In these mutants, insertion of the Tn5 element and a part or all of the plasmid had occurred. Application of this technique to the study of swarmer cell differentiation in P. mirabilis is discussed.     

Increased production of aspartase in Escherichia coli K-12 by use of stabilized aspA recombinant plasmid

Recombinant plasmid pYT471, which consists of the aspartase gene (aspA) and the multicopy vector pBR322, was lost from cells of Escherichia coli K-12 at high frequencies in medium in which aspartase was abundantly formed due to release from catabolite repression. This plasmid loss was not completely prevented by the selective pressure of antibiotic addition. To increase the stability of the aspA plasmid, pNK101 (pBR322::aspA-par) was constructed by using the partition locus (par) derived from the low-copy vector pSC101. In E. coli K-12 cells, pNK101 was lost at a frequency as low as 0.4% per cell generation in nonselective medium, whereas pYT471 was lost at a frequency as high as 8.5%. Cells harboring this stable plasmid produced ca. 30-fold more aspartase than did cells harboring the unstable plasmid after 30 cell generations. Thus, we could increase aspartase production by stabilizing the aspA recombinant plasmid.     

Increased production of aspartase in Escherichia coli K-12 by use of stabilized aspA recombinant plasmid.

Recombinant plasmid pYT471, which consists of the aspartase gene (aspA) and the multicopy vector pBR322, was lost from cells of Escherichia coli K-12 at high frequencies in medium in which aspartase was abundantly formed due to release from catabolite repression. This plasmid loss was not completely prevented by the selective pressure of antibiotic addition. To increase the stability of the aspA plasmid, pNK101 (pBR322::aspA-par) was constructed by using the partition locus (par) derived from the low-copy vector pSC101. In E. coli K-12 cells, pNK101 was lost at a frequency as low as 0.4% per cell generation in nonselective medium, whereas pYT471 was lost at a frequency as high as 8.5%. Cells harboring this stable plasmid produced ca. 30-fold more aspartase than did cells harboring the unstable plasmid after 30 cell generations. Thus, we could increase aspartase production by stabilizing the aspA recombinant plasmid.     

Performance and stability of ethanologenic Escherichia coli strain FBR5 during continuous culture on xylose and glucose

Escherichia coli FBR5 containing recombinant genes for ethanol production on plasmids that are also required for anaerobic growth was cultivated continuously on 50 g/l xylose or glucose in the absence of antibiotics and without the use of special measures to limit the entry of oxygen into the fermenter. Under chemostat conditions, stable ethanol yields of ca. 80–85% of the theoretical were obtained on both sugars over 26 days at dilution rates of 0.045/h (xylose) and 0.075/h (glucose), with average plasmid retention rates of 96% (xylose) and 97% (glucose). In a continuous fluidized bed fermenter, with the cells immobilized on porous glass beads, the extent of plasmid retention by the free cells fell rapidly, while that of the immobilized cells remained constant. This was shown to be due to diffusion of oxygen through the tubing used to recirculate the medium and free cells. A change to oxygen-impermeable tubing led to a stable high rate of plasmid retention (more than 96% of both the free and immobilized cells) with ethanol yields of ca. 80% on a 50 g/l xylose feed. The maximum permissible level of oxygen availability consistent with high plasmid retention by the strain appears to be of the order of 0.1 mmol per hour per gram dry biomass, based on measurements of the rate of oxygen penetration into the fermenters. Revertant colonies lacking the ethanologenic plasmid were easily detectable by their morphology which correlated well with their lack of ampicillin resistance upon transfer plating.     

Transfer of chromosomal genes and plasmids in Bacillus thuringiensis

A low frequency of chromosomal gene transfer from Bacillus thuringiensis to Bacillus cereus was detected by cell mating, with a tryptophan marker being the most frequently transferred gene among four that were tested. The process was resistant to DNase and was not mediated by cell filtrates. Among several B. thuringiensis subspecies tested, transfer was best with a derivative of B. thuringiensis subsp. kurstaki HD1, which lost several plasmids. All of the B. cereus recombinants contained at least one plasmid from the donor B. thuringiensis; frequently, it was a plasmid that encoded a protoxin gene. In matings with B. thuringiensis subsp. kurstaki HD1, a 29-megadalton plasmid that contained a ca. 2.5-kilobase region of homology with the chromosome was always transferred. No detectable transfer of chromosomal genes was found in B. thuringiensis subsp. kurstaki HD1 strains lacking this plasmid, suggesting that there may be chromosome mobilization.     

Plasmid-borne determinants of pigmentation and thiamine prototrophy in Erwinia herbicola.

Strains of Erwinia herbicola lost yellow pigmentation and thiamine prototrophy at high frequency when grown at elevated temperature (38 degrees C) or in the presence of sodium dodecyl sulfate. All pigmentless, thiamine-auxotrophic variants had lost a large plasmid (ca. 350 megadaltons). Conversely, all pigmented, thiamine-prototrophic strains contained the large plasmid. The evidence presented indicates that pigmentation and thiamine prototrophy are specified or controlled by genes carried on the 350-megadalton plasmid.     

Transfer of chromosomal genes and plasmids in Bacillus thuringiensis.

A low frequency of chromosomal gene transfer from Bacillus thuringiensis to Bacillus cereus was detected by cell mating, with a tryptophan marker being the most frequently transferred gene among four that were tested. The process was resistant to DNase and was not mediated by cell filtrates. Among several B. thuringiensis subspecies tested, transfer was best with a derivative of B. thuringiensis subsp. kurstaki HD1, which lost several plasmids. All of the B. cereus recombinants contained at least one plasmid from the donor B. thuringiensis; frequently, it was a plasmid that encoded a protoxin gene. In matings with B. thuringiensis subsp. kurstaki HD1, a 29-megadalton plasmid that contained a ca. 2.5-kilobase region of homology with the chromosome was always transferred. No detectable transfer of chromosomal genes was found in B. thuringiensis subsp. kurstaki HD1 strains lacking this plasmid, suggesting that there may be chromosome mobilization.     

hisT is part of a multigene operon in Escherichia coli K-12.

The Escherichia coli K-12 hisT gene has been cloned, and its organization and expression have been analyzed on multicopy plasmids. The hisT gene, which encodes tRNA pseudouridine synthase I (PSUI), was isolated on a Clarke-Carbon plasmid known to contain the purF gene. The presence of the hisT gene on this plasmid was suggested by its ability to restore both production of PSUI enzymatic activity and suppression of amber mutations in a hisT mutant strain. A 2.3-kilobase HindIII-ClaI restriction fragment containing the hisT gene was subcloned into plasmid pBR322, and the resulting plasmid (designated psi 300) was mapped with restriction enzymes. Complementation analysis with different kinds of hisT mutations and tRNA structural analysis confirmed that plasmid psi 300 contained the hisT structural gene. Enzyme assays showed that plasmid psi 300 overproduced PSUI activity by ca. 20-fold compared with the wild-type level. Subclones containing restriction fragments from plasmid psi 300 inserted downstream from the lac promoter established that the hisT gene is oriented from the HindIII site toward the ClaI site. Other subclones and derivatives of plasmid psi 300 containing insertion or deletion mutations were constructed and assayed for production of PSUI activity and production of proteins in minicells. These experiments showed that: (i) the proximal 1.3-kilobase HindIII-BssHII restriction fragment contains a promoter for the hisT gene and encodes a 45,000-dalton polypeptide that is not PSUI; (ii) the distal 1.0-kilobase BssHII-ClaI restriction fragment encodes the 31,000-dalton PSUI polypeptide; (iii) the 45,000-dalton polypeptide is synthesized in an approximately eightfold excess compared with PSUI; and (iv) synthesis of the two polypeptides is coupled, suggesting that the two genes are part of an operon. Insertion of mini-Mu d1 (lac Km) phage into plasmid psi 300 confirmed that the hisT gene is the downstream gene in the operon.     

A plasmid vector with temperature-controlled gene expression

A 169 b.p. fragment including the bla gene promoter p3 has been removed from pBR327 plasmid, and the deleted plasmid used for cloning the TaqI/BglII-fragment of the lambda c1857ind- DNA containing promoter pR and gene cI to obtain plasmid pCE119. Cells containing pCE119 produced a high level of beta-lactamase at 42 degrees C, the yield at 42 degrees C being 100 times higher than at 32 degrees C. For cloning and functional assays a pCEZ12 plasmid was constructed, in which promoter pR and repressor cI of lambda phage control the expression of the semi-synthetic beta-galactosidase gene. Yield of beta-galactosidase produced by pCEZ12 at 42 degrees C was ca. 300 times higher than at 32 degrees C.     

Characterization of cloned human alphoid satellite with an unusual monomeric construction: evidence for enrichment in HeLa small polydisperse circular DNA.

A recombinant DNA plasmid library was constructed from HeLa cell extrachromosomal circular DNA and the sequence organization of one family of clones, which contain sequences enriched in HeLa small polydisperse circular (spc) DNA, was studied by restriction mapping and base sequence analysis. Restriction mapping revealed each clone to be composed solely of imperfect tandem repeats of ca. 170 bp. The entire DNA sequence of one clone was determined and found to be alphoid satellite with a variant monomeric construction.     

Conservation of plasmid-encoded dibenzothiophene desulfurization genes in several rhodococci.

The cloned sulfur oxidation (desulfurization) genes (sox) for dibenzothiophene (DBT) from the prototype Rhodococcus sp. strain IGTS8 were used in Southern hybridization and PCR experiments to establish the DNA relatedness in six new rhodococcal isolates which are capable of utilizing DBT as a sole sulfur source for growth. The ability of these strains to desulfurize appears to be an exclusive property of a 4-kb gene locus on a large plasmid of ca. 150 kb in IGTS8 and ca. 100 kb in the other strains. Besides a difference in plasmid profile, IGTS8 is distinguishable from the other strains in at least the copy number of the insertion sequence IS1166, which is associated with the sox genes.