Plasmid contents of lactic acid bacteria isolated from different types of sour doughs

The plasmid contents of 13 lactic acid bacteria isolated from different types of sour doughs were examined and compared with the plasmid contents of 11 culture collection strains and one commercial pure starter culture for sour doughs. In addition, plasmid analysis was used as a tool to study the stability of a starter culture during sour dough fermentation in a bakery.The tested strains varied in plasmid content from no plasmid up to six plasmids, with molecular weights from 1.5 to 43 MDal. In most cases, the wild-type strains contained a higher number of plasmids than the culture collection strains. The ability of the strains to ferment different carbohydrates was also investigated, but no obvious correlations between the fermentation patterns and the plasmid patterns could be observed. During the fermentation of the bakery sour dough, strains other than the inoculated starter culture gradually became dominant in the microflora. These new strains contained 1–3 plasmids, contrary to the plasmidless starter culture, and they also fermented more carbohydrates than the starter culture.     

The use of cadmium resistance on the phage-resistance plasmid pNP40 facilitates selection for its horizontal transfer to industrial dairy starter lactococci

AIMS: To facilitate the horizontal transfer and selection of phage-resistance plasmids in industrial lactococci. METHODS AND RESULTS: Cadmium-resistance properties similar to those previously identified in Lactococcus were linked to the well-known phage-resistance plasmid pNP40. This finding was exploited to facilitate delivery of the plasmid to an industrial cheese starter Lactococcus lactis DPC4268. Additionally, 25 different cadmium-sensitive cheese starter lactococci were also identified as potential recipients for the phage-resistance plasmid pNP40, and also the plasmids pAH90/pAH82 which also encode cadmium resistance. All three plasmids were successfully conjugated to strain DPC4268. Cheddar cheese was manufactured in industry with the pNP40 phage-resistant transconjugant. SIGNIFICANCE AND IMPACT OF THE STUDY: Food-grade enhancement of phage resistance in industrial starter strains has been made simpler by the use of this selection, especially since the majority of potential recipient starter strains analysed were cadmium sensitive.     

Linear- and circular-plasmid copy numbers in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease agent, and other members of the spirochetal genus Borrelia have double-stranded linear plasmids in addition to supercoiled circular plasmids. The copy number relative to the chromosome was determined for 49- and 16-kb linear plasmids and a 27-kb circular plasmid of the type strain, B31, of B. burgdorferi. All three plasmids were present in low copy number, about one per chromosome equivalent, as determined by relative hybridizations of replicon-specific DNA probes. The low copy number of Borrelia plasmids suggests that initiation of DNA replication and partitioning are carefully controlled during the cell division cycle. The copy numbers of these three plasmids of strain B31 were unchanged after approximately 7,000 generations in continuous in vitro culture. A clone of B. burgdorferi B31 that did not contain the 16-kb linear plasmid was obtained after exposure of a culture to novobiocin, a DNA gyrase inhibitor. The plasmid-cured strain contains only one linear plasmid, the 49-kb plasmid, and thus has the smallest genome reported to date for B. burgdorferi.     

Evidence for diverse types of large plasmids in tumor-inducing strains of Agrobacterium.

Homology between the large plasmids of 15 pathogenic Agrobacterium strains isolated from various parts of the world has been measured and was found to vary over a wide range, from 3 to 100%. Two genetically distinct groups of plasmids can be identified: one closely related to the plasmid of A. tumefaciens A6, an octopine-utilizing strain, and the other closely related to the plasmid of A. tumefaciens C-58, a nopaline-utilizing strain. The plasmids of four Agrobacterium strains do not belong to either group. One of these four strains utilizes octopine, one utilizes nopaline, and two utilize neither. Three strains contained two large plasmids. In one of these strains, the two plasmids were not homologous to one another. Chromosomal homologies for the Agrobacterium strains surveyed also vary over a wide range, but do not correlate with plasmid homologies. Neither do plasmid homologies correlate with any numerical classification scheme. The significance of these plasmid homology studies for crown gall tumorigenesis is considered.     

Plasmids and transposons and their stability and mutability in bacteria isolated during an outbreak of hospital infection.

In an outbreak of hospital infection caused by Klebsiella aerogenes type K-16 isolates over a 3-month period carried, apparently unaltered, a cryptic 90-Megadalton (Md) plasmid (unclassified) and a multiple-resistance 65-Md plasmid of IncM. The IncM plasmid, identified in environmentally related strains of Citrobacter koseri and Escherichia coli, showed minor variations from that in the klebsiella vector. The IncM plasmids, as well as all wild host strains cured of the IncM plasmids, carried a transposable DNA sequence, encoding trimethoprim and, in every case but one, streptomycin resistance. This transposon appeared identical with Tn7, previously identified in unrelated plasmids in bacteria from different environments.     

Process of reductive evolution during 10 years in plasmids of a non-insect-transmissible phytoplasma

A non-insect-transmissible phytoplasma strain (OY-NIM) was obtained from insect-transmissible strain OY-M by plant grafting using no insect vectors. In this study, we analyzed for the gene structure of plasmids during its maintenance in plant tissue culture for 10 years. OY-M strain has one plasmid encoding orf3 gene which is thought to be involved in insect transmissibility. The gradual loss of OY-NIM plasmid sequence was observed in subsequent steps: first, the promoter region of orf3 was lost, followed by the loss of then a large region including orf3, and finally the entire plasmid was disappeared. In contrast, no mutation was found in a pseudogene on OY-NIM chromosome in the same period, indicating that OY-NIM plasmid evolved more rapidly than the chromosome-encoded gene tested. Results revealed an actual evolutionary process of OY plasmid, and provide a model for the stepwise process in reductive evolution of plasmids by environmental adaptation. Furthermore, this study indicates the great plasticity of plasmids throughout the evolution of phytoplasma.     

Identification of cell wall antigens associated with a large conjugative plasmid encoding phage resistance and lactose fermentation ability in lactic streptococci

In order to begin to analyze the gene products encoded by phage resistance plasmids in lactic streptococci, we identified phage-resistance plasmids by screening resistant strains from commercial starter cultures for the ability to carry out unselected cotransfer, by conjugation, of phage resistance with lactose fermentation ability (lac+). In this fashion, we identified a large (90 kilobases) plasmid, pCLP51R, that encodes the lac+ marker, resistance to a lytic phage called LP10G (1pr+), high-frequency conjugal donor ability (hft+), and clumpy growth of host bacteria in broth culture (clu+). The mechanism of resistance conferred by this plasmid appears to involve interference with a step in the phage replication cycle that occurs after the initial attachment of the phage. Polyacrylamide gel electrophoresis of cell surface extracts of isogenic strains, carrying or lacking pCLP51R, combined with immunoblotting analysis, showed that there were several plasmid-related differences in the banding pattern of low molecular weight proteins and that the plasmid resulted in production of several unique antigenic polypeptides in the size range of 15-30 kd, as well as modification of chromosomally encoded antigens to different molecular weight forms.     

Evaluation of the plasmid copy number in B. cereus spores, during germination, bacterial growth and sporulation using real-time PCR

Only a small number of studies have measured the plasmid copy number (PCN) variation during bacterial growth. Besides, information about the PCN in spores is still rare. In this work, we utilized a real-time PCR assay to evaluate the PCN of four different plasmids in Bacillus cereus. The PCN was measured in spores as well as during germination, active bacterial growth, and sporulation. Plasmid stability was also evaluated to ensure that plasmid loss does not affect the accuracy of the PCN measurement. We demonstrated that the PCN of low and high copy number plasmids varies with growth phase as well as culture media over B. cereus life cycle. The PCN was minimum during the germination and maximum during the stationary growth phase for all plasmids tested. We also demonstrated that the use of antibiotic in the culture media is not enough to ensure stable inheritance in spores of plasmids carrying antibiotic resistance genes. Moreover, we revealed that the PCN in spores is related to the PCN during endospores formation. Therefore, the plasmid partitioning during sporulation is not influenced by the unequal-size of the forespores and the mother cells, even for a plasmid distributed randomly.     

Sequence analysis and characterization of two cryptic plasmids derived from Lactobacillus buchneri CD034

Lactobacillus buchneri is probably the most beneficial microorganism for efficient preservation of animal feed silages made from grass, maize and other plant material against aerobic spoilage. Its obligatory heterofermentative nature, acid resistance and robustness have drawn attention to this species for applications as silage starter culture as well as for genetic engineering. For the first time, two cryptic plasmids present in the same L. buchneri strain, L. buchneri CD034, were isolated, sequenced and characterized. The larger plasmid, designated pCD034-1 was found to be 3424 bp in length with a G + C content of 38.36%. The smaller plasmid, designated pCD034-2 was found to be 2707 bp in length with a G + C content of 38.60%. On both plasmids we predicted three open reading frames. On pCD034-1, ORF 1 encodes a putative replication protein which shares 99% identity with the RepA protein of a Lactobacillus plantarum derived pC194/pUB110-family plasmid. ORF 2 encodes a putative protein of unknown function. ORF 1 and ORF 2 of pCD034-2 correspond to RepA and RepB proteins similar to those of plasmid pLB4 from L. plantarum. ORF 3 of both plasmids encodes a putative mobilization protein similar to that of the pediococcal plasmid pF8801. Double strand origins, putative single strand origins and typical mobilization start signals were identified. Both plasmids were shown to be maintained at relatively high plasmid copy numbers. Two shuttle vectors carrying the origins of replication of pCD034-1 and pCD034-2 were constructed and used to successfully transform two other species isolated from the same environment. Hence, we consider the two novel L. buchneri plasmids a valuable resource for the generation of shuttle and expression vectors for LAB.     

A method for plasmid purification directly from yeast

A rapid technique for purifying plasmids from yeast Saccharomyces cerevisiae is described that yields high-quality DNA suitable for bacterial transformation, yeast transformation, and direct DNA sequencing. The method requires only small culture volumes and proprietary bacterial plasmid miniprep kits that allow one to simultaneously prepare a large number of samples in a very short period of time while avoiding the use of toxic organic chemicals. Both yeast single-copy CEN/ARS and high-copy 2micro shuttle plasmids can be isolated using this method. This technique is useful for plasmid purification from yeast two-hybrid experiments as well as yeast genetics and molecular biology experiments.     

Plasmid DNA from methanogenic bacteria

In total, 73 strains of methanogen isolates from our laboratory and 6 from culture collections were examined for the presence of plasmid DNA. Five strains were found to contain detectable plasmids. Multiple plasmids were found in two isolates, while three strains contained only one plasmid each. A physical map of the plasmid pT3 was constructed by use of six different restriction endonucleases. All sites were aligned with a single BgII site, and the position of the restriction sites was determined by double or sequential digestion of the plasmid DNA.     

Occurrence of megaplasmids in halobacteria

Sixty-five halobacteria, including culture collection and freshly isolated strains from widely differing geographical areas, were examined for the presence of high molecular weight plasmids by agarose gel electrophoresis. Seventy-five per cent of all the strains were shown to harbour at least one plasmid. In the majority of strains three or four megaplasmids were detected. Approximate molecular weights of the plasmids were in the range < 100 to 300 megadaltons (Mdal). In most culture collection strains, two or three plasmids were demonstrated, except in two in which no plasmid was detected, and in two Haloarcula strains which were found to contain five and eight plasmids; four and six of the latter were more than 100 Mdal. No relationship between the plasmid profile of each strain and its taxonomic assignation nor its isolation source was found. Evidence is presented for the first time on the occurrence of megaplasmids in halobacteria.     

Isolation and characterization of sequences from mouse chromosomal DNA with ARS function in yeasts.

Fragments of chromosomal DNA from a variety of eucaryotes can act as ARSs (autonomously replicating sequence) in yeasts. ARSs enable plasmids to be maintained in extrachromosomal form, presumably because they function as initiation sites for DNA replication. We isolated eight different sequences from mouse chromosomal DNA which function as ARSs in Saccharomyces cerevisiae (bakers' yeast). Although the replication efficiency of the different mouse ARSs in yeasts appears to vary widely, about one-half of them functions as well as the yeast chromosomal sequence ARS1. Moreover, five of the ARSs also promote self replication of plasmids in Schizosaccharomyces pombe (fission yeast). Each of the ARSs was cloned into plasmids suitable for transformation of mouse tissue culture cells. Plasmids were introduced into thymidine kinase (TK)-deficient mouse L cells by the calcium phosphate precipitation technique in the absence of carrier DNA. In some experiments, the ARS plasmid contained the herpes simplex virus type 1 TK gene; in other experiments (cotransformations), the TK gene was carried on a separate plasmid used in the same transformation. In contrast to their behavior in yeasts, none of the ARS plasmids displayed a significant increase in transformation frequency in mouse cells compared with control plasmids. Moreover, only 1 of over 100 cell lines contained the original plasmid in extrachromosomal form. The majority of cell lines produced by transformation with an ARS TK plasmid contained multiple copies of plasmid integrated into chromosomal DNA. In most cases, results with plasmids used in cotransformations were similar to those for plasmids carrying TK. However, cell lines produced by cotransformations with plasmids containing any one of three of the ARSs (m24, m25, or m26) often contained extrachromosomal DNAs.     

Transfer and stability of drug resistance plasmids inEscherichia coli K12

Mating experiments between pairs of strains ofEscherichia coli containing either the compatible plasmids TP120 (Inc N) and R1 (Inc FII) or the incompatible plasmids TP125 (Inc B) and TP113 (Inc B) were undertaken in mixed continuous-flow cultures and in dialysis sacs suspended in pond water. Plasmid transfer was readily demonstrated between strains carrying compatible plasmids TP120 and R1 in both continuous-flow culture and pond water. In mixed cultures of strains carrying plasmids TP125 and TP113, transfer was only observed in continuous-flow culture systems. Strains ofE. coli containing aggregates of plasmids TP120 and R1 were shown to be stable over 5 months continuous cultivation under carbon limited conditions at a growth rate of 0.1 hours^–1 in the presence of drugs which select for the maintenance of both plasmids. In the strains containing plasmid aggregates, a gene dosage effect was observed with respect to the levels of resistance to drugs whose resistance was encoded by both plasmids. Chemostat experiments showed that no cointegrate plasmids were found from the strains ofE. coli initially containing both plasmid TP120 and plasmid R1.     

Production of toxin by Clostridium botulinum type A strains cured by plasmids.

Twelve strains of Clostridium botulinum type A and seven strains of Clostridium sporogenes were screened for plasmids by agarose gel electrophoresis of cleared lysates of cells from 5 ml of mid-log-phase culture. Nine type A strains had one or more plasmids of 4.3, 6.8, or 36 megadaltons (MDa); several strains showed a large plasmid of 61 MDa, but it was not consistently recovered. Four C. sporogenes strains had one or more plasmids of 4.3, 5.6 or 36 MDa. Isolates obtained from cultures of plasmid-containing C. botulinum type A strains grown in ionic detergent broth and from spontaneously arising variants were screened both for toxin production and for plasmid content. Toxigenicity of C. botulinum could not be correlated with the presence of any one plasmid.     

Production of toxin by Clostridium botulinum type A strains cured by plasmids

Twelve strains of Clostridium botulinum type A and seven strains of Clostridium sporogenes were screened for plasmids by agarose gel electrophoresis of cleared lysates of cells from 5 ml of mid-log-phase culture. Nine type A strains had one or more plasmids of 4.3, 6.8, or 36 megadaltons (MDa); several strains showed a large plasmid of 61 MDa, but it was not consistently recovered. Four C. sporogenes strains had one or more plasmids of 4.3, 5.6 or 36 MDa. Isolates obtained from cultures of plasmid-containing C. botulinum type A strains grown in ionic detergent broth and from spontaneously arising variants were screened both for toxin production and for plasmid content. Toxigenicity of C. botulinum could not be correlated with the presence of any one plasmid.     

Related plasmids found in an English Lake district stream

An examination of the distribution of plasmids carried by copper-tolerant bacteria from a freshwater stream revealed that >60% carried at least one plasmid, and that large plasmids (>100 kb) were predominant. A total of 10 copper-tolerant bacteria carrying the 54-kb plasmid, pFBA20, were detected at four sampling sites within the stream and, on consecutive occasions, at one site throughout a 1-year sampling period. The detection of this plasmid provides evidence that related plasmids can, under no apparent selective pressure, survive and disperse within the bacterial community. Two of the isolates that carried pFBA20 were phenotypically distinguishable. This would suggest that pFBA20 is transmissible.     

Molecular cloning in plasmid pBR322 giving altered expression of the tetracycline resistance gene

The two HindIII fragments of polyoma virus DNA were cloned in the HindIII site of plasmid pBR322, a site located in the RNA polymerase promoter involved in the expression of tetracycline resistance. Although insertion of foreign DNA into this site did not always result in the complete loss of tetracycline resistance, Escherichia coli K12 strain chi 1776 harbouring recombinant plasmids exhibited reduced growth properties in liquid culture with tetracycline and could easily be differentiated from bacteria transformed by non-recombinant plasmids. The formation of plasmid multimers increased the resistance to tetracycline at the level of the induction period, presumably as a result of a gene dosage effect.     

Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria

Multicopy plasmids are often chosen for the expression of recombinant genes in Escherichia coli. The high copy number is generally desired for maximum gene expression; however, the metabolic burden effects that usually result from multiple plasmid copies could prove to be detrimental for maximum productivity in certain metabolic engineering applications. In this study, low-copy mini-F plasmids were compared to high-copy pMB1-based plasmids for production of two metabolites in E. coli: polyphosphate (polyP) and lycopene derived from isopentenyl diphosphate (IPP). The stationary-phase accumulation of polyP on a per cell basis was enhanced approximately 80% when either high- or low-copy plasmids were used, from 120 micromol/g DCW without augmented polyP kinase (PPK) activity to approximately 220 micromol/g DCW. The cell density of the high-copy plasmid-containing culture at stationary phase was approximately 24% lower than the low-copy culture and 30% lower than the control culture. This difference in cell density is likely a metabolic burden effect and resulted in a lower overall product concentration for the high-copy culture (approximately 130 micromol/L culture) relative to the low-copy culture (approximately 160 micromol/L culture). When the gene for DXP (1-deoxy-D-xylulose 5-phosphate) synthase, the first enzyme in the IPP mevalonate-independent biosynthetic pathway, was expressed from the tac promoter on multicopy and low-copy plasmids, lycopene production was enhanced two- to threefold over that found in cells expressing the chromosomal copy only. Cell growth and lycopene production decreased substantially when isopropyl beta-D-thiogalactosidase (IPTG) was added to the high-copy plasmid-containing culture, suggesting that overexpression of DXP synthase was a significant metabolic burden. In the low-copy plasmid-containing culture, no differences in cell growth or lycopene production were observed with any IPTG concentrations. When dxs was placed under the control of the arabinose-inducible promoter (P(BAD)) on the low-copy plasmid, the amount of lycopene produced was proportional to the arabinose concentration and no significant changes in cell growth resulted. These results suggest that low-copy plasmids may be useful in metabolic engineering applications, particularly when one or more of the substrates used in the recombinant pathway are required for normal cellular metabolism.     

Conservation of plasmids among plant-pathogenic Pseudomonas syringae isolates of diverse origins

Thirty isolates of Pseudomonas syringae pv. tabaci, pv. angulata (pathogens on tobacco), pv. coronafaciens, and pv. striafaciens (pathogens on oats) were examined for plasmid DNAs. The strains were obtained from plants throughout the world, some over 50 years ago. Of the 22 tobacco pathogens, 16 contain predominantly one type of plasmid, the pJP27.00 type. The remaining six tobacco-specific strains do not harbor detectable plasmids. The oat pathogens contain one, two, or three plasmids. DNA homology studies indicate that the plasmid DNAs are highly conserved. More importantly, the plasmids harbored by strains isolated from one host plant are conserved most stringently; e.g., the plasmids from the tobacco pathogens are, with one exception, indistinguishable by restriction endonuclease digestion and Southern hybridization. There is also extensive homology among plasmids indigenous to the oat-specific P. syringae pv. coronafaciens and pv. striafaciens strains.