Transposition and transduction of plasmid DNA inStreptomyces spp.

Summary To expand the application of molecular genetics to many different streptomycete species, we have been developing two potentially widely applicable methodologies: transposon mutagenesis and plasmid transduction. We constructed three transposons from theStreptomyces lividans insertion sequence IS493. Tn5096 and Tn5097 contain an apramycin resistance gene inserted in different orientations between the two open reading frames of IS493. These transposons transpose from different plasmids into many different sites in theStreptomyces griseofuscus chromosome and into its resident linear plasmids. Tn5099 contains a promoterlessxylE gene and a hygromycin-resistance gene inserted in IS493 close to one end. Tn5099 transposes inS. griseofuscus giving operon fusions in some cases that drive expression of thexylE gene product, catechol deoxygenase, giving yellow colonies in the presence of catechol. We have also developed plasmid vectors that can be transduced into many streptomycete species by bacteriophage FP43. We describe the characterization of FP43 and mapping of several bacteriophage functions. The region of cloned FP43 DNA essential for plasmid transduction includes the origin for headful packaging.     

Intergeneric and intrageneric conjugal transfer of plasmid-encoded antibiotic resistance determinants in Leuconostoc spp.

Transfer of the broad-host-range resistance plasmids pIP501 and pAM beta 1 from Streptococcus faecalis to Leuconostoc dextranicum and Leuconostoc cremoris occurred between cells that were immobilized on nitrocellulose filters in the presence of DNase. Transfer of pIP501 to Leuconostoc spp. also occurred when Streptococcus sanguis and Streptococcus lactis were used as donors. In addition, transfer of pIP501 and pAM beta 1 was observed from L. cremoris and L. dextranicum transconjugants to S. sanguis and S. faecalis. Expression of the pAM beta 1 erythromycin and pIP501 erythromycin and chloramphenicol resistance determinants was essentially equivalent in donors and transconjugants. Frequencies of transfer generally ranged from 10(-4) to 10(-7) transconjugants per input donor cell. Intrageneric transfer of pIP501 and pAM beta 1 occurred between L. cremoris and L. dextranicum strains in the same approximate range. These data further extend the host range of pIP501 and pAM beta 1 and demonstrate another example of gene transfer in the genus Leuconostoc.     

肠球菌万古霉素耐药基因簇遗传特性

万古霉素耐药肠球菌自20世纪80年代后期被发现以来,已逐渐发展成为重要的医院感染病原菌。此类耐药肠球菌携带的万古霉素耐药基因簇编码产物可催化合成与万古霉素、替考拉宁等糖肽类抗生素亲和力极低的细胞壁前体导致耐药。目前已在肠球菌中发现的万古霉素耐药基因簇根据基因序列及构成不同分为9个型别;依据它们编码的连接酶合成产物不同又可分为D-Ala:D-Lac连接酶基因簇(VanA、VanB、VanD及VanM型)和D-Ala:D-Ser连接酶基因簇(VanC、VanE、VanG、VanL和VanN型)。这些耐药基因簇介导的耐药水平及其传播模式各有特点。文章综述了肠球菌中万古霉素耐药基因簇的类型、基因构成及传播特性。     

Intergeneric and intrageneric conjugal transfer of plasmid-encoded antibiotic resistance determinants in Leuconostoc spp

Transfer of the broad-host-range resistance plasmids pIP501 and pAM beta 1 from Streptococcus faecalis to Leuconostoc dextranicum and Leuconostoc cremoris occurred between cells that were immobilized on nitrocellulose filters in the presence of DNase. Transfer of pIP501 to Leuconostoc spp. also occurred when Streptococcus sanguis and Streptococcus lactis were used as donors. In addition, transfer of pIP501 and pAM beta 1 was observed from L. cremoris and L. dextranicum transconjugants to S. sanguis and S. faecalis. Expression of the pAM beta 1 erythromycin and pIP501 erythromycin and chloramphenicol resistance determinants was essentially equivalent in donors and transconjugants. Frequencies of transfer generally ranged from 10(-4) to 10(-7) transconjugants per input donor cell. Intrageneric transfer of pIP501 and pAM beta 1 occurred between L. cremoris and L. dextranicum strains in the same approximate range. These data further extend the host range of pIP501 and pAM beta 1 and demonstrate another example of gene transfer in the genus Leuconostoc.     

Metal resistance and plasmid DNA in Thiobacillus ferrooxidans

The minimal inhibitory concentrations of copper and nickel were determined for each of fifteen isolates of T. ferrooxidans native to a Cu/Ni tailings environment. Ten isolates were inhibited by 160 mM Cu,²⁺ or less, and ten were inhibited by 160 mM Ni²⁺or less. The isolates were screened for plasmid DNA using an alkaline lysis method and CCC plasmid forms were confirmed using the Hintermann technique. Two isolates were found to be devoid of plasmid DNA, and only one isolate contained more than two plasmids. Variability existed in plasmid size, although the majority were larger than the standard pBR322 (4.3 kbp). One plasmid was selected for further analysis using restriction endonucleases. EcoRI, HindIII and KpnI all cleaved the plasmid in two locations, and PstI cleaved the plasmid in six locations. PstI-digested fragments of the plasmid were ligated into pBR322, and the recombinant plasmids were transformed into Escherichia coli ATCC 8739. Four genetically-different transformants resulted, and each was grown in media containing 2.0 mM Cu²⁺ and compared to the growth of a control under similar conditions. There was no conferred copper resistance in E. coli, although one recombinant plasmid appeared to decrease the tolerance for E. coli ATCC 8739 to Cu²⁺.     

Enzymes of vancomycin resistance: the structure of D-alanine-D-lactate ligase of naturally resistant Leuconostoc mesenteroides

BACKGROUND: The bacterial cell wall and the enzymes that synthesize it are targets of glycopeptide antibiotics (vancomycins and teicoplanins) and beta-lactams (penicillins and cephalosporins). Biosynthesis of cell wall peptidoglycan requires a crosslinking of peptidyl moieties on adjacent glycan strands. The D-alanine-D-alanine transpeptidase, which catalyzes this crosslinking, is the target of beta-lactam antibiotics. Glycopeptides, in contrast, do not inhibit an enzyme, but bind directly to D-alanine-D-alanine and prevent subsequent crosslinking by the transpeptidase. Clinical resistance to vancomycin in enterococcal pathogens has been traced to altered ligases producing D-alanine-D-lactate rather than D-alanine-D-alanine. RESULTS: The structure of a D-alanine-D-lactate ligase has been determined by multiple anomalous dispersion (MAD) phasing to 2.4 A resolution. Co-crystallization of the Leuconostoc mesenteroides LmDdl2 ligase with ATP and a di-D-methylphosphinate produced ADP and a phosphinophosphate analog of the reaction intermediate of cell wall peptidoglycan biosynthesis. Comparison of this D-alanine-D-lactate ligase with the known structure of DdlB D-alanine-D-alanine ligase, a wild-type enzyme that does not provide vancomycin resistance, reveals alterations in the size and hydrophobicity of the site for D-lactate binding (subsite 2). A decrease was noted in the ability of the ligase to hydrogen bond a substrate molecule entering subsite 2. CONCLUSIONS: Structural differences at subsite 2 of the D-alanine-D-lactate ligase help explain a substrate specificity shift (D-alanine to D-lactate) leading to remodeled cell wall peptidoglycan and vancomycin resistance in Gram-positive pathogens.     

Identification of Carnobacterium spp. and Leuconostoc spp. in meat by genus-specific 16S rRNA probes

Oligonucleotide probes specific for Carnobacterium and Leuconostoc species were constructed from the variable regions of 16S rRNA obtained from the literature and sequence data bases. The probes were hybridized with crude nucleic acid extract from 32 type strains of lactic acid bacteria (LAB) commonly found on meat. Two of the probes hybridized only to the four Carnobacterium species whereas the other two hybridized only to five of the six Leuconostoc species tested. The probes were also hybridized with nucleic acids from unknown strains of LAB. The identification was consistent with the results of biochemical tests used to characterize the two genera.     

Transduction of plasmid DNA in Streptomyces spp. and related genera by bacteriophage FP43.

A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus. The transducing particles contained linear concatemers of plasmid DNA. Lysates of FP43 prepared on S. griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA. Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared. FP43 lysates prepared on S. griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.     

Kinetics of citrate uptake in growing cells of Leuconostoc spp.

Abstract Several yeast strains of the species Saccharomyces cerevisiae, S. bayanus and S. paradoxus , first identified by hybridization experiments and measurements of DNA/DNA homology, were characterized using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene. There was no exception to the agreement between this method and classical genetic analyses for any of the strains examined, so PCR/RFLP of the MET2 gene is a reliable and fast technique for delimiting S. cerevisiae and S. bayanus . Enological strains classified as S., bayanus , S. chevalieri , and S. capensis gave S. cerevisiae restriction patterns, whereas most S. uvarum strains belong to S. bayanus . Enologists should no longer use the name of S. bayanus for S. cerevisiae Gal strains, and should consider S. bayanus as a distinct species.     

Citrate Fermentation by Lactococcus and Leuconostoc spp

Citrate and lactose fermentation are subject to the same metabolic regulation. In both processes, pyruvate is the key intermediate. Lactococcus lactis subsp. lactis biovar diacetylactis homofermentatively converted pyruvate to lactate at high dilution (growth) rates, low pH, and high lactose concentrations. Mixed-acid fermentation with formate, ethanol, and acetate as products was observed under conditions of lactose limitation in continuous culture at pH values above 6.0. An acetoin/butanediol fermentation with alpha-acetolactate as an intermediate was found upon mild aeration in continuous culture and under conditions of excess pyruvate production from citrate. Leuconostoc spp. showed a limited metabolic flexibility. A typical heterofermentative conversion of lactose was observed under all conditions in both continuous and batch cultures. The pyruvate produced from either lactose or citrate was converted to d-lactate. Citrate utilization was pH dependent in both L. lactis and Leuconostoc spp., with maximum rates observed between pH 5.5 and 6.0. The maximum specific growth rate was slightly stimulated by citrate, in L. lactis and greatly stimulated by citrate in Leuconostoc spp., and the conversion of citrate resulted in increased growth yields on lactose for both L. lactis and Leuconostoc spp. This indicates that energy is conserved during the metabolism of citrate.     

Replication of antibiotic resistance plasmid R6K DNA in vitro.

A soluble extract prepared from cells of an Escherichia coli strain carrying the antibiotic resistance plasmid R6K is capable of carrying out the complete process of R6K DNA replication. DNA synthesis in vitro is dependent on the four deoxyribo- and ribonucleotide triphosphates and is sensitive to rifampin and streptolydigin, inhibitors of DNA-dependent RNA polymerase. The incorporation of deoxyribonucleotides into R6K DNA also is sensitive to actinomycin D, novobiocin, arabinofuranosyl-CTP, and N-ethylmaleimide. Kinetics of synthesis are linear for 60 to 120 min. Replication proceeds semiconservatively and supercoiled closed-circular DNA molecules are synthesized. Analysis by alkaline sucrose gradient centrifugation indicated that the early R6K DNA products contain DNA fragments of approximately 18 S in size, corresponding to the length between the R6K alpha origin of replication and the terminus of replication observed in vivo. Addition of exogenous supercoiled R6K DNA is inhibitory to the in vitro system, whereas the addition of R6K DNA in the form of relaxation complex stimulates R6K DNA synthesis to a small extent.     

Oxygen and pyruvate as external electron acceptors for Leuconostoc spp.

L. NURAIDA, I. GRIGOLAVA, J.D. OWENS AND G. CAMPBELL-PLATT. 1992. Leuconostoc mesenteroides NCDO 518, Leuc, mesenteroides NCIB 8710, Leur. mesenteroides NCIB 8023, Leuconostoc sp. Pz 45, Leuconostoc sp. Pz 34 and Leuconostoc sp. Pz 10 were grown in a chemically defined medium with glucose and different external electron acceptors. All strains, except Leuconostoc sp. Pz 10, formed acetate under aerobic conditions. Leuconostoc Pz 10 produced only small amounts of acetate under aerobic or anaerobic conditions. This strain was the only strain not possessing NADH oxidase. All strains produced acetate from a mixture of glucose plus pyruvate. None of the bacteria used glycerol. Oxygen and pyruvate as external electron acceptors increased the cell yield on glucose.     

Mobilization of vancomycin resistance by transposon-mediated fusion of a VanA plasmid with an Enterococcus faecium sex pheromone-response plasmid

A striking feature of recent outbreaks of vancomycin-resistant (Vm^R) enterococci is the apparent horizontal dissemination of resistance determinants. The plasmids pHKK702 and pHKK703 from Enterococcus faecium clinical isolate R7 have been implicated in the conjugal transfer of Vm^R. pHKK702 is a 41-kb plasmid that contains an element indistinguishable from the glycopeptide-resistance transposon Tn1546. pHKK703 is an approx. 55-kb putative sex pheromone-response plasmid that is required for conjugative mobilization of pHKK702. During experiments in which strain R7 was used as a donor, a highly conjugative Vm^R transconjugant was isolated that formed constitutive cellular aggregates. Restriction analyses and DNA hybridizations revealed that the transconjugant harbored a single plasmid of approx. 92 kb and this plasmid (pHKK701) was composed of DNA from both pHKK702 and pHKK703. Results from DNA sequence analyses showed that a 39-kb composite transposon (Tn5506) from pHKK702 had inserted into pHKK703. The left end of Tn5506 contained a single insertion sequence (IS) element, IS1216V2, whereas the right end was composed of a tandem IS structure consisting of the novel 1065-bp IS1252 nested within an IS1216V1 element. Transposition of Tn5506 from pHKK702 to pHKK703 created an 8-bp target sequence duplication at the site of insertion and interrupted an ORF (ORFX) that was 91% identical to that of prgX, a gene proposed to negatively regulate sex pheromone response of the E. faecalis plasmid, pCF10. We propose that the interruption of ORFX by Tn5506 led to the constitutive cellular aggregation phenotype and thereby enhanced the efficiency with which Vm^R was transferred. Similar IS1216V-mediated transposition events may contribute to the horizontal spread of glycopeptide resistance among enterococci in nature.     

First description of ColE-type plasmid in Aeromonas spp. carrying quinolone resistance (qnrS2) gene

Aims: Isolation and full sequence analysis of ColE‐type plasmid, which carries the qnrS2 gene. Methods and Results: Quinolone resistance (qnrS2) gene‐carrying plasmids were isolated from Aeromonas sobria and Aeromonas hydrophila strains, and plasmid sequencing was achieved by a primer‐walking approach. The total sizes of these plasmids (pAQ2‐1 and pAQ2‐2) were 6900 bp and 6903 bp, respectively, and they were 99·1% identical to each other. The genes (oriV and repA) for plasmid replication were organized similar to the corresponding genes in the ColE2‐type plasmids, pAsa3 and pAsa1, isolated from Aeromonas salmonicida subsp. salmonicida, but the gene (mobA) for mobilization was homologue to ColE1‐type plasmid (pAsa2) from Aer. salmonicida subsp. salmonicida. Additionally, the qnrS2 gene was part of a mobile insertion cassette element in the plasmid. Conclusions: Two plasmids were assumed to be the same plasmid, and this identification of a plasmid‐mediated qnrS2 gene from the two different strains underlines a possible diffusion of these resistance determinants in an aquaculture system. Significance and Impact of the Study: This is the first finding of the ColE‐type plasmid carrying the qnrS2 gene.     

Effect of Leuconostoc spp. on the formation of Streptococcus mutans biofilm

Insoluble glucans synthesized by Streptococcus mutans enhance the pathogenicity of oral biofilm by promoting the adherence and accumulation of cariogenic bacteria on the surface of the tooth. The objective of this study was to investigate the effect of Leuconostoc spp. on the in vitro formation of S. mutans biofilm. Three strains, Leuconostoc gelidum ATCC 49366, Leuconostoc mesenteroides ssp. cremoris ATCC 19254 and Leuconostoc mesenteroides ssp. mesenteroides ATCC 8293, were used in this study. They exhibited profound inhibitory effects on the formation of S. mutans biofilm and on the proliferation of S. mutans. The water-soluble polymers produced from sucrose were most strongly produced by L. gelidum, followed by L. mesenteroides ssp. cremoris and L. mesenteroides ssp. mesenteroides. The mean wet weights of the artificial biofilm of S. mutans were also significantly reduced as a result of the addition of the water-soluble polymers obtained from Leuconostoc cultures. According to the results of thin-layer chromatographic analysis, the hydrolysates of the water-soluble polymers produced by Leuconostoc were identical to those of dextran T-2000, forming predominately alpha-(1-6) glucose linkages. These results indicate that dextran-producing Leuconostoc strains are able to inhibit the formation of S. mutans biofilm in vitro.     

Influence of Lactobacillus spp. from an Inoculant and of Weissella and Leuconostoc spp. from Forage Crops on Silage Fermentation

Lactobacillus spp. from an inoculant and Weissella and Leuconostoc spp. from forage crops were characterized, and their influence on silage fermentation was studied. Forty-two lactic acid-producing cocci were obtained from forage crops and grasses. All isolates were gram-positive, catalase-negative cocci that produced gas from glucose, and produced more than 90% of their lactate in the d-isomer form. These isolates were divided into groups A and B by sugar fermentation patterns. Two representative strains from the two groups, FG 5 and FG 13, were assigned to the species Weissella paramesenteroides and Leuconostoc pseudomesenteroides, respectively, on the basis of DNA-DNA relatedness. Strains FG 5, FG 13, and SL 1 (Lactobacillus casei), isolated from a commercial inoculant, were used as additives to alfalfa and Italian ryegrass silage preparations. Lactic acid bacterium counts were higher in all additive-treated silages than in the control silage at an early stage of ensiling. During silage fermentation, inoculation with SL 1 more effectively inhibited the growth of aerobic bacteria and clostridia than inoculation with strain FG 5 or FG 13. SL 1-treated silages stored well. However, the control and FG 5- and FG 13-treated silages had a significantly (P < 0.05) higher pH and butyric acid and ammonia nitrogen contents and significantly (P < 0.05) lower lactate content than SL 1-treated silage. Compared with the control silage, SL 1 treatments reduced the proportion of d-(−)-lactic acid, gas production, and dry matter loss in two kinds of silage, but the FG 5 and FG 13 treatments gave similar values in alfalfa silages and higher values (P < 0.05) in Italian ryegrass silage. The results confirmed that heterofermentative strains of W. paramesenteroides FG 5 and L. pseudomesenteroides FG 13 did not improve silage quality and may cause some fermentation loss.Silage is now the most common preserved cattle feed in many countries, including Japan. It is well established that lactic acid bacteria (LAB) play an important role in silage fermentation. Epiphytic microflora, the microorganisms naturally present on forage crops, are responsible for silage fermentation and also influence silage quality (3, 11, 15). Lactobacilli and lactic acid-producing cocci, e.g., leuconostocs, lactococci, streptococci, pediococci, and Weissella species, are major components of the microbial flora in various types of forage crops (3). Stirling and Whittenbury (21) reported that leuconostocs were the most numerous and widely distributed on forages and that lactobacilli occurred mostly on grasses. Cai et al. (3) examined a large number of forage crops and grasses and also found that the predominant LAB were lactic acid-producing cocci and that lactobacilli were the least numerous and mostly homofermentative. Ruser (17) found that although all LAB groups were present in chopped-maize samples, homofermentative lactobacilli and heterofermentative leuconostocs were present in the highest numbers.In order to improve silage quality, many LAB-containing biological additives have been developed and are currently available (13, 20, 25). These inoculants may inhibit the growth of harmful bacteria and enhance lactic acid fermentation during ensiling periods. The epiphytic LAB influence the effectiveness of silage inoculants because the introduced bacteria must compete with these LAB (12). Therefore, the LAB species and their characteristics in the silage environment require further study. However, while an increasing number of studies have reported positive benefits from using some bacterial inoculants as silage additives, relatively few have reported the effect of epiphytic LAB, especially Leuconostoc and Weissella species, on silage fermentation. In the present study, the characterization of Leuconostoc and Weissella species isolated from forage crops and their influence on silage fermentation were examined.     

Insertional inactivation of a gene which controls expression of vancomycin resistance on plasmid pHKK100

Expression of inducible high level vancomycin resistance (Vmr) in enterococci appears to require other plasmid-encoded genes in addition to the previously described structural genes vanA and vanH. Tn917 mutagenesis was used to identify such a region in the Vmr plasmid pHKK100. Insertional inactivation of a 693-bp open reading frame upstream from vanH resulted in complete loss of Vmr. This putative 26,642-Da protein has been designated VanR.     

Dimerization of plasmid DNA accelerates selection for antibiotic resistance

Dimerization of multicopy plasmids is widely assumed to be disadvantageous both for plasmid maintenance and for the host cell. It is known that dimerization causes plasmid instability; dimer-containing cells grow slower than their monomer-containing counterparts. However, as we demonstrate here, under conditions of selective stress, dimers provide an advantage for bacteria. Dimers facilitate segregation of mutants from numerous copies of the parental plasmid. Accelerated segregation greatly increases the rate of accumulation of plasmids carrying mutations that are adaptive for bacteria. In contrast, resolution of dimers by site-specific recombination decreases, 10³-10⁵-fold, the efficiency of selection of spontaneous reversions in the tet gene of pBR327.