Characterization and molecular cloning in Escherichia coli of a plasmid from the mollicute Spiroplasma citri

Two plasmids, pMH1 with 7 kilobase pairs and pM41 with 8 kilobase pairs, were purified from the plant pathogen Spiroplasma citri and characterized by restriction mapping. Upon in vitro DNA recombination with plasmid pBR328 as a vector, we have cloned pMH1 in Escherichia coli. A radioactive probe obtained upon nick translation of the recombinant plasmid was used to further characterize and compare pMH1 and pM41.     

Identification of a single base change in ribosomal RNA leading to erythromycin resistance.

The molecular basis of a mutation conferring an erythromycin-resistance phenotype was explored, as an approach to the role of 23 S rRNA in the peptidyl-transferase activity of 50 S ribosomal subunits. Mutagenization of an Escherichia coli strain, which carried the multicopy plasmid pLC7-21 containing the rrnH operon, led to the production of an erythromycin-resistant strain. Plasmid pBFL1 isolated from this mutant was able to transform the sensitive RecA- strain EM4 and to induce a "dissociated" type of antibiotic resistance. Two ribosome populations occurred in EM4/pBFL1: normal particles coded for by the seven rrn chromosomal genes and mutated particles containing rRNA of plasmid origin. The latter particles displayed in vitro lower affinity and susceptibility to erythromycin than wild type particles. The mutation within plasmid pBFL1 was mapped by a multiple primer extension technique. Three synthetic primers were used to sequence the central loop in domain V of 23 S rRNA, leading to identification of a C to U transition at position 2611. This base change was proved to be responsible for the erythromycin-resistance phenotype by the plasmid-plasmid marker rescue technique. A molecular explanation for the rrn mutations leading, respectively, to undissociated and to dissociated types of resistance to the MLSb (macrolide-lincosamide-synergimycin B) group of antibiotics is proposed. These results and some literature data support the notion that rRNA bases involved in antibiotic resistance play a conformational role in the ribosomal binding sites for the MLSb antibiotics.     

Incompatibility behavior of a symbiotic plasmid pMH7653Rb in Mesorhizobium huakuii 7653R

Mesorhizobium huakuii strain 7653R harbored two indigenous plasmids named pMH7653Ra and pMH7653Rb.The larger plasmid pMH7653Rb (symbiotic plasmid) was transferred to M.huakuii HN308SR harboring three plasmids: pMHHN308a,pMHHN308b and pMHHN308c,and HN3015SR harboring three plasmids: pMHHN3015a,pMHHN3015b and pMHHN3015c by tri-parent mating.Two stable indigenous plasmids,pMHHN308b and pMHHN308c of HN308SR,were co-eliminated due to the introduction of pMH7653Rb,and the transconjugant was named HN308SRN14.The results implied that pMH7653Rb and pMHHN308b,pMHHN308c were incompatible and might have been ascribed to the same incompatible group.The plasmid profiles of transconjugant HN3015SRN14 showed that the second largest plasmid pMHHN3015b of HN3015SR was cured due to the introduction of pMH7653Rb.The results also implied that pMH7653Rb and pMHHN3015b were incompatible.Results from plant nodulation tests showed that pMH7653Rb could only maintain the nodulation ability in transconjugant HN308SRN14 and its nodule number was more than that of wild strain HN308SR,but could not replace the nitrogen fixation effect of pMHHN308b and pMHHN308c.The plasmid cured mutant HN308SRN14D harboring only pMHHN308a formed null nodules that demonstrated pMHHN308a was relevant to nodulation ability.HN3015SRN14 harboring pMH7653Rb,pMHHN3015a and pMHHN3015c formed null nodules while HN3015SRN14D containing pMHHN3015a and pMHHN3015c lost the nodulation ability.The plasmid replication repC-like gene sequences were detected by a polymerase chain reaction from 7653R,HN308,HN3015,HN308SRN14 and HN3015SRN14.The repC gene sequence similarities of the strains tested attained 99%.     

Biochemical transformation of thymidine kinase (TK)-deficient mouse cells by herpes simplex virus type 1 DNA fragments purified from hybrid plasmids.

The thymidine kinase (TK) gene of HSV-1 has been cloned in Escherichia coli K12 plasmids, pMH1, pMH1A, and pMH4. These plasmids contain a 1,92Obp HSV-1 TK DNA sequence, which replaces a 2,067 bp EcoR I to Pvu II sequence of plasmid pBR322 DNA. Superhelical DNAs of plasmids pMH1, pMH1A, and pMH4 as well as plasmid DNAs cleaved by EcoR I, Hinc II, Bg1 II, Sma I, and Pvu II transformed TK-deficient LM(TK-) cells to the TK+ phenotype. A 1,230bp EcoR I-Sma I fragment purified from pMH1 DNA (and from plasmid pAGO, DNA, the parent of pMH1) also transformed LM(TK-) cells. Serological and disc PAGE studies demonstrated that the TK activity expressed in biochemically transformed cells were HSV-1-specific. The experiments suggest that the HSV-1 TK coding region may be contained within a l.1kbp DNA sequence extending from about the Hinc II (or Bgl II) cleavage site to the Sma I site. 35S-methionine labeling experiments carried out on cell lines transformed by Hinc II-cleaved pMH1 DNA and by the EcoR I-Sma I fragment showed that the TKs purified from the transformed cells consisted of about 39-40,000 dalton polypeptides.     

Incompatibility behavior of a symbiotic plasmid pMH7653Rb in <Emphasis Type="Italic">Mesorhizobium huakuii</Emphasis> 7653R

Mesorhizobium huakuii strain 7653R harbored two indigenous plasmids named pMH7653Ra and pMH7653Rb. The larger plasmid pMH7653Rb (symbiotic plasmid) was transferred to M. huakuii HN308SR harboring three plasmids: pMHHN308a, pMHHN308b and pMHHN308c, and HN3015SR harboring three plasmids: pMHHN3015a, pMHHN3015b and pMHHN3015c by tri-parent mating. Two stable indigenous plasmids, pMHHN308b and pMHHN308c of HN308SR, were co-eliminated due to the introduction of pMH7653Rb, and the transconjugant was named HN308SRN14. The results implied that pMH7653Rb and pMHHN308b, pMHHN308c were incompatible and might have been ascribed to the same incompatible group. The plasmid profiles of transconjugant HN3015SRN14 showed that the second largest plasmid pMHHN3015b of HN3015SR was cured due to the introduction of pMH7653Rb. The results also implied that pMH7653Rb and pMHHN3015b were incompatible. Results from plant nodulation tests showed that pMH7653Rb could only maintain the nodulation ability in transconjugant HN308SRN14 and its nodule number was more than that of wild strain HN308SR, but could not replace the nitrogen fixation effect of pMHHN308b and pMHHN308c. The plasmid cured mutant HN308SRN14D harboring only pMHHN308a formed null nodules that demonstrated pMHHN308a was relevant to nodulation ability. HN3015SRN14 harboring pMH7653Rb, pMHHN3015a and pMHHN3015c formed null nodules while HN3015SRN14D containing pMHHN3015a and pMHHN3015c lost the nodulation ability. The plasmid replication repC-like gene sequences were detected by a polymerase chain reaction from 7653R, HN308, HN3015, HN308SRN14 and HN3015SRN14. The repC gene sequence similarities of the strains tested attained 99%.     

Integrative and free Spiroplasma citri oriC plasmids: expression of the Spiroplasma phoeniceum spiralin in Spiroplasma citri.

The replication region (oriC) of the Spiroplasma citri chromosome has been recently sequenced, and a 2-kbp DNA fragment was characterized as an autonomously replicating sequence (F. Ye, J. Renaudin, J. M. Bové, and F. Laigret, Curr. Microbiol. 29:23-29, 1994). In the present studies, we have combined this DNA fragment, containing the dnaA gene and the flanking dnaA boxes, with a ColE1-derived Escherichia coli replicon and the Tet M determinant, which confers resistance to tetracycline. The recombinant plasmid, named pBOT1, was introduced into S. citri cells, in which it replicated. Plasmid pBOT1 was shuttled from E. coli to S. citri and back to E. coli. In S. citri, replication of pBOT1 did not require the presence of a functional dnaA gene on the plasmid. However, the dnaA box region downstream of the dnaA gene was essential. Upon passaging of the S. citri transformants, the plasmid integrated into the spiroplasmal host chromosome by recombination at the replication origin. The integration process led to duplication of the oriC sequences. In contrast to the integrative pBOT1, plasmid pOT1, which does not contain the E. coli replicon, was stably maintained as a free extrachromosomal element. Plasmid pOT1 was used as a vector to introduce into S. citri the G fragment of the cytadhesin P1 gene of Mycoplasma pneumoniae and the spiralin gene of Spiroplasma phoeniceum. The recombinant plasmids, pOTPG with the G fragment and pOTPS with the spiralin gene, were stably maintained in spiroplasmal transformants. Expression of the heterologous S. phoeniceum spiralin in S. citri was demonstrated by Western immunoblotting.     

Conjugal Transfer of Lactose-Fermenting Ability Among Streptococcus cremoris and Streptococcus lactis Strains

Streptococcus cremoris C3 was found to transfer lactose-fermenting ability to LM2301, a Streptococcus lactis C2 lactose-negative streptomycin-resistant (Lac⁻ Str^r) derivative which is devoid of plasmid deoxyribonucleic acid (DNA); to LM3302, a Lac⁻ erythromycin-resistant (Ery^r) derivative of S. lactis ML3; and to BC102, an S. cremoris B₁ Lac⁻ Ery^r derivative which is devoid of plasmid DNA. S. cremoris strains R1, EB₇, and Z8 were able to transfer lactose-fermenting ability to LM3302 in solid-surface matings. Transduction and transformation were ruled out as mechanisms of genetic transfer. Chloroform treatment of donor cells prevented the appearance of recombinant clones, indicating that viable cell-to-cell contact was responsible for genetic transfer. Transfer of plasmid DNA was confirmed by agarose gel electrophoresis. Transconjugants recovered from EB₇ and Z8 matings with LM3302 exhibited plasmid sizes not observed in the donor strains. Transconjugants recovered from R1, EB₇, and Z8 matings with LM3302 were able to donate lactose-fermenting ability at a high frequency to LM2301. In S. cremoris R1, EB₇, and Z8 matings with LM2301, streptomycin resistance was transferred from LM2301 to the S. cremoris strains. The results confirm genetic transfer resembling conjugation between S. cremoris and S. lactis strains and present presumptive evidence for plasmid linkage of lactose metabolism in S. cremoris.     

Characterization of the plasmids comprising the “R Factor” R5 and their relationships to other R plasmids

The “R factor” R5 confers resistance to tetracycline (Tc), streptomycin (Sm) and spectinomycin (Sp), chloramphenicol (Cm), sulfonamides (Su), kanamycin (Km), and mercuric ion (Mer). This phenotype is mediated by the presence of two R plasmids: pMH1 and pMH2, having approximate weights of 18.5 and 62 megadaltons (Mdal), respectively. pMH1 encodes Sm, Su, Cm, Mer, and Km resistance, and is nonconjugative. pMH2 confers Sm, Su, Cm, Mer, and Tc resistance, is conjugative, and belongs to the FII incompatibility group. NR79 is a 63-Mdal R plasmid encoding the same resistances as “R5,” and was derived from the same geographical source. It belongs to the FII incompatibility group and is conjugative. Analysis of restriction endonuclease digestion patterns and polynucleotide sequence homologies indicate that pMH1, pMH2, and NR79 are closely related. In addition, pMH2 and NR79 exhibit nearly complete homology to R100. Restriction endonuclease maps and resistance gene locations for pMH1, pMH2, and NR79 have been derived and a model for the evolutionary relationships of these plasmids is presented.     

Conjugative R-plasmid of facultative methylotrophic bacteria Pseudomonas sp. M

50 Md conjugative plasmid, designated pM3, has been found in the cells from natural isolates of Pseudomonas sp M. The plasmid determines the resistance to tetracycline and streptomycin and is capable of conjugative transfer between the cells of Pseudomonas and Escherichia coli. The conjugative derivatives of pM3 deleted for 14 Md of molecular mass were isolated after acridine dyes treatment of cells harbouring plasmid pM3. The discovered plasmid was not shown to belong to IncP1 incompatibility group.     

Gene disruption through homologous recombination in Spiroplasma citri: an scm1-disrupted motility mutant is pathogenic

To determine whether homologous recombination could be used to inactivate selected genes in Spiroplasma citri, plasmid constructs were designed to disrupt the motility gene scm1. An internal scm1 gene fragment was inserted into plasmid pKT1, which replicates in Escherichia coli but not in S. citri, and into the S. citri oriC plasmid pBOT1, which replicates in spiroplasma cells as well as in E. coli. Electrotransformation of S. citri with the nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In contrast, spiroplasmal transformants were obtained with the replicative, pBOT1-derived plasmid pCJ32. During passaging of the transformants, the plasmid was found to integrate into the chromosome by homologous recombination either at the oriC region or at the scm1 gene. In the latter case, plasmid integration by a single crossover between the scm1 gene fragment carried by the plasmid and the full-length scm1 gene carried by the chromosome led to a nonmotile phenotype. Transmission of the scm1-disrupted mutant to periwinkle (Catharanthus roseus) plants through injection into the leafhopper vector (Circulifer haematoceps) showed that the motility mutant multiplied in the insects and was efficiently transmitted to plants, in which it induced symptoms similarly to the wild-type S. citri strain. These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, the S. citri oriC plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination in S. citri, a mollicute which probably lacks a functional RecA protein.     

Susceptibility of Pediococcus spp. to antimicrobial agents

AIM: To investigate the susceptibility of Pediococcus species to antimicrobial agents. METHODS AND RESULTS: The susceptibility to 14 antimicrobial agents of 31 genotypically distinct strains of six Pediococcus species was assessed by using Etests on ISO-sensitest agar supplemented with horse blood. The species included were Pediococcus acidilactici, Pediococcus damnosus, Pediococcus dextrinicus, Pediococcus inopinatus, Pediococcus parvulus and Pediococcus pentosaceus. For several antimicrobial agents, some species were more susceptible than others. The two industrially important species, P. acidilactici and P. pentosaceus, differed with respect to erythromycin and trovafloxacin susceptibility, and in general both species had higher minimum inhibitory concentrations than the other species. In an erythromycin-resistant P. acidilactici, an erythromycin resistance methylase B [erm(B)] gene was identified by PCR. Using a plasmid preparation from strain P. acidilactici 6990, a previously erythromycin-sensitive Lactococcus lactis strain was made resistant. Transformants harboured a single plasmid, sized at 11.6 kb through sequence analysis. In addition, the erm(B) gene was identified within the plasmid sequence. CONCLUSIONS: The phenotypic test indicated the absence of acquired antimicrobial resistance genes in 30 of the strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These results will help in selection of the best Pediococcus strains for use as starter cultures.     

Identification of a locus involved in meningococcal lipopolysaccharide biosynthesis by deletion mutagenesis

A novel method for insertion/deletion mutagenesis in meningococci was devised. This consisted of ligating a digest of total chromosomal DNA to a 1.1 kb restriction fragment containing an erythromycin-resistance marker ( ermC ), and subsequent transformation of the ligation mixture into the homologous meningococcal strain H44/76. Southern blotting of a number of the resulting erythromycin-resistant transformants demonstrated that all carried the ermC gene inserted at different positions in the chromosome. Mutants with a specific phenotype were identified by screening with the anti-lipopolysaccharide (LPS) monoclonal antibody MN4A8B2, which is specific for immunotype L3. In this way, two independent L3-negative mutant strains were isolated. In transformation experiments with chromosomal DNA from these mutants, erythromycin-resistance and lack of MN4A8B2 reactivity were always linked, showing that the insertion/deletion was in a locus involved in LPS biosynthesis. On SDS–PAGE, the mutant LPS displayed an electrophoretic mobility intermediate between that produced by the previously isolated galE and rfaF mutant strains. Chemical analysis of the mutant LPS revealed that the structure was probably lipid A–(KDO)₂–(Hep)₂. Chromosomal DNA flanking the ermC insertion in these two mutant strains was cloned, and used as probe for the isolation of the corresponding region of the wild-type strain. From hybridization and polymerase chain reaction (PCR) analysis, it could be concluded that both mutations map to the same locus. The affected gene probably encodes the glycosyltransferase necessary for adding N -acetylglucosamine to heptose.     

Characterization of the recA gene regions of Spiroplasma citri and Spiroplasma melliferum.

In previous studies (A. Marais, J. M. Bove, and J. Renaudin, J. Bacteriol. 178:862-870, 1996), we have shown that the recA gene of Spiroplasma citri R8A2 was restricted to the first 390 nucleotides of the N-terminal part. PCR amplification and sequencing studies of five additional strains of S. citri have revealed that these strains had the same organization at the recA region as the R8A2 strain. In contrast to S. citri, Spiroplasma melliferum was found to contain a full-length recA gene. However, in all five S. melliferum strains tested, a TAA stop codon was found within the N-terminal region of the recA reading frame. Our results suggest that S. melliferum, as well as S. citri, is RecA deficient. In agreement with the recA mutant genotype of S. citri and S. melliferum, we have shown that these organisms are highly sensitive to UV irradiation.     

Effect of Bacillus subtilis BsuM restriction-modification on plasmid transfer by polyethylene glycol-induced protoplast fusion

Polyethylene glycol (PEG)-induced cell fusion is a promising method to transfer larger DNA from one cell to another than conventional genetic DNA transfer systems. The laboratory strain Bacillus subtilis 168 contains a restriction (R) and modification (M) system, BsuM, which recognizes the sequence 5'-CTCGAG-3'. To study whether the BsuM system affects DNA transfer by the PEG-induced cell fusion between R(+)M(+) and R(-)M(-) strains, we examined transfer of plasmids pHV33 and pLS32neo carrying no and eight BsuM sites, respectively. It was shown that although the transfer of pLS32neo but not pHV33 from the R(-)M(-) to R(+)M(+) cells was severely restricted, significant levels of transfer of both plasmids from the R(+)M(+) to R(-)M(-) cells were observed. The latter result shows that the chromosomal DNA in the R(-)M(-) cell used as the recipient partially survived restriction from the donor R(+)M(+) cell, indicating that the BsuM R(-)M(-) strain is useful as a host for accepting DNA from cells carrying a restriction system(s). Two such examples were manifested for plasmid transfer from Bacillus circulans and Bacillus stearothermophilus strains to a BsuM-deficient mutant, B. subtilis RM125.     

Characterization of a plasmid from moderately halophilic eubacteria.

A plasmid has been isolated for the first time from moderately halophilic eubacteria. Halomonas elongata, Halomonas halmophila, Deleya halophila and Vibrio costicola were found to harbour an 11.5 kbp plasmid (pMH1). The plasmid was isolated and characterized after transformation into Escherichia coli JM101 cells. A restriction map was constructed, and unique restriction sites for EcoRI, EcoRV and ClaI were detected. The occurrence of such a plasmid in the original halophilic strains was confirmed by Southern hybridization. The plasmid carries genetic determinants that mediate resistance to kanamycin, tetracycline, and neomycin. This property, together with its relatively small size, its stability in E. coli cells, and the presence of unique restriction sites, makes pMH1 a good candidate for the development of a cloning vector for moderate halophiles.     

Spiroplasma citri virus SpV1-derived cloning vector: deletion formation by illegitimate and homologous recombination in a spiroplasmal host strain which probably lacks a functional recA gene.

We have previously described the use of the replicative form (RF) of Spiroplasma citri virus SpV1 as a vector for expressing an epitope of the P1 adhesin protein from Mycoplasma pneumoniae in S. citri (A. Marais, J. M. Bové, S.F. Dallo, J. B. Baseman, and J. Renaudin, J. Bacteriol. 175:2783-2787, 1993). We have now studied the structural instability of the recombinant RF leading to loss of the DNA insert. Analyses of viral clones with deletions have shown that both illegitimate and homologous recombination were involved in deletion formation. For one such clone, deletion has occurred via a double crossing-over exchange between the circular free viral RF and SpV1 viral sequences present in the S. citri host chromosome. The homologous recombination process usually requires the RecA protein. However, characterization of the recA gene of the S. citri R8A2 host strain revealed that over two-thirds of the open reading frame of the recA gene was deleted from the C-terminal part, indicating that this particular strain is probably RecA deficient.     

Improved method for electroporation of Staphylococcus aureus

We have developed a significantly improved method for the electroporation of plasmid DNA into Staphylococcus aureus. The highest transformation efficiency achieved with this procedure was 4.0 x 10(8) transformants per microgram of plasmid pSK265 DNA. This represents a 530-fold improvement over the previously reported optimum efficiency of 7.5 x 10(5) transformants per microgram of plasmid DNA after electroporation of S. aureus cells [9]. Identical results were obtained when electrocompetent cells, which had been stored frozen at -80 degrees C, were used. The improved efficiency is due primarily to the use of a modified medium (designated as B2 medium) and secondarily to the use of 0.1-cm cuvettes. Several other plasmids (pI258, pMH109, and pSK270) were also electrotransformed into competent cells using our procedure, and for each plasmid, the transformation efficiency was significantly reduced compared to that observed when pSK265 DNA was used. With respect to plasmid pI258, the transformation efficiency was 3500-fold higher than that reported previously for transformation of this plasmid into S. aureus RN4220 [9]. The optimized electroporation procedure was less successful in transforming other staphylococci. Electrocompetent cells of S. aureus ATCC 29213 and S. epidermidis ATCC 12228 produced 5.5 x 10(5) and 5 x 10(3) transformants per microgram of pSK265 DNA, respectively.