Isolation of transfer-negative nif-plasmids (pCE1) and their integration into the chromosome of Escherichia coli with the help of phage Mu

Summary Strain JC5466 of Escherichia coli K12 harbouring the nitrogen fixation plasmid pCE1 was lysogenized with bacteriophage Mu cts, followed by partial induction and infection with bacteriophage PRD1. This made it possible to obtain transfer-defective derivatives of pCE1, carrying Mu prophage. These derivatives could be mobilized by using the helper plasmid pME400 and it was possible to segregate the helper plasmid from the donor plasmid in the transconjugants.By incubating the strains 302 and 328 at 42°C, for induction of Mu prophage, derivatives with different plasmid contents could be obtained such as strains without plasmids, some with smaller or larger plasmids and others possessing plasmids without any visible alteration in size. Integration of the nitrogen-fixation (nif) genes into the chromosomes of the strains without plasmids and those containing a smaller plasmid, was confirmed by Southern hybridization using radioactive nifKDH DNA. Conjugation assays have shown that the plasmid is integrated into the chromosome as a unit but that it can also be excised.     

The maintenance affinities of KLF-1 proximal F merogenotes inEscherichia coli

Summary In conjugation with donor strains carrying proximal F merogenotes of KLF-1 type about 100-fold lower frequency ofLeu ⁺ orLac ⁺ recombinants was found. The determination of the level of β-galactosidase synthesis during the initial period of mating indicated that the transfer process of plasmid DNA was not impaired. Among the recombinants selected a large fraction have not expressed the plasmic fertility functions. This phenomenon was found to be replicon specific and was observed only with proximal F merogenotes but not with classical F'lac and F'ORF-1 elements or RI-19 plasmid. The expression of KLF-1 plasmid functions in the cell seems to be affected by a chromosomal gene of the proximal F merogenote closely linked toleu marker.     

Xanthomonas campestris as a host for the production of recombinantPseudomonas aeruginosa lipase

Recombinant plasmid pBP13, which expresses the alkaline lipase fromPseudomonas aeruginosa IGB83 under thetac promoter was transferred toXanthomonas campestris pvcampestris IBT148. Different fermentation conditions were tested for lipase productivity by strain IBT148 carrying plasmid pBP13, and a fermentation process was established in an instrumented bioreactor, where lipase production was increased more than 12-fold with respect to the initial culture conditions in shake flasks. Xanthan gum stabilized the activity of the alkaline lipase.     

Conjugal Transfer of Plasmid DNA fromEscherichia colito Enterococci: A Method to Make Insertion Mutations

Shuttle vector pAT18 was transferred by conjugation fromEscherichia coliS17-1 toEnterococcus faecalisOG1RF andEnterococcus faeciumSE34. Transfer was mediated by the transfer functions of plasmid RK2 inE. coliS17-1 and the origin of conjugal transfer (oriT) located on pAT18. TheoriTsequence was then inserted into two plasmids to generate vectors pTEX5235 and pTEX5236. These two vectors cannot replicate in gram-positive bacteria and can be used to make insertion mutants in gram-positive bacteria. An internal sequence from an autolysin gene ofE. faecalisOG1RF was cloned into pTEX5235 and transferred by conjugation fromE. coliS17-1 toE. faecalisOG1RF. The plasmid was found to integrate into the chromosome of OG1RF by a single crossover event, resulting in a disrupted autolysin gene. A cosmid carrying the pyrimidine gene cluster fromE. faecalis,with a transposon insertion inpyrC,was also transferred fromE. coliS17-1 toE. faecalisOG1RF. After selection for the transposon, it was found to have recombined into the recipient chromosome by a double crossover between the cosmid and the chromosome of OG1RF. This resulted in apyrCknockout mutant showing an auxotrophic phenotype.     

Transfer of plasmids fromEscherichia coli andPseudomonas aeruginosa to methylotrophic bacteria and their detection in the new hosts

Several plasmids of incompatibility group P were transferred fromEscherichia coli andPseudomonas aeruginosa strains toMethylophilus methylotrophus and two other methylotrophs to test their recipient ability. The presence of plasmids in transconjugants was confirmed by electrophoretic analysis. Optimal conditions for detection of plasmid DNA in the strains tested based on alkaline lysis of cells at elevated temperature were established. Special behaviour of plasmids carrying the Mu phage in methylotrophic hosts is described.     

Expression of virulence and antibiotic resistance in anEscherichia coli transconjugant carrying a large plasmid pCAT120 ofShigella dysenteriae type I and its spontaneous fragmentations

The role of a 120-kb plasmid in relation to virulence and drug resistance factor inShigella dysenteriae was studied. For characterization of plasmids, the mating system is a useful and efficient means of transferring both large and small plasmids to a new host. The conjugative transfer of a 120-kb (pCAT120) ampicillin-resistant plasmid ofS. dysenteriae toE. coli K-12 was not successful. Introduction of anE. coli fertility factor plasmid F, did not help to mobilize the plasmid. Low transfer frequencies of antibiotic markers toE. coli were achieved by treatment of the donorS. dysenteriae with N-methyl-N'-nitro-N-nitrosoguanidine. The transconjugants showed resistance to ampicillin, chloramphenicol, tetracycline and cadmium. A transconjugant carrying the 120-kb plasmid ofS. dysenteriae produced keratoconjunctivitis in guinea pigs. Repeated subculture of Clm^r transconjugant (pCAT120) on tryptic soya agar plates became Clm^S and showed four distinct DNA bands ranging from 3 to 10 kb in size on agarose gel electrophoresis. Utilization of organic acids, metal resistance (Cd), dye-binding properties (Crb⁺, Ebr⁺) and drug resistance (Amp, Tet) were identified on 10, 7, 4 and 3-kb plasmid DNA fragment of pCAT120 respectively. Crb⁺ 4-kb DNA fragment of pCAT120 was isolated, purified and transferred to an avirulentE. coli K12 by trans-formation. However, transformant (pET4) showed poor growth on solid media and its growth in liquid culture was only possible after supplementation of the unknown low-molar-mass thermolabile factor(s) secreted by the recipient strain. A 130-kDa outer membrane protein was synthesized by the transformant (pET4) carrying a 4-kb Congo red binding plasmid DNA fragment of pCAT120. A highly reduced rate of synthesis of a few low-molar-mass outer membrane proteins was also observed among the transformant (pET4) in relation to the recipient strain. Transconjugant carrying four plasmid DNA fragments of pCAT120 and Crb⁺ transformant (pET4) failed to produce keratoconjunctivitis in guinea pigs. Presented in part at the57th Annual Meeting of Society of Biological Chemists (India), New Delhi, October, 1988 (Abstr. No. 269 & 272) andIndo-UK Workshop on Diarrhoeal Diseases, Calcutta, January 1989 (Abstr. Page No. 215-217).     

Variable presence of the 1.94kb mitochondrial plasmid in maize S cytoplasm and its relationship to cytoplasmic male sterility

Mitochondrial DNA (mtDNA) was isolated from over 100 different maize nucleo-cytoplasmic combinations. DNA preparations were assayed for the presence of the 1.94kb mitochondrial plasmid by agarose gel electrophoresis and hybridization to a recombinant clone of the plasmid. The plasmid was present in all tested inbreds which carried N, male fertile, cytoplasm or the cytoplasmically male sterile (cms) groups,cms-T andcms-C. However, members of thecms-S group differed with respect to the presence of the plasmid. Cytoplasms I, J and S possessed the plasmid, whereas cytoplasms B, CA, D, G, H, IA, ME, ML, PS, RD and VG did not.Cms-S group lines which had spontaneously reverted to fertility (nuclear and cytoplasmic revertants) did not exhibit a concomitant change in 1.94kb plasmid levels, although all such lines showed the previously reported alteration in levels of the linear mtDNAs, S1 and S2. The presence or absence of the plasmid was not correlated with (i) frequency of reversion to fertility, (ii) the degree of male sterility expressed, (iii) the presence or absence of standard nuclear restorer to fertility genes and (iv) nuclear genotype. Latin American races carrying RU cytoplasm possessed the plasmid, as did sweet corn varieties. The relevance of the data tocms and evolution of thecms-S group is discussed.     

Increased efficiency of transformation ofBacillus stearothermophilus by a plasmid carrying a thermostable kanamycin resistance marker

Recombinant plasmids carrying either the wildtype kanamycin nucleotidyltransferase gene encoded originally by the mesophilic plasmid pUB110 or the gene encoding the thermostable TK101 mutant were constructed and introduced intoBacillus stearothermophilus by a protoplast transformation procedure. When kanamycin-resistant transformants were selected at 47°C, the transformation efficiency of the plasmid bearing the TK101 gene was nine times higher than that of the plasmid encoding the wildtype enzyme. The difference in transformation efficiencies between the two plasmids was increased when transformants were selected at higher temperatures, reflecting the difference in thermostabilities of the respective kanamycin nucleotidyltransferases. We conclude that, even though the pUB110 enzyme is sufficiently active at 47°C to confer kanamycin resistance toB. stearothermophilus, the additional stability of the TK101 mutant is advantageous in transformation ofB. stearothermophilus. The TK101 gene may also have broad utility as a marker for cloning vectors in other thermophiles.     

Isolation of nonsense suppressor mutants in Pseudomonas.

A strain of Escherichia coli harboring the drug resistance plasmid RP1 was treated with the mutagen N-methyl-N-nitro-N-nitro-N-nitrosoguanidine, and mutants were isolated in which ampicillin resistance had been lost due to an amber mutation in the plasmid. One of these mutants was again treated, and a strain was isolated in which tetracycline resistance was also lost due to an amber mutation in the plasmid. The plasmid containing amber mutations in the genes amp and tet was named pLM2. This plasmid could be transferred to strains of Pseudomonas aeruginosa, P. phaseolicola, and P. pseudoalcaligenes. Mutants resistant to ampicillin and tetracycline could not be obtained from P. phaseolicola carrying pLM2. However, strains of E. coli, P. aeruginosa, and P. pseudoalcaligenes carrying the plasmid did produce mutants simultaneously resistant to both antibiotics. All of the mutants of E. coli had developed nonsense suppressors since they became phenotypically lac+, although harboring a lac amber mutation, and formed plaques with amber mutants of phages PRR1 and PRD1 that attack organisms carrying RP1. Approximately 20% of the resistant mutants of P. aeruginosa and P. pseudoalcaligenes were sensitive to the amber mutant of PRD1. These mutants were of variable stability and grew somewhat more slowly than their parent strains. One of the suppressor mutants of P. pseudoalcaligenes, designated ERA(pLM2)S4, was used for the isolation of nonsense mutants of bacteriophage PHA6, a virus having a segmented genome of double-stranded ribonucleic acid and an envelope of lipids and proteins.     

Suppressor mutation in Pseudomonas aeruginosa.

Suppressor mutations were identified in Pseudomonas aeruginosa, and a comparison was made with Escherichia coli suppressor systems. A suppressor-sensitive (sus) derivative of a plasmid, RP4 trp, and several Sus mutants of IncP1 plasmid-specific phages, were isolated by using E. coli. Plasmid RP4 trp (sus) was transferred to P. aeruginosa strains carrying trp markers which did not complement RP4 trp(sus), and Trp+ variants were selected. Some, but not all such revertants, could propagate PRD1 Sus phages, and these mutants were found to be supressor positive. Plating efficiencies of various Sus phages on these strains were compared with on E. coli strains carrying known suppressor genes. The results suggested that the Pseudomonas suppressors were probably amber suppressors. In iddition, some Sus phages (PRD1sus-55, PRD1sus-56) were obtained which, although apparently of the amber type for E. coli, were able to propagate equally well on sup+ or sup strains of P. aeruginosa. On the other hand, several mutants of phage PRR1 which were suppressed in E. coli were not suppressed by the P. aeruginosa suppressor. Suppressor-sensitive mutants were also isolated with P. aeruginosa bacteriophages E79 and D3.     

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.     

Sequence Analysis of the Plasmid pRRI2 from the Rumen Bacterium Prevotella ruminicola 223/M2/7 and the Use of pRRI2 in Prevotella/Bacteroides Shuttle Vectors

pRRI2 is a small cryptic plasmid from the rumen bacterium Prevotella ruminicola 223/M2/7 which has been used for the construction of shuttle vectors (pRH3 and pRRI207) that replicate in many Bacteroides/Prevotella strains as well as in Escherichia coli. Sequence analysis of pRRI2 reveals that it is a 3240-bp plasmid carrying two clear open reading frames. Rep, encoded by ORF1, shows 48 and 47% amino acid sequence identity with RepA proteins from Bacteroides vulgatus and Bacteroides fragilis, respectively. ORF2, named Pre, shares 34% amino acid sequence identity with a putative plasmid recombination protein from the Flavobacterium spp. plasmid pFL1 and 30% amino acid sequence identity with BmpH from B. fragilis Tn5520. Disruption of ORF1 with HindIII prevents replication and maintenance in Bacteroides spp. hosts, but shuttle vectors carrying pRRI2 interrupted within ORF2, by EcoRI*, are able to replicate. pRRI2 shows no significant similarity with the only other P. ruminicola plasmid to have been studied previously, pRAM4.     

Characterization of the mutant recombinant strainPseudomonas putida IPM-36 exhibiting anticipating growth on a medium containing an inducer of thecry3A gene expression

Induction of the expression of the 8-endotoxin gene fromBacillus thuringiensis var.tenebrionis in the recombinant strainPseudomonas putida IPM-36 negatively affected the viability and the growth rate of the culture. In order to optimize the insecticide production by the recombinant strain, mutant clones exhibiting anticipating growth on an inducer-containing medium were selected and studied. These clones differed in such aspects as the localization of mutations (either in plasmid pBTN11, carrying thecry3A gene, or in the chromosome), growth rate, or the level of δ-endotoxin synthesis after induction. Several obtained mutants proved much superior toP. putida IPM-36 in their structural and segregation stability, although they were as efficient as the original strain with respect to the production of the insecticide protein Cry3A.     

Occurrence of plasmid DNA in the sepiolid squid symbiontVibrio fischeri

Because of the importance of plasmids in many bacterial associations with plants and animals, we determined the occurrence and distribution of plasmid DNA in symbioticVibrio fischeri from the light organ of the sepiolid squidEuprymna scolopes. Analyses of 225 isolates of symbioticV. fischeri from 25 individual squids revealed an overall plasmid-carriage rate of 56%. A large plasmid (39 kb) was detected in 96% of those isolates carrying plasmids, and multiple small plasmids were found to co-occur with one of the large plasmids in 81% of plasmid-carrying strains. In addition, these plasmids appear to be restricted toV. fischeri strains isolated fromE. scolopes and from seawater at sites of squid populations. We were unable to assign a role or function to these plasmids, but they do not carry genes required for the establishment of the light organ symbiosis. We conclude that the essential bacterial symbiotic determinants must be encoded on the chromosome and that the plasmids may carry genes that are important for the survival of theseV. fischeri strains outside of the symbiotic association.     

The Level of Expression of α-toxin by Different Strains ofClostridium perfringensis Dependent on Differences in Promoter Structure and Genetic Background

The control of expression of the α-toxin gene (cpaorplc) ofClostridium perfringenshas been studied in three strains shown to have high (NCTC8237), intermediate (strain 13) and low (NCTC8533) phospholipase C activity in the culture supernatant. The phospholipase C activity was shown to be related tocpamRNA levels. Primer extension studies were performed to locate thecpapromoter regions in strains NCTC8237 and 13. Differences in promoter sequences could account for the differences in α-toxin production between strains 13 and NCTC8237. In contrast, the differences in α-toxin production between strains NCTC8237 and NCTC8533 were unlikely to be due to promoter differences because the upstream promoter-containing sequences were identical in these strains. The recombinant plasmid carrying the NCTC8237cpagene was introduced into strains 13 and NCTC8533. The level of production of the α-toxin was 16-fold higher in strain 13, indicating the presence of strain-dependant regulatory systems.     

R transfer toS. panama in vitro and in vivo

Thein-vitro and thein-vivo transfer frequencies ofE.coli 50 (R1) carrying a phage-restricting R factor, and ofE.coli 71 (R2) carrying a non-restricting R factor, were measured. Thein-vitro transfer frequencies were found to be greatly dependent on the method of conjugation employed. The transfer,in vivo, of R factor R2 toS.panama was slightly more efficient than was the transfer of R1.     

Characterization of conjugative plasmids carrying antibiotic resistance genes encoding 16S rRNA methylase, extended-spectrum beta-lactamase, and/or plasmid-mediated AmpC beta-lactamase

In this study, we identified extended-spectrum β-lactamase (ESBL) and plasmid-mediated AmpC β-lactamase which were associated with 16S rRNA methylase gene on the conjugative plasmid. Among 82 clinical isolates of Enterobacteriaceae that carry 16S rRNA methylase gene (64 strains, armA, and 18 strains, rmtB), bla _SHV-12 was detected either alone or combined with bla _DHA-1, bla _CTX-M-3, and bla _CTX-M-14 in 30 strains carrying armA and 6 strains carrying rmtB. The bla _CTX-M-3 was detected in 13 of 64 strains carrying armA but no strains carrying rmtB. Whereas bla _CTX-M-14 was detected in 15 of 18 strains carrying rmtB but only 2 of 64 strains carrying armA. Overall, bla _SHV-12 and bla _CTX-M-14 was the most common ESBL gene which was associated with armA and rmtB, respectively. In addition, we found that bla _CTX-M-3 localized with armA on the same IncL/M plasmid and bla _CTX-M-14 localized with rmtB on the same IncA/C plasmid. Restriction fragment length polymorphism of conjugative plasmids and pulsed-field gel electrophoresis of genomic DNAs revealed that intercellular horizontal transfer of conjugative plasmid and clonal transmission have been occurred at the same time.     

Construction ofClostridium butyricum hybrid plasmids and transfer toBacillus subtilis

Summary Small plasmids were isolated from type strains ofClostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these crypticC. butyricum plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into theE. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from theStaphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance toBacillus subtilis. Hybrid plasmids (pHL105, pHL1051) derived from pCBU2 were identified, which are capable of replication and expression of theS. aureus drug resistance marker in bothE. coli andB. subtilis. No structural instability was detected upon retransformation of pHL105 fromB. subtilis intoE. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer betweenE. coli and a wide range of bacilli and clostridia.     

Cloning of a plasmid region specifying the N transfer system of bacterial conjugation in Escherichia coli

HindIII restriction sites were created artificially by the insertion of the transposon Tn.5 into the IncN plasmid pCU1 near a presumptive end of its conjugal transfer region (tra). This allowed cloning of an entire and continuous 19.4-kb region of this plasmid that specifies the N transfer system. The cloning vector was the nonconjugative plasmid pACYC184. The recombinant plasmid was as efficient in transfer as the parental N plasmid. Other clones and deletions extending into the tra region allowed localization of a 11.2-kb segment of this region that determines sensitivity to the N-specific bacteriophages IKe and PRD1. It could also be concluded that the ability of pCU1 to promote the killing of Klebsiella pneumoniae requires a 2-kb region that is not part of, but adjacent to the tra region.