Bacteria in the gular and paracloacal glands of the American alligator (Alligator mississippiensis; Reptila, Crocodilia)

Plasmid DNA of six strains of Rhizobium galegae was blotted onto nitrocellulose and hybridized with the 4.8 kb PstI fragment of pRme4lb, a megaplasmid carrying the nifH and the nifD genes of Rhizobium meliloti. DNA sequences homologous to the nif genes were localized on the megaplasmid or on the large plasmid bands of the R. galegae strains tested. In three of the strains analysed the nif genes were located on the megaplasmids. In the other three strains investigated, which also possessed megaplasmids, the nif genes were located on the smaller plasmids.     

Physiological and biochemical analysis of the transformants of aerobic methylobacteria expressing the dcm A gene of dichloromethane dehydrogenase

The transformants of Methylobacterium dichloromethanicum DM4 (DM4-2cr-/pME8220 and DM4-2cr-/pME8221) and of Methylobacterium extorquens AM1 (AM1/pME8220 and AM1/pME8221) that express the dcm A gene of dichloromethane dehalogenase undergo lysis when incubated in the presence of dichloromethane and are sensitive to acidic shock. The lysis of the transformants was found to be related neither to the accumulation of Cl- ions, CH2O, and HCOOH, nor to the impairment of glutathione synthesis or to the maintenance of intracellular pH. The (exo-) Klenow fragment-mediated incorporation of [alpha-32P]dATP into the DNA of the transformants DM4-2cr-/pME8220 and AM1/pME8220 was considerably greater when the transformed cells were incubated with CH2Cl2 than when they were incubated with CH3OH, indicating the occurrence of a significant increase in the total length of gaps. At the same time, the strain AM1 (which lacks dichloromethane dehalogenase) and the dichloromethane-degrading strain DM4 incubated with CH2Cl2 showed an insignificant increase in the total length of the gaps. The transformed cells are likely to lyse due to the relatively inefficient repair of DNA lesions that are induced in response to the alkylating action of S-chloromethylglutathione, an intermediate product of CH2Cl2 degradation. The data obtained suggest that the bacterial mineralization of dichloromethane requires an efficient DNA repair system.     

Transposon-mediated integration of the RP1 plasmid into chromosomes of methylotrophic bacteria

The homology region between the DNA of plasmid RP1ts::Tn601 and chromosome of the thermotolerant methylotrophic bacterium Methylobacterium sp. SKF240 has been constructed by transposon Tn601 translocation into the chromosome. The clones of Methylobacterium sp. SKF240 having integrated the plasmid RP1 into the chromosome have been obtained by conjugation on the basis of above mentioned genetic technique. The integration of plasmid RP1 into the chromosomal DNA of the methylotroph has been confirmed by the genetic and electrophoretic methods. Clones harbouring the integrated plasmid are able to transfer the chromosomal genes for methionine and isoleucine-valine synthesis to the recipient cells of P. aeruginosa PAO ML4262 by conjugation.     

Integration of replication-defective R68.45-like plasmids into the Pseudomonas aeruginosa chromosome

R68.45 and other similar broad-host-range (IncP) plasmids carrying a tandem repeat of the 2.1 kb insertion element IS21 mobilize the chromosome of many different Gram-negative bacteria. To analyse the structure of R68.45-chromosome cointegrates, whose involvement in the mobilization process had been postulated previously, we selected for the stable integration of R68.45-like plasmids into the Pseudomonas aeruginosa chromosome. Two plasmids were chosen: pME28, a transfer-deficient, mobilizable RP1 derivative with an inactive replication control (trfA) gene, and pME487, an R68.45 derivative with a trfA(ts) mutation causing temperature-sensitive replication. Chromosomally integrated pME28 and pME487 were found to be flanked by single IS21 elements. This structure is in agreement with a 'cut-and-paste' mode of R68.45 transposition. pME28 and pME487 showed a low specificity of insertion but rarely (less than 0.1%) induced auxotrophic mutations. Hfr (high-frequency-of-recombination) donors of P. aeruginosa could be obtained by chromosomal integration of pME487 or pME28; in the latter case, the transfer functions lacking from pME28 had to be provided in trans on an autonomous plasmid. Hfr donors gave higher conjugational linkage and transferred longer stretches of the P. aeruginosa chromosome than did R68.45 donors. This suggests that the integration of R68.45 into the donor chromosome is short-lived in P. aeruginosa.     

Physiological and Biochemical Analysis of the Transformants of Aerobic Methylobacteria Expressing the dcmA Gene of Dichloromethane Dehalogenase

Transformants of Methylobacterium dichloromethanicum DM4 (DM4-2cr^–/pME 8220 and DM4-2cr^–/pME8221) and of Methylobacterium extorquens AM1 (AM1/pME8220 and AM1/pME8221) that express the dcm A gene of dichloromethane dehalogenase undergo lysis when incubated in the presence of dichloromethane and are sensitive to acidic shock. The lysis of the transformants was found to be related neither to the accumulation of Cl^– ions, CH₂O, or HCOOH, nor to the impairment of glutathione synthesis or to the disturbance of intracellular pH homeostasis. The (exo^–) Klenow fragment–mediated incorporation of [-³²P]dATP into the DNA of the transformants DM4-2cr^–/pME8220 and AM1/pME8220 was considerably greater when the transformed cells were incubated with CH₂Cl₂ than when they were incubated with CH₃OH, indicating the occurrence of a significant increase in the total length of gaps. At the same time, the strain AM1 (which lacks dichloromethane dehalogenase) and the dichloromethane-degrading strain DM4 incubated with CH₂Cl₂ showed an insignificant increase in the total length of the gaps. The transformed cells are likely to lyse due to the relatively inefficient repair of DNA lesions that are induced in response to the alkylating action of S-chloromethylglutathione, an intermediate product of CH₂Cl₂ degradation. The data obtained suggest that the bacterial mineralization of dichloromethane requires an efficient DNA repair system.     

Detection of nif Genes in Nitrogen-Fixing Bacterial Isolate from Tomato (Lycopersicon esculentum Mill cv Pusa Ruby)

Nitrogen-fixing bacterial isolate from the intercellular spaces of tomato root cortical cells was studied for the location of nif genes on the chromosomal or plasmid DNA. The bacterial isolate showed two plasmids of approximate molecular sizes of 220 and 120 kb. Klebsiella pneumoniae nif HDK probe hybridized with the chromosomal DNA and not with the plasmid DNA thereby showing that nif genes are localised on the chromosomal DNA.     

Changes of chlorine isotope composition characterize bacterial dehalogenation of dichloromethane

Fractionation of dichloromethane (DCM) molecules with different chlorine isotopes by aerobic methylobacteria Methylobacterium dichloromethanicum DM4 and Albibacter nethylovorans DM10; cell-free extract of strain DM4; and transconjugant Methylobacterium evtorquens Al1/pME 8220, expressing the dcmA gene for DCM dehalogenase but unable to grow on DCM, was studied. Kinetic indices of DCM isotopomers for chlorine during bacterial dehalogenation and diffusion were compared. A two-step model is proposed, which suggests diffusional DCM transport to bacterial cells.     

An improved method for rapid purification of covalently closed circular plasmid DNA over a wide size range

An improved method has been developed for the large-scale purification of covalently closed circular (CCC) plasmid DNA molecules of sizes ranging from 4·3 to 73 kb. This protocol uses an alkaline-lysis procedure followed by acid-phenol extraction but with several modifications to previously reported methods. The principal modification is the replacement of NaCl by MgCl₂ in the extraction buffer to improve yield and to remove chromosomal and other non-CCC plasmid DNA. Plasmid DNA can be purified in less than 1 h and used successfully in restriction enzyme analysis and cloning experiments.     

嗜热硫杆菌启动子的克隆及定位

用DNA体外重组技术,将嗜热硫扦菌(Thiobacillus sp．)染色体DNA的Hind III片段插人启动子探测质粒pSDSI(Apr、Tc2)的Hind III位点,在四环素平板上获得一批转化子。四环素抗性能力测定表明,有12个转化子可以在含240／μg／ml四环素的平板上生长,有2个可以在360μg／ml的四环素平板上生长,对这二个抗性表达能力较高、分子量较小的质粒pSDH7和pSDH11作了限制性酶切图谱分析,并利用PstI位点,通过亚克隆将pSDH11插人片段上的启动子定位在0．25kb片段内。分子杂交实验证明克隆的启动子活性片段确实来源于嗜热硫杆菌染色体DNA。     

Methylobacterium Genome Sequences: A Reference Blueprint to Investigate Microbial Metabolism of C1 Compounds from Natural and Industrial Sources

Background

Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared.

Methodology/Principal Findings

The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name “island integration determinant” (iid).

Conclusion/Significance

These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.     

Intermolecular recE4-independent recombination in Bacillus subtilis: formation of plasmid pKBT1

Summary The plasmid pKBT1 was derived by in vivo recE4-independent recombinational event(s) yielding a structure containing regions of plasmid and chromosomal origin. BamHI digests of plasmid pUB110 (Kan^r/Neo^r) and Bg/II digests of pTL12 (Tmp^r, leuA) were mixed, ligated and used to transform competent cells of a recE4 strain of Bacillus subtilis. Kanamycin-resistant transformants were electrophoretically screened for hybrid plasmids. Plasmid pKBT1 (8.0 kb) was smaller than pTL12 (10.4 kb) but larger than monomeric pUB110 (4.5 kb). Plasmid PKBT1 was stably maintained in recE4 strains of B. subtilis and conferred kanamycin resistance but did not specify trimethoprim resistance or leucine prototrophy. At least 86% of the pUB110 monomer length was present in pKBT1 and was completely contained within a single 5.58 kb HindIII fragment. The other segment of pKBT1 was of chromosomal origin as evidenced by lack of homology to pTL12 and strong hybridization to B. subtilis chromosomal DNA. At least one of the in vivo recE4-independent event(s) which produced pKBT1 must have involved intermolecular recombination between transforming and chromosomal DNA. This finding differs from previous reports in which recE4-independent recombination involving pUB110 sequences was a strictly intramolecular event.     

Expression of an artificial yeast TRP-gene cluster in yeast and Escherichia coli

Summary All five tryptophan biosynthetic genes of Saccharomyces cerevisiae were unified on plasmid pME554, which is based on 2 m DNA and pBR322 sequences allowing for autonomous replication in yeast and E. coli. Homologous and heterologous expression of this artificial yeast TRP-gene cluster was studied. Plasmid pME554 allowed for nearly normal growth of a yeast strain bearing auxotrophic mutations in all five TRP-genes. The plasmid-borne genes TRP2 to TRP5 were expressed and regulated normally in the frame of the general control. Gene TRP1, carried on an EcoRI/BglII fragment lacking the ARS1 function, was expressed poorly and did not respond to the general control like the chromosomally-borne TRP1 gene.Plasmid pME554 allowed for poor growth of E. coli strain W3110 tna ^– trpEA2 on minimal medium. Marked stimulation was observed, however, when anthranilic acid or indole were added. Accordingly, poor expression of the first Trp-enzyme anthranilate synthase and the last enzyme tryptophan synthase was found, whereas the other three genes were moderately well expressed in E. coli.Abbreviations bp basepair - kb kilobase     

Plasmid content and localization of the genes encoding the denitrification enzymes in two strains of Rhodobacter sphaeroides

Plasmid content and localization of the genes encoding the reductases of the denitrification pathway were determined in the photosynthetic bacterium Rhodobacter sphaeroides forma sp. denitrificans by transverse alternating-field electrophoresis (TAFE) and hybridization with digoxigenin-labeled homologous probes. Two large plasmids of 102 and 115 kb were found. The genes encoding the various reductases are not clustered on a single genetic unit. The nap locus (localized with a napA probe), the nirK gene and the norCB genes encoding the nitrate, nitrite and nitric oxide reductases, respectively, were found on different AseI and SnaBI digested chromosomal DNA fragments, whereas the nos locus (localized with a nosZ probe), encoding the nitrous oxide reductase, was identified on the 115-kb plasmid. Furthermore, the genes encoding two proteins of unknown function, one periplasmic and the other cytoplasmic, but whose synthesis is highly induced by nitrate, were found on a different chromosomal fragment. For comparison, the same experiments were carried out on the well-characterized strain Rhodobacter sphaeroides 2.4.1.     

Transfer of prophage Mu into methylotrophic bacteria in the plasmid RP4

Bacteriophage Mu genome has been transferred into the cells of Pseudomonas methanolica and Methylobacterium sp. SKF240, that are naturally resistant to the bacteriophage, as a fragment of a hybrid plasmid RP4::Mu cts62. Temperature induction of the bacteriophage results in host cell lysis. Plasmid RP::Mu cis62 is maintained in methylotrophic cells presenting a cointegrative structure.The genetic and electrophoretic, analyses of the DNA isolated from transconjugant cells have confirmed the conclusion. Bacteriophage Mu propagation has been shown to be restricted in methylotrophic cells.     

Plasmid analysis in pink facultative methylotrophic bacteria using a modified acetone-alkaline hydrolysis method

Routine screening of indigenous and recombinant plasmids in pink facultative methylotrophic bacteria has been difficult, time-consuming, and yields variable results. We report a modified alkaline hydrolysis method for rapid plasmid isolation from these organisms that reproducibly results in good yields of closed circular plasmid DNA which can be readily digested with restriction enzymes. This method greatly facilitates direct screening of indigenous and introduced recombinant plasmids in the methylotrophic host strain. We have confirmed earlier findings that the original NCIB wild-type strain of Methylobacterium sp. strain AM1 (NCIB 9133) contains three cryptic plasmids. However, sizing of these plasmids by comparison to standards and by restriction fragment analysis suggests that they are larger than previously reported. We have designated these plasmids pAM1-1 (65 kb), pAM1-2 (40 kb) and pAM1-3 (33 kb). We have also shown that a rifamycin-resistant strain of Methylobacterium sp. strain AM1 used routinely in our laboratory lacks pAM1-2, although no phenotype has been associated with its loss. Finally, we have shown that another pink facultative methylotroph, Methylobacterium isolate (#YK1), contains three cryptic plasmids of approximately 43, 37 and 22 kb, respectively.     

Rhizobium meliloti carries two megaplasmids

In Rhizobium meliloti strain 41 the existence of a second megaplasmid (pRme41c) with a molecular weight similar to the sym megaplasmid pRme41b was demonstrated. Derivatives of the wild-type strain carrying pRme41b or pRme41c tagged with Tn5 allowed the examination of the transfer ability of both megaplasmids. The introduction of megaplasmids into the wild-type R. meliloti was not detected, probably because of the action of plasmid genes coding for entry exclusion of the same type of plasmid. However, transmissibility of both megaplasmids was observed in matings with Nod- or Fix- pRme41b deletion mutant recipients and with Agrobacterium tumefaciens at frequencies of 10(-6) - 10(-8). Introduction of the megaplasmids into the R. meliloti recipients resulted in the loss of the same plasmid. On the other hand, pRme41b and pRme41c were compatible. From the extent of deletions in various Nod- and Fix- mutants a DNA region carrying genes probably involved in "surface exclusion" on pRme41b was located. This DNA region is about 50 kb distant from the nod genes and exhibits strong homology with a DNA segment of pRme41c. Symbiotic genes on pRme41c were not identified.     

Genetic determination of degradation of ampholytic surfactants

Plasmid DNA was detected in Pseudomonas putida 141 and P. stutzeri AT strains which caused destruction of the ampholytic surfactants alkylamino-bis-propionate (AABP) and amidobetaine, respectively. As was demonstrated using genetic analytic procedures, the plasmids controlled AABP and amidobetaine destruction. No plasmid DNA was found in P. desmolytica C37 which caused cyclimide destruction or in Pseudomonas sp. 1 and Citrobacter freundii TO strains responsible for AABP destruction. Apparently, destruction of these xenobiotics was controlled by chromosomal genes.     

Diversity of carbofuran-degrading soil bacteria and detection of plasmid-encoded sequences homologous to the mcd gene

Abstract: Fifty-five bacterial isolates, from English and French soils with different histories of carbofuran field treatment, which hydrolysed the N -methylcarbamate insecticide carbofuran to carbofuran 7-phenol were characterised phenotypically and genetically. The isolates were compared by using 125 physiological tests and morphological features, plasmid profiles and restriction fragment length polymorphism (RFLP) patterns of total DNA using the rRNA operon of Escherichia coli as a DNA probe. Cluster analysis of both phenotypic characters and RFLP patterns showed a high degree of diversity amongst the bacteria. Ten distinct plasmid profiles with 2–4 plasmids ranging in size from 84 to about 438 kb were visualised in 50 isolates. The majority of isolates had one of two types of plasmid profiles. Plasmid profiles and Eco RI restricted total DNA patterns were hybridised with an internal fragment of the carbofuran hydrolase ( mcd ) gene and 22 diverse soil isolates exhibited sequence homology with this gene probe. Our results indicate that sequences homologous to the mcd gene are located on a conserved Eco RI fragment (12 or 14 kb) of a plasmid (100, 105, 115 or 124 kb) found in diverse soil isolates from geographically distant areas. Thirty-three isolates did not exhibit detectable homology to the mcd gene probe and the hydrolase enzymes and genes in these isolates need further investigation.     

Construction, transfer and properties of a novel temperature-sensitive integrable plasmid for genomic analysis of Staphyiococcus aureus

As an alternative approach to genetic transfer and analysis, a novel integrable plasmid system was developed that should prove useful for mapping and cloning various genes in Staphylococcus aureus and other Gram-positive bacteria. The use of a restriction-deficient recipient strain and an improved protocol for protoplast plasmid transformation facilitated direct cloning of a recombinant plasmid (pPQ126) in S. aureus NCTC 8325-4. Plasmid pPQ126 (13.6 kb) is a novel, temperature-sensitive integrable plasmid containing genes encoding resistance to erythromycin and chloramphenicol (from plasmid pTV1ts), and resistance to gentamicin (from transposon Tn4001). When introduced into an appropriate recipient strain at the permissive temperature (30 degrees C), pPQ126 replicates autonomously. Integration of pPQ126 is directed into homologous chromosomal target sequences (chromosomal insertions of Tn551 or Tn4001) by growing a population of cells containing autonomous pPQ126 in the presence of gentamicin, erythromycin, and chloramphenicol at 39 degrees C (nonpermissive temperature). Elevated temperature both selects for and maintains pPQ126 as an integrated replicon. Integration of pPQ126 occurs at significantly reduced frequency in a recombination-deficient host, and does not occur in the absence of host chromosomal homology. Integrated pPQ126 excises from the chromosome under permissive conditions (30 degrees C), and excision results in derivatives of pPQ126 that harbour DNA of chromosomal origin.