Isolation and characterization of altered plasmids in mutant strains of Pseudomonas putida NCIB 9816

The ability of P. putida NCIB 9816 to grow with naphthalene (Nah+) and salicylate (Sal+) is correlated with the presence of an 83 kilobase (kb) conjugative plasmid, pDTG1. Derivatives of pDTG1 were obtained from cells after exposure to halogenated analogs of naphthalene or salicylate. The selection of mutants having a Nah-Sal- or a Nah-Sal+ phenotype could be enhanced by the addition of triphenyltetrazolium chloride to the indicator medium. Structurally modified plasmids were characterized by restriction endonuclease digestion and Southern hybridization experiments. The region of pDTG1 DNA that encodes the enzymes responsible for the conversion of naphthalene to salicylate was identified. The structural changes in mutant plasmids were correlated with the absence of essential enzymatic activities.     

Isolation and characterization of naphthalene-catabolic genes and plasmids from oil-contaminated soil by using two cultivation-independent approaches

Two different cultivation-independent approaches were applied to isolate genes for naphthalene dioxygenase (NDO) from oil-contaminated soil in Japan. One approach was the construction of a broad-host-range cosmid-based metagenomic DNA library, and the other was the so-called exogenous plasmid isolation technique. Our screening of NDO genes in both approaches was based on the functional complementation of Pseudomonas putida strains which contained Tn4655K, a transposon carrying the entire set of naphthalene-catabolic (nah) genes but lacking the NDO-encoding gene. We obtained in the former approach a cosmid clone (pSLX928-6) that carried an nah upper pathway operon for conversion of naphthalene to salicylate, and this operon showed a significantly high level of similarity to the corresponding operon on an IncP-9 naphthalene-catabolic plasmid, pDTG1. In the latter approach, the microbial fraction from the soil was mated with a plasmid-free P. putida strain containing a chromosomal copy of Tn4655K, and transconjugants were obtained that received either a 200- or 80-kb plasmid containing all the nah genes for the complete degradation of naphthalene. Subsequent analysis revealed that (1) both plasmids belong to the IncP-9 incompatibility group; (2) their nah upper pathway operons are significantly similar, but not completely identical, to those of pDTG1 and pSLX928-6; and (3) these plasmids carried genes for the salicylate metabolism by the meta-cleavage pathway. A.O. and R.M. contributed equally to this work.     

Conjugal transfer and mobilization capacity of the completely sequenced naphthalene plasmid pNAH20 from multiplasmid strain Pseudomonas fluorescens PC20

The complete 83 042-bp nucleotide sequence of the IncP-9 naphthalene degradation plasmid pNAH20 from Pseudomonas fluorescens PC20 exhibits striking similarity in size and sequence to another naphthalene (NAH) plasmid pDTG1. However, the positions of insertion sequence (IS) elements significantly alter both catabolic and backbone functions provided by the two plasmids. In pDTG1, insertion of a pCAR1 IS Pre1 -like element disrupts expression of the lower naphthalene operon and this strain utilizes the chromosomal pathway for complete naphthalene degradation. In pNAH20, this operon is intact and functional. The transfer frequency of pNAH20 is 100 times higher than that of pDTG1 probably due to insertion of the pCAR1 IS Pre2 -like element into the mpfR gene coding for a putative repressor of the mpf operon responsible for mating pilus formation. We also demonstrate in situ plasmid transfer – we isolated a rhizosphere transconjugant strain of pNAH20, P. fluorescens NS8. The plasmid pNS8, a derivative of pNAH20, lacks the ability to self-transfer as a result of an additional insertion event of IS Pre2 -like element that disrupts the gene coding for VirB2-like major pilus protein MpfA. The characteristics of the strain PC20 and the conjugal transfer/mobilization capacity of pNAH20 (or its backbone) make this strain/plasmid a potentially successful tool for bioremediation applications.     

Transposon and spontaneous deletion mutants of plasmid-borne genes encoding polycyclic aromatic hydrocarbon degradation by a strain of Pseudomonas fluorescens

Pseudomonas fluorescens strain LP6a, isolated from petroleum condensate-contaminated soil, utilizes the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, anthracene and 2-methylnaphthalene as sole carbon and energy sources. The isolate also co-metabolically transforms a suite of PAHs and heterocycles including fluorene, biphenyl, acenaphthene, 1-methylnaphthalene, indole, benzothiophene, dibenzothiophene and dibenzofuran, producing a variety of oxidized metabolites. A 63 kb plasmid (pLP6a) carries genes encoding enzymes necessary for the PAH-degrading phenotype of P. fluorescens LP6a. This plasmid hybridizes to the classical naphthalene degradative plasmids NAH7 and pWW60, but has different restriction endonuclease patterns. In contrast, plasmid pLP6a failed to hybridize to plasmids isolated from several phenanthrene-utilizing strains which cannot utilize naphthalene. Plasmid pLP6a exhibits reproducible spontaneous deletions of a 38 kb region containing the degradative genes. Two gene clusters corresponding to the archetypal naphthalene degradation upper and lower pathway operons, separated by a cryptic region of 18 kb, were defined by transposon mutagenesis. Gas chromatographic-mass spectrometric analysis of metabolites accumulated by selected transposon mutants indicates that the degradative enzymes encoded by genes on pLP6a have a broad substrate specificity permitting the oxidation of a suite of polycyclic aromatic and heterocyclic substrates.     

The P-7 Incompatibility Group Plasmids Responsible for Biodegradation of Naphthalene and Salicylate in Fluorescent Pseudomonads

Analysis of seven plasmids (77 to 135 kb in size) of the P-7 incompatibility group that are responsible for the biodegradation of naphthalene and salicylate has shown that the main natural host of IncP-7 plasmids is the species Pseudomonas fluorescens. The IncP-7 plasmids are structurally diverse and do not form groups, as is evident from their cluster analysis. The naphthalene catabolism genes of six of the IncP-7 plasmids are conservative and homologous to the catabolic genes of NAH7 and pDTG1 plasmids. The pAK5 plasmid contains the classical nahA gene, which codes for naphthalene dioxygenase, and the salicylate 5-hydroxylase gene (nagG) sequence, which makes the conversion of salicylate to gentisate possible.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 342–348.Original Russian Text Copyright © 2005 by Izmalkova, Sazonova, Sokolov, Kosheleva, Boronin.     

Incompatibility group P-7 plasmids responsible for biodegradation of naphthalene and salicylate in fluorescent pseudomonads

Analysis of seven plasmids (77 to 135 kbp in size) of the P-7 incompatibility group that are responsible for the biodegradation of naphthalene and salicylate has shown that the main natural host of IncP-7 plasmids is the species Pseudomonas fluorescens. The IncP-7 plasmids are structurally diverse and do not form groups, as is evident from their cluster analysis. The naphthalene catabolism genes of six of the IncP-7 plasmids are conservative and homologous to the catabolic genes of NAH7 and pDTG1 plasmids. The pAK5 plasmid contains the classical nahA gene, which codes for naphthalene dioxygenase, and the salicylate 5-hydroxylase gene (nagG) sequence, which makes the conversion of salicylate to gentisate possible.     

Plasmid-mediated mineralization of naphthalene, phenanthrene, and anthracene.

The well-characterized plasmid-encoded naphthalene degradation pathway in Pseudomonas putida PpG7(NAH7) was used to investigate the role of the NAH plasmid-encoded pathway in mineralizing phenanthrene and anthracene. Three Pseudomonas strains, designated 5R, DFC49, and DFC50, were recovered from a polynuclear aromatic hydrocarbon-degrading inoculum developed from a manufactured gas plant soil slurry reactor. Plasmids pKA1, pKA2, and pKA3, approximately 100 kb in size, were isolated from these strains and characterized. These plasmids have homologous regions of upper and lower NAH7 plasmid catabolic genes. By conjugation experiments, these plasmids, including NAH7, have been shown to encode the genotype for mineralization of [9-14C]phenanthrene and [U-14C]anthracene, as well as [1-14C]naphthalene. One strain, Pseudomonas fluorescens 5RL, which has the complete lower pathway inactivated by transposon insertion in nahG, accumulated a metabolite from phenanthrene and anthracene degradation. This is the first direct evidence to indicate that the NAH plasmid-encoded catabolic genes are involved in degradation of polynuclear aromatic hydrocarbons other than naphthalene.     

Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

Bacteria Shigella spp. are highly contagious, severely harmful and gram-negative facultative intracellular pathogens. They may cause shigellosis characterized by fever, dehydration and hematochezia in clinic, and shigellosis has been remaining a leading cause of infant mortality in the world. Shigella belongs to the family Enterobacteriaceae and the group Escherichiaeae, which are divided into four species and at least 47 serotypes: Shigella dysenteriae (13 serotypes), Shigella flexneri (15 …     

Effect of transposons on expression of genes for naphthalene biodegradation in Pseudomonas putida BS202(NPL-1) and derivative strains

NPL-1 and its derivative plasmid pBS106, which control the degradation of naphthalene and salicylate, were found to contain class II transposons of the Tn3 family. These transposons are involved in intraplasmid rearrangements, such as deletions and inversions, and can influence the expression of the catabolic and regulatory genes borne by biodegradation plasmids. The formation of a strong NahR-independent constitutive promoter by the inversion of a DNA fragment may be responsible for changing the character of naphthalene dioxygenase synthesis from inducible (in the case of plasmid NPL-1) to constitutive (in the case of plasmid NPL-41). The stability of plasmids NPL-1 and NPL-41 in the Pseudomonas putida strains grown on different substrates depends on the expression of the nah and tnp genes.     

Nah-genes of Pseudomonas putida: molecular genetic analysis of the plasmid pBS286

The hybridization and restriction analysis of the plasmid pBS286 (73 Kb, the P-9 Inc group) as well as parental plasmids NPL-1, NPL-41 demonstrated that pBS286 plasmid (delta NPL-41::TnA) with the constitutive synthesis of naphthalene dioxygenase carried genes for naphthalene oxidation to salicylate and those participating in degradation of catechol. Restriction map of pBS286 using XhoI restriction endonuclease and that of the nah region using EcoRI, BamHI, SalI and XhoI were established. Structural peculiarities of nah genes from pBS286 are compared with previously described NAH7. Some nah genes were localized. An inverted DNA segment involved in nah gene regulation was shown to be closely linked to a proximal part of the nah1 operon or overlapped. Possible occurrence of a regulatory R locus in this region is suggested.     

Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.     

Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of <Emphasis Type="Italic">Shigella flexneri</Emphasis> 2a

The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.     

Sequence and Gene Expression Analyses of Plasmid pHPM8 from Helicobacter pylori Reveal the Presence of Two Operons with Putative Roles in Plasmid Replication and Antibiotic Activity

The DNA sequence of a 7.8-kb Helicobacter pylori plasmid, pHPM8, was determined. Six open reading frames (ORFs) were present. Ribonuclease protection studies showed that ORF1/ORF2 and ORF3/ORF4 genes are organized in operons possibly involved in plasmid replication and in production of a peptide with antibiotic activity, respectively. Finding areas of pHPM8 with a high level of identity to H. pylori chromosomal DNA supported the hypothesis that recombination occurs between plasmids and the chromosome of H. pylori.     

Naphthalene and Donor Cell Density Influence Field Conjugation of Naphthalene Catabolism Plasmids

We examined transfer of naphthalene-catabolic genes from donor microorganisms native to a contaminated site to site-derived, rifampin-resistant recipient bacteria unable to grow on naphthalene. Horizontal gene transfer (HGT) was demonstrated in filter matings using groundwater microorganisms as donors. Two distinct but similar plasmid types, closely related to pDTG1, were retrieved. In laboratory-incubated sediment matings, the addition of naphthalene stimulated HGT. However, recipient bacteria deployed in recoverable vessels in the field site (in situ) did not retrieve plasmids from native donors. Only when plasmid-containing donor cells and naphthalene were added to the in situ mating experiments did HGT occur.     

Naphthalene and donor cell density influence field conjugation of naphthalene catabolism plasmids

We examined transfer of naphthalene-catabolic genes from donor microorganisms native to a contaminated site to site-derived, rifampin-resistant recipient bacteria unable to grow on naphthalene. Horizontal gene transfer (HGT) was demonstrated in filter matings using groundwater microorganisms as donors. Two distinct but similar plasmid types, closely related to pDTG1, were retrieved. In laboratory-incubated sediment matings, the addition of naphthalene stimulated HGT. However, recipient bacteria deployed in recoverable vessels in the field site (in situ) did not retrieve plasmids from native donors. Only when plasmid-containing donor cells and naphthalene were added to the in situ mating experiments did HGT occur.