Development and Application of PCR Primers for the Detection of the tfd Genes in Delftia acidovorans P4a Involved in the Degradation of 2,4‐D

Primers specific for the genes tfdD, tfdE and tfdF, derived from conserved amino acid sequence motifs of the corresponding homologous enzymes, and primers specific for the genes tfdA and tfdB as well as tfdC taken from the literature were applied in PCR reactions using the genomic DNA of Delftia acidovorans P4a as the template. PCR products were obtained with all primer pairs that were similar in size to those found with the genomic DNA of strains harbouring plasmid pJP4 as the carrier of tfd genes. The nucleotide sequences and the corresponding amino acid sequences of the PCR products obtained with Strain P4a were compared with the sequence databases. According to BLAST analyses, the partial sequences of tfdA and tfdB exhibited a 94–99% degree of identity with the homologous sequences of the 2,4‐D‐degrading strains Achromobacter xylosoxidans subsp. denitrificans EST4002 (pEST4011), Burkholderia sp. RASC, Variovorax paradoxus TV1 (pTV1) and Burkholderia cepacia 2a (pIJB1), whereas the partial sequences of the tfdC, tfdD, tfdE and tfdF genes revealed a 96–100% degree of identity with the homologous sequences of the chlorobenzene‐utilizing strains Ralstonia eutropha NH9 (pENH91), Pseudomonas chlororaphis RW71 and Pseudomonas sp. P51 (pP51).     

The Chlorocatechol-Catabolic Transposon Tn5707 of Alcaligenes eutrophus NH9, Carrying a Gene Cluster Highly Homologous to That in the 1,2,4-Trichlorobenzene-Degrading Bacterium Pseudomonas sp. Strain P51, Confers the Ability To Grow on 3-Chlorobenzoate

Alcaligenes eutrophus (Ralstonia eutropha) NH9, isolated in Japan, utilizes 3-chlorobenzoate as its sole source of carbon and energy. Sequencing of the relevant region of plasmid pENH91 from strain NH9 revealed that the genes for the catabolic enzymes were homologous to the genes of the modified ortho-cleavage pathway. The genes from strain NH9 (cbnR-ABCD) showed the highest homology (89 to 100% identity at the nucleotide level) to the tcbR-CDEF genes on plasmid pP51 of the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51, which was isolated in The Netherlands. The structure of the operon, including the lengths of open reading frames and intervening sequences, was completely conserved between the cbn and tcb genes. Most nucleotide substitutions were localized within and proximal to the cbnB (tcbD) gene. The difference in the chloroaromatics that the two strains could use as growth substrates seemed to be due to differences in enzymes that convert substrates to chlorocatechols. The restriction map of plasmid pENH91 was clearly different from that of pP51 except in the regions that contained the cbnR-ABCD and tcbR-CDEF genes, respectively, suggesting that the chlorocatechol gene clusters might have been transferred as units. Two homologous sequences, present as direct repeats in both flanking regions of the cbnR-ABCD genes on pENH91, were found to be identical insertion sequences (ISs), designated IS1600, which formed a composite transposon designated Tn5707. Although the tcbR-CDEF genes were not associated with similar ISs, a DNA fragment homologous to IS1600 was cloned from the chromosome of strain P51. The sequence of the fragment suggested that it might be a remnant of an IS. The two sequences, together with IS1326 and nmoT, formed a distinct cluster on a phylogenetic tree of the IS21 family. The diversity of the sources of these IS or IS-like elements suggests the prevalence of ISs of this type.     

Evidence forCis- andTrans-acting element coevolution of the 2-μm circle genome inSaccharomyces cerevisiae

Summary We compared the DNA sequence of the yeas 2-μm plasmidcis-actingSTB andtrans-actingREP1 partition loci of laboratory haploid and industrial amphiploid strains. Several industrial strains had a uniqueSTB sequence (type 1) sharing only 70% homology with laboratorySTB (type 2). Type 1 plasmids had a REP1 protein with 6–10% amino acid substitutions when compared to REP1 of type 2 plasmids. All 2-μm variants that shared a similarSTB consensus sequence exhibited a high degree ofREP1 nucleotide and amino acid sequence conservation. These observations suggest molecular coevolution oftrans-acting elements with cognate target DNA structure. Based on DNA sequencing and Southern hybridization analyses, we classified 2-μm variants into two main evolutionary lineages that differ atSTB as well asREP1 loci. The role of molecular coevolution in yeast intra- and interspecies plasmid evolution was discussed.     

Heme content of recombinant catalase from Psychrobacter sp. T-3 altered by host Escherichia coli cell growth conditions

The catalase gene of Psychrobacter sp. T-3 was cloned, and the gene product (PktA) was overexpressed in Escherichia coli. The specific activity of the purified PktA was slightly lower than that of the native purified enzyme obtained from Psychrobacter sp. T-3. Spectrophotometric measurements of the purified enzymes suggested that the recombinant PktA contains a mixture of heme b and d, although the native enzyme contains the sole heme b. An addition of the heme precursor 5-aminolevulinic acid (ALA) to the medium increased the heme b content of the recombinant PktA, and the resulting enzyme showed higher specific activity than the native enzyme. This is the first report that shows the heme content of overproduced catalase altered by the host cell growth conditions.     

RepC_soli: a novel promiscuous plasmid type of Rhodobacteraceae mediates horizontal transfer of antibiotic resistances in the ocean

Alphaproteobacteria are typically characterized by a multipartite genome organization with a chromosome, stable chromids and accessory plasmids. Extrachromosomal elements determine the lifestyle of roseobacters and their horizontal transfer was previously correlated with rapid adaptations to novel ecological niches. We characterized the distribution and biology of a novel Rhodobacteraceae-specific plasmid type that was designated RepC_soli according to its diagnostic solitary replicase. This low copy number replicon exhibits an exceptional stability, which is likely ensured by non-canonical separate parA and parB partitioning genes. RepC_soli plasmids occur frequently in the surface-associated marine genus Phaeobacter and comparative genome analyses revealed the emergence of four compatibility groups. The universal presence of conserved type IV secretion systems in RepC_soli plasmids is indicative of their recurrent mobilization, a prediction that was experimentally validated by conjugation of the 57 kb Phaeobacter inhibens P72 plasmid (pP72_e) over genus borders. RepC_soli plasmids harbour a diverse collection of beneficial genes including transporters for heavy metal detoxification, prokaryotic defence systems and a conspicuous abundance of antibiotic resistance genes. The pP72_e-encoded efflux pump FloR conferred an about 50-fold increase of resistance against chloramphenicol. Its specific occurrence in Phaeobacter likely reflects a genetic footprint of (former) antimicrobial use in marine aquaculture.     

Purification and characterization of a novel organic solvent-tolerant and cold-adapted lipase from Psychrobacter sp. ZY124

By screening 25 different psychrophilic strains isolated from the Arctic habitat, we isolated a strain capable of producing lipase. We identified this strain as Psychrobacter sp. ZY124 based on the amplified 16S rDNA sequence. The lipase, named as Lipase ZC12, produced from the supernatant of Psychrobacter sp. ZY124 cultured at 15 °C was purified to homogeneity by ammonium sulfate precipitation followed by Phenyl Sepharose FF gel hydrophobic chromatography. Based on the obtained amino acid sequence, Lipase ZC12 is classified as a member of the Proteus/psychrophilic subfamily of lipase family I.1; it has a molecular weight of 37.9 kDa. We also determined that the apparent optimum temperature for Lipase ZC12 activity is 40 °C. Lipase ZC12 shows remarkable organic solvent tolerance by remaining more 50% after incubated with 10–90% different organic solvents. In addition, acyl chain esters with C12 or longer were confirmed to be preferable substrates for Lipase ZC12. Lipase ZC12 also shows better stereoselectivity for (R, S)-1-phenylethanol chiral resolution in n-hexane solvent with (S)-1-phenylethanol (ee_p 92%) and conversion rate (39%) by transesterification reactions. These properties may provide potential applications in biocatalysis and biotransformation in non-aqueous media, such as in detergent, transesterification or esterification and chiral resolution.

     

Two novel families of plasmids from hyperthermophilic archaea encoding new families of replication proteins

Thermococcales (phylum Euryarchaeota) are model organisms for physiological and molecular studies of hyperthermophiles. Here we describe three new plasmids from Thermococcales that could provide new tools and model systems for genetic and molecular studies in Archaea. The plasmids pTN2 from Thermococcus nautilus sp. 30-1 and pP12-1 from Pyrococcus sp. 12-1 belong to the same family. They have similar size (∼12 kb) and share six genes, including homologues of genes encoded by the virus PAV1 from Pyrococcus abyssi. The plasmid pT26-2 from Thermococcus sp. 26-2 (21.5 kb), that corresponds to another plasmid family, encodes many proteins having homologues in virus-like elements integrated in several genomes of Thermococcales and Methanococcales. Our analyses confirm that viruses and plasmids are evolutionary related and co-evolve with their hosts. Whereas all plasmids previously isolated from Thermococcales replicate by the rolling circle mechanism, the three plasmids described here probably replicate by the theta mechanism. The plasmids pTN2 and pP12-1 encode a putative helicase of the SFI superfamily and a new family of DNA polymerase, whose activity was demonstrated in vitro, whereas pT26-2 encodes a putative new type of helicase. This strengthens the idea that plasmids and viruses are a reservoir of novel protein families involved in DNA replication.     

Characterization of <Emphasis Type="Italic">Penicillium</Emphasis> Species by Ribosomal DNA Sequencing and BOX,ERIC and REP-PCR Analysis

The genus Penicillium is one of the largest and widely distributed fungal genera described to date. As a result, its taxonomic classification and species discrimination within this genus has become complicated. In this study, 52 isolates that belonged to the Penicillum genus and other related genera were characterized using two DNA-based methods: (i) analysis of the nucleotide sequences of internal transcribed spacers in ribosomal DNA and (ii) analysis of DNA fingerprints that were generated by polymerase chain reactions with specific primers for enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) sequences, and BOX elements. Using both methods, Penicillium species were discriminated from other fungal genera. Furthermore, Penicillium species that include strains which are used as biocontrol agents, such as P. glabrum, P. purpurogenum, and P. oxalicum, could be distinguished from other Penicillium species using these techniques. Based on our findings, we propose that a polyphasic approach that includes analysis of the nucleotide sequences of ribosomal DNA and detecting the presence of highly conserved, repeated nucleotide sequences can be used to determine the genetic relationships between different Penicillium species. Furthermore, we propose that our results can be used as a start point to develop a strategy to monitor the environmental presence of particular strains of Penicillium species when they are used as biocontrol agents.     

Genetic Organization of the Basic Replicon of Plasmid pMTH4 of a Facultatively Methylotrophic Bacterium <Emphasis Type="Italic">Paracoccus methylutens</Emphasis> DM12

Two functional regions within the basic replicon of plasmid pMTH4 of Paracoccus methylutens DM12 have been distinguished that are responsible for the replication of the plasmid (REP) and its stabilization (STA). In the REP region, a gene encoding the putative replication initiation protein RepA has been identified, with the highest similarity to the replication protein of plasmid pALC1 (Paracoccus alcaliphilus). The potential origin of replication (oriV), consisting of five long repeated sequences (iterons) as well as putative DnaA and IHF boxes, has been localized in the promoter region of the gene repA. The STA region was found to ensure stability for heterogeneous plasmid pABW3 that is unstable itself in paracocci. The mini-STA region (850 bp) contains two short open reading frames, one of which shows similarity to the RelB protein of Escherichia coli. Our investigations suggest that the stabilizing system of pMTH4 is based on the toxin and antidote principle.     

Pseudomonas C12B, an SDS degrading strain, harbours a plasmid coding for degradation of medium chain length n-alkanes

Pseudomonas C12B is able to degrade alkyl sulfates, alkylbenzene sulfonates, and linear alkanes and alkenes. Mitomycin C curing experiments and conjugation experiments demonstrated that the ability to utilize n-alkanes (C₉–C₁₂) and n-alkenes (C₁₀ and C₁₂) of medium chain length was plasmid-encoded. The plasmid was designated pDEC. Its size was estimated at several hundreds kb according to mobility in agarose gels. The plasmid did not confer resistance to the antibiotics tested. Analysis of alkylsulfatases P1 and P2 in original and cured strains confirmed that both enzymes are encoded by the chromosome. The ability of Pseudomonas C12B to utilize alkylbenzene sulfonates also appears to be encoded by the chromosome. pDEC could be transferred only to cured derivatives of Pseudomonas C12B, but not to strains of P. aeruginosa, P. putida, or Acinetobacter sp. Cured derivatives of Pseudomonas C12B could not serve as hosts for the broad host range plasmid CAM–OCT. The enzyme system encoded by the putative dec genes present on plasmid pDEC differs from the system coded by the alk genes of plasmid OCT in the size range of hydrocarbons preferentially used.     

Comparison of the nucleotide sequences of the meta-cleavage pathway genes of TOL plasmid pWW0 from Pseudomonas putida with other meta-cleavage genes suggests that both single and multiple nucleotide substitutions contribute to enzyme evolution

TOL plasmid pWW0 from Pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidation of toluene and xylenes. The structural genes for these catabolic enzymes are clustered into two operons, the xylCMABN operon, which encodes a set of enzymes required for the transformation of toluene/xylenes to benzoate/toluates, and the xylXYZLTEGFJQKIH operon, which encodes a set of enzymes required for the transformation of benzoate/toluates to Krebs cycle intermediates. The latter operon can be divided physically and functionally into two parts, the xylXYZL cluster, which is involved in the transformation of benzoate/toluates to (methyl)catechols, and the xylTEGFJQKIH cluster, which is involved in the transformation of (methyl)catechols to Krebs cycle intermediates. Genes isofunctional to xylXYZL are present in Acinetobacter calcoaceticus, and constitute a benzoate-degradative pathway, while xylTEGFJQKIH homologous encoding enzymes of a methylphenol-degradative pathway and a naphthalene-degradative pathway are present on plasmid pVI150 from P. putida CF600, and on plasmid NAH7 from P. putida PpG7, respectively. Comparison of the nucleotide sequences of the xylXYZLTEGFJQKIH genes with other isofunctional genes suggested that the xylTEGFJQKIH genes on the TOL plasmid diverged from these homologues 20 to 50 million years ago, while the xylXYZL genes diverged from the A. calcoaceticus homologues 100 to 200 million years ago. In codons where amino acids are not conserved, the substitution rate in the third base was higher than that in synonymous codons. This result was interpreted as indicating that both single and multiple nucleotide substitutions contributed to the amino acid-substituting mutations, and hence to enzyme evolution. This observation seems to be general because mammalian globin genes exhibit the same tendency.     

Production of volatile aroma compounds by bacterial strains isolated from different surface-ripened French cheeses

Twelve bacterial strains belonging to eight taxonomic groups: Brevibacterium linens, Microbacterium foliorum, Arthrobacter arilaitensis, Staphylococcus cohnii, Staphylococcus equorum, Brachybacterium sp., Proteus vulgaris and Psychrobacter sp., isolated from different surface-ripened French cheeses, were investigated for their abilities to generate volatile aroma compounds. Out of 104 volatile compounds, 54 volatile compounds (identified using dynamic headspace technique coupled with gas chromatography-mass spectrometry [GC-MS]) appeared to be produced by the different bacteria on a casamino acid medium. Four out of eight species used in this study: B. linens, M. foliorum, P. vulgaris and Psychrobacter sp. showed a high flavouring potential. Among these four bacterial species, P. vulgaris had the greatest capacity to produce not only the widest varieties but also the highest quantities of volatile compounds having low olfactive thresholds such as sulphur compounds. Branched aldehydes, alcohols and esters were produced in large amounts by P. vulgaris and Psychrobacter sp. showing their capacity to breakdown the branched amino acids. This investigation shows that some common but rarely mentioned bacteria present on the surface of ripened cheeses could play a major role in cheese flavour formation and could be used to produce cheese flavours.     

Parallel and Divergent Genotypic Evolution in Experimental Populations of Ralstonia sp.

Genetic rearrangements within a population of bacteria were analyzed to understand the degree of divergence occurring after experimental evolution. We used 18 replicate populations founded from Ralstonia sp. strain TFD41 that had been propagated for 1,000 generations with 2,4-dichlorophenoxyacetic acid (2,4-D) as the carbon source. Genetic divergence was examined by restriction fragment length polymorphism analysis of the incumbent plasmid that carries the 2,4-D catabolic genes and by amplification of random regions of the genome via PCR. In 18 evolved clones examined, we observed duplication within the plasmid, including the tfdA gene, which encodes a 2,4-D dioxygenase that catalyzes the first step in the 2,4-D catabolic pathway. In 71 of 72 evolved clones, a common 2.4-kb PCR product was lost when genomic fingerprints produced by PCR amplification using degenerate primers based on repetitive extragenic palindromic (REP) sequences (REP-PCR) were compared. The nucleotide sequence of the 2.4-kb PCR product has homology to the TRAP (tripartite ATP-independent periplasmic) solute transporter gene family. Hybridization of the 2.4-kb REP-PCR product from the ancestor to genomic DNA from the evolved populations showed that the loss of the PCR product resulted from deletions in the genome. Deletions in the plasmid and presence and/or absence of other REP-PCR products were also found in these clones but at much lower frequencies. The common and uncommon genetic changes observed show that both parallel and divergent genotypic evolution occurred in replicate populations of this bacterium.     

Comparison of pulsed‐field gel electrophoresis and three rep‐PCR methods for evaluating the genetic relatedness of Stenotrophomonas maltophilia isolates

Aims: In this study, three facile repetitive‐sequence PCR (rep‐PCR) techniques have been compared with the pulsed‐field gel electrophoresis (PFGE) method for differentiating the genetic relatedness of clinical Stenotrophomonas maltophilia isolates. Methods and Results: The dendrograms of 20 S. maltophilia isolates were constructed based on the data obtained from PFGE and three PCR‐based methods, i.e. enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR), BOX‐PCR and repetitive extragenic palindromic‐PCR (REP‐PCR). When compared with PFGE, ERIC‐PCR displayed a much lower discriminatory power, whereas BOX‐PCR and REP‐PCR had a comparable discriminatory power for close genetic‐related isolates. Conclusion: BOX‐PCR and REP‐PCR can be convenient and effective methods for evaluating the close genetic relatedness of clinical S. maltophilia isolates. Significance and Impact of the Study: A rapid method for determining S. maltophilia’s close genetic relatedness provides a convenient tool for understanding the epidemiology of S. maltophilia.     

Phylogeography of the Solanaceae-infecting Basidiomycota fungus <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG-3 based on sequence analysis of two nuclear DNA loci

Background  

The soil fungus Rhizoctonia solani anastomosis group 3 (AG-3) is an important pathogen of cultivated plants in the family Solanaceae. Isolates of R. solani AG-3 are taxonomically related based on the composition of cellular fatty acids, phylogenetic analysis of nuclear ribosomal DNA (rDNA) and beta-tubulin gene sequences, and somatic hyphal interactions. Despite the close genetic relationship among isolates of R. solani AG-3, field populations from potato and tobacco exhibit comparative differences in their disease biology, dispersal ecology, host specialization, genetic diversity and population structure. However, little information is available on how field populations of R. solani AG-3 on potato and tobacco are shaped by population genetic processes. In this study, two field populations of R. solani AG-3 from potato in North Carolina (NC) and the Northern USA; and two field populations from tobacco in NC and Southern Brazil were examined using sequence analysis of two cloned regions of nuclear DNA (pP42F and pP89).     

Nucleotide Sequence of Plasmid pAQ1 of Marine Cyanobacterium Synechococcus sp. PCC7002

We have determined the complete nucleotide sequence of pAQ1,the smallest plasmid of the unicellular marine cyanobacteriumSynechococcus sp. PCC7002. The plasmid consists of 4,809 bpand has at least four open reading frames that potentially encodepolypeptides of 50 or more amino acids. We found that a palindromicelement, the core sequence of which is G(G/A)CGATCGCC, is over-representednot only in plasmid pAQ1 but also in the accumulated cyanobacterialgenomic sequences from Synechococcus sp. PCC6301, PCC7002, PCC7942,vulcanus and Synechocystis sp. PCC6803 within GenBank and EMBLdatabases. It suggests that this sequence might mediate generearrangement, thus increasing genetic diversity, since recombinationevents are frequent in cyanobacteria.     

Environmentally co‐occurring mercury resistance plasmids are genetically and phenotypically diverse and confer variable context‐dependent fitness effects

Plasmids are important mobile elements that can facilitate genetic exchange and local adaptation within microbial communities. We compared the sequences of four co‐occurring pQBR family environmental mercury resistance plasmids and measured their effects on competitive fitness of a Pseudomonas fluorescens SBW25 host, which was isolated at the same field site. Fitness effects of carriage differed between plasmids and were strongly context dependent, varying with medium, plasmid status of competitor and levels of environmental mercury. The plasmids also varied widely in their rates of conjugation and segregational loss. We found that few of the plasmid‐borne accessory genes could be ascribed functions, although we identified a putative chemotaxis operon, a type IV pilus‐encoding cluster and a region encoding putative arylsulfatase enzymes, which were conserved across geographically distant isolates. One plasmid, pQBR55, conferred the ability to catabolize sucrose. Transposons, including the mercury resistance Tn5042, appeared to have been acquired by different pQBR plasmids by recombination, indicating an important role for horizontal gene transfer in the recent evolution of pQBR plasmids. Our findings demonstrate extensive genetic and phenotypic diversity among co‐occurring members of a plasmid community and suggest a role for environmental heterogeneity in the maintenance of plasmid diversity.     

Genetic Transformation System for a Psychrotrophic Deep-sea Bacterium: Isolation and Characterization of a Psychrotrophic Plasmid

A versatile system that permits genetic manipulation of a psychrotrophic deep-sea bacterium, Pseudoalteromonas sp. PS1M3, has been developed. A cryptic indigenous plasmid, pPS1M3, of 3.1 kb from the above strain was isolated and characterized. The nucleotide sequence analysis of plasmid pPS1M3 revealed the presence of one open reading frame, and its deduced amino acid sequence was identified as the essential protein for plasmid maintenance. Transformation with the pPS1M3 harboring antibiotic resistance genes by electroporation was fully successful using the pPS1M3-cured strain as a host. This plasmid was quite stable under nonselective culture conditions for about 100 generations at 4°C. The copy number of this plasmid in the cell was about 5 copies per chromosome. Received May 30, 2000; accepted October 11, 2000.