Evolution of genetic architecture and gene regulation in biphenyl/PCB-degrading bacteria.

A variety of bacteria in the environment can utilize xenobiotic compounds as a source of carbon and energy. The bacterial strains degrading xenobiotics are suitable models to investigate the adaptation and evolutionary processes of bacteria because they appear to have emerged relatively soon after the release of these compounds into the natural environment. Analyses of bacterial genome sequences indicate that horizontal gene transfer (HGT) is the most important contributor to the bacterial evolution of genetic architecture. Further, host bacteria that can use energy effectively by controlling the expression of organized gene clusters involved in xenobiotic degradation will have a survival advantage in harsh xenobiotic-rich environments. In this review, we summarize the current understanding of evolutionary mechanisms operative in bacteria, with a focus on biphenyl/PCB-degrading bacteria. We then discuss metagenomic approaches that are useful for such investigation.

A New ICEclc Subfamily Integrative and Conjugative Element Responsible for Horizontal Transfer of Biphenyl and Salicylic Acid Catabolic Pathway in the PCB-Degrading Strain Pseudomonas stutzeri KF716.

Integrative and conjugative elements (ICEs) are chromosomally integrated self-transmissible mobile genetic elements. Although some ICEs are known to carry genes for the degradation of aromatic compounds, information on their genetic features is limited. We identified a new member of the ICEclc family carrying biphenyl catabolic bph genes and salicylic acid catabolic sal genes from the PCB-degrading strain Pseudomonas stutzeri KF716. The 117-kb ICEbph-salKF716 contains common core regions exhibiting homology with those of degradative ICEclc from P. knackmussii B13 and ICEXTD from Azoarcus sp. CIB. A comparison of the gene loci collected from the public database revealed that several putative ICEs from P. putida B6-2, P, alcaliphila JAB1, P. stutzeri AN10, and P. stutzeri 2A20 had highly conserved core regions with those of ICEbph-salKF716, along with the variable region that encodes the catabolic genes for biphenyl, naphthalene, toluene, or phenol. These data indicate that this type of ICE subfamily is ubiquitously distributed within aromatic compound-degrading bacteria. ICEbph-salKF716 was transferred from P. stutzeri KF716 to P. aeruginosa PAO1 via a circular extrachromosomal intermediate form. In this study, we describe the structure and genetic features of ICEbph-salKF716 compared to other catabolic ICEs.

Biphenyl/PCB Degrading bph Genes of Ten Bacterial Strains Isolated from Biphenyl-Contaminated Soil in Kitakyushu, Japan: Comparative and Dynamic Features as Integrative Conjugative Elements (ICEs).

We sequenced the entire genomes of ten biphenyl/PCB degrading bacterial strains (KF strains) isolated from biphenyl-contaminated soil in Kitakyushu, Japan. All the strains were Gram-negative bacteria belonging to ß- and γ-proteobacteria. Out of the ten strains, nine strains carried a biphenyl catabolic bph gene cluster as integrative conjugative elements (ICEs), and they were classified into four groups based on the structural features of the bph genes. Group I (five strains) possessed bph genes that were very similar to the ones in Pseudomonasfurukawaii KF707 (formerly Pseudomonas pseudoalcaligenes KF707), which is one of the best characterized biphenyl-utilizing strains. This group of strains carried salicylate catabolic sal genes that were approximately 6-kb downstream of the bph genes. Group II (two strains) possessed bph and sal genes similar to the ones in KF707, but these strains lacked the bphX region between bphC and bphD, which is involved in the downstream catabolism of biphenyl. These bph-sal clusters in groups I and II were located on an integrative conjugative element that was larger than 110 kb, and they were named ICEbph-sal. Our previous study demonstrated that the ICEbph-sal of Pseudomonas putida KF715 in group II existed both in an integrated form in the chromosome (referred to as ICEbph-salKF715 (integrated)) and in a extrachromosomal circular form (referred to as ICEbph-sal (circular)) (previously called pKF715A, 483 kb) in the stationary culture. The ICEbph-sal was transferred from KF715 into P. putida AC30 and P. putida KT2440 with high frequency, and it was maintained stably as an extrachromosomal circular form. The ICEbph-salKF715 (circular) in these transconjugants was further transferred to P. putida F39/D and then integrated into the chromosome in one or two copies. Meanwhile, group III (one strain) possessed bph genes, but not sal genes. The nucleotide sequences of the bph genes in this group were less conserved compared to the genes of the strains belonging to groups I and II. Currently, there is no evidence to indicate that the bph genes in group III are carried by a mobile element. Group IV (two strains) carried bph genes as ICEs (59-61 kb) that were similar to the genes found in Tn4371 from Cupriavidus oxalacticus A5 and ICEKKS1024677 from the Acidovorax sp. strain KKS102. Our study found that bph gene islands have integrative functions, are transferred among soil bacteria, and are diversified through modification.

Pseudomonas furukawaii sp. nov., a polychlorinated biphenyl-degrading bacterium isolated from biphenyl-contaminated soil in Japan.

Strain KF707T was isolated from a biphenyl-contaminated site in Kitakyushu, Japan. Analysis of 16S rRNA gene sequences, retrieved from the whole-genome sequence, revealed that the isolate was closely related to members of the genus Pseudomonas, sharing the highest sequence similarities with Pseudomonas balearica strain SP1402T (DSM 6083) (97.8 %). The DNA G+C chromosome and plasmid content of strain KF707T were 65.5 and 60.5 mol%. The major cellular fatty acids were iso-C15 :  0 and C16 : 1ω7c/C16 : 1ω6c. Polyphasic analysis indicated that strain KF707T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas furukawaii sp. nov. is proposed. The type strain is KF707T (=DSM 10086T=NBRC 110670T).

Insights into the genomic plasticity of Pseudomonas putida KF715, a strain with unique biphenyl-utilizing activity and genome instability properties.

Pseudomonas putida KF715 exhibits unique properties in both catabolic activity and genome plasticity. Our previous studies revealed that the DNA region containing biphenyl and salycilate metabolism gene clusters (termed the bph-sal element) was frequently deleted and transferred by conjugation to closely related P. putida strains. In this study, we first determined the complete nucleotide sequence of the KF715 genome. Next, to determine the underlying cause of genome plasticity in KF715, we compared the KF715 genome with the genomes of one KF715 defective mutant, two transconjugants, and several P. putida strains available from public databases. The gapless KF715 genome sequence revealed five replicons: one circular chromosome, and four plasmids. Southern blot analysis indicated that most of the KF715 cell population carries the bph-sal element on the chromosome whereas a small number carry it on a huge plasmid, pKF715A. Moreover, the bph-sal element is present stably on the plasmid and did not integrate into the chromosome of its transconjugants. Comparative genome analysis and experiments showed that a number of diverse putative genetic elements are present in KF715 and are likely involved in genome rearrangement. These data provide insights into the genetic plasticity and adaptability of microorganisms for survival in various ecological niches.

Complete Genome Sequence of the Polychlorinated Biphenyl-Degrading Bacterium Pseudomonas putida KF715 (NBRC 110667) Isolated from Biphenyl-Contaminated Soil.

Pseudomonas putida KF715 (NBRC 110667) utilizes biphenyl as a sole source of carbon and degrades polychlorinated biphenyls (PCBs). Here, we report a complete genome sequence of the KF715 strain, which comprises a circular chromosome and four plasmids. Biphenyl catabolic genes were located on the largest plasmid, pKF715A.

Draft Genome Sequence of the Polychlorinated Biphenyl-Degrading Bacterium Pseudomonas stutzeri KF716 (NBRC 110668).

Pseudomonas stutzeri KF716 (NBRC 110668) utilizes biphenyl as a sole source of carbon and energy and degrades polychlorinated biphenyls. Here, we report the first draft genome sequence of a biphenyl-degrading strain of the species P. stutzeri.

Draft Genome Sequence of the Polychlorinated Biphenyl-Degrading Bacterium Comamonas testosteroni KF712 (NBRC 110673).

We present a 5.89-Mb draft genome sequence of Comamonas testosteroni KF712 (NBRC 110673), a polychlorinated biphenyl degrader. The genome sequence clarified that KF712 harbors the gene clusters coding for the catabolism of biphenyl and at least seven other aromatic compounds.

Draft Genome Sequence of Pseudomonas aeruginosa KF702 (NBRC 110665), a Polychlorinated Biphenyl-Degrading Bacterium Isolated from Biphenyl-Contaminated Soil.

Pseudomonas aeruginosa KF702 (NBRC 110665) utilizes biphenyl as a sole source of carbon and degrades polychlorinated biphenyls (PCBs). Here, we report the 7,167,540-bp draft genome sequence of KF702, which contains 6,714 coding sequences and a 65.8 mol% G+C content. The strain possesses genes for biphenyl catabolism and other genes that mediate degradation of various aromatic compounds.

Draft Genome Sequence of Pseudomonas abietaniphila KF701 (NBRC110664), a Polychlorinated Biphenyl-Degrading Bacterium Isolated from Biphenyl-Contaminated Soil.

Pseudomonas abietaniphila KF701 utilizes biphenyl as a sole source of carbon and degrades polychlorinated biphenyls (PCBs). Here, we report the 6,886,250-bp draft genome sequence of KF701, which contains 6,315 coding sequences and 59.4 mol% G+C content. The strain possesses genes for biphenyl catabolism and other genes that mediate the degradation of benzoate, salicylate, and phenol.

Draft Genome Sequence of Pseudomonas toyotomiensis KF710, a Polychlorinated Biphenyl-Degrading Bacterium Isolated from Biphenyl-Contaminated Soil.

Pseudomonas toyotomiensis KF710 utilizes biphenyl and degrades polychlorinated biphenyls (PCBs). Here, we report the genome sequence of the KF710 strain, consisting of 5,596,721 bp with 5,155 coding sequences. The biphenyl catabolic genes were almost identical to those of Pseudomonas pseudoalcaligenes KF707, one of the most well-characterized biphenyl-utilizing strains.

Draft Genome Sequence of Pseudomonas abietaniphila KF717 (NBRC 110669), Isolated from Biphenyl-Contaminated Soil in Japan.

Pseudomonas abietaniphila KF717 utilizes biphenyl as a sole source of carbon and energy and degrades polychlorinated biphenyls (PCBs). We report here the 6,930,016-bp genome sequence of this strain, which contains 6,323 predicted coding sequences (CDSs), including the biphenyl-utilizing bph gene cluster.

Draft Genome Sequence of the Polychlorinated Biphenyl-Degrading Bacterium Pseudomonas putida KF703 (NBRC 110666) Isolated from Biphenyl-Contaminated Soil.

Pseudomonas putida KF703 (NBRC 110666) utilizes biphenyl as a sole source of carbon and degrades polychlorinated biphenyls (PCBs). Here, we report the draft genome sequence of the KF703 strain, which provides insight into the molecular mechanisms of adaptation to an environment polluted by aromatic compounds.

Draft Genome Sequence of the Polychlorinated Biphenyl-Degrading Bacterium Cupriavidus basilensis KF708 (NBRC 110671) Isolated from Biphenyl-Contaminated Soil.

We report the draft genome sequence of Cupriavidus basilensis KF708 (NBRC 110671), which utilizes biphenyl as a sole carbon source and degrades polychlorinated biphenyls (PCBs). The KF708 strain possesses genes for biphenyl catabolism and other genes involved in various aromatic compounds.

Draft Genome Sequence of Cupriavidus pauculus Strain KF709, a Biphenyl-Utilizing Bacterium Isolated from Biphenyl-Contaminated Soil.

We report the draft genome sequence of Cupriavidus pauculus strain KF709, which comprises 6,826,799 bp with 6,272 coding sequences. The strain KF709 utilizes biphenyl and degrades low-chlorinated biphenyls; however, it possesses fewer coding sequences involved in the degradation of aromatic compounds than other strains belonging to the Betaproteobacteria.

Efficient screening of environmental isolates for Saccharomyces cerevisiae strains that are suitable for brewing.

We developed an efficient screening method for Saccharomyces cerevisiae strains from environmental isolates. MultiPlex PCR was performed targeting four brewing S. cerevisiae genes (SSU1, AWA1, BIO6, and FLO1). At least three genes among the four were amplified from all S. cerevisiae strains. The use of this method allowed us to successfully obtain S. cerevisiae strains.

Evaluation of the inhibitory effects of chloroform on ortho-chlorophenol- and chloroethene-dechlorinating Desulfitobacterium strains.

Organohalide-respiring Desulfitobacterium strains are believed to play an important role in the bioremediation and natural attenuation of chlorinated aliphatic and aromatic hydrocarbons. However, several studies have reported that chloroform significantly inhibits microbial reductive dechlorination of chloroethene. In this study, we examined the effect of chloroform on several Desulfitobacterium strains, including ortho-chlorophenol-dechlorinating Desulfitobacterium dehalogenans JW/IU-1 and Desulfitobacterium hafniense DCB-2, and also the chloroethene-dechlorinating strain D. hafniense TCE1. In medium containing 3-chloro-4-hydroxyphenylacetate as an electron acceptor, chloroform inhibited the growth of strains JW/IU-1 and DCB-2. Although chloroform did not directly inhibit dechlorination of 3-chloro-4-hydroxyphenylacetate by resting cells, cells cultivated with chloroform showed decreased dechlorination activity. Moreover, transcription of the gene encoding the reductive dehalogenase CprA decreased significantly in cells cultivated with chloroform. These results indicate that chloroform inhibits the growth and dechlorination activity of strains JW/IU-1 and DCB-2 via inhibition of cprA transcription. In contrast, cultivation of strain TCE1 in the presence of chloroform gave rise to a PceA reductive dehalogenase gene-deletion variant of strain TCE1; a similar phenomenon was observed in our previous study of chloroethene-dechlorinating D. hafniense strain Y51. Our results suggest that chloroform extensively inhibits the dechlorination activity of Desulfitobacterium strains, and that the inhibitory mechanism appears to differ between ortho-chlorophenol dechlorinators and chloroethene dechlorinators.

Hybrid pseudomonads engineered by two-step homologous recombination acquire novel degradation abilities toward aromatics and polychlorinated biphenyls.

Pseudomonas pseudoalcaligenes KF707 possesses a chromosomally encoded bph gene cluster responsible for the catabolism of biphenyl and polychlorinated biphenyls. Previously, we constructed chimeric versions of the bphA1 gene, which encodes a large subunit of biphenyl dioxygenase, by using DNA shuffling between bphA1 genes from P. pseudoalcaligenes KF707 and Burkholderia xenovorans LB400. In this study, we demonstrate replacement of the bphA1 gene with chimeric bphA1 sequence within the chromosomal bph gene cluster by two-step homologous recombination. Notably, some of the hybrid strains acquired enhanced and/or expanded degradation capabilities for specific aromatic compounds, including single aromatic hydrocarbons and polychlorinated biphenyls.

Microbial degradation of polychlorinated biphenyls: biochemical and molecular features.

It is more than 40 years since the environmental contamination of polychlorinated biphenyls (PCBs) was first reported in wildlife samples. Since then, a huge number of papers on PCBs have been published, which include the biodegradation of PCBs and toxicology of PCBs. The studies on the microbial degradation of PCBs during the few decades provided significant insight into the areas of microbial ecology, biochemistry, and molecular genetics.

Cross-regulation of biphenyl- and salicylate-catabolic genes by two regulatory systems in Pseudomonas pseudoalcaligenes KF707.

Pseudomonas pseudoalcaligenes KF707 grows on biphenyl and salicylate as sole sources of carbon. The biphenyl-catabolic (bph) genes are organized as bphR1A1A2(orf3)A3A4BCX0X1X2X3D, encoding the enzymes for conversion of biphenyl to acetyl coenzyme A. In this study, the salicylate-catabolic (sal) gene cluster encoding the enzymes for conversion of salicylate to acetyl coenzyme A were identified 6.6-kb downstream of the bph gene cluster along with a second regulatory gene, bphR2. Both the bph and sal genes were cross-regulated positively and/or negatively by the two regulatory proteins, BphR1 and BphR2, in the presence or absence of the effectors. The BphR2 binding sequence exhibits homology with the NahR binding sequences in various naphthalene-degrading bacteria. Based on previous studies and the present study we propose a new regulatory model for biphenyl and salicylate catabolism in strain KF707.