Molecular relationship of chromosomal genes encoding biphenyl/polychlorinated biphenyl catabolism: some soil bacteria possess a highly conserved bph operon.

All the genes we examined that encoded biphenyl/polychlorinated biphenyl (PCB) degradation were chromosomal, unlike many other degradation-encoding genes, which are plasmid borne. The molecular relationship of genes coding for biphenyl/PCB catabolism in various biphenyl/PCB-degrading Pseudomonas, Achromobacter, Alcaligenes, Moraxella, and Arthrobacter strains was investigated. Among 15 strains tested, 5 Pseudomonas strains and one Alcaligenes strain possessed the bphABC gene cluster on the XhoI 7.2-kilobase fragment corresponding to that of Pseudomonas pseudoalcaligenes KF707. More importantly, the restriction profiles of these XhoI 7.2-kilobase fragments containing bphABC genes were very similar, if not identical, despite the dissimilarity of the flanking chromosomal regions. Three other strains also possessed bphABC genes homologous with those of KF707, and five other strains showed weak or no significant genetic homology with bphABC of KF707. The immunological cross-reactivity of 2,3-dihydroxybiphenyl dioxygenases from various strains corresponded well to the DNA homology. On the other hand, the bphC gene of another PCB-degrading strain, Pseudomonas paucimobilis Q1, lacked genetic as well as immunological homology with any of the other 15 biphenyl/PCB degraders tested. The existence of the nearly identical chromosomal genes among various strains may suggest that a segment containing the bphABC genes has a mechanism for transferring the gene from one strain to another.     

Pseudomonas putida KF715 bphABCD operon encoding biphenyl and polychlorinated biphenyl degradation: cloning, analysis, and expression in soil bacteria.

We cloned the entire bphABCD genes encoding degradation of biphenyl and polychlorinated biphenyls to benzoate and chlorobenzoates from the chromosomal DNA of Pseudomonas putida KF715. The nucleotide sequence revealed two open reading frames corresponding to the bphC gene encoding 2,3-dihydroxybiphenyl dioxygenase and the bphD gene encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (ring-meta-cleavage compound) hydrolase.     

Vectors for transposon mutagenesis of non-enteric bacteria

Summary We have constructed a series of transposon delivery vectors derived from pRK2013. Since pRK2013 has a broad host range transfer system and a ColE1 replicon, it can be transferred to, but not replicated in, many nonenteric gram-negative bacteria. Thus pRK2013 provides an effective mechanism for the transient introduction of a transposon. Delivery vectors containing Tn7 (tmp str), Tn10 (tet), Tn10 HH104 (tet), or Tn5-132 (tet) have been constructed. When transposition in Caulobacter crescentus was examined, both Tn7 and Tn5-132 were found to transpose efficiently. In contrast, although the antibiotic resistances of Tn10 and Tn501 (mer) were expressed in C. crescentus, no transposition was observed with either transposon. However, transposition of Tn10 from the Tn10 vectors did occur in Acinetobacter calcoaceticus, and transposition of Tn501 from pMD100 has been demonstrated in Rhizobium japonicum (Bullerjahn and Benzinger 1984). Thus, transposon-host interactions play an important role in the determination of whether a particular transposon can transpose in a given host. Futhermore, the results with C. crescentus indicate that there must be different requirements for host interactions for Tn10 and Tn501 than for Tn5 and Tn7.     

Gene components responsible for discrete substrate specificity in the metabolism of biphenyl (bph operon) and toluene (tod operon).

bph operons coding for biphenyl-polychlorinated biphenyl degradation in Pseudomonas pseudoalcaligenes KF707 and Pseudomonas putida KF715 and tod operons coding for toluene-benzene metabolism in P. putida F1 are very similar in gene organization as well as size and homology of the corresponding enzymes (G. J. Zylstra and D. T. Gibson, J. Biol. Chem. 264:14940-14946, 1989; K. Taira, J. Hirose, S. Hayashida, and K. Furukawa, J. Biol. Chem. 267:4844-4853, 1992), despite their discrete substrate ranges for metabolism. The gene components responsible for substrate specificity between the bph and tod operons were investigated. The large subunit of the terminal dioxygenase (encoded by bphA1 and todC1) and the ring meta-cleavage compound hydrolase (bphD and todF) were critical for their discrete metabolic specificities, as shown by the following results. (i) Introduction of todC1C2 (coding for the large and small subunits of the terminal dioxygenase in toluene metabolism) or even only todC1 into biphenyl-utilizing P. pseudoalcaligenes KF707 and P. putida KF715 allowed them to grow on toluene-benzene by coupling with the lower benzoate meta-cleavage pathway. Introduction of the bphD gene (coding for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase) into toluene-utilizing P. putida F1 permitted growth on biphenyl. (ii) With various bph and tod mutant strains, it was shown that enzyme components of ferredoxin (encoded by bphA3 and todB), ferredoxin reductase (bphA4 and todA), and dihydrodiol dehydrogenase (bphB and todD) were complementary with one another. (iii) Escherichia coli cells carrying a hybrid gene cluster of todClbphA2A3A4BC (constructed by replacing bphA1 with todC1) converted toluene to a ring meta-cleavage 2-hydroxy-6-oxo-hepta-2,4-dienoic acid, indicating that TodC1 formed a functional multicomponent dioxygenase associated with BphA2 (a small subunit of the terminal dioxygenase in biphenyl metabolism), BphA3, and BphA4.     

Molecular cloning and characterization of catechol 2,3-dioxygenases from biphenyl/polychlorinated biphenyls-degrading bacteria.

Catechol 2,3-dioxygenases were cloned from Alcaligenes sp. KF711, Pseudomonas putida KF715, and Achromobacter xylosoxidans KF701 which are biphenyl/polychlorinated biphenyls-degrading bacteria. All of the cloned enzymes were purified by preparative polyacrylamide gel electrophoresis (PAGE). The purified catechol 2,3-dioxygenases were significantly different from one another in ring-fission activities to catechol and its derivatives. The catechol 2,3-dioxygenase from Alcaligenes sp. KF711 exhibited higher ring-fission activity to 4-chlorocatechol than those from P. putida KF715 and A. xylosoxidans KF701. In electrophoretic mobilities, the three enzymes were different from one another on nondenaturing PAGE but the same on SDS-PAGE.     

Enhanced biodegradation of polychlorinated biphenyls after site-directed mutagenesis of a biphenyl dioxygenase gene.

Biphenyl dioxygenase catalyzes the first step in the aerobic degradation of polychlorinated biphenyls (PCBs). The nucleotide and amino acid sequences of the biphenyl dioxygenases from two PCB-degrading strains (Pseudomonas sp. strain LB400 and Pseudomonas pseudoalcaligenes KF707) were compared. The sequences were found to be nearly identical, yet these enzymes exhibited dramatically different substrate specificities for PCBs. Site-directed mutagenesis of the LB400 bphA gene resulted in an enzyme combining the broad congener specificity of LB400 with increased activity against several congeners characteristic of KF707. These data strongly suggest that the BphA subunit of biphenyl dioxygenase plays an important role in determining substrate selectivity. Further alteration of this enzyme can be used to develop a greater understanding of the structural basis for congener specificity and to broaden the range of degradable PCB congeners.     

Characterization of the mgl operon of Escherichia coli by transposon mutagenesis and molecular cloning

We used transposon insertion mutagenesis, molecular cloning, and a novel procedure for in vitro construction of polar and nonpolar insertion mutations to characterize the genetic organization and gene products of the beta-methylgalactoside (Mgl) transport system, which utilizes the galactose-binding protein. The data indicate that the mgl operon contained three genes, which were transcribed in the order mglB, mglA, and mglC. The first gene coded for the 31,000 Mr galactose-binding protein, which was synthesized as a 3,000-dalton-larger precursor form. The mglA product was a 50,000 Mr protein which was tightly associated with the membrane, and the mglC product was a 38,000 Mr protein which was apparently loosely associated with the membrane and was probably located on the internal face of the cytoplasmic membrane. Identification of gene products was facilitated by in vitro insertion of a fragment of Tn5 containing the gene conferring kanamycin resistance into a restriction site in the operon. The fragment proved to have a polar effect on the expression of promoter-distal genes only when inserted in one of the two possible orientations. The three identified gene products were necessary and apparently sufficient for transport activity, but only the binding protein was required for chemotaxis towards galactose. The transport system appeared to contain the minimum number of components for a binding protein-related system: a periplasmic recognition component, a transmembrane protein, and a peripheral membrane protein that may be involved in energy linkage.     

Aerobic biodegradation of biphenyl and polychlorinated biphenyls by Arctic soil microorganisms.

We examined the degradation of biphenyl and the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1221 by indigenous Arctic soil microorganisms to assess both the response of the soil microflora to PCB pollution and the potential of the microflora for bioremediation. In soil slurries, Arctic soil microflora and temperate-soil microflora had similar potentials to mineralize [14C]biphenyl. Mineralization began sooner and was more extensive in slurries of PCB-contaminated Arctic soils than in slurries of uncontaminated Arctic soils. The maximum mineralization rates at 30 and 7 degrees C were typically 1.2 to 1.4 and 0.52 to 1.0 mg of biphenyl g of dry soil-1 day-1, respectively. Slurries of PCB-contaminated Arctic soils degraded Aroclor 1221 more extensively at 30 degrees C (71 to 76% removal) than at 7 degrees C (14 to 40% removal). We isolated from Arctic soils organisms that were capable of psychrotolerant (growing at 7 to 30 degrees C) or psychrophilic (growing at 7 to 15 degrees C) growth on biphenyl. Two psychrotolerant isolates extensively degraded Aroclor 1221 at 7 degrees C (54 to 60% removal). The soil microflora and psychrotolerant isolates degraded all mono-, most di-, and some trichlorobiphenyl congeners. The results suggest that PCB pollution selected for biphenyl-mineralizing microorganisms in Arctic soils. While low temperatures severely limited Aroclor 1221 removal in slurries of Arctic soils, results with pure cultures suggest that more effective PCB biodegradation is possible under appropriate conditions.     

Organisation of the bph gene cluster of transposon Tn4371, encoding enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds

Tn4371 is a 55 kb transposon which encodes enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds into benzoate and 4-chlorobenzo-ate derivatives. We constructed a cosmid library of Tn4371 DNA. The bph genes involved in biphenyl/4-chlorobiphenyl degradation were found to be clustered in the middle of the transposon. Sequencing revealed an organisation of the bph genes similar to that previously found in Pseudomonas sp. KKS102, i.e. the bphEGF genes are located upstream of bphA1A2A3 and bphA4 is separated from bphA1A2A3 by bphBCD. Consensus sequences for σ54-associated RNA polymerase were found upstream of bphA1 and bphEGF. Plasmid RP4::Tn4371 was transferred into a mutant of Alcaligenes eutrophus H16 lacking σ54. In contrast to wild-type H16 exconjugants, the σ54 mutant exconjugants could not grow on biphenyl, indicating the dependence of Tn4371bph gene expression on σ54. The Tn4371-encoded bph pathway was activated when biphenyl and various biphenyl-like compounds were present in the growth medium. Preliminary observations indicate the presence of a region outside the catabolic genes downstream of bphA4 which is involved in mediating at least the basal expression of BphC.     

Life cycle assessment of polychlorinated biphenyl contaminated soil remediation processes

Purpose  

A life-cycle assessment (LCA) was performed to evaluate the environmental impacts of the remediation of industrial soils contaminated by polychlorobiphenyl (PCB). Two new bioremediation treatment options were compared with the usual incineration process. In this attributional LCA, only secondary impacts were considered. The contaminated soil used for the experiments contained 200 mg of PCB per kilogram.     

Enhanced mineralization of polychlorinated biphenyls in soil inoculated with chlorobenzoate-degrading bacteria.

An Altamont soil containing no measurable population of chlorobenzoate utilizers was examined for the potential to enhance polychlorinated biphenyl (PCB) mineralization by inoculation with chlorobenzoate utilizers, a biphenyl utilizer, combinations of the two physiological types, and chlorobiphenyl-mineralizing transconjugants. Biphenyl was added to all soils, and biodegradation of 14C-Aroclor 1242 was assessed by disappearance of that substance and by production of 14CO2. Mineralization of PCBs was consistently greatest (up to 25.5%) in soils inoculated with chlorobenzoate degraders alone. Mineralization was significantly lower in soils receiving all other treatments: PCB cometabolizer (10.7%); chlorobiphenyl mineralizers (8.7 and 14.9%); and mixed inocula of PCB cometabolizers and chlorobenzoate utilizers (11.4 and 18.0%). However, all inoculated soils had higher mineralization than did the uninoculated control (3.1%). PCB disappearance followed trends similar to that observed with the mineralization data, with the greatest degradation occurring in soils inoculated with the chlorobenzoate-degrading strains Pseudomonas aeruginosa JB2 and Pseudomonas putida P111 alone. While the mechanism by which the introduction of chlorobenzoate degraders alone enhanced biodegradation of PCBs could not be elucidated, the possibility that chlorobenzoate inoculants acquired the ability to metabolize biphenyl and possibly PCBs was explored. When strain JB2, which does not utilize biphenyl, was inoculated into soil containing biphenyl and Aroclor 1242, the frequency of isolates able to utilize biphenyl and 2,5-dichlorobenzoate increased progressively with time from 3.3 to 44.4% between 15 and 48 days, respectively. Since this soil contained no measurable level of chlorobenzoate utilizers yet did contain a population of biphenyl utilizers, the possibility of genetic transfer between the latter group and strain JB2 cannot be excluded.     

红球菌R04联苯/多氯联苯代谢相关调控蛋白RHOGL007659的生理功能

【目的】研究红球菌(Rhodococcus sp.)R04调控蛋白RHOGL007659的生理功能及其缺陷菌株的代谢特性,初步探究红球菌R04降解联苯的调控机制。【方法】通过基因同源重组敲除红球菌R04联苯代谢相关基因RHOGL007659。比较红球菌R04(野生型)和缺陷型菌株R04Δ7659(基因RHOGL007659缺陷型的R04)在不同碳源培养下的生长情况,HPLC分析R04和R04Δ7659转化联苯的能力。提取R04和R04Δ7659的总RNA,实时荧光定量PCR检测联苯降解关键基因的转录表达。纯化Bph B(联苯降解脱氢酶)和Bph D(联苯降解水解酶),制备多克隆抗体。Western blot分析Bph B和Bph D蛋白在R04和R04Δ7659中的表达水平。【结果】获得了RHOGL007659基因的缺陷型菌株R04Δ7659,与R04相比,R04Δ7659在联苯培养条件下的生物量趋近于零。HPLC分析表明,RHOGL007659基因的缺失使红球菌R04丧失转化联苯的能力。实时荧光定量PCR结果表明,在联苯培养条件下,缺失RHOGL007659后的R04,其联苯降解关键基因均有不同程度的下调表达。Western blot分析显示RHOGL007659缺失后,联苯降解关键酶Bph B和Bph D表达量均降低,这与实时荧光定量PCR结果相一致。【结论】RHOGL007659是红球菌R04联苯降解关键基因簇的调控蛋白,该蛋白对红球菌R04代谢联苯过程具有正调控作用。     

Suicide vector for transposon mutagenesis in Pseudomonas solanacearum.

A suicide vector was constructed by cloning the transfer genes of the wide-host-range (IncW group) plasmid R388 into the BamHI site of pBR325. This plasmid can deliver Tn5 into Pseudomonas solanacearum at frequencies ranging from 10(-6) to 10(-9) per recipient.     

Pathological changes of experimental polychlorinated biphenyl poisoning in chickens.

Two experiments were carried out to study pathological changes of polychlorinated biphenyl (PCB) "KC 400" poisoning. In them chickens were administered with this agent for a relatively short time. Eleven experimental diets were prepared to contain 11 different PCB concentrations, 4,800 ppm being the highest, respectively. They were given to broiler chickens in initial feeding and later. All the chickens fed 300 ppm and higher concentrations of PCB died, showing the serious dilatation of uriniferous tubules and involution of the bursa of Fabricius, by the time when they reached 12 days of age. All the chickens, except a very few, fed 100 ppm remained alive at the end of the experimental period, when they were 41 days of age and exhibited such edematous changes as hydropericardium. Few chickens fed 50 ppm and less manifested pathological changes until the end of the experimental period, when they were 22 days of age. Two experimental diets were prepared to contain 600 and 200 ppm of PCB, respectively. They were given to layer chickens in initial feeding and later. These layers were sacrificed for autopsy every fourth day. A gain in body weight was retarded mostly in those fed 600 ppm. Dilatation of uriniferous tubules and involution of the bursa of Fabricius began in the very early stage of experimental feeding in both groups. Hydropericardium occurred to many of the chickens fed 200 ppm in the late stage of the experimental period. The results of both experiments indicated that there was no essential difference in changes of PCB poisoning between broiler and layer chickens. The principal changes of the poisoning were dilatation of uriniferous tubules, involution of the bursa of Fabricius, and edematous alterations. They appeared in a relatively early stage of the experimental period of PCB feeding. Their severity was strongly dependent upon the concentration of PCB contained in the diet.     

Site-selected transposon mutagenesis at the hcf106 locus in maize.

The High chlorophyll fluorescence106 (Hcf106) gene in maize is required for chloroplast membrane biogenesis, and the hcf106-mum1 allele is caused by the insertion of a Robertson's Mutator Mu1 element into the promoter of the gene. Seedlings homozygous for hcf106-mum1 are pale green and die 3 weeks after germination, but only in the presence of Mutator activity conferred by active, autonomous Mu regulatory transposons elsewhere in the genome. When Mutator activity is lost, the mutant phenotype is suppressed, and homozygous plants have an almost wild-type phenotype. To isolate derivative alleles at the hcf106 locus that no longer require Mutator activity for phenotypic expression, we have developed a method for site-selected transposon mutagenesis in maize. This procedure, first described for Caenorhabditis elegans and Drosophila, involves using polymerase chain reaction (PCR) to screen pools of individuals for insertions and deletions in genes of known sequence. Pools of seedlings segregating for the progenitor allele hcf106-mum1 were screened by PCR for insertions and deletions associated with Robertson's Mutator. In a 360-bp target region, two new insertions and one deletion were identified in only 700 Mu-active gametes screened. One of the insertions was in the progenitor hcf106-mum1 allele and the other was in the wild-type allele, but all three new alleles were found to have break-points at the same nucleotide in the first intron. Unlike the hcf-106-mum1 progenitor allele, the deletion and one of the insertions conferred pale green seedling lethal phenotypes in the absence of mutator activity. However, the second insertion had a weak, viable phenotype under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene-specific mutagenesis enables rapid continuous evolution of enzymes in vivo

Various in vivo mutagenesis methods have been developed to facilitate fast and efficient continuous evolution of proteins in cells. However, they either modify the DNA region that does not match the target gene, or suffer from low mutation rates. Here, we report a mutator, eMutaT7 (enhanced MutaT7), with very fast in vivo mutation rate and high gene-specificity in Escherichia coli. eMutaT7, a cytidine deaminase fused to an orthogonal RNA polymerase, can introduce up to ∼4 mutations per 1 kb per day, rivalling the rate in typical in vitro mutagenesis for directed evolution of proteins, and promotes rapid continuous evolution of model proteins for antibiotic resistance and allosteric activation. eMutaT7 provides a very simple and tunable method for continuous directed evolution of proteins, and suggests that the fusion of new DNA-modifying enzymes to the orthogonal RNA polymerase is a promising strategy to explore the expanded sequence space without compromising gene specificity.