Biodegradation of 4-nitrotoluene by Pseudomonas sp. strain 4NT

A strain of Pseudomonas spp. was isolated from nitrobenzene-contaminated soil on 4-nitrotoluene as the sole source of carbon, nitrogen, and energy. The organism also grew on 4-nitrobenzaldehyde, and 4-nitrobenzoate. 4-Nitrobenzoate and ammonia were detected in the culture fluid of glucose-grown cells after induction with 4-nitrotoluene. Washed suspensions of 4-nitrotoluene- or 4-nitrobenzoate-grown cells oxidized 4-nitrotoluene, 4-nitrobenzaldehyde, 4-nitrobenzyl alcohol, and protocatechuate. Extracts from induced cells contained 4-nitrobenzaldehyde dehydrogenase, 4-nitrobenzyl alcohol dehydrogenase, and protocatechuate 4,5-dioxygenase activities. Under anaerobic conditions, cell extracts converted 4-nitrobenzoate or 4-hydroxylaminobenzoate to protocatechuate. Conversion of 4-nitrobenzoate to protocatechuate required NADPH. These results indicate that 4-nitrotoluene was degraded by an initial oxidation of the methyl group to form 4-nitrobenzyl alcohol, which was converted to 4-nitrobenzoate via 4-nitrobenzaldehyde. The 4-nitrobenzoate was reduced to 4-hydroxylaminobenzoate, which was converted to protocatechuate. A protocatechuate 4,5-dioxygenase catalyzed meta-ring fission of the protocatechuate. The detection of 4-nitrobenzaldehyde and 4-nitrobenzyl alcohol dehydrogenase and 4-nitrotoluene oxygenase activities in 4-nitrobenzoate-grown cells suggests that 4-nitrobenzoate is an inducer of the 4-nitrotoluene degradative pathway.     

Regulation of growth of Acinetobacter calcoaceticus NCIB8250 on benzyl alcohol in batch culture

Formation of benzoate and catechol during oxidation of benzyl alcohol by washed suspensions of Acinetobacter calcoaceticus NCIB8250 confirmed earlier results indicating that this organism metabolizes benzyl alcohol via benzaldehyde, benzoate, and the 3-oxoadipate pathway. There was no evidence for feedback inhibition of benzyl alcohol dehydrogenase or benzaldehyde dehydrogenase II. Examination of growth curves and patterns of substrate utilization, as well as measurement of enzyme activities, showed that benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II are repressed when A. calcoaceticus utilizes L-mandelate or phenylglyoxylate. Growth of bacteria on L-mandelate prior to their inoculation into benzyl alcohol/salts medium leads to an exceptionally long lag period before benzyl alcohol is used at the maximum rate. Benzyl alcohol metabolism is also suppressed during growth on benzoate.     

Engineering of quasi-natural Pseudomonas putida strains for toluene metabolism through an ortho-cleavage degradation pathway

To construct a bacterial catalyst for bioconversion of toluene and several alkyl and chloro- and nitro-substituted derivatives into the corresponding benzoates, the upper TOL operon of plasmid pWW0 of Pseudomonas putida was fully reassembled as a single gene cassette along with its cognate regulatory gene, xylR. The corresponding DNA segment was then targeted to the chromosome of a P. putida strain by using a genetic technique that allows deletion of all recombinant tags inherited from previous cloning steps and leaves the otherwise natural strain bearing exclusively the DNA segment encoding the phenotype of interest. The resulting strains grew on toluene as the only carbon source through a two-step process: conversion of toluene into benzoate, mediated by the upper TOL enzymes, and further metabolism of benzoate through the housekeeping ortho-ring cleavage pathway of the catechol intermediate.     

Characterization of a spontaneously occurring mutant of the TOL20 plasmid in Pseudomonas putida MT20: possible regulatory implications

Pseudomonas putida MT20 carries a plasmid (TOL20) that codes for the enzymes responsible for the catabolism of toluene, m- and p-xylene to benzoate, and m- and p-toluate, respectively, followed by meta cleavage of the aromatic ring. Growth on 5 mM benzoate selects very strongly for (i) strains that have been cured of the plasmid and (ii) strains with an intermediate growth pattern (the B3 phenotype) that retain the ability to grow on toluene, m-xylene, and benzoate but are unable to grow on m-toluate. Both types of strains were selected because they are no longer able to oxidize benzoate by the plasmid pathway but instead use an alternative route, the ortho or beta-ketoadipate pathway, which is chromosomally coded and supports faster growth. Evidence that one strain with the B3 phenotype, MT20-B3, has a regulatory mutation that prevents induction of the meta-pathway enzymes by benzoate and m-toluate, but which enables them to be induced by toluene and m-xylene, is presented. The plasmid in this strain, as in most of the others with the same phenotype, is nonconjugative. Analysis of MT20-B3, together with revertants of it and other noninducible mutants, has led to a model for the regulation of the plasmid-coded enzymes in MT20, in which it is proposed that the early enzymes for degradation of m-toluate and benzoate are positively controlled by two regulator molecules, one of which interacts with toluene and m-xylene as inducers and the other of which interacts with benzoate and m-toluate. It is argued that MT20-B3 and strains with a similar phenotype arose as a result of a deletion of the gene coding for the second regulator molecule.     

The broad substrate chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase of Pseudomonas sp. strain P51 are linked evolutionarily to the enzymes for benzene and toluene degradation

The chlorobenzene degradation pathway of Pseudomonas sp. strain P51 is an evolutionary novelty. The first enzymes of the pathway, the chlorobenzene dioxygenase and the cis-chlorobenzene dihydrodiol dehydrogenase, are encoded on a plasmid-located transposon Tn5280. Chlorobenzene dioxygenase is a four-protein complex, formed by the gene products of tcbAa for the large subunit of the terminal oxygenase, tcbAb for the small subunit, tcbAc for the ferredoxin, and tcbAd for the NADH reductase. Directly downstream of tcbAd is the gene for the cis-chlorobenzene dihydrodiol dehydrogenase, tcbB. Homology comparisons indicated that these genes and gene products are most closely related to those for toluene (todC1C2BAD) and benzene degradation (bedC1C2BA and bnzABCD) and distantly to those for biphenyl, naphthalene, and benzoate degradation. Similar to the tod-encoded enzymes, chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase were capable of oxidizing 1,2-dichlorobenzene, toluene, naphthalene, and biphenyl, but not benzoate, to the corresponding dihydrodiol and dihydroxy intermediates. These data strongly suggest that the chlorobenzene dioxygenase and dehydrogenase originated from a toluene or benzene degradation pathway, probably by horizontal gene transfer. This evolutionary event left its traces as short gene fragments directly outside the tcbAB coding regions.     

Molecular analysis of the plasmid-borne bed gene cluster from Pseudomonas putida ML2 and cloning of the cis-benzene dihydrodiol dehydrogenase gene

Pseudomonas putida ML2 contains a large catabolic plasmid, pHMT112, carrying genes that encode the dioxygenase and dehydrogenase involved in the catabolism of benzene via the ortho or beta-ketoadipate pathway. pHMT112 was derived from a larger and less stable plasmid in P. putida ML2 following growth on succinate as carbon and energy source but was, however, stably maintained in P. putida even in the absence of selection for growth on benzene. Cleavage sites for the restriction endonucleases DraI, XbaI, and BamHI were mapped on the plasmid. A region of the plasmid, downstream of the benzene dioxygenase genes (bedC1C2BA), was found to encode the cis-benzene dihydrodiol dehydrogenase gene (bedD). Recombinant Escherichia coli containing bedC1C2BAD genes was found to express benzene dioxygenase and dehydrogenase activity, indicated by the production of catechol when incubated in the presence of benzene. Hybridization using benzene dioxygenase genes as probes was used to construct a restriction map of the 35.5-kb XhoI-DraI fragment on which the bed operon was located.     

Fowlpox virus encodes nonessential homologs of cellular alpha-SNAP, PC-1, and an orphan human homolog of a secreted nematode protein

The genome of fowlpox virus (FWPV), type species of the Avipoxviridae, is considerably rearranged compared with that of vaccinia virus (the prototypic poxvirus and type species of the Orthopoxviridae) and is 30% larger. It is likely that the genome of FWPV contains genes in addition to those found in vaccinia virus, probably involved with its replication and survival in the chicken. A 7,470-bp segment of the FWPV genome has five open reading frames (ORFs), two of which encode ankyrin repeat proteins, many examples of which have been found in poxviruses. The remaining ORFs encode homologs of cellular genes not reported in any other virus. ORF-2 encodes a homolog of the yeast Sec17p and mammalian SNAP proteins, crucial to vesicular transport in the exocytic pathway. ORF-3 encodes a homolog of an orphan human protein, R31240_2, encoded on 19p13.2. ORF-3 is also homologous to three proteins (YLS2, YMV6, and C07B5.5) from the free-living nematode Caenorhabditis elegans and to a 43-kDa antigen from the parasitic nematode Trichinella spiralis. ORF-5 encodes a homolog of the mammalian plasma cell antigen PC-1, a type II glycoprotein with exophosphodiesterase activity. The ORFs are present in the virulent precursor, HP1, of the sequenced attenuated virus (FP9) and are conserved in other strains of FWPV. They were shown, by deletion mutagenesis, to be nonessential to virus replication in tissue culture. RNA encoding the viral homolog of PC-1 is expressed strongly early and late in infection, but RNAs encoding the homologs of SNAP and R31240_2 are expressed weakly and late.     

Hemiacetal dehydrogenation activity of alcohol dehydrogenases in Saccharomyces cerevisiae

Some methylotrophic yeasts produce methyl formate from methanol and formaldehyde via hemiacetal formation. We investigated Saccharomyces cerevisiae to find whether this yeast has a carboxylate ester producing pathway that proceeds via hemiacetal dehydrogenation. We confirmed that the purified alcohol dehydrogenase (Adh) protein from S. cerevisiae can catalyze the production of esters. High specific activities were observed toward the hemiacetals corresponding to the primary alcohols when ether groups were substituted for methylene groups, resulting in the formation of formate esters. Both ADH and methyl formate synthesizing activities were sharply reduced in the delta adh1 delta adh2 mutant. The ADH1 and ADH2 genes encode the major Adh proteins in S. cerevisiae. Thus, it was concluded that the S. cerevisiae Adh protein catalyzes activities for the production of certain carboxylate esters.     

p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate

Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the further catabolism of p-cumate and is located, in turn, upstream of the tod (toluene catabolism) operon in P. putida F1. The sequences of an 11,236-bp DNA segment carrying the cym operon and a 915-bp DNA segment completing the sequence of the 2,673-bp DNA segment separating the cmt and tod operons have been determined and are discussed here. The cym operon contains six genes in the order cymBCAaAbDE. The gene products have been identified both by functional assays and by comparing deduced amino acid sequences to published sequences. Thus, cymAa and cymAb encode the two components of p-cymene monooxygenase, a hydroxylase and a reductase, respectively; cymB encodes p-cumic alcohol dehydrogenase; cymC encodes p-cumic aldehyde dehydrogenase; cymD encodes a putative outer membrane protein related to gene products of other aromatic hydrocarbon catabolic operons, but having an unknown function in p-cymene catabolism; and cymE encodes an acetyl coenzyme A synthetase whose role in this pathway is also unknown. Upstream of the cym operon is a regulatory gene, cymR. By using recombinant bacteria carrying either the operator-promoter region of the cym operon or the cmt operon upstream of genes encoding readily assayed enzymes, in the presence or absence of cymR, it was demonstrated that cymR encodes a repressor which controls expression of both the cym and cmt operons and is inducible by p-cumate but not p-cymene. Short (less than 350 bp) homologous DNA segments that are located upstream of cymR and between the cmt and tod operons may have been involved in recombination events that led to the current arrangement of cym, cmt, and tod genes in P. putida F1.     

Inhibition of rat lung mixed-function oxidase activity following repeated low-level toluene inhalation: possible role of toluene metabolites

Toluene is a commonly used solvent that has been shown to alter mixed-function oxidase (MFO) activity, in an organ- and isozyme-specific pattern, following intraperitoneal administration. The purpose of this study was to determine whether similar changes occurred following repeated, low-level inhalation exposure, and to investigate the role of toluene metabolites in these alterations. Exposure to 375 ppm toluene, 6 h/d for up to 5 d, resulted in significant inhibition of the activity of pulmonary arylhydrocarbon hydroxylase (AHH), cytochrome P-4502B1 (CYP2B1), and CYP4B1, but not CYP1A1. After exposure to lower toluene levels (125 ppm, 6 h/d, 3 d), the activities of lung AHH, CYP2B1, and CYP4B1 were also significantly decreased, but in a dose-related manner. MFO activity was not consistently altered in liver. Control pulmonary or liver microsomes were incubated with various concentrations (0.01-10 mM) of toluene or its metabolites and CYP2B1, CYP1A1, and/or CYP4B1 activities were subsequently determined. Benzaldehyde produced a significant dose-related inhibition in the activity of all three lung P-450s examined (IC50 10(-3) M). Toluene was found to be a more potent inhibitor of lung CYP2B1 and CYP1A1 (IC50, 10(-4) M) than benzaldehyde, but neither toluene nor benzyl alcohol was an effective inhibitor of lung CYP4B1. Toluene and its metabolites were weaker inhibitors of CYP1A1 than of CYP2B1. For CYP2B1 and CYP1A1, the order of inhibitory potency was toluene > benzaldehyde > benzyl alcohol and suggests that both the parent molecule and its metabolites may act in concert to inhibit catalytic activity of these cytochromes. The MFO inhibition seen after repeated low-level toluene inhalation exposure could result in altered metabolic profiles of other xenobiotics in an organ-specific fashion.     

The evaluation of the professional training of physicians specializing in the epidemiology of infectious diseases. A set of examination questions]

The nucleotide sequence of cbaC, the 1-carboxy-3-chloro-4,5-dihydroxycyclohexa-2,6-diene (cis-diol) dehydrogenase gene from the 3-chlorobenzoate (3-Cba) catabolic transposon Tn5271 was determined. The functional significance of the deduced open reading frame was evaluated by deletion of an internal BstEII restriction site in cbaC and by the creation of nested deletions using exonuclease III. Expression studies were carried out with Alcaligenes sp. strain BR6024, a chloramphenicol-resistant, tryptophan auxotroph derived from the wild-type isolate BR60. BR6024 hosts carrying complete cbaAB (3-Cba 3,4-(4,5)-dioxygenase and reductase) genes, with deletions of cbaC, metabolized 3Cba to the cis-4,5-diol metabolite. These mutants failed to grow on 3-Cba; however, they grew on 3,4-dichlorobenzoate, accumulating 5-chloroprotocatechuate transiently. These results indicated the cbaC dehydrogenase was not required for re-aromatization of the unstable 3,4-dCba cis-4,5-diol metabolite. Spontaneous elimination of HCl from this metabolite is proposed to generate 5-chloroprotocatechuate, which is a substrate for the protocatechuate metaring fission pathway in Alcaligenes sp. BR60. The relationship of the deduced amino acid sequence of cbaC with 15 other oxidoreductases and sequences of unknown function from bacteria, plants and animals revealed a conserved N-terminal GxxGxG dinucleotide-binding domain and a conserved region with a H(x11)KHVLxEKPxA consensus flanked by alpha-helical domains. o-Phthalate cis-diol dehydrogenase (Pseudomonas putida), glucose-fructose oxidoreductase (Zymomonas mobilis), myo-inositol-2-dehydrogenase (Bacillus subtilis) and D-galactose-1-dehydrogenase (Pseudomonas fluorescens) are related proteins. These dehydrogenases are unrelated to the Type I, II and III dehydrogenase superfamilies that include the cis-diol dehydrogenases involved in benzoate, toluene, biphenyl and naphthalene catabolism (Type II) and benzene catabolism (Type III).     

钯掺杂α-MnO2无溶剂下催化氧化苯甲醇的性能

通过共沉淀和原位煅烧转化方法, 将Pd掺杂δ-MnO₂前驱体煅烧后制备得到Pd掺杂α-MnO₂纳米棒催化材料.通过氮气物理吸附、X射线衍射、透射电子显微镜、扫描电子显微镜、热重分析、X射线光电子能谱等技术对催化材料进行了表征.扫描电镜和透射电镜结果显示, α-MnO₂纳米棒表面没有明显的Pd纳米颗粒, 表明Pd可能掺杂到α-MnO₂晶格中.纯α-MnO₂的还原温度在390℃左右, 但Pd掺杂可以极大地促进α-MnO₂还原, 还原温度可低至约200℃左右.研究了所制备催化剂在无溶剂条件下对于以分子氧为氧化剂选择性催化氧化苯甲醇为苯甲醛的催化性能.结果表明: 在无溶剂及用纯氧气为氧化剂条件下, Pd掺杂α-MnO₂纳米棒对苯甲醇氧化显示出增强的催化活性; 所掺杂的氧化态Pd物质可增强催化材料中的氧迁移率; 在这些Pd掺杂α-MnO₂催化材料中, 当以Pd (3%, 质量分数) -MnO₂为催化剂时, 在110℃反应4 h后, 苯甲醇的转化率为39%, 远高于同条件下以纯α-MnO₂为催化剂时18. 3%的苯甲醇转化率.      

Expression and substrate specificity of the toluene dioxygenase of Pseudomonas putida NCIMB 11767

Pseudomonas putida NCIMB 11767 oxidized phenol, monochlorophenols, several dichlorophenols and a range of alkylbenzenes (C1-C6) via an inducible toluene dioxygenase enzyme system. Biphenyl and naphthalene were also oxidized by this enzyme. Growth on toluene and phenol induced the meta-ring-fission enzyme, catechol 2,3-oxygenase, whereas growth on benzoate, which did not require expression of toluene dioxygenase, induced the ortho-ring-cleavage enzyme, catechol 1,2-oxygenase. Monochlorobenzoate isomers and 2,3,5-trichlorophenol were gratuitous inducers of toluene dioxygenase, whereas 3,4-dichlorophenol was a fortuitous oxidation substrate of the enzyme. The organism also grew on 2,4- and 2,5-dichloro isomers of both phenol and benzoate, on 2,3,4-trichlorophenol and on 1-phenylheptane. During growth on toluene in nitrogen-limited chemostat culture, expression of both toluene dioxygenase and catechol 2,3-oxygenase was positively correlated with increase in specific growth rate (0.11-0.74 h-1), whereas the biomass yield coefficient decreased. At optimal dilution rates, the predicted performance of a 1-m3 bioreactor supplied with 1 g nitrogen l-1 for removal of toluene was 57 g day-1 and for removal of trichloroethylene was 3.4 g day-1. The work highlights the oxidative versatility of this bacterium with respect to substituted hydrocarbons and shows how growth rate influences the production of competent cells for potential use as bioremediation catalysts.     

The trithorax group gene moira encodes a brahma-associated putative chromatin-remodeling factor in Drosophila melanogaster

The genes of the trithorax group (trxG) in Drosophila melanogaster are required to maintain the pattern of homeotic gene expression that is established early in embryogenesis by the transient expression of the segmentation genes. The precise role of each of the diverse trxG members and the functional relationships among them are not well understood. Here, we report on the isolation of the trxG gene moira (mor) and its molecular characterization. mor encodes a fruit fly homolog of the human and yeast chromatin-remodeling factors BAF170, BAF155, and SWI3. mor is widely expressed throughout development, and its 170-kDa protein product is present in many embryonic tissues. In vitro, MOR can bind to itself and it interacts with Brahma (BRM), an SWI2-SNF2 homolog, with which it is associated in embryonic nuclear extracts. The leucine zipper motif of MOR is likely to participate in self-oligomerization; the equally conserved SANT domain, for which no function is known, may be required for optimal binding to BRM. MOR thus joins BRM and Snf5-related 1 (SNR1), two known Drosophila SWI-SNF subunits that act as positive regulators of the homeotic genes. These observations provide a molecular explanation for the phenotypic and genetic relationships among several of the trxG genes by suggesting that they encode evolutionarily conserved components of a chromatin-remodeling complex.     

Establishment of new genetic traits in a microbial biofilm community

Conjugational transfer of the TOL plasmid (pWWO) was analyzed in a flow chamber biofilm community engaged in benzyl alcohol degradation. The community consisted of three species, Pseudomonas putida RI, Acinetobacter sp. strain C6, and an unidentified isolate, D8. Only P. putida RI could act as a recipient for the TOL plasmid. Cells carrying a chromosomally integrated lacIq gene and a lacp-gfp-tagged version of the TOL plasmid were introduced as donor strains in the biofilm community after its formation. The occurrence of plasmid-carrying cells was analyzed by viable-count-based enumeration of donors and transconjugants. Upon transfer of the plasmids to the recipient cells, expression of green fluorescence was activated as a result of zygotic induction of the gfp gene. This allowed a direct in situ identification of cells receiving the gfp-tagged version of the TOL plasmid. Our data suggest that the frequency of horizontal plasmid transfer was low, and growth (vertical transfer) of the recipient strain was the major cause of plasmid establishment in the biofilm community. Employment of scanning confocal laser microscopy on fixed biofilms, combined with simultaneous identification of P. putida cells and transconjugants by 16S rRNA hybridization and expression of green fluorescence, showed that transconjugants were always associated with noninfected P. putida RI recipient microcolonies. Pure colonies of transconjugants were never observed, indicating that proliferation of transconjugant cells preferentially took place on preexisting P. putida RI microcolonies in the biofilm.     

Identification of tandemly repeated type VI cellulose-binding domains in an endoglucanase from the aerobic soil bacterium Cellvibrio mixtus

We present results showing that several species in the plant genus Leavenworthia, in the Brassica family, have three alcohol dehydrogenase loci, unlike Arabidopsis thaliana, which has only a single classical (class P) alcohol dehydrogenase locus. Based on a portion of the sequence, the alcohol dehydrogenase loci of Leavenworthia show about 92%-93% amino acid sequence identity to that of the A. thaliana alcohol dehydrogenase. The great majority of the sequence differences from the A. thaliana Adh-coding sequence, and also between three different Leavenworthia species, are synonymous, suggesting that all are currently functional (or have been in the recent evolutionary past). The loci differ in the numbers of introns present, with one locus (Adh-3) having no introns present. RT-PCR tests detect expression of all three loci. Linkage data using variant alleles identified by single-strand conformation polymorphism analysis show that the three Leavenworthia loci are not closely linked. The results therefore suggest that the Adh-3 locus may have arisen via an mRNA intermediate but, despite loss of the introns, is expressed.