Cloning, Expression, and Nucleotide Sequence of the Pseudomonas aeruginosa 142 ohb Genes Coding for Oxygenolytic ortho Dehalogenation of Halobenzoates

We have cloned and characterized novel oxygenolytic ortho-dehalogenation (ohb) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degrading Pseudomonas aeruginosa 142. Among 3,700 Escherichia coli recombinants, two clones, DH5αF′(pOD22) and DH5αF′(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclone of pOD33, plasmid pE43, containing the 3,687-bp minimized ohb DNA region conferred to P. putida PB2440 the ability to grow on 2-CBA as a sole carbon source. Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA. Terminal oxidoreductase ISP_OHB structural genes ohbA and ohbB, which encode polypeptides with molecular masses of 20,253 Da (β-ISP) and 48,243 Da (α-ISP), respectively, were identified; these proteins are in accord with the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypeptides synthesized in E. coli and P. aeruginosa parental strain 142. The ortho-halobenzoate 1,2-dioxygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISP_OHB utilized electron transfer components provided by the heterologous hosts. ISP_OHB formed a new phylogenetic cluster that includes aromatic oxygenases featuring atypical structural-functional organization and is distant from the other members of the family of primary aromatic oxygenases. A putative IclR-type regulatory gene (ohbR) was located upstream of the ohbAB genes. An open reading frame (ohbC) of unknown function that overlaps lengthwise with ohbB but is transcribed in the opposite direction was found. The ohbC gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in E. coli. The ohb genes are flanked by an IS1396-like sequence containing a putative gene for a 39,715-Da transposase A (tnpA) at positions 4731 to 5747 and a putative gene for a 45,247-Da DNA topoisomerase I/III (top) at positions 346 to 1563. The ohb DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.     

Construction and characterization of two recombinant bacteria that grow on ortho- and para-substituted chlorobiphenyls

Cloning and expression of the aromatic ring dehalogenation genes in biphenyl-growing, polychlorinated biphenyl (PCB)-cometabolizing Comamonas testosteroni VP44 resulted in recombinant pathways allowing growth on ortho- and para-chlorobiphenyls (CBs) as a sole carbon source. The recombinant variants were constructed by transformation of strain VP44 with plasmids carrying specific genes for dehalogenation of chlorobenzoates (CBAs). Plasmid pE43 carries the Pseudomonas aeruginosa 142 ohb genes coding for the terminal oxygenase (ISPOHB) of the ortho-halobenzoate 1,2-dioxygenase, whereas plasmid pPC3 contains the Arthrobacter globiformis KZT1 fcb genes, which catalyze the hydrolytic para-dechlorination of 4-CBA. The parental strain, VP44, grew only on low concentrations of 2- and 4-CB by using the products from the fission of the nonchlorinated ring of the CBs (pentadiene) and accumulated stoichiometric amounts of the corresponding CBAs. The recombinant strains VP44(pPC3) and VP44(pE43) grew on, and completely dechlorinated high concentrations (up to 10 mM), of 4-CBA and 4-CB and 2-CBA and 2-CB, respectively. Cell protein yield corresponded to complete oxidation of both biphenyl rings, thus confirming mineralization of the CBs. Hence, the use of CBA dehalogenase genes appears to be an effective strategy for construction of organisms that will grow on at least some congeners important for remediation of PCBs.     

Cloning, characterization, and expression of a novel gene encoding a reversible 4-hydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum.

A novel gene, designated ohb1, which encodes the oxygen-sensitive and biotin-, ATP-, thiamin-, pyridoxal phosphate-, and metal-ion-independent, reversible 4-hydroxybenzoate decarboxylase (4-HOB-DC) from the obligate anaerobe Clostridium hydroxybenzoicum JW/Z-1(T) was sequenced (GenBank accession no. AF128880) and expressed. The 1,440-bp open reading frame (ORF) (ohb1) encodes 480 amino acids. Major properties of the heterologous enzyme (Ohb1) expressed in Escherichia coli DH5alpha were the same as those described for the native 4-HOB-DC (Z. He and J. Wiegel, J. Bacteriol. 178:3539-3543, 1996). The deduced amino acid sequence shows up to 57% identity and up to 74% similarity to hypothetical proteins deduced from ORFs in genomes from bacteria and archaea, suggesting a possible novel gene family.     

Cloning and heterologous expression of xylanase from Pichia stipitis in Escherichia coli

AIMS: The main goal of this study was to characterize the xylanase (xynA) gene from Pichia stipitis NRRL Y-11543. METHODS AND RESULTS: The xylanase gene was cloned into pUC19 in Escherichia coli DH5alphaF' and selected by growth on RBB-xylan. All functional clones contained a recombinant plasmid with an insert of 2.4 kbp, as determined by restriction mapping. The nucleotide sequence of the P. stipitis xylanase gene consisted of 1146 bp and encoded a protein of 381 amino acids with a molecular weight of 43 649 Da. The sequence contained a putative 20-amino acid N-terminal signal sequence and four N-linked glycosylation sites. The Km values for non-glycosylated and glycosylated xylanases were 1.4 mg ml-1 and 4.2 mg ml-1, respectively, and Vmax values were 0.8 and 0.082 micromol min-1 mg-1 protein, respectively. CONCLUSION: Xylanase, a rarely found enzyme in yeast species, has been characterized in detail. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study can be used to develop better xylanase-utilizing yeast strains.     

Aerobic mineralization of chlorobenzoates by a natural polychlorinated biphenyl-degrading mixed bacterial culture

A natural mixed aerobic bacterial culture, designated MIXE1, was found to be capable of degrading several low-chlorinated biphenyls when 4-chlorobiphenyl was used as a co-substrate. MIXE1 was capable of using all the three monochlorobenzoate (CBA) isomers tested as well as 2,5-, 3,4- and 3,5-dichlorobenzoate (dCBA) as the sole carbon and energy source. During MIXE1 growth on these substrates, a nearly stoichiometric amount of chloride was released: 0.5 g/l of each chlorobenzoate was completely mineralized by MIXE1 after 2 or 3 days of culture incubation. Two strains, namely CPE2 and CPE3, were selected from MIXE1: CPE2, referred to the Pseudomonas genus, was found to be capable of totally degrading both 2-CBA and 2,5-dCBA, whereas Alcaligenes strain CPE3 was capable of mineralizing 3-, 4-CBA and 3,4-dCBA. Substrate uptake studies carried out with whole cells of strain CPE2 suggested that 2-CBA was metabolized through catechol, while 2,5-dCBA was degraded via 4-chlorocatechol. 3-CBA, 4-CBA, and 3,4-dCBA appeared to be degraded through 3,4-dihydroxybenzoate by the CPE3 strain. MIXE1, which is capable of degrading several chlorobenzoates, should therefore be able to mineralize a number of low-chlorinated congeners of simple and complex polychlorinated biphenyl mixtures. Correspondence to: F. Fava     

Characterization of a plasmid carrying the Escherichia coli K-12 entD, fepA, fes, and entF genes

Abstract The plasmid pMS101 carries Escherichia coli K-12 genes ( entD, fes, entF ) essential for enterochelin-mediated iron transport [Laird, A.J. and Young, I.G., Gene 11 (1980) 359–366]. We have further characterized pMS101 and shown that it also contains the gene ( fepA ) for the 81 000 Da outer membrane ferrienterochelin receptor. Subcloning experiments in conjunction with complementation and maxicell studies demonstrated the gene order to be entD fepA fes entF . The entF - and fes -encoded polypeptides were found to be 115 000 and 42 000-Da soluble proteins, respectively. Plasmid-borne enterochelin cluster genes were overexpressed in iron-deficient conditions and their products were undetectable under iron-replete conditions.     

Cloning, characterization, and expression in Escherichia coli of a gene encoding Listeria seeligeri catalase, a bacterial enzyme highly homologous to mammalian catalases.

A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the gram-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DH5 alpha as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately 50 times that of the combined E. coli catalases. The nucleotide sequence was determined, and the deduced amino acid sequence revealed 43.2% amino acid sequence identity between bovine liver catalase and L. seeligeri catalase. Most of the amino acid residues which are involved in catalytic activity, the formation of the active center accession channel, and heme binding in bovine liver catalase were also present in L. seeligeri catalase at the corresponding positions. The recombinant protein contained 488 amino acid residues and had a calculated molecular weight of 55,869. The predicted isoelectric point was 5.0. Enzymatic and genetic analyses showed that there is most probably a single catalase of this type in L. seeligeri. A perfect 21-bp inverted repeat, which was highly homologous to previously reported binding sequences of the Fur (ferric uptake regulon) protein of E. coli, was detected next to the putative promoter region of the L. seeligeri catalase gene.     

Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli. Cloning and characterization of quinolinate synthesis genes nadA and nadB

The two genes, nadA and nadB, responsible for quinolinate biosynthesis from aspartate and dihydroxyacetone phosphate in Escherichia coli were cloned and characterized. Quinolinate (pyridine-2,3-dicarboxylate) is the biosynthetic precursor of the pyridine ring of NAD. Gene nadA was identified by complementation in three different nadA mutant strains. Sequence analysis provided an 840-bp open reading frame coding for a 31,555-Da protein. Gene nadB was identified by complementation in a nadB mutant strain and by the L-aspartate oxidase activity of its gene product. Sequence analysis showed a 1620-bp open reading frame coding for a 60,306-Da protein. For both genes, promoter regions and ribosomal binding sites were assigned by comparison to consensus sequences. The nadB gene product, L-aspartate oxidase, was purified to homogeneity and the N-terminal sequence of 19 amino acids was determined. The enzyme was shown to be specific for L-aspartate. High-copy-number vectors, carrying either gene nadA, nadB or nadA + nadB, increased quinolinate production 1.5-fold, 2.0-fold and 15-fold respectively. Both gene products seem to be equally rate-limiting in quinolinate synthesis.     

Identification of a purC gene from Streptococcus pneumoniae.

A gene encoding 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide synthetase was identified in Streptococcus pneumoniae as a 708-bp segment of the genome encoding a 27,001-Da protein with strong similarity to known PurC proteins. The S. pneumoniae purC gene, found immediately adjacent to the competence induction genes, comAB, was cloned and sequenced. The predicted protein product of purC displayed substantial (> 40%) identity to the entire sequence of the PurC proteins of Bacillus subtilis and Escherichia coli. Function of the S. pneumoniae gene product was demonstrated by complementation of E. coli purC mutations.     

Structures of the rfaB, rfaI, rfaJ, and rfaS genes of Escherichia coli K-12 and their roles in assembly of the lipopolysaccharide core.

Analysis of the sequence of a 4.1-kb rfa region downstream from rfaP revealed four genes. The first of these encodes a basic protein of 36,730 Da and does not correspond to any known rfa gene. It has been designated rfaS. The second gene was identified as rfaB on the basis of its ability to complement a Salmonella typhimurium rfaB mutant and encodes a 42,060-Da protein. The third and fourth genes encode proteins of 39,423 and 36,046 Da which are strongly homologous to the RfaI and RfaJ proteins of S. typhimurium. Escherichia coli K-12 restriction fragments carrying these genes complement an S. typhimurium rfaI mutant and, at lower efficiency, an rfaJ mutant. The difference in complementation efficiency suggests that the rfaI and rfaJ genes of E. coli K-12 have sugar and acceptor specificities different from those of S. typhimurium, as predicted from the different lipopolysaccharide (LPS) core structures of the two organisms. Defined mutations affecting all four genes were constructed in vitro and crossed onto the chromosome. The phenotypes of these mutations suggest that extension of the core may require protein-protein interactions between the enzymes involved in core completion as well as the interaction of these enzymes with their specific acceptor molecules. Mutants blocked at rfaI or genes encoding earlier steps in core biosynthesis exhibited a single predominant LPS band on gels while mutants blocked at rfaJ or genes encoding later steps produced multiple strong bands, indicating that one of the processes generating core heterogeneity requires a functional rfaI gene.     

Synthesis of F-pilin polypeptide in the absence of F traJ product

The products of a lambda transducing phage (ED lambda 101) which carries a segment of the F tra operon expressing F traA , traL , and traE activity from the lambda leftward promoter were examined using a uv-irradiated host system. After infection of an F- host, products of traE (19,500 Da) and traA (14,000 Da) were detectable among the lambda early proteins synthesized. Infection of an Flac host altered the pattern of polypeptides synthesized by the phage in that the 14,000-Da traA product became barely detectable and was replaced by a polypeptide which migrated at 7000 Da. A derivative of ED lambda 101 carrying the traA1 amber mutation was unable to synthesize either the 14,000-Da polypeptide in F- cells or the 7000-Da polypeptide in Flac cells. The 7000-Da polypeptide derived from ED lambda 101 was synthesized in the absence of traJ product in F- cells coinfected with a second transducing phage which carried a tra operon segment including traQ . It was also a product of ED lambda 134 which expresses genes traA through traH . The 7000-Da polypeptide, like F-pilin, associated primarily with the inner membrane, and could be immunoprecipitated with antiserum prepared against purified F-pili. Analysis of membranes from F- cells infected with ED lambda 101 indicated that the 14,000-Da traA product synthesized under these conditions accumulated in the inner membrane. These results show that both the 14,000-Da traA product might be processed to F-pilin in a traQ -dependent reaction which occurs in or on the inner membrane of the Escherichia coli host. However, the possibility that traQ encodes a regulatory product which affects expression of the traA sequence has not been excluded.     

Insertional inactivation of a gene which controls expression of vancomycin resistance on plasmid pHKK100

Expression of inducible high level vancomycin resistance (Vmr) in enterococci appears to require other plasmid-encoded genes in addition to the previously described structural genes vanA and vanH. Tn917 mutagenesis was used to identify such a region in the Vmr plasmid pHKK100. Insertional inactivation of a 693-bp open reading frame upstream from vanH resulted in complete loss of Vmr. This putative 26,642-Da protein has been designated VanR.