Strain MB1 Research Articles

Gene Cloning and Nucleotide Sequencing and Properties of a Cocaine Esterase fromRhodococcussp. Strain MB1

A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an M(r) of approximately 65,000. The apparent K(m) of the enzyme (mean +/- standard deviation) for cocaine was measured as 1.33 +/- 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.

Identification of three merB genes and characterization of a broad-spectrum mercury resistance module encoded by a class II transposon of Bacillus megaterium strain MB1.

The complete structure of a broad-spectrum mercury resistance module was shown by sequencing the Gram-positive bacterial transposon TnMERI1 of Bacillus megaterium MB1. The regions encoding organomercury resistance were identified. Upstream of a previously identified organomercurial lyase merB (merB1) region of TnMERI1, a second merR (merR2) and a second merB gene (merB2) were found. These genes constitute a second operon (mer operon 2) following a promoter/operator (P(merR2)) region. A third organomercurial lyase gene (merB3) was found immediately upstream of the mer operon (mer operon 1) followed by a promoter/operator (P(merB3)) region homologous to that of the mer operon 1 (P(merR1)-merR1-merE-like-merT-merP-merA). The complete genetic structure of the mercury resistance module is organized as P(merB3)-merB3-P(merR1)-merR1-merE-like-merT+ ++ -merP-merA-P(merR2)-merR2 -merB2-merB1. The subcloning analysis of these three merB genes showed distinct substrate specificity as different organomercury lyase genes.

Characterization of a Pseudomonas putida allylic alcohol dehydrogenase induced by growth on 2-methyl-3-buten-2-ol.

We have been working to develop an enzymatic assay for the alcohol 2-methyl-3-buten-2-ol (232-MB), which is produced and emitted by certain pines. To this end we have isolated the soil bacterium Pseudomonas putida MB-1, which uses 232-MB as a sole carbon source. Strain MB-1 contains inducible 3-methyl-2-buten-1-ol (321-MB) and 3-methyl-2-buten-1-al dehydrogenases, suggesting that 232-MB is metabolized by isomerization to 321-MB followed by oxidation. 321-MB dehydrogenase was purified to near-homogeneity and found to be a tetramer (151 kDa) with a subunit mass of 37,700 Da. It catalyzes NAD+-dependent, reversible oxidation of 321-MB to 3-methyl-2-buten-1-al. The optimum pH for the oxidation reaction was 10.0, while that for the reduction reaction was 5.4. 321-MB dehydrogenase oxidized a wide variety of aliphatic and aromatic alcohols but exhibited the highest catalytic specificity with allylic or benzylic substrates, including 321-MB, 3-chloro-2-buten-1-ol, and 3-aminobenzyl alcohol. The N-terminal sequence of the enzyme contained a region of 64% identity with the TOL plasmid-encoded benzyl alcohol dehydrogenase of P. putida. The latter enzyme and the chromosomally encoded benzyl alcohol dehydrogenase of Acinetobacter calcoaceticus were also found to catalyze 321-MB oxidation. These findings suggest that 321-MB dehydrogenase and other bacterial benzyl alcohol dehydrogenases are broad-specificity allylic and benzylic alcohol dehydrogenases that, in conjunction with a 232-MB isomerase, might be useful in an enzyme-linked assay for 232-MB.

A phylogenetic analysis of aerobic polychlorinated biphenyl-degrading bacteria.

Several bacterial isolates were characterized based on their abilities to degrade specific polychlorinated biphenyls (PCBs) and their 16S rRNA gene sequences. The members of one group of bacteria consisting of Alcaligenes species, including the PCB-degrading bacterium Alcaligenes eutrophus H850, had strong abilities to degrade a broad range of PCBs but not the di-para-chlorine-substituted congeners. The members of another group, which included the PCB-degrading bacterium originally classified as Corynebacterium sp. strain MB1, had strong abilities to degrade di-para-chlorine-substituted PCBs. These bacteria were most likely different members of Rhodococcus species.

A systematic study on equilibrium and kinetics of biosorptive accumulation. The case of Ag and Ni

Biosorptive metal uptake capacities have been quantitatively evaluated through a systematic experimental approach for silver and nickel and six selected strains of microbial biomass. Strains MB 127 and BP 7 26 have been identified to be the most efficient for silver sequestering. Biosorption of nickel by the strains tested is generally low. Overall kinetic experiments have been performed for the determination of the necessary contact time for the attainment of equilibrium. Systematic desorption experiments have shown that biosorption of silver and nickel by the strains tested is irreversible under the conditions examined. Simultaneous considerations of hydrolysis behaviour, chemical coordination, and stereochemical and redox characteristics have been proven to be very helpful in understanding the observed differences between silver and nickel biosorptive performances.

Reclassification of the Polychlorinated Biphenyl-Degraders Acinetobacter sp. Strain P6 and Corynebacterium sp. Strain MB1 as Rhodococcus globerulus

Summary Polychlorinated biphenyls-degrading isolates, previously identified as Acinetobacter sp. strain P6 and Corynebacterium sp. strain MB1, have been definitively determined to be almost identical strains of Rhodococcus globerulus by polyphasic analyses including phenotypic characterization, chemotaxonomic determination and characterization of cell wall amino acids and sugars, plasmid content, genomic DNA fingerprinting and 16S ribosomal DNA sequence determination.

A Gram-positive Polychlorinated Biphenyl-degrading Bacterium,Rhodococcus erythropolisStrain TA421, Isolated from a Termite Ecosystem

Gram-positive bacteria, identified as Rhodococcus erythropolis, were isolated from the ecosystem of the wood-feeding termite Reticulitermes speratus and found to aerobically degrade polychlorinated biphenyl (PCB) compounds. Rhodococcus erythropolis strain TA421 and strain TA431 were isolated by enrichment culture from termites obtained from different locations and each was found to be capable of degrading polychlorinated biphenyl (PCB) compounds to chlorobenzoates. These results suggest that the termite ecosystem is one possible habitat for biphenyl- and PCB-degrading Rhodococci. The spectrum of PCB-congeners degraded by strain TA421 is different from that of other, previously characterized PCB-degrading bacteria such as Rhodococcus globerulus strain P6 (formerly Corynebacterium sp. strain MB1) or Pseudomonas sp. strain LB400.

Three different 2,3-dihydroxybiphenyl-1,2-dioxygenase genes in the gram-positive polychlorobiphenyl-degrading bacterium Rhodococcus globerulus P6.

Rhodococcus globerulus P6 (previously designated Acinetobacter sp. strain P6, Arthrobacter sp. strain M5, and Corynebacterium sp. strain MB1) is able to degrade a wide range of polychlorinated biphenyl (PCB) congeners. The genetic and biochemical analyses of the PCB catabolic pathway reported here have revealed the existence of a PCB gene cluster--bphBC1D--and two further bphC genes--bphC2 and bphC3--that encode three narrow-substrate-specificity enzymes (2,3-dihydroxybiphenyl dioxygenases) that meta cleave the first aromatic ring. None of the bphC genes show by hybridization homology to each other or to bphC genes in other bacteria, and the three bphC gene products have different kinetic parameters and sensitivities to inactivation by 3-chlorocatechol. This suggests that there exists a wide diversity in PCB meta cleavage enzymes.

Evidence for novel mechanisms of polychlorinated biphenyl metabolism in Alcaligenes eutrophus H850.

Previous studies indicated that Alcaligenes eutrophus H850 attacks a different spectrum of polychlorinated biphenyl (PCB) congeners than do most PCB-degrading bacteria and that novel mechanisms of PCB degradation might be involved. To delineate this, we have investigated the differences in congener selectivity and metabolite production between H850 and Corynebacterium sp. strain MB1, an organism that apparently degrades PCBs via a 2,3-dioxygenase. H850 exhibited a superior ability to degrade congeners via attack on 2-, 2,4-, 2,5-, or 2,4,5-chlorophenyl rings in PCBs but an inferior ability to degrade congeners via attack on a 4-chlorophenyl ring. Reactivity preferences were also reflected in the products formed from unsymmetrical PCBs; thus MB1 attacked the 2,3-chlorophenyl ring of 2,3,2',5'-tetrachlorobiphenyl to yield 2,5-dichlorobenzoic acid, while H850 attacked the 2,5-chlorophenyl ring to yield 2,3-dichlorobenzoic acid and a novel metabolite, 2',3'-dichloroacetophenone. Furthermore, H850 oxidized 2,4,5,2',4',5'-hexachlorobiphenyl, a congener with no adjacent unsubstituted carbons, to 2',4',5'-trichloroacetophenone. The atypical congener selectivity pattern and novel metabolites produced suggest that A. eutrophus H850 may degrade certain PCB congeners by a new route beginning with attack by some enzyme other than the usual 2,3-dioxygenase.