Haloalkane degradation and assimilation by Rhodococcus rhodochrous NCIMB -13064

The bacterium Rhodococcus rhodochrous NCIMB 13064, isolated from an industrial site, could use a wide range of 1-haloalkanes as sole carbon source but apparently utilized several different mechanisms simultaneously for assimilation of substrate. Catabolism of 1-chlorobutane occurred mainly by attack at the C-1 atom by a hydrolytic dehalogenase with the formation of butanol which was metabolized via butyric acid. The detection of small amounts of gamma-butyrolactone in the medium suggested that some oxygenase attack at C-4 also occurred, leading to the formation of 4-chlorobutyric acid which subsequently lactonized chemically to gamma-butyrolactone. Although 1-chlorobutane-grown cells exhibited little dehalogenase activity on 1-chloroalkanes with chain lengths above C-10, the organism utilized such compounds as growth substrates with the release of chloride. Concomitantly, gamma-butyrolactone accumulated to 1 mM in the culture medium with 1-chlorohexadecane as substrate. Traces of 4-hydroxybutyric acid were also detected. It is suggested that attack on the long-chain chloroalkane is initiated by an oxygenase at the non-halogenated end of the molecule leading to the formation of an omega-chlorofatty acid. This is degraded by beta-oxidation to 4-chlorobutyric acid which is chemically lactonized to gamma-butyrolactone which is only slowly further catabolized via 4-hydroxybutyric acid and succinic acid. However, release of chloride into the medium during growth on long-chain chloroalkanes was insufficient to account for all the halogen present in the substrate. Analysis of the fatty acid composition of 1-chlorohexadecane-grown cells indicated that chlorofatty acids comprised 75% of the total fatty acid content with C-14:0, C-16:0, C-16:1, and C-18:1 acids predominating. Thus the incorporation of 16-chlorohexadecanoic acid, the product of oxygenase attack directly into cellular lipid represents a third route of chloroalkane assimilation. This pathway accounts at least in part for the incomplete mineralization of long-chain chloroalkane substrates. This is the first report of the coexistence of a dehalogenase and the ability to incorporate long-chain haloalkanes into the lipid fraction within a single organism and raises important questions regarding the biological treatment of haloalkane containing effluents.

Plasmid pRTL1 controlling 1-chloroalkane degradation by Rhodococcus rhodochrous NCIMB 13064

Rhodococcus rhodochrous NCIMB13064 can dehalogenate and use a wide range of 1-haloalkanes as sole carbon and energy source. The 1-chloroalkane degradation phenotype may be lost by cells spontaneously or after treatment with Mitomycin C. Two laboratory derivatives of the original strain exhibited differing degrees of stability of the chloroalkane degradation marker. Plasmids of approximately 100 kbp (pRTL1) and 80 kbp (pRTL2) have been found in R. rhodochrous NCIMB13064. pRTL1 was shown to be carrying at least some genes for the dehalogenation of 1-chloroalkanes with short chain lengths (C-3 to C-9). However, no connection was found between the utilization of 1-chloroalkanes with longer chain lengths (C-12 to C-18) and the presence of pRTL1. Three separate events were observed to lead to the inability of NCIMB13064 to dehalogenate the short-chain 1-chloroalkanes; the complete loss of pRTL1, the integration of pRTL1 into the chromosome, or the deletion of a 20-kbp fragment in pRTL1. High-frequency transfer of the 1-chloroalkane degradation marker associated with pRTL1 has been demonstrated in bacterial crosses between different derivatives of R. rhodochrous NCIMB13064, (C) 1995 Academic Press, Inc.

The purification and properties of phosphonoacetate hydrolase, a novel carbon-phosphorus bond-cleavage enzyme from Pseudomonas fluorescens 23F

A novel, inducible, carbon-phosphorus bond-cleavage enzyme, phosphonoacetate hydrolase, was purified from cells of Pseudomonas fluorescens 23F grown phosphonoacetate. The native enzyme had a molecular mass of approximately 80 kDa and, upon SDS/PAGE, yielded a homogenous protein band with an apparent molecular mass of about 38 kDa. Activity of purified phosphonoacetate hydrolase was Zn2+ dependent and showed pH and temperature optima of approximately 7.8 and 37 degrees C, respectively. The purified enzyme had an apparent K-m of 1.25 mM for its sole substrate phosphonoacetate, and was inhibited by the structural analogues 3-phosphonopropionate and phosphonoformate. The NH2-terminal sequence of the first 19 amino acids displayed no significant similarity to other databank sequences.

Degradation of 1,3-dichloropropene by Pseudomonas cichorii 170

The gram-negative bacterium Pseudomonas cichorii 170, isolated from soil that was repeatedly treated with the nematocide 1,3-dichloropropene, could utilize low concentrations of 1,3-dichloropropene as a sole carbon and energy source, Strain 170 was also able to grow on 3-chloroallyl alcohol, 3-chloroacrylic acid, and several 1-halo-n-alkanes. This organism produced at least three different dehalogenases: a hydrolytic haloalkane dehalogenase specific for haloalkanes and two 3-chloroacrylic acid dehalogenases, one specific for cis-3-chloroacrylic acid and the other specific for trans-3-chloroacrylic acid. The haloalkane dehalogenase and the trans-3-chloroacrylic acid dehalogenase were expressed constitutively, whereas the cis-3-chloroacrylic acid dehalogenase was inducible, The presence of these enzymes indicates that 1,3-dichloropropene is hydrolyzed to 3-chloroallyl alcohol, which is oxidized in two steps to 3-chloroacrylic acid. The latter compound is then dehalogenated, probably forming malonic acid semialdehyde. The haloalkane dehalogenase gene, which is involved in the conversion of 1,3-dichloropropene to 3-chloroallyl alcohol, was cloned and sequenced, and this gene turned out to be identical to the previously studied dhaA gene of the gram-positive bacterium Rhodococcus rhodochrous NCIMB13063, Mutants resistant to the suicide substrate 1,2-dibromoethane lacked haloalkane dehalogenase activity and therefore could not utilize haloalkanes for growth. PCR analysis showed that these mutants had lost at least part of the dhaA gene.

Cloning of new Rhodococcus extradiol dioxygenase genes and study of their distribution in different Rhodococcus strains

Four extradiol dioxygenase genes which encode enzymes active against catechol and substituted catechols were cloned from two different Rhodococcus strains, and their nucleotide sequences were determined. A catechol 2,3-dioxygenase gene (edoC) was shown to be identical to the previously described ipbC gene from the isopropylbenzene operon of Rhodococcus erythropolis. Amino acid sequences deduced from the three other genes (edoA, edoB and edoD) were shown to have various degrees of homology to different extradiol dioxygenases, The EdoA and EdoB dioxygenases were classified as belonging to the third family of type I oxygenases and represented two new subfamilies, whereas the EdoD dioxygenase was a type II enzyme. Analysis of six Rhodococcus strains revealed a wide distribution of the above dioxygenase genes. Rhodococcus sp. I1 was shown to harbour all four of the analysed dioxygenase genes. Nucleotide sequences homologous to the edoB gene were present in all of the strains, including R. erythropolis NCIMB 13065, which did not utilize any of the aromatic compounds analysed. The latter finding points to the existence of a silent pathway(s) for degradation of aromatic compounds in this Rhodococcus strain.

Degradation of carbon disulphide (CS2) in soils and groundwater from a CS2 -contaminated site

This study is the first investigation of biodegradation of carbon disulphide (CS₂) in soil that provides estimates of degradation rates and identifies intermediate degradation products and carbon isotope signatures of degradation. Microcosm studies were undertaken under anaerobic conditions using soil and groundwater recovered from CS₂-contaminated sites. Proposed degradation mechanisms were validated using equilibrium speciation modelling of concentrations and carbon isotope ratios. A first-order degradation rate constant of 1.25 × 10-2 h-1 was obtained for biological degradation with soil. Carbonyl sulphide (COS) and hydrogen sulphide (H₂S) were found to be intermediates of degradation, but did not accumulate in vials. A 13C/12C enrichment factor of -7.5 ± 0.8 ‰ was obtained for degradation within microcosms with both soil and groundwater whereas a 13C/12C enrichment factor of -23.0 ± 2.1 ‰ was obtained for degradation with site groundwater alone. It can be concluded that biological degradation of both CS2-contaminated soil and groundwater is likely to occur in the field suggesting that natural attenuation may be an appropriate remedial tool at some sites. The presence of biodegradation by-products including COS and H₂S indicates that biodegradation of CS₂ is occurring and stable carbon isotopes are a promising tool to quantify CS₂ degradation.

Structural and kinetic characterization of the LPS biosynthetic enzyme D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) from Escherichia coli

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

Structure and function of sedoheptulose-7-phosphate isomerase, a critical enzyme for lipopolysaccharide biosynthesis and a target for antibiotic adjuvants

The barrier imposed by lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria presents a significant challenge in treatment of these organisms with otherwise effective hydrophobic antibiotics. The absence of L-glycero-D-manno-heptose in the LPS molecule is associated with a dramatically increased bacterial susceptibility to hydrophobic antibiotics and thus enzymes in the ADP-heptose biosynthesis pathway are of significant interest. GmhA catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-manno-heptose 7-phosphate, the first committed step in the formation of ADP-heptose. Here we report structures of GmhA from Escherichia coli and Pseudomonas aeruginosa in apo, substrate, and product-bound forms, which together suggest that GmhA adopts two distinct conformations during isomerization through reorganization of quaternary structure. Biochemical characterization of GmhA mutants, combined with in vivo analysis of LPS biosynthesis and novobiocin susceptibility, identifies key catalytic residues. We postulate GmhA acts through an enediol-intermediate isomerase mechanism.

Functional characterization of the initiation enzyme of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-l-Rhap-(1-->], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three alpha-l-rhamnose residues, and a beta-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of Escherichia coli and Salmonella enterica serovar Typhimurium.

Outer membrane protein profiles and multilocus enzyme electrophoresis analysis for differentiation of clinical isolates of Proteus mirabilis and Proteus vulgaris

Outer membrane protein (MP) profiles and multilocus enzyme electrophoresis (MEE) analysis were used as tools for differentiating clinical isolates of Proteus spp. Fourteen distinct MP profiles were established by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis in 54 clinical isolates of Proteus spp. (44 strains identified as P. mirabilis and 10 strains identified as P. vulgaris). Forty-one isolates of P. mirabilis and eight isolates of P. vulgaris were grouped within six and three MP profiles, respectively. The remaining P. mirabilis and P. vulgaris isolates had unique profiles. MEE analysis was used to further discriminate among the strains belonging to the same MP groups. Thirty-five distinct electrophoretic types (ETs) were identified among P. mirabilis isolates. The isolates of P. mirabilis from the four most common MP groups were subgrouped into 30 ETs. All of the P. vulgaris strains had unique ETs. The results suggest that upon biochemical classification of Proteus isolates as P. mirabilis or P. vulgaris, further differentiation among strains of the same species can be obtained by the initial determination of MP profiles followed by MEE analysis of strains with identical MPs.

A PLATE ASSAY FOR THE DETECTION OF ORGANOPHOSPHONATE MINERALIZATION BY ENVIRONMENTAL BACTERIA, AND ITS MODIFICATION AS AN ACTIVITY STAIN FOR IDENTIFICATION OF THE CARBON-PHOSPHORUS BOND-CLEAVAGE ENZYME PHOSPHONOACETATE HYDROLASE

A replica plate screening technique, based on the acid molybdate assay for detection of phosphate has been developed to permit the detection of microorganisms capable of mineralizing organophosphonates. The method was further adapted as the basis of an activity stain for the detection of the carbon - phosphorus bond cleavage enzyme phosphonoacetate hydrolase in PAGE gels.

Development of a solid-phase extraction coupling chemiluminescent enzyme immunoassay for determination of organophosphorus pesticides in environmental water samples

Solid-phase extraction (SPE) and direct competitive chemiluminescence enzyme immunoassay (dcCL-EIA) were combined for the detection of organophosphorus pesticides (OPs) in environmental water samples. dcCL-EIA based on horseradish peroxidase labeled with a broad-specificity monoclonal antibody against OPs was developed, and the effects of several physicochemical parameters on dcCL-EIA performance were studied. SPE was used for the pretreatment of water samples to remove interfering substances and to concentrate the OP analytes. The coupling of SPE and dcCL-EIA can detect seven OPs (parathion, coumaphos, phoxim, quinalphos, triazophos, dichlofenthion, and azinphos-ethyl) with the limit of quantitation below 0.1 ng/mL. The recoveries of OPs from spiked water samples ranged from 62.5% to 131.7% by SPE-dcCL-EIA and 69.5% to 112.3% by SPE-HPLC-MS/MS. The screening of OP residues in real-world environmental water samples by the developed SPE-dcCL-EIA and their confirmatory analysis using SPE-HPLC-MS/MS demonstrated that the assay is ideally suited as a monitoring method for OP residues prior to chromatographic analysis.