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C S Harwood

Publications and source records attributed to C S Harwood.

At least 19 recordsLinked to original sources
PubMed2001

Reductive, coenzyme A-mediated pathway for 3-chlorobenzoate degradation in the phototrophic bacterium Rhodopseudomonas palustris.

We isolated a strain of Rhodopseudomonas palustris (RCB100) by selective enrichment in light on 3-chlorobenzoate to investigate the steps that it uses to accomplish anaerobic dechlorination. Analyses of metabolite pools as well as enzyme assays suggest that R. palustris grows on 3-chlorobenzoate by (i) converting it to 3-chlorobenzoyl coenzyme A (3-chlorobenzoyl-CoA), (ii) reductively dehalogenating 3-chlorobenzoyl-CoA to benzoyl-CoA, and (iii) degrading benzoyl-CoA to acetyl-CoA and carbon dioxide. R. palustris uses 3-chlorobenzoate only as a carbon source and thus incorporates the acetyl-CoA that is produced into cell material. The reductive dechlorination route used by R. palustris for 3-chlorobenzoate degradation differs from those previously described in that a CoA thioester, rather than an unmodified aromatic acid, is the substrate for complete dehalogenation.

Biodegradation, Environmental↗
PubMed2000

An aerotaxis transducer gene from Pseudomonas putida.

An aerotaxis gene, aer, was cloned from Pseudomonas putida PRS2000. A P. putida aer mutant displayed an altered aerotactic response in a capillary assay. Wild-type P. putida clustered at the air/liquid interface. In contrast, the aer mutant did not cluster at the interface, but instead formed a diffuse band at a distance from the meniscus. Wild-type aer, provided in trans, complemented the aer mutant to an aerotactic response that was stronger than wild-type. The P. putida Aer sequence is similar over its entire length to the aerotaxis (energy taxis) signal transducer protein, Aer, of Escherichia coli. The amino-terminus is similar to redox-sensing regulatory proteins, and the carboxy-terminus contains the highly conserved domain present in chemotactic transducers.

Carrier Proteins↗
PubMed2000

Toluene-degrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene.

The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene. Pseudomonas mendocina KR1 and P. putida PaW15 did not show a convincing response. The chemotactic responses of P. putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for P. putida F1, including benzene and ethylbenzene, were chemoattractants. P. putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P. putida F1. Mutant strains of P. putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P. putida F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.

Alkanes↗
PubMed2000

HbaR, a 4-hydroxybenzoate sensor and FNR-CRP superfamily member, regulates anaerobic 4-hydroxybenzoate degradation by Rhodopseudomonas palustris.

Under anaerobic conditions, structurally diverse aromatic compounds are catabolized by bacteria to form benzoyl-coenzyme A (benzoyl-CoA), the starting compound for a central reductive pathway for aromatic ring degradation. The structural genes required for the conversion of 4-hydroxybenzoate (4-HBA) to benzoyl-CoA by Rhodopseudomonas palustris have been identified. Here we describe a regulatory gene, hbaR, that is part of the 4-HBA degradation gene cluster. An hbaR mutant that was constructed was unable to grow anaerobically on 4-HBA. However, the mutant retained the ability to grow aerobically on 4-HBA by an oxygen-requiring pathway distinct from the anaerobic route of 4-HBA degradation. The effect of the HbaR protein on expression of hbaA encoding 4-HBA-CoA ligase, the first enzyme for 4-HBA degradation, was investigated by using hbaA::'lacZ transcriptional fusions. HbaR was required for a 20-fold induction of beta-galactosidase activity that was observed with a chromosomal hbaA::'lacZ fusion when cells grown anaerobically on succinate were switched to anaerobic growth on succinate and 4-HBA. HbaR also activated expression from a plasmid-borne hbaA-'lacZ fusion when it was expressed in aerobically grown Pseudomonas aeruginosa cells, indicating that the activity of this regulator is not sensitive to oxygen. The deduced amino acid sequence of HbaR indicates that it is a member of the FNR-CRP superfamily of regulatory proteins. It is most closely related to transcriptional activators that are involved in regulating nitrate reduction. Previously, it has been shown that R. palustris has an FNR homologue, called AadR, that is also required for 4-HBA degradation. Our evidence indicates that AadR activates expression of hbaR in response to anaerobiosis and that HbaR, in turn, activates expression of 4-HBA degradation in response to 4-HBA as an effector molecule.

Aerobiosis↗
PubMed2000

2-Hydroxycyclohexanecarboxyl coenzyme A dehydrogenase, an enzyme characteristic of the anaerobic benzoate degradation pathway used by Rhodopseudomonas palustris.

A gene, badH, whose predicted product is a member of the short-chain dehydrogenase/reductase family of enzymes, was recently discovered during studies of anaerobic benzoate degradation by the photoheterotrophic bacterium Rhodopseudomonas palustris. Purified histidine-tagged BadH protein catalyzed the oxidation of 2-hydroxycyclohexanecarboxyl coenzyme A (2-hydroxychc-CoA) to 2-ketocyclohexanecarboxyl-CoA. These compounds are proposed intermediates of a series of three reactions that are shared by the pathways of cyclohexanecarboxylate and benzoate degradation used by R. palustris. The 2-hydroxychc-CoA dehydrogenase activity encoded by badH was dependent on the presence of NAD(+); no activity was detected with NADP(+) as a cofactor. The dehydrogenase activity was not sensitive to oxygen. The enzyme has apparent K(m) values of 10 and 200 microM for 2-hydroxychc-CoA and NAD(+), respectively. Western blot analysis with antisera raised against purified His-BadH identified a 27-kDa protein that was present in benzoate- and cyclohexanecarboxylate-grown but not in succinate-grown R. palustris cell extracts. The active form of the enzyme is a homotetramer. badH was determined to be the first gene in an operon, termed the cyclohexanecarboxylate degradation operon, containing genes required for both benzoate and cyclohexanecarboxylate degradation. A nonpolar R. palustris badH mutant was unable to grow on benzoate or cyclohexanecarboxylate but had wild-type growth rates on succinate. Cells blocked in expression of the entire cyclohexanecarboxylate degradation operon excreted cyclohex-1-ene-1-carboxylate into the growth medium when given benzoate. This confirms that cyclohex-1-ene-1-carboxyl-CoA is an intermediate of anaerobic benzoate degradation by R. palustris. This compound had previously been shown not to be formed by Thauera aromatica, a denitrifying bacterium that degrades benzoate by a pathway that is slightly different from the R. palustris pathway. 2-Hydroxychc-CoA dehydrogenase does not participate in anaerobic benzoate degradation by T. aromatica and thus may serve as a useful indicator of an R. palustris-type benzoate degradation pathway.

Alcohol Oxidoreductases↗
PubMed2000

BenR, a XylS homologue, regulates three different pathways of aromatic acid degradation in Pseudomonas putida.

Pseudomonas putida converts benzoate to catechol using two enzymes that are encoded on the chromosome and whose expression is induced by benzoate. Benzoate also binds to the regulator XylS to induce expression of the TOL (toluene degradation) plasmid-encoded meta pathway operon for benzoate and methylbenzoate degradation. Finally, benzoate represses the ability of P. putida to transport 4-hydroxybenzoate (4-HBA) by preventing transcription of pcaK, the gene encoding the 4-HBA permease. Here we identified a gene, benR, as a regulator of benzoate, methylbenzoate, and 4-HBA degradation genes. A benR mutant isolated by random transposon mutagenesis was unable to grow on benzoate. The deduced amino acid sequence of BenR showed high similarity (62% identity) to the sequence of XylS, a member of the AraC family of regulators. An additional seven genes located adjacent to benR were inferred to be involved in benzoate degradation based on their deduced amino acid sequences. The benABC genes likely encode benzoate dioxygenase, and benD likely encodes 2-hydro-1,2-dihydroxybenzoate dehydrogenase. benK and benF were assigned functions as a benzoate permease and porin, respectively. The possible function of a final gene, benE, is not known. benR activated expression of a benA-lacZ reporter fusion in response to benzoate. It also activated expression of a meta cleavage operon promoter-lacZ fusion inserted in an E. coli chromosome. Third, benR was required for benzoate-mediated repression of pcaK-lacZ fusion expression. The benA promoter region contains a direct repeat sequence that matches the XylS binding site previously defined for the meta cleavage operon promoter. It is likely that BenR binds to the promoter region of chromosomal benzoate degradation genes and plasmid-encoded methylbenzoate degradation genes to activate gene expression in response to benzoate. The action of BenR in repressing 4-HBA uptake is probably indirect.

Amino Acid Sequence↗
PubMed1999

NahY, a catabolic plasmid-encoded receptor required for chemotaxis of Pseudomonas putida to the aromatic hydrocarbon naphthalene.

Pseudomonas putida G7 exhibits chemotaxis to naphthalene, but the molecular basis for this was not known. A new gene, nahY, was found to be cotranscribed with meta cleavage pathway genes on the NAH7 catabolic plasmid for naphthalene degradation. The nahY gene encodes a 538-amino-acid protein with a membrane topology and a C-terminal region that resemble those of chemotaxis transducer proteins. A P. putida G7 nahY mutant grew on naphthalene but was not chemotactic to this aromatic hydrocarbon. The protein NahY thus appears to function as a chemoreceptor for naphthalene or a related compound. The presence of nahY on a catabolic plasmid implies that chemotaxis may facilitate biodegradation.

Bacterial Proteins↗
PubMed1999

Conserved cytoplasmic loops are important for both the transport and chemotaxis functions of PcaK, a protein from Pseudomonas putida with 12 membrane-spanning regions.

Chemotaxis to the aromatic acid 4-hydroxybenzoate (4-HBA) by Pseudomonas putida is mediated by PcaK, a membrane-bound protein that also functions as a 4-HBA transporter. PcaK belongs to the major facilitator superfamily (MFS) of transport proteins, none of which have so far been implicated in chemotaxis. Work with two well-studied MFS transporters, LacY (the lactose permease) and TetA (a tetracycline efflux protein), has revealed two stretches of amino acids located between the second and third (2-3 loop) and the eighth and ninth (8-9 loop) transmembrane regions that are required for substrate transport. These sequences are conserved among most MFS transporters, including PcaK. To determine if PcaK has functional requirements similar to those of other MFS transport proteins and to analyze the relationship between the transport and chemotaxis functions of PcaK, we generated strains with mutations in amino acid residues located in the 2-3 and 8-9 loops of PcaK. The mutant proteins were analyzed in 4-HBA transport and chemotaxis assays. Cells expressing mutant PcaK proteins had a range of phenotypes. Some transported at wild-type levels, while others were partially or completely defective in 4-HBA transport. An aspartate residue in the 8-9 loop that has no counterpart in LacY and TetA, but is conserved among members of the aromatic acid/H(+) symporter family of the MFS, was found to be critical for 4-HBA transport. These results indicate that conserved amino acids in the 2-3 and 8-9 loops of PcaK are required for 4-HBA transport. Amino acid changes that decreased 4-HBA transport also caused a decrease in 4-HBA chemotaxis, but the effect on chemotaxis was sometimes slightly more severe. The requirement of PcaK for both 4-HBA transport and chemotaxis demonstrates that P. putida has a chemoreceptor that differs from the classical chemoreceptors described for Escherichia coli and Salmonella typhimurium.

Amino Acid Sequence↗
PubMed1999

BadR, a new MarR family member, regulates anaerobic benzoate degradation by Rhodopseudomonas palustris in concert with AadR, an Fnr family member.

A cluster of genes for the anaerobic degradation of benzoate has been described for the phototrophic bacterium Rhodopseudomonas palustris. Here we provide an initial analysis of the regulation of anaerobic benzoate degradation by examining the contributions of two regulators: a new regulator, BadR, encoded by the benzoate degradation gene cluster, and a previously described regulator, AadR, whose gene lies outside the cluster. Strains with single mutations in either badR or aadR grew slowly on benzoate but were relatively unimpaired in growth on succinate and several intermediates of benzoate degradation. A badR aadR double mutant was completely defective in anaerobic growth on benzoate. Effects of the regulators on transcriptional activation were monitored with an R. palustris strain carrying a chromosomal fusion of 'lacZ to the badE gene of the badDEFG operon. This operon encodes benzoyl-coenzyme A (benzoyl-CoA) reductase, an unusual oxygen-sensitive enzyme that catalyzes the benzene ring reduction reaction that is the rate-limiting step in anaerobic benzoate degradation. Expression of badE::'lacZ was induced 100-fold when cells grown aerobically on succinate were shifted to anaerobic growth on succinate plus benzoate. The aadR gene was required for a 20-fold increase in expression that occurred in response to anaerobiosis, and badR was responsible for a further 5-fold increase in expression that occurred in response to benzoate. Further studies with the badE::'lacZ fusion strain grown with various kinds of aromatic acids indicated that BadR probably responds to benzoyl-CoA acting as an effector molecule. Sequence information indicates that BadR is a member of the MarR family of transcriptional regulators. These studies expand the range of functions regulated by MarR family members to include anaerobic aromatic acid degradation and provide an example of a MarR-type protein that acts as a positive regulator rather than as a negative regulator, as do most MarR family members. AadR resembles the Escherichia coli Fnr regulator in sequence and contains cysteine residues that are spaced appropriately to serve in the capacity of a redox-sensing protein.

Anaerobiosis↗
PubMed1998

Identification of a chemotaxis gene region from Pseudomonas putida.

Pseudomonas putida is chemotactic to a range of organic compounds, including several aromatic compounds. Genes involved in this behavioral response were identified by Tn5 mutagenesis of P. putida PRS2000, resulting in a strain that was nonchemotactic to all chemoattractants tested. Cloning and sequencing of the DNA at the DNA at the Tn5 insertion site revealed a 13-kb region that contained 12 open reading frames, 9 of which are homologous to chemotaxis, flagellar and motility genes in other bacterial species. This indicates that the basic chemotaxis machinery of P. putida is similar to that of other bacterial systems, even though some of the compounds that are sensed as attractants are different.

Chemotaxis↗
PubMed1998

2-Ketocyclohexanecarboxyl coenzyme A hydrolase, the ring cleavage enzyme required for anaerobic benzoate degradation by Rhodopseudomonas palustris.

2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium Rhodopseudomonas palustris by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography. The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the badI gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of badI indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins. Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration. This indicates that the native form of the enzyme is a homotetramer. The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 micromol min(-1) mg of protein(-1). Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate. An R. palustris mutant, carrying a chromosomal disruption of badI, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate. These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by R. palustris.

Acyl Coenzyme A↗
PubMed1997

A cluster of bacterial genes for anaerobic benzene ring biodegradation.

A reductive benzoate pathway is the central conduit for the anaerobic biodegradation of aromatic pollutants and lignin monomers. Benzene ring reduction requires a large input of energy and this metabolic capability has, so far, been reported only in bacteria. To determine the molecular basis for this environmentally important process, we cloned and analyzed genes required for the anaerobic degradation of benzoate and related compounds from the phototrophic bacterium, Rhodopseudomonas palustris. A cluster of 24 genes was identified that includes twelve genes likely to be involved in anaerobic benzoate degradation and additional genes that convert the related compounds 4-hydroxybenzoate and cyclohexanecarboxylate to benzoyl-CoA. Genes encoding benzoyl-CoA reductase, a novel enzyme able to overcome the resonance stability of the aromatic ring, were identified by directed mutagenesis. The gene encoding the ring-cleavage enzyme, 2-ketocyclohexanecarboxyl-CoA hydrolase, was identified by assaying the enzymatic activity of the protein expressed in Escherichia coli. Physiological data and DNA sequence analyses indicate that the benzoate pathway consists of unusual enzymes for ring reduction and cleavage interposed among enzymes homologous to those catalyzing fatty acid degradation. The cloned genes should be useful as probes to identify benzoate degradation genes from other metabolically distinct groups of anaerobic bacteria, such as denitrifying bacteria and sulfate-reducing bacteria.

Aerobiosis↗
PubMed1997

Chemotaxis of Pseudomonas spp. to the polyaromatic hydrocarbon naphthalene.

Two naphthalene-degrading bacteria, Pseudomonas putida G7 and Pseudomonas sp. strain NCIB 9816-4, were chemotactically attracted to naphthalene in drop assays and modified capillary assays. Growth on naphthalene or salicylate induced the chemotactic response. P. putida G7 was also chemotactic to biphenyl; other polyaromatic hydrocarbons that were tested did not appear to be chemoattractants for either Pseudomonas strain. Strains that were cured of the naphthalene degradation plasmid were not attracted to naphthalene.

Biodegradation, Environmental↗
PubMed1997

PcaK, a high-affinity permease for the aromatic compounds 4-hydroxybenzoate and protocatechuate from Pseudomonas putida.

PcaK is a transporter and chemoreceptor protein from Pseudomonas putida that is encoded as part of the beta-ketoadipate pathway regulon for aromatic acid degradation. When expressed in Escherichia coli, PcaK was localized to the membrane and catalyzed the accumulation of two aromatic substrates, 4-hydroxybenzoate and protocatechuate, against a concentration gradient. Benzoate inhibited 4-hydroxybenzoate uptake but was not a substrate for PcaK-catalyzed transport. A P. putida pcaK mutant was defective in its ability to accumulate micromolar amounts of 4-hydroxybenzoate and protocatechuate. The mutant was also impaired in growth on millimolar concentrations of these aromatic acids. In contrast, the pcaK mutant grew at wild-type rates on benzoate. The Vmax for uptake of 4-hydroxybenzoate was at least 25 nmol/min/mg of protein, and the Km was 6 microM. PcaK-mediated transport is energized by the proton motive force. These results show that although aromatic acids in the undissociated (uncharged) form can diffuse across bacterial membranes, high-specificity active transport systems probably also contribute to the ability of bacteria to grow on the micromolar concentrations of these compounds that are typically present in soil. A variety of aromatic molecules, including naturally occurring lignin derivatives and xenobiotics, are metabolized by bacteria and may be substrates for transport proteins. The characterization of PcaK provides a foundation for understanding active transport as a critical step in the metabolism of aromatic carbon sources.

Bacterial Proteins↗
PubMed1997

4-hydroxybenzoyl coenzyme A reductase (dehydroxylating) is required for anaerobic degradation of 4-hydroxybenzoate by Rhodopseudomonas palustris and shares features with molybdenum-containing hydroxylases.

The anaerobic degradation of 4-hydroxybenzoate is initiated by the formation of 4-hydroxybenzoyl coenzyme A, with the next step proposed to be a dehydroxylation to benzoyl coenzyme A, the starting compound for a central pathway of aromatic compound ring reduction and cleavage. Three open reading frames, divergently transcribed from the 4-hydroxybenzoate coenzyme A ligase gene, hbaA, were identified and sequenced from the phototrophic bacterium Rhodopseudomonas palustris. These genes, named hbaBCD, specify polypeptides of 17.5, 82.6, and 34.5 kDa, respectively. The deduced amino acid sequences show considerable similarities to a group of hydroxylating enzymes involved in CO, xanthine, and nicotine metabolism that have conserved binding sites for [2Fe-2S] clusters and a molybdenum cofactor. Cassette disruption of the hbaB gene yielded a mutant that was unable to grow anaerobically on 4-hydroxybenzoate but grew normally on benzoate. The hbaB mutant cells did not accumulate [14C]benzoyl coenzyme A during short-term uptake of [14C]4-hydroxybenzoate, but benzoyl coenzyme A was the major radioactive metabolite formed by the wild type. In addition, crude extracts of the mutant failed to convert 4-hydroxybenzoyl coenzyme A to benzoyl coenzyme A. This evidence indicates that the hbaBCD genes encode the subunits of a 4-hydroxybenzoyl coenzyme A reductase (dehydroxylating). The sizes of the specified polypeptides are similar to those reported for 4-hydroxybenzoyl coenzyme A reductase isolated from the denitrifying bacterium Thauera aromatica. The amino acid consensus sequence for a molybdenum cofactor binding site is in HbaC. This cofactor appears to be an essential component because anaerobic growth of R. palustris on 4-hydroxybenzoate, but not on benzoate, was retarded unless 0.1 microM molybdate was added to the medium. Neither tungstate nor vanadate replaced molybdate, and tungstate competitively inhibited growth stimulation by molybdate.

Amino Acid Sequence↗
PubMed1996

The beta-ketoadipate pathway and the biology of self-identity.

The beta-ketoadipate pathway is a chromosomally encoded convergent pathway for aromatic compound degradation that is widely distributed in soil bacteria and fungi. One branch converts protocatechuate, derived from phenolic compounds including p-cresol, 4-hydroxybenzoate and numerous lignin monomers, to beta-ketoadipate. The other branch converts catechol, generated from various aromatic hydrocarbons, amino aromatics, and lignin monomers, also to beta-ketoadipate. Two additional steps accomplish the conversion of beta-ketoadipate to tricarboxylic acid cycle intermediates. Enzyme studies and amino acid sequence data indicate that the pathway is highly conserved in diverse bacteria, including Pseudomonas putida, Acinetobacter calcoaceticus, Agrobacterium tumefaciens, Rhodococcus erythropolis, and many others. The catechol branch of the beta-ketoadipate pathway appears to be the evolutionary precursor for portions of the plasmid-borne ortho-pathways for chlorocatechol degradation. However, accumulating evidence points to an independent and convergent evolutionary origin for the eukaryotic beta-ketoadipate pathway. In the face of enzyme conservation, the beta-ketoadipate pathway exhibits many permutations in different bacterial groups with respect to enzyme distribution (isozymes, points of branch convergence), regulation (inducing metabolites, regulatory proteins), and gene organization. Diversity is also evident in the behavioral responses of different bacteria to beta-ketoadipate pathway-associated aromatic compounds. The presence and versatility of transport systems encoded by beta-ketoadipate pathway regulons is just beginning to be explored in various microbial groups. It appears that in the course of evolution, natural selection has caused the beta-ketoadipate pathway to assume a characteristic set of features or identity in different bacteria. Presumably such identities have been shaped to optimally serve the diverse lifestyles of bacteria.

Adipates↗