The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM-1

4-Chlorobenzoate:CoA ligase, the first enzyme in the pathway for 4-chlorobenzoate dissimilation, has been partially purified from Arthrobacter sp. strain TM-1, by sequential ammonium sulphate precipitation and chromatography on DEAE-Sepharose and Sephacryl S-200. The enzyme, a homodimer of subunit molecular mass approximately 56 kD, is dependent on Mg2+-ATP and coenzyme A, and produces 4-chlorobenzoyl CoA and AMP. Besides Mg2+, Mn2+, Co2+, Fe2+ and Zn2+ are also stimulatory, but not Ca2+. Maximal activity is exhibited at pH 7.0 and 25 degrees C. The ligase demonstrates broad specificity towards other halobenzoates, with 4-chlorobenzoate as best substrate. The apparent Michaelis constants (Km) of the enzyme for 4-chlorobenzoate, CoA and ATP were determined as 3.5, 30 and 238 microM respectively. 4-Chlorobenzoyl CoA dehalogenase, the second enzyme, has been purified to homogeneity by sequential column chromatography on hydroxyapatite, DEAE-Sepharose and Sephacryl S-200. It is a homotetramer of 33 kD subunits with an isoelectric point of 6.4. At pH 7.5 and 30 degrees C, Km and kcat for 4-CBCoA are 9 microM and 1 s(-1) respectively. The optimum pH is 7.5, and maximal enzymic activity occurs at 45 degrees C. The properties of this enzyme are compared with those of the 4-chlorobenzoyl CoA dehalogenases from Arthrobacter sp. strain 4-CB1 and Pseudomonas sp. strain CBS-3, which differ variously in their N-terminal amino acid sequences, optimal pH values, pI values and/or temperatures of maximal activity.     

Catabolic degradation of 4-chlorobiphenyl byPseudomonas sp. DJ-12 via consecutive reaction ofmeta-cleavage and hydrolytic dechlorination

Pseudomonas sp. strain DJ-12 is a bacterial isolate capable of degrading 4-chlorobiphenyl (4CBP) as a carbon and energy source. The catabolic degradation of 4CBP by the strain DJ-12 was studied along with the genetic organization of the genes responsible for the crucial steps of the catabolic degradation. The catabolic pathway was characterized as being conducted by consecutive reactions of themeta-cleavage of 4CBP, hydrolytic dechlorination of 4-chlorobenzoate (4CBA), hydroxylation of 4-hydroxybenzoate, andmeta-cleavage of protocatechuate. ThepcbC gene responsible for themeta-cleavage of 4CBP only showed a 30 to 40% homology in its deduced amino acid sequence compared to those of the corresponding genes from other strains. The amino acid sequence of 4CBA-CoA dechlorinase showed an 86% homology with that ofPseudomonas sp. CBS3, yet only a 50% homology with that ofArthrobacter spp. However, thefcb genes for the hydrolytic dechlorination of 4CBA inPseudomonas sp. DJ-12 showed an uniquely different organization from those of CBS3 and other reported strains. Accordingly, these results indicate that strain DJ-12 can degrade 4CBP completely viameta-cleavage and hydrolytic dechlorination using enzymes that are uniquely different in their amino acid sequences from those of other bacterial strains with the same degradation activities.     

Dehalogenation of 4-chlorobenzoate

Pseudomonas sp. CBS3 is capable of growing with 4-chlorobenzoate as sole source of carbon and energy. The removal of the chlorine of 4-chlorobenzoate is performed in the first degradation step by an enzyme system consisting of three proteins. A 4-halobenzoate-coenzyme A ligase activates 4-chlorobenzoate in a coenzyme A, ATP and Mg²⁺ dependent reaction to 4-chlorobenzoyl-coenzyme A. This thioester intermediate is dehalogenated by the 4-chlorobenzoyl-coenzyme A dehalogenase. Finally coenzyme A is split off by a 4-hydroxybenzoyl-CoA thioesterase to form 4-hydroxybenzoate. The involved 4-chlorobenzoyl-coenzyme A dehalogenase was purified to apparent homogeneity by a five-step purification procedure. The native enzyme had an apparent molecular mass of 120,000 and was composed of four identical polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.7. The maximal initial rate of catalysis was achieved at pH 10 at 60 °C. The apparent K _m value for 4-chlorobenzoyl-coenzyme A was 2.4–2.7 µM. V _max was 1.1 × 10^–7 M sec^–1 (2.2 µmol min^–1 mg^–1 of protein). The NH₂-terminal amino acid sequence was determined. All 4-halobenzoyl-coenzyme A thioesters, except 4-fluorobenzoyl-coenzyme A, were dehalogenated by the 4-chlorobenzoyl-CoA dehalogenase.Abbreviations CBA chlorobenzoate - CoA coenzyme A - HBA hydroxybenzoate - DTT dithiothreitol - HPLC high performance liquid chromatography - PAGE polyacrylamide gel electrophoresis     

Structure and denaturation of 4-chlorobenzoyl coenzyme A dehalogenase from <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. strain TM-1

The secondary structure of the trimeric protein 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1, the second of three enzymes involved in the dechlorination of 4-chlorobenzoate to form 4-hydroxybenzoate, has been examined. E_mM for the enzyme was 12.59. Analysis by circular dichroism spectrometry in the far uv indicated that 4-chlorobenzoyl coenzyme A dehalogenase was composed mostly of α-helix (56%) with lesser amounts of random coil (21%), β-turn (13%) and β-sheet (9%). These data are in close agreement with a computational prediction of secondary structure from the primary amino acid sequence, which indicated 55.8% α-helix, 33.7% random coil and 10.5% β-sheet; the enzyme is, therefore, similar to the 4-chlorobenzoyl coenzyme A dehalogenase from Pseudomonas sp. CBS-3. The three-dimensional structure, including that of the presumed active site, predicted by computational analysis, is also closely similar to that of the Pseudomonas dehalogenase. Study of the stability and physicochemical properties revealed that at room temperature, the enzyme was stable for 24 h but was completely inactivated by heating to 60°C for 5 min; thereafter by cooling at 1°C min⁻¹ to 45°C, 20.6% of the activity could be recovered. Mildly acidic (pH 5.2) or alkaline (pH 10.1) conditions caused complete inactivation, but activity was fully recovered on returning the enzyme to pH 7.4. Circular dichroism studies also indicated that secondary structure was little altered by heating to 60°C, or by changing the pH from 7.4 to 6.0 or 9.2. Complete, irreversible destruction of, and maximal decrease in the fluorescence yield of the protein at 330–350 nm were brought about by 4.5 M urea or 1.1 M guanidinium chloride. Evidence was obtained to support the hypothetical three-dimensional model, that residues W140 and W167 are buried in a non-polar environment, whereas W182 appears at or close to the surface of the protein. At least one of the enzymes of the dehalogenase system (the combined 4-chlorobenzoate:CoA ligase, the dehalogenase and 4-hydroxybenzoyl coenzyme A thioesterase) appears to be capable of association with the cell membrane.

Pathways for 3-chloro- and 4-chlorobenzoate degradationin Pseudomonas aeruginosa 3mT

A bacterial isolate, Pseudomonas aeruginosa 3mT, exhibited the ability to degrade high concentrations of 3-chlorobenzoate (3-CBA, 8 g l^-1) and 4-chlorobenzoate (4-CBA 12 g l^-1) (Ajithkumar 1998). In this study, by delineating the initial biochemical steps involved in the degradation of these compounds, we investigated how this strain can do so well. Resting cells, permeabilised cells as well as cell-free extracts failed to dechlorinate both 3-CBA and 4-CBA under anaerobic conditions, whereas the former two readily degraded both compounds under aerobic conditions. Accumulation of any intermediary metabolite was not observed during growth as well as reaction with resting cells under highly aerated conditions. However, on modification of reaction conditions, 3-chlorocatechol (3-CC) and 4-chlorocatechol (4-CC) accumulated in 3-CBA and 4-CBA flasks, respectively. Fairly high titres of pyrocatechase II (chlorocatechol 1,2-dioxygenase) activity were obtained in extracts of cells grown on 3-CBA and 4-CBA. Meta-pyrocatechase (catechol 2,3-dioxygenase) activity against4-CC and catechol, but not against 3-CC, was also detected in low titres. Accumulation of small amounts of 2-chloro-5-hydroxy muconic semialdehyde, the meta-cleavage product of 4-CC, was detected in the medium, when 4-CBA concentration was 4 mM or greater, indicating the presence of a minor meta-pathway in strain 3mT. However, 3-CBA exclusively, and more than 99% of 4-CBA were degraded through the formation of the respective chlorocatechol, via a modified ortho-pathway. This defies the traditional view that the microbes that follow chlorocatechol pathways are not very good degraders of chlorobenzoates. 4-Hydroxybenzoatewas readily (and 3-hydroxybenzoate to a lesser extent) degraded by the strain, through the formation of protocatechuate and gentisate, respectively, as intermediary dihydroxy metabolites.     

Identification of 4-chlorobenzoyl-coenzyme A as intermediate in the dehalogenation catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3

The intermediate in the reaction catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 was identified as 4-chlorobenzoyl-CoA. One component of 4-chlorobenzoate debalogenase worked as a a 4-chlorobenzoyl-CoA ligase catalysing the formation of 4-chlorobenzoyl-CoA from 4-chlorobenzoate, coenzyme A and ATP. This intermediate was detected spectrophotometrically and by HPLC. 4-chlorobenzoyl-CoA was the substrate for the dehalogenase component, which catalysed the conversion to 4-hydroxybenzoate with concomitant release or coenzyme A.     

Identification of genes coding for hydrolytic dehalogenation in the metagenome derived from a denitrifying 4-chlorobenzoate degrading consortium.

A metagenomic approach was taken to investigate the genetic basis for the ability of an anaerobic consortium to grow on either 4-chlorobenzoate or 4-bromobenzoate under denitrifying conditions. Degenerate PCR primers were designed for the family of 4-chlorobenzoyl-CoA dehalogenase genes. The primers were utilized to screen a metagenome library and two overlapping clones were identified which yield a PCR product. The complete sequence of one metagenome clone was determined and genes encoding 4-chlorobenzoyl-CoA ligase (FcbA) and 4-chlorobenzoyl-CoA dehalogenase (FcbB) were identified. Analysis of the ORFs present in the nucleotide sequence suggests that the metagenome clone originated from an uncultured denitrifying microorganism belonging to the Betaproteobacteria. Interestingly, unlike similar gene clusters reported in aerobes, a gene encoding 4-hydroxybenzoyl-CoA thioesterase was not present in the gene cluster. This suggests that 4-hydroxybenzoyl-CoA is further degraded via the anaerobic reduction pathway in the corresponding microorganism instead of through thioester hydrolysis to yield 4-hydroxybenzoate.     

Soil microbial population dynamics following bioaugmentation with a 3-chlorobenzoate-degrading bacterial culture

Changes in microbial populations were evaluated following inoculation of contaminated soil with a 3-chlorobenzoate degrader. Madera sandy loam was amended with 0, 500, or 1000 g 3-chlorobenzoate g^-1 dry soil. Selected microcosms were inoculated with the degrader Comamonas testosteroni BR60. Culturable bacterial degraderswere enumerated on minimal salts media containing 3-chlorobenzoate. Culturableheterotrophic bacteria were enumerated on R2A. Isolated degraders were grouped by enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction fingerprints and identified based on 16S ribosomal-DNA sequences. Bioaugmentation increased the rate of degradation at both levels of 3-chlorobenzoate. In both the 500 and 1000 g 3-chlorobenzoate g^-1 dry soil inoculated microcosms, degradersincreased from the initial inoculum and decreased following degradation of 3-CB.Inoculation delayed the development of indigenous 3-chlorobenzoate degrading populations. It is unclear if inoculation altered the composition of indigenous degrader populations. In the uninoculated soil, degraders increased from undetectable levels to 6.6 × 10⁷ colony-forming-units g^-1 dry soil in the 500 g 3-chlorobenzoate g^-1 dry soil microcosms, but none were detected in the 1000 g 3-chlorobenzoate g^-1 dry soil microcosms. Degraders isolated from uninoculated soil were identified as one of two distinct Burkholderia species.In the uninoculated soil, numbers of culturable heterotrophic bacteria initially decreased following addition of 1000 g 3-chlorobenzoate g^-1 dry soil. Inoculation with C. testosteroni reduced this negative impact on culturable bacterial numbers. The results indicate that bioaugmentation may not only increase the rate of 3-chlorobenzoate degradation but also reduce the deleterious effects of 3-chlorbenzoate on indigenous soil microbial populations.     

Evidence for a new pathway in the bacterial degradation of 4-fluorobenzoate

Six bacterial strains able to use 4-fluorobenzoic acid as their sole source of carbon and energy were isolated by selective enrichment from various water and soil samples from the Stuttgart area. According to their responses in biochemical and morphological tests, the organisms were assigned to the genera Alcaligenes, Pseudomonas, and Aureobacterium. To elucidate the degradation pathway of 4-fluorobenzoate, metabolic intermediates were identified. Five gram-negative isolates degraded this substrate via 4-fluorocatechol, as described in previous studies. In growth experiments, these strains excreted 50 to 90% of the fluoride from fluorobenzoate. Alcaligenes sp. strains RHO21 and RHO22 used all three isomers of monofluorobenzoate. Alcaligenes sp. strain RHO22 also grew on 4-chlorobenzoate. Aureobacterium sp. strain RHO25 transiently excreted 4-hydroxybenzoate into the culture medium during growth on 4-fluorobenzoate, and stoichiometric amounts of fluoride were released. In cell extracts from this strain, the enzymes for the conversion of 4-fluorobenzoate, 4-hydroxybenzoate, and 3,4-dihydroxybenzoate could be detected. All these enzymes were inducible by 4-fluorobenzoate. These data suggest a new pathway for the degradation of 4-fluorobenzoate by Aureobacterium sp. strain RHO25 via 4-hydroxybenzoate and 3,4-dihydroxybenzoate.     

Evidence for a new pathway in the bacterial degradation of 4-fluorobenzoate.

Six bacterial strains able to use 4-fluorobenzoic acid as their sole source of carbon and energy were isolated by selective enrichment from various water and soil samples from the Stuttgart area. According to their responses in biochemical and morphological tests, the organisms were assigned to the genera Alcaligenes, Pseudomonas, and Aureobacterium. To elucidate the degradation pathway of 4-fluorobenzoate, metabolic intermediates were identified. Five gram-negative isolates degraded this substrate via 4-fluorocatechol, as described in previous studies. In growth experiments, these strains excreted 50 to 90% of the fluoride from fluorobenzoate. Alcaligenes sp. strains RHO21 and RHO22 used all three isomers of monofluorobenzoate. Alcaligenes sp. strain RHO22 also grew on 4-chlorobenzoate. Aureobacterium sp. strain RHO25 transiently excreted 4-hydroxybenzoate into the culture medium during growth on 4-fluorobenzoate, and stoichiometric amounts of fluoride were released. In cell extracts from this strain, the enzymes for the conversion of 4-fluorobenzoate, 4-hydroxybenzoate, and 3,4-dihydroxybenzoate could be detected. All these enzymes were inducible by 4-fluorobenzoate. These data suggest a new pathway for the degradation of 4-fluorobenzoate by Aureobacterium sp. strain RHO25 via 4-hydroxybenzoate and 3,4-dihydroxybenzoate.     

Mycolytic effect of extracellular enzymes of antagonistic microbes to Colletotrichum falcatum,red rot pathogen of sugarcane

Strains of selected bacteria and Trichoderma harzianum isolated from sugarcane rhizosphere and endosphere regions were tested for the production of chitinolytic enzymes and their involvement in the suppression of Colletotrichum falcatum, red rot pathogen of sugarcane. Among several strains tested for chitinolytic activity, 12 strains showed a clearing zone on chitin-amended agar medium. Among these, bacterial strains AFG2, AFG 4, AFG 10, FP7 and VPT4 and all the tested T. harzianum strains produced clearing zones of a size larger than 10 mm. The antifungal activity of these strains increased when chitin was incorporated into the medium. Trichoderma harzianum strain T5 showed increased levels of activity of N-acetylglucosaminidase and -1,3-glucanase when grown on minimal medium containing chitin or cell wall of the pathogen. Lytic enzymes of bacterial strains AFG2, AFG4, VPT4 and FP7 and T. harzianum T5 inhibited conidial germination and mycelial growth of the pathogen. Enzymes from T. harzianum T5 were found to be the most effective in inhibiting the fungus. When mycelial discs of the pathogen were treated with the enzymes, electrolytes were released from fungal mycelia. The results indicated that antagonistic T. harzianum T5 caused a higher level of lysis of the pathogen mycelium, and the inhibitory effect was more pronounced when the lytic enzymes were produced using chitin or cell wall of the pathogen as carbon source.     

Versatility of soil column experiments to study biodegradation of halogenated compounds under environmental conditions

Soil column experiments were performed to obtain insight in the different biological and physico-chemical processes affecting biodegradation of halogenated compounds under natural conditions in a water infiltration site. Lower chlorinated aromatic compounds could be degraded under aerobic conditions, whereas highly chlorinated compounds and chlorinated aliphatic compounds were mainly transformed under anaerobic conditions. Microorganisms which derive energy from reductive dechlorination were enriched and characterized. It was found that microbes could adapt to using chlorinated benzenes by evolution of new enzyme specificities and by exchange of genetic material. For halogenated pollutants, which are generally hydrophobic, sorption processes control the concentration available for biodegradation. The effects of very low concentrations of halogenated compounds on their biodegradability are described. The use of isolated bacterial strains to enhance biodegradation was evaluated with respect to their temperature-related activity and to their adhesion properties.Abbreviations 3-CB 3-chlorobenzoate - DCB dichlorobenzene - HCH hexachlorocyclohexane - IS insertion sequence - PER tetrachloroethylene - S_min minimal substrate concentration for growth - TCB trichlorobenzene - TRI trichloroethylene - filtration coefficient     

Anaerobic oxidation of phenylacetate and 4-hydroxyphenylacetate to benzoyl-coenzyme A and CO2 in denitrifying Pseudomonas sp.

Anaerobic degradation of (4-hydroxy)phenylacetate in denitrifying Pseudomonas sp. was investigated. Evidence is presented for -oxidation of the coenzyme A (CoA)-activated carboxymethyl side chain, a reaction which has not been described. The C₆–C₂ compounds are degraded to benzoyl-CoA and furtheron to CO₂ via the following intermediates: Phenylacetyl-CoA, phenylglyoxylate, benzoyl-CoA plus CO₂; 4-hydroxyphenylacetyl-CoA, 4-hydroxyphenylglyoxylate, 4-hydroxybenzoyl-CoA plus CO₂, benzoyl-CoA. Trace amounts of mandelate possibly derived from mandelyl-CoA were detected during phenylacetate degradation in vitro. The reactions are catalyzed by (i) phenylacetate-CoA ligase which converts phenylacetate to phenylacetyl-CoA and by a second enzyme for 4-hydroxyphenylacetate; (ii) a (4-hydroxy)-phenylacetyl-CoA dehydrogenase system which oxidizes phenylacetyl-CoA to (4-hydroxy)phenylglyoxylate plus CoA; and (iii) (4-hydroxy)phenylglyoxylate: acceptor oxidoreductase (CoA acylating) which catalyzes the oxidative decarboxylation of (4-hydroxy)phenylglyoxylate to (4-hydroxy)benzoyl-CoA and CO₂. (iv) The degradation of 4-hydroxyphenylacetate in addition requires the reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA, catalyzed by 4-hydroxybenzoyl-CoA reductase (dehydroxylating). The whole cell regulation of these enzyme activities supports the proposed pathway. An ionic mechanism for anaerobic -oxidation of the CoA-activated carboxymethyl side chain is proposed. Phenylacetic acids are plant constituents and in addition are formed from a large variety of natural aromatic compounds by microorganisms; their degradation therefore plays a significant role in nature, as illustrated in the preceding paper (Mohamed and Fuchs 1993). We have investigated and purified an enzyme which catalyzes the first step in the anaerobic degradation of phenylacetate in a denitrifying Pseudomonas sp. Phenylacetate is converted to phenylacetyl-CoA by phenylacetate-CoA ligase (AMP forming). The postulated function of this enzyme is corroborated by the strict regulation of its expression. 4-Hydroxyphenylacetate appears to be similarly activated by an independent enzyme prior to further degradation.We have suggested before that phenylacetyl-CoA is anaerobically converted by -oxidation of the side chain to phenylglyoxylate¹, which is oxidatively decarboxylated to benzoyl-CoA plus CO₂ (Seyfried et al. 1991; Dangel et al. 1991). 4-Hydroxyphenylacetate was proposed to be similarly oxidized to 4-hydroxybenzoyl-CoA plus CO₂, followed by reductive dehydroxylation to benzoyl-CoA. The evidence was not presented in full, and the crucial -oxidation was not demonstrated in vitro. We present here ample evidence for this pathway. A hypothetical mechanism is proposed by which the oxidation of the -methylene group to an -carbonyl group may occur.     

Total biodegradation of 4-chlorobiphenyl (4 CB) by a two-membered bacterial culture

Summary Several bacterial strains that can oxidize mono- and dichlorinated biphenyls with one unsubstituted ring have already been described. The major route for this biodegradation leads ultimately to the corresponding chlorobenzoic acid, but several other minor chlorinated metabolites that might possibly be of concern for the environment have also been described previously. Since none of the bacterial strains that are able to oxidize these chlorinated biphenyls in pure culture are known to degrade chlorobenzoic acid, the oxidation of these substrates by axenic cultures always generates chlorobenzoates plus several other metabolites. In the present study, we have estimated the biodegradation of 4-chlorobiphenyl (4CB) by a two-membered bacterial culture containing one strain able to grow on 4CB and to transform it into 4-chlorobenzoate (4CBA) and one strain able to degrade 4CBA. The results were encouraging, since it was shown that the degradation of 4CB was more rapid and complete with the double bacterial culture.     

Induction of the halobenzoate catabolic pathway and cometabolism of ortho-chlorobenzoates in Pseudomonas aeruginosa 142 grown on glucose-supplemented media

The aerobic cometabolism of ortho-substitutedchlorobenzoates by Pseudomonas aeruginosa strain 142 growing on glucose-supplemented medium was analyzed. The strain, which can use 2-chlorobenzoate (2-CBA) and 2,4-dichlorobenzoate (2,4-DCBA) as sole carbon and energy sources, showed high rates of 2-CBA metabolism in glucose-fed cells. In contrast, 2,4-DCBA was metabolized only after extended incubation of the full grown culture and depletion of glucose.In addition to the ortho-dehalogenation (ohb ₁₄₂) genes encoding the and subunits of the oxygenase component of a 2-halobenzoate dioxygenase, strain 142 harbours a closely relatedohbABCDFG gene cluster previously identified inP. aeruginosa JB2 (ohb _JB2). The genes for the chlorocatechol ortho-catabolic pathway were identified andsequenced in this strain, showing a near complete identity with the clcABD operon of the pAC27 plasmid. Relative quantification of mRNA by RT-PCR shows apreferential induction of ohb ₁₄₂ by 2-CBA, which is abolished in glucose-grown cultures. The alternate ohb _JB2and clc genes were expressed preferentially in 2,4-DCBAgrown cultures. Only ohb _JB2appears to be expressed in the presence of the carbohydrate. Detection of chlorocatechol-1,2-dioxygenase activity in 2,4-DCBA plus glucose grown cultures suggests the presence of an alternate system for the ortho-cleavage of chlorobenzoates. The recruitment of elements from two halobenzoate dioxygenase systems with different induction patterns, together with achlorocatechol degradative pathway not repressed by carbon catabolite, may allow P. aeruginosa 142 to cometabolize haloaromatics in carbohydrate grown cultures.     

Biodegradation and chemotaxis of polychlorinated biphenyls,biphenyls, and their metabolites by <Emphasis Type="Italic">Rhodococcus</Emphasis> spp.

Two biphenyl-degrading bacterial strains, SS1 and SS2, were isolated from polychlorinated biphenyl (PCB)-contaminated soil. They were identified as Rhodococcus ruber and Rhodococcus pyridinivorans based on the 16S rRNA gene sequence, as well as morphological, physiological and biochemical characteristics. SS1 and SS2 exhibited tolerance to 2000 and 3000 mg/L of biphenyl. And they could degrade 83.2 and 71.5% of 1300 mg/L biphenyl within 84 h, respectively. In the case of low-chlorinated PCB congeners, benzoate and 3-chlorobenzoate, the degradation activities of SS1 and SS2 were also significant. In addition, these two strains exhibited chemotactic response toward TCA-cycle intermediates, benzoate, biphenyl and 2-chlorobenzoate. This study indicated that, like the flagellated bacteria, non-flagellated Rhodococcus spp. might actively seek substrates through the process of chemotaxis once the substrates are depleted in their surroundings. Together, these data provide supporting evidence that SS1 and SS2 might be good candidates for restoring biphenyl/PCB-polluted environments.     

Inducibility of benzoate oxidizing cell activities in Acinetobacter calcoaceticus strain Bs 5 by chlorobenzoates as influenced by the position of chlorine atoms and the inducer concentration

Summary Among four chlorobenzoates tested, only 3-chlorobenzoate and 4-chlorobenzoate were capable of inducing benzoate oxidizing cell activities in Acinetobacter calcoaceticus strain Bs 5, whereas 2-chlorobenzoate and 2,6-dichlorobenzoate were not. With the monochlorobenzoates, this inducing capability decreased with increasing proximity of the chlorine atom to the carboxyl group, i.e. in the order: 4-chlorobenzoate > 3-chlorobenzoate > 2-chlorobenzoate. It is therefore supposed that the induction of benzoate oxidizing cell activities is inhibited primarily be sterical influences of the chlorine substituents of the various chlorobenzoates.With decreasing concentration of 3-chlorobenzoate and 4-chlorobenzoate, the induction of benzoate oxidizing cell activities decreased. Below a critical concentration of 1 M, these activities were no longer detectable in the cells of Acinetobacter calcoaceticus, with the consequence that below this concentration limit, the degradation of 3-chlorobenzoate and 4-chlorobenzoate was no longer possible.     

Microbial sensors for determination of aromatics and their chloroderivatives I. Determination of 3-chlorobenzoate using a Pseudomonas-containing biosensor

Summary An amperometric biosensor for the determination of benzoate and 3-chlorobenzoate using Pseudomonas has been developed. The influence of preincubation of the biosensor with desired substrates on sensitivity and specificity was investigated. The Pseudomonas sensor was more sensitive to benzoate and 3-chlorobenzoate than to 2- and 4-chloro-, 2,4-dichlorobenzoate and 2,4-dichlorophenol. Incubation of the sensor with benzoate and 3-chlorobenzoate enhanced the activity of the microbial sensor. Other chlorobenzoates tested caused a decrease of sensitivity. A linear relationship between the current range and the concentration of benzoate was observed up to 160 mol/l. In the case of 3-chlorobenzoate a linear relationship was observed for concentrations up to 200 mol/l. The signal is reproducible within 5.5% when the test solution contains 40 mol/l of 3-chlorobenzoate. Offprint requests to: K. Riedel     

New perspectives on bacterial ferredoxin evolution

Summary Recent evidence indicates that a gene transposition event occurred during the evolution of the bacterial ferredoxins subsequent to the ancestral intrasequence gene duplication. In light of this new information, the relationships among the bacterial ferredoxins were reexamined and an evolutionary tree consistent with this new understanding was derived. The bacterial ferredoxins can be divided into several groups based on their sequence properties; these include the clostridial-type ferredoxins, theAzotobacter-type ferredoxins, and a group containing the ferredoxins from the anaerobic, green, and purple sulfur bacteria. Based on sequence comparison, it was concluded that the amino-terminal domain of theAzotobacter-type ferredoxins, which contains the novel 3Fe3S cluster binding site, is homologous with the carboxyl-terminal domain of the ferredoxins from the anaerobic photosynthetic bacteria.A number of ferredoxin sequences do not fit into any of the groups described above. Based on sequence properties, these sequences can be separated into three groups: a group containingMethanosarcina barkeri ferredoxin andDesulfovibrio desulfuricans ferredoxin II, a group containingDesulfovibrio gigas ferredoxin andClostridium thermoaceticum ferredoxin, and a group containingDesulfovibrio africanus ferredoxin I andBacillus stearothermophilus ferredoxin. The last two groups differ from all of the other bacterial ferredoxins in that they bind only one FeS cluster per polypeptide, whereas the others bind two. Sequence examination indicates that the second binding site has been either partially or completely lost from these ferredoxins.Methanosarcina barkeri ferredoxin andDesulfovibrio desulfuricans ferredoxin II are of interest because, of all the ferredoxins whose sequences are presently known, they show the strongest evidence of internal gene duplication. However, the derived evolutionary tree indicates that they diverged from theAzotobacter-type ferredoxins well after the ancestral internal gene duplication. This apparent discrepancy is explained by postulating a duplication of one halfchain sequence and a deletion of the other halfchain. TheClostridium thermoaceticum andBacillus stearothermophilus groups diverged from this line and subsequently lost one of the FeS binding sites.It has recently become apparent that gene duplication is ubiquitous among the ferredoxins. Several organisms are now known to have a variety of ferredoxins with widely divergent properties. Unfortunately, in only one case are the sequences of more than one ferredoxin from the same organism known. Thus, although the major features of the bacterial ferredoxin tree are now understood, a complete bacterial phylogeny cannot be inferred until more sequence information is available.     

Immunofluorescent Localization of Z-Nin in the Z-Disk

Of 14 coryneform and 2 Micrococcus strains tested, Arthrobacter globiformis IFO 12137, A. simplex IFO 12069, and Brevibacterium helvolum IFO 12073 utilized l-arginine as a sole carbon and nitrogen source, and synthesized the enzymes specific for the arginine oxygenase pathway when grown on l-arginine. The first step reaction was stimulated by FAD and aeration, and the enzyme responsible was shown to be arginine 2-monooxygenase (EC 1.13.12.1). High activities of five enzymes, including guanidinobutyramidase and ganidinobutyrase (EC 3.5.3.7), were detected in the extract of l-arginine-grown A. simplex cells. The enzymes in the last two steps, 4-aminobutyrate aminotransferase (EC 2.6.1.19) and succinate-semialdehyde dehydrogenase (EC 1.2.1.16), of B. helvolum were also induced by putrescine. These results indicate that some bacteria belonging to the coryneform group employ the arginine oxygenase pathway as a major route for l-arginine metabolism, l-arginine being degraded to succinate via 4-guanidinobutyramide and 4-guanidinobutyrate. The last part of the pathway may be common to the pathway for putrescine degradation.