Microbial degradation of lipid by Acinetobacter sp. strain SOD-1

Acinetobacter sp. strain SOD-1, capable of rapidly degrading salad oil, was isolated from soil. Strain SOD-1 showed good growth and degraded 68.7+/-2.7 and 83.0% of an initial 3000 ppm salad oil suspension in 24 h at 20 degrees C and pH 7.0 and at 35 degrees C and pH 8.0, respectively. The degradation rate depended on pH, temperature, phosphate concentration, and initial cell density.     

Novel partial reductive pathway for 4-chloronitrobenzene and nitrobenzene degradation in Comamonas sp. strain CNB-1

Comamonas sp. strain CNB-1 grows on 4-chloronitrobenzene (4-CNB) and nitrobenzene as sole carbon and nitrogen sources. In this study, two genetic segments, cnbB-orf2-cnbA and cnbR-orf1-cnbCaCbDEFGHI, located on a newly isolated plasmid, pCNB1 (ca. 89 kb), and involved in 4-CNB/nitrobenzene degradation, were characterized. Seven genes (cnbA, cnbB, cnbCa, cnbCb, cnbD, cnbG, and cnbH) were cloned and functionally expressed in recombinant Escherichia coli, and they were identified as encoding 4-CNB nitroreductase (CnbA), 1-hydroxylaminobenzene mutase (CnbB), 2-aminophenol 1,6-dioxygenase (CnbCab), 2-amino-5-chloromuconic semialdehyde dehydrogenase (CnbD), 2-hydroxy-5-chloromuconic acid (2H5CM) tautomerase, and 2-amino-5-chloromuconic acid (2A5CM) deaminase (CnbH). In particular, the 2A5CM deaminase showed significant identities (31 to 38%) to subunit A of Asp-tRNAAsn/Glu-tRNAGln amidotransferase and not to the previously identified deaminases for nitroaromatic compound degradation. Genetic cloning and expression of cnbH in Escherichia coli revealed that CnbH catalyzed the conversion of 2A5CM into 2H5CM and ammonium. Four other genes (cnbR, cnbE, cnbF, and cnbI) were tentatively identified according to their high sequence identities to other functionally identified genes. It was proposed that CnbH might represent a novel type of deaminase and be involved in a novel partial reductive pathway for chloronitrobenzene or nitrobenzene degradation.     

Metabolism of 2-chloro-4-nitrophenol in a gram negative bacterium, Burkholderia sp. RKJ 800

A 2-chloro-4-nitrophenol (2C4NP) degrading bacterial strain designated as RKJ 800 was isolated from a pesticide contaminated site of India by enrichment method and utilized 2C4NP as sole source of carbon and energy. The stoichiometric amounts of nitrite and chloride ions were detected during the degradation of 2C4NP. On the basis of thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectrometry, chlorohydroquinone (CHQ) and hydroquinone (HQ) were identified as major metabolites of the degradation pathway of 2C4NP. Manganese dependent HQ dioxygenase activity was observed in the crude extract of 2C4NP induced cells of the strain RKJ 800 that suggested the cleavage of the HQ to γ-hydroxymuconic semialdehyde. On the basis of the 16S rRNA gene sequencing, strain RKJ 800 was identified as a member of genus Burkholderia. Our studies clearly showed that Burkholderia sp. RKJ 800 degraded 2-chloro-4-nitrophenol via hydroquinone pathway. The pathway identified in a gram negative bacterium, Burkholderia sp. strain RKJ 800 was differed from previously reported 2C4NP degradation pathway in another gram-negative Burkholderia sp. SJ98. This is the first report of the formation of CHQ and HQ in the degradation of 2C4NP by any gram-negative bacteria. Laboratory-scale soil microcosm studies showed that strain RKJ 800 is a suitable candidate for bioremediation of 2C4NP contaminated sites.     

Biotransformation of 4-chloro-2-nitrophenol into 5-chloro-2-methylbenzoxazole by a marine Bacillus sp. strain MW-1

Decolourization, detoxification and biotransformation of 4-chloro-2-nitrophenol (4C2NP) by Bacillus sp. strain MW-1 were studied. This strain decolorized 4C2NP only in the presence of an additional carbon source. On the basis of thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), 4-chloro-2-aminophenol, 4-chloro-2-acetaminophenol and 5-chloro-2-methylbenzoxazole were identified as metabolites. Resting cells depleted 4C2NP with stoichiometric formation of 5-chloro-2-methyl benzoxazole. This is the first report of the formation of 5-chloro-2-methylbenzoxazole from 4C2NP by any bacterial strain.     

Biodegradation and metabolic pathway of sulfamethoxazole by a novel strain Acinetobacter sp.

Applied Microbiology and Biotechnology - In this study, a novel strain capable of degrading sulfamethoxazole (SMX) was isolated and identified as Acinetobacter sp. The effect of influencing...     

Reductive dehalogenation mediated initiation of aerobic degradation of 2-chloro-4-nitrophenol (2C4NP) by Burkholderia sp. strain SJ98

Burkholderia sp. strain SJ98 (DSM 23195) was previously isolated and characterized for degradation and co-metabolic transformation of a number nitroaromatic compounds. In the present study, we evaluated its metabolic activity on chlorinated nitroaromatic compounds (CNACs). Results obtained during this study revealed that strain SJ98 can degrade 2-chloro-4-nitrophenol (2C4NP) and utilize it as sole source of carbon, nitrogen, and energy under aerobic conditions. The cells of strain SJ98 removed 2C4NP from the growth medium with sequential release of nearly stoichiometric amounts of chloride and nitrite in culture supernatant. Under aerobic degradation conditions, 2C4NP was transformed into the first intermediate that was identified as p-nitrophenol by high-performance liquid chromatography, LCMS-TOF, and GC-MS analyses. This transformation clearly establishes that the degradation of 2C4NP by strain SJ98 is initiated by "reductive dehalogenation"; an initiation mechanism that has not been previously reported for microbial degradation of CNAC under aerobic conditions.     

Enhanced phenol degradation by immobilized Acinetobacter sp. strain AQ5NOL 1

A locally isolated Acinetobacter sp. Strain AQ5NOL 1 was encapsulated in gellan gum and its ability to degrade phenol was compared with the free cells. Optimal phenol degradation was achieved at gellan gum concentration of 0.75% (w/v), bead size of 3 mm diameter (estimated surface area of 28.26 mm²) and bead number of 300 per 100 ml medium. At phenol concentration of 100 mg l⁻¹, both free and immobilized bacteria exhibited similar rates of phenol degradation but at higher phenol concentrations, the immobilized bacteria exhibited a higher rate of degradation of phenol. The immobilized cells completely degrade phenol within 108, 216 and 240 h at 1,100, 1,500 and 1,900 mg l⁻¹ phenol, respectively, whereas free cells took 240 h to completely degrade phenol at 1,100 mg l⁻¹. However, the free cells were unable to completely degrade phenol at higher concentrations. Overall, the rates of phenol degradation by both immobilized and free bacteria decreased gradually as the phenol concentration was increased. The immobilized cells showed no loss in phenol degrading activity after being used repeatedly for 45 cycles of 18 h cycle. However, phenol degrading activity of the immobilized bacteria experienced 10 and 38% losses after the 46 and 47th cycles, respectively. The study has shown an increased efficiency of phenol degradation when the cells are encapsulated in gellan gum.     

Regulation of the 4-hydroxyphenylacetic acid meta-cleavage pathway in an Acinetobacter sp

Lactate-grown cultures of $\text{Acinetobacter}$ sp. strain 3B-1 synthesize constitutively all enzymes except the 4-hydroxyphenylacetic acid-3-hydroxylase. All enzymes are further synthesized when strain 3B-1 is grown with 4-hydroxyphenylacetic acid. Induction studies with two mutant strains, one defective in the 3-hydroxylase, and the other defective in the dehydrogenase, indicate that 4-hydroxyphenylacetic acid induces the 3-hydroxylase only, and the second metabolite 3,4-dihydroxyphenylacetic acid appears to induce 3,4-dihydroxyphenylacetic acid-2,3-dioxygenase and subsequent enzymes. Thus, the enzymes of the 4-hydroxyphenylacetic acid $\text{meta}$-cleavage pathway are synthesized following at least two sequential inductive events.     

Cometabolism of 3,4-dichlorobenzoate by Acinetobacter sp. strain 4-CB1

When Acinetobacter sp. strain 4-CB1 was grown on 4-chlorobenzoate (4-CB), it cometabolized 3,4-dichlorobenzoate (3,4-DCB) to 3-chloro-4-hydroxybenzoate (3-C-4-OHB), which could be used as a growth substrate. No cometabolism of 3,4-DCB was observed when Acinetobacter sp. strain 4-CB1 was grown on benzoate. 4-Carboxyl-1,2-benzoquinone was formed as an intermediate from 3,4-DCB and 3-C-4-OHB in aerobic and anaerobic resting-cell incubations and was the major transient intermediate found when cells were grown on 3-C-4-OHB. The first dechlorination step of 3,4-DCB was catalyzed by the 4-CB dehalogenase, while a soluble dehalogenase was responsible for dechlorination of 3-C-4-OHB. Both enzymes were inducible by the respective chlorinated substrates, as indicated by oxygen uptake experiments. The dehalogenase activity on 3-C-4-OHB, observed in crude cell extracts, was 109 and 44 nmol of 3-C-4-OHB min-1 mg of protein-1 under anaerobic and aerobic conditions, respectively. 3-Chloro-4-hydroxybenzoate served as a pseudosubstrate for the 4-hydroxybenzoate monooxygenase by effecting oxygen and NADH consumption without being hydroxylated. Contrary to 4-CB metabolism, the results suggest that 3-C-4-OHB was not metabolized via the protocatechuate pathway. Despite the ability of resting cells grown on 4-CB or 3-C-4-OHB to carry out all of the necessary steps for dehalogenation and catabolism of 3,4-DCB, it appeared that 3,4-DCB was unable to induce the necessary 4-CB dehalogenase for the initial p-dehalogenation step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cometabolism of 3,4-dichlorobenzoate by Acinetobacter sp. strain 4-CB1.

When Acinetobacter sp. strain 4-CB1 was grown on 4-chlorobenzoate (4-CB), it cometabolized 3,4-dichlorobenzoate (3,4-DCB) to 3-chloro-4-hydroxybenzoate (3-C-4-OHB), which could be used as a growth substrate. No cometabolism of 3,4-DCB was observed when Acinetobacter sp. strain 4-CB1 was grown on benzoate. 4-Carboxyl-1,2-benzoquinone was formed as an intermediate from 3,4-DCB and 3-C-4-OHB in aerobic and anaerobic resting-cell incubations and was the major transient intermediate found when cells were grown on 3-C-4-OHB. The first dechlorination step of 3,4-DCB was catalyzed by the 4-CB dehalogenase, while a soluble dehalogenase was responsible for dechlorination of 3-C-4-OHB. Both enzymes were inducible by the respective chlorinated substrates, as indicated by oxygen uptake experiments. The dehalogenase activity on 3-C-4-OHB, observed in crude cell extracts, was 109 and 44 nmol of 3-C-4-OHB min-1 mg of protein-1 under anaerobic and aerobic conditions, respectively. 3-Chloro-4-hydroxybenzoate served as a pseudosubstrate for the 4-hydroxybenzoate monooxygenase by effecting oxygen and NADH consumption without being hydroxylated. Contrary to 4-CB metabolism, the results suggest that 3-C-4-OHB was not metabolized via the protocatechuate pathway. Despite the ability of resting cells grown on 4-CB or 3-C-4-OHB to carry out all of the necessary steps for dehalogenation and catabolism of 3,4-DCB, it appeared that 3,4-DCB was unable to induce the necessary 4-CB dehalogenase for the initial p-dehalogenation step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aerobic degradation of 2-picolinic acid by a nitrobenzene-assimilating strain: Streptomyces sp. Z2

Streptomyces sp. Z2 was isolated from nitrobenzene contaminated activated sludge, which utilized nitrobenzene as a sole source of carbon, nitrogen, and energy under aerobic condition. It was found that besides nitrobenzene strain Z2 can degrade 2-picolinic acid. Strain Z2 completely degraded 2-picolinic acid with initial concentration of 500 mg/L, 1000 mg/L, 1500 mg/L, 2000 mg/L, 2500 mg/L, and 3000 mg/L within 36 h, 50 h, 72 h, 100 h, 136 h, and 180 h, respectively. Kinetics of 2-picolinic acid degradation was described using the Andrews equation. The kinetic parameters were as follows: q_max = 3.81 h^?1, K_s = 83.10 mg/L, and K_i = 252.11 mg/L. During the biodegradation process, Z2 transformed 2-picolinic acid into a product which was identified as 6-hydroxy picolinic acid by UV–vis spectrometry, ¹H nuclear magnetic resonance spectroscopy, and mass spectrometry. 6-Hydroxy picolinic acid was then cleaved and mineralized with release of ammonia.     

Mineralization of 2-chloro- and 2,5-dichlorobiphenyl by Pseudomonas sp. strain UCR2

Pseudomonas sp. strain UCR2 was isolated from a multi-chemostat mating experiment between a chlorobenzoate-degrader, Pseudomonas aeruginosa strain JB2, and a chlorobiphenyl-degrader, Arthrobacter sp. strain B1Barc. Strain UCR2 differed from either of the parental organisms in that it grew on both 2-chloro- and 2,5-dichlorobiphenyl with concomitant release of chloride. Phenotypic typing by the Biolog system indicated that strain UCR2 shared greater similarity with strain JB2 (88%) than strain B1Barc (3%). In DNA:DNA hybridization experiments, genomic DNA from strain UCR2 hybridized with both strain JB2 and strain B1Barc, with the former pairing yielding a much stronger signal than the latter. In contrast, no hybridization whatsoever was observed when the parental organisms strains JB2 and B1Barc were probed against each other.     

Evidence for a novel pathway in the degradation of fluorene by Pseudomonas sp. strain F274.

A fluorene-utilizing microorganism, identified as a species of Pseudomonas, was isolated from soil severely contaminated from creosote use and was shown to accumulate six major metabolites from fluorene in washed-cell incubations. Five of these products were identified as 9-fluorenol, 9-fluorenone, (+)-1,1a-dihydroxy-1-hydro-9-fluorenone, 8-hydroxy-3,4-benzocoumarin, and phthalic acid. This last compound was also identified in growing cultures supported by fluorene. Fluorene assimilation into cell biomass was estimated to be approximately 50%. The structures of accumulated products indicate that a previously undescribed pathway of fluorene catabolism is employed by Pseudomonas sp. strain F274. This pathway involves oxygenation of fluorene at C-9 to give 9-fluorenol, which is then dehydrogenated to the corresponding ketone, 9-fluorenone. Dioxygenase attack on 9-fluorenone adjacent to the carbonyl group gives an angular diol, 1,1a-dihydroxy-1-hydro-9-fluorenone. Identification of 8-hydroxy-3,4-benzocoumarin and phthalic acid suggests that the five-membered ring of the angular diol is opened first and that the resulting 2'-carboxy derivative of 2,3-dihydroxy-biphenyl is catabolized by reactions analogous to those of biphenyl degradation, leading to the formation of phthalic acid. Cell extracts of fluorene-grown cells possessed high levels of an enzyme characteristic of phthalate catabolism, 4,5-dihydroxyphthalate decarboxylase, together with protocatechuate 4,5-dioxygenase. On the basis of these findings, a pathway of fluorene degradation is proposed to account for its conversion to intermediary metabolites. A range of compounds with structures similar to that of fluorene was acted on by fluorene-grown cells to give products consistent with the initial reactions proposed.     

A novel pathway for the biodegradation of gamma-hexachlorocyclohexane by a Xanthomonas sp. strain ICH12

AIM: To isolate gamma-hexachlorocyclohexane (HCH)-degrading bacteria from contaminated soil and characterize the metabolites formed and the genes involved in the degradation pathway. METHODS AND RESULTS: A bacterial strain Xanthomonas sp. ICH12, capable of biodegrading gamma- HCH was isolated from HCH-contaminated soil. DNA-colony hybridization method was employed to detect bacterial populations containing specific gene sequences of the gamma-HCH degradation pathway. linA (dehydrodehalogenase), linB (hydrolytic dehalogenase) and linC (dehydrogenase) from a Sphingomonas paucimobilis UT26, reportedly possessing gamma-HCH degradation activity, were used as gene probes against isolated colonies. The isolate was found to grow and utilize gamma-HCH as the sole carbon and energy source. The 16S ribosomal RNA gene sequence of the isolate resulted in its identification as a Xanthomonas species, and we designated it as strain ICH12. During the degradation of gamma-HCH by ICH12, formation of two intermediates, gamma-2,3,4,5,6-pentachlorocyclohexene (gamma-PCCH), and 2,5-dichlorobenzoquinone (2,5-DCBQ), were identified by gas chromatography-mass spectrometric (GC-MS) analysis. While gamma-PCCH was reported previously, 2,5-dichlorohydroquinone was a novel metabolite from HCH degradation. CONCLUSIONS: A Xanthomonas sp. for gamma-HCH degradation from a contaminated soil was isolated. gamma-HCH was utilized as sole source of carbon and energy, and the degradation proceeds by successive dechlorination. Two degradation products gamma-PCCH and 2,5-DCBQ were characterized, and the latter metabolite was not known in contrasts with the previous studies. The present work, for the first time, demonstrates the potential of a Xanthomonas species to degrade a recalcitrant and widespread pollutant like gamma-HCH. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the isolation and characterization of a novel HCH-degrading bacterium. Further results provide an insight into the novel degradation pathway which may exist in diverse HCH-degrading bacteria in contaminated soils leading to bioremediation of gamma-HCH.     

Novel intermediates of acenaphthylene degradation by Rhizobium sp. strain CU-A1: evidence for naphthalene-1,8-dicarboxylic acid metabolism

The acenaphthylene-degrading bacterium Rhizobium sp. strain CU-A1 was isolated from petroleum-contaminated soil in Thailand. This strain was able to degrade 600 mg/liter acenaphthylene completely within three days. To elucidate the pathway for degradation of acenaphthylene, strain CU-A1 was mutagenized by transposon Tn5 in order to obtain mutant strains deficient in acenaphthylene degradation. Metabolites produced from Tn5-induced mutant strains B1, B5, and A53 were purified by thin-layer chromatography and silica gel column chromatography and characterized by mass spectrometry. The results suggested that this strain cleaved the fused five-membered ring of acenaphthylene to form naphthalene-1,8-dicarboxylic acid via acenaphthenequinone. One carboxyl group of naphthalene-1,8-dicarboxylic acid was removed to form 1-naphthoic acid which was transformed into salicylic acid before metabolization to gentisic acid. This work is the first report of complete acenaphthylene degradation by a bacterial strain.     

Identification of novel genes involved in long-chain n-alkane degradation by Acinetobacter sp. strain DSM 17874

Acinetobacter sp. strain DSM 17874 is capable of utilizing n-alkanes with chain lengths ranging from that of decane (C10H22) to that of tetracontane (C40H82) as a sole carbon source. Two genes encoding AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, have been shown to be involved in the degradation of n-alkanes with chain lengths of from 10 to 20 C atoms in this strain. Here, we describe a novel high-throughput screening method and the screening of a transposon mutant library to identify genes involved in the degradation of n-alkanes with C chain lengths longer than 20, which are solid at 30 degrees C, the optimal growth temperature for Acinetobacter sp. strain DSM 17874. A library consisting of approximately 6,800 Acinetobacter sp. strain DSM 17874 transposon mutants was constructed and screened for mutants unable to grow on dotriacontane (C32H66) while simultaneously showing wild-type growth characteristics on shorter-chain n-alkanes. For 23 such mutants isolated, the genes inactivated by transposon insertion were identified. Targeted inactivation and complementation studies of one of these genes, designated almA and encoding a putative flavin-binding monooxygenase, confirmed its involvement in the strain's metabolism of long-chain n-alkanes. To our knowledge, almA represents the first cloned gene shown to be involved in the bacterial degradation of long-chain n-alkanes of 32 C's and longer. Genes encoding AlmA homologues were also identified in other long-chain n-alkane-degrading Acinetobacter strains.     

Novel pathway for degradation of protocatechuic acid in Bacillus species.

A species of Bacillus, tentatively identified as B. circulans, degrades protocatechuic acid by a novel reaction involving meta-fission between C2 and C3 of the benzene nucleus. 2-Hydroxymuconic semialdehyde is then degraded to pyruvate and acetaldehyde by enzymatic reactions described in previous work. Protocatechuate 2,3-oxygenase exhibits a rather narrow substrate specificity; the methyl and ethyl esters of protocatechuic acid are oxidized, but other substrates for ring-fission oxygenases, notably catechol, gallic acid, and homoprotocatechuic acid, are not attached.     

Degradation of 5-chloro-2-hydroxynicotinic acid by Mycobacterium sp. BA

Bacteria with the ability to use 5-chloro-2-hydroxynicotinic acid as sole source of carbon and energy have been isolated from enrichment cultures with 2-hydroxynicotinic acid and 5-chloro-2-hydroxynicotinic acid. According to their morphological and physiological properties, these bacteria have been classified as Mycobacterium sp. The first metabolite in the degradation pathway is 5-chloro-2,6-dihydroxynicotinic acid. 2-Hydroxynicotinic acid had an inductive effect on the degrading enzymes. Chloride was released from 5-chloro-2-hydroxynicotinic acid, chloromaleic acid and chlorofumaric acid. A degradation pathway is proposed.