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Biodegradation of pyridine pollutant by microorganisms is one of the economical and effective methods to solve the environmental pollution of pyridine under high salinity conditions. To this end, screening of microorganisms with pyridine degradation capability and high salinity tolerance is an important prerequisite. In this paper, a salt-resistant pyridine degradation bacterium was isolated from the activated sludge of Shanxi coking wastewater treatment plant, and identified as a bacterium belonging to Rhodococcus on the basis of colony morphology and 16S rDNA gene phylogenetic analysis. Salt tolerance experiment showed that strain LV4 could grow and degrade pyridine with the initial concentration of 500 mg/L completely in 0%-6% saline environment. However, when the salinity was higher than 4%, strain LV4 grew slowly and the degradation time of pyridine by strain LV4 was significantly prolonged. Scanning electron microscopy showed that the cell division of strain LV4 became slower, and more granular extracellular polymeric substance (EPS) was induced to secrete in high salinity environment. When the salinity was not higher than 4%, strain LV4 responded to the high salinity environment mainly through increasing the protein content in EPS. The optimum conditions for pyridine degradation by strain LV4 at 4% salinity were 30 ℃, pH 7.0 and 120 r/min (DO 10.30 mg/L). Under these optimal conditions, strain LV4 could completely degrade pyridine with an initial concentration of 500 mg/L at a maximum rate of (29.10±0.18) mg/(L·h) after 12 h adaptation period, and the total organic carbon (TOC) removal efficiency reached 88.36%, indicating that stain LV4 has a good mineralization effect on pyridine. By analyzing the intermediate products in pyridine degradation process, it was speculated that strain LV4 achieved pyridine ring opening and degradation mainly through two metabolic pathways: pyridine-ring hydroxylation and pyridine-ring hydrogenation. The rapid degradation of pyridine by strain LV4 in high salinity environment indicates its application potential in the pollution control of high salinity pyridine environment.

Microorganisms are the dominant players driving the degradation and transformation of chloramphenicol (CAP) in the environment. However, little bacterial strains are able to efficiently degrade and mineralize CAP, and the CAP degrading pathways mediated by oxidative reactions remain unclear. In this study, a highly efficient CAP-degrading microbial consortium, which mainly consists of Rhodococcus (relative abundance >70%), was obtained through an enrichment process using CAP-contaminated activated sludge as the inoculum. A bacterial strain CAP-2 capable of efficiently degrading CAP was isolated from the consortium and identified as Rhodococcus sp. by 16S rRNA gene analysis. Strain CAP-2 can efficiently degrade CAP under different nutrient conditions. Based on the biotransformation characteristics of the detected metabolite p-nitrobenzoic acid and the reported metabolites p-nitrobenzaldehyde and protocatechuate by strain CAP-2, a new oxidative pathway for the degradation of CAP was proposed. The side chain of CAP was oxidized and broken to generate p-nitrobenzaldehyde, which was further oxidized to p-nitrobenzoic acid. Strain CAP-2 can be used to further study the molecular mechanism of CAP catabolism, and has the potential to be used in in situ bioremediation of CAP-contaminated environment.

As self-subunit swapping chaperones or metallochaperones, the activators assist nitrile hydratases to take up metal ions and they are essential for active expression of nitrile hydratases. Compared with nitrile hydratases, the activators have a low sequence identity. Study of the activation characteristics and the relationships between structures and functions of the activators is of great significance for understanding the maturation mechanism of nitrile hydratase. We co-expressed low-molecular-mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1 with four heterologous activators respectively and determined their activation abilities. Then we made sequence analysis and structure modelling, and studied the functions of the important domains of the activators. Results showed that all four heterologous activators could activate L-NHase, however, the specific activities of L-NHases were different after activation. L-NHase showed the highest specific activity after being activated by activator A, which was 97.79% of that of the original enzyme, but the specific activity of L-NHase after being activated by activator G was only 23.94% of that of the original enzyme. Activator E and activator G had conserved domains (TIGR03889), and deletion of their partial sequences resulted in a substantial loss of activation abilities for both activators. Replacing the N-terminal sequence of activator G with the N-terminal sequence of activator E, and adding the C-terminal sequence of activator E to the C-terminus of activator G could increase the specific activity of L-NHase by 178.40%. The activation by nitrile hydratase activators was universal and specific, and the conserved domains of activators were critical for activation, while the N-terminal domain and C-terminal domain also had important effects on activation.

Objective: The physiological function of membrane protein RHOGL009301 in Rhodococcus sp. R04 and the metabolic properties of the mutant strain were studied to determine the relationship between the physiological function of the membrane protein and the transport of benzoate., Methods: The RHOGL009301 gene and the green fluorescent protein gene were fused for expressing in Rhodococcus erythropolis, and the location of RHOGL009301 was observed by Delta Vision. The RHOGL009301 gene was knocked out by homologous recombination, and the growth of wild strain and deficient strain in different carbon sources were compared. The internal and external metabolites of the wild strain and the deficient strain when grown on biphenyl and benzoate were measured by HPLC, and the changes of metabolite concentration in different growth conditions were analyzed., Results: A fusion gene that contained RHOGL009301 gene and the green fluorescent protein gene was co-expressed in Rhodococcus erythropolis and localized on the cell membrane. The deficient strain R04ΔMP of RHOGL009301 gene was obtained. The biomass of the deficient strain was significantly reduced in biphenyl and benzoate culture, and its growth rate was slowed down. HPLC analysis showed that the deletion of RHOGL009301 gene inhibited the transport of benzoate., Conclusion: RHOGL009301 membrane protein is one of the proteins involved in metabolism and transport of benzoate. Based on sequence homology analysis, we can conclude that the membrane protein is a novel benzoate transport protein.

Objective: Studying transcriptional characteristics of Rhodococcus sp. R04 is to find the genes that participate in the transportation, metabolism and regulation of polychlorinated biphenyls (PCBs), and disclose the molecular mechanism of biodegradation of PCBs., Methods: Strain R04 was separately cultivated on ethanol, glucose and biphenyl. The total RNA of the above different cultures was extracted, and the cDNA was obtained by reverse transcription and determined by high-throughput sequencing. The data obtained by sequencing were analyzed to find the correlation among the PCBs metabolic network, gene transcription regulation and metabolic response., Results: The sequencing results showed that 375 genes were up-regulated during grown on biphenyl, relative to growth on glucose, and 332 genes were up-regulated, relative to growth on ethanol. Those genes were found to participate in multiple biological processes of biphenyl metabolite. Among the genes relative to biphenyl/PCBs degradation, the genes located on the gene cluster in the upper biphenyl pathway were significantly up-regulated, while bphC4 and bphD2 were up-regulated slightly. By contrast, the other genes encoding BphC and BphD isozymes in the biphenyl pathway were down-regulated even if growth on biphenyl., Conclusion: Transcriptomic analysis suggested that benzoate was degraded via ortho cleavage pathway, meta cleavage pathway or protocatechuic acid pathway, which provide more valuable data for us to reveal the characteristics and regulation of downstream metabolic pathways of biphenyl and PCBs.

Objective: We characterizeda manganese catalase lacking n-terminal ( MnCAT-C), to revealits roles in bacterial growth, reactive oxygen species ( ROS) removal and degradation of polychlorinated biphenyls ( PCBs) in Rhodococcus sp. R04., Methods: Manganese catalase (Mn-CAT) sequence of the strain R04 was aligned with that of Rhodococcus sp. R11101. Mn-CAT and MnCAT-C were expressed in E. coli BL21 (DE3), and the target protein was purified with Q-sepharose and ammonium sulphate precipitation. Knockout strain was obtained by homologous recombination. ROS was measured by fluorescence polarization, and the degradation rate of PCBs was measured by HPLC. [Results] MnCAT-C protein was purified, and SDS-PAGE analysis showed that its molecular weight was 23 kDa. Compared with wild strains, the ROS concentration increased, and the growth rate was inhibited in knockout strains. Moreover, the degradationrate of PCBs decreased. [Conclusion] MnCAT-C retained the majority of the active properties of the original enzyme, including ROS clearance. The lack of MnCAT-C gene affected the growth rate and the PCBsdegradationrate instrain R04.

Objective: The removal of phenolic compounds was a key problem for coking wastewater treatment. This study focused on the isolation and characterization of anefficient phenol-degrading bacterial strain from coking wastewater., Methods: Phenol-degrading bacterium was screened by using phenol as the sole carbon source, strain was identified according to the morphological properties and 16S rRNA sequence analysis, the phenol-degrading characteristics and the potential for removal of phenol in the coking wastewater were evaluated., Results: Strain P1 was identified as the genus Rhodococcus sp. with morphological properties and 16S rRNA gene sequence. This strain could survive at the presence of phenol with concentration up to 1400 mg/L. The optimal conditions for biodegradation of 600 mg/L phenol in mineral medium were at 32 - 42 degrees C, pH 7.0 and 0 - 4% NaCl. The phenol-degrading efficiency by P1 strain was different in response to various heavy metal ions, Ni2+, Mn2+ and low concentration of Pb2+ had no effect on the phenol degradation. However, Cu2+, Ni2+ and Cd2+ seriously inhibited phenol degradation by strain P1. The 279.9 mg/L phenols from 1/3 coking wastewater were completely degraded after incubation for 2 days., Conclusion: Strain P1 is an efficient phenol-degrading bacterium and has of the potential for removing phenolic compounds in coking wastewater.

Objective: We characterized alcohol dehydrogenase from Rhodococcus erytropolis to catalyze ketoesters or ketones., Methods: We cloned alcohol dehydrogenase gene (adh) of 1047 bp from Rhodococcus erythropolis ATCC 4277, inserted the open reading frame of adh into vector pET-22b(+) and expressed in auto-inducing media for 24 h at 15 degrees C. The enzyme activity was determined at 30 degrees C using acetophenone as substrate., Results: Under the above conditions, the specific enzyme activity of crude extract was 2.6 U/mg. The optimal pH was between 6.0 and 6.5 and the enzyme can survived up to 60 degrees C. After incubation at 60 degrees C for 5 h, 80% enzyme activity remained. The optimal substrate among beta-ketoesters examined was ethyl acetoaetate. Ethyl 4-chloroacetoacetate was catalyzed by whole cell in aqueous phase. After chiral liquid chromatography, the product showed (R)-enantioselective., Conclusion: The study shows that the enzyme might have potential in beta-ketoesters transformation on industrial scale.

Unlabelled: Two linear plasmids, pNSL1 and pNSL1, were detected from Rhodocuccus sp. NS1., Objective: Cloning, sequencing and identification of replication origin of the Rhodococcus linear plasmid pNSL1., Methods: Large amount of linear plasmid DNA was recovered from pulsed-field gels for shotgun-cloning and sequencing, and identification of its replication locus., Results: The complete nucleotide sequence of pNSL1 consisted of 117252 bp, including the conserved 1282-bp telomere sequences among Rhodococcus linear plasmids. pNSL1 encoded 103 open reading frames, including functions of replication, maintenance and transfer etc. A locus, pNSL1. 038 and upstream 767-bp non-coding sequence, was identified for autonomous replication by cloning in an E. coli vector and introduced by electroporation into Nocardia coralline 4. 1037., Conclusion: Cloning and sequencing of Rhodococcus linear plasmid pNSL1, and identification of its replication origin.

The genus Rhodococcus is of considerable interest in recent years, stemming from their diverse applications in biodegradation, bioremediation, biotransformation and biosurfactant. Using Nocardia/Rhodococcus-Escherichia coli shuttle plasmid pNV18.1 as the backbone vector, we tested the driven efficiency of promoters Ptac and PlacZ of E. coli and Pami-1/Pami-2 of R. ruber in host R. rhodochrous ATCC 33278 by overexpression of nitrile hydratase. Results showed that the specific activity of nitrile hydratase per dry cell weight in engineered Rhodococcus strains driven by Ptac, Pami-1, Pami-2 and PlacZ was 7.5, 6.3, 5.3 and 1.8 times of that in the wild, respectively. It indicated that these promoters could be well recognized by RNA polymerase of Rhodococcus. We further expressed the beta-galactosidase reporter gene (lacZ) in R. ruber driven by promoter PlacZ. Results indicated that lacZ was an appropriate reporter gene for genetic or metabolic engineering research of Rhodococcus.

Gordonia sp. C-6 can desulfurize benzothiophene (BT) as the pathway similar to the "4S" pathway of dibenzothiophene (DBT)-desulfurizing, but the strain can not grow with DBT as the sole sulfur source. At current, there were not related reports on BT-desulfurizing genes at home or abroad. The DBT-desulfurizing genes of Rhodocossus erythropolis DS-3, dszA, dszB, dszC and dszABC were introduced into Gordonia sp. C-6 respectively using a Rhodococcus-E. coli shuttle vector, to construct new recombinant strains Gordonia sp. CRA, Gordonia sp. CRB, Gordonia sp. CRC and Gordonia sp. CRABC, the enzyme activities of which was respectively 76.8 micromol x (g x h)(-1), 51.6 micromol x (g x h)(-1) and 62.4 micromol x (g x h)(-1), increasing by 1.5 times compared with 35.2 micromol x (g x h)(-1), 21.3 micromol x (g x h)(-1) and 25.5 micromol x (g x h)(-1) of wild strain Rhodocossus erythropolis DS-3. Of the recombinant strains, only recombinant strains Gordonia sp. CRC with DszC and Gordonia sp. CRABC with DszABC exhibite significant growth with DBT as the sole sulfur source, Gordonia sp. CRA with DszA and Gordonia sp. CRB with DszB could not live with DBT as the sole sulfur source. The results show that, DBT monoxygenase and BT monoxygenase, catalyzed the first two steps of DBT and BT oxidation, respectively, are the key enzymes responsible for substrate-recognition, however, the enzymes catalyzed the last two steps have the similar substrate specificity. The active sites of the two monoxygenase could be predicted by comparing with the sequence differences of their amino acids.

A quinoline-degrading bacterium QL2, which utilizes quinoline as sole source of carbon, nitrogen and energy, was isolated from activated sludge in a coke-plant wastewater biological treatment system. According to the morphological characteristics, physiological and biochemical characteristics, and sequence analysis of 16S rRNA, the strain was identified as Rhodococcus sp.. The optimal temperature, initial pH, and shaker rotary speed for strain QL2 utilizing quinoline are 35-42 degrees C, pH 8-9, and 150 r/min, respectively. Extra nitrogen sources stimulate the isolate growth on quinoline, and inorganic nitrogen better than organic nitrogen, NH4+ -N better than NO3(-) -N. The degradation reaction of quinoline by strain QL2 can be described with zero order kinetic equation within the initial quinoline concentrations of 60-680 mg/L. When the initial concentration was 150 mg/L, quinoline was degraded completely in 8 hours and TOC removal efficiency was 70% in 14 hours. This bacterium produced pigmented compounds, and ring nitrogen was released into the growth medium as ammonium. The main intermediate in the degradation pathway was 2-hydroxyquinoline by the analysis of HPLC and GC/MS. With a broad range of substrate utilization, the strain can degrade phenol, naphthalene, pyridine, and some other kinds of aromatic compounds.

A quantitative competitive PCR (QC-PCR) system was developed to quantify the number and analyze the function of the Rhodococcus ruber Em1 strain in a wastewater treatment system in Nanchong oil refinery plant. Strain Em1 was able to degrade various kinds of hydrocarbons and aromatic compounds with high efficiency and produce bioemulsifier, so it was introduced into the waste liquid petroleum-disposing system. The sediment samples were collected from the disposing system in the range of 5 months, and then the numbers of strain Eml and degrading efficiencies were studied. The results showed that the primers based on 16S rRNA gene sequence of strain Em1 were specific at species level. The PCR products amplified from sediment total DNA with the specific primers were cloned and sequenced, in which 62.2% were the fragments of 16S rRNA gene of strain Em1. Furthermore, the number of Em1 strain ranging from 3.4 x 10(5) - 4.3 x 10(8) CFU/g in the sediment samples were detected, which indicated that the strain Eml added into purposely did exist stably and reproduced well in the waste-deposing system during a long period. The high relativity, with relative coefficient R2 of 0.89, between Eml cell number and the amount of COD (Chemical Oxygen Demand) removal proved that the strain Em1 played an important role in this bio-augmentation treatment system.

Polyphasic evidence for the reclassification of Nocardia nostocoides AS4.1186 and Nocardia salmonicolor var. aurantiaca AS4.1187 indicated that strain AS4.1186 was closely related to Saccharothrix texasensis and strain AS4.1187 was very closely related to Rhodococcus ruber. Binary 16S rDNA sequence for the pair AS4.1186 and Saccharothrix texasensis NRRL B-16134T, and the pair AS4.1187 and Rhodococcus ruber DSM 43338T exhibited 99.3% and 99.5% similarity, respectively. Corresponding DNA-DNA reassociation values were significantly higher than 70%, 77.6% and 82.9%, respectively. Results of chemotaxonomic analyses of cell wall, mycolic acid, principal menaquinones, phospholipid type and the G + C content of the DNA supported the conclusions of the genotypic analyses. The very similar morphological and physiological characteristics agreed with the high degree of relatedness. On the basis of phylogenetic analyses, DNA-DNA reassociation values, chemotaxonomic properties, morphological and physiological characteristics, it is concluded that strain AS4.1186 and AS4.1187 should be removed from the genus Nocardia. We propose to transfer Nocardia nostocoides AS4.1186 to the genus Saccharothrix, combine with the species S. texasensis, and combine Nocardia salmonicolor var. aurantiaca AS4.1187 with the species Rhodococcus ruber.

The developed course and reaction mechanisms of petroleum biodesulfurization were introduced. The recent development of genetic engineering technology, which used in desulfuration strain's construction, reconstruction and other fields, was summarized emphatically. Its current research situation internal and overseas and the developing prospect were simply analyzed, and our research designs were submitted.