In situ bioremediation of petroleum hydrocarbon-contaminated soil: isolation and application of a Rhodococcus strain.

Due to low consumption and high efficiency, in situ microbial remediation of petroleum hydrocarbons (PHs)-contaminated sites in in-service petrochemical enterprises has attracted more and more attention. In this study, a degrading strain was isolated from oil depot-contaminated soil with soil extract (PHs) as the sole carbon source, identified and named Rhodococcus sp. OBD-3. Strain OBD-3 exhibited wide adaptability and degradability over a wide range of temperatures (15-37 °C), pH (6.0-9.0), and salinities (1-7% NaCl) to degrade 60.6-86.6% of PHs. Under extreme conditions (15 °C and 3-7% salinity), PHs were degraded by 60.6 ± 8.2% and more than 82.1% respectively. In OBD-3, the alkane monooxygenase genes alkB1 and alkB2 (GenBank accession numbers: MZ688386 and MZ688387) were found, which belonged to Rhodococcus by sequence alignment. Moreover, strain OBD-3 was used in lab scale remediation in which the contaminated soil with OBD-3 was isolated as the remediation object. The PHs were removed at 2,809 ± 597 mg/kg within 2 months, and the relative abundances of Sphingobium and Pseudomonas in soil increased more than fivefold. This study not only established a system for the isolation and identification of indigenous degrading strains that could efficiently degrade pollutants in the isolated environment but also enabled the isolated degrading strains to have potential application prospects in the in situ bioremediation of PHs-contaminated soils.

Promoting effect of the recombinant resuscitation promoting factors-2 of Rhodococcus erythropolis on petroleum degradation and cultivable bacterial diversities of the oil-contaminated soils.

Resuscitation-promoting factors (Rpfs) belong to peptidoglycan hydrolases, which participate in recovery of dormant cells and promoting bacteria growth. In this study, the resuscitation promoting factor rpf2 gene of Rhodococcus erythropolis KB1 was expressed in Escherichia coli and purified by Ni2+ affinity chromatography. The purified recombinant fusion protein Rpf2 showed a closely 50 kDa band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The protein showed muralytic activity, with a specific activity of 1503 ± 123 U mg-1 when determined with 4-methylumbelliferyl-ß-d-N, N',N″-triacetotri-ylchitoside as substrate. It also showed protease activity when measured with azocasein as substrate, with a specific activity of 1528 ± 411 U mg-1 . The addition of the recombinant Rpf2 protein significantly increased petroleum degradation efficiency of the indigenous micro-organisms and the petroleum degradation rates increased from 30·86 to 43·45%, 45·20 and 49·23% in the treatment groups. The recombinant protein also increased the petroleum-degrading bacterial diversities enriched from the contaminated soils. The cultivable bacterial flora of the treatment groups supplemented with different concentrations of Rpf2 increased from 82 genera in 9 phyla to 116 genera in 16 phyla and 138 genera in 16 phyla respectively. Thirteen extra petroleum-degrading bacteria strains were isolated from the petroleum-contaminated soils in the groups containing the recombinant Rpf2.

Bioballs carrying a syntrophic Rhodococcus and Mycolicibacterium consortium for simultaneous sorption and biodegradation of fuel oil in contaminated freshwater.

Nonpathogenic effective bacterial hydrocarbon degraders, Rhodococcus ruber S103, Mycolicibacterium parafortuitum J101 and Mycolicibacterium austroafricanum Y502, were isolated from mixed polycyclic aromatic hydrocarbon (PAH)-enriched river sediments. They possessed broad substrate specificities toward various PAHs and aliphatic compounds as sole carbon sources. These strains exhibited promising characteristics, including biosurfactant production, high cell hydrophobicity, biofilm formation and no antagonistic interactions, and contained genes encoding hydrocarbon-degrading enzymes. The mixed bacterial consortium combining S103, J101 and Y502, showed more effective syntrophic degradation of two types of refined petroleum products, diesel and fuel oils, than monocultures. The defined consortium immobilized on plastic balls achieved over 50% removal efficiency of high fuel oil concentration (3000 mg L-1) in a synthetic medium and contaminated freshwater. Furthermore, the immobilized cells simultaneously degraded more than 46% of total fuel oil adsorbed on plastic balls in both culture systems. SEM imaging confirmed that the immobilized consortium exhibited biofilm formation with the bacterial community covering most of the bioball surface, resulting in high bacterial survival against toxic contaminants. The results of this study showed the potential use of the cooperative interaction between Rhodococcus and Mycolicibacterium as immobilized bioballs for the bioremediation of fuel oil-contaminated environments. Additionally, this research has motivated further investigations into the development of bioremediation products for fuel oil degradation.

Catabolic enzyme activities during biodegradation of three-ring PAHs by novel DTU-1Y and DTU-7P strains isolated from petroleum-contaminated soil.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants having health hazards. PAH-utilizing bacterial strains were isolated from petroleum-contaminated soil from siding area, Bijwasan supply location of BPCL, Delhi, India. Bacterial strains with different morphology were isolated and acclimatized to a mixture of low molecular weight PAH compounds in the concentration range of 50-10,000 mg/L. Two bacterial strains surviving at 10,000 mg/L PAH concentration were identified as Kocuria flava and Rhodococcus pyridinivorans, based on 16S rRNA gene sequencing and phylogenetic analysis over MEGA X, are reported for the first time for PAH degradation. The strain K. flava could degrade phenanthrene, anthracene, and fluorene with efficiency of 55.13%, 59.01%, and 63.46%, whereas R. pyridinivorans exhibited 62.03%, 64.99%, and 66.79% degradation for respective PAHs at initial PAH concentration of 10 mg/L. Slightly lower degradation of phenanthrene could be attributed to its more stable chemical structure. The consortium of both the strains degraded 61.32%, 64.72%, and 66.64%, of 10 mg/L of phenanthrene, anthracene, and fluorene, respectively, in 15 days of incubation period indicating no synergistic or antagonistic effect towards degradation. Catechol 2,3-dioxygenase (C23O), dehydrogenase and peroxidase enzyme activities during PAH degradation coincided with degradation of PAHs, thus highlighting the role of these enzymes in catabolising three-ring PAHs. This is the first investigation confirming the participation of C23O, dehydrogenase and peroxidases enzyme profiles throughout the period of degradation. The study concludes that these strains can play significant role in microbial remediation of PAH-contaminated environment.

A Set of Active Promoters with Different Activity Profiles for Superexpressing Rhodococcus Strain.

Rhodococcus bacteria are a promising platform for biodegradation, biocatalysis, and biosynthesis, but the use of rhodococci is hampered by the insufficient number of both platform strains for expression and promoters that are functional and thoroughly studied in these strains. To expand the list of such strains and promoters, we studied the expression capability of the Rhodococcus rhodochrous M33 strain, and the functioning of a set of recombinant promoters in it. We showed that the strain supports superexpression of the target enzyme (nitrile hydratase) using alternative inexpensive feedings-acetate and urea-without growth factor supplementation, thus being a suitable expression platform. The promoter set included Ptuf (elongation factor Tu) and Psod (superoxide dismutase) from Corynebacterium glutamicum ATCC13032, Pcpi (isocitrate lyase) from Rhodococcus erythropolis PR4, and Pnh (nitrile hydratase) from R. rhodochrous M8. Activity levels, regulation possibilities, and growth-phase-dependent activity profiles of these promoters were studied in derivatives of the M33 strain. The activities of the promoters were significantly different (Pcpi < Psod ≪ Ptuf < Pnh), covering 103-fold range, and the most active Pnh and Ptuf produced up to a 30-50% portion of target protein in soluble intracellular proteins. On the basis of the mRNA quantification and amount of target protein, the production level of Pnh was positioned close to the theoretical upper limit of expression in a bacterial cell. A selection method for the laboratory evolution of such active promoters directly in Rhodococcus was also proposed. Concerning regulation, Ptuf could not be regulated (2-fold change), while others were tunable (6-fold for Psod, 79-fold for Pnh, and 44-fold for Pcpi). The promoters possessed four different activity profiles, including three with peak of activity at different growth phases and one with constant activity throughout the growth phases. Ptuf and Pcpi did not change their activity profile under different growth conditions, whereas the Psod and Pnh profiles changed depending on the growth media. The results allow flexible construction of Rhodococcus strains using the studied promoters, and demonstrate a valuable approach for complex characterization of promoters intended for biotechnological strain construction.

Complete genome sequencing and comparative CAZyme analysis of Rhodococcus sp. PAMC28705 and PAMC28707 provide insight into their biotechnological and phytopathogenic potential.

Study of carbohydrate-active enzymes (CAZymes) can reveal information about the lifestyle and behavior of an organism. Rhodococcus species is well known for xenobiotic metabolism; however, their carbohydrate utilization ability has been less discussed till date. This study aimed to present the CAZyme analysis of two Rhodococcus strains, PAMC28705 and PAMC28707, isolated from lichens in Antarctica, and compare them with other Rhodococcus, Mycobacterium, and Corynebacterium strains. Genome-wide computational analysis was performed using various tools. Results showed similarities in CAZymes across all the studied genera. All three genera showed potential for significant polysaccharide utilization, including starch, cellulose, and pectin referring their biotechnological potential. Keeping in mind the pathogenic strains listed across all three genera, CAZymes associated to pathogenicity were analyzed too. Cutinase enzyme, which has been associated with phytopathogenicity, was abundant in all the studied organisms. CAZyme gene cluster of Rhodococcus sp. PAMC28705 and Rhodococcus sp. PAMC28707 showed the insertion of cutinase in the cluster, further supporting their possible phytopathogenic properties.

Close-up on a bacterial informational war in the geocaulosphere.

The geocaulosphere is home to microbes that establish communication between themselves and others that disrupt them. These cell-to-cell communication systems are based on the synthesis and perception of signaling molecules, of which the best known belong to the N-acyl-homoserine lactone (AHL) family. Among indigenous bacteria, certain Gram-positive actinobacteria can sense AHLs produced by soft-rot Gram-negative phytopathogens and can degrade the quorum-sensing AHL signals to impair the expression of virulence factors. We mimicked this interaction by introducing dual-color reporter strains suitable for monitoring both the location of the cells and their quorum-sensing and -quenching activities, in potato tubers. The exchange of AHL signals within the pathogen's cell quorum was clearly detected by the presence of bright green fluorescence instead of blue in a portion of Pectobacterium-tagged cells. This phenomenon in Rhodococcus cells was accompanied by a change from red fluorescence to orange, showing that the disappearance of signaling molecules is due to rhodococcal AHL degradation rather than the inhibition of AHL production. Rhodococci are victorious in this fight for the control of AHL-based communication, as their jamming activity is powerful enough to prevent the onset of disease symptoms.

Biodegradation of naphthenic acids: identification of Rhodococcus opacus R7 genes as molecular markers for environmental monitoring and their application in slurry microcosms.

Nowadays, the increase of the unconventional oil deposit exploitation and the amount of oil sands process-affected waters (OSPW) in tailing ponds emerges the importance of developing bio-monitoring strategies for the restoration of these habitats. The major constituents of such deposits are naphthenic acids (NAs), emerging contaminant mixtures with toxic and recalcitrant properties. With the aim of developing bio-monitoring strategies based on culture-independent approach, we identified genes coding for enzymes involved in NA degradation from Rhodococcus opacus R7 genome, after the evaluation of its ability to mineralize model NAs. R. opacus R7 whole-genome analysis unveiled the presence of pobA and chcpca gene clusters putatively involved in NAs degradation. Gene expression analysis demonstrated the specific induction of R7 aliA1 gene, encoding for a long-chain-fatty-acid-CoA ligase, in the presence of cyclohexanecarboxylic acid (CHCA) and hexanoic acid (HA), selected as representative compounds for alicyclic and linear NAs, respectively. Therefore, aliA1 gene was selected as a molecular marker to monitor the biodegradative potential of slurry-phase sand microcosms in different conditions: spiked with CHCA, in the presence of R. opacus R7, the autochthonous microbial community, and combining these factors. Results revealed that the aliA1-targeting culture-independent approach could be a useful method for bio-monitoring of NA degradation in a model laboratory system.

Beyond N and P: The impact of Ni on crude oil biodegradation.

N and P are the key limiting nutrients considered most important for the stimulation of crude oil degradation but other trace nutrients may also be important. Experimental soil microcosms were setup to investigate crude oil degradation in the context of Ni amendments. Amended Nickel as NiO, NiCl2, or, a porphyrin complex either inhibited, had no effect, or, enhanced aerobic hydrocarbon degradation in an oil-contaminated soil. Biodegradation was significantly (95% confidence) enhanced (70%) with low levels of Ni-Porph (12 mg/kg) relative to an oil-only control; whereas, NiO (200 and 350 mg/kg) significantly inhibited (36 and 87%) biodegradation consistent with oxide particle induced reactive oxygen stress. Microbial community compositions were also significantly affected by Ni. In 16S rRNA sequence libraries, the enriched hydrocarbon degrading genus, Rhodococcus, was partially replaced by a Nocardia sp. in the presence of low levels of NiO (12 and 50 mg/kg). In contrast, the highest relative and absolute Rhodococcus abundances were coincident with the maximal rates of oil degradation observed in the Ni-Porph-amended soils. Growth dependent constitutive requirements for Ni-dependent urease or perhaps Ni-dependent superoxide dismutase enzymes (found in Rhodococcus genomes) provided a mechanistic explanation for stimulation. These results suggest biostimulation technologies, in addition to N and P, should also consider trace nutrients such as Ni tacitly considered adequately supplied and available in a typical soil.

Screening, partial purification of antivibriosis metabolite sterol-glycosides from Rhodococcus sp. against aquaculture associated pathogens.

The present study probed the antimicrobial potential of a rare mangrove associated actinomycetes against an array of aquatic bacterial pathogens causing disease outbreak in fin and shellfish. Antibacterial activity results implied that the mangrove associated actinomycetes RAS7 exhibited striking inhibitory activity against the tested aquatic bacterial pathogens. Identification of strain RAS7 through polyphasic and 16S rRNA sequencing affirmed that the strain belongs to Rhodococcus sp. Optimization of culture conditions for antibacterial activity by Rhodococcus sp. inferred that it grew well and exerted notable antagonistic activity in medium supplied with 1% galactose and peptone as carbon and nitrogen sources. Similarly, the strain grown in 0.1% tyrosine, 1% NaCl, pH 7.5 and temperature 35 °C recorded maximum bioactivity against the test pathogens. The crude ethyl acetate extract of Rhodococcus sp. at 200  µg/ml recorded markedly pronounced growth inhibitory activity ranged between 14 and 29 mm. The cytotoxic effect of crude extract against brine shrimp Artemia salina nauplii registered LC50 value of 134.294 µg/ml after 24 h of exposure. The secondary metabolite was separated using Ethyl acetate: Methanol (7:3) as solvent system through TLC. The TLC autobiogram mapped the active spot in TLC with Rf value of 0.84. Analysis of chemical constituents and FT-IR spectral analysis substantiated that the active principle in bioassay guided fraction was sterol-glycosides.

Biodegradation of Tetralin: Genomics, Gene Function and Regulation.

Tetralin (1,2,3,4-tetrahydonaphthalene) is a recalcitrant compound that consists of an aromatic and an alicyclic ring. It is found in crude oils, produced industrially from naphthalene or anthracene, and widely used as an organic solvent. Its toxicity is due to the alteration of biological membranes by its hydrophobic character and to the formation of toxic hydroperoxides. Two unrelated bacteria, Sphingopyxis granuli strain TFA and Rhodococcus sp. strain TFB were isolated from the same niche as able to grow on tetralin as the sole source of carbon and energy. In this review, we provide an overview of current knowledge on tetralin catabolism at biochemical, genetic and regulatory levels in both strains. Although they share the same biodegradation strategy and enzymatic activities, no evidences of horizontal gene transfer between both bacteria have been found. Moreover, the regulatory elements that control the expression of the gene clusters are completely different in each strain. A special consideration is given to the complex regulation discovered in TFA since three regulatory systems, one of them involving an unprecedented communication between the catabolic pathway and the regulatory elements, act together at transcriptional and posttranscriptional levels to optimize tetralin biodegradation gene expression to the environmental conditions.

Rhodococcus daqingensis sp. nov., isolated from petroleum-contaminated soil.

A novel Gram-stain positive, aerobic, non-motile bacterial strain, designated Z1T, was isolated from a sample of petroleum-contaminated soil collected in Daqing, Heilongjiang province, China and characterised with a series of taxonomic approaches. The morphological and chemotaxonomic properties of the isolate were typical of those of members of the genus Rhodococcus. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Z1T belongs to the genus Rhodococcus and clustered with Rhodococcus maanshanensis DSM 44675T (99.2%, sequence similarity) and Rhodococcus tukisamuensis JCM 11308T (97.9%), respectively. However, the DNA-DNA hybridizations between strain Z1T and R. maanshanensis DSM 44675T and R. tukisamuensis JCM 11308T were both less than 70%. The optimal growth temperature and pH for strain Z1T were found to be at 28 °C and at pH 7.0. The peptidoglycan was found to contain meso-diaminopimelic acid; arabinose, galactose and glucose were detected as diagnostic sugars. The main polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unidentified lipid; MK-8(H2) was found as the major menaquinone. The major fatty acids were identified as C16:0, 10-methyl C18:0 and C18:1ω9c. Mycolic acids were found to be present. The G + C content of the genomic DNA was determined to be 66.7 mol%. Based on a comparative analysis of phenotypic and genotypic characteristics, in combination with DNA-DNA hybridization results, strain Z1T can be distinguished from the type strains of its two close neighbours as a novel species of the genus Rhodococcus, for which the name Rhodococcus daqingensis sp. nov. is proposed. The type strain is Z1T (= CGMCC 1.13630T = DSM 107227T).

Oil-degrading properties of a psychrotolerant bacterial strain, Rhodococcus sp. Y2-2, in liquid and soil media.

The aim of this study was to investigate oil-degrading ability of newly isolated strain Rhodococcus Y2-2 at low temperature. Rhodococcus sp. Y2-2 was isolated from oil-contaminated soil sampled at the end of winter using a newly developed transwell plate method. In the liquid phase, the oil-degradation efficiency of strain Rhodococcus sp. Y2-2 was about 84% with an initial concentration of 1500 ppm TPH (500 ppm each of kerosene, gasoline, and diesel) when incubated for 2 weeks under optimal conditions: 10 °C, pH 7, and 0.5 g L- 1 inoculum. In the soil phase, the isolate showed 80% oil degradation efficiency using glucose as a carbon source, with an initial concentration of 4000 ppm TPH and the addition of water during 14 days of incubation at 10 °C. Additionally, the degradation efficiency of the isolate was increased by the addition of mixture of surfactant alpha olefin sulfonate and gelatin, although strain Y2-2 also produced many biosurfactant components. This study shows Rhodococcus sp. Y2-2 can degrade oil components both in liquid and soil media by consuming kerosene, gasoline, and diesel as a carbon and energy source. Therefore, the crude oil-degrading ability of Rhodococcus sp. Y2-2 at low temperature provides proper bioremediation tool to clean up oil-contaminated sites especially in cold area or during winter season.

Characterization of biosurfactants produced by the oil-degrading bacterium Rhodococcus erythropolis S67 at low temperature.

Production of trehalolipid biosurfactants by Rhodococcus erythropolis S67 depending on the growth temperature was studied. R. erythropolis S67 produced glycolipid biosurfactants such as 2,3,4-succinoyl-octanoyl-decanoyl-2'-decanoyl trehalose and 2,3,4-succinoyl-dioctanoyl-2'-decanoyl trehalose during the growth in n-hexadecane medium at 26 and 10 °C, despite the different aggregate state of the hydrophobic substrate at low temperature. The surface tension of culture medium was found being reduced from 72 to 27 and 45 mN m-1, respectively. Production of trehalolipid biosurfactants by R. erythropolis S67 at low temperature could be useful for the biodegradation of petroleum hydrocarbons at low temperatures by enhancing the bioremediation performance in cold regions.

Characterization of the Rhodococcus sp. MK1 strain and its pilot application for bioremediation of diesel oil-contaminated soil.

Petroleum hydrocarbons and derivatives are widespread contaminants in both aquifers and soil, their elimination is in the primary focus of environmental studies. Microorganisms are key components in biological removal of pollutants. Strains capable to utilize hydrocarbons usually appear at the contaminated sites, but their metabolic activities are often restricted by the lack of nutrients and/or they can only utilize one or two components of a mixture. We isolated a novel Rhodococcus sp. MK1 strain capable to degrade the components of diesel oil simultaneously. The draft genome of the strain was determined and besides the chromosome, the presence of one plasmid could be revealed. Numerous routes for oxidation of aliphatic and aromatic compounds were identified. The strain was tested in ex situ applications aiming to compare alternative solutions for microbial degradation of hydrocarbons. The results of bioaugmentation and biostimulation experiments clearly demonstrated that - in certain cases - the indigenous microbial community could be exploited for bioremediation of oil-contaminated soils. Biostimulation seems to be efficient for removal of aged contaminations at lower concentration range, whereas bioaugmentation is necessary for the treatment of freshly and highly polluted sites.

Occurrence of diverse alkane hydroxylase alkB genes in indigenous oil-degrading bacteria of Baltic Sea surface water.

Formation of specific oil degrading bacterial communities in diesel fuel, crude oil, heptane and hexadecane supplemented microcosms of the Baltic Sea surface water samples was revealed. The 475 sequences from constructed alkane hydroxylase alkB gene clone libraries were grouped into 30 OPFs. The two largest groups were most similar to Pedobacter sp. (245 from 475) and Limnobacter sp. (112 from 475) alkB gene sequences. From 56 alkane-degrading bacterial strains 41 belonged to the Pseudomonas spp. and 8 to the Rhodococcus spp. having redundant alkB genes. Together 68 alkB gene sequences were identified. These genes grouped into 20 OPFs, half of them being specific only to the isolated strains. Altogether 543 diverse alkB genes were characterized in the brackish Baltic Sea water; some of them representing novel lineages having very low sequence identities with corresponding genes of the reference strains.

Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium.

Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. Single strains and four bacterial consortia designed by mixing the single bacterial cultures respectively in the following ratios: (Alcanivorax: Pseudomonas, 1:1), (Alcanivorax: Rhodococcus, 1:1), (Pseudomonas: Rhodococcus, 1:1), and (Alcanivorax: Pseudomonas: Rhodococcus, 1:1:1), were analyzed in order to evaluate their oil degrading capability. All experiments were carried out in microcosms systems containing seawater (with and without addition of inorganic nutrients) and crude oil (unique carbon source). Measures of total and live bacterial abundance, Card-FISH and quali-, quantitative analysis of hydrocarbons (GC-FID) were carried out in order to elucidate the co-operative action of mixed microbial populations in the process of biodegradation of crude oil. All data obtained confirmed the fundamental role of bacteria belonging to Alcanivorax genus in the degradation of linear hydrocarbons in oil polluted environments.

Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium

Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. Single strains and four bacterial consortia designed by mixing the single bacterial cultures respectively in the following ratios: (Alcanivorax: Pseudomonas, 1:1), (Alcanivorax: Rhodococcus, 1:1), (Pseudomonas: Rhodococcus, 1:1), and (Alcanivorax: Pseudomonas: Rhodococcus, 1:1:1), were analyzed in order to evaluate their oil degrading capability. All experiments were carried out in microcosms systems containing seawater (with and without addition of inorganic nutrients) and crude oil (unique carbon source). Measures of total and live bacterial abundance, Card-FISH and quali-, quantitative analysis of hydrocarbons (GC-FID) were carried out in order to elucidate the co-operative action of mixed microbial populations in the process of biodegradation of crude oil. All data obtained confirmed the fundamental role of bacteria belonging to Alcanivorax genus in the degradation of linear hydrocarbons in oil polluted environments.

Transcriptome of the quorum-sensing signal-degrading Rhodococcus erythropolis responds differentially to virulent and avirulent Pectobacterium atrosepticum.

Social bacteria use chemical communication to coordinate and synchronize gene expression via the quorum-sensing (QS) regulatory pathway. In Pectobacterium, a causative agent of the blackleg and soft-rot diseases on potato plants and tubers, expression of the virulence factors is collectively controlled by the QS-signals N-acylhomoserine lactones (NAHLs). Several soil bacteria, such as the actinobacterium Rhodococcus erythropolis, are able to degrade NAHLs, hence quench the chemical communication and virulence of Pectobacterium. Here, next-generation sequencing was used to investigate structural and functional genomics of the NAHL-degrading R. erythropolis strain R138. The R. erythropolis R138 genome (6.7 Mbp) contained a single circular chromosome, one linear (250 kbp) and one circular (84 kbp) plasmid. Growth of R. erythropolis and P. atrosepticum was not altered in mixed-cultures as compared with monocultures on potato tuber slices. HiSeq-transcriptomics revealed that no R. erythropolis genes were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the avirulent P. atrosepticum mutant expI, which is defective for QS-signal synthesis. By contrast 50 genes (<1% of the R. erythropolis genome) were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the NAHL-producing virulent P. atrosepticum. Among them, quantitative real-time reverse-transcriptase-PCR confirmed that the expression of some alkyl-sulfatase genes decreased in the presence of a virulent P. atrosepticum, as well as deprivation of organic sulfur such as methionine, which is a key precursor in the synthesis of NAHL by P. atrosepticum.

Crystal structures of apo-DszC and FMN-bound DszC from Rhodococcus erythropolis D-1.

UNLABELLED: The release of SO2 from petroleum products derived from crude oil, which contains sulfur compounds such as dibenzothiophene (DBT), leads to air pollution. The '4S' metabolic pathway catalyzes the sequential conversion of DBT to 2-hydroxybiphenyl via three enzymes encoded by the dsz operon in several bacterial species. DszC (DBT monooxygenase), from Rhodococcus erythropolis D-1 is involved in the first two steps of the '4S' pathway. Here, we determined the first crystal structure of FMN-bound DszC, and found that two distinct conformations occur in the loop region (residues 131-142) adjacent to the active site. On the basis of the DszC-FMN structure and the previously reported apo structures of DszC homologs, the binding site for DBT and DBT sulfoxide is proposed. DATABASE: The atomic coordinates and structure factors for apo-DszC (PDB code: 3X0X) and DszC-FMN (PDB code: 3X0Y) have been deposited in the Protein Data Bank (http://www.rcsb.org).