This study gives the first picture of whole RNA-Sequencing analysis of a PCB-degrading microbe, Rhodococcus jostii RHA1. Genes that were highly expressed in biphenyl-grown cells, compared with pyruvate-grown cells, were chosen based on the Reads Per Kilobase Million (RPKM) value and were summarized based on the criteria of RPKM ≥100 and fold change ≥2.0. Consequently, 266 total genes were identified as genes expressed particularly for the degradation of biphenyl. After comparison with previous microarray data that identified highly-expressed genes, based on a fold change ≥2.0 and p-value ≤0.05, 62 highly-expressed genes from biphenyl-grown cells were determined from both analytical platforms. As these 62 genes involve known PCB degradation genes, such as bph, etb, and ebd, the genes identified in this study can be considered as essential genes for PCB/biphenyl degradation. In the 62 genes, eleven genes encoding hypothetical proteins were highly expressed in the biphenyl-grown cells. Meanwhile, we identified several highly-expressed unannotated DNA regions on the opposite strand. In order to verify the encoded proteins, two regions were cloned into an expression vector. A protein was successfully obtained from one region at approximately 25 kDa from the unannotated strand. Thus, the genome sequence with transcriptomic analysis gives new insight, considering re-annotation of the genome of R. jostii RHA1, and provides a clearer picture of PCB/biphenyl degradation in this strain.
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.
Study of the potential of Antarctic microorganisms for use in bioremediation is of increasing interest due to their adaptations to harsh environmental conditions and their metabolic potential in removing a wide variety of organic pollutants at low temperature. In this study, the psychrotolerant bacterium Rhodococcus sp. strain AQ5-07, originally isolated from soil from King George Island (South Shetland Islands, maritime Antarctic), was found to be capable of utilizing phenol as sole carbon and energy source. The bacterium achieved 92.91% degradation of 0.5 g/L phenol under conditions predicted by response surface methodology (RSM) within 84 h at 14.8 °C, pH 7.05, and 0.41 g/L ammonium sulphate. The assembled draft genome sequence (6.75 Mbp) of strain AQ5-07 was obtained through whole genome sequencing (WGS) using the Illumina Hiseq platform. The genome analysis identified a complete gene cluster containing catA, catB, catC, catR, pheR, pheA2, and pheA1. The genome harbours the complete enzyme systems required for phenol and catechol degradation while suggesting phenol degradation occurs via the β-ketoadipate pathway. Enzymatic assay using cell-free crude extract revealed catechol 1,2-dioxygenase activity while no catechol 2,3-dioxygenase activity was detected, supporting this suggestion. The genomic sequence data provide information on gene candidates responsible for phenol and catechol degradation by indigenous Antarctic bacteria and contribute to knowledge of microbial aromatic metabolism and genetic biodiversity in Antarctica.
Poly-β-hydroxybutyrate (PHB) is a biodegradable polymer, synthesized as carbon and energy reserve by bacteria and archaea. To the best of our knowledge, this is the first report on PHB production by a rare actinomycete species, Rhodococcus pyridinivorans BSRT1-1. Response surface methodology (RSM) employing central composite design, was applied to enhance PHB production in a flask scale. A maximum yield of 3.6 ± 0.5 g/L in biomass and 43.1 ± 0.5 wt% of dry cell weight (DCW) of PHB were obtained when using RSM optimized medium, which was improved the production of biomass and PHB content by 2.5 and 2.3-fold, respectively. The optimized medium was applied to upscale PHB production in a 10 L stirred-tank bioreactor, maximum biomass of 5.2 ± 0.5 g/L, and PHB content of 46.8 ± 2 wt% DCW were achieved. Furthermore, the FTIR and 1H NMR results confirmed the polymer as PHB. DSC and TGA analysis results revealed the melting, glass transition, and thermal decomposition temperature of 171.8, 4.03, and 288 °C, respectively. In conclusion, RSM can be a promising technique to improve PHB production by a newly isolated strain of R. pyridinivorans BSRT1-1 and the properties of produced PHB possessed similar properties compared to commercial PHB.
In this work, we report on the isolation of a phenol-degrading Rhodococcus sp. with a high tolerance towards phenol. The isolate was identified as Rhodococcus sp. strain AQ5NOL 2, based on 16S rDNA analysis. The strain degraded phenol using the meta pathway, a trait shared by many phenol-degraders. In addition to phenol biodegradation, the strain was also capable of degrading diesel. Strain AQ5NOL 2 exhibited a broad optimum temperature for growth on phenol at between 20 °C and 35 °C. The best nitrogen sources were ammonium sulphate, glycine or phenylalanine, followed by proline, nitrate, leucine, and alanine (in decreasing efficiency). Strain AQ5NOL 2 showed a high tolerance and degradation capacity of phenol, for it was able to register growth in the presence of 2000 mg l(-1) phenol. The growth of this strain on phenol as sole carbon and energy source were modeled using Haldane kinetics with a maximal specific growth rate (μ(max)) of 0.1102 hr(-1), a half-saturation constant (K(s) ) of 99.03 mg l(-1) or 1.05 mmol l(-1), and a substrate inhibition constant (K(i)) of 354 mg l(-1) or 3.76 mmol l(-1). Aside from phenol, the strain could utilize diesel, 2,4-dinitrophenol and ρ-cresol as carbon sources for growth. Strain AQ5NOL 2 exhibited inhibition of phenol degradation by Zn(2+), Cu(2+), Cr(6+), Ag(+) and Hg(2+) at 1 mg l(-1).
Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.