Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m-and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3-and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.
Soil is an important environmental reservoir of antibiotic resistance genes (ARGs), which are increasingly recognized as environmental contaminants. Methods to assess the risks associated with the acquisition or transfer of resistance mechanisms are still underdeveloped. Quantification of background levels of antibiotic resistance genes and what alters those is a first step in understanding our environmental resistome. Toward this goal, 62 samples were collected over 3 years from soils near the 30-year old Gondwana Research Station and for 4 years before and during development of the new Jang Bogo Research Station, both at Terra Nova Bay in Antarctica. These sites reflect limited and more extensive human impact, respectively. A qPCR array with 384 primer sets targeting antibiotic resistance genes and mobile genetic elements (MGEs) was used to detect and quantify these genes. A total of 73 ARGs and MGEs encompassing eight major antibiotic resistance gene categories were detected, but most at very low levels. Antarctic soil appeared to be a common reservoir for seven ARGs since they were present in most samples (42%-88%). If the seven widespread genes were removed, there was a correlation between the relative abundance of MGEs and ARGs, more typical of contaminated sites. There was a relationship between ARG content and distance from both research stations, with a significant effect at the Jang Bogo Station especially when excluding the seven widespread genes; however, the relative abundance of ARGs did not increase over the 4 year period. Silt, clay, total organic carbon, and SiO were the top edaphic factors that correlated with ARG abundance. Overall, this study identifies that human activity and certain soil characteristics correlate with antibiotic resistance genes in these oligotrophic Antarctic soils and provides a baseline of ARGs and MGEs for future comparisons.
Alkylbenzene-degrading Rhodococcus sp. strain DK17 is able to utilize phthalate and terephthalate as growth substrates. The genes encoding the transformation of phthalate and terephthalate to protocatechuate are organized as two separate operons, located 6.7kb away from each other. Interestingly, both the phthalate and terephthalate operons are induced in response to terephthalate while expression of the terephthalate genes is undetectable in phthalate-grown cells. In addition to two known plasmids (380-kb pDK1 and 330-kb pDK2), a third megaplasmid (750-kb pDK3) was newly identified in DK17. The phthalate and terephthalate operons are duplicated and are present on both pDK2 and pDK3. RT-PCR experiments, coupled with sequence analysis, suggest that phthalate and terephthalate degradation in DK17 proceeds through oxygenation at carbons 3 and 4 and at carbons 1 and 2 to form 3,4-dihydro-3,4-dihydroxyphthalate and 1,2-dihydro-1,2-dihydroxyterephthalate, respectively. The 3,4-dihydroxyphthalate pathway was further corroborated through colorometric tests. Apparently, the two dihydrodiol metabolites are subsequently dehydrogenated and decarboxylated to form protocatechuate, which is further degraded by a protocatechuate 3,4-dioxygenase as confirmed by a ring-cleavage enzyme assay.
The genus Rhodococcus is a phylogenetically and catabolically diverse group that has been isolated from diverse environments, including polar and alpine regions, for its versatile ability to degrade a wide variety of natural and synthetic organic compounds. Their metabolic capacity and diversity result from their diverse catabolic genes, which are believed to be obtained through frequent recombination events mediated by large catabolic plasmids. Many rhodococci have been used commercially for the biodegradation of environmental pollutants and for the biocatalytic production of high-value chemicals from low-value materials. Recent studies of their physiology, metabolism, and genome have broadened our knowledge regarding the diverse biotechnological applications that exploit their catabolic enzymes and pathways.
This study presents taxonomic description of two novel diesel-degrading, psychrophilic strains: Kopri-42T and Kopri-43, isolated during screening of oil-degrading psychrotrophs from oil-contaminated Arctic soil. A preliminary 16S rRNA gene sequence and phylogenetic tree analysis indicated that these Arctic strains belonged to the genus Flavobacterium, with the nearest relative being Flavobacterium psychrolimnae LMG 22018T (98.9% sequence similarity). The pairwise 16S rRNA gene sequence identity between strains Kopri-42T and Kopri-43 was 99.7%. The DNA-DNA hybridization value between strain Kopri-42T and Kopri-43 was 88.6 ± 2.1% indicating that Kopri-42T and Kopri-43 represents two strains of the same genomospecies. The average nucleotide identity and in silico DNA-DNA hybridization values between strain Kopri-42T and nearest relative F. psychrolimnae LMG 22018T were 92.4% and 47.9%, respectively. These values support the authenticity of the novel species and confirmed the strain Kopri-42T belonged to the genus Flavobacterium as a new member. The morphological, physiological, biochemical and chemotaxonomic data also distinguished strain Kopri-42T from its closest phylogenetic neighbors. Based on the polyphasic data, strains Kopri-42T and Kopri-43 represents a single novel species of the genus Flavobacterium, for which the name Flavobacterium petrolei sp. nov. is proposed. The type strain is Kopri-42T (=KEMB 9005-710T = KACC 19625T = NBRC 113374T).
A bacterium with lipolytic activity was isolated from the Chukchi Sea within the Arctic Ocean. The lipase BpL5 from the isolate, Bacillus pumilus ArcL5, belongs to subfamily 4 of lipase family I. The optimum pH and temperature of the recombinant enzyme BpL5, as expressed in Escherichia coli, were 9.0 and 20 °C, respectively. The enzyme retained 85 % of its activity at 5 °C. There was a significant difference between temperatures for maximal activity (20 °C) and for protein denaturation (approx. 45 °C). The enzyme preferred middle-chain (C8) p-nitrophenyl substrates. Two mutants, S139A and S139Y, were rationally designed based on the 3D-structure model, and their activities were compared with that of the wild type. The both mutants showed significantly improved activity against tricaprylin.
Chromohalobacter sp. strain HS-2 was isolated from salted fermented clams and analyzed for the ability to grow on benzoate and p-hydroxybenzoate as the sole carbon and energy source. HS-2 was characterized as moderately halophilic, with an optimal NaCl concentration of 10%. The genes encoding the benzoate metabolism were cloned into a cosmid vector, sequenced, and then analyzed to reveal the benzoate (benABCD) and catechol (catBCA) catabolic genes, both of which are flanked on either side by LysR-type transcriptional regulator (catR) and membrane transport protein for benzoate (benE) in the gene order catRBCAbenABCDE. Near the large cat-ben cluster, a p-hydroxybenzoate hydroxylase gene (pobA) and two putative regulatory genes (pcaQ and pobR) were additionally detected. The HS-2 genes involved in benzoate and p-hydroxybenzoate degradation are tightly clustered within a c. 19 kb region, and show quite a different genetic organization from those of other benzoate catabolic genes. Reverse transcriptase-PCR experiments show that benzoate induces the expression of benzoate 1,2-dioxygenase, catechol 1,2-dioxygenase, and protocatechuate 3,4-dioxygenase while p-hydroxybenzoate only induced the expression of p-hydroxybenzoate hydroxylase. When expressed in Escherichia coli, benzoate 1,2-dioxygenase (BenABC) and p-hydroxybenzoate hydroxylase (PobA) transformed benzoate and p-hydroxybenzoate into cis-benzoate dihydrodiol and protocatechuate, respectively.
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