New cblA gene participates in regulation of cobalt-dependent transcription of nitrile hydratase genes in Rhodococcus rhodochrous

Lavrov, K. V.; Shemyakina, A. O.; Grechishnikova, Elena G.; Novikov, A. D.; Derbikov, D. D.; Kalinina, Tatyana I; Yanenko, A. S.

doi:10.1016/j.resmic.2018.03.006

Cited by 14 publications

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“…In view of the high proportion of the NHase protein, it was interesting to characterize the level of NHase mRNA in the cells of the M33 strain. We used the results of the RNA-seq approach to estimate relative levels of transcripts of most genes of the M33 strain during growth on minimal medium with cobalt, an inducer of NHase transcription. , The reads per kilobase per million (RPKMs) (see Table S1) are presented in Figure C as a pie-diagram of RPKMs of particular transcripts.…”

Section: Resultsmentioning

confidence: 99%

“…The R. rhodochrous M33 strain was chosen because of its ability to superproduce intracellular enzymes without the use of heterologous genetic parts such as the T7 phage expression system . This ability was demonstrated earlier using M33 (nitrile hydratase) , and its derivative strains (acylamidase, nitrilase, and amidase). In addition, these strains are involved in one of the two above-mentioned fields of biotechnological applications of Rhodococcus , namely the biocatalytic production of acrylic monomers. ,,,, In this work we focused on both the further characterization of the strain and establishing the functionality of a set of promoters in it.…”

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confidence: 99%

“…A set of promoters selected for this study included P tuf ( t hermo u nstable f actor of translation elongation, also reported as P eftu ) and P sod ( s uper o xide d ismutase) from Corynebacterium glutamicum ATCC13032, P cpi ( c onstitutive p romoter region of i socitrate lyase promoter) from R. erythropolis PR4, and P nh ( n itrile h ydratase) from R. rhodochrous M8 . The choice of promoters (more correctly, DNA fragments with promoter activity) was based on several simple criteria.…”

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

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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 P tuf (elongation factor Tu) and P sod (superoxide dismutase) from Corynebacterium glutamicum ATCC13032, P cpi (isocitrate lyase) from Rhodococcus erythropolis PR4, and P nh (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 (P cpi < P sod ≪ P tuf < P nh ), covering 10 3 -fold range, and the most active P nh and P tuf 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 P nh 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, P tuf could not be regulated (2-fold change), while others were tunable (6-fold for P sod , 79-fold for P nh , and 44-fold for P cpi ). 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. P tuf and P cpi did not change their activity profile under different growth conditions, whereas the P sod and P nh 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.

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

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“…4b, in the front of R. rhodochrous M8 NHase encoding gene nhmB and nhmA , two comparable regulators, NhmC and NhmD, appeared and a downstream regulator CblA acting as repressor was indispensable for cobalt‐dependent regulation of transcription independently in R. rhodochrous M8 NHase (Lavrov et al . 2018). However, for E. adhaerens TMX‐23, the expression of Nhase encoding genes could not be induced by cobalt, and the comparable cobalt‐induced regulator CblA not occurred in the downstream of nhcA and nhpA .…”

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Copper stimulates neonicotinoid insecticide thiacloprid degradation by Ensifer adhaerens TMX‐23

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Aims Aims of this study are to elucidate the molecular mechanism of copper‐improved thiacloprid (THI) degradation by Ensifer adhaerens TMX‐23 and characterize copper resistance of this strain. Methods and Results Resting cells of E. adhaerens TMX‐23 were used to degrade THI, with formation of THI amide and 98·31% of 0·59 mmol l−1 THI was degraded in 100 min. The addition of copper improved the degradation of THI and showed little inhibitory effects on the growth of E. adhaerens TMX‐23. E. adhaerens TMX‐23 degraded THI to THI amide by nitrile hydratases (NhcA and NhpA). QPCR analysis indicated that the expression of nhpA was up‐regulated in the presence of copper. E. adhaerens TMX‐23 nitrile hydratases were purified, and enzyme assay of NhpA exhibited the highest NHase activity toward THI. The addition of copper activated the activity of NhcA. Soil degradation experiment indicated that E. adhaerens TMX‐23 could quickly eliminate THI residual in copper‐added soil. Conclusions Copper improved THI degradation by E. adhaerens TMX‐23 was attributed to the induced expression of nhpA and activated NhcA. Significance and Impact of the Study This study broadens the investigation of regulatory mechanism of NHase expression and provided theoretical basis for using metal‐resistant microbes to degrade pesticide in heavy metal co‐contaminated environments.

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“…The effort to close this genome was undertaken because the strain is actually a platform for the development of biocatalysts for acrylic monomer production (several biocatalysts were derived from it earlier; see references 2-4 and also see the information about biocatalytic production of acrylic monomers [5,6]). Additionally, the strain is one of the model strains for basic research on the genetics of rhodococci (1,(7)(8)(9). Further basic and applied research based on the strain requires precise genome information.…”

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Complete Genome Sequence of Rhodococcus sp. Strain M8, a Platform Strain for Acrylic Monomer Production

Novikov

Lavrov

Kasianov

et al. 2021

Microbiol Resour Announc

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New cblA gene participates in regulation of cobalt-dependent transcription of nitrile hydratase genes in Rhodococcus rhodochrous

Cited by 14 publications

References 41 publications

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

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

Copper stimulates neonicotinoid insecticide thiacloprid degradation by Ensifer adhaerens TMX‐23

Complete Genome Sequence of Rhodococcus sp. Strain M8, a Platform Strain for Acrylic Monomer Production

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