Calcimycin is a unique ionophoric antibiotic that is widely used in biochemical and pharmaceutical applications, but the genetic basis underlying the regulatory mechanisms of calcimycin biosynthesis are unclear. Here, we identified the calR3 gene, which encodes a novel TetR family transcriptional regulator and exerts a negative effect on calcimycin biosynthesis. Disruption of calR3 in Streptomyces chartreusis NRRL 3882 led to significantly increased calcimycin and its intermediate cezomycin. Gene expression analysis showed that the transcription of calR3 and its adjacent calT gene were dramatically enhanced (30- and 171-fold, respectively) in GLX26 (ΔcalR3) mutants compared with the wild-type strains. Two CalR3-binding sites within the bidirectional calR3-calT promoter region were identified using a DNase I footprinting assay, indicating that CalR3 directly repressed the transcription of its own gene and the calT gene. In vitro electrophoretic mobility shift assays suggested that both calcimycin and cezomycin can act as CalR3 ligands to induce CalR3 to dissociate from its binding sites. These findings indicate negative feedback for the regulation of CalR3 in calcimycin biosynthesis and suggest that calcimycin production can be improved by manipulating its biosynthetic machinery.

In order to solve the defects of traditional coal spontaneous combustion prevention technology in a closed goaf, a strain of aerobic endogenous bacteria was isolated from coal and used as a blocking raw material. Based on the metabolic and reproductive characteristics of microorganisms, the experimental study on the inhibition of coal spontaneous combustion by microorganisms was carried out. The colonies were isolated and purified by the dilution concentration plate method and the scribing plate method. The growth morphology of microorganisms was analyzed, and the growth curve was determined. The strains were identified by seamless cloning technology for high-throughput sequencing. The surface morphology of coal was analyzed by SEM, the differences of oxidation characteristic temperature points were analyzed by TG–DTG–DSC images, a programmed heating experiment was used to analyze the concentration of the indicator gas CO, and the changes in microscopic groups before and after microbial action were analyzed by FTIR and XPS spectra. Therefore, the inhibition of coal oxidation by endogenous bacteria was verified from macroscopic and microscopic perspectives. The results show that the coal bacteria isolated from the coal is Lysinibacilus sp. After the culture of Lysinibacilus sp., the surface of the coal demonstrated less detritus, and was relatively smooth. In the early stage of low temperature oxidation of coal spontaneous combustion, the characteristic temperature point of coal oxidation and the reaction between coal and O₂ could be delayed by Lysinibacilus sp., and the total heat release was reduced in the combustion process. Not only that, Lysinibacilus sp. could also reduce the CO concentration during coal heating. After the coal was decomposed by Lysinibacilus sp., the C=C thick ring skeleton structure had little effect; however, the aromatic substitution pattern changed. This bacterium had an effect on the C-O bond, reducing the percentage of -CH₂- and increasing the percentage of -CH₃. It might also use the crystalline water in coal for life activities. The carboxyl carbon in coal changed the most, with a decrease of 12.03%, so it might become the carbon source required for microbial growth. The reproductive metabolism of microorganisms also affected the form of nitrogen, and the percentage of pyridine nitrogen in coal was reduced. The ratio of single-bond carbon to double-bond carbon in raw coal was about 3:2, but after this bacterial action, the ratio of the two was about 1:1. The analytical conclusions of XPS and FTIR spectra were consistent, and the results supported each other.

Toxin-antitoxin (TA) systems are the major mechanism for persister formation in Mycobacterium tuberculosis (Mtb). Previous studies found that HigBA2 (Rv2022c-Rv2021c), a predicted type II TA system of Mtb, could be activated for transcription in response to multiple stresses such as anti-tuberculosis drugs, nutrient starvation, endure hypoxia, acidic pH, etc. In this study, we determined the binding site of HigA2 (Rv2021c), which is located in the coding region of the upstream gene higB2 (Rv2022c), and the conserved recognition motif of HigA2 was characterized via oligonucleotide mutation. Eight binding sites of HigA2 were further found in the Mtb genome according to the conserved motif. RT-PCR showed that HigA2 can regulate the transcription level of all eight of these genes and three adjacent downstream genes. DNA pull-down experiments showed that twelve functional regulators sense external regulatory signals and may regulate the transcription of the HigBA2 system. Of these, Rv0903c, Rv0744c, Rv0474, Rv3124, Rv2603c, and Rv3583c may be involved in the regulation of external stress signals. In general, we identified the downstream target genes and possible upstream regulatory genes of HigA2, which paved the way for the illustration of the persistence establishment mechanism in Mtb.

Mycobacterium tuberculosis (Mtb), as a typical intracellular pathogen, possesses several putative restriction–modification (R-M) systems, which restrict exogenous DNA’s entry, such as bacterial phage infection. Here, we investigate Rv2528c, a putative Mrr-like type IV restriction endonuclease (REase) from Mtb H37Rv, which is predicted to degrade methylated DNA that contains m6A, m5C, etc. Rv2528c shows significant cytotoxicity after being expressed in Escherichia coli BL21(DE3)pLysS strain. The Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay indicates that Rv2528c cleaves genomic DNA in vivo. The plasmid transformation efficiency of BL21(DE3)pLysS strain harboring Rv2528c gene was obviously decreased after plasmids were in vitro methylated by commercial DNA methyltransferases such as M.EcoGII, M.HhaI, etc. These results are consistent with the characteristics of type IV REases. The in vitro DNA cleavage condition and the consensus cleavage/recognition site of Rv2528c still remain unclear, similar to that of most Mrr-family proteins. The possible reasons mentioned above and the potential role of Rv2528c for Mtb were discussed.