Screening and identification of novel isolate Streptomyces sp., NLKPB45 from Nellore costal region for its biomedical applications.

Actinobacteria, which are the prolific producers of antibiotics and significant suppliers to the pharmaceutical industry, can produce a wide variety of bioactive metabolites. An actinomycete strain designated NLKPB45 was isolated from mangrove soils samples of Nellore coastal regions Andhra Pradesh and assessed for antibiotic production and activity against pathogenic bacteria. From a total of 9 mangrove soil samples, 143 acinomycetes were isolated. Among the isolated them 6 actinomycetes strains showed potential antibacterial activity against at two tested pathogens gram positive and gram negative bacteria E. coli and S. aureus. The potent strain NLKPB45 was identified by 16S gene isolation and sequencing to the Streptomyces genus. The ethyl acetate extracts also as shown excellent antimicrobial activity against Salmonella sp., staphylococcus aureus, E. coli, and B. subtilus were detected in both the supernatant extract samples from fermentations of culture NLKPB45. The anticancer activity of extracts in the HeLa with IC50 value of 37.1924 µg/ml, MCF-7 IC50 value of 40.9177 µg/ml and HT 29 IC50 value of 43.3758 µg/ml.

A NusG paralogue from Mycobacterium tuberculosis, Rv0639, has evolved to interact with ribosomal protein S10 (Rv0700) but not to function as a transcription elongation-termination factor.

NusG, a well-conserved protein in all the three forms of life, is involved in transcription elongation and termination, as well as in the process of transcription-translation coupling. The existence of species-specific functional, as well as conformational, divergences in NusG makes it an attractive transcription factor to study, especially if it originates from a pathogen. Here, we report functional and conformational characterizations of the Mycobacterium tuberculosis (Mtb) protein Rv0639 that has been annotated as a homologue of Escherichia coli NusG. Rv0639 failed to complement the in vivo functions of E. coli NusG (Ec NusG) and did not exhibit any signature of a transcription elongation-termination factor. However, it retained the ability to bind to its cognate ribosomal protein S10 (Rv0700). Compared with Ec NusG, Rv0639 possesses unique conformational features characterized by altered secondary structures in the C-terminal domain (CTD), an unusually long and disordered linker region between the N-terminal domain (NTD) and CTD, and a folding of its NTD over its CTD. This unusual folded conformation could have imparted specialized functions to this protein, required to adapt the physiology of Mtb. We speculate that in the absence of a bona fide RfaH, a NusG paralogue that is involved in pathogenicity in E. coli, Rv0639 functions as an RfaH-like factor and is involved in pathogenicity using unidentified ops-like sequences in the Mtb genome. And hence, we reannotate Rv0639 as a paralogue of NusG, instead of a homologue.

Interaction with the nascent RNA is a prerequisite for the recruitment of Rho to the transcription elongation complex in vitro.

In the conventional model of the Rho-dependent transcription termination, the terminator Rho binds to the rut (Rho utilization) site and translocates along the nascent RNA prior to making possible interactions with the elongating RNA polymerase (RNAP). Even though the interaction between Rho and isolated RNAs was studied in great detail, the same has never been shown with the nascent RNA emerging from the transcription elongation complex (EC). Direct demonstration and requirement of the Rho-nascent RNA binding become even more important because of the recently proposed alternative model where Rho loads onto the RNAP prior to the formation of the nascent RNA. Here, we have measured the direct association of Rho in vitro with the free RNAP, RNAP-promoter binary complex and stalled ECs with varied length of RNA. We observed the association of Rho only with the ECs having the rut-site-containing long nascent RNA. This association was significantly reduced when either a Rho mutant, Y80C, defective for RNA binding or an antisense oligo to the rut site was used or when the rut site was eliminated by RNase digestion or replacement with a random RNA sequence. The presence of EC-bound NusG, the binding partner of Rho, did not facilitate this association. RNase footprinting of the Rho-EC complex revealed a clear Rho-mediated protection of the rut sites on the nascent RNA. We concluded that the nascent RNA loading of Rho and its interaction with the rut site are mandatory and prerequisites for its recruitment to the EC under in vitro experimental conditions.

A bacterial transcription terminator with inefficient molecular motor action but with a robust transcription termination function.

Molecular motors such as helicases/translocases are capable of translocating along the single-stranded nucleic acids and unwinding DNA or RNA duplex substrates using the energy derived from their ATPase activity. The bacterial transcription terminator, Rho, is a hexameric helicase and releases RNA from the transcription elongation complexes by an unknown mechanism. It has been proposed, but not directly demonstrated, that kinetic energy obtained from its molecular motor action (helicase/translocase activities) is instrumental in dissociating the transcription elongation complex. Here we report a hexameric Rho analogue (Rv1297, M. tb. Rho) from Mycobacterium tuberculosis having poor RNA-dependent ATP hydrolysis and inefficient DNA-RNA unwinding activities. However, compared to Escherichia coli Rho, it exhibited very robust and earlier transcription termination from the elongation complexes of E. coli RNA polymerase. Bicyclomycin, an inhibitor of ATPase as well as RNA release activities of E. coli Rho, inhibited the ATPase activity of M. tb. Rho with comparable efficiency but was not efficient in inhibiting its transcription termination function. Unlike E. coli Rho, M. tb. Rho was capable of releasing RNA in the presence of nonhydrolyzable analogues of ATP quite efficiently. Also, this termination function most likely does not require NusG, an RNA-release facilitator, as this Rho was incapable of binding to NusG either of M. tb. (Rv0639) or E. coli. These results strongly suggest that the ATPase activity of M. tb. Rho is uncoupled from its transcription termination function and this function may not be dependent on its helicase/translocase activity.