In the present study we aimed to perform the first analysis of antimicrobial activity of bacteria isolated from Krubera-Voronja Cave, with the main focus on their activity against Grampositive bacteria, including Gram-positive pathogens. Using five different media, in total 874 heterotrophic cultures were isolated from water and sediment samples collected in Krubera-Voronja Cave at a depth from 220 m to 1640 m. 14.0% of all isolates demonstrated antibacterial activity against Gram-positive and Gram-negative test microorganisms. Our results show that this percentage was not uniform; it increased with the sampling depth and was the highest in the lower part of the cave. 24 isolates were active exclusively against Gram-positive test strains Micrococcus luteus and Bacillus thuringiensis. Two isolates, namely strains 1350R2-TSA30-6 and 1410WF1-TSA30-2, were chosen for the further work because of the high and comparable activity against both Gram-positive test microorganisms. It was determined that both strains belong to the family Bacillaceae in phylum Firmicutes. The detailed bioactivity analysis of these two Gram-positive strains revealed the different mixtures of volatile compounds with antibacterial activity. The main antibacterial compounds of the strain 1350R2-TSA30-6 are pyrrolopyrazines pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-and pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)-. The main antibacterial compound of the strain 1410WF1-TSA30-2 is 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester. Mixtures of the volatile antimicrobial compounds of both strains were antagonistic against Gram-positive strains isolated from Krubera-Voronja Cave, and their activity against Gram-positive pathogenic bacteria substantially differed. antimicrobial, Bacillus, cave microorganisms, Krubera-Voronja Cave, volatile compound

Identification of novel bioactive compounds represents an important field in modern biomedical research. Microorganisms of the underexplored environments, such as deserts, hot springs, oceans, and caves are highly promising candidates for screening such metabolites. Screening for biosynthetic genes is the most effective strategy to characterize bioactivity in a certain environment. However, knowledge is either scant or non-existent about the expression of the biosynthetic genes encoding for various bioactive compounds in the microorganisms from the caves. The aim of the current study was to screen for the genes of polyketide synthases and non-ribosomal peptide synthetases in Krubera–Voronja Cave (43.4184 N 40.3083 E, Western Caucasus) bacterial isolates as well as to evaluate the expression of these genes under laboratory conditions. In total, 91 bacterial strains isolated from the cave were screened for the presence of polyketide synthase and non-ribosomal peptide synthetase genes. Phenotypically inactive strains were the main focus (the test group) of our study, while the strains with the identified antibacterial activity served as the control group. Our PCR-based screening clearly showed that the majority of the strains harbored at least one biosynthetic gene. Prediction of the putative products allowed us to identify bioactive compounds with antibacterial, anticancer, antifungal, anti-inflammatory, antimycoplasmic, antiviral, insecticidal, and thrombolytic activity. For most polyketide synthases and non-ribosomal peptide synthetases, putative products could not be predicted; they are unknown. Qualitative transcriptional analysis did not show substantial differences between the test group and the control group of the strains. One to four biosynthetic genes were constitutively expressed in all the tested strains, irrespective of the group. Quantitative transcriptional analysis of the constitutively expressed biosynthetic genes demonstrated that the expression of a particular gene could be affected by both the amount of the nutrients in the culture medium and the growth phase.