Iva Tomova

Diversity and biosynthetic potential of culturable aerobic heterotrophic bacteria isolated from Magura Cave, Bulgaria

Biocapacity of bacteria inhabiting karstic caves to produce valuable biologically active compounds is still slightly investigated. A total of 46 culturable heterotrophic bacteria were isolated under aerobic conditions from the Gallery with pre-historical drawings in Magura Cave, Bulgaria. Phylogenetic analysis revealed that most of bacterial isolates aff iliated with Proteobacteria (63%), followed by Actinobacteria (10.9%), Bacteroidetes (10.9%), and Firmicutes (6.5%). A strong domination of Gram-negative bacteria (total 81%) belonging to nine genera: Serratia, Pseudomonas, Enterobacter, Sphingobacterium, Stenotrophomonas, Commamonas, Acinetobacter, Obesumbacterium, and Myroides, was observed. Gram-positive isolates were represented by the genera Bacillus, Arthrobacter, and Micrococcus. One isolate showed a signif icant phylogenetic distance to the closest neighbor and could represent а novel species. Heterotrophic bacterial isolates from Magura Cave were investigated for hydrolytic enzymes production, antimicrobial and hemolytic activity. Predominance of producers of protease (87%), followed by xanthan lyase (64%), lipase (40%), β-glycosidase (40%), and phytase (21%) was observed. Over 75% of the isolates demonstrated antimicrobial and hemolytic activity. The results suggest that heterotrophic bacteria isolated from Magura Cave could be a valuable source of industrially relevant psychrotolerant enzymes and bioactive metabolites. This study is a f irst report on the taxonomic composition and biological activity of culturable bacteria inhabiting a cave in Bulgaria.

Antimicrobial activity and resistance to heavy metals and antibiotics of heterotrophic bacteria isolated from sediment and soil samples collected from two Antarctic islands

In this study, 24 Antarctic bacteria, isolated from sediment and soil samples from Deception and Galindez Islands, were characterized for their antimicrobial activity and response to 13 antibiotics and seven heavy metals. Multiple antibiotic resistance was observed for 67% of sediment isolates and 92% of soil isolates, suggesting medium anthropogenic impact in these Antarctic regions. The results revealed a varying response of the Antarctic bacteria to the tested heavy metals. All isolates showed multiple metal resistance towards two to six heavy metals, with minimum inhibitory concentrations ranging from 1.0 mM to 23.2 mM. The majority of the strains in both groups were resistant to lead, nickel, copper and zinc. Plasmids were detected in 21% of isolates. A consortium of highly metal-resistant bacteria could be developed with potential application for biological treatment of wastewaters. Strains that are highly sensitive to cadmium would be promising for developing biosensors to detect this highly toxic heavy metal in environmental samples. All Antarctic bacteria were found to inhibit the growth of one to all eight of the tested indicator bacteria, and 87% inhibited the growth of one to all four of the yeast indicator cultures. Promising psychrotolerant strains were detected as a valuable source of new antimicrobial compounds.

Myroides guanonis sp. nov., isolated from prehistoric paintings

A novel psychrotolerant, strictly aerobic, non-motile, rod-shaped bacterial strain, designated IM13(T), was isolated from a sample taken from prehistoric guano paintings in Magura Cave, northwest Bulgaria and subjected to a polyphasic taxonomic study. Strain IM13(T) formed yellow colonies on LB agar plates and was Gram-staining-negative, heterotrophic and alkalitolerant. It grew optimally at pH 7.5 and 30 °C in the absence of NaCl. Phylogenetic analysis of the whole 16S rRNA gene revealed that strain IM13(T) branched with representatives of the genus Myroides with sequence similarity of 93-94 % with other species of the genus. The novel isolate contained iso-C15 : 0 (49.1 %), iso-C17 : 1ω9c (18.2 %) and iso-C17 : 0 3-OH (14.0 %) as dominant fatty acids. The DNA G+C content of strain IM13(T) was 33.5 mol%. Based on phylogenetic inference and phenotypic characteristics, it was concluded that strain IM13(T) represents a novel species of the genus Myroides, for which the name Myroides guanonis sp. nov. is proposed. The type strain is IM13(T) ( = DSM 26542(T) = NBIMCC 8736(T)).

Archaeal Diversity at Two Hot Springs, Rupi Basin, Bulgaria

ABSTRACT Archaea is the newest and the least understood domain which diversity and physiology are predominantly suggested after investigation of natural ecosystems. Recently many new archaeal groups have been detected by molecular phylogenetic methods. Diversity of thermophilic archaea in two hot springs at Rupi basin was studied by a culture-independent approach and high degree of covering biological diversity was reached in both clone libraries (RB and RB2). The 16S rRNA gene analysis of 379 clones grouped in 33 restriction profiles for each library showed presence of eleven groups from the domain Archaea, five of them being common for both springs. The retrieved sequences grouped together predominantly with environmental sequences and a small number of already known culturable Archaea was detected. A strong domination of HWCG III (Hot Water Crenarcheotic Group) was observed in both springs revealing strong domination of ammonia oxidizing metabolism. Although the difference in temperature between the two springs was only seven degrees, presence of hyperthermophiles was registered in the warmer spring. Four of the RB and nine of the RB2 sequences showed less than 97% similarity to the closest neighbour and were referred as representing new phylogenetic units.

Unusually High Archaeal Diversity in a Crystallizer Pond, Pomorie Salterns, Bulgaria, Revealed by Phylogenetic Analysis

Recent studies on archaeal diversity in few salterns have revealed heterogeneity between sites and unique structures of separate places that hinder drawing of generalized conclusions. Investigations on the archaeal community composition in P18, the biggest crystallizer pond in Pomorie salterns (PS) (34% salinity), demonstrated unusually high number of presented taxa in hypersaline environment. Archaeal clones were grouped in 26 different operational taxonomic units (OTUs) assigned to 15 different genera from two orders, Halobacteriales and Haloferacales. All retrieved sequences were related to culturable halophiles or unculturable clones from saline (mostly hypersaline) niches. New sequences represented 53.9% of archaeal OTUs. Some of them formed separate branches with 90% similarity to the closest neighbor. Present results significantly differed from the previous investigations in regard to the number of presented genera, the domination of some genera not reported before in such extreme niche, and the identification of previously undiscovered 16S rRNA sequences.

Molecular Characterization of the Archaeal Diversity in Vlasa Hot Spring, Bulgaria, by using 16S rRNA and Glycoside Hydrolase Family 4 Genes

ABSTRACT Biodiversity in the archaeal community from Vlasa hot spring, Velingrad, Bulgaria was investigated by sequence analysis of PCR amplified fragments of 16S rDNA and a metabolic gene of glycoside hydrolase 4 family (GH4). The 16S rRNA gene analysis demonstrated that the spring was inhabited predominantly by Crenarchaeota affiliated to two orders, Desulfurococcales (families Pyrodictiaceae and Desulfurococcaceae) and Thermoproteales. Almost half of the 16S rDNA clones were affiliated with hyperthermophilic anaerobic sulfate reducer Thermosphaera aggregans. Five 16S rDNA sequences were under cut off value of 97% homology to those of Genbank database and suggested the existence of novel phylogenetic units in the community. The archaeal diversity of the studied hot spring was further analyzed through sequence analysis of metagenomicaly cloned GH4 gene fragments. The comparative 16SrDNA and GH4 phylogenetic analyses demonstrated good correlation of the phylogenetic tree topology from both approaches, corresponding to the affiliation of the identified 16S rDNA sequences predominantly to organotrophic metabolizing taxons. The opportunity for simultaneous application of two molecular approaches, 16S rDNA and metabolite genes analyses for in dept characterization of environmental samples and directed metagenomic identification and cloning of metabolite genes of industrial interest is discussed.

HIGH PHYLOGENETIC DIVERSITY OF BACTERIA IN THE AREA OF PREHISTORIC PAINTINGS IN MAGURA CAVE, BULGARIA

Magura Cave, situated in the northwest of Bulgaria and containing prehistoric paintings, is a famous tourist site. The present study is the first report on bacterial diversity in guano paintings in a Bulgarian cave using molecular methods. We identified 68 taxa, which is an unusually high number for oligotrophic niches. They are affiliated with eight phyla representing more than half of the bacterial divisions identified in caves. As in many other caves, Proteobacteria dominated in this type of ecosystem (about 40%), followed by Nitrospirae (22.5%) and Acidobacteria (21.5%). Weakly represented were Actinobacteria (6.4%), Chloroflexi (3.2%), Planctomycetes (2.2%), Firmicutes (2.2%), and Gemmatimonadetes (2.2%). About one third of all DNA sequences recovered in this study were new. Some of them had more than 10% divergence from the closest neighbor, which suggests the existence of new taxa of at least genus level. Bacteria identified in the community expressed various types of metabolism; lithoautotrophic, organotrophic, and methylotrophic.

Thermophiles as a Promising Source of Exopolysaccharides with Interesting Properties

Thermophilic niches offer diversity of prokaryotic microorganisms enormous part of which is still unknown. Thermophiles represent a vast natural resource of various bioactive compounds, among which exopolysaccharides (EPSs) attracted major interest and attention in last two decades due to the significant variety in their structural and functional properties despite of the limited knowledge in the field. Thermophilic EPS producers were isolated from both, Bacteria (Thermotoga, Thermus, Bacillus Geobacillus, Brevibacillus, Aeribacillus) and Archaea (Thermococcus, Sulfolobus) domains. Advantages in using thermophilic processes for EPS production and potential use of the synthesized EPSs are discussed. Their biotechnologically interesting properties, like high molecular weight, stability of their molecules, good synergism with other hydrocolloids, biological activity against cytotoxic compounds, antiviral and immunostimulating activities determine their possible future applications.

Phylogenetic analysis of the bacterial community in a crystallizer pond, Pomorie salterns, Bulgaria

ABSTRACT The aim of this study was to investigate the bacterial community habituating P18, the biggest crystallizer pond in Pomorie salterns (34% salinity). The obtained results showed that the bacterial community differs from many previous reports of low bacterial diversity in hypersaline environments and demonstrates unusually high diversity of presented taxa, some unusual domination of diverse genera not reported before as dominant and identification of previously unknown 16S rRNA sequences. The retrieved 23 bacterial operational taxonomic units (OTUs) affiliated with 15 bacterial genera from four phyla – Firmicutes, 47.5%; Proteobacteria, 23.1%; Bacteroidetes, 22%; Deinococcus–Thermus, 2.4%; and one-candidate division SR1, 4.8%. Representatives of the phylum Firmicutes predominated in the bacterial community with almost half of the retrieved sequences. Almost all clones branched together with cultured halophiles or uncultured clones retrieved from saline niches. Despite of the high salt concentration, some of the closest phylogenetic neighbours were moderate halophiles. New sequences represented 42.3% of bacterial OTUs. Some of them formed separate branches with similarity less than 85%.

Diversity of Heterotrophic Halophilic Bacteria Isolated from Coastal Solar Salterns, Bulgaria and Their Ability to Synthesize Bioactive Molecules with Biotechnological Impact

Investigations on the microbial life in several coastal solar salterns have revealed the presence of novel organisms and synthesis of unusual molecules active in extreme conditions which might be useful in different biotechnological industries. Biodiversity of heterotrophic aerobic bacteria isolated from two salterns, Pomorie salterns and Burgas salterns located at Burgas Bay, Black Sea coast, Bulgaria, as well as ability of the isolates to synthesize biotechnologically valuable compounds were investigated. The results revealed high taxonomic and metabolic bacterial diversity—we isolated 20 morphologically different moderately halophilic and two halotolerant strains affiliated with 11 species from eight genera referred to the phyla Proteobacteria, Firmicutes, and Actinobacteria. Gram-negative bacteria belonged to the genera Halomonas, Chromohalobacter, Salinivibrio, Cobetia, and Nesiotobacter, and gram-positive strains were representatives of the genera Virgibacillus, Salinicoccus, and Brevibacterium. All isolates were found to be alkalitolerant, and 41% of them were psychrotolerant. The strains degraded nine of the tested 18 substrates; polygalacturonase, catalase, phytase, and lipase producers were predominant. This is the first reported detection of xanthan lyase, gellan lyase, arabinase, and phytase activities in halophilic bacteria. Nine of the strains belonging to five different genera were found to produce exopolysaccharides (EPS). The highest level of EPS was observed in Chromohalobacter canadensis strain 28. More than a half of the strains displayed antimicrobial activity against one to five test bacteria and yeasts. The present study is the first report on halophilic bacteria isolated from salterns at the Black Sea coast indicating that the investigated area is an untapped resource of halophilic bacteria with biotechnological potential.