svetlana boycheva - Academia.edu (original) (raw)

Papers by svetlana boycheva

Scripta Scientifica Pharmaceutica, Aug 22, 2017

The advent of metagenomic approaches revolutionized our understanding of the role of gut microbia... more The advent of metagenomic approaches revolutionized our understanding of the role of gut microbial communities and their interactions with the host. However, due to the dominant perception that the majority of bacteria are unculturable, their functional and phenotypic characteristics remain unknown. To address this problem, we developed a novel platform for high-throughput specific phenotypic selection of environmental microbial strains and communities. We collected samples from gastrointestinal tract of the herbivorous fish Kyphosus sydneyanus, which is a hindgut fermenter. Applying the two main approaches, 16S ribosomal RNA gene amplicons and shotgun metagenomics, we profiled the taxonomic composition and functional diversity of the microbial species and compared the ones presented in the original samples to those that grew in the lab. A total of 96 strains were isolated, representing 52 novel species from 10 families belonging to the phyla Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria In addition, of the 52 species cultured, 47 were strict anaerobes. The metabolic analysis also revealed that the variance in dietary algae, may influence the gut microbiota, due to the difference in nutritional composition between algal groups. In conclusion, our approach enables not only the large-scale culturing and phenotyping of novel microbial species but also unlocks the fish intestinal microbiota for metabolic capabilities. The potential application of seaweed degrading microbes for fishmeal protein production will be discussed.Acknowledgements: New Zealand Government Fundin

Molecular Ecology, 2022

Many marine herbivorous fishes harbor diverse microbial communities in the hindgut that can play ... more Many marine herbivorous fishes harbor diverse microbial communities in the hindgut that can play important roles in host health and nutrition. Kyphosus sydneyanus is a temperate marine herbivorous fish that feeds predominantly on brown seaweeds. We employed 16S rRNA gene amplicon sequencing and gas chromatography to characterize microbial communities and their metabolites in different hindgut regions of six K. sydneyanus. Measurements were confined to three distal sections of the intestine, labelled III, IV and V from anterior to posterior. A total of 625 operational taxonomic units from 20 phyla and 123 genera were obtained. Bacteroidota, Firmicutes and Proteobacteria were the major phyla in mean relative abundance, which varied along the gut. Firmicutes (76 %) was the most dominant group in section III, whereas Bacteroidota (69.3%) dominated section V. Total short-chain fatty acid (SCFA) concentration was highest in sections IV and V, confirming active fermentation in these two most distal sections. The abundance of Bacteroidota correlated with propionate concentration in section V, while Firmicutes positively correlated with formate in sections III and IV. Acetate levels were highest in sections IV and V, which correlated with abundance of Bacteroidota. Despite differences in gut microbial community composition, SCFA profiles were consistent between individual fish in the different hindgut regions of K. sydneyanus, although proportions of SCFAs differed among gut sections. These findings demonstrate functional compartmentalization of the hindgut microbial community, highlighting the need for regional sampling when interpreting overall microbiome function. These results support previous work suggesting that hindgut microbiota in marine herbivorous fish are important to nutrition in some host species by converting dietary carbohydrates into metabolically useful SCFAs.

Journal of Microbiological Methods, 2015

Environmental isolates belonging to the genus Acidovorax play a crucial role in degrading a wide ... more Environmental isolates belonging to the genus Acidovorax play a crucial role in degrading a wide range of pollutants. Studies on Acidovorax are currently limited for many species due to the lack of genetic tools. Here, we described the use of the replicon from a small, cryptic plasmid indigenous to Acidovorx temperans strain CB2, to generate stably maintained shuttle vectors. In addition, we have developed a scarless gene knockout technique, as well as establishing green fluorescent protein (GFP) reporter and complementation systems. Taken collectively, these tools will improve genetic manipulations in the genus Acidovorax.

Motivation: The effect of two neighboring codons (codon pairs) on gene expression is mediated via... more Motivation: The effect of two neighboring codons (codon pairs) on gene expression is mediated via the interaction of their cognate tRNAs occupying the two functional ribosomal sites during the translation elongation step. For steric reasons it is reasonable to assume that not all combinations of codons and therefore of tRNAs are equally favorable when situated on the ribosome surface. Aiming of identifying preferential and rare codon pairs, we have determined the frequency of occurrence of all possible combinations of codon pairs in the entire genome of Escherichia coli (E.coli). Results: The frequency of occurrence of the 3904 codon pairs comprising both sense:sense and sense:stop codon pairs in the full set of E.coli 4289 ORFs was found to vary from zero to 4913 times. For most of the pairs we have observed a significant difference between the real and statistically predicted frequency of occurrence. The analysis of 334 highly expressed and 303 poorly expressed E.coli genes showed...

Biochemistry Usa, Feb 19, 2008

A recombinant form of the prototypic diheme bacterial cytochrome c peroxidase (BCCP) from Pseudom... more A recombinant form of the prototypic diheme bacterial cytochrome c peroxidase (BCCP) from Pseudomonas aeruginosa (PsaCCP) has been expressed in Escherichia coli and purified to homogeneity. This material was used to carry out the first integrated biochemical, spectroscopic and structural investigation of the factors leading to reductive activation of this class of enzymes. A single, tightly bound, Ca2+ ion (K = 3 x 10(10) M-1) found at the domain interface of both the fully oxidized and mixed-valence forms of the enzyme is absolutely required for catalytic activity. Reduction of the electron-transferring (high-potential) heme in the presence of Ca2+ ions triggers substantial structural rearrangements around the active-site (low-potential) heme to allow substrate binding and catalysis. The enzyme also forms a mixed-valence state in the absence of Ca2+ ions, but a combination of electronic absorption, and EPR spectroscopies suggests that under these circumstances the low potential heme remains six-coordinate, unable to bind substrate and therefore catalytically inactive. Our observations strongly suggest that the two mixed-valence forms of native PsaCCP reported previously by Foote and colleagues (Foote, N., Peterson, J., Gadsby, P., Greenwood, C., and Thomson, A. (1985) Biochem. J. 230, 227-237) correspond to the Ca2+-loaded and -depleted forms of the enzyme.

Biotechnology & Biotechnological Equipment, 2002

Journal of Inorganic Biochemistry, 2007

Mutagenesis studies have been used to investigate the role of a heme ligand containing protein lo... more Mutagenesis studies have been used to investigate the role of a heme ligand containing protein loop (67-79) in the activation of diheme peroxidases. Two mutant forms of the cytochrome c peroxidase of Pseudomonas aeruginosa have been produced. One mutant (loop mutant) is devoid of the protein loop and the other (H71G) contains a non-ligating Gly at the normal histidine ligand site. Spectroscopic data show that in both mutants the distal histidine ligand of the peroxidatic heme in the un-activated enzyme is lost or is exchangeable. The un-activated H71G and loop mutants show, respectively, 75% and 10% of turnover activity of the wild-type enzyme in the activated form, in the presence of hydrogen peroxide and the physiological electron donor cytochrome c 551. Both mutant proteins show the presence of constitutive reactivity with peroxide in the normally inactive, fully oxidised, form of the enzyme and produce a radical intermediate. The radical product of the constitutive peroxide reaction appears to be located at different sites in the two mutant proteins. These results show that the loss of the histidine ligand from the peroxidatic heme is, in itself, sufficient to produce peroxidatic activity by providing a peroxide binding site and that the formation of radical intermediates is very sensitive to changes in protein structure. Overall, these data are consistent with a major role for the protein loop 67-79 in the activation of di-heme peroxidases and suggest a ''charge hopping'' mechanism may be operative in the process of intra-molecular electron transfer.

Current Microbiology, 2004

In a previous study, we have identified four types of 3Ј terminal codon pairs depending on their ... more In a previous study, we have identified four types of 3Ј terminal codon pairs depending on their frequency of occurrence in the Escherichia coli genome: overrepresented, moderately represented, underrepresented, and missing. In this study, the influence of eight codon pairs belonging to these four groups on the efficiency of chloramphenicol acetyltransferase (cat) gene expression in E. coli is examined. Our results show that the missing codon pairs CCU:UAG (Pro:Stop) and CCC:UAG (Pro: Stop) had decreasing effect, whereas another missing pair CCU:AGG (Pro:Arg) had an opposite effect on the yield of CAT protein in comparison with the wild-type cat gene.

ChemBioChem, 2007

Mutant forms of the enzyme cytochrome c peroxidase from Pseudomonas aeruginosa, in which the pero... more Mutant forms of the enzyme cytochrome c peroxidase from Pseudomonas aeruginosa, in which the peroxidatic haem ligand (H71) and putative haem-bridging amino acid (W94) have been mutated, were produced in an E. coli expression system as a means of investigating possible mechanisms of intramolecular electron transfer within the enzyme. EPR spectroscopy indicated the presence of a high-spin, presumably five-coordinate, peroxidatic haem site in the H71G and H71G/W94A mutants, whilst the W94A mutant apparently retained the normal six-coordinate haem structures. In turnover experiments, these mutants show 55, 4, and &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;1% activity, respectively, as compared to the wild-type enzyme. The W94A mutant shows essentially no activity in turnover experiments. Circular dichroism spectroscopy indicates no measurable difference in the secondary structure of the H71G mutant from that of the native enzyme, whilst some small differences are observed for the double mutant. Treatment of the oxidised mutant proteins with hydrogen peroxide, in the absence of preactivation or exogenous reductants, yields products that suggest the formation of a tryptophan radical species in the case of the H71 mutant and the production of a porphyrin radical in the case of the double mutant. These results are discussed in terms of the intramolecular electron transfer in this enzyme.

Biochemistry, 2008

Bioinformatics, 2003

Escherichia coli genome contains 4290 open reading j-ames (ORFs). Using own computer programme, t... more Escherichia coli genome contains 4290 open reading j-ames (ORFs). Using own computer programme, the full set of ORFs was analyzed for occurrence of all possible combinations of codons in doublets (codon pairs) except for the combinations stop:stop and stop:sense codons. Thus 19 missing pairs were identified among the overall 1358854 analyzed codon pairs. Two of the missing pairs CCU:AGG and ACU:AGA represented combinations betJ.veen sense codons and 17 were combinations of sense and stop codons. The stop codons in the latter group of missing pairs were definitely biased. Except for one codon pair (ACU: UGA), where the stop codon was UGA, the stop codon in all the rest was UAG. Surprisingly, there was not a single missing codon pair containing the stop codon UAA. The sense codons found in most of the missing codon pairs belonged to the category of rare codons.