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The Shine-Dalgarno (SD) sequence is a key element directing the translation to initiate at the authentic start codons and also enabling translation initiation to proceed in 5′ untranslated mRNA regions (5′-UTRs) containing moderately strong secondary structures. Bioinformatic analysis of almost forty genomes from the major bacterial phylum Bacteroidetes revealed, however, a general absence of SD sequence, drop in GC content and consequently reduced tendency to form secondary structures in 5′-UTRs. The experiments using the Prevotella bryantii TC1-1 expression system were in agreement with these findings: neither addition nor omission of SD sequence in the unstructured 5′-UTR affected the level of the reporter protein, non-specific nuclease NucB. Further, NucB level in P. bryantii TC1-1, contrary to hMGFP level in Escherichia coli, was five times lower when SD sequence formed part of the secondary structure with a folding energy -5,2 kcal/mol. Also, the extended SD sequences did not affect protein levels as in E. coli. It seems therefore that a functional SD interaction does not take place during the translation initiation in P. bryanttii TC1-1 and possibly other members of phylum Bacteroidetes although the anti SD sequence is present in 16S rRNA genes of their genomes. We thus propose that in the absence of the SD sequence interaction, the selection of genuine start codons in Bacteroidetes is accomplished by binding of ribosomal protein S1 to unstructured 5′-UTR as opposed to coding region which is inaccessible due to mRNA secondary structure. Additionally, we found that sequence logos of region preceding the start codons may be used as taxonomical markers. Depending on whether complete sequence logo or only part of it, such as information content and base proportion at specific positions, is used, bacterial genera or families and in some cases even bacterial phyla can be distinguished.

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Two novel strains AV382 and AV436 were isolated from a submerged industrial bioreactor for production of apple cider vinegar in Kopivnik (Slovenia). Both strains showed very high (≥98.2%) 16S rRNA gene sequence similarities with Komagataeibacter species, but lower 16S–23S rRNA gene internal transcribed spacer (ITS). The highest similarity of the 16S–23S rRNA gene ITS of AV382 was to Komagataeibacter kakiaceti LMG 26206T (91.6%), of AV436 to Komagataeibacter xylinus LMG 1515T (93.9%). The analysis of genome sequences confirmed that AV382 is the most closely related to K. kakiaceti (ANIb 88.2%) and AV436 to K. xylinus (ANIb 91.6%). Genome to genome distance calculations exhibit for both strains ≤47.3% similarity to all type strains of the genus Komagataeibacter. The strain AV382 can be differentiated from its closest relatives K. kakiaceti and Komagataeibacter saccharivorans by its ability to form 2-keto and 5-keto-D-gluconic acids from glucose, incapability to grow in the presence of 30% glucose, formation of C19:0 cyclo ω8c fatty acid and tolerance of up to 5% acetic acid in the presence of ethanol. The strain AV436 can be differentiated from its closest relatives K. xylinus, Komagataeibacter sucrofermentans, and Komagataeibacter nataicola by its ability to form 5-keto-D-gluconic acid, growth on 1-propanol, efficient synthesis of cellulose, and tolerance to up to 5% acetic acid in the presence ethanol. The major fatty acid of both strains is C18:1ω7c. Based on a combination of phenotypic, chemotaxonomic and phylogenetic features, the strains AV382T and AV436T represent novel species of the genus Komagataeibacter, for which the names Komagataeibactermelaceti sp. nov. and Komagataeibacter melomenusus are proposed, respectively. The type strain of Komagataeibacter melaceti is AV382T (= ZIM B1054T = LMG 31303T = CCM 8958T) and of Komagataeibacter melomenusus AV436T (= ZIM B1056T = LMG 31304T = CCM 8959T).

Campylobacter-specific bacteriophages (phages) are considered as an alternative intervention strategy to decrease the level of poultry contamination with Campylobacter, a leading cause of gastroenteritis worldwide. Eradication efficiency depends primarily on phage-host interaction mediated by phage tail-spike proteins and bacterial receptors. Here, this interaction was characterised using tail-spike gene sequence analysis, phage neutralisation by antiserum and host range analysis of newly isolated group III Campylobacter phages with 68 Campylobacter jejuni and Campylobacter coli strains. Three different groups of phages were obtained using antibody neutralisation assay, and they were further divided according to polymorphisms observed within tail fibre sequences and host range. Only moderate congruence was observed between these criteria with notable exception of two phages. The infection relied on capsule in all phages isolated, and flagella were found to influence phage propagation on agar plates, but not in broth. Their specificity was more C. jejuni oriented with tendency to lyse human isolates more efficiently. Additionally, natural resistance of C. jejuni to phages did not correlate with their antibiotic resistance patterns. These findings provide new insights into Campylobacter-phage interaction.

The restriction endonucleases PbrTI and Pru2I, isoschizomers of Sau3AI and HaeIII, were partially purified and characterized from anaerobic rumen bacteria Prevotella bryantii TC1-1 and Prevotella ruminicola 23, respectively. These are the first type II restriction endonucleases discovered in strains of the genus Prevotella, and they represent one of the barriers hindering gene transfer in these microorganisms. Heterologous DNA was protected against the action of the PbrTI or Pru2I by incubation in a cell-free extract of the respective strain which contained 20 mM EDTA. This led to the development of a protocol enabling successful electrotransformation of the P. bryantii TC1-1 strain with a pRH3 Bacteroides--Escherichia coli shuttle vector containing up to 7-kb long DNA inserts. Plasmid DNA isolated from the transformed strain facilitated the transfer with further increased efficiency and made possible the introduction of ligation reaction products directly to P. bryantii TC1-1 without passing them first through E. coli.

Intestinal phages are abundant and important components of gut microbiota, yet the isolated and characterized representatives that infect abundant gut bacteria are sparse. Here we describe the isolation of human intestinal phages infecting Bacteroidesuniformis. Bacteroides is one of the most common bacterial groups in the global human gut microbiota; however, to date not many Bacteroides specific phages are known. Phages isolated in this study belong to a novel viral genus, Bacuni, within the Siphoviridae family. Their genomes encode diversity-generating retroelements (DGR), which were shown in other bacteriophages to promote phage adaptation to rapidly changing environmental conditions and to broaden their host range. Three isolated phages showed 99.83% genome identity but one of them infected a distinct B. uniformis strain. The tropism of Bacuni phages appeared to be dependent on the interplay of DGR mediated sequence variations of gene encoding putative phage fimbrial tip proteins and mutations in host genes coding for outer-membrane proteins. We found prophages with up to 85% amino acid similarity over two-thirds of the Bacuni phage genome in the B. acidifaciens and Prevotella sp. genomes. Despite the abundance of Bacteroides within the human microbiome, we found Bacuni phages only in a limited subset of published gut metagenomes.