Global analysis of the RNA-RNA interactome in Acinetobacter baumannii AB5075 uncovers a small regulatory RNA repressing the virulence-related outer membrane protein CarO.

Acinetobacter baumannii is an opportunistic Gram-negative pathogen that infects critically ill patients. The emergence of antimicrobial resistant A. baumannii has exacerbated the need to characterize environmental adaptation, antibiotic resistance and pathogenicity and their genetic regulators to inform intervention strategies. Critical to adaptation to changing environments in bacteria are small regulatory RNAs (sRNAs), however, the role that sRNAs play in the biology of A. baumannii is poorly understood. To assess the regulatory function of sRNAs and to uncover their RNA interaction partners, we employed an RNA proximity ligation and sequencing method (Hi-GRIL-seq) in three different environmental conditions. Forty sRNAs were ligated to sRNA-RNA chimeric sequencing reads, suggesting that sRNA-mediated gene regulation is pervasive in A. baumannii. In-depth characterization uncovered the sRNA Aar to be a post-transcriptional regulator of four mRNA targets including the transcript encoding outer membrane protein CarO. Aar initiates base-pairing with these mRNAs using a conserved seed region of nine nucleotides, sequestering the ribosome binding sites and inhibiting translation. Aar is differentially expressed in multiple stress conditions suggesting a role in fine-tuning translation of the Aar-target molecules. Our study provides mechanistic insights into sRNA-mediated gene regulation in A. baumannii and represents a valuable resource for future RNA-centric research endeavours.

Recombinant domains III of Tick-Borne Encephalitis Virus envelope protein in combination with dextran and CpGs induce immune response and partial protectiveness against TBE virus infection in mice.

BACKGROUND: E protein of tick-borne encephalitis virus (TBEV) and other flaviviruses is located on the surface of the viral particle. Domain III of this protein seems to be a promising component of subunit vaccines for prophylaxis of TBE and kits for diagnostics of TBEV. METHODS: Three variants of recombinant TBEV E protein domain III of European, Siberian and Far Eastern subtypes fused with dextran-binding domain of Leuconostoc citreum KM20 were expressed in E. coli and purified. The native structure of domain III was confirmed by ELISA antibody kit and sera of patients with tick-borne encephalitis. Immunogenic and protective properties of the preparation comprising these recombinant proteins immobilized on a dextran carrier with CpG oligonucleotides as an adjuvant were investigated on the mice model. RESULTS: All 3 variants of recombinant proteins immobilized on dextran demonstrate specific interaction with antibodies from the sera of TBE patients. Thus, constructed recombinant proteins seem to be promising for TBE diagnostics. The formulation comprising the 3 variants of recombinant antigens immobilized on dextran and CpG oligonucleotides, induces the production of neutralizing antibodies against TBEV of different subtypes and demonstrates partial protectivity against TBEV infection. CONCLUSIONS: Studied proteins interact with the sera of TBE patients, and, in combination with dextran and CPGs, demonstrate immunogenicity and limited protectivity on mice compared with reference "Tick-E-Vac" vaccine.

Solitary restriction endonucleases in prokaryotic genomes.

Prokaryotic restriction-modification (R-M) systems defend the host cell from the invasion of a foreign DNA. They comprise two enzymatic activities: specific DNA cleavage activity and DNA methylation activity preventing cleavage. Typically, these activities are provided by two separate enzymes: a DNA methyltransferase (MTase) and a restriction endonuclease (RE). In the absence of a corresponding MTase, an RE of Type II R-M system is highly toxic for the cell. Genes of the R-M system are linked in the genome in the vast majority of annotated cases. There are only a few reported cases in which the genes of MTase and RE from one R-M system are not linked. Nevertheless, a few hundreds solitary RE genes are present in the Restriction Enzyme Database (http://rebase.neb.com) annotations. Using the comparative genomic approach, we analysed 272 solitary RE genes. For 57 solitary RE genes we predicted corresponding MTase genes located distantly in a genome. Of the 272 solitary RE genes, 99 are likely to be fragments of RE genes. Various explanations for the existence of the remaining 116 solitary RE genes are also discussed.

Plasmid-encoded lactose metabolism and mobilized colistin resistance (mcr-9) genes in Salmonella enterica serovars isolated from dairy facilities in the 1980s.

Horizontal gene transfer by plasmids can confer metabolic capabilities that expand a host cell's niche. Yet, it is less understood whether the coalescence of specialized catabolic functions, antibiotic resistances and metal resistances on plasmids provides synergistic benefits. In this study, we report whole-genome assembly and phenotypic analysis of five Salmonella enterica strains isolated in the 1980s from milk powder in Munich, Germany. All strains exhibited the unusual phenotype of lactose-fermentation and encoded either of two variants of the lac operon. Surprisingly, all strains encoded the mobilized colistin resistance gene 9 (mcr-9), long before the first report of this gene in the literature. In two cases, the mcr-9 gene and the lac locus were linked within a large gene island that formed an IncHI2A-type plasmid in one strain but was chromosomally integrated in the other strain. In two other strains, the mcr-9 gene was found on a large IncHI1B/IncP-type plasmid, whereas the lac locus was encoded on a separate chromosomally integrated plasmidic island. The mcr-9 sequences were identical and genomic contexts could not explain the wide range of colistin resistances exhibited by the Salmonella strains. Nucleotide variants did explain phenotypic differences in motility and exopolysaccharide production. The observed linkage of mcr-9 to lactose metabolism, an array of heavy-metal detoxification systems, and other antibiotic resistance genes may reflect a coalescence of specialized phenotypes that improve the spread of colistin resistance in dairy facilities, much earlier than previously suspected.

PLANdbAffy: probe-level annotation database for Affymetrix expression microarrays.

Standard Affymetrix technology evaluates gene expression by measuring the intensity of mRNA hybridization with a panel of the 25-mer oligonucleotide probes, and summarizing the probe signal intensities by a robust average method. However, in many cases, signal intensity of the probe does not correlate with gene expression. This could be due to the hybridization of the probe to a transcript of another gene, mapping of the probe to an intron, alternative splicing, single nucleotide polymorphisms and other reasons. We have developed a database, PLANdbAffy (available at http://affymetrix2.bioinf.fbb.msu.ru), that contains the results of the alignment of probe sequences from five Affymetrix expression microarrays to the human genome. We have determined the probes matching the transcript-coding regions in the correct orientation. For each such probe alignment region, we determined the mRNA and EST sequences that contain the probe sequence. In the textual part of the database interface we summarize the data on the sequences that cover the probe alignment region and SNPs that are located inside it. The graphical part of our database interface is implemented as custom tracks to the UCSC genome browser that allows one to utilize all the data that are offered by UCSC browser.

Enhanced C/EBP binding to G·T mismatches facilitates fixation of CpG mutations in cancer and adult stem cells.

Somatic mutations in regulatory sites of human stem cells affect cell identity or cause malignant transformation. By mining the human genome for co-occurrence of mutations and transcription factor binding sites, we show that C/EBP binding sites are strongly enriched with [C > T]G mutations in cancer and adult stem cells, which is of special interest because C/EBPs regulate cell fate and differentiation. In vitro protein-DNA binding assay and structural modeling of the CEBPB-DNA complex show that the G·T mismatch in the core CG dinucleotide strongly enhances affinity of the binding site. We conclude that enhanced binding of C/EBPs shields CpG·TpG mismatches from DNA repair, leading to selective accumulation of [C > T]G mutations and consequent deterioration of the binding sites. This mechanism of targeted mutagenesis highlights the effect of a mutational process on certain regulatory sites and reveals the molecular basis of putative regulatory alterations in stem cells.

Draft Genome Sequence and Annotation of Acinetobacter soli AS15, Isolated from an Irish Hospital.

We report the draft genome sequence of Acinetobacter soli AS15, which was isolated in 2018 from a rectal screen of a patient at St. Vincent's University Hospital (Dublin, Ireland). The draft genome sequence is 3,589,002 bp and was assembled into 82 contigs.

Probe-Level Universal Search (PLUS) algorithm for gender differentiation in affymetrix datasets.

Affymetrix microarrays measure gene expression based on the intensity of hybridization of a panel of oligonucleotide probes (probe set) with mRNA. The signals from all probes within a probe set are converted into a single measure that represents the expression value of a gene. This step diminishes the number of independently measured parameters and eliminates from consideration individual "good-working" probes. We propose a new feature selection algorithm (Probe Level Universal Search or PLUS algorithm) for probe-level analysis of gene expression datasets. The algorithm evaluates the intensities of perfect-match Affymetrix probes individually and selects probes that allow one to distinguish two given classes of samples. The algorithm was used to differentiate the samples according to their gender ("gender differentiation"). The universal gender differentiating set of 3' Gene Affymetrix microarray probes was selected; the set consists of 38 probes from XIST gene of X-chromosome and 17 probes from five Y-chromosome genes: RPS4Y1, EIF1A, DDX3Y, JARID1D and USP9Y. The selection procedure based on the probes selected by PLUS algorithm differentiates the sex chromosome karyotype of the sample, reveals samples with incorrect gender labels and samples from patients with hereditary syndromes or cancer-associated chromosome abnormalities.