Accelerating hydrogen evolution at neutral pH by destabilization of water with a conducting oxophilic metal oxide

Self-supporting Ni4Mo–V2O3 nanosheets, which combine oxophilic V2O3 as a water dissociation center with Ni4Mo as a proton recombination site, display an extremely low overpotential (39.3 mV at 10 mA cm−2) for hydrogen evolution at neutral pH.

Finding a Tandem Repeats Motifs in the Completed Genomes of Human Coronavirus (hku1) Which is Identified as a Hotspot Region for the Viruses Recombination by Using Regular Expression Language

A lot of research studies have been surveyed the completed genomes of prokaryotic and eukaryotic and focused on the correlation between the percentage of microsatellite sequences in completed genomes and the whole size of the organism genomes. There are fewer studies made in repetitive sequences otherwise simple sequence repeats or long tandem repeats of virus genomes. simple sequence repeats (SSRs) are the most important regions for recombination and moving repeats blocks from site to another site in the genomes. A tool was programmed and designed by visual basic 6.0 to find the long tandem repeats in DNA sequences of the small genomes. The tool named “Repeater Finder Regular Expression”, (RFRE) Version 1.0, 2016. The tool was utilized to discover different pattern of long tandem repeats (LTR) motifs on the completed genomes of human corona virus strains by using a joined regular expression language. In this study, a twenty-nine accession numbers of human coronavirus completed genomes, (hku1) strains were retrieved from the Genbank. The researcher can write a different regular expression patterns and joined regular expression patterns through the designed tool to search and find a specific motif of nucleotide sequences inside the complete genomes. The RFRE tool searched and found three different total lengths of a perfect long tandem repeats (240bp, 300bp and 480bp). A Dot plot gave a picture view for the long tandem repeat sequences in the completed genome sequence (KF430201.1) of human coronavirus. The genomic dot plot tool YASS was used as a genomic similarity searching tool to check for the uninterrupted repeats and confirm the sensitivity of the (RFRE) tool. To identify the recombination site in the genomes of human coronavirus the RAT tool was applied to find the recombination sites between the completed genomes of human corona viruses .The RAT tool recognized the recombination site in the nucleotide position (3012) and at the same time this recombination site position (3012) was also recognized as a beginning position of a long tandem repeat. A precise motif was predicted from the translated repeats of Human Corona Virus which found by PRATT tool. There was a relationship between the total length of long tandem repeats and genome size of Human Corona Virus and the correlation value R2 was equal to (0,451). In conclusion, this study presented the importance of finding the long tandem repeats of human coronavirus and gives a relationship between the completed genome size of human coronavirus types and long tandem repeats. The nucleotide position (3012) was a hot spot site for a recombination among the complete genomes of human coronavirus and also identified as a repetitive site in the genomes of human corona virus (hku1). The repeats in human coronavirus (hku1) were predicated to be a main major role of virus evolution.

A potential genomic recombination site upstream of the rfb locus in Leptospira interrogans is associated with serogroup Serjoe and serovar Hardjo classification

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of the genus Leptospira. It has a global distribution and affects domestic animals, including cattle. In livestock production, Leptospira interrogans serogroup Sejroe serovar Hardjo is the major reproductive disease leading to economic losses. The whole-genome sequence of the first Brazilian clinical isolate classified as L. interrogans serogroup Sejroe serovar Hardjo strain Norma enabled the evaluation of its genomic features. Here, we investigated particularities of this isolate, obtained from a leptospirosis outbreak. Bioinformatic analysis using the L. interrogans serovar Hardjo str. Norma was applied as a reference for genomic evaluation and comparative analysis among L. interrogans and L. borgpetersenii serovars. Our data suggest the occurrence of genomic recombination in L. interrogans serovar Hardjo str. Norma encompassing 45 Kb located upstream of the rfb locus. A hallmark of genetic evolution was predicted through an orthologue analysis that identified that sugar enzymes associated with carbohydrate and lipid biosynthesis and metabolism composed this genetic module. Comparative genomics revealed a wide range of relatedness among the bacterial strains of serogroup Sejroe that are classified as L. interrogans and L. borgpetersenii species. Furthermore, identification of an IS3 family suggests a genetic recombination site in L. interrogans serovar Hardjo str. Norma that is distinct among L. interrogans serovars and may contribute to clarify the taxonomic classification of Leptospira spp.Impact StatementLeptospirosis remains an important neglected disease with worldwide distribution. This zoonotic disease impacts in the livestock production and the bovine infection is currently associated to species L. borgpetersenii and L. interrogans serovar Hardjo. L.interrogans serovar Hardjo infection is recognized as reproductive disease associated with abortion and economic lost. In this context, we studied a unique whole genome sequence of L. interrogans serovar Hardjo subtype Hardjo-prajitno isolated from bovine leptospirosis outbreaks in Brazilian dairy farm, one of the greatest country of milk production in world. We compared L. interrogans and L. borgpetersenii genomes with L. interrogans serogroup Sejroe serovar Hardjo subtype Hardjo-prajitno focusing on rfb locus and sugars biosynthesis. Leptospira spp. taxonomy and serology information are strictly associated with rfb locus and we found high correlation among bacterial strains classified in serogroup Sejroe. Although L. interrogans and L. borgpetersenii classified in serogroup Sejroe possess a greater genetic correlation, we uniquely identified that serovar Hardjo strains often possess identical loci carrying predicted sugar biossinthesis genes and mobile elements. The Sru (Sejroe specific Rfb Upstream locus) locus associated to rfb locus probably contribute to Leptospira spp. genetic information concerning serogroup and serovar degrees of taxonomic and serology in this microbiology field.Data SummaryAll Leptospira spp. genome sequences used in this study were retrieved from National Center for Biotechnology Information (NCBI) (Table 1) with NCBI ID: NZ_CP006723.1, NZ_CP012603.1, NC_004342.2, NZ_CP011934.1, NZ_AKXA02000040.1, NZ_CP013147.1, NZ_CP012029.1, NZ_CP015048.1, NC_008508.1, GCA_000216175.3,GCA_000244115.3, NC005823.1, GCA_000244395.3, GCA_000346975.1.HighlightsThe study identified potential molecular features associated with serovar Hardjo subtype Hardjo-prajitno, present in both L. interrogans and L. borgpetersenii, by comparative genomics.A new potential recombinant site found upstream of the rfb locus contains proteins that correlate with serogroup taxonomy in the Leptospira genus.The proteins encoded in the recombinant locus are associated with the synthesis of serological surface determinants such as carbohydrates and lipopolysaccharides.

ORBIT: a New Paradigm for Genetic Engineering of Mycobacterial Chromosomes

ABSTRACTTwo efficient recombination systems were combined to produce a versatile method for chromosomal engineering that obviates the need to prepare double-stranded DNA (dsDNA) recombination substrates. A synthetic “targeting oligonucleotide” is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annealase. This oligonucleotide contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a “payload plasmid” that contains a cognate recombination site and a selectable marker. The targeting oligonucleotide and payload plasmid are cotransformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate gene knockouts, promoter replacements, or C-terminal tags. This new system is called ORBIT (for “oligonucleotide-mediatedrecombineering followed byBxb1integrasetargeting”) and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions, or fusions in a bacterial chromosome. We demonstrate the utility of this “drag and drop” strategy by the construction of insertions or deletions in over 100 genes inMycobacteriumtuberculosisandM. smegmatis.IMPORTANCEWe sought to develop a system that could increase the usefulness of oligonucleotide-mediated recombineering of bacterial chromosomes by expanding the types of modifications generated by an oligonucleotide (i.e., insertions and deletions) and by making recombinant formation a selectable event. This paper describes such a system for use inM. smegmatisandM. tuberculosis. By incorporating a single-stranded DNA (ssDNA) version of the phage Bxb1attPsite into the oligonucleotide and coelectroporating it with a nonreplicative plasmid that carries anattBsite and a drug selection marker, we show both formation of a chromosomalattPsite and integration of the plasmid in a single transformation. No target-specific dsDNA substrates are required. This system will allow investigators studying mycobacterial diseases, including tuberculosis, to easily generate multiple mutants for analysis of virulence factors, identification of new drug targets, and development of new vaccines.

ORBIT: a new paradigm for genetic engineering of mycobacterial chromosomes

ABSTRACTCurrent methods for genome engineering in mycobacteria rely on relatively inefficient recombination systems that require the laborious construction of a long double-stranded DNA substrate for each desired modification. We combined two efficient recombination systems to produce a versatile method for high-throughput chromosomal engineering that obviates the need for the preparation of double-stranded DNA recombination substrates. A synthetic “targeting oligonucleotide” is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annelase. This oligo contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a “payload plasmid” that contains a cognate recombination site and selectable marker. The targeting oligo and payload plasmid are co-transformed into a RecT‐ and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate knockouts and promoter replacements, or to fuse the C-terminal-encoding regions of target genes with tags of various functionalities. This new system is called ORBIT (Oligo-mediated Recombineering followed by Bxb1 Integrase Targeting) and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions or fusions in a target bacterium. We demonstrate the utility of ORBIT by the construction of insertions or deletions in over 100 genes inM. tuberculosisandM. smegmatis. The report describes the first genetic engineering technique for making selectable chromosomal fusions and deletions that does not require the construction of target‐ or modification-specific double-stranded DNA recombination substrates.

reconstructIon of recombination sites in genomic structures of the strains of genotype 6 of hepatitis c virus

The encoded portion of the complete genomes of 46 strains of the genotype 6 of hepatitis C virus through bioinformat-ics RDP programs complex group of 6 recombinants strains was identified, in which 7 recombination sites were fixed. Strains correspond to the three-recombinant HCV subtypes: 6a, 6b and 6I. For each of the identified recombinant we defined parent strains from which they can be obtained. Three recombinants were obtained from parent strains of the same subtype (homologous inside subgenotypic recombination). For the remaining three recombinants parent strains were members of three different subtypes (between subgenotypic recombination). In one strain we identified a unique recombination site in a highly conservative NS3 gene. Most of the recombination sites occurred in the region of the structural genes C, E1 and E2, and in the area of non-structural genes NS5a and NS5b. In the recombinant strain DQ480518-6a two recombination site were identified. One site is located in the structural and nonstructural genes (E2 + NS1 + NS2), and a second one in non-structural region. Dimensions of recombination sites can vary from 86 to 1072 nucleotide bases. The study identified “hot spots” of recombination in the strains of genotype 6 of hepatitis C virus. The recombinants were found in the population of the three countries: the United States (from the serum of an immigrant), Hong Kong and China.The encoded portion of the complete genomes of 46 strains of the genotype 6 of hepatitis C virus through bioinformat-ics RDP programs complex group of 6 recombinants strains was identified, in which 7 recombination sites were fixed. Strains correspond to the three-recombinant HCV subtypes: 6a, 6b and 6I. For each of the identified recombinant we defined parent strains from which they can be obtained. Three recombinants were obtained from parent strains of the same subtype (homologous inside subgenotypic recombination). For the remaining three recombinants parent strains were members of three different subtypes (between subgenotypic recombination). In one strain we identified a unique recombination site in a highly conservative NS3 gene. Most of the recombination sites occurred in the region of the structural genes C, E1 and E2, and in the area of non-structural genes NS5a and NS5b. In the recombinant strain DQ480518-6a two recombination site were identified. One site is located in the structural and nonstructural genes (E2 + NS1 + NS2), and a second one in non-structural region. Dimensions of recombination sites can vary from 86 to 1072 nucleotide bases. The study identified “hot spots” of recombination in the strains of genotype 6 of hepatitis C virus. The recombinants were found in the population of the three countries: the United States (from the serum of an immigrant), Hong Kong and China.

Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa

The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5’ untranslated region (UTR) and a 3’ recombination site (attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-blaGES-1/aacA4 gene cassette array, which harbours a fused gene cassette represented by blaGES-1/aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of blaGES-1/aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, blaGES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette blaGES-1/aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW), had a higher transcription level than blaGES-1/aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream blaGES-1/aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-blaGES-1/aacA4 free circular forms, but not to blaGES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription.