Preparations of 2-epi-fortimicins A from 2-epi-fortimicin B by intramolecular base-catalyzed 2-O-acylation of 1,2′,6′-tri-N-benzyloxycarbonyl-2-epi-fortimicin B

Preparations of 2-epi-fortimicin A (4) from 2-epi-fortimicin B (3) are described. In contrast to the previously reported, selective 4-N-acylation of 1,2′,6′-tri-N-benzyloxycarbonylfortimicin B (8) with N-(N-benzyloxycarbonylglycyloxy)succinimide, 1,2′,6′-tri-N-benzyloxycarbonyl-2-epi-fortimicin B (5) underwent predominant 2-O,4-N-diacylation under similar conditions. Proof of the structure of the diacylated product is presented, with evidence that the diacylated product is formed by initial intramolecular, base-catalyzed 2-O-acylation. The in vitro antibacterial activities of 2-epi-fortimicin A (4), 2-O-glycyl-2-epi-fortimicin A (11), 1-N-glycyl-2-epi-fortimicin A (12), and 5-deoxy-2-epi-fortimicin A (13) are reported.     

A five-nucleotide sequence protects DNA from exonucleolytic degradation by AddAB, the RecBCD analogue of Bacillus subtilis

Homologous recombination in Bacillus subtilis requires the product of the addA and addB genes, the AddAB enzyme. This enzyme, which is both a helicase and a powerful nuclease, is thought to be the counterpart of the Escherichia coli RecBCD enzyme. From this analogy, it is expected that the nuclease activity of AddAB can be downregulated by a specific DNA sequence, which would correspond to the chi site in E. coli . Using protection of linear double-stranded DNA as a criterion, we identified the five-nucleotide sequence 5'-AGCGG-3', or its complement 5'-CCGCT-3', as being sufficient for AddAB nuclease attenuation. We have shown further that this attenuation occurs only if the sequence is properly oriented with respect to the translocating AddAB enzyme. Finally, inspection of the complete B. subtilis genome revealed that this five-nucleotide sequence is over-represented and is, in a majority of cases, co-oriented with DNA replication. Based on these observations, we propose that 5'-AGCGG-3', or its complement, is the B. subtilis analogue of the E. coli chi sequence.     

Characterization of a region of the Enterococcus faecalis plasmid pAMβ1 which enhances the segregational stability of pAMβ1-derived cloning vectors in Bacillus subtilis

The nucleotide sequence of a 2.13-kb EcoRI-HindIII, pAMβ1-derived fragment, isolated from the gram-positive cloning vector pHV1431, has been determined and shown to encode two ORFs. ORF H encodes for a protein of 23,930 Da which exhibits substantial homology to bacterial site-specific recombinases, particularly the resolvases of the gram-positive transposons Tn917 (30.3% identity) and Tn552 (31.6% identity) and the clostridial plasmid pIP404 (27.1% identity). The second ORF (I) is incomplete and encodes a polypeptide which has significant homology with Escherichia coli topoisomerase I (26.0% identity). Insertion of either the entire 2.13-kb EcoRI-HindIII fragment or a 0.73-kb EcoRI-DraI subfragment encoding only the resolvase into the pAMβ1-based cloning vector pMTL500E causes a significant enhancement of segregational stability (from 6.5 × 10⁻² to 3.0–4.0 × 10⁻³ plasmid loss per cell per generation). Improved segregational stability is mirrored by a reduction in plasmid polymerization. The introduction of a stop codon into the resolvase coding region negates its ability to promote segregational stability. It is proposed that the identified determinant stabilizes pAMβ1-based vectors in Bacillus subtilis by maintaining the plasmid population in the monomeric state, thereby reducing the chances of producing plasmid-free segregants.     

Systematic analysis of gut microbiome reveals the role of bacterial folate and homocysteine metabolism in Parkinson’s disease

1. Download : Download high-res image (187KB)
2. Download : Download full-size image

     

Prediction of surface exposed proteins in Streptococcus pyogenes,with a potential application to other Gram‐positive bacteria

The in silico prediction of bacterial surface exposed proteins is of growing interest for the rational development of vaccines and in the study of bacteria–host relationships, whether pathogenic or host beneficial. This interest is driven by the increase in the use of DNA sequencing as a major tool in the early characterization of pathogenic bacteria and, more recently, even of complex ecosystems at the host–environment interface in metagenomics approaches. Current protein localization protocols are not suited to this prediction task as they ignore the potential surface exposition of many membrane‐associated proteins. Therefore, we developed a new flow scheme, SurfG+, for the processing of protein sequence data with the particular aim of identification of potentially surface exposed (PSE) proteins from Gram‐positive bacteria, which was validated for Streptococcus pyogenes. The results of an exploratory case study on closely related lactobacilli of the acidophilus group suggest that the yogurt bacterium Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) dedicates a relatively important fraction of its coding capacity to secreted proteins, while the probiotic gastrointestinal (GI) tract bacteria L. johnsonii and L. gasseri appear to encode a larger variety of PSE proteins, that may play a role in the interaction with the host.     

Genetic Variability, Differentiation, and Founder Effect in Golden Jackals (Canis aureus) from Serbia as Revealed by Mitochondrial DNA and Nuclear Microsatellite Loci

We analyzed 121 golden jackals (Canis aureus) from six sample sites in Serbia with regard to genetic variability and differentiation as revealed by mitochondrial control region sequences and eight nuclear microsatellite loci. There was no variation at all in the mtDNA sequences, and nuclear variability was very low (average observed and expected heterozygosity of 0.29 and 0.34, respectively). This is in line with the considerable recent range expansion of this species in the Balkans and indicates a strong founder effect in the recently established Serbian population. We did not find evidence of differentiation between the northeastern jackals and those from the plain of Srem or those in between. F-statistics and Bayesian Structure analyses, however, were indicative of a low degree of overall differentiation in the Serbian population. A vagrant Austrian jackal that was also analyzed was genetically indistinguishable from its Serbian conspecifics.     

Sequence Analysis of the Bacillus subtilis 168 Chromosome Region between the sspC and odhA Loci (184{degrees}-180{degrees})

The nucleotide sequence of 45,389 bp in the 184°-;180°region of the Bacillus subtilis chromosome, containing the cgecluster, which is controlled by the sporulation regulatory proteinGerE, was determined. Fifty-four putative ORFs with putativeribosome-binding sites were recognized. Seven of them correspondto previously characterized genes: cgeB, cgeA, cgeC, cgeD, cgeE,ctpA, and odhA. The deduced products of 25 ORFs were found todisplay significant similarities to proteins in the data banks.We have identified genes involved in detoxification, cell walls,and in the metabolism of biotins, purines, fatty acids, carbohydratesand amino acids. The remaining 22 ORFs showed no similarityto known proteins. Both an attachment site of the SPßprophage and 2 new putative DNA replication terminators wereidentified in this region.