scholarly journals Genetic Characterization of Optochin-Susceptible Viridans Group Streptococci

2003 ◽  
Vol 47 (10) ◽  
pp. 3187-3194 ◽  
Author(s):  
Antonio J. Martín-Galiano ◽  
Luz Balsalobre ◽  
Asunción Fenoll ◽  
Adela G. de la Campa
Keyword(s):  
Amino Acid ◽  
Chromosomal Region ◽  
Genetic Characterization ◽  
Amino Acid Identity ◽  
Detailed Characterization ◽  
Genes Encoding ◽  
Α Helix ◽  
Viridans Group Streptococci ◽  
Type Strains

ABSTRACT Two clinical isolates of viridans group streptococci (VS) with different degrees of susceptibility to optochin (OPT), i.e., fully OPT-susceptible (Opts) VS strain 1162/99 (for which the MIC was equal to that for Streptococcus pneumoniae, 0.75 μg/ml) and intermediate Opts VS strain 1174/97 (MIC, 6 μg/ml) were studied. Besides being OPT susceptible, they showed characteristics typical of VS, such as bile insolubility; lack of reaction with pneumococcal capsular antibodies; and lack of hybridization with rRNA (AccuProbe)-, lytA-, and pnl-specific pneumococcal probes. However, these VS Opts strains and VS type strains hybridized with ant, a gene not present in S. pneumoniae. A detailed characterization of the genes encoding the 16S rRNA and SodA classified isolates 1162/99 and 1174/97 as Streptococcus mitis. Analysis of the atpCAB region, which encodes the c, a, and b subunits of the F0F1 H+-ATPase, the target of optochin, revealed high degrees of similarity between S. mitis 1162/99 and S. pneumoniae in atpC, atpA, and the N terminus of atpB. Moreover, amino acid identity between S. mitis 1174/97 and S. pneumoniae was found in α helix 5 of the a subunit. The organization of the chromosomal region containing the atp operon of the two Opts VS and VS type strains was spr1284-atpC, with spr1284 being located 296 to 556 bp from atpC, whereas in S. pneumoniae this distance was longer than 68 kb. In addition, the gene order in S. pneumoniae was IS1239-74 bp-atpC. The results suggest that the full OPT susceptibility of S. mitis 1162/99 is due to the acquisition of atpC, atpA, and part of atpB from S. pneumoniae and that the intermediate OPT susceptibility of S. mitis 1174/97 correlates with the amino acid composition of its a subunit.

Cloning and Characterization of Three Genes Encoding LMW-GSs from Triticum aestivum, Cultival Cheyenne

2012 ◽  
Vol 554-556 ◽  
pp. 1116-1120 ◽  
Author(s):  
Mei Rong Chen ◽  
Xing Shen ◽  
Lin Li ◽  
Song Qing Hu
Keyword(s):  
Amino Acid ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Amino Acid Residues ◽  
Repetitive Domain ◽  
Genebank Accession ◽  
Genes Encoding ◽  
Α Helix ◽  
Β Sheet

Three low molecular weight subunit genes, named LMW-CND1 (GeneBank accession JQ780048), LMW-CND2 (GeneBank accession JQ779840), LMW-CND3 (GeneBank accession JQ779841), with a ORF of 1053 bp, 903 bp, 969 bp, respectively, were isolated from cv. Cheyenne and characterized detailed in molecular level. The proteins encoded by the genes, with 350, 300, 322 amino acid residues respectively, differ only in repetitive domain of sequences due to insertion or deletion of repeats in this domain. Highly similarity in amino-acid sequence between these three subunits and other published LMW-GSs was also observed, showing that all three genes published here are typical LMW-GS genes and closely related to the genes on chromosome 1D. Besides, secondary structure prediction of proteins indicated that, in the three LMW-GSs, random loop accounts for no less than 70 %, α-helix amounts to 26 %, average, and only 1.4 %~1.7 % is β-sheet.

Functional and Structural Characterization of Acquired Pan-Aminoglycoside Resistance 16S rRNA Methyltransferase NpmB1

2021 ◽  
Author(s):  
Akito Kawai ◽  
Masahiro Suzuki ◽  
Kentaro Tsukamoto ◽  
Yusuke Minato ◽  
Yohei Doi
Keyword(s):  
Amino Acid ◽  
16S Rrna ◽  
Amino Acid Identity ◽  
Single Amino Acid ◽  
Aminoglycoside Resistance ◽  
E Coli ◽  
The United Kingdom ◽  
Amino Acid Variant ◽  
A Site

Post-translational methylation of the A site of 16S rRNA at position A1408 leads to pan-aminoglycoside resistance encompassing both 4,5- and 4,6-disubstituted 2-deoxystreptamine (DOS) aminoglycosides. To date, NpmA is the only acquired enzyme with such function. Here, we present function and structure of NpmB1 whose sequence was identified in Escherichia coli genomes registered from the United Kingdom. NpmB1 possesses 40% amino acid identity with NpmA1 and confers resistance to all clinically relevant aminoglycosides including 4,5-DOS agents. Phylogenetic analysis of NpmB1 and NpmB2, its single amino acid variant, revealed that the encoding gene was likely acquired by E. coli from a soil bacterium. The structure of NpmB1 suggests that it requires a structural change of the β6/7 linker in order to bind to 16S rRNA. These findings establish NpmB1 and NpmB2 as the second group of acquired pan-aminoglycoside resistance 16S rRNA methyltransferases.

scholarly journals A50 Whole-genome sequencing of African swine fever isolates from Sardinia

2019 ◽  
Vol 5 (Supplement_1) ◽  
Author(s):  
C Torresi ◽  
F Granberg ◽  
L Bertolotti ◽  
A Oggiano ◽  
B Colitti ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Data ◽  
Temporal Distribution ◽  
African Swine Fever ◽  
Amino Acid Identity ◽  
Genotype I ◽  
Acid Identity ◽  
Wide Range ◽  
Intergenic Sequences ◽  
Genes Encoding

Abstract In order to assess the molecular epidemiology of African swine fever (ASF) in Sardinia, we analyzed a wide range of isolates from wild and domestic pigs over a 31-year period (1978–2009) by genotyping sequence data from the genes encoding the p54 and the p72 proteins and the CVR. On this basis, the analysis of the B602L gene revealed a minor difference, placing the Sardinian isolates into two clusters according to their temporal distribution. As an extension of this study, in order to achieve a higher level of discrimination, three further variable genome regions, namely p30, CD2v, and I73R/I329L, of a large number of isolates collected from outbreaks in the years 2002–14 have been investigated. Sequence analysis of the CD2v region revealed a temporal subdivision of the viruses into two subgroups. These data, together with those from the B602L gene analysis, demonstrated that the viruses circulating in Sardinia belong to p72/genotype I, but since 1990 have undergone minor genetic variations in respect to its ancestor, thus making it impossible to trace isolates, enabling a more accurate assessment of the origin of outbreaks, and extending knowledge of virus evolution. To solve this problem, we have sequenced and annotated the complete genome of nine ASF isolates collected in Sardinia between 1978 and 2012. This was achieved using sequence data determined by next-generation sequencing. The results showed a very high identity with range of nucleotide similarity among isolates of 99.5 per cent to 99.9 per cent. The ASF virus (ASFV) genomes were composed of terminal inverted repeats and conserved and non-conserved ORFs. Among the conserved ORFs, B385R, H339R, and O61R-p12 showed 100 per cent amino acid identity. The same was true for the hypervariable ORFs, with regard to X69R, DP96R, DP60R, EP153R, B407L, I10L, and L60L genes. The EP402R and B602L genes showed, as expected, an amino acid identity range of 98.5 per cent to 100 per cent and 91 per cent to 100 per cent, respectively. In addition, all of the isolates displayed variable intergenic sequences. As a whole, the results from our studies confirmed a remarkable genetic stability of the ASFV/p72 genotype I viruses circulating in Sardinia.

scholarly journals Identification of Streptococci to Species Level by Sequencing the Gene Encoding the Manganese-Dependent Superoxide Dismutase

1998 ◽  
Vol 36 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Claire Poyart ◽  
Gilles Quesne ◽  
Stephane Coulon ◽  
Patrick Berche ◽  
Patrick Trieu-Cuot
Keyword(s):  
Superoxide Dismutase ◽  
Pcr Assay ◽  
Degenerate Primers ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Type Strains ◽  
Internal Fragment ◽  
Rrna Sequences ◽  
16S Rrna Sequences

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodAint ) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus,S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus,S. canis, S. cricetus, S. downei,S. dysgalactiae, S. equi subsp.equi, S. equi subsp. zooepidemicus,S. equinus, S. gordonii, S. iniae,S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis,S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis,S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodAint fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of theirsodAint fragments on the phylogenetic tree of the sodAint fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.

scholarly journals Prevalence and Genetic Characterization of Two Mitochondrial Gene Sequences of Strobilocercus Fasciolaris in the Livers of Brown Rats (Rattus norvegicus) in Heilongjiang Province in Northeastern China

2020 ◽  
Vol 10 ◽  
Author(s):  
Fengnian Zhao ◽  
Yun Zhou ◽  
Yanchen Wu ◽  
Kexin Zhou ◽  
Aiqin Liu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Intraspecific Variation ◽  
Rattus Norvegicus ◽  
Genetic Characterization ◽  
Northeastern China ◽  
Amino Acid Sequences ◽  
Morphological Observation ◽  
Heilongjiang Province ◽  
Gene Sequences

Rodents constitute the largest and most successful group of mammals worldwide. Brown rats (Rattus norvegicus) are one of the most common rodent species, and they serve as intermediate hosts of Hydatigera taeniaeformis. Although there have been a few studies reporting on the presence of the larval form of H. taeniaeformis (strobilocercus fasciolaris) in brown rats worldwide, little information is available on the genetic characterization of this parasite, with no molecular data from China. Therefore, from April 2014 to March 2016, this study was carried out to understand the prevalence and genetic characters of strobilocercus fasciolaris in brown rats captured in Heilongjiang Province in northeastern China. The livers of brown rats were collected and examined for the presence of cysts. Each cyst was identified based on morphological observation: the larvae with the naked eye and the scolexes under a microscope. The results were confirmed by polymerase chain reaction (PCR) and sequencing of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 4 (nad4) genes. At the investigated sites, 11.8% (13/110) of the brown rats were infected with strobilocercus fasciolaris. Based on sequence analysis, there were 10 and six haplotypes regarding the cox1 and the nad4 loci, with 24 and 42 polymorphic sites, respectively (degree of intraspecific variation: 0.3%–4.4% and 0.6%–4.7%, respectively). Twelve nucleotide sequences (six of the 10 at the cox1 locus and all six at the nad4 locus) have not previously been described. Base differences in three of the six novel cox1 gene sequences and five of the six novel nad4 gene sequences caused amino acid changes. Phylogenetic analyses of the cox1 and nad4 gene sequences based on neighbor-joining and Bayesian inference trees indicated that all the strobilocercus fasciolaris isolates belonged to Hydatigera taeniaeformis sensu stricto (s.s.). This is the first report on the genetic characterization of strobilocercus fasciolaris in brown rats in China. The findings of novel cox1 and nad4 nucleotide and amino acid sequences may reflect the region-specific genetic characterization of the parasite. The data will be useful to explore the biological and epidemiological significance of the intraspecific variation within H. taeniaeformis s.s.

MUTANT AND CHIMAERIC RECOMBINANT PLASMINOGEN ACTIVATORS

1987 ◽  
Author(s):  
L Piérard ◽  
P Jacobs ◽  
D Gheysen ◽  
M Hoylaerts ◽  
A Cravador ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activators ◽  
Tissue Type ◽  
Single Chain ◽  
Type Plasminogen Activator ◽  
Genes Encoding ◽  
A Chain ◽  
Mutant Forms ◽  
Kringle 2

In order to produce plasminogen activators (PA) more specific and more active than their natural counterparts, we designed recombinant genes encoding mutant forms of urokinase (u-PA) and chimaeric molecules combining fragments of tissue type plasminogen activator (t-PA) and of u-PA. The following constructs have been realized : 1°) u-PA where amino acids Arg156 and Lys158 have been replaced by Thr. The purpose of this approach was to obtain a prourokinase molecule displaying similar properties as the natural single chain urokinase (scu-PA) but resistant to the cleavage by plasmin ; 2°) u-PA where the second cleavage site, Lys135-Lys136, was also eliminated either by replacing amino acid 132 to amino acid 147 by a shorter link (Ser-Thr) as found in t-PA, or by replacing the two lysines by glutamine residues. The resulting molecules correspond thus to completely uncleavable scu-PA forms ; 3°) an hybrid composed of the finger domain of t-PA and of the B-chain of u-PA ; 4°) an hybrid made of the A-chain of t-PA and of the B-chain of u-PA ; 5°) an hybrid where the kringle 2 of t-PA has been inserted between the kringle domain and the B-chain of u-PA. The last three constructs have been made to confer the fibrin binding specificity of t-PA to the B-chain of u-PA.All recombinant DNAs were introduced, via an expression vector, into R1610 and CosI cells. Secretion of the recombinant products was monitored by ELISA and activities were assayed in an immobilized system involving a monoclonal antibody (AAU2) raised against 33K u-PA, plasminogen and the specific chromogenic substrate S2251. In this assay, all recombinant products, except the plasmin resistant (156-158) scu-PA, showed apparent specific activities comparable to the activity of natural two-chain u-PA. Potential interest of these new plasminogen activators in therapy will be discussed and further characterization of the new molecules will-be presented.

Primary structure, expression and localization of two intermediate subunit lectins ofEntamoeba disparthat contain multiple CXXC motifs

Parasitology ◽  
2007 ◽  
Vol 134 (14) ◽  
pp. 1989-1999 ◽  
Author(s):  
H. TACHIBANA ◽  
X.-J. CHENG ◽  
S. KOBAYASHI ◽  
Y. OKADA ◽  
J. ITOH ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Confocal Microscopy ◽  
Phenotypic Expression ◽  
Surface Proteins ◽  
Rt Pcr ◽  
Entamoeba Dispar ◽  
Genes Encoding ◽  
Sequence Identities

SUMMARYWe have recently identified 2 surface proteins inEntamoeba histolyticaas intermediate subunits of galactose- andN-acetyl-D-galactosamine-inhibitable lectin (EhIgl1 and EhIgl2); these proteins both contain multiple CXXC motifs. Here, we report the molecular characterization of the corresponding proteins inEntamoeba dispar, which is neither pathogenic nor invasive. TwoIglgenes encoding 1110 and 1106 amino acids (EdIgl1 and EdIgl2) were cloned from 2 strains ofE. dispar. The amino acid sequence identities were 79% between EdIgl1 and EdIgl2, 75–76% between EdIgl1 and EhIgl1, and 73–74% between EdIgl2 and EhIgl2. However, all the CXXC motifs were conserved in the EdIgl proteins, suggesting that the fold conferred by this motif is important for function. Comparison of the expression level of theIglgenes by real-time RT-PCR showed 3–5 times higher expression ofEdIgl1compared toEdIgl2. Most EdIgl1 and EdIgl2 proteins were co-localized on the surface and in the cytoplasm of trophozoites, based on confocal microscopy. However, a different localization of EdIgl1 and EdIgl2 in intracellular vacuoles and a different level of phenotypic expression of the two Igls were also observed. These results demonstrate that Igls are important proteins even in non-pathogenic amoeba and that Igl1 and Igl2 may possess different functions.

scholarly journals Biochemical and Genetic Characterization of Mundticin KS, an Antilisterial Peptide Produced by Enterococcus mundtii NFRI 7393

2002 ◽  
Vol 68 (8) ◽  
pp. 3830-3840 ◽  
Author(s):  
Shinichi Kawamoto ◽  
Jun Shima ◽  
Rumi Sato ◽  
Tomoko Eguchi ◽  
Sadahiro Ohmomo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Characterization ◽  
Solid Phase ◽  
Exchange Chromatography ◽  
Nucleotide Sequencing ◽  
Lactobacillus Curvatus ◽  
Amino Acid Peptide ◽  
Enterococcus Mundtii

ABSTRACT Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.

scholarly journals Purification and Characterization of the Coniferyl Aldehyde Dehydrogenase from Pseudomonas sp. Strain HR199 and Molecular Characterization of the Gene

1998 ◽  
Vol 180 (17) ◽  
pp. 4387-4391 ◽  
Author(s):  
Sandra Achterholt ◽  
Horst Priefert ◽  
Alexander Steinbüchel
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ferulic Acid ◽  
Aldehyde Dehydrogenase ◽  
Genomic Library ◽  
Aromatic Aldehydes ◽  
Amino Acid Identity ◽  
Reading Frame ◽  
Relative Molecular Weight

ABSTRACT The coniferyl aldehyde dehydrogenase (CALDH) ofPseudomonas sp. strain HR199 (DSM7063), which catalyzes the NAD+-dependent oxidation of coniferyl aldehyde to ferulic acid and which is induced during growth with eugenol as the carbon source, was purified and characterized. The native protein exhibited an apparent molecular mass of 86,000 ± 5,000 Da, and the subunit mass was 49.5 ± 2.5 kDa, indicating an α2 structure of the native enzyme. The optimal oxidation of coniferyl aldehyde to ferulic acid was obtained at a pH of 8.8 and a temperature of 26°C. The Km values for coniferyl aldehyde and NAD+ were about 7 to 12 μM and 334 μM, respectively. The enzyme also accepted other aromatic aldehydes as substrates, whereas aliphatic aldehydes were not accepted. The NH2-terminal amino acid sequence of CALDH was determined in order to clone the encoding gene (calB). The corresponding nucleotide sequence was localized on a 9.4-kbp EcoRI fragment (E94), which was subcloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The partial sequencing of this fragment revealed an open reading frame of 1,446 bp encoding a protein with a relative molecular weight of 51,822. The deduced amino acid sequence, which is reported for the first time for a structural gene of a CALDH, exhibited up to 38.5% amino acid identity (60% similarity) to NAD+-dependent aldehyde dehydrogenases from different sources.

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