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

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.

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Characterization of diatom–cyanobacteria symbioses on the basis of nifH, hetR and 16S rRNA sequences

Environmental Microbiology ◽

10.1111/j.1462-2920.2006.01068.x ◽

2006 ◽

Vol 8 (11) ◽

pp. 1913-1925 ◽

Cited By ~ 93

Author(s):

Rachel A. Foster ◽

Jonathan P. Zehr

Keyword(s):

16S Rrna ◽

Rrna Sequences ◽

16S Rrna Sequences

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New Strategy for Identification of Novel cry-Type Genes from Bacillus thuringiensis Strains

Applied and Environmental Microbiology ◽

10.1128/aem.71.2.761-765.2005 ◽

2005 ◽

Vol 71 (2) ◽

pp. 761-765 ◽

Cited By ~ 23

Author(s):

Corina M. Berón ◽

Leonardo Curatti ◽

Graciela L. Salerno

Keyword(s):

Bacillus Thuringiensis ◽

Phylogenetic Distance ◽

Cloning And Sequencing ◽

Degenerate Primers ◽

Dna Templates ◽

Cry Proteins ◽

New Strategy ◽

Genes Encoding ◽

Encoding Protein

ABSTRACT We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.

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Characterization of Enterococcus spp. from Human and Animal Feces Using 16S rRNA Sequences, theespGene, and PFGE for Microbial Source Tracking in Korea

Environmental Science & Technology ◽

10.1021/es903282p ◽

2010 ◽

Vol 44 (9) ◽

pp. 3423-3428 ◽

Cited By ~ 12

Author(s):

Sei-Yoon Kim ◽

Jung Eun Lee ◽

Sunghee Lee ◽

Hee Tae Lee ◽

Ho-Gil Hur ◽

...

Keyword(s):

16S Rrna ◽

Microbial Source Tracking ◽

Source Tracking ◽

Enterococcus Spp ◽

Animal Feces ◽

Rrna Sequences ◽

16S Rrna Sequences

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Construction and Characterization of Shuttle Vectors for Succinic Acid-Producing Rumen Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.01382-07 ◽

2007 ◽

Vol 73 (17) ◽

pp. 5411-5420 ◽

Cited By ~ 22

Author(s):

Yu-Sin Jang ◽

Young Ryul Jung ◽

Sang Yup Lee ◽

Ji Mahn Kim ◽

Jeong Wook Lee ◽

...

Keyword(s):

Succinic Acid ◽

Shuttle Vector ◽

Green Fluorescence Protein ◽

Expression Vectors ◽

Rumen Bacteria ◽

Gene Encoding ◽

Shuttle Vectors ◽

Genes Encoding ◽

Fluorescence Protein

ABSTRACT Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 × 106 and 7.1 × 106 transformants/μg DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.

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Cloning and characterization of a 150 kDa microsphere antigen of Theileria parva that is immunologically cross-reactive with the polymorphic immunodominant molecule (PIM)

Parasitology ◽

10.1017/s0031182098003163 ◽

1998 ◽

Vol 117 (4) ◽

pp. 321-330 ◽

Cited By ~ 21

Author(s):

R. A. SKILTON ◽

R. P. BISHOP ◽

C. W. WELLS ◽

P. R. SPOONER ◽

E. GOBRIGHT ◽

...

Keyword(s):

Immunoelectron Microscopy ◽

Signal Sequence ◽

Peptide Sequence ◽

Theileria Parva ◽

Repeat Region ◽

Gene Encoding ◽

Genes Encoding ◽

Immunodominant Antigens ◽

Proline Rich Region

To identify the genes encoding novel immunodominant antigens of Theileria parva a λgt11 library of piroplasm genomic DNA was immunoscreened with bovine recovery serum and a gene encoding a 150 kDa antigen (p150) was identified. The predicted polypeptide contains an N-terminal secretory signal sequence and a proline-rich region of repeated amino acid motifs. The repeat region is polymorphic between stocks of T. parva in both copy number and sequence, and analysis of the repeat region from 10 stocks of T. parva revealed 5 p150 variants. A monoclonal antibody (mAb) against the T. parva polymorphic immunodominant molecule (PIM) cross-reacted with the recombinant p150. The p150 has sequence homology with a PIM peptide sequence containing the anti-PIM mAb epitope. Immunoelectron microscopy demonstrated that the p150 antigen, like PIM, is located in the microspheres of the sporozoites and is exocytosed following sporozoite invasion of the host lymphocyte. By immunoelectron microscopy p150 was subsequently transiently detectable on the sporozoite surface and in the lymphocyte cytosol. Immunoblotting showed that p150 is also expressed by the schizont stage, but at much lower levels compared to the sporozoite. These results suggest a major role for p150 in the early events of host–sporozoite interaction.

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Cloning, nucleotide sequence and characterization of a gene encoding superoxide dismutase from Campylobacter jejuni and Campylobacter coli

Microbiology ◽

10.1099/13500872-140-5-1203 ◽

1994 ◽

Vol 140 (5) ◽

pp. 1203-1208 ◽

Cited By ~ 38

Author(s):

D. Purdy ◽

S. F. Park

Keyword(s):

Superoxide Dismutase ◽

Nucleotide Sequence ◽

Campylobacter Jejuni ◽

Campylobacter Coli ◽

Gene Encoding

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Two Alternative Pathways for the Synthesis of the Rare Compatible Solute Mannosylglucosylglycerate in Petrotoga mobilis

Journal of Bacteriology ◽

10.1128/jb.01424-09 ◽

2010 ◽

Vol 192 (6) ◽

pp. 1624-1633 ◽

Cited By ~ 16

Author(s):

Chantal Fernandes ◽

Vitor Mendes ◽

Joana Costa ◽

Nuno Empadinhas ◽

Carla Jorge ◽

...

Keyword(s):

Sequence Homology ◽

Thermotoga Maritima ◽

Functional Characterization ◽

Compatible Solute ◽

Homologous Gene ◽

Gene Encoding ◽

Alternative Pathways ◽

Cell Extracts ◽

Genes Encoding

ABSTRACT The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and d-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis.

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cumA Multicopper Oxidase Genes from Diverse Mn(II)-Oxidizing and Non-Mn(II)-OxidizingPseudomonas Strains

Applied and Environmental Microbiology ◽

10.1128/aem.67.9.4272-4278.2001 ◽

2001 ◽

Vol 67 (9) ◽

pp. 4272-4278 ◽

Cited By ~ 88

Author(s):

Chris A. Francis ◽

Bradley M. Tebo

Keyword(s):

Phylogenetic Analysis ◽

16S Rrna ◽

Multicopper Oxidase ◽

Copper Binding ◽

Gene Sequences ◽

Degenerate Primers ◽

Oxidase Gene ◽

Alternative Function ◽

Rrna Sequences ◽

16S Rrna Sequences

ABSTRACT A multicopper oxidase gene, cumA, required for Mn(II) oxidation was recently identified in Pseudomonas putida strain GB-1. In the present study, degenerate primers based on the putative copper-binding regions of the cumAgene product were used to PCR amplify cumA gene sequences from a variety of Pseudomonas strains, including both Mn(II)-oxidizing and non-Mn(II)-oxidizing strains. The presence of highly conserved cumA gene sequences in several apparently non-Mn(II)-oxidizing Pseudomonasstrains suggests that this gene may not be expressed, may not be sufficient alone to confer the ability to oxidize Mn(II), or may have an alternative function in these organisms. Phylogenetic analysis of both CumA and 16S rRNA sequences revealed similar topologies between the respective trees, including the presence of several distinct phylogenetic clusters. Overall, our results indicate that both thecumA gene and the capacity to oxidize Mn(II) occur in phylogenetically diverse Pseudomonas strains.

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Generation of Markerless Deletions in the Nosocomial PathogenClostridium difficileby Induction of DNA Double-Strand Breaks

Applied and Environmental Microbiology ◽

10.1128/aem.02055-18 ◽

2018 ◽

Vol 85 (3) ◽

Cited By ~ 3

Author(s):

Elena-Stella Theophilou ◽

Prerna Vohra ◽

Maurice P. Gallagher ◽

Ian R. Poxton ◽

Garry W. Blakely

Keyword(s):

Clostridium Difficile ◽

Regulatory Elements ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Gene Encoding ◽

Strand Breaks ◽

Allelic Exchange ◽

Genes Encoding

ABSTRACTClostridium difficileis an important nosocomial pathogen associated with potentially fatal disease induced by the use of antibiotics. Genetic characterization of such clinically important bacteria is often hampered by lack of availability of suitable tools. Here, we describe the use of I-SceI to induce DNA double-strand breaks, which increase the frequency of allelic exchange and enable the generation of markerless deletions inC. difficile. The usefulness of the system is illustrated by the deletion of genes encoding putative AddAB homologues. The ΔaddABmutants are sensitive to ultraviolet light and the antibiotic metronidazole, indicating a role in homologous recombination and the repair of DNA breaks. Despite the impairment in recombination, the mutants are still proficient for induction of the SOS response. In addition, deletion of thefliCgene, and subsequent complementation, reveals the importance of potential regulatory elements required for expression of a downstream gene encoding the flagellin glycosyltransferase.IMPORTANCEMost sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution inClostridium difficile. Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions inC. difficileand expands the toolbox available for genetic analysis of this important anaerobic pathogen.

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