DNase test as a novel approach for the routine screening of Corynebacterium diphtheriae

Aims: To examine the value of the DNase test as an alternative procedure for differentiating Corynebacterium diphtheriae from Corynebacterium‐like colonies. Methods and Results: DNase test medium was inoculated by spotting a loopful of bacterial growth and incubated aerobically at 37°C. The DNase production was detectable following both 24 and 48 h incubation periods. The DNase activity was detected in all 91 C. diphtheriae (37 toxigenic and 54 nontoxigenic) strains examined, previously identified by both conventional biochemical methods and API Coryne System. Conversely, DNase test results were negative in 93·9% of the 564 nondiphtherial Gram‐positive rod clinical strains. Conclusions: The DNase test emerged as an easily interpretable and cost‐effective alternative screening procedure for C. diphtheriae laboratory identification. Significance and Impact of the Study: The method should facilitate routine laboratory diagnosis of toxigenic and nontoxigenic C. diphtheriae.     

Identification of the emerging skin pathogen Corynebacterium amycolatum using PCR-amplification of the essential divIVA gene as a target

The actinomycete Corynebacterium amycolatum is a saprophytic bacterium usually associated with the human skin, but it is at present considered an emergent pathogen as it is isolated from nosocomial settings from samples of immunosuppressed patients. The conventional method to distinguish C. amycolatum from closely related species is mainly based on phenotypic or chemotaxonomic studies. We developed a molecular method to identify rapidly C. amycolatum based on the use of different primers for amplification of the cell division divIVA gene using conventional or real-time PCR. This technique was used for the first time to distinguish C. amycolatum from the closely related Corynebacterium striatum, Corynebacterium minutissimum and Corynebacterium xerosis, without the requirement of further molecular analysis. The suitability of the identification method was tested on 51 clinical isolates belonging to the nonlipophilic fermentative group of corynebacteria (cluster C. striatum/C. amycolatum), which were accurately characterized by sequencing a 0.8 kb fragment of the 16S rRNA gene.     

Rapid and informative assays for Yd2, the barley yellow dwarf virus resistance gene, based on the nucleotide sequence of a closely linked gene

This paper describes the isolation of the cDNA encoding a protein previously shown to be indicative of the disease-resistance phenotype mediated by the Yd2 gene in barley (Hordeum vulgare L.). Amino acid sequences of four peptides obtained after isolation of the protein on two-dimensional polyacrylamide gels were completely homologous to sequences occurring within subunit E of barley vacuolar proton-translocating ATPase. Nucleotide sequence data of cloned cDNAs from both Yd2 and non-Yd2 barley varieties showed an amino acid change arising from a single-base-pair polymorphism. This was predicted to result in the shift in isoelectric point used previously to differentiate the protein in Yd2 and non-Yd2 barleys. Earlier work had indicated very close linkage between the gene from which this cDNA is derived, which we have named Ylp, and Yd2, the barley yellow dwarf virus resistance gene. We report here the development of PCR-based assays which discriminate between the two alleles of Ylp and thereby act as valuable predictors of Yd2 for barley breeders and others looking to study this important gene in cereal crops. The validity of each assay was tested with an extensive survey of over 100 barley varieties currently under cultivation in Australia or of importance to Australian barley breeding programmes. Complete agreement was observed between the allele of Ylp detected by the assay and the known Yd2 status of the barleys. A dominant PCR marker for the Yd2-associated allele of Ylp was subsequently developed using an allele-specific primer pair. This fast and economical assay will have broad application in the marker-assisted selection of Yd2-containing lines.     

Cloning and nucleotide sequence of the mphB gene for macrolide 2'-phosphotransferase II in Escherichia coli

Abstract Macrolide 2'-phosphotransferase II [MPH(2')II] inactivates macrolide antibiotics. The mphB gene for MPH(2')II was cloned from Escherichia coli and sequenced. Analysis of the nucleotide sequence indicated that mphB encoded a protein of 302 amino acids with a molecular mass of 34483 Da. The carboxy-terminal region of the deduced protein contained a sequence that resembled a conserved functional domain in aminoglycoside phosphotransferases.     

Analysis of DNA from a Beta procumbens chromosome fragment in sugar beet carrying a gene for nematode resistance

Summary We have begun to apply techniques for the preparation and anaylsis of large DNA segments from sugar beet (Beta vulgaris) addition lines carrying a mitotically stable chromosome fragment from B. procumbens that confers monogenic resistance to the nematode Heterodera schachtii, with a view towards isolating the resistance gene. DNA probes specific for this chromosome fragment were selected, and various methods for cloning genome-specific fragments, including probes from megabase DNA separated in pulsed-field slab gels, are compared. Probes that display high homology to B. procumbens have been used for hybridization of a representative genomic library and for initial step in mapping the chromosome fragment via pulsed-field gel electrophoresis after restriction with infrequently cutting enzymes. Our data indicate that DNA molecules from the entire chomosome fragment can be separated from protoplast DNA lysates.     

Identification of a deletion in the LDL receptor gene A Finnish type of mutation

A cDNA probe for the low density lipoprotein (LDL) receptor gene was used to screen DNA samples from 52 unrelated Finnish patients with the heterozygous form of familial hypercholesterolemia (FH) and 51 healthy controls. Southern blot analysis using the restriction enzyme PvuII revealed an abnormal 11 kb (kilo base-pair) restriction fragment in 16 (31%) of the patients but none of the controls. A more detailed restriction enzyme analysis of the DNA from patients revealed a mutation which apparently is due to an 8 kb deletion extending from intron 15 to exon 18 of the LDL receptor gene. Co-segregation of FH with the mutated gene was demonstrated in three families. These data are consistent with a ‘founder gene effect’ and support the assumption that recombinant DNA methods may have great impact on the diagnostics of FH in genetically homogeneous populations.     

Identification of resistance gene analogs linked to a powdery mildew resistance locus in grapevine

Oligonucleotide primers, designed to conserved regions of nucleotide binding site (NBS) motifs within previously cloned pathogen resistance genes, were used to amplify resistance gene analogs (RGAs) from grapevine. Twenty eight unique grapevine RGA sequences were identified and subdivided into 22 groups on the basis of nucleic acid sequence-identity of approximately 70% or greater. Representatives from each group were used in a bulked segregant analysis strategy to screen for restriction fragment length polymorphisms linked to the powdery mildew resistance locus, Run1, introgressed into Vitis vinifera L. from the wild grape species Muscadinia rotundifolia. Three RGA markers were found to be tightly linked to the Run1 locus. Of these markers, two (GLP1–12 and MHD145) cosegregated with the resistance phenotype in 167 progeny tested, whereas the third marker (MHD98) was mapped to a position 2.4 cM from the Run1 locus. The results demonstrate the usefulness of RGA sequences, when used in combination with bulked segregant analysis, to rapidly generate markers tightly linked to resistance loci in crop species. Received: 2 May 2001 / Accepted: 3 August 2001     

Identification of a STS marker linked to the Aegilops speltoides-derived leaf rust resistance gene Lr28 in wheat

 A sequence-tagged-site (STS) marker is reported linked to Lr28, a leaf rust resistance gene in wheat. RAPD (random amplified polymorphic DNA) analysis of near-isogenic lines (NILs) of Lr28 in eight varietal backgrounds was carried out using random primers. Genomic DNA enriched for low-copy sequences was used for RAPD analysis to overcome the lack of reproducibility due to the highly repetitive DNA sequences present in wheat. Of 80 random primers tested on the enriched DNA, one RAPD marker distinguished the NILs and the donor parent from the susceptible recurrent parents. The additional band present in resistant lines was cloned, sequenced, and STS primers specific for Lr28 were designed. The STS marker (Indian patent pending: 380 Del98) was further confirmed by bulk segregation analysis of F₃ families. It was consistently present in the NILs, the resistant F₃ bulk and the resistant F₃ lines, but was absent in recurrent parents, the susceptible F₃ bulk and the susceptible F₃ lines. Received: 20 February 1998 / Accepted: 4 March 1998     

The Corynebacterium glutamicum gene pmt encoding a glycosyltransferase related to eukaryotic protein-O-mannosyltransferases is essential for glycosylation of the resuscitation promoting factor (Rpf2) and other secreted proteins

Two-dimensional gel electrophoresis and immunoassays revealed several proteins of the secretory subproteome of Corynebacterium glutamicum to be glycosylated. By genome-wide searches for genes involved in glycosylation, the C. glutamicum gene cg1014 was found to exhibit significant similarity to eukaryotic protein-O-mannosyltransferases (PMTs) and to a recently identified orthologue of Mycobacterium tuberculosis, Rv1002c, which is responsible for protein-O-mannosylation. The putative membrane protein Cg1014 showed the same predicted transmembrane topology as Saccharomyces cerevisiae PMT1 and M. tuberculosis Rv1002c along with conserved amino acid residues responsible for catalytic activity. Deletion of the C. glutamicum pmt gene (cg1014) caused a complete loss of glycosylation of secreted proteins including the resuscitation promoting factor 2 (Rpf2), which is involved in intercellular communication and growth stimulation of C. glutamicum. Because the gene pmt as well as rpf genes are present in the genomes of all actinobacteria sequenced so far, this work provides new insights into bacterial protein glycosylation and new opportunities to elucidate the molecular mechanisms of Rpf activity in pathogenic growth and infection.     

Identification of a novel fosfomycin resistance gene (fosA2) in Enterobacter cloacae from the Salmon River, Canada

Aims: To investigate the occurrence of fosfomycin‐resistant (fos^R) bacteria in aquatic environments. Methods and Results: A fos^R strain of Enterobacter cloacae was isolated from a water sample collected at a site (50°41′33·44″N, 119°19′49·50″W) near the mouth of the Salmon River at Salmon Arm, in south‐central British Columbia, Canada. The strain was identified by PCR screening for plasmid‐borne, fosA‐family amplicons, followed by selective plating. Sequencing of the resistance gene cloned using PCR primers to conserved flanking DNA revealed a new allele (95% amino acid identity to fosA), and I‐Ceu I PFGE showed that it was chromosomally located. In Escherichia coli, the cloned DNA conferred a greater resistance to fosfomycin than its fosA counterpart. Conclusions: Gene fosA2 conferred fosfomycin resistance in an environmental isolate of Ent. cloacae. Significance and Impact of the Study: The repurposing of older antibiotics should be considered in the light of existing reservoirs of resistance genes in the environment.     

Identification of an amino acid residue required for differential recognition of a viral movement protein by the Tomato mosaic virus resistance gene Tm-2(2)

The Tm-2 gene of tomato and its allelic gene, Tm-2², confer resistance to Tomato mosaic virus (ToMV) and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class of plant resistance (R) genes. Despite exhibiting only four amino acid differences between the products of Tm-2 and Tm-2², Tm-2² confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7. An Agrobacterium-mediated transient expression system was used to study the mechanism of differential recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7 by Tm-2 and Tm-2². Although resistance induced by Tm-2 and Tm-2² is not usually accompanied by hypersensitive response (HR), Tm-2 and Tm-2² induced HR-like cell death by co-expression with MP of a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-2² but not Tm-2 induced cell death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR of Tm-2² is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-2² are involved in HR cell death.