Subject(s)
Bacterial Proteins/chemistry , Campylobacter lari/chemistry , Hexosyltransferases/chemistry , Lipopolysaccharides/chemistry , Membrane Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Campylobacter lari/enzymology , Campylobacter lari/genetics , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Glycosylation , Hexosyltransferases/genetics , Hexosyltransferases/metabolism , Kinetics , Lipopolysaccharides/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Molecular , Peptides/chemistry , Peptides/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
Full-length tlyA gene and its adjacent genetic loci from the urease-positive thermophilic Campylobacter (UPTC) CF89-12 [approximately 15,000 base pairs (bp) in length], as well as a reference strain Campylobacter lari RM2100 (approximately 9,000 bp), were analyzed. The possible open-reading frame of tlyA from UPTC CF89-12 was shown to have 720 bp with a calculated molecular mass of approximately 26.7 kDa. Using a primer pair designed in silico, a total of approximately 1.1 kbp consisting of putative promoter region, structural gene for tlyA, and its adjacent genetic loci were identified in all 17 C. lari isolates [n = 13 for UPTC; n = 4 for urease-negative (UN) C. lari]. Although sequence differences were demonstrated at approximately 20 loci within the 90 bp non-coding (NC) region, including the putative promoter structure candidates immediately upstream of the tlyA gene among the 18 isolates including C. lari RM2100, no sequence differences were identified within the NC region among the five UN C. lari isolates examined. A start codon ATG and a probable ribosome-binding site, AGGC(T)GG(A), for the tlyA gene were identified in all 18 isolates, including C. lari RM2100. The putative intrinsic ρ-independent transcriptional terminator structure candidate was also identified for the tlyA gene in both UPTC CF89-12 and C. lari RM2100. Additionally, the hemolysis assay was performed with some of the C. lari isolates. The tlyA gene nucleotide sequence data may possibly be useful for discrimination between UN C. lari and UPTC organisms, as well as for the differentiation among the four thermophilic Campylobacter species.
Subject(s)
Bacterial Proteins/genetics , Campylobacter Infections/microbiology , Campylobacter Infections/veterinary , Campylobacter lari/genetics , Hemolysin Proteins/genetics , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Base Sequence , Birds/microbiology , Bivalvia/microbiology , Campylobacter lari/chemistry , Campylobacter lari/classification , Campylobacter lari/isolation & purification , Chickens/microbiology , Environmental Microbiology , Hemolysin Proteins/chemistry , Molecular Sequence Data , Open Reading Frames , Phylogeny , Promoter Regions, Genetic , Sequence AlignmentABSTRACT
Amplified fragment-length polymorphism analysis (AFLP) has been shown to be a suitable method for subtyping of bacteria belonging to the genus Campylobacter. Campylobacter lari is a phenotypically and genotypically diverse species that comprises the classical nalidixic acid-resistant thermophilic campylobacters and the biochemical C. lari variants, urease-positive, nalidixic acid-susceptible, and urease producing nalidixic acid-susceptible strains. AFLP profiling and whole-cell protein profile analysis are suitable methods for studying the taxonomic and epidemiological relationships among strains of the C. lari variants. Numerical analysis of AFLP profiles and of partial protein profiles allows the discrimination of distinct C. lari genogroups. No correlation of these genogroups with different sources of the strains has been identified until now.
Subject(s)
Bacterial Proteins/analysis , Campylobacter lari/chemistry , Campylobacter lari/classification , Polymorphism, Restriction Fragment Length , Animals , Bacterial Typing Techniques , Campylobacter lari/genetics , Campylobacter lari/metabolism , DNA, Bacterial/analysis , Humans , Reproducibility of Results , Species SpecificityABSTRACT
Multiple strains of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis isolated from animal, clinical, or food samples have been analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Whole bacterial cells were harvested from colonies or confluent growth on agar and transferred directly into solvent and then to a spot of dried 3-methoxy-4-hydroxycinnamic acid (matrix). Multiple ions in the 5,000- to 15,000-Da mass range were evident in spectra for each strain; one or two ions in the 9,500- to 11,000-Da range were consistently high intensity. "Species-identifying" biomarker ions (SIBIs) were evident from analyses of multiple reference strains for each of the six species, including the genome strains C. jejuni NCTC 11168 and C. jejuni RM1221. Strains grown on nine different combinations of media and atmospheres yielded SIBI masses within +/-5 Da with external instrument calibration. The highest-intensity C. jejuni SIBIs were cytosolic proteins, including GroES, HU/HCj, and RplL. Multiple intraspecies SIBIs, corresponding probably to nonsynonymous nucleotide polymorphisms, also provided some intraspecies strain differentiation. MALDI-TOF MS analysis of 75 additional Campylobacter strains isolated from humans, poultry, swine, dogs, and cats revealed (i) associations of SIBI type with source, (ii) strains previously speciated incorrectly, and (iii) "strains" composed of more than one species. MALDI-TOF MS provides an accurate, sensitive, and rapid method for identification of multiple Campylobacter species relevant to public health and food safety.
