Campylobacter jejuni, from the delta-epsilon group of proteobacteria, is a microaerophilic, Gram-negative, flagellate, spiral bacterium—properties it shares with the related gastric pathogen Helicobacter pylori. It is the leading cause of bacterial food-borne diarrhoeal disease throughout the world1. In addition, infection with C. jejuni is the most frequent antecedent to a form of neuromuscular paralysis known as Guillain–Barre syndrome2. Here we report the genome sequence of C. jejuni NCTC11168. C. jejuni has a circular chromosome of 1,641,481 base pairs (30.6% G+C) which is predicted to encode 1,654 proteins and 54 stable RNA species. The genome is unusual in that there are virtually no insertion sequences or phage-associated sequences and very few repeat sequences. One of the most striking findings in the genome was the presence of hypervariable sequences. These short homopolymeric runs of nucleotides were commonly found in genes encoding the biosynthesis or modification of surface structures, or in closely linked genes of unknown function. The apparently high rate of variation of these homopolymeric tracts may be important in the survival strategy of C. jejuni.

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The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences

Rapid cycle DNA amplification was continuously monitored by three different fluorescence techniques. Fluorescence was monitored by (i) the double-strand-specific dye SYBR Green I, (ii) a decrease in fluorescein quenching by rhodamine after exonuclease cleavage of a dual-labeled hydrolysis probe and (iii) resonance energy transfer of fluorescein to Cy5 by adjacent hybridization probes. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy number. The sensitivity of SYBR Green I detection is limited by nonspecific product formation. Use of a single exonuclease hydrolysis probe or two adjacent hybridization probes offers increasing levels of specificity. In contrast to fluorescence measurement once per cycle, continuous monitoring throughout each cycle monitors the temperature dependence of fluorescence. The cumulative, irreversible signal of hydrolysis probes can be distinguished easily from the temperature-dependent, reversible signal of hybridization probes. By using SYBR Green I, product denaturation, annealing and extension can be followed within each cycle. Substantial product-to-product annealing occurs during later amplification cycles, suggesting that product annealing is a major cause of the plateau effect. Continuous within-cycle monitoring allows rapid optimization of amplification conditions and should be particularly useful in developing new, standardized clinical assays.

BioTechniques 30th Anniversary GEM Continuous Fluorescence Monitoring of Rapid Cycle DNA Amplification

In a recent study, immunoglobulin G in human plasma was identified as a major inhibitor of diagnostic PCR (W. Abu Al-Soud, L. J. Jonsson, and P. Radstrom. J. Clin. Microbiol. 38:345–350, 2000). In this study, two major PCR inhibitors in human blood cells were purified using size exclusion and anion-exchange chromatographic procedures. Based on N-terminal amino acid sequencing and electrophoretic analysis of the purified polypeptides, hemoglobin and lactoferrin were identified as PCR-inhibitor components in erythrocytes and leukocytes, respectively. When different concentrations of hemoglobin or lactoferrin were added to PCR mixtures of 25 μl containing 10 different thermostable DNA polymerases and 1 ng of Listeria monocytogenes DNA as template DNA, AmpliTaq Gold, Pwo, and Ultma were inhibited in the presence of ≤1.3 μg of hemoglobin and ≤25 ng of lactoferrin, while rTth and Tli were found to resist inhibition of at least 100 μg of hemoglobin. In addition, the quantitative effects of seven low-molecular-mass inhibitors, present in blood samples or degradation products of hemoglobin, on real-time DNA synthesis of rTth using the LightCycler Instrument were investigated. A reaction system based on a single-stranded poly(dA) template with an oligo(dT) primer annealed to the 3′ end was used. It was found that the addition of 0.25 to 0.1 mg of bile per ml, 2.5 mM CaCl2, 0.25 mM EDTA, 5 μM FeCl3, and 0.01 IU of heparin per ml reduced the fluorescence to approximately 76, 70, 46, 17, and 51%, respectively. Finally, the effects of nine amplification facilitators were studied in the presence of hemoglobin and lactoferrin. Bovine serum albumin (BSA) was the most efficient amplification facilitator, so that the addition of 0.4% (wt/vol) BSA allowed AmpliTaq Gold to amplify DNA in the presence of 20 instead of 1 μg of hemoglobin and 500 instead of 5 ng of lactoferrin. Including 0.02% (wt/vol) gp32, a single-stranded-DNA binding protein, in the reaction mixture of AmpliTaq Gold was also found to reduce the inhibitory effects of hemoglobin and lactoferrin.

Purification and Characterization of PCR-Inhibitory Components in Blood Cells

Background
Gene duplications have a major role in the evolution of new biological functions. Theoretical studies often assume that a duplication per se is selectively neutral and that, following a duplication, one of the gene copies is freed from purifying (stabilizing) selection, which creates the potential for evolution of a new function.

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Selection in the evolution of gene duplications

Since the eradication of polio in most parts of the world, Guillain-Barre syndrome (GBS) has become the most common cause of acute flaccid paralysis. GBS is an autoimmune disorder of the peripheral nervous system characterized by weakness, usually symmetrical, evolving over a period of several days or more. Since laboratories began to isolate Campylobacter species from stool specimens some 20 years ago, there have been many reports of GBS following Campylobacter infection. Only during the past few years has strong evidence supporting this association developed. Campylobacter infection is now known as the single most identifiable antecedent infection associated with the development of GBS. Campylobacter is thought to cause this autoimmune disease through a mechanism called molecular mimicry, whereby Campylobacter contains ganglioside-like epitopes in the lipopolysaccharide moiety that elicit autoantibodies reacting with peripheral nerve targets. Campylobacter is associated with several pathologic forms of GBS, including the demyelinating (acute inflammatory demyelinating polyneuropathy) and axonal (acute motor axonal neuropathy) forms. Different strains of Campylobacter as well as host factors likely play an important role in determining who develops GBS as well as the nerve targets for the host immune attack of peripheral nerves. The purpose of this review is to summarize our current knowledge about the clinical, epidemiological, pathogenetic, and laboratory aspects of campylobacter-associated GBS.

https://www.ccrc.uga.edu/~rcarlson/GBSyn.pdf

Campylobacter Species and Guillain-Barré Syndrome

Construction of new Campylobacter cloning vectors and a new mutational cat cassette.

Campylobacter coli VC167 T2 has two flagellin genes, flaA and flaB, which share 91.9% sequence identity. The flaA gene is transcribed from a o-28 promoter, and the flaB gene from a o-54 promoter. Gene replacement mutagenesis techniques were used to generate flaA+ flaB and flaA flaB+ mutants. Both gene products are capable of assembling independently into functional filaments. A flagellar filament composed exclusively of the flaA gene product is indistinguishable in length from that of the wild type and shows a slight reduction in motility. The flagellar filament composed exclusively of the flaB gene product is severely truncated in length and greatly reduced in motility. Thus, while both flagellins are not necessary for motility, both products are required for a fully active flagellar filament. Although the wild-type flagellar filament is a heteropolymer of the flaA and flaB gene products, immunogold electron microscopy suggests that flaB epitopes are poorly surface exposed along the length of the wild-type filament.

Role of two flagellin genes in Campylobacter motility.

Two genes have been identified in Campylobacter coli VC167 which are required for the biosynthesis of post-translational modifications on flagellin proteins. The ptmA gene encodes a protein of predicted M(r) 28,486 which shows significant homology to a family of alcohol dehydrogenases from a variety of bacteria. The ptmB gene encodes a protein of predicted M(r) 26,598 with significant homology to CMP-N-acetylneuraminic acid synthetase enzymes involved in sialic acid capsular biosynthesis in Neisseria meninigitidis and Escherichia coli K1. Site-specific mutation of either ptmA or ptmB caused loss of reactivity with antisera specific to the post-translational modifications and a change in the isoelectric focusing fingerprints relative to the parent strains. Mutation of ptmB, but not of ptmA, caused a change in apparent M(r) of the flagellin subunit in SDS-PAGE gels. The ptmA and ptmB genes are present in other strains of Campylobacter. In a rabbit model the ptmA mutant showed a reduced ability to elicit protection against subsequent challenge with heterologous strains of the same Lior serotype compared to the parental wild-type strain. This suggests that the surface-exposed post-translational modifications may play a significant role in the protective immune response.

Identification and characterization of genes required for post-translational modification of Campylobacter coli VC167 flagellin.

The complex flagellum of Campylobacter coli VC167 is encoded by two tandemly oriented flagellin genes which are transcribed as two discrete transcriptional units from two different classes of promoters. The flaB gene, which encodes the minor FlaB filament protein, is controlled by a sigma 54 promoter. A transcriptional fusion between a promoterless chloramphenicol acetyltransferase (CAT) reporter gene cartridge and C. coli VC167 DNA carrying flaB transcription and translation signals, including the typical position -13-to-(-)26 flaB sigma 54 consensus promoter sequence, was constructed. When carried on plasmid pRIC1013, the sigma 54-CAT fusion expressed chloramphenicol resistance in Escherichia coli, and CAT production was affected by the pH of the growth medium, the composition of the growth atmosphere, and the growth temperature, with production being significantly higher at 42 degrees C. A conjugative suicide vector, pRIC1028, containing the sigma 54-CAT fusion was constructed and used to recombine the flaB-CAT fusion back into the C. coli chromosome in the correct position with respect to the flaA gene and its transcription terminator. CAT production from the flaB sigma 54 promoter in the C. coli transconjugant VC167-T2/28-1 was shown to peak at mid-log phase and to be modulated by growth medium pH, growth temperature, and the concentration of certain inorganic salts and divalent cations in the growth medium. Under growth conditions which promoted elevated flaB sigma 54 promoter activity, a flaA flaB+ mutant of C. coli VC167 produced increased amounts of FlaB flagellar protein and displayed increased motility.

The Campylobacter sigma 54 flaB flagellin promoter is subject to environmental regulation.

The complex flagellar filaments of the LIO8 serogroup member Campylobacter coli VC167 are composed of two highly related subunit proteins encoded by the flaA and flaB genes which share 92% identity. Using oligonucleotide primers based on the known DNA sequence of both the flaA and flaB genes from C. coli VC167 in the polymerase chain reaction, we have shown conservation of both fla genes among isolates within the LIO8 heat-labile serogroup by digestion of the amplified product with PstI and EcoRI restriction endonucleases. Amplification and subsequent restriction analysis of the flaA flagellin gene from Campylobacter isolates belonging to 13 different LIO serogroups further identified 10 unique polymorphic groups. Within most of the serogroups examined, isolates appeared to contain flaA genes with conserved primary structures. Only in serogroups LIO11 and LIO29 did independent isolates possess flagellin genes with different primary structures. Furthermore, by employing primers specific for the flaB gene of C. coli VC167, all serogroups examined contained a second fla gene corresponding to flaB. In all serogroups except the LIO5 and LIO6 isolates which were identical to each other, the polymorphic pattern of this flaB gene was identical to that of the corresponding flaA gene. These data indicate that the presence of a second highly homologous flagellin gene is widespread throughout Campylobacter isolates and that in most instances, the primary structure of the two fla genes is conserved within isolates belonging to the same heat-labile LIO serogroup. This may represent the presence of clonal evolutionary groups in Campylobacter spp.

R A Alm

Papers

Construction of new Campylobacter cloning vectors and a new mutational cat cassette.

Role of two flagellin genes in Campylobacter motility.

Identification and characterization of genes required for post-translational modification of Campylobacter coli VC167 flagellin.

The Campylobacter sigma 54 flaB flagellin promoter is subject to environmental regulation.

Distribution and polymorphism of the flagellin genes from isolates of Campylobacter coli and Campylobacter jejuni.