Molecular and in silico typing of the lipooligosaccharide biosynthesis gene cluster in Campylobacter jejuni and Campylobacter coli.

The extensive genetic variation in the lipooligosaccharide (LOS) core biosynthesis gene cluster has led to the development of a classification system; with 8 classes (I-VIII) for Campylobacter coli (C. coli) LOS region and with 23 classes (A-W) or four groups (1-4) for Campylobacter jejuni (C. jejuni) LOS region. PCR based LOS locus type identification for C. jejuni clinical isolates from a UK hospital as well as in silico LOS locus analysis for C. jejuni and C. coli genome sequences from GenBank was carried out to determine the frequencies of various LOS genotypes in C. jejuni and C. coli. Analysis of LOS gene content in 60 clinical C. jejuni isolates and 703 C. jejuni genome sequences revealed that class B (Group 1) was the most abundant LOS class in C. jejuni. The hierarchy of C. jejuni LOS group prevalence (group 1 > group 2 > group 3 > group 4) as well as the hierarchy of the frequency of C. jejuni LOS classes present within the group 1 (B > C > A > R > M > V), group 2 (H/P > O > E > W), group 3 (F > K > S) and group 4 (G > L) was identified. In silico analysis of LOS gene content in 564 C. coli genome sequences showed class III as the most abundant LOS locus type in C. coli. In silico analysis of LOS gene content also identified three novel LOS types of C. jejuni and previously unknown LOS biosynthesis genes in C. coli LOS locus types I, II, III, V and VIII. This study provides C. jejuni and C. coli LOS loci class frequencies in a smaller collection of C. jejuni clinical isolates as well as within the larger, worldwide database of C. jejuni and C. coli.

An Updated Classification System and Review of the Lipooligosaccharide Biosynthesis Gene Locus in Campylobacter jejuni.

Lipooligosaccharide (LOS) is an integral component of the Campylobacter cell membrane with a structure of core oligosaccharides forming inner and outer core regions and a lipid A moiety. The gene content of the LOS core biosynthesis cluster exhibits extensive sequence variation, which leads to the production of variable cell surface LOS structures in Campylobacter. Some LOS outer core molecules in Campylobacter jejuni are molecular mimics of host structures (such as neuronal gangliosides) and are thought to trigger neuronal disorders (particularly Guillain-Barré syndrome and Miller Fisher syndrome) in humans. The extensive genetic variation in the LOS biosynthesis gene cluster, a majority of which occurs in the LOS outer core biosynthesis gene content present between lgtF and waaV, has led to the development of a classification system with 23 classes (A-W) and four groups (1-4) for the C. jejuni LOS region. This review presents an updated and simplified classification system for LOS typing alongside an overview of the frequency of C. jejuni LOS biosynthesis genotypes and structures in various C. jejuni populations.

RitR is an archetype for a novel family of redox sensors in the streptococci that has evolved from two-component response regulators and is required for pneumococcal colonization.

To survive diverse host environments, the human pathogen Streptococcus pneumoniae must prevent its self-produced, extremely high levels of peroxide from reacting with intracellular iron. However, the regulatory mechanism(s) by which the pneumococcus accomplishes this balance remains largely enigmatic, as this pathogen and other related streptococci lack all known redox-sensing transcription factors. Here we describe a two-component-derived response regulator, RitR, as the archetype for a novel family of redox sensors in a subset of streptococcal species. We show that RitR works to both repress iron transport and enable nasopharyngeal colonization through a mechanism that exploits a single cysteine (Cys128) redox switch located within its linker domain. Biochemical experiments and phylogenetics reveal that RitR has diverged from the canonical two-component virulence regulator CovR to instead dimerize and bind DNA only upon Cys128 oxidation in air-rich environments. Atomic structures show that Cys128 oxidation initiates a "helical unravelling" of the RitR linker region, suggesting a mechanism by which the DNA-binding domain is then released to interact with its cognate regulatory DNA. Expanded computational studies indicate this mechanism could be shared by many microbial species outside the streptococcus genus.

High Throughput Method for Analysis of Repeat Number for 28 Phase Variable Loci of Campylobacter jejuni Strain NCTC11168.

Mutations in simple sequence repeat tracts are a major mechanism of phase variation in several bacterial species including Campylobacter jejuni. Changes in repeat number of tracts located within the reading frame can produce a high frequency of reversible switches in gene expression between ON and OFF states. The genome of C. jejuni strain NCTC11168 contains 29 loci with polyG/polyC tracts of seven or more repeats. This protocol outlines a method-the 28-locus-CJ11168 PV-analysis assay-for rapidly determining ON/OFF states of 28 of these phase-variable loci in a large number of individual colonies from C. jejuni strain NCTC11168. The method combines a series of multiplex PCR assays with a fragment analysis assay and automated extraction of fragment length, repeat number and expression state. This high throughput, multiplex assay has utility for detecting shifts in phase variation states within and between populations over time and for exploring the effects of phase variation on adaptation to differing selective pressures. Application of this method to analysis of the 28 polyG/polyC tracts in 90 C. jejuni colonies detected a 2.5-fold increase in slippage products as tracts lengthened from G8 to G11 but no difference between tracts of similar length indicating that flanking sequence does not influence slippage rates. Comparison of this observed slippage to previously measured mutation rates for G8 and G11 tracts in C. jejuni indicates that PCR amplification of a DNA sample will over-estimate phase variation frequencies by 20-35-fold. An important output of the 28-locus-CJ11168 PV-analysis assay is combinatorial expression states that cannot be determined by other methods. This method can be adapted to analysis of phase variation in other C. jejuni strains and in a diverse range of bacterial species.

A novel Sit4 phosphatase complex is involved in the response to ceramide stress in yeast.

Ceramide is a building block for complex sphingolipids in the plasma membrane, but it also plays a significant role in secondary signalling pathways regulating cell proliferation and apoptosis in response to stress. Ceramide activated protein phosphatase activity has been previously observed in association with the Sit4 protein phosphatase. Here we find that sit4Δ mutants have decreased ceramide levels and display resistance to exogenous ceramides and phytosphingosine. Mutants lacking SIT4 or KTI12 display a shift towards nonhydroxylated forms of long chain bases and sphingolipids, suggesting regulation of hydroxylase (SUR2) or ceramide synthase by Sit4p. We have identified novel subunits of the Sit4 complex and have also shown that known Sit4 regulatory subunits-SAP proteins-are not involved in the ceramide response. This is the first observation of separation of function between Sit4 and SAP proteins. We also find that the Sit4p target Elongator is not involved in the ceramide response but that cells deficient in Kti12p-an accessory protein with an undefined regulatory role-have similar ceramide phenotypes to sit4Δ mutants. Therefore, Kti12p may play a similar secondary role in the ceramide response. This evidence points to a novel Sit4-dependent regulatory mechanism in response to ceramide stress.

Ferric reductase genes involved in high-affinity iron uptake are differentially regulated in yeast and hyphae of Candida albicans.

The pathogenic yeast Candida albicans possesses a reductive iron uptake system which is active in iron-restricted conditions. The sequestration of iron by this mechanism initially requires the reduction of free iron to the soluble ferrous form, which is catalysed by ferric reductase proteins. Reduced iron is then taken up into the cell by a complex of a multicopper oxidase protein and an iron transport protein. Multicopper oxidase proteins require copper to function and so reductive iron and copper uptake are inextricably linked. It has previously been established that Fre10 is the major cell surface ferric reductase in C. albicans and that transcription of FRE10 is regulated in response to iron levels. We demonstrate here that Fre10 is also a cupric reductase and that Fre7 also makes a significant contribution to cell surface ferric and cupric reductase activity. It is also shown, for the first time, that transcription of FRE10 and FRE7 is lower in hyphae compared to yeast and that this leads to a corresponding decrease in cell surface ferric, but not cupric, reductase activity. This demonstrates that the regulation of two virulence determinants, the reductive iron uptake system and the morphological form of C. albicans, are linked.

Copper-dependent transcriptional regulation by Candida albicans Mac1p.

We have previously shown that copper uptake and regulation in the opportunistic pathogen Candida albicans has some similarities to those in Saccharomyces cerevisiae, including the activation of the copper transporter gene CaCTR1 under low-copper conditions by the transcription factor CaMac1p. However, in this study, further analysis has shown that the actual mechanism of regulation by CaMac1p is different from that of its S. cerevisiae homologue. We demonstrate for the first time, to our knowledge, that the CaMAC1 gene is transcriptionally autoregulated in a copper-dependent manner, in contrast to ScMAC1, which is constitutively transcribed. We also demonstrate that the presence of one copper response element in the promoters of CaCTR1, CaMAC1 and the ferric/cupric reductase gene CaFRE7 is sufficient for normal levels of copper-responsive transcription. In contrast, two promoter elements are essential for normal levels of copper-dependent transcriptional activation by ScMac1p. CaMac1p is also involved in the regulation of the iron-responsive transcriptional repressor gene SFU1 and the alternative oxidase gene AOX2. This work describes a key feature of the copper uptake system in C. albicans that distinguishes it from similar processes in the model yeast S. cerevisiae. The importance of copper uptake in the environment of the human host and the implications for the disease process are discussed.