Dataset of the phospholipidome and transcriptome of Campylobacter jejuni under different growth conditions.

The membrane phospholipid composition is not a stable bacterial characteristic but can change in response to altered environmental conditions. Here we provide the dataset of the phospholipidome and transcriptome of the microaerophilic human pathogen Campylobacter jejuni under different environmental conditions. These data have been used in Cao (2020), The unique phospholipidome of the enteric pathogen C. jejuni: Lysolipids are required for motility at low oxygen availability. Here the abundance of each phospholipid is shown during the growth of C. jejuni for 0-108 h under low and high oxygen conditions (0.3 vs 10% O2). The phospholipid data were obtained by applying high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS). The transcriptomic data obtained by RNA-seq show the differential expressed genes between logarithmic and stationary grown bacteria. In addition, our data might serve as a reference information for further in-depth investigation to understand the relation between specific phospholipids and the activity of membrane associated proteins.

The Unique Phospholipidome of the Enteric Pathogen Campylobacter jejuni: Lysophosholipids Are Required for Motility at Low Oxygen Availability.

In response to changes in their environment bacteria need to change both their protein and phospholipid repertoire to match environmental requirements, but the dynamics of bacterial phospholipid composition under different growth conditions is still largely unknown. In the present study, we investigated the phospholipidome of the bacterial pathogen Campylobacter jejuni. Transcription profiling on logarithmic and stationary phase grown cells of the microaerophilic human pathogen C. jejuni using RNA-seq revealed differential expression of putative phospholipid biosynthesis genes. By applying high-performance liquid chromatography tandem-mass spectrometry, we identified 203 phospholipid species representing the first determination of the phospholipidome of this pathogen. We identified nine different phospholipid classes carrying between one and three acyl chains. Phospholipidome analysis on bacteria of different ages (0-5 days) showed rapid changes in the ratio of phospholipids containing ethanolamine, or glycerol as phospholipid head group and in the number of cyclopropane bond containing fatty acids. Oxygen concentration influenced the percentage of lysophospholipids, and cyclo-propane bonds containing acyl chains. We show that large amounts of the phospholipids are lysophospholipids (30-45%), which mutant studies reveal are needed for normal C. jejuni motility at low oxygen conditions. C. jejuni possesses an unusual phospholipidome that is highly dynamic in response to environmental changes.

Generation of the membrane potential and its impact on the motility, ATP production and growth in Campylobacter jejuni.

The generation of a membrane potential (Δψ), the major constituent of the proton motive force (pmf), is crucial for ATP synthesis, transport of nutrients and flagellar rotation. Campylobacter jejuni harbors a branched electron transport chain, enabling respiration with different electron donors and acceptors. Here, we demonstrate that a relatively high Δψ is only generated in the presence of either formate as electron donor or oxygen as electron acceptor, in combination with an acceptor/donor respectively. We show the necessity of the pmf for motility and growth of C. jejuni. ATP generation is not only accomplished by oxidative phosphorylation via the pmf, but also by substrate-level phosphorylation via the enzyme AckA. In response to a low oxygen tension, C. jejuni increases the transcription and activity of the donor complexes formate dehydrogenase (FdhABC) and hydrogenase (HydABCD) as well as the transcription of the alternative respiratory acceptor complexes. Our findings suggest that in the gut of warm-blooded animals, C. jejuni depends on at least formate or hydrogen as donor (in the anaerobic lumen) or oxygen as acceptor (near the epithelial cells) to generate a pmf that sustains efficient motility and growth for colonization and pathogenesis.

Function and Regulation of the C4-Dicarboxylate Transporters in Campylobacter jejuni.

C4-dicarboxylates are important molecules for the human pathogen C.jejuni, as they are used as carbon and electron acceptor molecules, as sugars cannot be utilized by this microaerophilic organism. Based on the genome analysis, C. jejuni may possess five different C4-dicarboxylate transporters: DctA, DcuA, DcuB, and two homologs of DcuC. Here, we investigated the regulation and function of various C4-dicarboxylate transporters in C. jejuni. Transcription of the dctA and dcuC homologs is constitutive, while dcuA and dcuB are both directly regulated by the two-component RacR/RacS system in response to limited oxygen availability and the presence of nitrate. The DctA transporter is the only C4-dicarboxylate transporter to allow C. jejuni to grow on C4-carbon sources such as aspartate, fumarate, and succinate at high oxygen levels (10% O2) and is indispensable for the uptake of succinate from the medium under these conditions. Both DcuA and DcuB can sequester aspartate from the medium under low-oxygen conditions (0.3% O2). However, under these conditions, DcuB is the only transporter to secrete succinate to the environment. Under low-oxygen conditions, nitrate prevents the secretion of succinate to the environment and was able to overrule the phenotype of the C4-transporter mutants, indicating that the activity of the aspartate-fumarate-succinate pathway in C. jejuni is strongly reduced by the addition of nitrate in the medium.

The Campylobacter jejuni†RacRS system regulates fumarate utilization in a low oxygen environment.

The natural environment of the human pathogen Campylobacter jejuni is the gastrointestinal tract of warm-blooded animals. In the gut, the availability of oxygen is limited; therefore, less efficient electron acceptors such as nitrate or fumarate are used by C. jejuni. The molecular mechanisms that regulate the activity of the highly branched respiratory chain of C. jejuni are still a mystery mainly because C. jejuni lacks homologues of transcription factors known to regulate energy metabolism in other bacteria. Here we demonstrate that dependent on the available electron acceptors the two-component system RacRS controls the production of fumarate from aspartate, as well as its transport and reduction to succinate. Transcription profiling, DNAse protection and functional assays showed that phosphorylated RacR binds to and represses at least five promoter elements located in front of genes involved in the uptake and synthesis of fumarate. The RacRS system is active in the presence of nitrate and trimethyl-amine-N-oxide under oxygen-limited conditions when fumarate is less preferred as an alternative electron acceptor. In the inactive state, RacRS allows utilization of fumarate for respiration. The unique C. jejuni RacRS regulatory system illustrates the disparate evolution of Campylobacter and aids the survival of this pathogen.

Regulation of energy metabolism by the extracytoplasmic function (ECF) σ factors of Arcobacter butzleri.

The extracytoplasmic function (ECF) σ factors are fundamental for bacterial adaptation to distinct environments and for survival under different stress conditions. The emerging pathogen Arcobacter butzleri possesses seven putative pairs of σ/anti-σ factors belonging to the ECF family. Here, we report the identification of the genes regulated by five out of the seven A. butzleri ECF σ factors. Three of the ECF σ factors play an apparent role in transport, energy generation and the maintenance of redox balance. Several genes like the nap, sox and tct genes are regulated by more than one ECF σ factor, indicating that the A. butzleri ECF σ factors form a network of overlapping regulons. In contrast to other eubacteria, these A. butzleri ECF regulons appear to primarily regulate responses to changing environments in order to meet metabolic needs instead of an obvious role in stress adaptation.

Growth phase-dependent activation of the DccRS regulon of Campylobacter jejuni.

Two-component systems are widespread prokaryotic signal transduction devices which allow the regulation of cellular functions in response to changing environmental conditions. The two-component system DccRS (Cj1223c-Cj1222c) of Campylobacter jejuni is important for the colonization of chickens. Here, we dissect the DccRS system in more detail and provide evidence that the sensor DccS selectively phosphorylates the cognate effector, DccR. Microarray expression profiling, real-time reverse transcription-PCR (RT-PCR), electrophoretic mobility shift assay, and primer extension analyses revealed that the DccRS regulon of strain 81116 consists of five promoter elements, all containing the consensus direct repeat sequence WTTCAC-N6-TTCACW covering the putative -35 promoter regions. One of these promoters is located in front of an operon encoding a putative macrolide efflux pump while the others are in front of genes coding for putative periplasmic or membrane proteins. The DccRS-regulated genes in C. jejuni strain 81116 are needed to enhance early in vivo growth of C. jejuni in 7-day-old chickens. The DccRS system is activated in the late stationary bacterial growth phase, probably by released metabolic products. Whole-genome mRNA profiling and real-time RT-PCR analysis under these conditions demonstrated that the system has no influence on the transcription of genes outside the DccRS regulon.

Temperature-dependent FlgM/FliA complex formation regulates Campylobacter jejuni flagella length.

Regulation of the biosynthesis of the flagellar filament in bacteria containing multiple flagellin genes is not well understood. The major food-borne pathogen Campylobacter jejuni possesses on both poles a flagellum that consists of two different flagellin subunits, FlaA and FlaB. Here we identify the protein Cj1464 as a regulator of C. jejuni flagellin biosynthesis. The protein shares characteristics of the FlgM family of anti-sigma factor proteins: it represses transcription of sigma(28)-dependent genes, forms a complex with sigma factor FliA, and is secreted through the flagellar filament. However, unlike other FlgM proteins, the interaction of C. jejuni FlgM with FliA is regulated by temperature and the protein does not inhibit FliA activity during the formation of the hook-basal body complex (HBB). Instead, C. jejuni FlgM limits the length of the flagellar filament by suppressing the synthesis of both the sigma(28)- and the sigma(54)-dependent flagellins. The main function of the C. jejuni FlgM therefore is not to silence sigma(28)-dependent genes until the HBB is completed, but to prevent unlimited elongation of the flagellum, which otherwise leads to reduced bacterial motility.

A cell-free assay for the functional analysis of variants of the mismatch repair protein MLH1.

The hereditary colon and endometrium cancer predisposition Lynch Syndrome (also called HNPCC) is caused by a germ-line mutation in one of the DNA mismatch repair (MMR) genes. A significant fraction of the gene alterations detected in suspected Lynch Syndrome patients is comprised of amino acid substitutions. The relevance for cancer risk of these variants is difficult to assess, as currently no time- and cost-effective, validated, and widely applicable functional assays for the measurement of MMR activity are available. Here we describe a rapid, cell-free, and easily quantifiable MMR activity assay for the diagnostic assessment of variants of the MLH1 MMR protein. This assay allows the parallel generation and functional analysis of a series of variants of the MLH1 protein in vitro using readily available, or preprepared, reagents. Using this assay we have tested 26 MLH1 variants and of these, 15 had lost activity. These results are in concordance with those obtained from first-generation assays and with in silico and pathology data. After its multifocal technical and clinical validation this assay could have great impact for the diagnosis and counseling of carriers of an MLH1 variant and their relatives.

Functional analysis of a Campylobacter jejuni alkaline phosphatase secreted via the Tat export machinery.

Bacterial alkaline phosphatases (PhoA) hydrolyse phosphate-containing substrates to provide the preferred phosphorus source inorganic phosphate (P(i)). Campylobacter jejuni does not contain a typical PhoA homologue but contains a phosphatase that is regulated by the two-component system PhosS/PhosR. Here we describe the characterization of the enzyme, its secretion pathway and its function in the bacterium's biology. Phosphatase assays showed that the enzyme utilizes exclusively phosphomonoesters as a substrate, requires Ca(2+) for its activity, and displays maximum activity at a pH of 10. Gene disruption revealed that it is the sole alkaline phosphatase in C. jejuni. The protein contained a twin-arginine motif (RR) at its N terminus, typical of substrates of the Tat secretion system. Substitution of the twin-arginine residues showed that they are essential for enzyme activity. C. jejuni genome analysis indicated the presence of four ubiquitously expressed Tat components that may form a functional Tat secretion system as well as 11 putative Tat substrates, including the alkaline phosphatase (PhoA(Cj)) and the nitrate reductase NapA. Inactivation of tatC caused defects in both PhoA(Cj) and NapA activity as well as a reduction in bacterial growth that were all restored by complementation in trans with an intact tatC copy. The atypical overall features of the PhoA(Cj) compared to Escherichia coli PhoA support the existence in prokaryotes of a separate group of Tat-dependent alkaline phosphatases, classified as the PhoX family.

The Campylobacter jejuni PhosS/PhosR operon represents a non-classical phosphate-sensitive two-component system.

The bacterial pathogen Campylobacter jejuni carries several putative two-component signal transduction systems of unknown function. Here we report that the PhosS (Cj0889) and PhosR (Cj0890) proteins constitute a two-component system that is activated by phosphate limitation. Microarray analysis, real-time RT-PCR, and primer extension experiments indicated that this system regulates 12 genes (including the pstSCAB genes) present in three transcriptional units. Gel shift assays confirmed that recombinant PhosR protein bound DNA fragments containing the promoter regions upstream of these three transcriptional units. Although functionally similar, the PhosS/PhosR does not exhibit sequence homology with the classical PhoBR systems, has a different pho box (5'-GTTTCNAAAANGTTTC-3') recognized by the C. jejuni response regulator, and is not autoregulated. Because of these atypical properties, we designated the Cj0889-Cj0890 operon as the C. jejuni PhosS/PhosR system (phosphate sensor/phosphate response regulator) and the phosphate-regulated genes as the pho regulon of C. jejuni.

Antigenic variation within the subunit protein of members of the colonization factor antigen I group of fimbrial proteins in human enterotoxigenic Escherichia coli.

Colonization factor antigens (CFAs) of human enterotoxigenic Escherichia coli can be divided in groups based on the N-terminal amino acid sequence of their major subunit protein. One of the groups that has been distinguished in this way, is the CFA/I group of fimbriae. The sequence of the fimbrial subunit genes in the operons encoding the antigens CS4, CS14 and CS17, all members of this group, was determined. A duplication of the fimbrial subunit gene (csuA) was found in the CS14 operon, both genes encoding very similar proteins. Purified CS14 fimbriae consist of two proteins with different molecular masses (15.5 and 17.0 kDa) but identical N-terminal amino acid sequences, which strongly suggests that both csuA genes are transcribed. A phylogenetic tree derived from the amino acid sequences of the CFA/I, CS1, CS2, CS4, CS14, CS17 and CS19 subunit proteins shows that CS1, CS17 and CS19 belong to the same subgroup. CFA/I, CS4 and CS14 belong to a second subgroup, while CS2 is distinct within the CFA/I group of fimbriae. The genetic similarity between CS1, CS17 and CS19 is reflected in the substantial immunological cross-reactivity observed, both between their protein subunits and intact fimbriae.

Haemolytic Escherichia coli isolated from dogs with diarrhea have characteristics of both uropathogenic and necrotoxigenic strains.

Twenty-four haemolytic Escherichia coli strains were isolated from dogs with diarrhea. The strains were serotyped and analysed by polymerase chain reaction (PCR) for genes encoding virulence factors associated with E. coli that cause diarrhea in animals. Adhesion antigen production was deduced from haemagglutination experiments. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of heat extracts was also used as an indication for the production of adhesive structures. The majority of the strains was shown to produce this type of virulence factor. Adhesion and invasion tests of the strains and Caco-2 cells showed that all strains adhered and that two were invasive. The two invasive strains were positive in the intimin PCR and one of them also contained genes encoding CS31A. The PCR for heat stable toxin (ST) was positive in only four strains, as was the presence of F17 fimbrial genes. Surprisingly, 19 strains had intact P fimbrial operons, coding for an adhesin involved in urinary tract infection (UTI). The cytotoxic necrotising factor 1 (CNF1) gene, also mainly found in UTI was likewise detected in these 19 strains. Cytolethal distending toxin (Cdt) genes were found in five strains. The high number of strains positive for CNF1 and P fimbriae prompted us to test the strains in a multiplex PCR used to test E. coli isolated from UTI in various species for 30 virulence associated genes. The data showed that the majority of the diarrhea isolates have virulence factor profiles highly similar to UTI E. coli isolates from dogs. This raises the question whether these isolates are real intestinal pathogens or "innocent bystanders". However, since CNF1 producing necrotoxic E. coli (NTEC) strains isolated from humans, pigs and calves with diarrhea appear to be highly related to our strains, it might be that in dogs this type of isolate is capable of causing not only UTI, but also diarrhea. If this is the case and this type of isolate is "bifunctional", domestic animals likely constitute a reservoir of NTEC strains which can be also pathogenic for humans.