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DNA sequence heterogeneity of Campylobacter jejuni CJIE4 prophages and expression of prophage genes.

ABSTRACT: Campylobacter jejuni carry temperate bacteriophages that can affect the biology or virulence of the host bacterium. Known effects include genomic rearrangements and resistance to DNA transformation. C. jejuni prophage CJIE1 shows sequence variability and variability in the content of morons. Homologs of the CJIE1 prophage enhance both adherence and invasion to cells in culture and increase the expression of a specific subset of bacterial genes. Other C. jejuni temperate phages have so far not been well characterized. In this study we describe investigations into the DNA sequence variability and protein expression in a second prophage, CJIE4. CJIE4 sequences were obtained de novo from DNA sequencing of five C. jejuni isolates, as well as from whole genome sequences submitted to GenBank by other research groups. These CJIE4 DNA sequences were heterogenous, with several different insertions/deletions (indels) in different parts of the prophage genome. Two variants of a 3-4 kb region inserted within CJIE4 had different gene content that distinguished two major conserved CJIE4 prophage families. Additional indels were detected throughout the prophage. Detection of proteins in the five isolates characterized in our laboratory in isobaric Tags for Relative and Absolute Quantitation (iTRAQ) experiments indicated that prophage proteins within each of the two large indel variants were expressed during growth of the bacteria on Mueller Hinton agar plates. These proteins included the extracellular DNase associated with resistance to DNA transformation and prophage repressor proteins. Other proteins associated with known or suspected roles in prophage biology were also expressed from CJIE4, including capsid protein, the phage integrase, and MazF, a type II toxin-antitoxin system protein. Together with the results previously obtained for the CJIE1 prophage these results demonstrate that sequence variability and expression of moron genes are both general properties of temperate bacteriophages in C. jejuni.

SUBMITTER: Clark CG

PROVIDER: S-EPMC3995785 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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DNA sequence heterogeneity of Campylobacter jejuni CJIE4 prophages and expression of prophage genes.

Clark Clifford G CG Chong Patrick M PM McCorrister Stuart J SJ Mabon Philip P Walker Matthew M Westmacott Garrett R GR

PloS one 20140422 4

Campylobacter jejuni carry temperate bacteriophages that can affect the biology or virulence of the host bacterium. Known effects include genomic rearrangements and resistance to DNA transformation. C. jejuni prophage CJIE1 shows sequence variability and variability in the content of morons. Homologs of the CJIE1 prophage enhance both adherence and invasion to cells in culture and increase the expression of a specific subset of bacterial genes. Other C. jejuni temperate phages have so far not be ...[more]

PMID: 24756024

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Project description:Temperate bacteriophages (prophages) have recently been demonstrated in Campylobacter jejuni. However, what they do there is largely unknown. In the series of studies that are the subject of these submissions we have investigated the relative expression levels of proteins in C. jejuni isolates that differ in the presence or absence of the CJIE1 prophage. At the time of the initial investigations whole genome sequence data were not available for the isolates used, though DNA microarray data indicated that the isolates were very closely related. The overall project was carried out through four separate experiments. The first set of experiments (experiment 1) therefore compared protein expression in 4-plex iTRAQ experiments using three isolates carrying the (previously sequenced) prophage with a fourth isolate that was epidemiologically and genetically related to these isolates but which naturally lacked the CJIE1 prophage. Three replicate experiments were done. These isolates were grown on medium containing blood to obtain optimal growth and determine whether any differences were found. Three replicate experiments were done. However, since these growth conditions may not mimic in vivo growth of the organism the results may not be considered relevant or very interesting. Previous work in the scientific literature indicated that growth on medium lacking blood but containing sodium deoxycholate induced the expression of at least some proteins associated with virulence and provided data thought to be of relevance to the virulence of the bacterium. Therefore two isolates were chosen, one with and one without the CJIE1 prophage. Each of three replicates of a 4-plex iTRAQ experiment compared protein expression of the two isolates (00-2425 and 00-2426) after growth on Mueller-Hinton agar with and without 0.1% sodium deoxycholate. There was pretty good overlap between the proteins expressed in the first two experiments but, because all results were expressed as log2 change values, it was difficult to determine whether the expression of some proteins was completely absent in one or the other isolate. As well, protein expression overall showed quite extensive differences compared with the first experiment that we attributed to the lack of blood in the growth medium; we thought we might be seeing a response to limited iron as well. Finally, the response to the deoxycholate was extensive, and we wished to further explore this phenomenon as a means of determining or verifying what effects were due to the presence of the CJIE1 prophage and which were due to other differences. Further 4-plex iTRAQ experiments compared protein expression for each of isolate after growth on medium containing blood, without blood, without blood plus sodium deoxycholate, and without blood plus 2.5% Oxgall. Comparison of the results for isolate 00-2425 (with the CJIE1 prophage) and 00-2426 (without the prophage) identified a small number of proteins that could not be detected in one or the other isolate and allowed discrimination of the effects of the prophage from the responses to iron limitation and bile salt preparations. This dataset is associated with the first experiment, experiment 1, which compares C. jejuni isolates carrying the CJIE1 prophage (00-2425, 00-2538, 00-2544) with a very closely related isolate lacking the prophage (00-2426)

Project description:BACKGROUND: Campylobacter is the most commonly reported bacterial cause of enteritis in humans in the EU Member States and other industrialized countries. One significant source of infection is broilers and consumption of undercooked broiler meat. Campylobacter jejuni is the Campylobacter sp. predominantly found in infected humans and colonized broilers. Sequence analysis of the 16S rRNA gene is very useful for identification of bacteria to genus and species level. The objectives in this study were to determine the degree of intraspecific variation in the 16S rRNA genes of C. jejuni and C. coli and to determine whether the 16S rRNA sequence types correlated with genotypes generated by PFGE analysis of SmaI restricted genomic DNA of the strains. METHODS: The 16S rRNA genes of 45 strains of C. jejuni and two C. coli strains isolated from broilers were sequenced and compared with 16S rRNA sequences retrieved from the Ribosomal Database Project or GenBank. The strains were also genotyped by PFGE after digestion with SmaI. RESULTS: Sequence analyses of the 16S rRNA genes revealed nine sequence types of the Campylobacter strains and the similarities between the different sequence types were in the range 99.6-99.9%. The number of nucleotide substitutions varied between one and six among the nine 16S rRNA sequence types. One of the nine 16S rRNA sequence profiles was common to 12 of the strains from our study and two of these were identified as Campylobacter coli by PCR/REA. The other 10 strains were identified as Campylobacter jejuni. Five of the nine sequence types were also found among the Campylobacter sequences deposited in GenBank. The three 16S rRNA genes in the analysed strains were identical within each individual strain for all 47 strains. CONCLUSION: C. jejuni and C. coli seem to lack polymorphisms in their 16S rRNA gene, but phylogenetic analysis based on 16S rRNA sequences was not always sufficient for differentiation between C. jejuni and C. coli. The strains were grouped in two major clusters according to 16S rRNA, one cluster with only C. jejuni and the other with both C. jejuni and C. coli. Genotyping of the 47 strains by PFGE after digestion with SmaI resulted in 22 subtypes. A potential correlation was found between the SmaI profiles and the 16S rRNA sequences, as a certain SmaI type only appeared in one of the two major phylogenetic groups.

Project description:Campylobacter jejuni is a major cause of human gastroenteritis yet there is limited knowledge of how disease is caused. Molecular genetic approaches are vital for research into the virulence mechanisms of this important pathogen. Vectors that allow expression of genes in C. jejuni via recombination onto the chromosome are particularly useful for genetic complementation of insertional knockout mutants and more generally for expression of genes in particular C. jejuni host backgrounds.A series of three vectors that allow integration of genes onto the C. jejuni chromosome were constructed by standard cloning techniques with expression driven from three different strong promoters. Following integration onto the C. jejuni chromosome expression levels were quantified by fluorescence measurements and cells visualized by fluorescence microscopy.We have created plasmid, pCJC1, designed for recombination-mediated delivery of genes onto the C. jejuni chromosome. This plasmid contains a chloramphenicol resistance cassette (cat) with upstream and downstream restriction sites, flanked by regions of the C. jejuni pseudogene Cj0223. Cloning of genes immediately upstream or downstream of the cat gene allows their subsequent introduction onto the C. jejuni chromosome within the pseudogene. Gene expression can be driven from the native gene promoter if included, or alternatively from the cat promoter if the gene is cloned downstream of, and in the same transcriptional orientation as cat. To provide increased and variable expression of genes from the C. jejuni chromosome we modified pCJC1 through incorporation of three relatively strong promoters from the porA, ureI and flaA genes of C. jejuni, Helicobacter pylori and Helicobacter pullorum respectively. These promoters along with their associated ribosome binding sites were cloned upstream of the cat gene on pCJC1 to create plasmids pCJC2, pCJC3 and pCJC4. To test their effectiveness, a green fluorescent protein (gfp) reporter gene was inserted downstream of each of the three promoters and following integration of promoter-gene fusions onto the C. jejuni host chromosome, expression levels were quantified. Expression from the porA promoter produced the highest fluorescence, from flaA intermediate levels and from ureI the lowest. Expression of gfp from the porA promoter enabled visualization by fluorescent microscopy of intracellular C. jejuni cells following invasion of HeLa cells.The plasmids constructed allow stable chromosomal expression of genes in C. jejuni and, depending on the promoter used, different expression levels were obtained making these plasmids useful tools for genetic complementation and high level expression.

Dataset Information

DNA sequence heterogeneity of Campylobacter jejuni CJIE4 prophages and expression of prophage genes.

Publications

DNA sequence heterogeneity of Campylobacter jejuni CJIE4 prophages and expression of prophage genes.

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