Analysis of the Campylobacter jejuni genome by SMRT DNA sequencing identifies restriction-modification motifs

Abstract

Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. The goal of this study was to analyze the C. jejuni F38011 strain, recovered from an individual with severe enteritis, at a genomic and proteomic level to gain insight into microbial processes. The C. jejuni F38011 genome is comprised of 1,691,939 bp, with a mol.% (G+C) content of 30.5%. PacBio sequencing coupled with REBASE analysis was used to predict C. jejuni F38011 genomic sites and enzymes that may be involved in DNA restriction-modification. A total of five putative methylation motifs were identified as well as the C. jejuni enzymes that could be responsible for the modifications. Peptides corresponding to the deduced amino acid sequence of the C. jejuni enzymes were identified using proteomics. This work sets the stage for studies to dissect the precise functions of the C. jejuni putative restriction-modification enzymes. Taken together, the data generated in this study contributes to our knowledge of the genomic content, methylation profile, and encoding capacity of C. jejuni.

Fig 1. Genomic map of the C. jejuni F38011 strain.

A 1.69 Mbp circular chromosome of the C. jejuni F38011 strain, beginning with DnaA and encoding a putative 1613 CDSs. Direction of the predicted protein coding sequences (CDS), transfer RNAs (tRNA), ribosomal RNAs (rRNA), mol.% (G+C) content, and GC skew are indicated.

Fig 2. The C. jejuni F38011 genome shares similarity to the NCTC 11168 genome.

Panel A) Analysis of the C. jejuni F38011 and NCTC 11168 genomes using Artemis Comparison Tool (ACT) shows the gene order and organization of the C. jejuni F38011 genome is similar to the NCTC 11168 strain. Blocks of similarity are indicated with red and inversions are indicated with blue between strains. Panel B) Comparison of the seven hypervariable plasticity regions (PR1—PR7) in the genomes of the C. jejuni F38011 and NCTC 11168 strains. Genes are shown in blue with the gray bar below each PR indicating the gap fraction of pairwise alignment with black columns representing absence of genetic information in the C. jejuni F38011 strain. PR parameters were determined as described elsewhere [33].

Fig 3. The methylome of C. jejuni F38011 contains 5 dominant methylation motifs.

The methylation consensus sequences identified by PacBio with adenine methylations found in motifs 1, 2, 3, and 4 (motifs 1, 2 and 4 have a partner motif; RAATTY partner motif not shown) and cytosine methylation found in motif 5. Consensus sequences for each motif is represented as logos, where the height of each stack indicates conservation of sequence (bits) and the height of the symbols represent the relative frequency of the base. An asterisk below a base indicates the modified nucleotide in each consensus sequence. The consensus sequence on the circular genome is indicated with a black line. The numbers within each genome represent methylated sequences compared to the total number of each identified consensus sequence.

Fig 4. Analysis of the proteins synthesized by C. jejuni F38011.

Panel A) Full proteomic analysis of C. jejuni F38011 proteins identified by LC-MS/MS analysis that scored greater than the 1% false discovery rate (FDR) arranged by subcellular localization, as assessed by PSORT. Panel B) Analysis of 485 enzymes grouped by functional classes synthesized by C. jejuni F38011, as derived from UniProt.

Fig 5. Genomic sites of methyl-cytosine modification is not correlated near putative methionine initiation codons.

The methyl-cytosine consensus sequence is distributed throughout the genome with no correlation of proximity near the putative methionine initiation codon.