Identification of Campylobacter jejuni ATCC 43431-specific genes by whole microbial genome comparisons

Abstract

This study describes a novel approach to identify unique genomic DNA sequences from the unsequenced strain C. jejuni ATCC 43431 by comparison with the sequenced strain C. jejuni NCTC 11168. A shotgun DNA microarray was constructed by arraying 9,600 individual DNA fragments from a C. jejuni ATCC 43431 genomic library onto a glass slide. DNA fragments unique to C. jejuni ATCC 43431 were identified by competitive hybridization to the array with genomic DNA of C. jejuni NCTC 11168. The plasmids containing unique DNA fragments were sequenced, allowing the identification of up to 130 complete and incomplete genes. Potential biological roles were assigned to 66% of the unique open reading frames. The mean G+C content of these unique genes (26%) differs significantly from the G+C content of the entire C. jejuni genome (30.6%). This suggests that they may have been acquired through horizontal gene transfer from an organism with a G+C content lower than that of C. jejuni. Because the two C. jejuni strains differ by Penner serotype, a large proportion of the unique ATCC 43431 genes encode proteins involved in lipooligosaccharide and capsular biosynthesis, as expected. Several unique open reading frames encode enzymes which may contribute to genetic variability, i.e., restriction-modification systems and integrases. Interestingly, many of the unique C. jejuni ATCC 43431 genes show identity with a possible pathogenicity island from Helicobacter hepaticus and components of a potential type IV secretion system. In conclusion, this study provides a valuable resource to further investigate Campylobacter diversity and pathogenesis.

FIG. 1.

Conserved and divergent C. jejuni NCTC 111168 ORFs in C. jejuni ATCC 43431 identified by microarray analysis (row 1), DNA sequencing (row 2), and PCR analysis (row 3). ORFs are listed with respect to their location on the NCTC 11168 genome horizontally from left to right in 21 sections of 80 ORFs. It should be noticed that the gene order might be different in the genome of C. jejuni ATCC 43431. For the microarray data (row 1), the color scale is located at the bottom of the figure, with the brightest blue representing ORFs found to be present with high certainty, the brightest yellow indicating ORFs found to be absent with high certainty, and black corresponding to ORFs found to be uncertain or slightly divergent. For the sequencing data (row 2), sequenced ORFs are indicated in blue, while green indicates missing data. For the PCR analysis (row 3), ORFs successfully amplified are represented in blue, ORFs with failed PCR amplification are indicated in yellow, and untested ORFs are indicated in green.

FIG. 1.

Conserved and divergent C. jejuni NCTC 111168 ORFs in C. jejuni ATCC 43431 identified by microarray analysis (row 1), DNA sequencing (row 2), and PCR analysis (row 3). ORFs are listed with respect to their location on the NCTC 11168 genome horizontally from left to right in 21 sections of 80 ORFs. It should be noticed that the gene order might be different in the genome of C. jejuni ATCC 43431. For the microarray data (row 1), the color scale is located at the bottom of the figure, with the brightest blue representing ORFs found to be present with high certainty, the brightest yellow indicating ORFs found to be absent with high certainty, and black corresponding to ORFs found to be uncertain or slightly divergent. For the sequencing data (row 2), sequenced ORFs are indicated in blue, while green indicates missing data. For the PCR analysis (row 3), ORFs successfully amplified are represented in blue, ORFs with failed PCR amplification are indicated in yellow, and untested ORFs are indicated in green.

FIG. 2.

PFGE of SmaI-digested (A) and SalI-digested (B) C. jejuni chromosomal DNA. Lanes: M, lambda 50-kb markers; 1, restriction fragments obtained from digestion of C. jejuni NCTC 11168 DNA; 2, restriction fragments obtained from digestion of C. jejuni ATCC 43431 DNA. The ladder sizes are indicated on the left of each panel. For SalI, the gel running conditions allowed the entire 50-kb ladder (from 50 kb to 1 Mb) to be separated, whereas the SmaI conditions were optimized for separation up to 550 kb.

FIG. 3.

Comparison of the LOS cluster from NCTC 11828/81116 and ATCC 43431 suggests the existence of a fourth class of LOS gene clusters, designated class D. The 11828 sequences from 12960 to waaF were from AF343914 (33). The 11828/81116 sequences from waaC to rmlB were from AJ131360 (11). The sequence data for ATCC 43431 from waaC to rmlB are from AF411225 (13). The gene order and direction in the LOS locus of ATCC 43431 were confirmed by PCR and DNA sequencing of the junctions between each contig identified by this study. The entire DNA sequence from the LOS cluster of this strain has been deposited in the NCBI database under accession number AY501976. This cluster of genes is anchored by waaC and waaF, similarly to the class A, B and C clusters (14), but differs substantially from these by lacking the neu genes and the cst-encoded sialyltransferase. Where relevant, both the suggested gene name and the ORF designations as described previously (14) are shown. The striking finding of numerous conserved genes in ATCC 43431 linked as in 81116 suggests that these two strains represent the same LOS cluster class. One of the 81116 genes was not found (absent) in the sequencing of unique ATCC 43431 ORFs. The glycosyltransferase designated wlaNA was also not identified in this study but was previously reported by Gilbert et al. (13) to be contiguous with waaC, htrB, and rmlAB.