Novel molecular features of the fibrolytic intestinal bacterium Fibrobacter intestinalis not shared with Fibrobacter succinogenes as determined by suppressive subtractive hybridization

Abstract

Suppressive subtractive hybridization was conducted to identify unique genes coding for plant cell wall hydrolytic enzymes and other properties of the gastrointestinal bacterium Fibrobacter intestinalis DR7 not shared by Fibrobacter succinogenes S85. Subtractive clones from F. intestinalis were sequenced and assembled to form 712 nonredundant contigs with an average length of 525 bp. Of these, 55 sequences were unique to F. intestinalis. The remaining contigs contained 764 genes with BLASTX similarities to other proteins; of these, 80% had the highest similarities to proteins in F. succinogenes, including 30 that coded for carbohydrate active enzymes. The expression of 17 of these genes was verified by Northern dot blot analysis. Of genes not exhibiting BLASTX similarity to F. succinogenes, 30 encoded putative transposases, 6 encoded restriction modification genes, and 45% had highest similarities to proteins in other species of gastrointestinal bacteria, a finding suggestive of either horizontal gene transfer to F. intestinalis or gene loss from F. succinogenes. Analysis of contigs containing segments of two or more adjacent genes revealed that only 35% exhibited BLASTX similarity and were in the same orientation as those of F. succinogenes, indicating extensive chromosomal rearrangement. The expression of eight transposases, and three restriction-modification genes was confirmed by Northern dot blot analysis. These data clearly document the maintenance of carbohydrate active enzymes in F. intestinalis necessitated by the preponderance of polysaccharide substrates available in the ruminal environment. It also documents substantive changes in the genome from that of F. succinogenes, which may be related to the introduction of the array of transposase and restriction-modification genes.

FIG. 1.

Summary of the similarities of F. intestinalis contigs to genes present in the GenBank nonredundant amino acid database and the F. succinogenes S85 genome translated amino acid sequence database. The distribution of contigs is based on the gene in each contig with the lowest E-value. A possible second gene in a contig was not taken into account. The DNA inserts from clones were assembled into contigs, and redundant sequences were eliminated prior to BLAST analysis. (A) Similarity based on BLASTN search; (B) similarity based on BLASTX, tBLASTX, conserved domain database search, and PSI-BLAST search. Abbreviations: nraa, nonredundant amino acid database (which contains GenBank CDS translations, RefSeq Proteins, PDB, SwissProt, PIR and PRF); GFS, Genome of F. succinogenes database; GFS-aa, GFS amino acid sequence database; GFS-nraa, combined database contains both nraa and GFS-aa. Superscript a, total nucleic acid base pairs.

FIG. 2.

Dendrogram of putative transposases and their closest relatives identified from the Swiss-Prot database (http://www.expasy.org/), showing that they belong to different insertion sequence families. The genes from F. intestinalis were highlighted as RSGA plus the number. The genes closest to the F. intestinalis clones were selected by using a BLASTP search against the Swiss-Prot database. The accession number and name of organisms are shown in the figure. The tree was constructed by using CLUSTAL W (version 1.83) and TreeView (version 1.6.6). The dashed line separates the different transposase families. The genes are clustered in different branches based on their sequence similarities. However, conclusive phylogenetic relationships between the genes cannot be accurately determined because the SSH transposase genes are incomplete sequences.

FIG. 3.

Dendrogram of the relationships between the codon usage of F. succinogenes cellulolytic enzymes and F. intestinalis cellulolytic enzymes and transposases. Clones of SSH from F. intestinalis DR7 were named as RSGA plus the number for cellulolytic enzymes and Tra plus the number for transposases. The transposases showing the highest similarity to the ISPg7 transposase of P. gingivalis are represented by Tra201 and Tra532 because of their nearly identical DNA sequences. Genes from F. succinogenes were labeled with ORF numbers. Most of the genes from F. succinogenes were clustered in regions A and B as shown in the figures. Region A contains 33 F. succinogenes genes and includes ORFs 106, 267, 924, 1077, 1107, 1635, 1685, 1708, 1766, 2210, 2280, 2282, 2283, 2284, 2321, 2325, 2552, 2754, 2861, 2862, 3023, 3555, 3598, 3766, 3767, 3815, 3983, 4138, 4190, 4208, 4500, 4614, and A00451. Region B contains 41 F. succinogenes genes and includes ORFs 65, 143, 241, 279, 369, 601, 925, 990, 1079, 1086, 1088, 1258, 1538, 1785, 1918, 2003, 2115, 2519, 2520, 2686, 2715, 2748, 3025, 3444, 3674, 3685, 3687, 3690, 3711, 3713, 3720, 3726, 3727, 4081, 4082, 4341, 4367, 4534, 4628, A00235, and A00267.