Genome size determination and coding capacity of Sodalis glossinidius, an enteric symbiont of tsetse flies, as revealed by hybridization to Escherichia coli gene arrays

Abstract

Recent molecular characterization of various microbial genomes has revealed differences in genome size and coding capacity between obligate symbionts and intracellular pathogens versus free-living organisms. Multiple symbiotic microorganisms have evolved with tsetse fly, the vector of African trypanosomes, over long evolutionary times. Although these symbionts are indispensable for tsetse fecundity, the biochemical and molecular basis of their functional significance is unknown. Here, we report on the genomic aspects of the secondary symbiont Sodalis glossinidius. The genome size of Sodalis is approximately 2 Mb. Its DNA is subject to extensive methylation and based on some of its conserved gene sequences has an A+T content of only 45%, compared to the typically AT-rich genomes of endosymbionts. Sodalis also harbors an extrachromosomal plasmid about 134 kb in size. We used a novel approach to gain insight into Sodalis genomic contents, i.e., hybridizing its DNA to macroarrays developed for Escherichia coli, a closely related enteric bacterium. In this analysis we detected 1,800 orthologous genes, corresponding to about 85% of the Sodalis genome. The Sodalis genome has apparently retained its genes for DNA replication, transcription, translation, transport, and the biosynthesis of amino acids, nucleic acids, vitamins, and cofactors. However, many genes involved in energy metabolism and carbon compound assimilation are apparently missing, which may indicate an adaptation to the energy sources available in the only nutrient of the tsetse host, blood. We present gene arrays as a rapid tool for comparative genomics in the absence of whole genome sequence to advance our understanding of closely related bacteria.

FIG. 1

Sodalis genome size determination. (A) Bacterial DNA samples embedded in agarose plugs were subjected to CHEF electrophoresis using pulse times of 150 to 200 s for 20 h at 200 V and 14°C to obtain chromosomal DNA devoid of plasmids. Sizes on the left are indicated in kilobases. (B) Sodalis genomic DNA devoid of its plasmid was analyzed by CHEF gel electrophoresis. PmeI and PacI fragments were resolved at pulse times of 18.3 to 26.3 s over 35 h at 200 V. Three different pulse times were used to resolve the SwaI fragments in different size ranges: 18.3 to 26.3 s for 35 h (a), 6.8 to 12.9 s for 33 h (b), and 1 to 6 s and 6 to 15 s for 15 h each (c).

FIG. 2

Sodalis plasmid size determination. Plasmid DNA fragments were resolved by CHEF gel electrophoresis at 2-s pulse time for 12 h at 170 V. Lanes 1 and 5, molecular weight markers (lambda ladder and lambda/HindIII, respectively); lanes 2 to 4, purified Sodalis plasmid DNA digested with restriction enzymes EcoRI, HindIII, and PstI, respectively.

FIG. 3

E. coli gene array hybridization analysis of Sodalis DNA. The autoradiogram shows the 1,800 signals detected by hybridization of Sodalis chromosomal DNA to Panorama macroarrays containing 4,290 E. coli ORFs. Each gene is spotted in duplicate over three panels.

FIG. 4

List of genes detected in Sodalis by E. coli array hybridization analysis. A.A.B.&M., amino acid biosynthesis and metabolism; B.C.P.&C., biosynthesis of cofactors, prosthetic groups, and carriers; C.C.C., carbon compound catabolism; C.I.M., central intermediary metabolism; C.P., cell processes; C.S., cell structure; D.R.R.M.&R., DNA replication, recombination, modification, and repair; E.M., energy metabolism; F.A.&P.M., fatty acid and phospholipid metabolism; M.P., membrane proteins; N.B.&M., nucleotide biosynthesis and metabolism; T.&P.T.M., translation and posttranslational modification; T.R.P.&D., transcription, RNA processing, and degradation; T.&B.P., transport and binding proteins, R.F., regulatory function; P.R.P., putative regulatory proteins; P.T./P., phage, transposon, or plasmid; H.U.U., hypothetical, unclassified, unknown.

FIG. 4

List of genes detected in Sodalis by E. coli array hybridization analysis. A.A.B.&M., amino acid biosynthesis and metabolism; B.C.P.&C., biosynthesis of cofactors, prosthetic groups, and carriers; C.C.C., carbon compound catabolism; C.I.M., central intermediary metabolism; C.P., cell processes; C.S., cell structure; D.R.R.M.&R., DNA replication, recombination, modification, and repair; E.M., energy metabolism; F.A.&P.M., fatty acid and phospholipid metabolism; M.P., membrane proteins; N.B.&M., nucleotide biosynthesis and metabolism; T.&P.T.M., translation and posttranslational modification; T.R.P.&D., transcription, RNA processing, and degradation; T.&B.P., transport and binding proteins, R.F., regulatory function; P.R.P., putative regulatory proteins; P.T./P., phage, transposon, or plasmid; H.U.U., hypothetical, unclassified, unknown.

FIG. 4

List of genes detected in Sodalis by E. coli array hybridization analysis. A.A.B.&M., amino acid biosynthesis and metabolism; B.C.P.&C., biosynthesis of cofactors, prosthetic groups, and carriers; C.C.C., carbon compound catabolism; C.I.M., central intermediary metabolism; C.P., cell processes; C.S., cell structure; D.R.R.M.&R., DNA replication, recombination, modification, and repair; E.M., energy metabolism; F.A.&P.M., fatty acid and phospholipid metabolism; M.P., membrane proteins; N.B.&M., nucleotide biosynthesis and metabolism; T.&P.T.M., translation and posttranslational modification; T.R.P.&D., transcription, RNA processing, and degradation; T.&B.P., transport and binding proteins, R.F., regulatory function; P.R.P., putative regulatory proteins; P.T./P., phage, transposon, or plasmid; H.U.U., hypothetical, unclassified, unknown.

FIG. 5

Numbers of genes in different functional categories in the known genome of E. coli compared to the numbers of putative genes detected in Sodalis on the basis of gene array analysis.

FIG. 6

Methylation status of Sodalis DNA. Two pairs of isoschizomers that are diagnostic for Dcm (BstNI and EcoRII) and Dam (Sau3AI and MboI) methylation status of DNA were used to digest total (A) and plasmid (B) DNA preparations. (A) M, lambda/HindIII molecular weight marker; lane 1, Sodalis total DNA uncut; lanes 2 to 5, Sodalis total DNA digested with BstNI, EcoRII, Sau3AI, and MboI, respectively. (B) Lane 1, Sodalis plasmid DNA uncut; lanes 2 to 5, Sodalis plasmid DNA digested with BstNI, EcoRII, Sau3AI, and MboI, respectively.