A novel application of gene arrays: Escherichia coli array provides insight into the biology of the obligate endosymbiont of tsetse flies

Abstract

Symbiotic associations with microorganisms are pivotal in many insects. Yet, the functional roles of obligate symbionts have been difficult to study because it has not been possible to cultivate these organisms in vitro. The medically important tsetse fly (Diptera: Glossinidae) relies on its obligate endosymbiont, Wigglesworthia glossinidia, a member of the Enterobacteriaceae, closely related to Escherichia coli, for fertility and possibly nutrition. We show here that the intracellular Wigglesworthia has a reduced genome size smaller than 770 kb. In an attempt to understand the composition of its genome, we used the gene arrays developed for E. coli. We were able to identify 650 orthologous genes in Wigglesworthia corresponding to approximately 85% of its genome. The arrays were also applied for expression analysis using Wigglesworthia cDNA and 61 gene products were detected, presumably coding for some of its most abundant products. Overall, genes involved in cell processes, DNA replication, transcription, and translation were found largely retained in the small genome of Wigglesworthia. In addition, genes coding for transport proteins, chaperones, biosynthesis of cofactors, and some amino acids were found to comprise a significant portion, suggesting an important role for these proteins in its symbiotic life. Based on its expression profile, we predict that Wigglesworthia may be a facultative anaerobic organism that utilizes ammonia as its major source of nitrogen. We present an application of E. coli gene arrays to obtain broad genome information for a closely related organism in the absence of complete genome sequence data.

Figure 1

Genome size characterization of Wigglesworthia by contour-clamped homogeneous electric fields (CHEF) gel electrophoresis. (A) W. brevipalpis chromosome digested with ApaI and SmaI restriction enzymes were run at pulse times ranging from 5–40 s for 27 h and 20–100 s for 24 h, respectively. (B) Wigglesworthia pallidipes and (C) Wigglesworthia palpalis chromosomes were digested with ApaI restriction enzyme and run at pulse times ranging from 3–60 s for 48 h and 5–30 s at 23 h, respectively.

Figure 2

Gene array hybridization analysis. The autoradiogram shows the signals detected by hybridization of W. pallidipes DNA to Panorama E. coli macroarrays under stringent conditions. Each gene is spotted in duplicate and 4,290 genes are distributed over three panels. The enlarged insert shows some of the genes (and their products in parenthesis) identified in this region: 1, fusA (protein elongation factor G); 2, tufA (elongation factor EF-Tu); 3, hslU (heat shock protein); 4, prlC (oligopeptidase A); 5, groEL (chaperonin); 6, b0669 (function unknown); 7, lipA (lipoic acid synthetase); 8, b0667 (function unknown); 9 and 10, quadruple control spots containing 10 and 5 ng of E. coli genomic DNA, respectively.

Figure 3

DNA methylation status of W. pallidipes. Lane 1, λ molecular weight marker; lane 2, Wigglesworthia total DNA uncut; lanes 3–6, Wigglesworthia DNA digested with restriction enzymes BstNI, EcoRII, Sau3AI, and MboI, respectively.

Figure 4

AmtB expression in bacteriome tissue. The Western blot was probed with antibodies against Gp63 (GroEL) (Top) and AmtB (Bottom), successively. Lane 1, female bacteriome; lane 2, male bacteriome; lane 3, muscle tissue; lane 4, protein extract from an in vitro culture of Sodalis.