TonB-Dependent Heme Iron Acquisition in the Tsetse Fly Symbiont Sodalis glossinidius

Abstract

bSodalis glossinidius is an intra-and extracellular symbiont of the tsetse fly (Glossina sp.), which feeds exclusively on vertebrate blood. S. glossinidius resides in a wide variety of tsetse tissues and may encounter environments that differ dramatically in iron content. The Sodalis chromosome encodes a putative TonB-dependent outer membrane heme transporter (HemR) and a putative periplasmic/inner membrane ABC heme permease system (HemTUV). Because these gene products mediate iron acquisition processes by other enteric bacteria, we characterized their regulation and physiological role in the Sodalis/tsetse system. Our results show that the hemR and tonB genes are expressed by S. glossinidius in the tsetse fly. Furthermore, transcription of hemR in Sodalis is repressed in a high-iron environment by the iron-responsive transcriptional regulator Fur. Expression of the S. glossinidius hemR and hemTUV genes in an Escherichia coli strain unable to use heme as an iron source stimulated growth in the presence of heme or hemoglobin as the sole iron source. This stimulation was dependent on the presence of either the E. coli or Sodalis tonB gene. Sodalis tonB and hemR mutant strains were defective in their ability to colonize the gut of tsetse flies that lacked endogenous symbionts, while wild-type S. glossinidius proliferated in this same environment. Finally, we show that the Sodalis HemR protein is localized to the bacterial membrane and appears to bind hemin. Collectively, this study provides strong evidence that TonB-dependent, HemR-mediated iron acquisition is important for the maintenance of symbiont homeostasis in the tsetse fly, and it provides evidence for the expression of bacterial high-affinity iron acquisition genes in insect symbionts.A ll insects house endogenous microorganisms that mediate critical aspects of their host's physiology. Tsetse flies (Diptera: Glossinidae) harbor three phylogenetically distinct endosymbiotic bacteria, including parasitic Wolbachia spp., obligate Wigglesworthia sp., and commensal Sodalis glossinidius, that are maternally transmitted during the fly's unique viviparous mode of reproduction (reviewed in references 1 and 2). Sodalis glossinidius, a Gram-negative gammaproteobacterium in the family Enterobacteriaceae, resides both intra-and extracellularly in a wide range of tsetse tissues, including the midgut, muscle, fat body, milk gland (a gland that supplies nutrients to developing intrauterine larvae), and salivary glands. Genomic data indicate that genes whose products facilitate a free-living lifestyle, such as those involved in the biosynthesis and transport of nutrients, are retained in Sodalis. However, the high number of pseudogenes (972) present in the S. glossinidius genome suggests that this bacterium is actively transitioning from a free-living to symbiotic lifestyle (3). Sodalis can be cultured outside its tsetse host, suggesting that it has not undergone a high degree of reductive evolution characteristic of ancient obligate bacterial symbioses. The ability to cul...

Symbiont-induced odorant binding proteins mediate insect host hematopoiesis

“…All blood meals (three per week), throughout the course of the entire experiment, also contained vitamin-rich yeast extract (1% w/v) to restore fertility associated with the absence of Wigglesworthia (Weiss et al, 2012). Ten days post-copulation, 4 cohorts of symbiont-cured females were regularly fed a diet supplemented with either 1) Wigglesworthia -containing bacteriome extracts (obtained by dissecting bacteriomes from Gmm WT females), 2) Wigglesworthia -free bacteriome extracts (derived from the offspring of females fed a diet supplemented with ampicillin, which results in the production of progeny that lack Wigglesworthia but still harbor Sodalis; Pais et al, 2008; Weiss et al, 2011), 3) Sodalis cell extracts (derived from Sodalis maintained in culture; Hrusa et al, 2015), and 4) bacteriome extracts harvested from aposymbiotic females. Offspring (symbiont-free) of these extract supplemented females were designated Gmm bact/ Wgm + , Gmm bact/ Wgm - , Gmm Sgm+ and Gmm bact/Apo , respectively.…”

Thermal stress responses of Sodalis glossinidius, an indigenous bacterial symbiont of hematophagous tsetse flies

“…We tried insertions at four different locations in dnaK using Targetron mutagenesis, as well as deletion of dnaK by allelic change. Notably, these mutagenesis techniques have been successfully used to make mutations in numerous other Sodalis genes (53–55). As Sodalis transitions from a free-living lifestyle to a mutualistic one (33), growth outside the tsetse host (on agar plates or in liquid BHI) may generate low-grade oxidative stress that requires DnaK for an appropriate cellular response that is critical for bacterial survival.…”

Conjugal DNA transfer in the maternally inherited symbiont of tsetse fliesSodalis glossinidius

“…Although S. glossinidius has been isolated in axenic culture (4) and its genome has been sequenced (20), this bacterium has been proven refractory to artificial DNA transformation techniques. To date, two artificial transformation methods have been employed to introduce of exogenous plasmid DNA into S. glossinidius: heat-shock transformation and electroporation (8, 10, 13, 14, 16, 17, 21–25). While these transformation procedures were originally developed for Escherichia coli and popularized by the use of this organism as the workhorse of molecular biology (26, 27), they have proven to be both unreliable and inefficient as DNA delivery methods for S. glossinidius .…”