Regulation of High-Affinity Iron Acquisition Homologues in the Tsetse Fly Symbiont Sodalis glossinidius

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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...

Section: Construction Of Sodalis Mutantsmentioning

confidence: 99%

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confidence: 99%

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

Hrusa

Farmer

Weiss

et al. 2015

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“…In blood-feeding insects, iron is a component of blood meal hemoglobin and is sequestered within host storage molecules, including heme, transferrin, ferritin, and iron-containing proteins (11)(12)(13). Sodalis has several putative iron acquisition systems, including a siderophore system that synthesizes and transports the siderophore achromobactin (14,15). Siderophores are iron-chelating molecules secreted by microbes.…”

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confidence: 99%

“…Thus, expression of iron acquisition systems is repressed when sufficient iron is present in the cell (18). Although Sodalis was initially reported to lack the fur gene (17), further annotation of the genome indicated that the commensal bacterium does contain a functional fur gene whose product regulates the expression of putative heme and Sit iron acquisition systems (14).…”

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confidence: 99%

Characterization of the Achromobactin Iron Acquisition Operon in Sodalis glossinidius

Smith

Weiss

Aksoy

et al. 2013

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bSodalis glossinidius is a facultative, extra-and intracellular symbiont found in most tissues of the tsetse fly (Glossinia sp.). Sodalis has a putative achromobactin siderophore iron acquisition system on the pSG1 plasmid. Reverse transcription (RT)-PCR analysis revealed that the achromobactin operon is transcribed as a single polycistronic molecule and is expressed when Sodalis is within the tsetse fly. Expression of the achromobactin operon was repressed under iron-replete conditions; in a mutant that lacks the iron-responsive transcriptional repressor protein Fur, expression was aberrantly derepressed under these iron-replete conditions, indicating that the Fur protein repressed achromobactin gene expression when iron was plentiful. A putative Fur binding site within the Sodalis achromobactin promoter bound Fur in Escherichia coli Fur titration assays. Wild-type Sodalis produced detectable siderophore in vitro, but a mutation in the putative achromobactin biosynthesis gene acsD eliminated detectable siderophore production in Sodalis. Reduced growth of the siderophore synthesis mutant was reconstituted by addition of exogenous achromobactin, suggesting the strain retains a functional siderophore transport system; however, reduced growth of a Sodalis ferric-siderophore outer membrane receptor mutant with a mutation in acr was not reconstituted by exogenous siderophore due to its defective transporter. The Sodalis siderophore synthesis mutant showed reduced growth in tsetse that lacked endogenous symbionts (aposymbiotic) when the flies were inoculated with Sodalis intrathoracically, but not when inoculated per os. Our findings suggest that Sodalis siderophores play a role in iron acquisition in certain tsetse fly tissues and provide evidence for the regulation of iron acquisition mechanisms in insect symbionts.

“…Whereas this process has a streamlining effect, eliminating genes that are no longer required for the symbiotic lifestyle, it also drives a reduction in metabolic plasticity, yielding fastidious bacteria that have proven to be difficult to manipulate in the laboratory due to their low growth rate, high level of susceptibility to contamination, and complex nutritional requirements (12). To date, only a few studies have utilized genetic techniques to explore the nature of host-symbiont interactions (4,5,14), and the implementation of these techniques has proved arduous and unreliable over the long term. In the current study, we report on the adaptation and optimization of the lambda Red recombineering strategy (6) for the genetic manipulation of the tsetse fly endosymbiont Sodalis glossinidius (Fig.…”

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confidence: 99%

Lambda Red-Mediated Genetic Modification of the Insect Endosymbiont Sodalis glossinidius

Pontes

Dale

2011

In the current study, we adapted and optimized the lambda Red recombineering strategy to genetically manipulate the fastidious insect endosymbiont Sodalis glossinidius. This work greatly facilitates the application of genetics to the study of insect symbionts and should also prove useful in the context of long-awaited paratransgenic insect control strategies.The lifestyle switch from facultative to obligate host association is often accompanied by a process of bacterial genome degeneration and size reduction. Whereas this process has a streamlining effect, eliminating genes that are no longer required for the symbiotic lifestyle, it also drives a reduction in metabolic plasticity, yielding fastidious bacteria that have proven to be difficult to manipulate in the laboratory due to their low growth rate, high level of susceptibility to contamination, and complex nutritional requirements (12). To date, only a few studies have utilized genetic techniques to explore the nature of host-symbiont interactions (4,5,14), and the implementation of these techniques has proved arduous and unreliable over the long term. In the current study, we report on the adaptation and optimization of the lambda Red recombineering strategy (6) for the genetic manipulation of the tsetse fly endosymbiont Sodalis glossinidius (Fig. 1).A plasmid harboring lambda Red functions under the control of the P BAD arabinose-inducible promoter (pKD46) (6) was transformed into S. glossinidius using heat shock (7). Sodalis glossinidius is a fastidious bacterium that grows optimally in rich medium formulations containing glucose or N-acetyl-D-glucosamine (NAG) as a carbon source. Since S. glossinidius cannot use arabinose as a carbon source (3) and glucose and NAG can potentially interfere with the expression of genes under the regulation of the P BAD promoter through catabolite repression (8), we examined the expression of the bet lambda Red gene by quantitative PCR (4) in the presence of different sugars and 3Ј-5Ј-cyclic AMP (cAMP). As expected, we found that the expression of the bet gene is catabolite repressed by glucose and NAG but that catabolite repression can be overcome by the addition of cAMP (Fig. 2). Indeed, we were unable to obtain any recombinants following lambda Red recombineering without the addition of cAMP.In comparison to model organisms such as Escherichia coli, S. glossinidius divides very slowly under standard culture conditions. This is largely due to the fact that S. glossinidius cultures are maintained under anaerobic or microaerophilic conditions because the bacterium fails to grow on agar plates under atmospheric levels of oxygen (3). However, in a recent study, we found that S. glossinidius has a quorum-sensing system that regulates a large number of genes involved in the bacterial response to oxidative stress (11). Subsequently, we found that it was possible to significantly increase the growth rate of S. glossinidius by placing cultures in a shaking incubator once they had reached a cell density sufficient to ensure activa...