Complex Iron Uptake by the Putrebactin-Mediated and Feo Systems in Shewanella oneidensis

ABSTRACT Shewanella oneidensis is an extensively studied bacterium capable of respiring minerals, including a variety of iron ores, as terminal electron acceptors (EAs). Although iron plays an essential and special role in iron respiration of S. oneidensis, little has been done to date to investigate the characteristics of iron transport in this bacterium. In this study, we found that all proteins encoded by the pub-putA-putB cluster for putrebactin (S. oneidensis native siderophore) synthesis (PubABC), recognition-transport of Fe3+-putrebactin across the outer membrane (PutA), and reduction of ferric putrebactin (PutB) are essential to putrebactin-mediated iron uptake. Although homologs of PutA are many, none can function as its replacement, but some are able to work with other bacterial siderophores. We then showed that Fe2+-specific Feo is the other primary iron uptake system, based on the synthetical lethal phenotype resulting from the loss of both iron uptake routes. The role of the Feo system in iron uptake appears to be more critical, as growth is significantly impaired by the absence of the system but not of putrebactin. Furthermore, we demonstrate that hydroxyl acids, especially α-types such as lactate, promote iron uptake in a Feo-dependent manner. Overall, our findings underscore the importance of the ferrous iron uptake system in metal-reducing bacteria, providing an insight into iron homeostasis by linking these two biological processes. IMPORTANCE S. oneidensis is among the first- and the best-studied metal-reducing bacteria, with great potential in bioremediation and biotechnology. However, many questions regarding mechanisms closely associated with those applications, such as iron homeostasis, including iron uptake, export, and regulation, remain to be addressed. Here we show that Feo is a primary player in iron uptake in addition to the siderophore-dependent route. The investigation also resolved a few puzzles regarding the unexpected phenotypes of the putA mutant and lactate-dependent iron uptake. By elucidating the physiological roles of these two important iron uptake systems, this work revealed the breadth of the impacts of iron uptake systems on the biological processes.

Investigating theCampylobacter jejuniTranscriptional Response to Host Intestinal Extracts Reveals the Involvement of a Widely Conserved Iron Uptake System

ABSTRACTCampylobacter jejuniis a pathogenic bacterium that causes gastroenteritis in humans yet is a widespread commensal in wild and domestic animals, particularly poultry. Using RNA sequencing, we assessedC. jejunitranscriptional responses to medium supplemented with human fecal versus chicken cecal extracts and in extract-supplemented medium versus medium alone.C. jejuniexposed to extracts had altered expression of 40 genes related to iron uptake, metabolism, chemotaxis, energy production, and osmotic stress response. In human fecal versus chicken cecal extracts,C. jejunidisplayed higher expression of genes involved in respiration (fdhTU) and in known or putative iron uptake systems (cfbpA,ceuB,chuC, andCJJ81176_1649–1655[here designated1649–1655]). The1649–1655genes and downstream overlapping gene1656were investigated further. Uncharacterized homologues of this system were identified in 33 diverse bacterial species representing 6 different phyla, 21 of which are associated with human disease. The1649and1650(p19) genes encode an iron transporter and a periplasmic iron binding protein, respectively; however, the role of the downstream1651–1656genes was unknown. A Δ1651–1656deletion strain had an iron-sensitive phenotype, consistent with a previously characterized Δp19mutant, and showed reduced growth in acidic medium, increased sensitivity to streptomycin, and higher resistance to H2O2stress. In iron-restricted medium, the1651–1656andp19genes were required for optimal growth when using human fecal extracts as an iron source. Collectively, this implicates a function for the1649–1656gene cluster inC. jejuniiron scavenging and stress survival in the human intestinal environment.IMPORTANCEDirect comparative studies ofC. jejuniinfection of a zoonotic commensal host and a disease-susceptible host are crucial to understanding the causes of infection outcome in humans. These studies are hampered by the lack of a disease-susceptible animal model reliably displaying a similar pathology to human campylobacteriosis. In this work, we compared the phenotypic and transcriptional responses ofC. jejunito intestinal compositions of humans (disease-susceptible host) and chickens (zoonotic host) by using human fecal and chicken cecal extracts. The mammalian gut is a complex and dynamic system containing thousands of metabolites that contribute to host health and modulate pathogen activity. We identifiedC. jejunigenes more highly expressed during exposure to human fecal extracts in comparison to chicken cecal extracts and differentially expressed in extracts compared with medium alone, and targeted one specific iron uptake system for further molecular, genetic, and phenotypic study.

Iron Acquisition in Mycobacterium avium subsp. paratuberculosis

ABSTRACTMycobacterium aviumsubsp.paratuberculosisis a host-adapted pathogen that evolved from the environmental bacteriumM. aviumsubsp.hominissuisthrough gene loss and gene acquisition. Growth ofM. aviumsubsp.paratuberculosisin the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system inM. aviumsubsp.paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed thatM. aviumsubsp.paratuberculosisis impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, inM. aviumsubsp.paratuberculosisgenes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on aM. aviumsubsp.paratuberculosis-specific genomic island, LSPP15. We obtained a transposon (Tn) mutant with a disruption in the LSPP15 geneMAP3776cfor targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775ctoMAP3772c[MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressingMAP3775-2cin wild-typeM. aviumsubsp.paratuberculosis. These data indicate that the horizontally acquired LSPP15 genes contribute to iron acquisition byM. aviumsubsp.paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen.IMPORTANCEMany microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception isMycobacterium aviumsubsp.paratuberculosis, a fastidious, slow-growing animal pathogen whose growth needs to be supported by exogenous mycobacterial siderophore (mycobactin) in the laboratory. Data presented here demonstrate that, compared to other closely relatedM. aviumsubspecies, mycobactin production and iron uptake are different inM. aviumsubsp.paratuberculosis, and these phenotypes may be caused by numerous deletions in its mycobactin biosynthesis pathway. Using a genomic approach, supplemented by targeted genetic and biochemical studies, we identified that LSPP15, a horizontally acquired genomic island, may encode an alternative iron uptake system. These findings shed light on the potential physiological consequence of horizontal gene transfer inM. aviumsubsp.paratuberculosisevolution.

Fur-Regulated Iron Uptake System of Edwardsiella ictaluri and Its Influence on Pathogenesis and Immunogenicity in the Catfish Host

ABSTRACTThe ability of bacterial pathogens to take up iron from the host during infection is necessary for their multiplication within the host. However, host high-affinity iron binding proteins limit levels of free iron in fluids and tissues. To overcome this deficiency of iron during infection, bacterial pathogens have developed iron uptake systems that are upregulated in the absence of iron, typically tightly controlled by the ferric uptake regulator (Fur) protein. The iron uptake system ofEdwardsiella ictaluri, a host-restricted pathogen of channel catfish (Ictalurus punctatus) and the main pathogen of this fish in aquaculture, is unknown. Here we describe theE. ictaluriFur protein, the iron uptake machinery controlled by Fur, and the effects offurgene deletion on virulence and immunogenicity in the fish host. Analysis of theE. ictaluriFur protein shows that it lacks the N-terminal region found in the majority of pathogen-encoded Fur proteins. However, it is fully functional in regulated genes encoding iron uptake proteins.E. ictalurigrown under iron-limited conditions upregulates an outer membrane protein (HemR) that shows heme-hemoglobin transport activity and is tightly regulated by Fur.In vivostudies showed that anE. ictaluriΔfurmutant is attenuated and immune protective in zebrafish (Danio rerio) and catfish (Ictalurus punctatus), triggering systemic immunity. We conclude that anE. ictaluriΔfurmutant could be an effective component of an immersion-oral vaccine for the catfish industry.