Structural basis of membrane bending by the N-BAR protein endophilin.

Functioning as key players in cellular regulation of membrane curvature, BAR domain proteins bend bilayers and recruit interaction partners through poorly understood mechanisms. Using electron cryomicroscopy, we present reconstructions of full-length endophilin and its N-terminal N-BAR domain in their membrane-bound state. Endophilin lattices expose large areas of membrane surface and are held together by promiscuous interactions between endophilin's amphipathic N-terminal helices. Coarse-grained molecular dynamics simulations reveal that endophilin lattices are highly dynamic and that the N-terminal helices are required for formation of a stable and regular scaffold. Furthermore, endophilin accommodates different curvatures through a quantized addition or removal of endophilin dimers, which in some cases causes dimerization of endophilin's SH3 domains, suggesting that the spatial presentation of SH3 domains, rather than affinity, governs the recruitment of downstream interaction partners.

Lethal and Sub-lethal Implications of Sodium Chloride Exposure for Adult Unionid Mussel Species: Eurynia dilatata and Lasmigona costata.

The elevated use of salt as a de-icing agent on roads in Canada is causing an increase in the chloride concentration of freshwater ecosystems. Freshwater Unionid mussels are a group of organisms that are sensitive to increases in chloride levels. Unionids have greater diversity in North America than anywhere else on Earth, but they are also one of the most imperiled groups of organisms. This underscores the importance of understanding the effect that increasing salt exposure has on these threatened species. There are more data on the acute toxicity of chloride to Unionids than on chronic toxicity. This study investigated the effect of chronic sodium chloride exposure on the survival and filtering activity of two Unionid species (Eurynia dilatata, and Lasmigona costata) and assessed the effect on the metabolome in L. costata hemolymph. The concentration causing mortality after 28 days of exposure was similar for E. dilatata (1893 mg Cl-/L) and L. costata (1903 mg Cl-/L). Significant changes in the metabolome of the L. costata hemolymph were observed for mussels exposed to non-lethal concentrations. For example, several phosphatidylethanolamines, several hydroxyeicosatetraenoic acids, pyropheophorbide-a, and alpha-linolenic acid were significantly upregulated in the hemolymph of mussels exposed to 1000 mg Cl-/L for 28 days. While no mortality occurred in the treatment, elevated metabolites in the hemolymph are an indicator of stress.

Effects of municipal wastewater effluents on the digestive gland microbiome of wild freshwater mussels (Lasmigona costata).

Gut microbial communities are vital for maintaining host health, and are sensitive to diet, environment, and chemical exposures. Wastewater treatment plants (WWTPs) release effluents containing antimicrobials, pharmaceuticals, and other contaminants that may negatively affect the gut microbiome of downstream organisms. This study investigated changes in the diversity and composition of the digestive gland microbiome of flutedshell mussels (Lasmigona costata) from upstream and downstream of two large (service >100,000) WWTPs. Mussel digestive gland microbiome was analyzed following the extraction, PCR amplification, and sequencing of bacterial DNA using the V3-V4 hypervariable regions of the 16 S rRNA gene. Bacterial alpha diversity decreased at sites downstream of the second WWTP and these sites were dissimilar in beta diversity from sites upstream and downstream of the first upstream WWTP. The microbiomes of mussels collected downstream of the first WWTP had increased relative abundances of Actinobacteria, Bacteroidetes, and Firmicutes, with a decrease in Cyanobacteria, compared to upstream mussels. Meanwhile, those collected downstream of the second WWTP increased in Proteobacteria and decreased in Actinobacteria, Bacteroidetes, and Tenericutes. Increased Proteobacteria has been linked to adverse effects in mammals, but their functions in mussels is currently unknown. Finally, effluent-derived bacteria were found in the microbiome of mussels downstream of both WWTPs but not in those from upstream. Overall, results show that the digestive gland microbiome of mussels collected upstream and downstream of WWTPs differed, which has implications for altered host health and the transport of WWTP-derived bacteria through aquatic ecosystems.

Salt-Laden Winter Runoff and Freshwater Mussels; Assessing the Effect on Early Life Stages in the Laboratory and Wild Mussel Populations in Receiving Waters.

The widespread use of road salt for winter road maintenance has led to an increase in the salinity of surface water in many seasonally cold areas. Freshwater mussels have a heightened sensitivity to salt, which is a concern, because many Canadian mussel species at risk have ranges limited to southern Ontario, Canada's most road-dense region. This study examined the effect of winter road runoff on freshwater mussels. The impact of two bridges that span mussel habitat in the Thames River watershed (Ontario, Canada), the second most species-rich watershed for mussels in Canada, were studied. During a winter melt event, bridge runoff, as well as creek surface water surrounding the bridges were collected. Chloride concentrations in samples from bridge deck and tile drains varied (99-8250 mg/L). In general, survival of Lampsilis fasciola glochidia exposed to those samples reflected chloride levels (e.g. 84% at 99 mg/L; 0% at 8250 mg/L), although potassium (60 mg/L) may have at least contributed to toxicity in one sample. Serial dilution exposures with the two most toxic runoff samples revealed 48-h glochidia EC50s of 44% (McGregor Creek Tile Drain) and 26% (Baptiste Creek Deck Drain). During the melt event, the chloride concentrations in creek surface waters downstream of the bridges ranged from 69 to 179 mg Cl-/L; effects on glochidia (viability 77-91%) exposed to those waters was minimal. There were no live mussels surrounding one bridge (Baptiste Creek), likely due to poor habitat. At the other targeted bridge (McGregor Creek), fewer mussels were found close (< 100 m up- or downstream) to the bridge than further (> 200 m) away. However, other contributing factors, including agriculture, were present at both study areas.

Investigation of clearance rate as an endpoint in toxicity testing with freshwater mussels (Unionidae).

The implementation of ecologically relevant sub-lethal endpoints in toxicity testing with freshwater mussels can provide valuable information during risk assessment, especially since these organisms are often exposed to low levels of contaminants. This study examined how to optimize quantifying the filtering capacity or clearance rate (CR) of mussels after exposure to a reference toxicant, sodium chloride (NaCl). CR was defined as the number of algal cells an individual mussel can remove from the overlying water by filtration over time and was determined using spectrophotometric absorbance and direct microscopic examination. Optimization included consideration of the following factors: concentration of algae mixture at test initiation, duration of CR assay, and statistical power. Experimental vessels contained either juvenile (ten, ~ 4 months old) or adult (one, ~ 2.5 years old) Lampsilis siliquoidea. To detect a 10% change in filtering capacity, the optimized adult CR assay was run for 48 h with 2.7 × 107 cells/mL of algae added at test initiation and a minimum of 6 replicates per treatment. The optimized juvenile mussel CR assay was run for 48 h with 1.77 × 107 cells/mL of algae added at test initiation; however, 13 replicates would be required to detect a 10% change to satisfy each method. To reduce the number of juvenile mussels used in testing, a minimum of 4 replicates per treatment was recommended to detect a 25% change in CR. After exposure to a reference toxicant (NaCl), EC50s from the optimized CR assay were compared to two other mussel toxicity endpoints: survival and burial (ability of mussels to bury in clean sand). CR by direct microscopic examination was slightly more sensitive than survival and burial in juveniles and only slightly more sensitive than survival in adults. No significant differences (p > 0.05) were detected between the EC/LC50 values determined from CR and the less labour-intensive survival and burial endpoints. The present study suggests the CR for juvenile and adult L. siliquoidea remained largely unaffected in mussels that survived a 7-day NaCl exposure.

Freshwater mussels in an impacted watershed: Influences of pollution from point and non-point sources.

The Grand River watershed in a densely populated region of Ontario supports one of the richest assemblages of freshwater mussels in Canada. However, water quality in this watershed is influenced by urban development, agriculture, and industry. Mussel populations and water chemistry in the lower Grand River and the Boston Creek tributary were evaluated to determine whether point sources of pollution such as discharges of domestic wastewater and industrial effluent, and non-point sources of pollution are affecting mussel distribution and population structure. Semi-quantitative population surveys conducted at 9 study sites identified 20 mussel species, including 3 Species at Risk. Mussel abundance (34-160 mussels/search hour) and species richness indicated that mussel populations in the lower Grand River watershed are continuing to recover from historical lows reported in the 1970s. However, changes in populations at some sites were consistent with altered water chemistry. Most notable was that the three most abundant mussel species in the Boston Creek tributary downstream of a gypsum plant discharge were significantly smaller in length than those upstream of this site. The water chemistry in this habitat was characterized by elevated conductivity (∼2000 µS/cm) and calcium (∼500 mg/L), as well as concentrations of sulfate (∼1000 mg/L) that can be toxic to freshwater mussels. In the Grand River downstream of the confluence with Boston Creek, there tended to be (p > 0.05) fewer mussels (mean 34 ± 20/search h) compared to upstream (mean 67 ± 15/search h) and this corresponded to altered water chemistry, including elevated sulfate (239 mg/L) downstream of the confluence relative to upstream (58 mg/L). These data indicate that chronic exposures to high levels of major ions is likely driving changes to mussel population structure. In addition, the discharges of wash water from a gypsum plant may be impacting sensitive biota in the main stem Grand River well beyond the immediate tributary receiving environment.

The acute toxicity of bitumen-influenced groundwaters from the oil sands region to aquatic organisms.

The extraction of surface mined bitumen from oil sands deposits in northern Alberta, Canada produces large quantities of liquid tailings waste, termed oil sands process-affected water (OSPW), which are stored in large tailings ponds. OSPW-derived chemicals from several tailings ponds migrating past containment structures and through groundwater systems pose a concern for surface water contamination. The present study investigated the toxicity of groundwater from near-field sites adjacent to a tailings pond with OPSW influence and far-field sites with only natural oil sands bitumen influence. The acute toxicity of unfractionated groundwater and isolated organic fractions was assessed using a suite of aquatic organisms (Pimephales promelas, Oryzias latipes, Daphnia magna, Hyalella azteca, Lampsilis spp., Ceriodaphnia dubia, Hexagenia spp., and Vibrio fischeri). Assessment of unfractionated groundwater demonstrated toxicity towards all invertebrates in at least one far-field sample, with both near-field and far-field samples with bitumen influence toxic towards P. promelas, while no toxicity was observed for O. latipes. When assessing the unfractionated groundwater and isolated organic fractions from near-field and far-field groundwater sites, P. promelas and H. azteca were the most sensitive to organic components, while D. magna and L. cardium were most sensitive to the inorganic components. Groundwater containing appreciable amounts of dissolved organics exhibited similar toxicities to sensitive species regardless of an OSPW or natural bitumen source. The lack of a clear distinction in relative acute toxicities between near-field and far-field samples indicates that the water-soluble chemicals associated with bitumen are acutely toxic to several aquatic organisms. This result, combined with the similarities in chemical profiles between bitumen-influenced groundwater originating from OSPW and/or natural sources, suggests that the industrial bitumen extraction processes corresponding to the tailings pond in this study are not contributing unique toxic substances to groundwater, relative to natural bitumen compounds present in groundwater flow systems.

Method Development for a Short-Term 7-Day Toxicity Test with Unionid Mussels.

The US Environmental Protection Agency's short-term freshwater effluent test methods include a fish (Pimephales promelas), a cladoceran (Ceriodaphnia dubia), and a green alga (Raphidocelis subcapitata). There is a recognized need for additional taxa to accompany the three standard species for effluent testing. An appropriate additional taxon is unionid mussels because mussels are widely distributed, live burrowed in sediment and filter particles from the water column for food, and exhibit high sensitivity to a variety of contaminants. Multiple studies were conducted to develop a relevant and robust short-term test method for mussels. We first evaluated the comparative sensitivity of two mussel species (Villosa constricta and Lampsilis siliquoidea) and two standard species (P. promelas and C. dubia) using two mock effluents prepared by mixing ammonia and five metals (cadmium, copper, nickel, lead, and zinc) or a field-collected effluent in 7-day exposures. Both mussel species were equally or more sensitive (more than two-fold) to effluents compared with the standard species. Next, we refined the mussel test method by first determining the best feeding rate of a commercial algal mixture for three age groups (1, 2, and 3 weeks old) of L. siliquoidea in a 7-day feeding experiment, and then used the derived optimal feeding rates to assess the sensitivity of the three ages of juveniles in a 7-day reference toxicant (sodium chloride [NaCl]) test. Juvenile mussels grew substantially (30%-52% length increase) when the 1- or 2-week-old mussels were fed 2 ml twice daily and the 3-week-old mussels were fed 3 ml twice daily. The 25% inhibition concentrations (IC25s) for NaCl were similar (314-520 mg Cl/L) among the three age groups, indicating that an age range of 1- to 3-week-old mussels can be used for a 7-day test. Finally, using the refined test method, we conducted an interlaboratory study among 13 laboratories to evaluate the performance of a 7-day NaCl test with L. siliquoidea. Eleven laboratories successfully completed the test, with more than 80% control survival and reliable growth data. The IC25s ranged from 296 to 1076 mg Cl/L, with a low (34%) coefficient of variation, indicating that the proposed method for L. siliquoidea has acceptable precision. Environ Toxicol Chem 2021;40:3392-3409. © 2021 SETAC.

Toxicity of the pharmaceuticals finasteride and melengestrol acetate to benthic invertebrates.

The toxicity of endocrinologically active pharmaceuticals finasteride (FIN) and melengestrol acetate (MGA) was assessed in freshwater mussels, including acute (48 h) aqueous tests with glochidia from Lampsilis siliquoidea, sub-chronic (14 days) sediment tests with gravid female Lampsilis fasciola, and chronic (28 days) sediment tests with juvenile L. siliquoidea, and in chronic (42 days) sediment tests with the amphipod Hyalella azteca and the mayfly Hexagenia spp. Finasteride was not toxic in acute aqueous tests with L. siliquoidea glochidia (up to 23 mg/L), whereas significant toxicity to survival and burial ability was detected in chronic sediment tests with juvenile L. siliquoidea (chronic value (ChV, the geometric mean of LOEC and NOEC) = 58 mg/kg (1 mg/L)). Amphipods (survival, growth, reproduction, and sex ratio) and mayflies (growth) were similarly sensitive (ChV = 58 mg/kg (1 mg/L)). Melengestrol acetate was acutely toxic to L. siliquoidea glochidia at 4 mg/L in aqueous tests; in sediment tests, mayflies were the most sensitive species, with significant growth effects observed at 37 mg/kg (0.25 mg/L) (ChV = 21 mg/kg (0.1 mg/L)). Exposure to sublethal concentrations of FIN and MGA had no effect on the (luring and filtering) behaviour of gravid L. fasciola, or the viability of their brooding glochidia. Based on the limited number of measured environmental concentrations of both chemicals, and their projected concentrations, no direct effects are expected by these compounds individually on the invertebrates tested. However, organisms are exposed to contaminant mixtures in the aquatic environment, and thus, the effects of FIN and MGA as components of these mixtures require further investigation.

Sensitivity of multiple life stages of 2 freshwater mussel species (Unionidae) to various pesticides detected in Ontario (Canada) surface waters.

Freshwater mussels contribute important ecological functions to aquatic systems. The water filtered by mussel assemblages can improve water quality, and the mixing of sediments by burrowing mussels can improve oxygen content and release nutrients. However, nearly 70% of North American freshwater mussel species are listed as either endangered, threatened, or in decline. In Ontario, 28 species are in decline or in need of protection. Even though freshwater mussels have a heightened sensitivity to some contaminants, few studies have investigated the risks that various pesticide classes pose to one freshwater mussel species or among life stages. Lampsilis siliquoidea and Villosa iris were the focus of the present study, with the latter currently listed as of "special concern" in Canada. A potential risk to the recovery of freshwater mussel species is the presence and persistence of pesticides in Ontario surface waters. Acute (48 h) toxicity tests were performed with V. iris glochidia to determine the effect on viability (surrogate for survival) following exposure to 4 fungicides (azoxystrobin, boscalid, metalaxyl, and myclobutanil), 3 neonicotinoids (clothianidin, imidacloprid, and thiamethoxam), 2 carbamates (carbaryl and malathion), 1 organophosphate (chlorpyrifos), and 1 butenolide (flupyradifurone). Juvenile and adult L. siliquoidea were also exposed to azoxystrobin, clothianidin, imidacloprid (juvenile only), and carbaryl (adult only). Our study found in general that all life stages were insensitive to the pesticides tested, with median effect and lethal concentrations >161 µg/L. The pesticides tested likely represent a minimal risk (hazard quotients <5.4 × 10-3 ) to freshwater mussel viability and survival in acute (48 h) and subchronic (28 d) exposures, respectively, in Ontario streams where pesticide concentrations were considerably lower than those tested in the present study. Environ Toxicol Chem 2018;37:2871-2880. © 2018 SETAC.

X-ray structure of the T. aquaticus FtsY:GDP complex suggests functional roles for the C-terminal helix of the SRP GTPases.

FtsY and Ffh are structurally similar prokaryotic Signal Recognition Particle GTPases that play an essential role in the Signal Recognition Particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The two GTPases assemble in a GTP-dependent manner to form a heterodimeric SRP targeting complex. We report here the 2.1 A X-ray structure of FtsY from T. aquaticus bound to GDP. The structure of the monomeric protein reveals, unexpectedly, canonical binding interactions for GDP. A comparison of the structures of the monomeric and complexed FtsY NG GTPase domain suggests that it undergoes a conformational change similar to that of Ffh NG during the assembly of the symmetric heterodimeric complex. However, in contrast to Ffh, in which the C-terminal helix shifts independently of the other subdomains, the C-terminal helix and N domain of T. aquaticus FtsY together behave as a rigid body during assembly, suggesting distinct mechanisms by which the interactions of the NG domain "module" are regulated in the context of the two SRP GTPases.

Structure of a GDP:AlF4 complex of the SRP GTPases Ffh and FtsY, and identification of a peripheral nucleotide interaction site.

The signal recognition particle (SRP) GTPases Ffh and FtsY play a central role in co-translational targeting of proteins, assembling in a GTP-dependent manner to generate the SRP targeting complex at the membrane. A suite of residues in FtsY have been identified that are essential for the hydrolysis of GTP that accompanies disengagement. We have argued previously on structural grounds that this region mediates interactions that serve to activate the complex for disengagement and term it the activation region. We report here the structure of a complex of the SRP GTPases formed in the presence of GDP:AlF4. This complex accommodates the putative transition-state analog without undergoing significant change from the structure of the ground-state complex formed in the presence of the GTP analog GMPPCP. However, small shifts that do occur within the shared catalytic chamber may be functionally important. Remarkably, an external nucleotide interaction site was identified at the activation region, revealed by an unexpected contaminating GMP molecule bound adjacent to the catalytic chamber. This site exhibits conserved sequence and structural features that suggest a direct interaction with RNA plays a role in regulating the activity of the SRP targeting complex.

Freshwater mussels in an urban watershed: Impacts of anthropogenic inputs and habitat alterations on populations.

The substantial increase in urbanization worldwide has resulted in higher emissions of wastewater to riverine systems near urban centers, which often impairs aquatic populations and communities. This study examined the effect of urbanization on freshwater mussel populations, including Species at Risk in two rivers receiving wastewater. The influence of anthropogenic activities was assessed in a watershed in the Laurentian Great Lakes basin, one that historically supported one of the most diverse mussel faunas in Canada. In the Grand River (ON), four sites along a 60km reach spanning from an upstream reference site to an urban-impacted downstream area were examined. In the Speed River, mussel populations at six sites along a 10km reach, selected to bracket specific anthropogenic inputs and structures were assessed. A semi-quantitative visual search method revealed that catch per unit effort in the Grand River declined by >60% from the upstream reference site to the area downstream of an urban center. The size (length) frequency distribution of the most abundant species, Lasmigona costata, was significantly (p≤0.008) different upstream of the majority of urban inputs (45-130mm) compared to downstream of the cities (85-115mm). In the Speed River, impoundments and wastewater treatment plants (WWTP) reduced both the diversity and catch per effort. Most striking were 84 and 95% changes in the number of mussels found on either side of two impoundments, and a 98% drop in mussels immediately downstream of a WWTP outfall. These population level effects of decreased abundance and underrepresentation of smaller mussels downstream of the urban area correspond to previously documented impacts at the biochemical and whole organism level of biological organization in wild mussels at this location. Our results demonstrate that poor water quality and physical barriers in urban environments continue to impair susceptible populations and communities of aquatic animals.

Municipal wastewater treatment plant effluent-induced effects on freshwater mussel populations and the role of mussel refugia in recolonizing an extirpated reach.

Global human population and urbanization continually increase the volume of wastewater entering aquatic environments. Despite efforts to treat these effluents, they contribute a diverse suite of substances that enter watersheds at concentrations that have the potential to elicit adverse effects on aquatic organisms. The relationship between wastewater treatment plant (WWTP) effluent exposure and biological responses within aquatic ecosystems remains poorly understood, especially at the population level. To examine the effect of WWTP effluents on sentinel invertebrates, freshwater mussels were assessed in the Grand River, Ontario, in populations associated with the outfall of a major WWTP. This watershed, within the Laurentian Great Lakes basin, has a diverse community of twenty-five species of mussels, including nine Species at Risk, and is representative of many habitats that receive WWTP effluents regionally as well as globally. Surveys were conducted to assess the presence and species richness of freshwater mussels. In total, 55 sites downstream of the WWTP were examined using timed visual searches with one or 2 h of effort spent searching 100 m segments. Although seven species of mussels were found in moderate abundance (mean of 8 mussels per hour of searching across 2 sites) upstream of the WWTP outfall, no live mussels were observed for 7.0 km downstream of the WWTP. Long-term water quality monitoring data indicate that ammonia and nitrite concentrations along with large seasonal declines in diel dissolved oxygen were associated with the extirpation of mussels downstream of the WWTP. The first live mussels found downstream were below the confluence with a major tributary indicating that in addition to an improvement in water quality to a state that enables mussels (and/or their fish hosts) to survive, a nearby mussel refuge may have facilitated the recolonization of the depauperate WWTP-impacted zone.

The effects of pharmaceuticals on a unionid mussel (Lampsilis siliquoidea): An examination of acute and chronic endpoints of toxicity across life stages.

The toxicity and bioconcentration of 3 pharmaceuticals (amitriptyline, iopamidol, and sertraline) were examined using multiple life stages (larval, juvenile, and adult) of the unionid mussel Lampsilis siliquoidea. The endpoints examined varied with life stage but included survival, behavior (algal clearance rate, filtering frequency), and oxidative stress. Iopamidol was not toxic at concentrations up to 101 mg/L. Sertraline was the most toxic chemical (50% lethal concentrations [LC50] and effect concentrations [EC50] = 0.02-0.04 mg/L), but exposure did not induce oxidative stress. Glochidia and juveniles were more sensitive than adult mussels. Algal clearance rate in juvenile mussels was the most sensitive endpoint assessed, similar to or lower than the LC50 values for glochidia. However, the compounds examined were not toxic at concentrations detected in the environment. The relative bioconcentration factors were sertraline > amitriptyline > iopamidol. These results suggest that glochidia toxicity could be a screening tool for rapidly assessing the toxicity of chemicals of concern to freshwater mussels. Environ Toxicol Chem 2017;36:1572-1583. © 2016 SETAC.

Structure of the GMPPNP-stabilized NG domain complex of the SRP GTPases Ffh and FtsY.

Ffh and FtsY are GTPase components of the signal recognition particle co-translational targeting complex that assemble during the SRP cycle to form a GTP-dependent and pseudo twofold symmetric heterodimer. Previously the SRP GTPase heterodimer has been stabilized and purified for crystallographic studies using both the non-hydrolysable GTP analog GMPPCP and the pseudo-transition state analog GDP:AlF4, revealing in both cases a buried nucleotide pair that bridges and forms a key element of the heterodimer interface. A complex of Ffh and FtsY from Thermus aquaticus formed in the presence of the analog GMPPNP could not be obtained, however. The origin of this failure was previously unclear, and it was thought to have arisen from either instability of the analog, or, alternatively, from differences in its interactions within the tightly conscribed composite active site chamber of the complex. Using insights gained from the previous structure determinations, we have now determined the structure of the SRP GTPase targeting heterodimer stabilized by the non-hydrolysable GTP analog GMPPNP. The structure demonstrates how the different GTP analogs are accommodated within the active site chamber despite slight differences in the geometry of the phosphate chain. It also reveals a K+ coordination site at the highly conserved DARGG loop at the N/G interdomain interface.

Xenorhabdus nematophila requires an intact iscRSUA-hscBA-fdx operon to colonize Steinernema carpocapsae nematodes.

An insertion between iscA and hscB of the Xenorhabdus nematophila iscRSUA-hscBA-fdx locus, predicted to encode Fe-S assembly machinery, prevented colonization of Steinernema carpocapsae nematodes. The insertion disrupted cotranscription of iscA and hscB, but did not reduce hscBA expression, suggesting that X. nematophila requires coordinated expression of the isc-hsc-fdx locus for colonization.