Development of a Markerless Deletion Mutagenesis System in Nitrate-Reducing Bacterium Rhodanobacter denitrificans.

Rhodanobacter has been found as the dominant genus in aquifers contaminated with high concentrations of nitrate and uranium in Oak Ridge, TN, USA. The in situ stimulation of denitrification has been proposed as a potential method to remediate nitrate and uranium contamination. Among the Rhodanobacter species, Rhodanobacter denitrificans strains have been reported to be capable of denitrification and contain abundant metal resistance genes. However, due to the lack of a mutagenesis system in these strains, our understanding of the mechanisms underlying low-pH resistance and the ability to dominate in the contaminated environment remains limited. Here, we developed an in-frame markerless deletion system in two R. denitrificans strains. First, we optimized the growth conditions, tested antibiotic resistance, and determined appropriate transformation parameters in 10 Rhodanobacter strains. We then deleted the upp gene, which encodes uracil phosphoribosyltransferase, in R. denitrificans strains FW104-R3 and FW104-R5. The resulting strains were designated R3_Δupp and R5_Δupp and used as host strains for mutagenesis with 5-fluorouracil (5-FU) resistance as the counterselection marker to generate markerless deletion mutants. To test the developed protocol, the narG gene encoding nitrate reductase was knocked out in the R3_Δupp and R5_Δupp host strains. As expected, the narG mutants could not grow in anoxic medium with nitrate as the electron acceptor. Overall, these results show that the in-frame markerless deletion system is effective in two R. denitrificans strains, which will allow for future functional genomic studies in these strains furthering our understanding of the metabolic and resistance mechanisms present in Rhodanobacter species. IMPORTANCE Rhodanobacter denitrificans is capable of denitrification and is also resistant to toxic heavy metals and low pH. Accordingly, the presence of Rhodanobacter species at a particular environmental site is considered an indicator of nitrate and uranium contamination. These characteristics suggest its future potential application in bioremediation of nitrate or concurrent nitrate and uranium contamination in groundwater ecosystems. Due to the lack of genetic tools in this organism, the mechanisms of low-pH and heavy metal resistance in R. denitrificans strains remain elusive, which impedes its use in bioremediation strategies. Here, we developed a genome editing method in two R. denitrificans strains. This work marks a crucial step in developing Rhodanobacter as a model for studying the diverse mechanisms of low-pH and heavy metal resistance associated with denitrification.

Characterization of subsurface media from locations up- and down-gradient of a uranium-contaminated aquifer.

The processing of sediment to accurately characterize the spatially-resolved depth profiles of geophysical and geochemical properties along with signatures of microbial density and activity remains a challenge especially in complex contaminated areas. This study processed cores from two sediment boreholes from background and contaminated core sediments and surrounding groundwater. Fresh core sediments were compared by depth to capture the changes in sediment structure, sediment minerals, biomass, and pore water geochemistry in terms of major and trace elements including pollutants, cations, anions, and organic acids. Soil porewater samples were matched to groundwater level, flow rate, and preferential flows and compared to homogenized groundwater-only samples from neighboring monitoring wells. Groundwater analysis of nearby wells only revealed high sulfate and nitrate concentrations while the same analysis using sediment pore water samples with depth was able to suggest areas high in sulfate- and nitrate-reducing bacteria based on their decreased concentration and production of reduced by-products that could not be seen in the groundwater samples. Positive correlations among porewater content, total organic carbon, trace metals and clay minerals revealed a more complicated relationship among contaminant, sediment texture, groundwater table, and biomass. The fluctuating capillary interface had high concentrations of Fe and Mn-oxides combined with trace elements including U, Th, Sr, Ba, Cu, and Co. This suggests the mobility of potentially hazardous elements, sediment structure, and biogeochemical factors are all linked together to impact microbial communities, emphasizing that solid interfaces play an important role in determining the abundance of bacteria in the sediments.

The selective pressures on the microbial community in a metal-contaminated aquifer.

In many environments, toxic compounds restrict which microorganisms persist. However, in complex mixtures of inhibitory compounds, it is challenging to determine which specific compounds cause changes in abundance and prevent some microorganisms from growing. We focused on a contaminated aquifer in Oak Ridge, Tennessee, USA that has large gradients of pH and widely varying concentrations of uranium, nitrate, and many other inorganic ions. In the most contaminated wells, the microbial community is enriched in the Rhodanobacter genus. Rhodanobacter abundance is positively correlated with low pH and high concentrations of uranium and 13 other ions and we sought to determine which of these ions are selective pressures that favor the growth of Rhodanobacter over other taxa. Of these ions, low pH and high UO22+, Mn2+, Al3+, Cd2+, Zn2+, Co2+, and Ni2+ are both (a) selectively inhibitory of a Pseudomonas isolate from an uncontaminated well vs. a Rhodanobacter isolate from a contaminated well, and (b) reach toxic concentrations (for the Pseudomonas isolate) in the Rhodanobacter-dominated wells. We used mixtures of ions to simulate the groundwater conditions in the most contaminated wells and verified that few isolates aside from Rhodanobacter can tolerate these eight ions. These results clarify which ions are likely causal factors that impact the microbial community at this field site and are not merely correlated with taxonomic shifts. Furthermore, our general high-throughput approach can be applied to other environments, isolates, and conditions to systematically help identify selective pressures on microbial communities.

Microbial Functional Gene Diversity Predicts Groundwater Contamination and Ecosystem Functioning.

Contamination from anthropogenic activities has significantly impacted Earth's biosphere. However, knowledge about how environmental contamination affects the biodiversity of groundwater microbiomes and ecosystem functioning remains very limited. Here, we used a comprehensive functional gene array to analyze groundwater microbiomes from 69 wells at the Oak Ridge Field Research Center (Oak Ridge, TN), representing a wide pH range and uranium, nitrate, and other contaminants. We hypothesized that the functional diversity of groundwater microbiomes would decrease as environmental contamination (e.g., uranium or nitrate) increased or at low or high pH, while some specific populations capable of utilizing or resistant to those contaminants would increase, and thus, such key microbial functional genes and/or populations could be used to predict groundwater contamination and ecosystem functioning. Our results indicated that functional richness/diversity decreased as uranium (but not nitrate) increased in groundwater. In addition, about 5.9% of specific key functional populations targeted by a comprehensive functional gene array (GeoChip 5) increased significantly (P < 0.05) as uranium or nitrate increased, and their changes could be used to successfully predict uranium and nitrate contamination and ecosystem functioning. This study indicates great potential for using microbial functional genes to predict environmental contamination and ecosystem functioning.IMPORTANCE Disentangling the relationships between biodiversity and ecosystem functioning is an important but poorly understood topic in ecology. Predicting ecosystem functioning on the basis of biodiversity is even more difficult, particularly with microbial biomarkers. As an exploratory effort, this study used key microbial functional genes as biomarkers to provide predictive understanding of environmental contamination and ecosystem functioning. The results indicated that the overall functional gene richness/diversity decreased as uranium increased in groundwater, while specific key microbial guilds increased significantly as uranium or nitrate increased. These key microbial functional genes could be used to successfully predict environmental contamination and ecosystem functioning. This study represents a significant advance in using functional gene markers to predict the spatial distribution of environmental contaminants and ecosystem functioning toward predictive microbial ecology, which is an ultimate goal of microbial ecology.

Dynamic Succession of Groundwater Sulfate-Reducing Communities during Prolonged Reduction of Uranium in a Contaminated Aquifer.

To further understand the diversity and dynamics of SRB in response to substrate amendment, we sequenced genes coding for the dissimilatory sulfite reductase (dsrA) in groundwater samples collected after an emulsified vegetable oil (EVO) amendment, which sustained U(VI)-reducing conditions for one year in a fast-flowing aquifer. EVO amendment significantly altered the composition of groundwater SRB communities. Sequences having no closely related-described species dominated (80%) the indigenous SRB communities in nonamended wells. After EVO amendment, Desulfococcus, Desulfobacterium, and Desulfovibrio, known for long-chain-fatty-acid, short-chain-fatty-acid and H2 oxidation and U(VI) reduction, became dominant accounting for 7 ± 2%, 21 ± 8%, and 55 ± 8% of the SRB communities, respectively. Succession of these SRB at different bioactivity stages based on redox substrates/products (acetate, SO4-2, U(VI), NO3-, Fe(II), and Mn(II)) was observed. Desulfovibrio and Desulfococcus dominated SRB communities at 4-31 days, whereas Desulfobacterium became dominant at 80-140 days. By the end of the experiment (day 269), the abundance of these SRB decreased but the overall diversity of groundwater SRB was still higher than non-EVO controls. Up to 62% of the SRB community changes could be explained by groundwater geochemical variables, including those redox substrates/products. A significant (P < 0.001) correlation was observed between groundwater U(VI) concentrations and Desulfovibrio abundance. Our results showed that the members of SRB and their dynamics were correlated significantly with slow EVO biodegradation, electron donor production and maintenance of U(VI)-reducing conditions in the aquifer.

A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium.

Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. IMPORTANCE: Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Herein, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. While other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by an S-layer complex. The UBC provides a potential tool for the microbiological sequestration of uranium for the cleaning of contaminated environments.

System-Wide Adaptations of Desulfovibrio alaskensis G20 to Phosphate-Limited Conditions.

The prevalence of lipids devoid of phosphorus suggests that the availability of phosphorus limits microbial growth and activity in many anoxic, stratified environments. To better understand the response of anaerobic bacteria to phosphate limitation and starvation, this study combines microscopic and lipid analyses with the measurements of fitness of pooled barcoded transposon mutants of the model sulfate reducing bacterium Desulfovibrio alaskensis G20. Phosphate-limited G20 has lower growth rates and replaces more than 90% of its membrane phospholipids by a mixture of monoglycosyl diacylglycerol (MGDG), glycuronic acid diacylglycerol (GADG) and ornithine lipids, lacks polyphosphate granules, and synthesizes other cellular inclusions. Analyses of pooled and individual mutants reveal the importance of the high-affinity phosphate transport system (the Pst system), PhoR, and glycolipid and ornithine lipid synthases during phosphate limitation. The phosphate-dependent synthesis of MGDG in G20 and the widespread occurrence of the MGDG/GADG synthase among sulfate reducing ∂-Proteobacteria implicate these microbes in the production of abundant MGDG in anaerobic environments where the concentrations of phosphate are lower than 10 µM. Numerous predicted changes in the composition of the cell envelope and systems involved in transport, maintenance of cytoplasmic redox potential, central metabolism and regulatory pathways also suggest an impact of phosphate limitation on the susceptibility of sulfate reducing bacteria to other anthropogenic or environmental stresses.

Natural bacterial communities serve as quantitative geochemical biosensors.

UNLABELLED: Biological sensors can be engineered to measure a wide range of environmental conditions. Here we show that statistical analysis of DNA from natural microbial communities can be used to accurately identify environmental contaminants, including uranium and nitrate at a nuclear waste site. In addition to contamination, sequence data from the 16S rRNA gene alone can quantitatively predict a rich catalogue of 26 geochemical features collected from 93 wells with highly differing geochemistry characteristics. We extend this approach to identify sites contaminated with hydrocarbons from the Deepwater Horizon oil spill, finding that altered bacterial communities encode a memory of prior contamination, even after the contaminants themselves have been fully degraded. We show that the bacterial strains that are most useful for detecting oil and uranium are known to interact with these substrates, indicating that this statistical approach uncovers ecologically meaningful interactions consistent with previous experimental observations. Future efforts should focus on evaluating the geographical generalizability of these associations. Taken as a whole, these results indicate that ubiquitous, natural bacterial communities can be used as in situ environmental sensors that respond to and capture perturbations caused by human impacts. These in situ biosensors rely on environmental selection rather than directed engineering, and so this approach could be rapidly deployed and scaled as sequencing technology continues to become faster, simpler, and less expensive. IMPORTANCE: Here we show that DNA from natural bacterial communities can be used as a quantitative biosensor to accurately distinguish unpolluted sites from those contaminated with uranium, nitrate, or oil. These results indicate that bacterial communities can be used as environmental sensors that respond to and capture perturbations caused by human impacts.

Dynamic Succession of Groundwater Functional Microbial Communities in Response to Emulsified Vegetable Oil Amendment during Sustained In Situ U(VI) Reduction.

A pilot-scale field experiment demonstrated that a one-time amendment of emulsified vegetable oil (EVO) reduced groundwater U(VI) concentrations for 1 year in a fast-flowing aquifer. However, little is known about how EVO amendment stimulates the functional gene composition, structure, and dynamics of groundwater microbial communities toward prolonged U(VI) reduction. In this study, we hypothesized that EVO amendment would shift the functional gene composition and structure of groundwater microbial communities and stimulate key functional genes/groups involved in EVO biodegradation and reduction of electron acceptors in the aquifer. To test these hypotheses, groundwater microbial communities after EVO amendment were analyzed using a comprehensive functional gene microarray. Our results showed that EVO amendment stimulated sequential shifts in the functional composition and structure of groundwater microbial communities. Particularly, the relative abundance of key functional genes/groups involved in EVO biodegradation and the reduction of NO3 (-), Mn(IV), Fe(III), U(VI), and SO4 (2-) significantly increased, especially during the active U(VI) reduction period. The relative abundance for some of these key functional genes/groups remained elevated over 9 months. Montel tests suggested that the dynamics in the abundance, composition, and structure of these key functional genes/groups were significantly correlated with groundwater concentrations of acetate, NO3 (-), Mn(II), Fe(II), U(VI), and SO4 (2-). Our results suggest that EVO amendment stimulated dynamic succession of key functional microbial communities. This study improves our understanding of the composition, structure, and function changes needed for groundwater microbial communities to sustain a long-term U(VI) reduction.

Independence of nitrate and nitrite inhibition of Desulfovibrio vulgaris Hildenborough and use of nitrite as a substrate for growth.

Sulfate-reducing microbes, such as Desulfovibrio vulgaris Hildenborough, cause "souring" of petroleum reservoirs through produced sulfide and precipitate heavy metals, either as sulfides or by alteration of the metal reduction state. Thus, inhibitors of these microbes, including nitrate and nitrite ions, are studied in order to limit their impact. Nitrite is a potent inhibitor of sulfate reducers, and it has been suggested that nitrate does not inhibit these microbes directly but by reduction to nitrite, which serves as the ultimate inhibitor. Here we provide evidence that nitrate inhibition of D. vulgaris can be independent of nitrite production. We also show that D. vulgaris can use nitrite as a nitrogen source or terminal electron acceptor for growth. Moreover, we report that use of nitrite as a terminal electron acceptor requires nitrite reductase (nrfA) as a D. vulgaris nrfA mutant cannot respire nitrite but remains capable of utilizing nitrite as a nitrogen source. These results illuminate previously uncharacterized metabolic abilities of D. vulgaris that may allow niche expansion in low-sulfate environments. Understanding these abilities may lead to better control of sulfate-reducing bacteria in industrial settings and more accurate prediction of their interactions in the environment.

Stochasticity, succession, and environmental perturbations in a fluidic ecosystem.

Unraveling the drivers of community structure and succession in response to environmental change is a central goal in ecology. Although the mechanisms shaping community structure have been intensively examined, those controlling ecological succession remain elusive. To understand the relative importance of stochastic and deterministic processes in mediating microbial community succession, a unique framework composed of four different cases was developed for fluidic and nonfluidic ecosystems. The framework was then tested for one fluidic ecosystem: a groundwater system perturbed by adding emulsified vegetable oil (EVO) for uranium immobilization. Our results revealed that groundwater microbial community diverged substantially away from the initial community after EVO amendment and eventually converged to a new community state, which was closely clustered with its initial state. However, their composition and structure were significantly different from each other. Null model analysis indicated that both deterministic and stochastic processes played important roles in controlling the assembly and succession of the groundwater microbial community, but their relative importance was time dependent. Additionally, consistent with the proposed conceptual framework but contradictory to conventional wisdom, the community succession responding to EVO amendment was primarily controlled by stochastic rather than deterministic processes. During the middle phase of the succession, the roles of stochastic processes in controlling community composition increased substantially, ranging from 81.3% to 92.0%. Finally, there are limited successional studies available to support different cases in the conceptual framework, but further well-replicated explicit time-series experiments are needed to understand the relative importance of deterministic and stochastic processes in controlling community succession.

Metagenomes of tropical soil-derived anaerobic switchgrass-adapted consortia with and without iron.

Tropical forest soils decompose litter rapidly with frequent episodes of anoxia, making it likely that bacteria using alternate terminal electron acceptors (TEAs) such as iron play a large role in supporting decomposition under these conditions. The prevalence of many types of metabolism in litter deconstruction makes these soils useful templates for improving biofuel production. To investigate how iron availability affects decomposition, we cultivated feedstock-adapted consortia (FACs) derived from iron-rich tropical forest soils accustomed to experiencing frequent episodes of anaerobic conditions and frequently fluctuating redox. One consortium was propagated under fermenting conditions, with switchgrass as the sole carbon source in minimal media (SG only FACs), and the other consortium was treated the same way but received poorly crystalline iron as an additional terminal electron acceptor (SG + Fe FACs). We sequenced the metagenomes of both consortia to a depth of about 150 Mb each, resulting in a coverage of 26× for the more diverse SG + Fe FACs, and 81× for the relatively less diverse SG only FACs. Both consortia were able to quickly grow on switchgrass, and the iron-amended consortium exhibited significantly higher microbial diversity than the unamended consortium. We found evidence of higher stress in the unamended FACs and increased sugar transport and utilization in the iron-amended FACs. This work provides metagenomic evidence that supplementation of alternative TEAs may improve feedstock deconstruction in biofuel production.

Engineering robust control of two-component system phosphotransfer using modular scaffolds.

Synthetic biology applies engineering principles to facilitate the predictable design of biological systems. Biological systems composed of modular parts with clearly defined interactions are generally easier to manipulate than complex systems exhibiting a large number of subtle interactions. However, recreating the function of a naturally complex system with simple modular parts can increase fragility. Here, inspired by scaffold-directed signaling in higher organisms, we modularize prokaryotic signal transduction to allow programmable redirection of phosphate flux from a histidine kinase to response regulators based on targeting by eukaryotic protein-protein interaction domains. Although scaffold-directed colocalization alone was sufficient to direct signaling between components, this minimal system suffered from high sensitivity to changing expression levels of each component. To address this fragility, we demonstrate how to engineer autoinhibition into the kinase so that phosphotransfer is possible only upon binding to the scaffold. This system, in which scaffold performs the dual functions of activating this autoinhibited kinase and directing flux to the cotargeted response regulator, was significantly more robust to varying component concentrations. Thus, we demonstrate that design principles inspired by the complex signal-transduction pathways of eukaryotes may be generalized, abstracted, and applied to prokaryotes using well-characterized parts.

Draft genome sequences for two metal-reducing Pelosinus fermentans strains isolated from a Cr(VI)-contaminated site and for type strain R7.

Pelosinus fermentans 16S rRNA gene sequences have been reported from diverse geographical sites since the recent isolation of the type strain. We present the genome sequence of the P. fermentans type strain R7 (DSM 17108) and genome sequences for two new strains with different abilities to reduce iron, chromate, and uranium.

Microbial community succession during lactate amendment and electron acceptor limitation reveals a predominance of metal-reducing Pelosinus spp.

The determination of the success of in situ bioremediation strategies is complex. By using controlled laboratory conditions, the influence of individual variables, such as U(VI), Cr(VI), and electron donors and acceptors on community structure, dynamics, and the metal-reducing potential can be studied. Triplicate anaerobic, continuous-flow reactors were inoculated with Cr(VI)-contaminated groundwater from the Hanford, WA, 100-H area, amended with lactate, and incubated for 95 days to obtain stable, enriched communities. The reactors were kept anaerobic with N(2) gas (9 ml/min) flushing the headspace and were fed a defined medium amended with 30 mM lactate and 0.05 mM sulfate with a 48-h generation time. The resultant diversity decreased from 63 genera within 12 phyla to 11 bacterial genera (from 3 phyla) and 2 archaeal genera (from 1 phylum). Final communities were dominated by Pelosinus spp. and to a lesser degree, Acetobacterium spp., with low levels of other organisms, including methanogens. Four new strains of Pelosinus were isolated, with 3 strains being capable of Cr(VI) reduction while one also reduced U(VI). Under limited sulfate, it appeared that the sulfate reducers, including Desulfovibrio spp., were outcompeted. These results suggest that during times of electron acceptor limitation in situ, organisms such as Pelosinus spp. may outcompete the more-well-studied organisms while maintaining overall metal reduction rates and extents. Finally, lab-scale simulations can test new strategies on a smaller scale while facilitating community member isolation, so that a deeper understanding of community metabolism can be revealed.

Evidence-based annotation of transcripts and proteins in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough.

We used high-resolution tiling microarrays and 5' RNA sequencing to identify transcripts in Desulfovibrio vulgaris Hildenborough, a model sulfate-reducing bacterium. We identified the first nucleotide position for 1,124 transcripts, including 54 proteins with leaderless transcripts and another 72 genes for which a major transcript initiates within the upstream protein-coding gene, which confounds measurements of the upstream gene's expression. Sequence analysis of these promoters showed that D. vulgaris prefers -10 and -35 boxes different from those preferred by Escherichia coli. A total of 549 transcripts ended at intrinsic (rho-independent) terminators, but most of the other transcripts seemed to have variable ends. We found low-level antisense expression of most genes, and the 5' ends of these transcripts mapped to promoter-like sequences. Because antisense expression was reduced for highly expressed genes, we suspect that elongation of nonspecific antisense transcripts is suppressed by transcription of the sense strand. Finally, we combined the transcript results with comparative analysis and proteomics data to make 505 revisions to the original annotation of 3,531 proteins: we removed 255 (7.5%) proteins, changed 123 (3.6%) start codons, and added 127 (3.7%) proteins that had been missed. Tiling data had higher coverage than shotgun proteomics and hence led to most of the corrections, but many errors probably remain. Our data are available at http://genomics.lbl.gov/supplemental/DvHtranscripts2011/.

Novel insights into iron metabolism by integrating deletome and transcriptome analysis in an iron deficiency model of the yeast Saccharomyces cerevisiae.

BACKGROUND: Iron-deficiency anemia is the most prevalent form of anemia world-wide. The yeast Saccharomyces cerevisiae has been used as a model of cellular iron deficiency, in part because many of its cellular pathways are conserved. To better understand how cells respond to changes in iron availability, we profiled the yeast genome with a parallel analysis of homozygous deletion mutants to identify essential components and cellular processes required for optimal growth under iron-limited conditions. To complement this analysis, we compared those genes identified as important for fitness to those that were differentially-expressed in the same conditions. The resulting analysis provides a global perspective on the cellular processes involved in iron metabolism. RESULTS: Using functional profiling, we identified several genes known to be involved in high affinity iron uptake, in addition to novel genes that may play a role in iron metabolism. Our results provide support for the primary involvement in iron homeostasis of vacuolar and endosomal compartments, as well as vesicular transport to and from these compartments. We also observed an unexpected importance of the peroxisome for growth in iron-limited media. Although these components were essential for growth in low-iron conditions, most of them were not differentially-expressed. Genes with altered expression in iron deficiency were mainly associated with iron uptake and transport mechanisms, with little overlap with those that were functionally required. To better understand this relationship, we used expression-profiling of selected mutants that exhibited slow growth in iron-deficient conditions, and as a result, obtained additional insight into the roles of CTI6, DAP1, MRS4 and YHR045W in iron metabolism. CONCLUSION: Comparison between functional and gene expression data in iron deficiency highlighted the complementary utility of these two approaches to identify important functional components. This should be taken into consideration when designing and analyzing data from these type of studies. We used this and other published data to develop a molecular interaction network of iron metabolism in yeast.