Discriminating Susceptibility of Xanthine Oxidoreductase Family to Metals.

The xanthine oxidoreductase (XOR) family are metal-containing enzymes that use the molybdenum cofactor (Moco), 2Fe-2S clusters, and flavin adenine dinucleotide (FAD) for their catalytic activity. This large molybdoenzyme family includes xanthine, aldehyde, and CO dehydrogenases. XORs are widely distributed from bacteria to humans due to their key roles in the catabolism of purines, aldehydes, drugs, and xenobiotics, as well as interconversions between CO and CO2. Assessing the effect of excess metals on the Rubrivivax gelatinosus bacterium, we found that exposure to copper (Cu) or cadmium (Cd) caused a dramatic decrease in the activity of a high-molecular-weight soluble complex exhibiting nitroblue tetrazolium reductase activity. Mass spectrometry and genetic analyses showed that the complex corresponds to a putative CO dehydrogenase (pCOD). Using mutants that accumulate either Cu+ or Cd2+ in the cytoplasm, we show that Cu+ or Cd2+ is a potent inhibitor of XORs (pCOD and the xanthine dehydrogenase [XDH]) in vivo. This is the first in vivo demonstration that Cu+ affects Moco-containing enzymes. The specific inhibitory effect of these compounds on the XOR activity is further supported in vitro by direct addition of competing metals to protein extracts. Moreover, emphasis is given on the inhibitory effect of Cu on bovine XOR, showing that the XOR family could be a common target of Cu. Given the conservation of XOR structure and function across the tree of life, we anticipate that our findings could be transferable to other XORs and organisms. IMPORTANCE The high toxicity of Cu, Cd, Pb, As, and other metals arises from their ability to cross membranes and target metalloenzymes in the cytoplasm. Identifying these targets provides insights into the toxicity mechanisms. The vulnerability of metalloenzymes arises from the accessibility of their cofactors to ions. Accordingly, many enzymes whose cofactors are solvent exposed are likely to be targets of competing metals. Here, we describe for the first time, with in vivo and in vitro experiments, a direct effect of excess Cu on the xanthine oxidoreductase family (XOR/XDH/pCOD). We show that toxic metal affects these Moco enzymes, and we suggest that access to the Moco center by Cu ions could explain the Cu inhibition of XORs in living organisms. Human XOR activity is associated with hyperuricemia, xanthinuria, gout arthritis, and other diseases. Our findings in vivo highlight XOR as a Cu target and thus support the potential use of Cu in metal-based therapeutics against these diseases.

New insights into the mechanism of iron transport through the bacterial Ftr system present in pathogens.

Iron is an essential nutrient in bacteria. Its ferrous form, mostly present in low oxygen and acidic pH environments, can be imported using the specific Ftr-type transport system, which encompasses the conserved FtrABCD system found in pathogenic bacteria such as Bordetella, Brucella and Burkholderia. The nonpathogenicity and versatile metabolism of Rubrivivax gelatinosus make it an ideal model to study the FtrABCD system. Here, we report a new aspect of its regulation and the role of the periplasmic proteins FtrA and FtrB using in vivo and in vitro approaches. We investigated the metal binding mode and redox state of copper and iron to FtrA by crystallography and biophysical methods. An 'as isolated' FtrA protein from the bacterial periplasm contained a copper ion (Cu+ ) identified by electron paramagnetic resonance (EPR). Copper is coordinated by four conserved side chains (His and Met) in the primary metal site. Structural analysis of R. gelatinosus FtrA and FtrA homologues revealed that copper binding induces a rearrangement of the His95 imidazole ring, releasing thereafter space, as well as both Asp45 and Asp92 side chains, for iron binding in the secondary metal site. EPR highlighted that FtrA can oxidize the bound ferrous ion into the ferric form by reducing the bound Cu2+ into Cu+ , both metal sites being separated by 7 Å. Finally, we showed that FtrB binds iron and not copper. These results provide new insights into the mechanism of ferrous iron utilization by the conserved FtrABCD iron transporter for which we propose a new functional model.

A periplasmic cupredoxin with a green CuT1.5 center is involved in bacterial copper tolerance.

The importance of copper resistance pathways in pathogenic bacteria is now well recognized, since macrophages use copper to fight bacterial infections. Additionally, considering the increase of antibiotic resistance, growing attention is given to the antimicrobial properties of copper. It is of primary importance to understand how bacteria deal with copper. The Cu-resistant cuproprotein CopI is present in many human bacterial pathogens and environmental bacteria and crucial under microaerobiosis (conditions for most pathogens to thrive within their host). Hence, understanding its mechanism of function is essential. CopI proteins share conserved histidine, cysteine, and methionine residues that could be ligands for different copper binding sites, among which the cupredoxin center could be involved in the protein function. Here, we demonstrated that Vibrio cholerae and Pseudomonas aeruginosa CopI restore the Cu-resistant phenotype in the Rubrivivax gelatinosus ΔcopI mutant. We identified that Cys125 (ligand in the cupredoxin center) and conserved histidines and methionines are essential for R. gelatinosus CopI (RgCopI) function. We also performed spectroscopic analyses of the purified RgCopI protein and showed that it is a green cupredoxin able to bind a maximum of three Cu(II) ions: (i) a green Cu site (CuT1.5), (ii) a type 2 Cu binding site (T2) located in the N-terminal region, and (iii) a third site with a yet unidentified location. CopI is therefore one member of the poorly described CuT1.5 center cupredoxin family. It is unique, since it is a single-domain cupredoxin with more than one Cu site involved in Cu resistance.

Additive effects of metal excess and superoxide, a highly toxic mixture in bacteria.

Heavy metal contamination is a serious environmental problem. Understanding the toxicity mechanisms may allow to lower concentration of metals in the metal-based antimicrobial treatments of crops, and reduce metal content in soil and groundwater. Here, we investigate the interplay between metal efflux systems and the superoxide dismutase (SOD) in the purple bacterium Rubrivivax gelatinosus and other bacteria through analysis of the impact of metal accumulation. Exposure of the Cd2+ -efflux mutant ΔcadA to Cd2+ caused an increase in the amount and activity of the cytosolic Fe-Sod SodB, thereby suggesting a role of SodB in the protection against Cd2+ . In support of this conclusion, inactivation of sodB gene in the ΔcadA cells alleviated detoxification of superoxide and enhanced Cd2+ toxicity. Similar findings were described in the Cu+ -efflux mutant with Cu+ . Induction of the Mn-Sod or Fe-Sod in response to metals in other bacteria, including Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Vibrio cholera and Bacillus subtilis, was also shown. Both excess Cd2+ or Cu+ and superoxide can damage [4Fe-4S] clusters. The additive effect of metal and superoxide on the [4Fe-4S] could therefore explain the hypersensitive phenotype in mutants lacking SOD and the efflux ATPase. These findings underscore that ROS defence system becomes decisive for bacterial survival under metal excess.

Increasing the copper sensitivity of microorganisms by restricting iron supply, a strategy for bio-management practices.

Pollution by copper (Cu2+ ) extensively used as antimicrobial in agriculture and farming represents a threat to the environment and human health. Finding ways to make microorganisms sensitive to lower metal concentrations could help decreasing the use of Cu2 + in agriculture. In this respect, we showed that limiting iron (Fe) uptake makes bacteria much more susceptible to Cu2 + or Cd2+ poisoning. Using efflux mutants of the purple bacterium Rubrivivax gelatinosus, we showed that Cu+ and Cd2+ resistance relies on the expression of the Fur-regulated FbpABC and Ftr iron transporters. To support this conclusion, inactivation of these Fe-importers in the Cu+ or Cd2+ -ATPase efflux mutants gave rise to hypersensitivity towards these ions. Moreover, in metal overloaded cells the expression of FbpA, the periplasmic iron-binding component of the ferric ion transport FbpABC system was induced, suggesting that cells perceived an 'iron-starvation' situation and responded to it by inducing Fe-importers. In this context, the Fe-Sod activity increased in response to Fe homoeostasis dysregulation. Similar results were obtained for Vibrio cholerae and Escherichia coli, suggesting that perturbation of Fe-homoeostasis by metal excess appeared as an adaptive response commonly used by a variety of bacteria. The presented data support a model in which metal excess induces Fe-uptake to support [4Fe-4S] synthesis and thereby induce ROS detoxification system.

Cadmium and Copper Cross-Tolerance. Cu+ Alleviates Cd2 + Toxicity, and Both Cations Target Heme and Chlorophyll Biosynthesis Pathway in Rubrivivax gelatinosus.

Cadmium, although not redox active is highly toxic. Yet, the underlying mechanisms driving toxicity are still to be characterized. In this study, we took advantage of the purple bacterium Rubrivivax gelatinosus strain with defective Cd2 +-efflux system to identify targets of this metal. Exposure of the ΔcadA strain to Cd2 + causes a decrease in the photosystem amount and in the activity of respiratory complexes. As in case of Cu+ toxicity, the data indicated that Cd2 + targets the porphyrin biosynthesis pathway at the level of HemN, a S-adenosylmethionine and CxxxCxxC coordinated [4Fe-4S] containing enzyme. Cd2 + exposure therefore results in a deficiency in heme and chlorophyll dependent proteins and metabolic pathways. Given the importance of porphyrin biosynthesis, HemN represents a key metal target to account for toxicity. In the environment, microorganisms are exposed to mixture of metals. Nevertheless, the biological effects of such mixtures, and the toxicity mechanisms remain poorly addressed. To highlight a potential cross-talk between Cd2 + and Cu+ -efflux systems, we show (i) that Cd2 + induces the expression of the Cd2 +-efflux pump CadA and the Cu+ detoxification system CopA and CopI; and (ii) that Cu+ ions improve tolerance towards Cd2 +, demonstrating thus that metal mixtures could also represent a selective advantage in the environment.

Silver and Copper Acute Effects on Membrane Proteins and Impact on Photosynthetic and Respiratory Complexes in Bacteria.

Silver (Ag+) and copper (Cu+) ions have been used for centuries in industry, as well as antimicrobial agents in agriculture and health care. Nowadays, Ag+ is also widely used in the field of nanotechnology. Yet, the underlying mechanisms driving toxicity of Ag+ ions in vivo are poorly characterized. It is well known that exposure to excess metal impairs the photosynthetic apparatus of plants and algae. Here, we show that the light-harvesting complex II (LH2) is the primary target of Ag+ and Cu+ exposure in the purple bacterium Rubrivivax gelatinosus Ag+ and Cu+ specifically inactivate the 800-nm absorbing bacteriochlorophyll a (B800), while Ni2+ or Cd2+ treatment had no effect. This was further supported by analyses of CuSO4- or AgNO3-treated membrane proteins. Indeed, this treatment induced changes in the LH2 absorption spectrum related to the disruption of the interaction of B800 molecules with the LH2 protein. This caused the release of B800 molecules and subsequently impacted the spectral properties of the carotenoids within the 850-nm absorbing LH2. Moreover, previous studies have suggested that Ag+ can affect the respiratory chain in mitochondria and bacteria. Our data demonstrated that exposure to Ag+, both in vivo and in vitro, caused a decrease of cytochrome c oxidase and succinate dehydrogenase activities. Ag+ inhibition of these respiratory complexes was also observed in Escherichia coli, but not in Bacillus subtilisIMPORTANCE The use of metal ions represents a serious threat to the environment and to all living organisms because of the acute toxicity of these ions. Nowadays, silver nanoparticles are one of the most widely used nanoparticles in various industrial and health applications. The antimicrobial effect of nanoparticles is in part related to the released Ag+ ions and their ability to interact with bacterial membranes. Here, we identify, both in vitro and in vivo, specific targets of Ag+ ions within the membrane of bacteria. This include complexes involved in photosynthesis, but also complexes involved in respiration.

Biogenesis of the bacterial cbb3 cytochrome c oxidase: Active subcomplexes support a sequential assembly model.

The cbb3 oxidase has a high affinity for oxygen and is required for growth of bacteria, including pathogens, in oxygen-limited environments. However, the assembly of this oxidase is poorly understood. Most cbb3 are composed of four subunits: the catalytic CcoN subunit, the two cytochrome c subunits (CcoO and CcoP) involved in electron transfer, and the small CcoQ subunit with an unclear function. Here, we address the role of these four subunits in cbb3 biogenesis in the purple bacterium Rubrivivax gelatinosus Analyses of membrane proteins from different mutants revealed the presence of active CcoNQO and CcoNO subcomplexes and also showed that the CcoP subunit is not essential for their assembly. However, CcoP was required for the oxygen reduction activity in the absence of CcoQ. We also found that CcoQ is dispensable for forming an active CcoNOP subcomplex in membranes. CcoNOP exhibited oxygen reductase activity, indicating that the cofactors (hemes b and copper for CcoN and cytochromes c for CcoO and CcoP) were present within the subunits. Finally, we discovered the presence of a CcoNQ subcomplex and showed that CcoN is the required anchor for the assembly of the full CcoNQOP complex. On the basis of these findings, we propose a sequential assembly model in which the CcoQ subunit is required for the early maturation step: CcoQ first associates with CcoN before the CcoNQ-CcoO interaction. CcoP associates to CcoNQO subcomplex in the late maturation step, and once the CcoNQOP complex is fully formed, CcoQ is released for degradation by the FtsH protease. This model could be conserved in other bacteria, including the pathogenic bacteria lacking the assembly factor CcoH as in R. gelatinosus.

c-Type Cytochrome Assembly Is a Key Target of Copper Toxicity within the Bacterial Periplasm.

UNLABELLED: In the absence of a tight control of copper entrance into cells, bacteria have evolved different systems to control copper concentration within the cytoplasm and the periplasm. Central to these systems, the Cu(+) ATPase CopA plays a major role in copper tolerance and translocates copper from the cytoplasm to the periplasm. The fate of copper in the periplasm varies among species. Copper can be sequestered, oxidized, or released outside the cells. Here we describe the identification of CopI, a periplasmic protein present in many proteobacteria, and show its requirement for copper tolerance in Rubrivivax gelatinosus. The ΔcopI mutant is more susceptible to copper than the Cu(+) ATPase copA mutant. CopI is induced by copper, localized in the periplasm and could bind copper. Interestingly, copper affects cytochrome c membrane complexes (cbb3 oxidase and photosystem) in both ΔcopI and copA-null mutants, but the causes are different. In the copA mutant, heme and chlorophyll synthesis are affected, whereas in ΔcopI mutant, the decrease is a consequence of impaired cytochrome c assembly. This impact on c-type cytochromes would contribute also to the copper toxicity in the periplasm of the wild-type cells when they are exposed to high copper concentrations. IMPORTANCE: Copper is an essential cation required as a cofactor in enzymes involved in vital processes such as respiration, photosynthesis, free radical scavenging, and pathogenesis. However, copper is highly toxic and has been implicated in disorders in all organisms, including humans, because it can catalyze the production of toxic reactive oxygen species and targets various biosynthesis pathways. Identifying copper targets, provides insights into copper toxicity and homeostatic mechanisms for copper tolerance. In this work, we describe for the first time a direct effect of excess copper on cytochrome c assembly. We show that excess copper specifically affects periplasmic and membrane cytochromes c, thus suggesting that the copper toxicity targets c-type cytochrome biogenesis.

Oxygen-dependent copper toxicity: targets in the chlorophyll biosynthesis pathway identified in the copper efflux ATPase CopA deficient mutant.

Characterization of a copA(-) mutant in the purple photosynthetic bacterium Rubrivivax gelatinosus under low oxygen or anaerobic conditions, as well as in the human pathogen Neisseria gonorrhoeae identified HemN as a copper toxicity target enzyme in the porphyrin synthesis pathway. Heme synthesis is, however, unaffected by copper under high oxygen tension because of the aerobic coproporphyrinogen III oxidase HemF. Nevertheless, in the copA(-) mutant under aerobiosis, we show that the chlorophyll biosynthesis pathway is affected by excess copper resulting in a substantial decrease of the photosystem. Analyses of pigments and enzyme activity showed that under low copper concentrations, the mutant accumulated protochlorophyllide, suggesting that the protochlorophyllide reductase activity is affected by excess copper. Increase of copper concentration led to a complete lack of chlorophyll synthesis as a result of the loss of Mg-chelatase activity. Both enzymes are widely distributed from bacteria to plants; both are [4Fe-4S] proteins and oxygen sensitive; our data demonstrate their in vivo susceptibility to copper in the presence of oxygen. Additionally, our study provides the understanding of molecular mechanisms that may contribute to chlorosis in plants when exposed to metals. The role of copper efflux systems and the impact of copper on heme and chlorophyll biosynthesis in phototrophs are addressed.

EmbRS a new two-component system that inhibits biofilm formation and saves Rubrivivax gelatinosus from sinking.

Photosynthetic bacteria can switch from planktonic lifestyle to phototrophic biofilm in mats in response to environmental changes. The mechanisms of phototrophic biofilm formation are, however, not characterized. Herein, we report a two-component system EmbRS that controls the biofilm formation in a photosynthetic member of the Burkholderiales order, the purple bacterium Rubrivivax gelatinosus. EmbRS inactivation results in cells that form conspicuous bacterial veils and fast-sinking aggregates in liquid. Biofilm analyses indicated that EmbRS represses the production of an extracellular matrix and biofilm formation. Mapping of transposon mutants that partially or completely restore the wild-type (WT) phenotype allowed the identification of two gene clusters involved in polysaccharide synthesis, one fully conserved only in Thauera sp., a floc-forming wastewater bacterium. A second two-component system BmfRS and a putative diguanylate cyclase BdcA were also identified in this screen suggesting their involvement in biofilm formation in this bacterium. The role of polysaccharides in sinking of microorganisms and organic matter, as well as the importance and the evolution of such regulatory system in phototrophic microorganisms are discussed.

Coproporphyrin III excretion identifies the anaerobic coproporphyrinogen III oxidase HemN as a copper target in the Cu⁺-ATPase mutant copA⁻ of Rubrivivax gelatinosus.

Two genes encoding structurally similar Copper P1B -type ATPases can be identified in several genomes. Notwithstanding the high sequence and structural similarities these ATPases held, it has been suggested that they fulfil distinct physiological roles. In deed, we have shown that the Cu(+) -ATPase CtpA is required only for the activity of cuproproteins in the purple bacterium Rubrivivax gelatinosus; herein, we show that CopA is not directly required for cytochrome c oxidase but is vital for copper tolerance. Interestingly, excess copper in the copA(-) mutant resulted in a substantial decrease of the cytochrome c oxidase and the photosystem under microaerobic and anaerobic conditions together with the extrusion of coproporphyrin III. The data indicated that copper targeted the tetrapyrrole biosynthesis pathway at the level of the coproporphyrinogen III oxidase HemN and thereby affects the oxidase and the photosystem. This is the first in vivo demonstration that copper, like oxygen, affects tetrapyrrole biosynthesis presumably at the level of the SAM and [4Fe-4S] containing HemN enzyme. In light of these results and similar findings in Escherichia coli, the potential role of copper ions in the evolution of [4Fe-4S] enzymes and the Cu(+) -ATPases is discussed.

In situ dynamics of O2, pH and cyanobacterial transcripts associated with CCM, photosynthesis and detoxification of ROS.

The relative abundance of transcripts encoding proteins involved in inorganic carbon concentrating mechanisms (CCM), detoxification of reactive oxygen species (ROS) and photosynthesis in the thermophilic cyanobacterium Synechococcus OS-B' was measured in hot spring microbial mats over two diel cycles, and was coupled with in situ determinations of incoming irradiance and microenvironmental dynamics of O(2) and pH. Fluctuations in pH and O(2) in the mats were largely driven by the diel cycle of solar irradiance, with a pH variation from ∼7.0 to ∼9.5, and O(2) levels ranging from anoxia to supersaturation during night and day, respectively. Levels of various transcripts from mat cyanobacteria revealed several patterns that correlated with incident irradiance, O(2) and pH within the mat matrix. Transcript abundances for most genes increased during the morning dark-light transition. Some transcripts remained at a near constant level throughout the light period, whereas others showed an additional increase in abundance as the mat underwent transition from low-to-high light (potentially reflecting changes in O(2) concentration and pH), followed by either a decreased abundance in the early afternoon, or a gradual decline during the early afternoon and into the evening. One specific transcipt, psbA1, was the lowest during mid-day under high irradiance and increased when the light levels declined. We discuss these complex in situ transcriptional patterns with respect to environmental and endogenous cues that might impact and regulate transcription over the diel cycle.

Adaptation to oxygen: role of terminal oxidases in photosynthesis initiation in the purple photosynthetic bacterium, Rubrivivax gelatinosus.

The appearance of oxygen in the Earth's atmosphere via oxygenic photosynthesis required strict anaerobes and obligate phototrophs to cope with the presence of this toxic molecule. Here we show that in the anoxygenic phototroph Rubrivivax gelatinosus, the terminal oxidases (cbb(3), bd, and caa(3)) expand the range of ambient oxygen tensions under which the organism can initiate photosynthesis. Unlike the wild type, the cbb(3)(-)/bd(-) double mutant can start photosynthesis only in deoxygenated medium or when oxygen is removed, either by sparging cultures with nitrogen or by co-inoculation with strict aerobes bacteria. In oxygenated environments, this mutant survives nonphotosynthetically until the O(2) tension is reduced. The cbb(3) and bd oxidases are therefore required not only for respiration but also for reduction of the environmental O(2) pressure prior to anaerobic photosynthesis. Suppressor mutations that restore respiration simultaneously restore photosynthesis in nondeoxygenated medium. Furthermore, induction of photosystem in the cbb(3)(-) mutant led to a highly unstable strain. These results demonstrate that photosynthetic metabolism in environments exposed to oxygen is critically dependent on the O(2)-detoxifying action of terminal oxidases.

Organization and expression of photosynthesis genes and operons in anoxygenic photosynthetic proteobacteria.

Genes belonging to the same metabolic route are usually organized in operons in microbial genomes. For instance, most genes involved in photosynthesis were found clustered and organized in operons in photosynthetic Alpha- and Betaproteobacteria. The discovery of Gammaproteobacteria with a conserved photosynthetic gene cluster revives the questions on the role and the maintenance of such organization in proteobacteria. In this paper, we report the analysis of the structure and expression of the 14 kb cluster (crtEF-bchCXYZ-pufBALMC-crtADC) in the photosynthetic betaproteobacterium Rubrivivax gelatinosus, with the purpose of understanding the reasons and the biological constraints that might have led to the clustering of photosynthesis genes. The genetic analyses are substantiated by reverse transcription-PCR data which reveal the presence of a transcript encompassing the 14 genes and provide evidence of a polycistronic 'super-operon' organization starting at crtE and ending 14 kb downstream at the crtC gene. Furthermore, genetic analyses suggest that one of the selection pressures that may have driven and maintained the photosynthesis operons/super-operons in proteobacteria could very likely be the coexpression and regulation of the clustered genes/operon.

Regulation of nif gene expression and the energetics of N2 fixation over the diel cycle in a hot spring microbial mat.

Nitrogen fixation, a prokaryotic, O2-inhibited process that reduces N2 gas to biomass, is of paramount importance in biogeochemical cycling of nitrogen. We analyzed the levels of nif transcripts of Synechococcus ecotypes, NifH subunit and nitrogenase activity over the diel cycle in the microbial mat of an alkaline hot spring in Yellowstone National Park. The results showed a rise in nif transcripts in the evening, with a subsequent decline over the course of the night. In contrast, immunological data demonstrated that the level of the NifH polypeptide remained stable during the night, and only declined when the mat became oxic in the morning. Nitrogenase activity was low throughout the night; however, it exhibited two peaks, a small one in the evening and a large one in the early morning, when light began to stimulate cyanobacterial photosynthetic activity, but O2 consumption by respiration still exceeded the rate of O2 evolution. Once the irradiance increased to the point at which the mat became oxic, the nitrogenase activity was strongly inhibited. Transcripts for proteins associated with energy-producing metabolisms in the cell also followed diel patterns, with fermentation-related transcripts accumulating at night, photosynthesis- and respiration-related transcripts accumulating during the day and late afternoon, respectively. These results are discussed with respect to the energetics and regulation of N2 fixation in hot spring mats and factors that can markedly influence the extent of N2 fixation over the diel cycle.

A novel two domain-fusion protein in cyanobacteria with similarity to the CAB/ELIP/HLIP superfamily: evolutionary implications and regulation.

Vascular plants contain abundant, light-harvesting complexes in the thylakoid membrane that are non-covalently associated with chlorophylls and carotenoids. These light-harvesting chlorophyll a/b binding (LHC) proteins are members of an extended CAB/ELIP/HLIP superfamily of distantly related polypeptides, which have between one and four transmembrane helices (TMH). This superfamily includes the single TMH, high-light-inducible proteins (Hlips), found in cyanobacteria that are induced by various stress conditions, including high light, and are considered ancestral to the LHC proteins. The roles of, and evolutionary relationships between, these superfamily members are of particular interest, since they function in both light harvesting and photoprotection and may have evolved through tandem gene duplication and fusion events. We have investigated the Hlips (hli gene family) in the thermophilic unicellular cyanobacterium Synechococcus OS-B'. The five hli genes present on the genome of Synechococcus OS-B' are relatively similar, but transcript analyses indicate that there are different patterns of transcript accumulation when the cells are exposed to various growth conditions, suggesting that different Hlips may have specific functions. Hlip5 has an additional TMH at the N-terminus as a result of a novel fusion event. This additional TMH is very similar to a conserved hypothetical, single membrane-spanning polypeptide present in most cyanobacteria. The evolutionary significance of these results is discussed.

Population level functional diversity in a microbial community revealed by comparative genomic and metagenomic analyses.

In microbial mat communities of Yellowstone hot springs, ribosomal RNA (rRNA) sequence diversity patterns indicate the presence of closely related bacterial populations along environmental gradients of temperature and light. To identify the functional bases for adaptation, we sequenced the genomes of two cyanobacterial (Synechococcus OS-A and OS-B') isolates representing ecologically distinct populations that dominate at different temperatures and are major primary producers in the mat. There was a marked lack of conserved large-scale gene order between the two Synechococcus genomes, indicative of extensive genomic rearrangements. Comparative genomic analyses showed that the isolates shared a large fraction of their gene content at high identity, yet, differences in phosphate and nitrogen utilization pathways indicated that they have adapted differentially to nutrient fluxes, possibly by the acquisition of genes by lateral gene transfer or their loss in certain populations. Comparisons of the Synechococcus genomes to metagenomic sequences derived from mats where these Synechococcus stains were originally isolated, revealed new facets of microbial diversity. First, Synechococcus populations at the lower temperature regions of the mat showed greater sequence diversity than those at high temperatures, consistent with a greater number of ecologically distinct populations at the lower temperature. Second, we found evidence of a specialized population that is apparently very closely related to Synechococcus OS-B', but contains genes that function in the uptake of reduced ferrous iron. In situ expression studies demonstrated that these genes are differentially expressed over the diel cycle, with highest expression when the mats are anoxic and iron may be in the reduced state. Genomic information from these mat-specific isolates and metagenomic information can be coupled to detect naturally occurring populations that are associated with different functionalities, not always represented by isolates, but which may nevertheless be important for niche partitioning and the establishment of microbial community structure.

Responses of a thermophilic Synechococcus isolate from the microbial mat of Octopus Spring to light.

Thermophilic cyanobacteria of the genus Synechococcus are major contributors to photosynthetic carbon fixation in the photic zone of microbial mats in Octopus Spring, Yellowstone National Park. Synechococcus OS-B' was characterized with regard to the ability to acclimate to a range of different light irradiances; it grows well at 25 to 200 micromol photons m(-2) s(-1) but dies when the irradiance is increased to 400 micromol photons m(-2) s(-1). At 200 micromol photons m(-2) s(-1) (high light [HL]), we noted several responses that had previously been associated with HL acclimation of cyanobacteria, including cell bleaching, reduced levels of phycobilisomes and chlorophyll, and elevated levels of a specific carotenoid. Synechococcus OS-B' synthesizes the carotenoids zeaxanthin and beta,beta-carotene and a novel myxol-anhydrohexoside. Interestingly, 77-K fluorescence emission spectra suggest that Synechococcus OS-B' accumulates very small amounts of photosystem II relative to that of photosystem I. This ratio further decreased at higher growth irradiances, which may reflect potential photodamage following exposure to HL. We also noted that HL caused reduced levels of transcripts encoding phycobilisome components, particularly that for CpcH, a 20.5-kDa rod linker polypeptide. There was enhanced transcript abundance of genes encoding terminal oxidases, superoxide dismutase, tocopherol cyclase, and phytoene desaturase. Genes encoding the photosystem II D1:1 and D1:2 isoforms (psbAI and psbAII/psbAIII, respectively) were also regulated according to the light regimen. The results are discussed in the context of how Synechococcus OS-B' may cope with high light irradiances in the high-temperature environment of the microbial mat.

In situ analysis of nitrogen fixation and metabolic switching in unicellular thermophilic cyanobacteria inhabiting hot spring microbial mats.

Genome sequences of two Synechococcus ecotypes inhabiting the Octopus Spring microbial mat in Yellowstone National Park revealed the presence of all genes required for nitrogenase biosynthesis. We demonstrate that nif genes of the Synechococcus ecotypes are expressed in situ in a region of the mat that varies in temperature from 53.5 degrees C to 63.4 degrees C (average 60 degrees C); transcripts are only detected at the end of the day when the mat becomes anoxic. Nitrogenase activity in mat samples was also detected in the evening. Hitherto, N2 fixation in hot spring mats was attributed either to filamentous cyanobacteria (not present at >50 degrees C in these mats) or to heterotrophic bacteria. To explore how energy-generating processes of the Synechococcus ecotypes track natural light and O2 conditions, we evaluated accumulation of transcripts encoding proteins involved in photosynthesis, respiration, and fermentation. Transcripts from photosynthesis (cpcF, cpcE, psaB, and psbB) and respiration (coxA and cydA) genes declined in the evening. In contrast, transcripts encoding enzymes that may participate in fermentation fell into two categories; some (ldh, pdhB, ald, and ackA) decreased in the evening, whereas others (pflB, pflA, adhE, and acs) increased at the end of the day and remained high into the night. Energy required for N2 fixation during the night may be derived from fermentation pathways that become prominent as the mat becomes anoxic. In a broader context, our data suggest that there are critical regulatory switches in situ that are linked to the diel cycle and that these switches alter many metabolic processes within the microbial mat.