Rubrivivax Gelatinosus Research Articles

Investigating MerR’s Selectivity: The Crosstalk Between Cadmium and Copper Under Elevated Stress Conditions

Bacteria respond to metal pollution through sensors that control the uptake and the detoxification machineries. Specificity in metal recognition is therefore a prerequisite for triggering the appropriate response, particularly when facing a mixture of metals. In response to Cu+, the purple bacterium Rubrivivax gelatinosus induces the efflux Cu+-ATPase CopA by the Cu+ regulator CopR. However, genetic analyses have suggested the presence of additional regulators. Here, we show that CadR, the Cd2+ sensor, is involved in Cd2+ and Cu+ tolerance and demonstrate that CopR and CadR share common target genes. Interestingly, expression of the Cu+ detoxification and efflux (CopI/CopA) system was induced by Cd2+ and downregulated in the double mutant copRcadR−. This double mutant was more sensitive to low Cu+ concentration than the single copR− mutant, and accumulation of coproporphyrin III pointed to a significantly decreased expression of CopA. Furthermore, analyses of Cd2+ toxicity in the cadR− mutant suggested that although CopR is Cu+ selective, CopR is involved in Cd2+ response since the addition of Cu+ alleviates Cd2+ toxicity. Based on our current knowledge of metal transport across the inner membrane, Cd2+ and Cu+ do not share common efflux routes nor do they share common regulators. Nevertheless, the crosstalk between Cd2+ and Cu+ tolerance systems is demonstrated in the present study. The modulation of Cu+ detoxification by a Cd2+ regulator in vivo places emphasis on the relaxed selectivity, under elevated metal concentration, in MerR regulators.

Probing ligands to reaction centers to limit the photocycle in photosynthetic bacterium Rubrivivax gelatinosus

Light-induced electron flow between reaction center and cytochrome bc1 complexes is mediated by quinones and electron donors in purple photosynthetic bacteria. Upon high-intensity excitation, the contribution of the cytochrome bc1 complex is limited kinetically and the electron supply should be provided by the pool of reduced electron donors. The kinetic limitation of electron shuttle between reaction center and cytochrome bc1 complex and its consequences on the photocycle were studied by tracking the redox changes of the primary electron donor (BChl dimer) via absorption change and the opening of the closed reaction center via relaxation of the bacteriochlorophyll fluorescence in intact cells of wild type and pufC mutant strains of Rubrivivax gelatinosus. The results were simulated by a minimum model of reversible binding of different ligands (internal and external electron donors and inhibitors) to donor and acceptor sides of the reaction center. The calculated binding and kinetic parameters revealed that control of the rate of the photocycle is primarily due to 1) the light intensity, 2) the size and redox state of the donor pool, and 3) the unbinding rates of the oxidized donor and inhibitor from the reaction center. The similar kinetics of strains WT and pufC lacking the tetraheme cytochrome subunit attached to the reaction center raise the issue of the physiological importance of this subunit discussed from different points of view. SignificanceA crucial factor for the efficacy of electron donors in photosynthetic photocycle is not just the substantial size of the pool and large binding affinity (small dissociation constant KD = koff/kon) to the RC, but also the mean residence time (koff)−1 in the binding pocket. This is an important parameter that regulates the time of re-activation of the RC during multiple turnovers. The determination of koff has proven challenging and was performed by simulation of widespread experimental data on the kinetics of P+ and relaxation of fluorescence. This work is a step towards better understanding the complex pathways of electron transfer in proteins and simulation-based design of more effective electron transfer components in natural and artificial systems.

Biochemical investigations of polyphenol degradation enzymes in the phototrophic bacterium Rubrivivax gelatinosus.

Phloroglucinol (1,3,5-trihydroxybenzene) is an important intermediate in the degradation of flavonoids and tannins by anaerobic bacteria. Recent studies have shed light on the enzymatic mechanism of phloroglucinol degradation in butyrate-forming anaerobic bacteria, including environmental and intestinal bacteria such as Clostridium and Flavonifractor sp. Phloroglucinol degradation gene clusters have also been identified in other metabolically diverse bacteria, although the polyphenol metabolism of these microorganisms remain largely unexplored. Here, we describe biochemical studies of polyphenol degradation enzymes found in the purple non-sulfur bacterium Rubrivivax gelatinosus IL144, an anaerobic photoheterotroph reported to utilize diverse organic compounds as carbon sources for growth. In addition to the phloroglucinol reductase and dihydrophloroglucinol cyclohydrolase that catalyze phloroglucinol degradation, we characterize a Mn2+-dependent phloretin hydrolase that catalyzes the cleavage of phloretin into phloroglucinol and phloretic acid. We also report a Mn2+-dependent decarboxylase (DeC) that catalyzes the reversible decarboxylation of 2,4,6-trihydroxybenzoate to form phloroglucinol. A bioinformatics search led to the identification of DeC homologs in diverse soil and gut bacteria, and biochemical studies of a DeC homolog from the human gut bacterium Flavonifractor plautii demonstrated that it is also a 2,4,6-trihydroxybenzoate decarboxylase. Our study expands the range of enzymatic mechanisms for phloroglucinol formation, and provides further biochemical insight into polyphenol metabolism in the anaerobic biosphere.

Genes and Pathway Reactions Related to Carotenoid Biosynthesis in Purple Bacteria.

In purple bacteria, the genes of the carotenoid pathways are part of photosynthesis gene clusters which were distributed among different species by horizontal gene transfer. Their close organisation facilitated the first-time cloning of carotenogenic genes and promoted the molecular investigation of spheroidene and spirilloxanthin biosynthesis. This review highlights the cloning of the spheroidene and spirilloxanthin pathway genes and presents the current knowledge on the enzymes involved in the carotenoid biosynthesis of purple sulphur and non-sulphur bacteria. Mostly, spheroidene or spirilloxanthin biosynthesis exists in purple non-sulphur bacteria but both pathways operate simultaneously in Rubrivivax gelatinosus. In the following years, genes from other bacteria including purple sulphur bacteria with an okenone pathway were cloned. The individual steps were investigated by kinetic studies with heterologously expressed pathway genes which supported the establishment of the reaction mechanisms. In particular, the substrate and product specificities revealed the sequential order of the speroidene and spiriloxanthin pathways as well as their interactions. Information on the enzymes involved revealed that the phytoene desaturase determines the type of pathway by the formation of different products. By selection of mutants with amino acid exchanges in the putative substrate-binding site, the neurosporene-forming phytoene desaturase could be changed into a lycopene-producing enzyme and vice versa. Concerning the oxygen groups in neurosporene and lycopene, the tertiary alcohol group at C1 is formed from water and not by oxygenation, and the C2 or C4 keto groups are inserted differently by an oxygen-dependent or oxygen-independent ketolation reaction, respectively.

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.

Light and oxygen facilitating the directly treatment food wastewater and poly-β-hydroxybutyrate, 5-aminolevulinic acid, pigment productions by Rubrivivax gelatinosus.

Rubrivivax gelatinosus has the advantage of using wastewater to realize biomass recovery. However, they still cannot be applied large scale because they cannot directly treat the wastewater containing macromolecular organics. Thus, this article investigated the effects of light-oxygen conditions on R. gelatinosus by directly recycling wastewater containing macromolecular organics to produce biomass, poly-β-hydroxybutyrate (PHB), 5-aminolevulinic acid (5-ALA), and pigment. Results showed that R. gelatinosus directly treated the macromolecule organic (soybean protein and starch) wastewaters and achieved biomass recovery under light-anaerobic and light-micro-oxygen in six conditions. Chemical oxygen demand, protein, and starch removals for two wastewaters all reached above 70%. Renewable bio-resources such as biomass, PHB, 5-ALA, and pigment production were 10 times the initial content. Theoretical analysis indicated that light activated the synthesis of protease and amylase. However, oxygen concentration decided the number of enzymes. When oxygen was at micro-oxygen or anaerobic, the aforementioned expression and synthesis were conducted. In summary, this study expanded the viewpoint ignored by traditional theory. It was realized that R. gelatinosus directly treated wastewater and accumulated nutrients (biomass, PHB, pigment, and 5-ALA) for recycling, which reduced the secondary pollution of excess sludge into the environment.

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.

Photo-fermentative hydrogen production performance of a newly isolated Rubrivivax gelatinosus YP03 strain with acid tolerance

Screening and excavating new photosynthetic bacteria with excellent hydrogen production performance is extremely important for improving the photo-fermentative hydrogen production. A new photosynthetic bacterium YP03 was isolated and identified to be Rubrivivax gelatinosus by morphological characterization and phylogenetic analysis. The effects of several key factors on hydrogen production performance were carried out. The results indicated that YP03 strain showed a preference for the carbon sources, and 5375 ± 398 mL/L of maximum hydrogen yield was obtained using butyrate medium. Meanwhile, YP03 strain could use several nitrogen sources to produce hydrogen, and glutamic acid was the optimum nitrogen source for hydrogen produced. Furthermore, YP03 exhibited better hydrogen production performance at initial pH 7.0, reaction temperature 33 °C and light intensity 5000 lux, and the maximum hydrogen production rate was 108.3 ± 12.4 mL/(Lh), which was relatively high compared with the previous reports by R. gelatinosus. Especially, the proper pH for hydrogen production by YP03 ranged from weak acid to neutral (6.5–7.0) and it still could produce hydrogen at pH 5.5 showing the characteristic of acid tolerance. It suggested that YP03 is a potential candidate for the integration of dark- and photo-fermentative hydrogen production. These findings contribute to our understanding of YP03 strain and provide a prospective photosynthetic bacterium for efficient hydrogen production in future research.

Variation in Soil Denitrification among Fertilization Regimes and Its Microbial Mechanism

A 27-year field experiment with various fertilization regimes was chosen for evaluating how fertilization regimes influence soil denitrification potential (SDP), to determine whether the microbial mechanism of SDP variation varies with fertilization regimes. The results showed that the compost (OM) fertilizer treatment presented the highest SDP followed by half compost plus half chemical fertilizer (NPKOM) treatment. Both SDPs were 3–25 times higher than those of the other treatments, and the SDP was higher in the OM than NPKOM, indicating an increase in SDP with the application of compost. The higher SDPs from OM and NPKOM were closely associated with higher abundances of functional genes, and enrichments of Rubrivivax gelatinosus and Azospirillum sp. TSO28-1, is mainly determined by the organic carbon (SOC), total nitrogen (TN), and dissolved organic carbon (DOC). For the treatments without compost, SDP mainly followed the order of chemical fertilizers of N and P (NP) > chemical fertilizers of N and K (NK) > unfertilized (Nil) > chemical fertilizers of N, P, and K (NPK) > chemical fertilizers of P and K (PK), in which the variations had a close association with different specific microbial species. That is, relative to Nil, the enhanced SDP in NP and NK was coupled with the enrichment of Ideonella sp. NC3L-43b and R. gelatinosus, likely due to their higher NO3 − contents, while the reduced SDP in NPK was linked to the depletions of R. gelatinosus and Azospira sp. NC3H-14, and that in PK to the depletion of R. gelatinosus and Azospirillum sp. TSO28-1, probably as a result of their lower N:P ratios. Microbial mechanisms for SDP differences varied with fertilization regimes, and R. gelatinosus can be regarded as a universal microbial indicator for SDP differences across the fertilization treatments.

Illumination promoting biomass production in wastewater treatment for Rubrivivax gelatinosus

In order to improve the reuse degree of Rubrivivax gelatinosus (R. gelatinosus) resource in soybean producing wastewater (SPW) treatment, the impacts of diverse illumination strength on R. gelatinosus biomass and organic contaminant abatement were discussed. It was reported that with the best illumination strength (6,000 lux), the production (3,800 mg/L) of biomass was increased by sixty percent of dark group. COD removal and protein removal rate got 85 and 80%. Influence of diverse illumination strength on R. gelatinosus growth were divided into three aspects, including illumination limitation (0−5,000 lux), illumination saturation (5,000−7,000 lux), illumination inhibition (8,000 lux or higher). Meanwhile, at 6,000 lux, bacteriochlorophyll and carotenoids contents, ATP production were increased by 200, 200, 43.9% severally. Appropriate illumination improved ATP production by increasing the generation of bacteriochlorophyll and carotenoids in photosynthesis. Augment of energy production provoked the biomass production and wastewater treatment. Nevertheless, low or high illumination strength was un-advantage for R. gelatinosus growth due to the illumination deficiency or restrian. Moreover, 6,000 lux shortened the treatment time from 96 h to 48 h.

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.

Artificial humic substances improve microbial activity for binding CO2

SummaryHumic substances (HS) are an indicator of fertile soils, but more and more soils keep losing there humic matter. This is mostly due to anthropogenic over-cultivation. Artificial humic acid (A-HA) and artificial fulvic acid were synthesized from agricultural litter, with high similarity to natural HS extracted from soil. These samples were added to black soils, and soil activity and nutrients availability were analyzed. The results demonstrate that the content of dissolved organic matter and total organic carbon (TOC) largely increased. The increase in TOC 28 days after addition of A-HA was 21.4 g/kg. This was much higher than the amount of the added A-HA carbon, which was 0.3 g/kg. As a “secondary” benefit, nutrient availability is increased, promoting the growth of plants.Using high-throughput sequencing we revealed that A-HA strongly supports the growth of photosynthetic Rubrivivax gelatinosus, which induced the carbon sequestration. Thus, application of artificial HS shows potential for biologically amplified carbon sequestration within black soils.

Optimization of carriers and packaging for effective biofertilizers to enhance Oryza sativa L. growth in paddy soil

For a biofertilizer to promote plant growth after application, cell survival during storage must be ensured, while the carrier formulation and packaging are important factors during storage. This study aimed to optimize the ratio of rubber wood ash (RWA), decanter cake (DCC), rice husk ash (RHA), and spent coffee grounds (SCG) as a mixed carrier of biofertilizer of purple non-sulfur bacteria (PNSB), Rhodopseudomonas palustris and Rubrivivax gelatinosus. Different packaging materials, and different sealing methods were investigated. The phytotoxicity of PNSB biofertilizers in a solid form, and their effectiveness to enhance rice (Oryza sativa L.) seedling growth in paddy soil were also investigated in comparison with a liquid form. A mixed carrier of RWA, DCC, RHA and SCG at a ratio of 3:4:2:1 most effectively maintained viability. After 6-month storage at 25 ± 3 °C, PNSB population of a mixed carrier containing R. palustris at roughly 10 log CFU/g in nylon-linear low density polyethylene (nylon-LLDPE) bags that were vacuum sealed at 500 W decreased by only 3 log cycles compared with 7 log cycles in the control. The PNSB biofertilizers, each genus and a mixed culture at 1: 1, either in solid or liquid form at their optimal dilutions showed no phytotoxicity. However, the mixed carrier itself as a control was slightly toxic. At these optimal dilutions, all PNSB biofertilizers in both liquid and solid formulations significantly increased rice growth in paddy soil compared with controls. A suitable mix of agro-industrial waste and suitable packing prolonged the shelf-life and potency of PNSB biofertilizers.

How the O2-dependent Mg-protoporphyrin monomethyl ester cyclase forms the fifth ring of chlorophylls.

Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase catalyses the formation of the isocyclic ring, the hallmark of chlorins and bacteriochlorins, producing protochlorophyllide a and contributing significantly to the absorption properties of chlorophylls and bacteriochlorophylls. The O2-dependent cyclase is found in oxygenic phototrophs and in some purple bacteria. We overproduced the simplest form of the cyclase, AcsF from Rubrivivax gelatinosus, in Escherichia coli. In biochemical assays the diiron cluster within AcsF is reduced by ferredoxin furnished by NADPH and ferredoxin:NADP+ reductase or by direct coupling to Photosystem I photochemistry, linking cyclase to the photosynthetic electron transport chain. Kinetic analyses yield a k cat of 0.9 min-1, a K M of 7.0 µM for MgPME, and a K d for MgPME of 0.16 µM. Mass spectrometry identified 131-hydroxy-MgPME and 131-keto-MgPME as intermediates in the formation of the isocyclic ring, revealing the reaction chemistry that converts porphyrins to chlorins, and completing the work originated by Sam Granick in 1950.

Artificial Humic Substances Improve Microbial Activity for Binding CO <sub>2</sub>

Abundance of humic substances (HS) are the secret of fertile soils, but not all soils have enough humic matter, also due to anthropogenic over-cultivation. Artificial humic acid (A-HA) and artificial fulvic acid (A-FA) were synthesized from agricultural litter, with high similarity to natural extracts. These samples were added to black soils, and soil activity and nutrients availability were analyzed. The results demonstrate that the content of dissolved organic matter (DOC) and total organic carbon (TOC) largely increased. The increment of C in the soil was up to 21.4 g/kg, much higher than the carbon added via the A-HA. As a “secondary” benefit, nutrient availability is increased, promoting the growth of plants. Using high-throughput sequencing we revealed that A-HA strongly supports the growth of a photosynthetic Rubrivivax gelatinosus, which induced the carbon sequestration. Thus, application of A-HS shows potential for biologically amplified carbon sequestration within black soils.

Protochlorophyllide synthesis by recombinant cyclases from eukaryotic oxygenic phototrophs and the dependence on Ycf54.

The unique isocyclic E ring of chlorophylls contributes to their role as light-absorbing pigments in photosynthesis. The formation of the E ring is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase, and the O2-dependent cyclase in prokaryotes consists of a diiron protein AcsF, augmented in cyanobacteria by an auxiliary subunit Ycf54. Here, we establish the composition of plant and algal cyclases, by demonstrating the in vivo heterologous activity of O2-dependent cyclases from the green alga Chlamydomonas reinhardtii and the model plant Arabidopsis thaliana in the anoxygenic photosynthetic bacterium Rubrivivax gelatinosus and in the non-photosynthetic bacterium Escherichia coli. In each case, an AcsF homolog is the core catalytic subunit, but there is an absolute requirement for an algal/plant counterpart of Ycf54, so the necessity for an auxiliary subunit is ubiquitous among oxygenic phototrophs. A C-terminal ∼40 aa extension, which is present specifically in green algal and plant Ycf54 proteins, may play an important role in the normal function of the protein as a cyclase subunit.

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

SummaryPollution 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.

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

SummaryHeavy 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.

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.

Carbonic Anhydrase in Anoxygenic Phototrophic Bacteria

The genes encoding carbonic anhydrase (CA) were found in all anoxygenic purple bacteria. The genes of α- and β-CA were found in purple nonsulfur bacteria of the class Alphaproteobacteria:Rhodospirillum rubrum, Rhodospirillum fulvum, Rhodoblastus acidophilus, and Rhodopseudomonas palustris. The alphaproteobacteria Rhodomicrobium vannielii, Blastochlorisviridis, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodobacter veldkampii, Rhodovulumeuryhalinum, and Rhodovulum sulfidophilum possessed only the β-CA genes. Both nonsulfur purple bacteria of the class Betaproteobacteria (Rubrivivax gelatinosus) and purple sulfur bacteria (class Gammaproteobacteria) contained the α- and β-CA. No CA genes were found in green sulfur bacteria Chlorobaculum limnaeum and Chlorobaculum parvum, as well as in filamentous green nonsulfur bacteria Chloroflexus aurantiacus. However, the β-CA gene was revealed in Oscillochloris trichoides, which belonged to the latter taxonomic group. No γ-CA genes were detected in the genomes of the phototrophic bacteria studied in the present work. Although CA genes were present in all purple bacteria, the α- and β-CA activity was observed only in four species of purple nonsulfur Alphaproteobacteria: Rhodospirillum rubrum, Rhodopseudomonas palustris, Rhodoblastusacidophilus, and Rhodospirillum fulvum. These bacteria are able to synthesize CA under both photoautotrophic and photoheterotrophic conditions in the media with acetate, malate, or fructose. These bacteria (except for Rhodospirillum fulvum, which is unable to grow under aerobic conditions), also exhibit CA activity when grown under aerobic conditions in the dark.