Genome mining to unravel potential metabolic pathways linked to gallium bioleaching ability of bacterial mine isolates.

Gallium (Ga) is considered a high-tech Critical Metal, used in the manufacture of several microelectronic components containing either gallium arsenide (GaAs) or gallium nitride (GaN). The current high demand for this critical metal urges the development of effective recovery processes from secondary resources such as mine tailings or electronic recycling material. The importance of bioleaching as a biotechnological process to recover metals prompted this study, where an integrative approach combining experimental and genomic analysis was undertaken to identify potential mechanisms involved in bioleaching ability and strategies to cope with high metal(loid)s concentrations in five mine isolates. The Clusters of Orthologous Group (COG) annotation showed that the "amino acid transport and metabolism" [E] was the most predominant functional category in all genomes. In addition, the KEEG pathways analysis also showed predicted genes for the biosynthetic pathways of most amino acids, indicating that amino acids could have an important role in the Ga leaching mechanism. The presence of effective resistance mechanisms to Ga and arsenic (As) was particularly important in GaAs bioleaching batch assays, and might explain the divergence in bioleaching efficiency among the bacterial strains. Rhodanobacter sp. B2A1Ga4 and Sphingomonas sp. A2-49 with higher resistance, mainly to As, were the most efficient bioleaching strains under these conditions. In bioleaching assays using cell-free spent medium Arthrobacter silviterrae A2-55 with lower As resistance outperformed all the other stains. Overall, higher efficiency in Ga leaching was obtained in bioleaching assays using GaAs when compared to GaN.

Post-measurement compressed calibration for ICP-MS-based metal quantification in mine residues bioleaching.

Bioleaching is an actual economical alternative to treat residues, which allows, depending on the chosen strategy, two possible outcomes: (1) a leachate enriched with target metals, or (2) a residue enriched in target metals through the leaching of interfering components (IC). This work aimed to study the metals released by bioprocessing the Panasqueira mine tailings, as a strategy to increase critical metals' relative concentration in residues. Biostimulation of the local microbiota was compared to a bioaugmentation approach using the autochthonous Diaphorobacter polyhydroxybutyrativorans strain B2A2W2. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was selected to study the metals released in the leachate through multi-element external standards. A new data treatment method was developed to use a preliminary sweep of intensities to quantify the non-initial target metals concentration in the leachate, based on preliminary ICP-MS intensity measurements. The results demonstrated that biostimulation was an efficient bioleaching strategy for the IC silicon, aluminium, magnesium, selenium, manganese, zinc, iron, and copper, by decreasing concentration, resulting in a relative increase in the gallium and yttrium (10x) levels in the treated residue. The strategy followed to quantify a large number of elements with ICP-MS using a reduced number of data points for calibration proved valid and speeded up the analytical process.

Potential of tellurite resistance in heterotrophic bacteria from mining environments.

Untreated mining wastes and improper disposal of high-tech devices generate an environmental increase of bioavailable metalloids, exerting stress on autochthonous microbial populations. Tellurium is a metalloid, an element with raising economic importance; nevertheless, its interaction with living organisms is not yet fully understood. Here we characterized aerobic heterotrophic bacteria, isolated from high metal-content mining residues, able to resist/reduce tellurite into tellurium structures and to determine the presence of confirmed tellurite resistance genetic determinants in resistant strains. We identified over 50 tellurite-resistant strains, among 144 isolates, eight strains reduced tellurite to tellurium at different rates, with the concomitant production of tellurium deposits. Most tellurite resistance genes were found in strains from Bacillales, with the prevalence of genes of the ter operon. This work demonstrated that bacterial isolates, from environments with a persistent selective pressure, are potential candidates for uncovering strategies for tellurite resistance and/or production of valuable Te-containing materials.

Impact of Tellurite on the Metabolism of Paenibacillus pabuli AL109b With Flagellin Production Explaining High Reduction Capacity.

Tellurium (Te) is a metalloid with scarce and scattered abundance but with an increased interest in human activity for its uses in emerging technologies. As is seen for other metals and metalloids, the result of mining activity and improper disposal of high-tech devices will lead to niches with increased abundance of Te. This metalloid will be more available to bacteria and represent an increasing selective pressure. This environmental problem may constitute an opportunity to search for microorganisms with genetic and molecular mechanisms of microbial resistance to Te toxic anions. Organisms from Te-contaminated niches could provide tools for Te remediation and fabrication of Te-containing structures with added value. The objective of this study was to determine the ability of a high metal-resistant Paenibacillus pabuli strain ALJ109b, isolated from high metal content mining residues, to reduce tellurite ion, and to evaluate the formation of metallic tellurium by cellular reduction, isolate the protein responsible, and determine the metabolic response to tellurite during growth. P. pabuli ALJ109b demonstrated to be resistant to Te (IV) at concentrations higher than reported for its genus. It can efficiently remove soluble Te (IV) from solution, over 20% in 8 h of growth, and reduce it to elemental Te, forming monodisperse nanostructures, verified by scattering electron microscopy. Cultivation of P. pabuli ALJ109b in the presence of Te (IV) affected the general protein expression pattern, and hence the metabolism, as demonstrated by high-throughput proteomic analysis. The Te (IV)-induced metabolic shift is characterized by an activation of ROS response. Flagellin from P. pabuli ALJ109b demonstrates high Te (0) forming activity in neutral to basic conditions in a range of temperatures from 20°C to 37°C. In conclusion, the first metabolic characterization of a strain of P. pabuli response to Te (IV) reveals a highly resistant strain with a unique Te (IV) proteomic response. This strain, and its flagellin, display, all the features of potential tools for Te nanoparticle production.

Mutant Strains of Escherichia coli and Methicillin-Resistant Staphylococcus aureus Obtained by Laboratory Selection To Survive on Metallic Copper Surfaces.

Artificial laboratory evolution was used to produce mutant strains of Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) able to survive on antimicrobial metallic copper surfaces. These mutants were 12- and 60-fold less susceptible to the copper-mediated contact killing process than their respective parent strains. Growth levels of the mutant and its parent in complex growth medium were similar. Tolerance to copper ions of the mutants was unchanged. The mutant phenotype remained stable over about 250 generations under nonstress conditions. The mutants and their respective parental strains accumulated copper released from the metallic surfaces to similar extents. Nevertheless, only the parental strains succumbed to copper stress when challenged on metallic copper surfaces, suffering complete destruction of the cell structure. Whole-genome sequencing and global transcriptome analysis were used to decipher the genetic alterations in the mutant strains; however, these results did not explain the copper-tolerance phenotypes on the systemic level. Instead, the mutants shared features with those of stressed bacterial subpopulations entering the early or "shallow" persister state. In contrast to the canonical persister state, however, the ability to survive on solid copper surfaces was adopted by the majority of the mutant strain population. This indicated that application of solid copper surfaces in hospitals and elsewhere has to be accompanied by strict cleaning regimens to keep the copper surfaces active and prevent evolution of tolerant mutant strains.IMPORTANCE Microbes are rapidly killed on solid copper surfaces by contact killing. Copper surfaces thus have an important role to play in preventing the spread of nosocomial infections. Bacteria adapt to challenging natural and clinical environments through evolutionary processes, for instance, by acquisition of beneficial spontaneous mutations. We wish to address the question of whether mutants can be selected that have evolved to survive contact killing on solid copper surfaces. We isolated such mutants from Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) by artificial laboratory evolution. The ability to survive on solid copper surfaces was a stable phenotype of the mutant population and not restricted to a small subpopulation. As a consequence, standard operation procedures with strict hygienic measures are extremely important to prevent the emergence and spread of copper-surface-tolerant persister-like bacterial strains if copper surfaces are to be sustainably used to limit the spread of pathogenic bacteria, e.g., to curb nosocomial infections.

Tailings microbial community profile and prediction of its functionality in basins of tungsten mine.

In a circular economy concept, where more than 300 million tons of mining and quarrying wastes are produced annually, those are valuable resources, supplying metals that are extracted today by other processes, if innovative methods and processes for efficient extraction of these elements are applied. This work aims to assess microbiological and chemical spatial distribution within two tailing basins from a tungsten mine, using a MiSeq approach targeting the 16S rRNA gene, to relate microbial composition and function with chemical variability, thus, providing information to enhance the efficiency of the exploitation of these secondary sources. The tailings sediments core microbiome comprised members of family Anaerolineacea and genera Acinetobacter, Bacillus, Cellulomonas, Pseudomonas, Streptococcus and Rothia, despite marked differences in tailings physicochemical properties. The higher contents of Al and K shaped the community of Basin 1, while As-S-Fe contents were correlated with the microbiome composition of Basin 2. The predicted metabolic functions of the microbiome were rich in genes related to metabolism pathways and environmental information processing pathways. An in-depth understanding of the tailings microbiome and its metabolic capabilities can provide a direction for the management of tailings disposal sites and maximize their potential as secondary resources.

Two plant-hosted whole-cell bacterial biosensors for detection of bioavailable Cr(VI).

Metal whole-cell biosensors (WCBs) have been reported as very useful tools to detect and quantify the presence of bioavailable fractions of certain metals in water and soil samples. In the current work, two bacterial WCBs able to report Cr(VI) presence and plants growing on Cr(VI)-enriched soil/medium were used to assess the potential transfer of this metal to organisms of higher trophic levels, and the risk of transfer to the food chain. To do it, the functionality of the WCBs within tissues of inoculated plants in contact with Cr(VI)-contaminated soil and water was studied in vitro and in a controlled greenhouse environment. One WCB was the previously described Ochrobactrum tritici pCHRGFP2 and the second, Nitrospirillum amazonense pCHRGFP2, is a newly engineered naturally-occurring endophytic microorganism. Three rice varieties (IAC 4440, BRS 6 CHUÍ, IRGA 425) and one maize variety (1060) were tested as hosts and subjected to Cr(VI) treatments (25 µM), with different results obtained. Inoculation of each WCB into plants exposed to Cr(VI) showed GFP expression within plant tissues. WCBs penetrated the root tissues and later colonized the shoots and leaves. In general, a higher fluorescence signal was detected in roots, together with a higher Cr content and denser WCB colonization. Best fluorescence intensities per plant biomass of shoots were obtained for plant host IRGA 425. Therefore, by analyzing colonized tissues, both WCBs allowed the detection of Cr(VI) contamination in soils and its transfer to plants commonly used in crops for human diet.

Impact of plant-associated bacteria biosensors on plant growth in the presence of hexavalent chromium.

Cr(VI) is a highly toxic metal produced by anthropogenic activity which may impact the environment, affecting plants and animals. In plants, chromium both as Cr(III) or Cr(VI) can be absorbed by roots, is poorly translocated and affects negatively plant growth. Plants used in phytoremediation need to cope with chromium toxicity. This work aimed to evaluate strains of Ochrobactrum tritici and Nitrospirillum amazonense, resistant and modified in order to become chromate whole-cell biosensors, as plant-protectors enabling plants to withstand contaminated soils. In vitro tests were performed in three rice varieties and one maize variety. Initial evaluations of Cr(VI) toxicity to plants showed that plants had different sensitivities and BRS 6 CHUÍ rice variety was the most resistant. The metal affected plant growth and development, essentially in roots which were totally inhibited in rice varieties at 500 µM. This effect was plant-dependent. Modified N. amazonense proved to protect maize plants independently of the inoculation dose but O. tritici showed plant specificity and some toxicity when inoculated at high numbers, inhibiting rice development but not maize. Inoculants were directly responsible for growth improvements of specific plant varieties at 1.25 ppm Cr(VI), a concentration which corresponds to a weak soil contamination. Improvements were observed relatively to the Cr(VI)-treated controls, but also relative to the untreated controls, i.e., the benefits went beyond a simple neutralization of inhibition brought by Cr(VI) toxicity.

The Microbiome of Endophytic, Wood Colonizing Bacteria from Pine Trees as Affected by Pine Wilt Disease.

Pine wilt disease (PWD) is a devastating forest disease present worldwide. In this study we analyzed the effects of the invasion of the pinewood nematode Bursaphelenchus xylophilus, the major pathogen causing PWD, on the endophytic microbiome of adult P. pinaster trees. Wood samples from trees with different degrees of PWD disease were collected at two sites (A and M) in Portugal. Endophytic bacteria were characterized based on directly extracted DNA by fingerprinting and barcoding using the 16S rRNA gene as marker. Furthermore, cultivation-based approaches were used to obtain isolates of the major taxa to study their ecophysiology. The endophytic microbiome from P. pinaster trees differed significantly between the two sampling sites. Main bacterial OTUs belonged to the Proteobacteria (39% (site M) - 97% (site A)), and Firmicutes (0.70% (site A) - 44% (site M)). However, consequences of the invasion with the pathogen were comparable. Interestingly diversity of wood endophytic bacteria increased with the severity of the diseases, with highest diversity levels observed in in the most affected trees. Our results suggest that in the first stages of the disease, the defence mechanisms of plants are repressed by the pathogen, resulting in a colonization of the wood interior by soil microorganisms.

Natural hot spots for gain of multiple resistances: arsenic and antibiotic resistances in heterotrophic, aerobic bacteria from marine hydrothermal vent fields.

Microorganisms are responsible for multiple antibiotic resistances that have been associated with resistance/tolerance to heavy metals, with consequences to public health. Many genes conferring these resistances are located on mobile genetic elements, easily exchanged among phylogenetically distant bacteria. The objective of the present work was to isolate arsenic-, antimonite-, and antibiotic-resistant strains and to determine the existence of plasmids harboring antibiotic/arsenic/antimonite resistance traits in phenotypically resistant strains, in a nonanthropogenically impacted environment. The hydrothermal Lucky Strike field in the Azores archipelago (North Atlantic, between 11°N and 38°N), at the Mid-Atlantic Ridge, protected under the OSPAR Convention, was sampled as a metal-rich pristine environment. A total of 35 strains from 8 different species were isolated in the presence of arsenate, arsenite, and antimonite. ACR3 and arsB genes were amplified from the sediment's total DNA, and 4 isolates also carried ACR3 genes. Phenotypic multiple resistances were found in all strains, and 7 strains had recoverable plasmids. Purified plasmids were sequenced by Illumina and assembled by EDENA V3, and contig annotation was performed using the "Rapid Annotation using the Subsystems Technology" server. Determinants of resistance to copper, zinc, cadmium, cobalt, and chromium as well as to the antibiotics ß-lactams and fluoroquinolones were found in the 3 sequenced plasmids. Genes coding for heavy metal resistance and antibiotic resistance in the same mobile element were found, suggesting the possibility of horizontal gene transfer and distribution of theses resistances in the bacterial population.

Nematicidal bacteria associated to pinewood nematode produce extracellular proteases.

Bacteria associated with the nematode Bursaphelenchus xylophilus, a pathogen of trees and the causal agent of pine wilt disease (PWD) may play a role in the disease. In order to evaluate their role (positive or negative to the tree), strains isolated from the track of nematodes from infected Pinus pinaster trees were screened, in vitro, for their nematicidal potential. The bacterial products, from strains more active in killing nematodes, were screened in order to identify and characterize the nematicidal agent. Forty-seven strains were tested and, of these, 21 strains showed capacity to produce extracellular products with nematicidal activity. All Burkholderia strains were non-toxic. In contrast, all Serratia strains except one exhibited high toxicity. Nematodes incubated with Serratia strains showed, by SEM observation, deposits of bacteria on the nematode cuticle. The most nematicidal strain, Serratia sp. A88copa13, produced proteases in the supernatant. The use of selective inhibitors revealed that a serine protease with 70 kDa was majorly responsible for the toxicity of the supernatant. This extracellular serine protease is different phylogenetically, in size and biochemically from previously described proteases. Nematicidal assays revealed differences in nematicidal activity of the proteases to different species of Bursaphelenchus, suggesting its usefulness in a primary screen of the nematodes. This study offers the basis for further investigation of PWD and brings new insights on the role bacteria play in the defense of pine trees against B. xylophilus. Understanding all the factors involved is important in order to develop strategies to control B. xylophilus dispersion.

Metal-induced phosphate extracellular nanoparticulate formation in Ochrobactrum tritici 5bvl1.

Hexavalent chromium (Cr(VI)) is a toxic environmental contaminant which detoxification consists in reduction to Cr(III). In this work, the Cr(VI)-resistant and reducing Ochrobactrum tritici 5bvl1 produced phosphate nanoparticles upon exposure to Cr(VI) and Fe(III), effectively removing chromium from solution. Under Cr(VI) stress, higher siderophore production by strain 5bvl1 was observed. Cr(VI) toxicity was decreased in presence of Fe(III), increasing the growth and Cr(VI)-reduction rates in cell cultures, lowering the amount of morphologically compromised cells and promoting chromium immobilization as insoluble extracellular phosphate complexes. The formation of phosphate nanoparticles increased with Cr(VI) and Fe(III) concentrations and was also stimulated by Ni(II). Under these experimental conditions, nanoparticle formation occurred together with enhanced inorganic phosphate consumption by cells and increased polyphosphate kinase (PPK) activity. NMR analysis of the particles showed the presence of both polyphosphate and phosphonate together with orthophosphate, and FT-IR supported these results, also showing evidences of Cr(III) coordination. This work demonstrated that O. tritici 5bvl1 possesses protection mechanisms against chromium toxicity other than the presence of the Cr(VI) pump and SOD related enzymes previously described. Future assessment of the molecular regulation of production of these nanoparticles will open new perspectives for remediation of metal contaminated environments.

Oral propranolol as a new treatment for facial infantile hemangioma: case report.

Hemangiomas are the most common benign tumors of infancy. Despite their self-limited course, infantile capillary hemangiomas can impair vital or sensory functions as vision and cause cosmetic deformity. The usual treatments include oral/intralesional steroids, alpha interferon, cytotoxins, pulsed dye laser and cosmetic surgery resection. These treatments are not free of multiple complications and toxic side effects. This report describes the case of a 3-month-old female baby with progressively increasing hemangioma of the left upper eyelid impinging over the visual field. The hemangioma promptly responded to low-dose oral propranolol. A clinical response was noticed few days after the beginning of the treatment, with regression to 1/4 of its original size in 45 days of treatment, and to less than 1/10 after 8 months, free of any major side effects.

Oral propranolol as a new treatment for facial infantile hemangioma: case report / Propranolol oral: novo tratamento para hemangioma facial em bebês: relato de caso

Hemangiomas are the most common benign tumors of infancy. Despite their self-limited course, infantile capillary hemangiomas can impair vital or sensory functions as vision and cause cosmetic deformity. The usual treatments include oral/intralesional steroids, alpha interferon, cytotoxins, pulsed dye laser and cosmetic surgery resection. These treatments are not free of multiple complications and toxic side effects. This report describes the case of a 3-month-old female baby with progressively increasing hemangioma of the left upper eyelid impinging over the visual field. The hemangioma promptly responded to low-dose oral propranolol. A clinical response was noticed few days after the beginning of the treatment, with regression to 1/4 of its original size in 45 days of treatment, and to less than 1/10 after 8 months, free of any major side effects.

Hemangiomas são os tumores benignos mais comuns durante o primeiro ano de vida. Apesar do seu curso autolimitado, os hemangiomas capilares podem prejudicar funções vitais ou sensoriais como a visão e causar alteração estética. O tratamento usual inclui esteróides orais ou intralesionais, interferon alfa, citotoxinas, laser e ressecção cirúrgica. Entretanto estes tratamentos não estão livres de complicações e efeitos adversos. Este relato descreve o caso de um bebê feminino de 3 meses com um hemangioma rapidamente progressivo na pálpebra superior esquerda, causando obstrução no eixo visual. O hemangioma respondeu rapidamente a uma baixa dose oral de propranolol. A resposta clínica foi notada poucos dias após o início do tratamento, com regressão a 1/4 do seu tamanho original após 45 dias de tratamento, e a menos de 1/10 após 8 meses, sem ter apresentado nenhum efeito adverso.

Chromium resistance strategies and toxicity: what makes Ochrobactrum tritici 5bvl1 a strain highly resistant.

Large-scale industrial use of chromium (Cr) resulted in widespread environmental contamination with hexavalent chromium (Cr(VI)). The ability of microorganisms to survive in these environments and detoxify chromate requires the presence of specific resistance systems. Several Cr(VI) resistant species, belonging to a variety of genera, have been isolated in recent years. Ochrobactrum tritici strain 5bvl1 is a model for a highly Cr(VI)-resistant and reducing microorganism, with different strategies to cope with chromium. The strain contains the transposon-located (TnOtChr) chromate resistance genes chrB, chrA, chrC, chrF. The chrB and chrA genes were found to be essential for the establishment of high resistance but not chrC or chrF genes. Other mechanisms involved in chromium resistance in this strain were related to strategies such as specific or unspecific Cr(VI) reduction, free-radical detoxifying activities, and repairing DNA damage. Expression of the chrB, chrC or chrF genes was related to increased resistance to superoxide-generating agents. Genetic analyses also showed that, the ruvB gene is related to chromium resistance in O. tritici 5bvl1. The RuvABC complex probably does not form when ruvB gene is interrupted, and the repair of DNA damage induced by chromium is prevented. Aerobic or anaerobic chromate reductase activity and other unspecific mechanisms for chromium reduction have been identified in different bacteria. In the strain O. tritici 5bvl1, several unspecific mechanisms were found. Dichromate and chromate have different effects on the physiology of the chromium resistant strains and dichromate seems to be more toxic. Toxicity of Cr(VI) was evaluated by following growth, reduction, respiration, glucose uptake assays and by comparing cell morphology.

Diversity of bacteria associated with Bursaphelenchus xylophilus and other nematodes isolated from Pinus pinaster trees with pine wilt disease.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, has been thought to be the only causal agent of pine wilt disease (PWD), however, since bacteria have been suggested to play a role in PWD, it is important to know the diversity of the microbial community associated to it. This study aimed to assess the microbial community associated with B. xylophilus and with other nematodes isolated from pine trees, Pinus pinaster, with PWD from three different affected forest areas in Portugal. One hundred and twenty three bacteria strains were isolated from PWN and other nematodes collected from 14 P. pinaster. The bacteria strains were identified by comparative analysis of the 16S rRNA gene partial sequence. All except one gram-positive strain (Actinobacteria) belonged to the gram-negative Beta and Gammaproteobacteria. Most isolates belonged to the genus Pseudomonas, Burkholderia and to the family Enterobacteriaceae. Species isolated in higher percentage were Pseudomonas lutea, Yersinia intermedia and Burkholderia tuberum. The major bacterial population associated to the nematodes differed according to the forest area and none of the isolated bacterial species was found in all different forest areas. For each of the sampled areas, 60 to 100% of the isolates produced siderophores and at least 40% produced lipases. The ability to produce siderophores and lipases by most isolates enables these bacteria to have a role in plant physiological response. This research showed a high diversity of the microbial community associated with B. xylophilus and other nematodes isolated from P. pinaster with PWD.

Different physiological responses to chromate and dichromate in the chromium resistant and reducing strain Ochrobactrum tritici 5bvl1.

Studies of Cr(VI) toxicity are generally performed using chromate salts in solution, both when studying the effects on prokaryotes and eukaryotes. Some studies on human carcinogenesis and toxicology on bacteria were done using dichromate, but comparison with chromate was never reported before, and dichromate existence was never taken into consideration and usually overlooked. This paper studied comparatively the effect of dichromate and chromate on the physiology of Ochrobactrum tritici strain 5bvl1, a highly Cr(VI)-resistant and reducing microorganism. This study demonstrated that the addition of chromate or dichromate sodium salts to growth medium at neutral pH ended-up in two different solutions with a different balance of chemical species. Cr(VI) was toxic to O. tritici strain 5bvl1, as clearly shown on growth, reduction, respiration, glucose accumulation assays and by comparing cell morphology. Moreover, the addition of sodium dichromate was always more toxic to cells when compared to chromate and achieved a higher inhibition of every parameter studied. The toxicity differences between the two Cr(VI) oxyanions indicate the possibility of a different impact of Cr(VI) contamination on the environment. This may be of major importance, considering the slight acidity of most of the arable lands which favours the presence of dichromate, the more toxic species.

Identification of an aox system that requires cytochrome c in the highly arsenic-resistant bacterium Ochrobactrum tritici SCII24.

Microbial biotransformations have a major impact on environments contaminated with toxic elements, including arsenic, resulting in an increasing interest in strategies responsible for how bacteria cope with arsenic. In the present work, we investigated the metabolism of this metalloid in the bacterium Ochrobactrum tritici SCII24. This heterotrophic organism contains two different ars operons and is able to oxidize arsenite to arsenate. The presence of arsenite oxidase genes in this organism was evaluated, and sequence analysis revealed structural genes for an As(III) oxidase (aoxAB), a c-type cytochrome (cytC), and molybdopterin biosynthesis (moeA). Two other genes coding for a two-component signal transduction pair (aoxRS) were also identified upstream from the previous gene cluster. The involvement of aox genes in As(III) oxidation was confirmed by functionally expressing them into O. tritici 5bvl1, a non-As(III) oxidizer. Experiments showed that the As(III) oxidation process in O. tritici requires not only the enzyme arsenite oxidase but also the cytochrome c encoded in the operon. The fundamental role of this cytochrome c, reduced in the presence of arsenite in strain SCII24 but not in an O. tritici DeltaaoxB mutant, is surprising, since to date this feature has not been found in other organisms. In this strain the presence of an aox system does not seem to confer an additional arsenite resistance capability; however, it might act as part of an As(III)-detoxifying strategy. Such mechanisms may have played a crucial role in the development of early stages of life on Earth and may one day be exploited as part of a potential bioremediation strategy in toxic environments.

Leucobacter luti sp. nov., and Leucobacter alluvii sp. nov., two new species of the genus Leucobacter isolated under chromium stress.

Two strains designated RF6(T) and RB10(T) were isolated, from activated sludge and from river sediments, respectively, both systems receiving chromium contaminated water. Phylogenetic analysis showed that strain RF6(T)and strain RB10(T) represented two new species of the genus Leucobacter. Strain RB10(T) can be distinguished from RF6(T) by its ability to grow at 37 degrees C, by showing a different optimum pH, by cell wall amino acids different relative amount and by having the fatty acid strait C16:0 as the third most abundant fatty acid. On the basis of the distinct peptidoglycan composition, 16S ribosomal DNA sequence analysis, DNA-DNA reassociation values, and phenotypic characteristics we are of the opinion that strain RF6(T) represents a new species of the genus Leucobacter for which we propose the name Leucobacter luti (CIP 108818(T)=LMG 23118) and that strain RB10(T) represents an additional new species of the same genus for which we propose the name Leucobacter alluvii (CIP 108819(T)=LMG 23117).

Leucobacter chromiireducens sp. nov, and Leucobacter aridicollis sp. nov., two new species isolated from a chromium contaminated environment.

Two strains designated strains L-1T and L-9T were isolated from activated sludge of a treatment plant that receives wastewater from the tannery industry contaminated with chromium. Phylogenetic analysis showed that the organisms represented two new species of the genus Leucobacter. Strains L-1T and L-9T could be distinguished from the type strain of L. komagatae and from the type strain of "L. albus" by the B-type peptidoglycan composition, fatty acid composition, several phenotypic and physiological characteristics. The major fatty acids of the organisms were iso- and anteiso-branched C15:0 and C17:0, straight-chain C16:0 was also found in relatively high proportions. The organisms were halotolerant, grew in medium containing 9% NaCl, and all strains, including the type strain of L. komagatae grew in medium containing 5 mM Cr(VI). On the basis of the distinct peptidoglycan composition, 16S ribosomal DNA sequence analysis, percentage of DNA-DNA reassociation values, and phenotypic characteristics we are of the opinion that strain L-1T represents a new species of the genus Leucobacter for which we propose the name Leucobacter chromiireducens and that strain L-9T represents an additional new species of the same genus for which we propose the name Leucobacter aridicollis.