Patrick Billard

Population structure and abundance of arsenite-oxidizing bacteria along an arsenic pollution gradient in waters of the Upper Isle River Basin, France.

ABSTRACT Denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR) were successfully developed to monitor functional aoxB genes as markers of aerobic arsenite oxidizers. DGGE profiles showed a shift in the structure of the aoxB-carrying bacterial population, composed of members of the Alpha-, Beta- and Gammaproteobacteria, depending on arsenic (As) and Eh levels in Upper Isle River Basin waters. The highest aoxB gene densities were found in the most As-polluted oxic surface waters but without any significant correlation with environmental factors. Arsenite oxidizers seem to play a key role in As mobility in As-impacted waters.

Characterization of the ars Gene Cluster from Extremely Arsenic-Resistant Microbacterium sp. Strain A33

ABSTRACT The arsenic resistance gene cluster of Microbacterium sp. A33 contains a novel pair of genes (arsTX) encoding a thioredoxin system that are cotranscribed with an unusual arsRC2 fusion gene, ACR3, and arsC1 in an operon divergent from arsC3. The whole ars gene cluster is required to complement an Escherichia coli ars mutant. ArsRC2 negatively regulates the expression of the pentacistronic operon. ArsC1 and ArsC3 are related to thioredoxin-dependent arsenate reductases; however, ArsC3 lacks the two distal catalytic cysteine residues of this class of enzymes.

Functional Role of Lanthanides in Enzymatic Activity and Transcriptional Regulation of Pyrroloquinoline Quinone-Dependent Alcohol Dehydrogenases in Pseudomonas putida KT2440

ABSTRACT The oxidation of alcohols and aldehydes is crucial for detoxification and efficient catabolism of various volatile organic compounds (VOCs). Thus, many Gram-negative bacteria have evolved periplasmic oxidation systems based on pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) that are often functionally redundant. Here we report the first description and characterization of a lanthanide-dependent PQQ-ADH (PedH) in a nonmethylotrophic bacterium based on the use of purified enzymes from the soil-dwelling model organism Pseudomonas putida KT2440. PedH (PP_2679) exhibits enzyme activity on a range of substrates similar to that of its Ca2+-dependent counterpart PedE (PP_2674), including linear and aromatic primary and secondary alcohols, as well as aldehydes, but only in the presence of lanthanide ions, including La3+, Ce3+, Pr3+, Sm3+, or Nd3+. Reporter assays revealed that PedH not only has a catalytic function but is also involved in the transcriptional regulation of pedE and pedH, most likely acting as a sensory module. Notably, the underlying regulatory network is responsive to as little as 1 to 10 nM lanthanum, a concentration assumed to be of ecological relevance. The present study further demonstrates that the PQQ-dependent oxidation system is crucial for efficient growth with a variety of volatile alcohols. From these results, we conclude that functional redundancy and inverse regulation of PedE and PedH represent an adaptive strategy of P. putida KT2440 to optimize growth with volatile alcohols in response to the availability of different lanthanides. IMPORTANCE Because of their low bioavailability, lanthanides have long been considered biologically inert. In recent years, however, the identification of lanthanides as a cofactor in methylotrophic bacteria has attracted tremendous interest among various biological fields. The present study reveals that one of the two PQQ-ADHs produced by the model organism P. putida KT2440 also utilizes lanthanides as a cofactor, thus expanding the scope of lanthanide-employing bacteria beyond the methylotrophs. Similar to the system described in methylotrophic bacteria, a complex regulatory network is involved in lanthanide-responsive switching between the two PQQ-ADHs encoded by P. putida KT2440. We further show that the functional production of at least one of the enzymes is crucial for efficient growth with several volatile alcohols. Overall, our study provides a novel understanding of the redundancy of PQQ-ADHs observed in many organisms and further highlights the importance of lanthanides for bacterial metabolism, particularly in soil environments. IMPORTANCE Because of their low bioavailability, lanthanides have long been considered biologically inert. In recent years, however, the identification of lanthanides as a cofactor in methylotrophic bacteria has attracted tremendous interest among various biological fields. The present study reveals that one of the two PQQ-ADHs produced by the model organism P. putida KT2440 also utilizes lanthanides as a cofactor, thus expanding the scope of lanthanide-employing bacteria beyond the methylotrophs. Similar to the system described in methylotrophic bacteria, a complex regulatory network is involved in lanthanide-responsive switching between the two PQQ-ADHs encoded by P. putida KT2440. We further show that the functional production of at least one of the enzymes is crucial for efficient growth with several volatile alcohols. Overall, our study provides a novel understanding of the redundancy of PQQ-ADHs observed in many organisms and further highlights the importance of lanthanides for bacterial metabolism, particularly in soil environments.

Arsenite-induced changes in abundance and expression of arsenite transporter and arsenite oxidase genes of a soil microbial community

We describe a real-time PCR assay for the quantitative detection of arsB and ACR3(1) arsenite transporter gene families, two ubiquitous and key determinants of arsenic resistance in prokaryotes. The assay was applied in batch growth experiments using a wasteland soil bacterial community as an inoculum to investigate the effect of increasing arsenite [As(III)] concentrations on genes and transcript abundances. The aioA gene encoding the large subunit of arsenite oxidase was monitored in parallel. Results showed that arsB and ACR3(1) gene abundances correlated positively with the As(III) concentration. Both genes showed similar transcription patterns and strong upregulation by arsenic. Microbial As(III) oxidation occurred in As(III) spiked cultures and was associated with expression of the aioA gene in most cases. However, aioA was also expressed in several non-amended culture replicates. Analysis of cDNA clone libraries revealed that Pseudomonas was the dominant metabolically active genus whatever the As(III) concentration. Expressed arsB and ACR3(1) gene sequences were also affiliated with those from Pseudomonas, while expressed aioA sequences were more taxonomically diverse. The study suggests that arsenite transporter genes are appropriate biomarkers of arsenic stress that may be suitable for further exploring the adaptive response of bacterial communities to arsenic in contaminated environments.

Bio-dissolution of colloidal-size clay minerals entrapped in microporous silica gels.

Four colloidal-size fractions of strongly anisotropic particles of nontronite (NAu-2) having different ratios of basal to edge surfaces were incubated in the presence of heterotrophic soil bacteria to evaluate how changes in mineral surface reactivity influence microbial dissolution rate of minerals. To avoid any particle aggregation, which could change the reactive surface area available for dissolution, NAu-2 particles were immobilized in a biocompatible TEOS-derived silica matrix. The resulting hybrid silica gels support bacterial growth with NAu-2 as the sole source of Fe and Mg. Upon incubation of the hybrid material with bacteria, between 0.3% and 7.5% of the total Fe included in the mineral lattice was released with a concomitant pH decrease. For a given pH value, the amount of released Fe varied between strains and was two to twelve-fold higher than under abiotic conditions. This indicates that complexing agents produced by bacteria play an important role in the dissolution process. However, in contrast with proton-promoted NAu-2 dissolution (abiotic incubations) that was negatively correlated with particle size, bacterial-enhanced dissolution was constant for all size fractions used. We conclude that bio-dissolution of nontronite particles under acidic conditions seems to be controlled by bacterial metabolism rather than by the surface reactivity of mineral.

Dissolution of anisotropic colloidal mineral particles: evidence for basal surface reactivity of nontronite.

Anisotropic textural and crystallographic properties of phyllosilicate particles often influence the mineral weathering rate. The purpose of this study was to investigate how the changes in mineral surfaces (basal vs. edge) as a result of changes in crystal size control the dissolution of the mineral. Different nano-size fractions of Na-exchanged nontronites (NAu2 and NAu1) were immobilized in a silica gel and then incubated under acidic conditions using HNO(3) at 28 degrees C for 5 days. For each sample, the dissolution behavior was analyzed by measuring the amount of iron released from the mineral lattice. The results showed that for a given pH, a decrease in particle size significantly increased NAu2 and NAu1 dissolution. At pH 1.5, 7.2% of the total iron of the highest size sample of NAu2 was released in solution whereas this proportion increased up to 25% for the smallest size fraction. The percentage of total iron extracted from NAu1 at the same pH (1.5) was less important: 3.5% and 6.5% for higher and smaller size fractions, respectively. The observed increase in dissolution was not directly correlated to the increase in the amount of edge faces, suggesting that all mineral surfaces contributed to mineral dissolution. In the present case this may be related to the fact that 8% and 2% of total iron of NAu2 and NAu1, respectively, are located in the tetrahedral sheet. In conclusion, the basal surface of nontronites plays an important role in the weathering process.

Stability and toxicity of ZnO quantum dots: Interplay between nanoparticles and bacteria

The toxicity of quantum dots (QDs) has been commonly attributed to the release of metal ions from the core as well as to the production of reactive oxygen species. However, the information related to the stability of the nanoparticles are relatively scarce although this parameter may strongly influence their toxicity. The stability of aminosilane-capped ZnO QDs, here used as model nanoparticles, was investigated by inductively coupled plasma-optical emission spectrometer (ICP-OES) and whole cell biosensors using a dialysis setup to separate the QDs from the leaked Zn(2+) ions. The integrity of the ZnO QDs appeared strongly affected by their dilution in aqueous medium, whereas the nanoparticles were slightly stabilized by bacteria. Our results demonstrate some inadequacy between the implementation and use of whole cell biosensors, and the monitoring of metal release from QDs.

Comparative analysis of the genomic DNA terminal regions of the lactococcal bacteriophages from species c2

In an attempt to compare the cos intergenic region and bordering ORFs from Lactococcus lactis bacteriophages of the species c2, the nucleotide sequence of a 2479-bp fragment containing the cos site of phage P001 DNA was determined and compared with the corresponding regions of phages c2, bIL67 and P6 (partial sequence), which belong to species c2. This comparative analysis revealed that some characteristic features of the cos intergenic region are conserved in all members of species c2. Some of them are specific to species c2, as is the case for a GC-rich repeat in phase with the double helix that is located close to cos. One conserved motif seems to be more general, as it is found in all the cos regions of L. lactis bacteriophages that have been sequenced. It consists in a 4-nt indirect repeat TCAN/NACT located in a 15-bp fragment containing cos. This motif may be related to terminase specificity, as most of the cos asymmetric cleavages identified up to now are located within, or at the border of, these indirectly repeated sequences. Finally, some of the conserved DNA motifs of the species c2 cos-intergenic region seem to be even more general, as they are homologous to the lambda-R sites known to be involved in the maturation and the encapsidation of phage lambda DNA. Our comparative analysis also showed that within c2 phage DNAs, large blocks of sequences, i.e. the intergenic cos region and ORF/17 on the one hand, and ORF/16 on the other hand, evolved as distinct entities, probably by block recombination between phage DNAs of the same species.

Bioweathering of nontronite colloids in hybrid silica gel: implications for iron mobilization

This study aimed to study biotic iron dissolution using a new hybrid material constituted of well‐dispersed mineral colloids in a silica gel matrix. This permitted to prevent adsorption of colloidal mineral particles on bacteria. Hybrid silica gel (HSG) permitted to study bioweathering mechanisms by diffusing molecules.

Bacterial community structure and functional arrA gene diversity associated with arsenic reduction and release in an industrially contaminated soil

ABSTRACT This study aimed at evaluating potential arsenic (As) mobility in an industrially contaminated soil (64 mg/kg of As) of the Meuse River basin, and at identifying key bacterial groups that drive soil As dynamics. Both speciation and release of As from this soil was followed under anaerobic conditions using a laboratory batch experiment. In the presence of exogenous carbon sources, AsV initially present in the soil matrix and/or adsorbed on synthetic hydrous ferric oxides were solubilized and mainly reduced to AsIII by indigenous soil microflora. After a 1-month incubation period in these biotic conditions, AsIII accounted for 80–85% of the total dissolved As and more than 60% of the solid-phase As. Bacterial community structure (i.e., 16S rDNA-based capillary electrophoresis single-strand conformation polymorphism profiles) changed with incubation time and As amendment. The detection of distantly related arsenate respiratory reductase genes (arrA), as functional markers of AsV respirers, indicates that novel dissimilatory AsV-reducing bacteria may be involved in As biotransformation and mobility in anoxic soils. Since As and iron were concomitantly released, a crucial role of indirect As-mobilizing bacteria on As behavior was also revealed. Our results show that the majority of As within the soil matrix was bioavailable and bioaccessible for heterotrophic AsV reduction to AsIII, which may increase As toxicity and mobility in the contaminated soils.