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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.

A gene for high-affinity glucose transport, HGT1, has been isolated from the lactose-assimilating yeast Kluyveromyces lactis. Disruption strains showed much-reduced uptake of glucose at low concentrations and growth was particularly affected in low-glucose medium. The HGT1 nucleotide sequence implies that it encodes a typical transmembrane protein with 12 hydrophobic domains and with 26 to 31% amino acid identity with the Hxtp family of glucose transport elements in Saccharomyces cerevisiae. Expression is constitutive (in contrast to RAG1, the major gene for low-affinity glucose uptake in K. lactis) and is controlled by several genes also known to affect expression of RAG1. These include RAG5 (which codes for the single hexokinase of K. lactis), which is required for HGT1 transcription, and RAG4, which has a negative effect. The double mutant ⌬hgt1⌬rag1 showed further reduced glucose uptake but still grew quite well on 2% glucose and was not completely impaired even on 0.1% glucose.Eukaryotic and bacterial sugar transport proteins are related in protein sequence and cellular topology, forming the sugar permease superfamily (1). Within this superfamily of proteins are found transporters that act either by facilitated diffusion or by proton symport mechanisms (17

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 E h 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.Arsenic (As) occurs naturally as a local geological constituent of the soils surrounding the Upper Isle River Basin (Massif Central, France) due to natural geochemical anomalies but is also released from Au/As deposits of disused gold mines (4,11,12,33). Important variations in dissolved As concentrations are found in the Isle River and depend on the hydrogeological season, with maximum values in spring and summer generally detected during low-flow conditions (12,22), and probably on temperature-controlled microbial As(V) reduction and/or microbial dissolution of solid As carrier phases (22). Two toxic inorganic forms of As are usually detected in aquatic system: arsenite, As(III), which is found mainly under anaerobic conditions and is more mobile than arsenate, As(V), which typically occurs under aerobic conditions and tends to associate with oxyhydroxides and clay minerals (11,34). Although bacteria are known to play a key role in speciation, mobility, and bioavailability of As in the environment, they have never been considered in previous studies of As mobility in the Isle River system. Indeed, former investigations of As cycling were focused on geochemical studies (4,11,12,22,33).As(III)-oxidizing bacteria can contribute to a natural attenuation of As pollution by decreasing its bioavailability and can help remove As from mine wastewaters through bioprocessing (1, 2). Many As(III) oxidizers have been isolated from various environments, especially mesophilic ecosystems (3,5,8,16,25,27,32,38). They belong to more than 25 genera, mainly of the Proteobacteria phylum (3, 32, 38), and are related to organisms unable to oxidize As(III) based on 16S rRNA phylogeny. Diverse primer sets have been successfully developed to specifically target the functional aoxB gene (9,14,17,25,26), encoding the large molybdenum-bearing catalytic subunit of As(III)-oxidase (EC 1.20.98.1), an enzyme of the dimethyl sulfoxide (DMSO) reductase family. Using cloning-sequencing approaches, the aoxB gene has proven to be a reliable molecular marker for diversity studies of the polyphyletic aerobic As(III) oxidizers in As-impacted soil and water systems (17, 25). The genetic fingerprinting denaturing gradient gel electrophoresis (DGGE) technique is one useful tool for spatial, temporal, and geographical monitoring of complex bacterial population structure (23, 24). Quantitative real-time PCR (qPCR) p...

To determine the amount of metals detectable by bacteria, two plasmids were constructed in which the metal-inducible zntA and copA promoters from Escherichia coli were fused to a promoterless Vibrio fischeri luxCDABE operon. The luminescence response of E. coli bearing these constructs was studied as a function of the concentration of several heavy metals and was shown to be influenced by cell growth phase. The zntAp::lux fusion is induced mainly by salts of cadmium, lead, mercury and zinc, with significant induction by other metal ions, whereas the specificity of copA induction is restricted to copper and silver. In optimized assay conditions, metals could be detected at threshold concentrations ranging from nanomolar to micromolar, with maximal induction observed after only 60-100 min incubation. The ability of these biosensor strains to distinguish bioavailable quantities of metals in a sample makes them good candidates as useful tools to monitor metal contamination in environmental samples.

By combining our knowledge of the crystal structure of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the sequence of the photosynthetic NADP-dependent GAPDH of the chloroplast, two particular amino acid residues were predicted as the principal determinants of differing coenzyme specificity. By use of site-directed mutagenesis, the amino acids Leu 187 and Pro 188 of GAPDH from Bacillus stearothermophilus have been replaced with Ala 187 and Ser 188, which occur in the sequence from the chloroplast enzyme. The resulting mutant was shown to be catalytically active not only with its natural coenzyme NAD but also with NADP, thus confirming the initial hypothesis. This approach has not only enabled us to alter the coenzyme specificity by minimal amino acid changes but also revealed factors that control the relative affinity of the enzyme for NAD and NADP.

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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.Arsenic is widely dispersed in the environment and occurs primarily in two oxidation states, arsenate [As(V)] and arsenite [As(III)], and both are toxic to the majority of living organisms. The frequent abundance of arsenic in all environmental compartments has guided the evolution of detoxification systems in almost all microorganisms. Of these, the arsenic resistance system (ars) appears to be widely distributed among prokaryotes. It involves an arsenate reductase (ArsC), an arsenite efflux pump (ArsB or ACR3), and a transcriptional repressor (ArsR) (32), encoded by a set of genes that display large variations in their number and genomic organization. The early identified ars system of Escherichia coli plasmid R773 (41) has two additional components, ArsA, which acts as the catalytic subunit of the ArsAB arsenite extrusion pump (33), and ArsD, a metallochaperone protein that transfers As(III) to ArsA (18). In addition to these well-studied ars components, a variety of ars clusters contain additional genes whose functions in arsenic resistance have not been clearly established in many cases (31).Members of the Microbacterium lineage of actinobacteria that can tolerate various metals, including nickel, chromium, and uranium (1,16,25), have been isolated from metal-rich environments. New examples of arsenic-resistant isolates of Microbacterium are continuously being reported (1,2,8,10,12,21). In each case, however, the tolerance mechanism was not investigated, probably due to the lack of efficient genetic systems in this genus. Among actinobacteria, only Streptomyces sp. FR-008 (40) and Corynebacterium glutamicum ATCC 13032 (28) have been subjected to molecular characterization of determinants of defense against arsenic. In the former, the linear plasmid pHZ227 carries an arsenic resistance gene cluster with two novel genes, the arsO and arsT genes, which encode a putative flavin-binding monooxygenase and a putative thioredoxin reductase, respectively (40). The latter strain was recently shown to possess two members of a new class of arsenate reductases (Cg_ArsC1 and Cg_ArsC2) (30) and a transcriptional repressor (Cg_ArsR1) with a metalloid binding site unrelated to other previously characterized members of the ArsR/SmtB metalloregulatory proteins (29).The present study focuses on Microbacterium sp. strain A33, a soil isolate previously shown to tolerate high concentrations of arsenite and arsenate (2). Here, we report on the isolation and functional characteriz...