We report a novel pathway for arsenic detoxification in the legume symbiont Sinorhizobium meliloti. Although a majority of ars operons consist of three genes, arsR (transcriptional regulator), arsB [As(OH) 3 /H ؉ antiporter], and arsC (arsenate reductase), the S. meliloti ars operon includes an aquaglyceroporin (aqpS) in place of arsB. The presence of AqpS in an arsenic resistance operon is interesting, since aquaglyceroporin channels have previously been shown to adventitiously facilitate uptake of arsenite into cells, rendering them sensitive to arsenite. To understand the role of aqpS in arsenic resistance, S. meliloti aqpS and arsC were disrupted individually. Disruption of aqpS resulted in increased tolerance to arsenite but not arsenate, while cells with an arsC disruption showed selective sensitivity to arsenate. The results of transport experiments in intact cells suggest that AqpS is the only protein of the S. meliloti ars operon that facilitates transport of arsenite. Coexpression of S. meliloti aqpS and arsC in a strain of E. coli lacking the ars operon complemented arsenate but not arsenite sensitivity. These results imply that, when S. meliloti is exposed to environmental arsenate, arsenate enters the cell through phosphate transport systems and is reduced to arsenite by ArsC. Internally generated arsenite flows out of the cell by downhill movement through AqpS. Thus, AqpS confers arsenate resistance together with ArsC-catalyzed reduction. This is the first report of an aquaglyceroporin with a physiological function in arsenic resistance.Arsenic compounds are widespread in the biosphere, arising from both natural and anthropomorphic sources. The two biologically relevant oxidation states of inorganic arsenic are arsenite [As(III)] and arsenate [As(V)], the former being more toxic than the later. The primary mechanism of arsenite toxicity is due to its ability to react with protein sulfhydryl groups, thereby affecting their function. By itself, arsenate has low toxicity as a phosphate analogue, and its main toxicity is the result of its conversion to arsenite.In response to toxicity, microorganisms have evolved mechanisms for arsenic resistance. Arsenic resistance (ars) genes are common in microbes and are localized to ars operons on either the chromosome or plasmid (11). Many, if not most, ars operons consist of three genes: arsR, arsB, and arsC. ArsR is a trans-acting repressor (23, 25) that senses environmental As(III) and controls the expression of ArsB and ArsC. ArsC is a reductase that reduces As(V) to As(III) (14), while ArsB extrudes As(III) out of the cells by functioning as an As(OH) 3 /H ϩ antiporter (10). Therefore, expression of both ArsB and ArsC provides resistance to both As(III) and As(V). In addition to the three-gene chromosomal ars operon, some ars operons such as those carried by Escherichia coli plasmids R773 and R46 have five genes, arsRDABC, that encode two additional proteins, ArsD and ArsA. ArsD exhibits weak As(III)-responsive transcriptional repressor activity (4), andArsA...

As a means of investigating gene function, we developed a robust transcription fusion reporter vector to measure gene expression in bacteria. The vector, pTH1522, was used to construct a random insert library for the Sinorhizobium meliloti genome. pTH1522 replicates in Escherichia coli and can be transferred to, but cannot replicate in, S. meliloti. Homologous recombination of the DNA fragments cloned in pTH1522 into the S. meliloti genome generates transcriptional fusions to either the reporter genes gfp ؉ and lacZ or gusA and rfp, depending on the orientation of the cloned fragment. Over 12,000 fusion junctions in 6,298 clones were identified by DNA sequence analysis, and the plasmid clones were recombined into S. meliloti. Reporter enzyme activities following growth of these recombinants in complex medium (LBmc) and in minimal medium with glucose or succinate as the sole carbon source allowed the identification of genes highly expressed under one or more growth condition and those expressed at very low to background levels. In addition to generating reporter gene fusions, the vector allows Flp recombinase-directed deletion formation and gene disruption, depending on the nature of the cloned fragment. We report the identification of genes essential for growth on complex medium as deduced from an inability to recover recombinants from pTH1522 clones that carried fragments internal to gene or operon transcripts. A database containing all the gene expression activities together with a web interface showing the precise locations of reporter fusion junctions has been constructed (www.sinorhizobium.org).

The accessory protein CooC, which contains a nucleotide-binding domain (P-loop) near the N terminus, participates in the maturation of the nickel center of carbon monoxide dehydrogenase (CODH). In this study, CooC was purified from the chromatophore membranes of Rhodospirillum rubrum with a 3,464-fold purification and a 0.8% recovery, and its biochemical properties were characterized. CooC is a homodimer with a molecular mass of 61-63 kDa, contains less than 0.1 atom of Ni 2؉ or Fe 2؉ per dimer, and has a max at 277.5 nm (⑀ 277.5 32.1 mM ؊1 cm ؊1 ) with no absorption peaks at the visible region. CooC catalyzes the hydrolysis of ATP and GTP with K m values of 24.4 and 26.0 M and V max values of 58.7 and 3.7 nmol/min/mg protein for ATP and GTP hydrolysis, respectively. The P-loop mutated form of K13Q CooC was generated by site-specific replacement of lysine by glutamine and was purified according to the protocol for wild-type CooC purification. The K13Q CooC was inactive both in ATP hydrolysis and in vivo nickel insertion. In vitro nickel activation of apoCODH in the cell extracts from UR2 (wild type) and UR871 (K13Q CooC) showed that activation of nickel-deficient CODH was enhanced by CooC and dependent upon ATP hydrolysis. The overall results suggest that CooC couples ATP hydrolysis with nickel insertion into apoCODH. On the basis of our results and models for analogous systems, the functional roles of CooC in nickel processing into the active site of CODH are presented.Rhodospirillum rubrum, a purple nonsulfur photosynthetic bacterium, can use CO as the sole energy and carbon source during anaerobic growth in the dark (1, 2). The cooS-encoded carbon monoxide dehydrogenase (CODH) 1 from R. rubrum catalyzes the reversible oxidation of CO to CO 2 (3, 4). CODH contains a nickel-iron-sulfur cluster (C-center) and an ironsulfur cluster (B-center). CO oxidation occurs at the C-center, and the B-center mediates the transfer of electrons from the C-center to external electron acceptors (5). A nickel-deficient apoCODH, which contains all of the iron clusters of holoCODH yet no CO-oxidation activity, is obtained by growing wild-type R. rubrum on nickel-depleted medium (6). The apoCODH can be activated both in vivo and in vitro by the addition of nickel (6, 7).In contrast to the extensive knowledge about the catalytic (8) and spectroscopic properties (9, 10) of CODH, information about the biosynthesis of the nickel cluster is very limited. The basic studies on CO-dependent growth (11) and 63 Ni transport (12) demonstrate that three accessory proteins encoded by cooCTJ genes are involved in nickel incorporation into a nickel site. The cooT gene encodes a 7.1-kDa protein that shows marginal similarity to chaperone-type HypC protein required for the maturation of hydrogenase from Escherichia coli (13). The cooJ gene encodes a soluble, 12.6-kDa protein that has a histidine-rich nickel-binding domain. CooJ has been purified by IMAC from R. rubrum and has been shown to bind 4 Ni A mutant lacking a functional cooC gene requires ...

Functional metagenomics is a powerful experimental approach for studying gene function, starting from the extracted DNA of mixed microbial populations. A functional approach relies on the construction and screening of metagenomic libraries—physical libraries that contain DNA cloned from environmental metagenomes. The information obtained from functional metagenomics can help in future annotations of gene function and serve as a complement to sequence-based metagenomics. In this Perspective, we begin by summarizing the technical challenges of constructing metagenomic libraries and emphasize their value as resources. We then discuss libraries constructed using the popular cloning vector, pCC1FOS, and highlight the strengths and shortcomings of this system, alongside possible strategies to maximize existing pCC1FOS-based libraries by screening in diverse hosts. Finally, we discuss the known bias of libraries constructed from human gut and marine water samples, present results that suggest bias may also occur for soil libraries, and consider factors that bias metagenomic libraries in general. We anticipate that discussion of current resources and limitations will advance tools and technologies for functional metagenomics research.

Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon (12C) or stable-isotope-labeled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the 13C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes.