Genome sequence of the deltaproteobacterial strain NaphS2 and analysis of differential gene expression during anaerobic growth on naphthalene.

BACKGROUND: Anaerobic polycyclic hydrocarbon (PAH) degradation coupled to sulfate reduction may be an important mechanism for in situ remediation of contaminated sediments. Steps involved in the anaerobic degradation of 2-methylnaphthalene have been described in the sulfate reducing strains NaphS3, NaphS6 and N47. Evidence from N47 suggests that naphthalene degradation involves 2-methylnaphthalene as an intermediate, whereas evidence in NaphS2, NaphS3 and NaphS6 suggests a mechanism for naphthalene degradation that does not involve 2-methylnaphthalene. To further characterize pathways involved in naphthalene degradation in NaphS2, the draft genome was sequenced, and gene and protein expression examined. RESULTS: Draft genome sequencing, gene expression analysis, and proteomic analysis revealed that NaphS2 degrades naphthoyl-CoA in a manner analogous to benzoyl-CoA degradation. Genes including the previously characterized NmsA, thought to encode an enzyme necessary for 2-methylnaphthalene metabolism, were not upregulated during growth of NaphS2 on naphthalene, nor were the corresponding protein products. NaphS2 may possess a non-classical dearomatizing enzyme for benzoate degradation, similar to one previously characterized in Geobacter metallireducens. Identification of genes involved in toluene degradation in NaphS2 led us to determine that NaphS2 degrades toluene, a previously unreported capacity. The genome sequence also suggests that NaphS2 may degrade other monoaromatic compounds. CONCLUSION: This study demonstrates that steps leading to the degradation of 2-naphthoyl-CoA are conserved between NaphS2 and N47, however while NaphS2 possesses the capacity to degrade 2-methylnaphthalene, naphthalene degradation likely does not proceed via 2-methylnaphthalene. Instead, carboxylation or another form of activation may serve as the first step in naphthalene degradation. Degradation of toluene and 2-methylnaphthalene, and the presence of at least one bss-like and bbs-like gene cluster in this organism, suggests that NaphS2 degrades both compounds via parallel mechanisms. Elucidation of the key genes necessary for anaerobic naphthalene degradation may provide the ability to track naphthalene degradation through in situ transcript monitoring.

Anode biofilm transcriptomics reveals outer surface components essential for high density current production in Geobacter sulfurreducens fuel cells.

The mechanisms by which Geobacter sulfurreducens transfers electrons through relatively thick (>50 microm) biofilms to electrodes acting as a sole electron acceptor were investigated. Biofilms of Geobacter sulfurreducens were grown either in flow-through systems with graphite anodes as the electron acceptor or on the same graphite surface, but with fumarate as the sole electron acceptor. Fumarate-grown biofilms were not immediately capable of significant current production, suggesting substantial physiological differences from current-producing biofilms. Microarray analysis revealed 13 genes in current-harvesting biofilms that had significantly higher transcript levels. The greatest increases were for pilA, the gene immediately downstream of pilA, and the genes for two outer c-type membrane cytochromes, OmcB and OmcZ. Down-regulated genes included the genes for the outer-membrane c-type cytochromes, OmcS and OmcT. Results of quantitative RT-PCR of gene transcript levels during biofilm growth were consistent with microarray results. OmcZ and the outer-surface c-type cytochrome, OmcE, were more abundant and OmcS was less abundant in current-harvesting cells. Strains in which pilA, the gene immediately downstream from pilA, omcB, omcS, omcE, or omcZ was deleted demonstrated that only deletion of pilA or omcZ severely inhibited current production and biofilm formation in current-harvesting mode. In contrast, these gene deletions had no impact on biofilm formation on graphite surfaces when fumarate served as the electron acceptor. These results suggest that biofilms grown harvesting current are specifically poised for electron transfer to electrodes and that, in addition to pili, OmcZ is a key component in electron transfer through differentiated G. sulfurreducens biofilms to electrodes.

Insights into genes involved in electricity generation in Geobacter sulfurreducens via whole genome microarray analysis of the OmcF-deficient mutant.

Geobacter sulfurreducens effectively produces electricity in microbial fuel cells by oxidizing acetate with an electrode serving as the sole electron acceptor. Deletion of the gene encoding OmcF, a monoheme outer membrane c-type cytochrome, substantially decreased current production. Previous studies demonstrated that inhibition of Fe(III) reduction in the OmcF-deficient mutant could be attributed to poor transcription of the gene for OmcB, an outer membrane c-type cytochrome that is required for Fe(III) reduction. However, a mutant in which omcB was deleted produced electricity as well as wild type. Microarray analysis of the OmcF-deficient mutant versus the wild type revealed that many of the genes with the greatest decreases in transcript levels were genes whose expression was previously reported to be upregulated in cells grown with an electrode as the sole electron acceptor. These included genes with putative functions related to metal efflux and/or type I secretion and two hypothetical proteins. The outer membrane cytochromes, OmcS and OmcE, which previous studies have demonstrated are required for optimal current generation, were not detected on the outer surface of the OmcF-deficient mutant even though the omcS and omcE genes were still transcribed, suggesting that the putative secretion system could be involved in the export of outer membrane proteins necessary for electron transfer to the fuel cell anode. These results suggest that the requirement for OmcF for optimal current production is not because OmcF is directly involved in extracellular electron transfer but because OmcF is required for the appropriate transcription of other genes either directly or indirectly involved in electricity production.

Genome-wide gene expression patterns and growth requirements suggest that Pelobacter carbinolicus reduces Fe(III) indirectly via sulfide production.

Although Pelobacter species are closely related to Geobacter species, recent studies suggested that Pelobacter carbinolicus may reduce Fe(III) via a different mechanism because it lacks the outer-surface c-type cytochromes that are required for Fe(III) reduction by Geobacter sulfurreducens. Investigation into the mechanisms for Fe(III) reduction demonstrated that P. carbinolicus had growth yields on both soluble and insoluble Fe(III) consistent with those of other Fe(III)-reducing bacteria. Comparison of whole-genome transcript levels during growth on Fe(III) versus fermentative growth demonstrated that the greatest apparent change in gene expression was an increase in transcript levels for four contiguous genes. These genes encode two putative periplasmic thioredoxins; a putative outer-membrane transport protein; and a putative NAD(FAD)-dependent dehydrogenase with homology to disulfide oxidoreductases in the N terminus, rhodanese (sulfurtransferase) in the center, and uncharacterized conserved proteins in the C terminus. Unlike G. sulfurreducens, transcript levels for cytochrome genes did not increase in P. carbinolicus during growth on Fe(III). P. carbinolicus could use sulfate as the sole source of sulfur during fermentative growth, but required elemental sulfur or sulfide for growth on Fe(III). The increased expression of genes potentially involved in sulfur reduction, coupled with the requirement for sulfur or sulfide during growth on Fe(III), suggests that P. carbinolicus reduces Fe(III) via an indirect mechanism in which (i) elemental sulfur is reduced to sulfide and (ii) the sulfide reduces Fe(III) with the regeneration of elemental sulfur. This contrasts with the direct reduction of Fe(III) that has been proposed for Geobacter species.

c-Type cytochromes in Pelobacter carbinolicus.

Previous studies failed to detect c-type cytochromes in Pelobacter species despite the fact that other close relatives in the Geobacteraceae, such as Geobacter and Desulfuromonas species, have abundant c-type cytochromes. Analysis of the recently completed genome sequence of Pelobacter carbinolicus revealed 14 open reading frames that could encode c-type cytochromes. Transcripts for all but one of these open reading frames were detected in acetoin-fermenting and/or Fe(III)-reducing cells. Three putative c-type cytochrome genes were expressed specifically during Fe(III) reduction, suggesting that the encoded proteins may participate in electron transfer to Fe(III). One of these proteins was a periplasmic triheme cytochrome with a high level of similarity to PpcA, which has a role in Fe(III) reduction in Geobacter sulfurreducens. Genes for heme biosynthesis and system II cytochrome c biogenesis were identified in the genome and shown to be expressed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of protein extracted from acetoin-fermenting P. carbinolicus cells contained three heme-staining bands which were confirmed by mass spectrometry to be among the 14 predicted c-type cytochromes. The number of cytochrome genes, the predicted amount of heme c per protein, and the ratio of heme-stained protein to total protein were much smaller in P. carbinolicus than in G. sulfurreducens. Furthermore, many of the c-type cytochromes that genetic studies have indicated are required for optimal Fe(III) reduction in G. sulfurreducens were not present in the P. carbinolicus genome. These results suggest that further evaluation of the functions of c-type cytochromes in the Geobacteraceae is warranted.

Mutations in Arabidopsis yellow stripe-like1 and yellow stripe-like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds.

Here, we describe two members of the Arabidopsis (Arabidopsis thaliana) Yellow Stripe-Like (YSL) family, AtYSL1 and AtYSL3. The YSL1 and YSL3 proteins are members of the oligopeptide transporter family and are predicted to be integral membrane proteins. YSL1 and YSL3 are similar to the maize (Zea mays) YS1 phytosiderophore transporter (ZmYS1) and the AtYSL2 iron (Fe)-nicotianamine transporter, and are predicted to transport metal-nicotianamine complexes into cells. YSL1 and YSL3 mRNAs are expressed in both root and shoot tissues, and both are regulated in response to the Fe status of the plant. Beta-glucuronidase reporter expression, driven by YSL1 and YSL3 promoters, reveals expression patterns of the genes in roots, leaves, and flowers. Expression was highest in senescing rosette leaves and cauline leaves. Whereas the single mutants ysl1 and ysl3 had no visible phenotypes, the ysl1ysl3 double mutant exhibited Fe deficiency symptoms, such as interveinal chlorosis. Leaf Fe concentrations are decreased in the double mutant, whereas manganese, zinc, and especially copper concentrations are elevated. In seeds of double-mutant plants, the concentrations of Fe, zinc, and copper are low. Mobilization of metals from leaves during senescence is impaired in the double mutant. In addition, the double mutant has reduced fertility due to defective anther and embryo development. The proposed physiological roles for YSL1 and YSL3 are in delivery of metal micronutrients to and from vascular tissues.

Arabidopsis Yellow Stripe-Like2 (YSL2): a metal-regulated gene encoding a plasma membrane transporter of nicotianamine-metal complexes.

The Yellow Stripe-Like (YSL) family of proteins has been identified based on sequence similarity to maize Yellow Stripe1 (YS1), the transporter responsible for the primary uptake of iron from the soil. YS1 transports iron that is complexed by specific plant-derived Fe(III) chelators called phytosiderophores (PS). Non-grass species of plants neither make nor use PS, yet YSL family members are found in non-grass species (monocot, dicot, gymnosperm, and moss species) including Arabidopsis thaliana. YSLs in non-grasses have been hypothesized to transport metals complexed by nicotianamine (NA), an iron chelator that is structurally similar to PS and which is found in all higher plants. Here we show that Arabidopsis YSL2 (At5g24380) transports iron and copper when these metals are chelated by NA. YSL2 is expressed in many cell types in both roots and shoots, suggesting that diverse cell types obtain metals as metal-NA complexes. YSL2 transcription is regulated by the levels of iron and copper in the growth medium. Based on its expression pattern, a major function of the YSL2 appears to be in the lateral movement of metals in the vasculature.

Arabidopsis ALF4 encodes a nuclear-localized protein required for lateral root formation.

Lateral root formation, the primary way plants increase their root mass, displays developmental plasticity in response to environmental changes. The aberrant lateral root formation (alf)4-1 mutation blocks the initiation of lateral roots, thus greatly altering root system architecture. We have positionally cloned the ALF4 gene and have further characterized its phenotype. The encoded ALF4 protein is conserved among plants and has no similarities to proteins from other kingdoms. The gene is present in a single copy in Arabidopsis. Using translational reporters for ALF4 gene expression, we have determined that the ALF4 protein is nuclear localized and that the gene is expressed in most plant tissues; however, ALF4 expression and ALF4's subcellular location are not regulated by auxin. These findings taken together with further genetic and phenotypic characterization of the alf4-1 mutant suggest that ALF4 functions independent from auxin signaling and instead functions in maintaining the pericycle in the mitotically competent state needed for lateral root formation. Our results provide genetic evidence that the pericycle shares properties with meristems and that this tissue plays a central role in creating the developmental plasticity needed for root system development.