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The pili of Geobacter sulfurreducens are of interest because of the apparent importance of the type IV pili in extracellular electron transfer. A strain of G. sulfurreducens, designated strain MA, produced many more pili than the previously studied DL-1 strain even though genome resequencing indicated that the MA and DL-1 genome sequences were identical. Filaments that looked similar to type IV pili in transmission electron micrographs were abundant even after the gene encoding PilA, the structural pilin protein, was deleted. The results of proteinase K treatment indicated that the filaments were proteinaceous. The simultaneous deletion of several genes encoding homologues of type II pseudopilins was required before the filaments were significantly depleted. The pilA-deficient MA strain attached to glass as well as the wild-type MA did, but strains in which three or four pseudopilin genes were deleted in addition to pilA had impaired attachment capabilities. These results demonstrate that there are several proteins that can yield pilin-like filaments in G. sulfurreducens and that some means other than microscopic observation is required before the composition of filaments can be unambiguously specified.

The mechanism of fumarate reduction in Geobacter sulfurreducens was investigated. The genome contained genes encoding a heterotrimeric fumarate reductase, FrdCAB, with homology to the fumarate reductase of Wolinella succinogenes and the succinate dehydrogenase of Bacillus subtilis . Mutation of the putative catalytic subunit of the enzyme resulted in a strain that lacked fumarate reductase activity and was unable to grow with fumarate as the terminal electron acceptor. The mutant strain also lacked succinate dehydrogenase activity and did not grow with acetate as the electron donor and Fe(III) as the electron acceptor. The mutant strain could grow with acetate as the electron donor and Fe(III) as the electron acceptor if fumarate was provided to alleviate the need for succinate dehydrogenase activity in the tricarboxylic acid cycle. The growth rate of the mutant strain under these conditions was faster and the cell yields were higher than for wild type grown under conditions requiring succinate dehydrogenase activity, suggesting that the succinate dehydrogenase reaction consumes energy. An orthologous frdCAB operon was present in Geobacter metallireducens , which cannot grow with fumarate as the terminal electron acceptor. When a putative dicarboxylic acid transporter from G. sulfurreducens was expressed in G. metallireducens , growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms, G. sulfurreducens and possibly G. metallireducens use the same enzyme for both fumarate reduction and succinate oxidation in vivo.

Pollen tube elongation depends on the secretion of large amounts of membrane and cell wall materials at the pollen tube tip to sustain rapid growth. A large family of RAS-related small GTPases, Rabs or Ypts, is known to regulate both anterograde and retrograde trafficking of transport vesicles between different endomembrane compartments and the plasma membrane in mammalian and yeast cells. Studies on the functional roles of analogous plant proteins are emerging. We report here that a tobacco pollen-predominant Rab2, NtRab2, functions in the secretory pathway between the endoplasmic reticulum and the Golgi in elongating pollen tubes. Green fluorescent protein–NtRab2 fusion protein localized to the Golgi bodies in elongating pollen tubes. Dominant-negative mutations in NtRab2 proteins inhibited their Golgi localization, blocked the delivery of Golgi-resident as well as plasmalemma and secreted proteins to their normal locations, and inhibited pollen tube growth. On the other hand, when green fluorescent protein–NtRab2 was over-expressed in transiently transformed leaf protoplasts and epidermal cells, in which NtRab2 mRNA have not been observed to accumulate to detectable levels, these proteins did not target efficiently to Golgi bodies. Together, these observations indicate that NtRab2 is important for trafficking between the endoplasmic reticulum and the Golgi bodies in pollen tubes and may be specialized to optimally support the high secretory demands in these tip growth cells.

Microbial solar cells (MSCs) are microbial fuel cells (MFCs) that generate their own oxidant and/or fuel through photosynthetic reactions. Here, we present electrochemical analyses and biofilm 16S rRNA gene profiling of biocathodes of sediment/seawater-based MSCs inoculated from the biocathode of a previously described sediment/seawater-based MSC. Electrochemical analyses indicate that for these second-generation MSC biocathodes, catalytic activity diminishes over time if illumination is provided during growth, whereas it remains relatively stable if growth occurs in the dark. For both illuminated and dark MSC biocathodes, cyclic voltammetry reveals a catalytic-current–potential dependency consistent with heterogeneous electron transfer mediated by an insoluble microbial redox cofactor, which was conserved following enrichment of the dark MSC biocathode using a three-electrode configuration. 16S rRNA gene profiling showed Gammaproteobacteria , most closely related to Marinobacter spp., predominated in the enriched biocathode. The enriched biocathode biofilm is easily cultured on graphite cathodes, forms a multimicrobe-thick biofilm (up to 8.2 μm), and does not lose catalytic activity after exchanges of the reactor medium. Moreover, the consortium can be grown on cathodes with only inorganic carbon provided as the carbon source, which may be exploited for proposed bioelectrochemical systems for electrosynthesis of organic carbon from carbon dioxide. These results support a scheme where two distinct communities of organisms develop within MSC biocathodes: one that is photosynthetically active and one that catalyzes reduction of O 2 by the cathode, where the former partially inhibits the latter. The relationship between the two communities must be further explored to fully realize the potential for MSC applications.

Stylar proteins involved in the self-incompatible (SI) response ofLycopersicon hirsutum have been identified and mapped to the locus that controls SI (S locus).L. esculentum, a self-compatible (SC) species of cultivated tomato, does not display these proteins. Hybrids between SCL. esculentum and SIL. hirsutum are self-sterile despite these individuals bearing pollen containing theS allele ofL. esculentum. In progeny derived from backcrossing the hybrids toL. esculentum, there was a strong correlation between the presence of theS allele fromL. hirsutum and self-infertility. However, this relationship was uncoupled in a number of backcross (BC) progeny. The SI response appeared to be nonexistent in two self-fertile BC individuals that were heterozygous for theS allele ofL. hirsutum, based on Mendelian segregation of a tightly linked DNA marker,CD15, in selfed progeny. Among these progeny self-fertile individuals that were homozygous for theL. hirsutum allele of the linked marker were also determined to be homozygous for anS-related protein ofL. hirsutum through test crosses withL. esculentum. Therefore, plants were produced that were homozygous for a functionalS allele but were self-fertile. This result and other evidence suggest that theS-related proteins are not sufficient to elicit a self-incompatible response inL. esculentum and that there is a mutation(s) inL. esculentum somewhere other than theS locus that leads to self-compatibility.

Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs) were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.

Single domain antibodies (sdAb) are the recombinantly expressed variable regions from the heavy-chain-only antibodies found in camelids and sharks. SdAb are able to bind antigens with high affinity, and most are capable of refolding after heat or chemical denaturation to bind antigen again. Starting with our previously isolated ricin binding sdAb determined to bind to four non-overlapping epitopes, we constructed a series of sdAb pairs, which were genetically linked through peptides of different length. We designed the series so that the sdAb are linked in both orientations with respect to the joining peptide. We confirmed that each of the sdAb in the constructs was able to bind to the ricin target, and have evidence that they are both binding ricin simultaneously. Through this work we determined that the order of genetically linked sdAb seems more important than the linker length. The genetically linked sdAb allowed for improved ricin detection with better limits of detection than the best anti-ricin monoclonal we evaluated, however they were not able to refold as well as unlinked component sdAb.

Further insight into the metabolic status of cells within anode biofilms is essential for understanding the functioning of microbial fuel cells and developing strategies to optimize their power output. Cells throughout anode biofilms of Geobacter sulfurreducens reduced the metabolic stains: 5-cyano-2,3-ditolyl tetrazolium chloride and Redox Green, suggesting metabolic activity throughout the biofilm. To compare the metabolic status of cells growing close to the anode versus cells in the outer portion of the anode biofilm, anode biofilms were encased in resin and sectioned into inner (0-20 microm from anode surface) and outer (30-60 microm) fractions. Transcriptional analysis revealed that, at a twofold threshold, 146 genes had significant (P0.05) differences in transcript abundance between the inner and outer biofilm sections. Only 1 gene, GSU0093, a hypothetical ATP-binding cassette transporter, had significantly higher transcript abundances in the outer biofilm. Genes with lower transcript abundance in the outer biofilm included genes for ribosomal proteins and NADH dehydrogenase, suggesting lower metabolic rates. However, differences in transcript abundance were relatively low (threefold) and the expression of genes for the tricarboxylic acid cycle enzymes was not significantly lower. Lower expression of genes involved in stress responses in the outer biofilm may reflect the development of low pH near the surface of the anode. The results of this study suggest that cells throughout the biofilm are metabolically active and can potentially contribute to current production. The microtoming/microarray strategy described here may be useful for evaluating gene expression with depth in a diversity of microbial biofilms.

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.

Previous studies have shown that Geobacter sulfurreducens requires the outer-membrane, multicopper protein OmpB for Fe(III) oxide reduction. A homologue of OmpB, designated OmpC, which is 36 % similar to OmpB, has been discovered in the G. sulfurreducens genome. Deletion of ompC inhibited reduction of insoluble, but not soluble Fe(III). Analysis of multiple Geobacter and Pelobacter genomes, as well as in situ Geobacter, indicated that genes encoding multicopper proteins are conserved in Geobacter species but are not found in Pelobacter species. Levels of ompB transcripts were similar in G. sulfurreducens at different growth rates in chemostats and during growth on a microbial fuel cell anode. In contrast, ompC transcript levels increased at higher growth rates in chemostats and with increasing current production in fuel cells. Constant levels of Geobacter ompB transcripts were detected in groundwater during a field experiment in which acetate was added to the subsurface to promote in situ uranium bioremediation. In contrast, ompC transcript levels increased during the rapid phase of growth of Geobacter species following addition of acetate to the groundwater and then rapidly declined. These results demonstrate that more than one multicopper protein is required for optimal Fe(III) oxide reduction in G. sulfurreducens and suggest that, in environmental studies, quantifying OmpB/OmpC-related genes could help alleviate the problem that Pelobacter genes may be inadvertently quantified via quantitative analysis of 16S rRNA genes. Furthermore, comparison of differential expression of ompB and ompC may provide insight into the in situ metabolic state of Geobacter species in environments of interest.

Extracellular electron transfer onto Fe(III) oxides in Geobacter sulfurreducens is considered to require proteins that must be exported to the outer surface of the cell. In order to investigate this, the putative gene for OxpG, the pseudopilin involved in a type II general secretion pathway of Gram-negative bacteria, was deleted. The mutant was unable to grow with insoluble Fe(III) oxide as the electron acceptor. Growth on soluble Fe(III) was not affected. An analysis of proteins that accumulated in the periplasm of the oxpG mutant, but not in the wild-type, led to the identification of a secreted protein, OmpB. OmpB is predicted to be a multicopper protein, with highest homology to the manganese oxidase, MofA, from Leptothrix discophora. OmpB contains a potential Fe(III)-binding site and a fibronectin type III domain, suggesting a possible role for this protein in accessing Fe(III) oxides. OmpB was localized to the membrane fraction of G. sulfurreducens and in the supernatant of growing cultures, consistent with the type II secretion system exporting OmpB. A mutant in which ompB was deleted had the same phenotype as the oxpG mutant, suggesting that the failure to export OmpB was responsible for the inability of the oxpG-deficient mutant to reduce Fe(III) oxide. This is the first report that proposes a role for a multicopper oxidase-like protein in an anaerobic organism. These results further emphasize the importance of outer-membrane proteins in Fe(III) oxide reduction and suggest that outer-membrane proteins other than c-type cytochromes are required for Fe(III) oxide reduction in Geobacter species.

Outer membrane cytochromes are often proposed as likely agents for electron transfer to extracellular electron acceptors, such as Fe(III). The omcF gene in the dissimilatory Fe(III)-reducing microorganism Geobacter sulfurreducens is predicted to code for a small outer membrane monoheme c -type cytochrome. An OmcF-deficient strain was constructed, and its ability to reduce and grow on Fe(III) citrate was found to be impaired. Following a prolonged lag phase (150 h), the OmcF-deficient strain developed the ability to grow in Fe(III) citrate medium with doubling times and yields that were ca. 145% and 70% of those of the wild type, respectively. Comparison of the c -type cytochrome contents of outer membrane-enriched fractions prepared from wild-type and OmcF-deficient cultures confirmed the outer membrane association of OmcF and revealed multiple changes in the cytochrome content of the OmcF-deficient strain. These changes included loss of expression of two previously characterized outer membrane cytochromes, OmcB and OmcC, and overexpression of a third previously characterized outer membrane cytochrome, OmcS, during growth on Fe(III) citrate. The omcB and omcC transcripts could not be detected in the OmcF-deficient mutant by either reverse transcriptase PCR or Northern blot analyses. Expression of the omcF gene in trans restored both the capacity of the OmcF-deficient mutant to reduce Fe(III) and wild-type levels of omcB and omcC mRNA and protein. Thus, elimination of OmcF may impair Fe(III) reduction by influencing expression of OmcB, which has previously been demonstrated to play a critical role in Fe(III) reduction.

Self-incompatibility (SI) is the inability of otherwise fertile gametes to produce viable zygotes upon self-fertilization. The S locus of chromosome 1 in Lycopersicon is thought to be the main controlling factor in SI. However, the significance of other chromosome segments in the control of SI or the effect of a foreign genetic background on the S locus has not been thoroughly explored. In addition, the relationship between SI and wider interspecific crossing barriers remains unclear. Using DNA and protein markers for chromosome 1, we have created a series of backcross lines that contain either 1) the SI locus and flanking chromosome region from a SI species in a SC species background or 2) the same chromosome region from a SC species in a SI species background. The reproductive behavior of these plants will be discussed.

Pollination involves a series of interactions between the surface of the male gametophyte and the extracellular matrix of several distinct sporophytic tissues in the pistil. Pollen germinates on the stigmatic surface and develops a tube that elongates in the transmitting tract of the style to transport the male gametes to the embryo sac inside the ovary. Pollen tube growth is fuelled by cytosolic activities within the pollen, but female tissues facilitate this process to ensure that pollen tubes arrive at the ovary when the ovules are the most receptive for maximum reproductive success. Interactions between pollen and pistil factors, probably both physical and biochemical, must be transmitted to the pollen cytosol to elicit the appropriate pollen cellular activities. Our research focuses on identifying the molecules that are involved in these interactions, understanding their biochemical and cellular bases, transmittal of these interactive signals into the pollen cytosol and the pollen cellular responses.