[Show abstract][Hide abstract] ABSTRACT: Sulfate-reducing bacteria such as Desulfovibrio vulgaris Hildenborough are often found in environments with limiting growth nutrients. Using lactate as the electron donor and carbon source, and sulfate as the electron acceptor, wild type D. vulgaris shows motility on soft agar plates. We evaluated this phenotype with mutants resulting from insertional inactivation of genes potentially related to motility. Our study revealed that the cheA3 (DVU2072) kinase mutant was impaired in the ability to form motility halos. Insertions in two other cheA loci did not exhibit a loss in this phenotype. The cheA3 mutant was also non-motile in capillary assays. Complementation with a plasmid-borne copy of cheA3 restores wild type phenotypes. The cheA3 mutant displayed a flagellum as observed by electron microscopy, grew normally in liquid medium, and was motile in wet mounts. In the growth conditions used, the D. vulgaris ΔfliA mutant (DVU3229) for FliA, predicted to regulate flagella-related genes including cheA3, was defective both in flagellum formation and in forming the motility halos. In contrast, a deletion of the flp gene (DVU2116) encoding a pilin-related protein was similar to wild type. We conclude that wild type D. vulgaris forms motility halos on solid media that are mediated by flagella-related mechanisms via the CheA3 kinase. The conditions under which the CheA1 (DVU1594) and CheA2 (DVU1960) kinase function remain to be explored.
Frontiers in Microbiology 01/2014; 5:77. · 3.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Due to their adjacent location in the genomes of Desulfovibrio species and their potential for formation of an electron transfer pathway in sulfate-reducing prokaryotes, adenosyl phosphosulfate (APS) reductase (Apr) and quinone-interacting membrane bound oxidoreductase (Qmo) have been thought to interact together during the reduction of APS. This interaction was recently verified in Desulfovibrio desulfuricans (Ramos, et al., 2012). Membranes proteins of Desulfovibrio vulgaris Hildenborough ΔqmoABCD JW9021, a deletion mutant were compared to the parent strain using Blue-Native polyacrylamide gel electrophoresis to determine whether Qmo formed a complex with Apr or other proteins. In the parent strain of D. vulgaris, a unique band was observed which contained all 4 Qmo subunits and another band contained 3 subunits of Qmo as well as AprA and AprB. Similar results were observed with bands excised from membrane preparations of D. alaskensis strain G20. These results are in support of the formation of a physical complex between the two proteins. A result which was further confirmed by the co-purification of QmoA/B and AprA/B from affinity tagged DvH strains (AprA, QmoA and QmoB) regardless of which subunit had been tagged. This provides clear evidence for the presence of a Qmo-Apr complex that is at least partially stable in protein extracts of D. vulgaris and D. alaskensis .
[Show abstract][Hide abstract] ABSTRACT: The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA "parts" to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.
Applied and Environmental Microbiology 09/2011; 77(21):7595-604. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Project Goals: The ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) project aims to elucidate the mechanisms and key processes that enable microorganisms and their communities to function in metal-contaminated soil sites. The ENIGMA Biotechnology Component focuses its efforts on providing cross-cutting technologies that will support all other ENIGMA components with a particular focus on biological imaging at different scales of bacteria and microbial communities. These data will help to develop models of microbial community activity and principles of community organization in an effort to predict the role that microbial species and their interactions play in the dynamics of geochemical transformations in a changing environment. Abstract: Microbial physiology is inherently a multiscale biological process that coordinates complex processes such as extracellular metal reduction and response to environmental stresses and competing species. Bacteria often assemble into sustainable communities that allow individual bacteria to coordinate their respective behavior and thus optimizing the efficiency of biological processes, which may enhance the chances for species survival. ENIGMA is addressing the complexity of multiscale spatiotemporal biofilm organization through a combination of expertise in traditional structural biology and modern multimodal imaging. SAXS (Rambo et al. 2010) and single particle cryo-EM are proven technologies to determine protein complex stoichiometry and shape, allowing the fitting of high-resolution structures into the intermediate resolution density envelope (Han et al. 2009). Cryo-electron tomography of bacterial whole mount samples can detect intra-and extracelluar specializations e.g. those important for metal reduction. Cryo-EM analysis is complemented by widefield 2D section TEM and advanced 3D SEM imaging approaches (FIB/SEM & SBF/SEM) of cryo-preserved, freeze substituted and resin-embedded samples. With these novel EM imaging approaches, we have begun to examine large areas and volumes of biofilms in DvH and other soil bacteria. We have found outer membrane vesicles, vesicle chains and cell-cell connections (Palsdottir et al. 2009, Remis et al. 2010, Remis et al. submitted), as well as compartmentalization of metal precipitation (Auer, unpublished observation). These observations suggest a an intricate set of interactions and possibly coordination of function between community members. X-ray and EM-based imaging approaches are complemented by tag-based labeling of proteins both at the light and electron microscopy level, and allow the study of cell-to-cell variations in protein abundance and protein localization (Chabra et al. 2010). Advanced optical super-resolution imaging methods (including PALM and STORM) allow high precision localization and counting (Betzig et al 2006). Further integration of small molecule mass spectrometry imaging, while at a somewhat larger size scale, promises to link structural observation and protein localization with metabolic activity of biofilm regions. Through the integrated application of these imaging modalities ENIGMA is deconstructing a mechanistic understanding of biofilm function.