Large-scale genetic characterization of the model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough.

Sulfate-reducing bacteria (SRB) are obligate anaerobes that can couple their growth to the reduction of sulfate. Despite the importance of SRB to global nutrient cycles and their damage to the petroleum industry, our molecular understanding of their physiology remains limited. To systematically provide new insights into SRB biology, we generated a randomly barcoded transposon mutant library in the model SRB Desulfovibrio vulgaris Hildenborough (DvH) and used this genome-wide resource to assay the importance of its genes under a range of metabolic and stress conditions. In addition to defining the essential gene set of DvH, we identified a conditional phenotype for 1,137 non-essential genes. Through examination of these conditional phenotypes, we were able to make a number of novel insights into our molecular understanding of DvH, including how this bacterium synthesizes vitamins. For example, we identified DVU0867 as an atypical L-aspartate decarboxylase required for the synthesis of pantothenic acid, provided the first experimental evidence that biotin synthesis in DvH occurs via a specialized acyl carrier protein and without methyl esters, and demonstrated that the uncharacterized dehydrogenase DVU0826:DVU0827 is necessary for the synthesis of pyridoxal phosphate. In addition, we used the mutant fitness data to identify genes involved in the assimilation of diverse nitrogen sources and gained insights into the mechanism of inhibition of chlorate and molybdate. Our large-scale fitness dataset and RB-TnSeq mutant library are community-wide resources that can be used to generate further testable hypotheses into the gene functions of this environmentally and industrially important group of bacteria.

Deletion Mutants, Archived Transposon Library, and Tagged Protein Constructs of the Model Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough.

The dissimilatory sulfate-reducing deltaproteobacterium Desulfovibrio vulgaris Hildenborough (ATCC 29579) was chosen by the research collaboration ENIGMA to explore tools and protocols for bringing this anaerobe to model status. Here, we describe a collection of genetic constructs generated by ENIGMA that are available to the research community.

LurR is a regulator of the central lactate oxidation pathway in sulfate-reducing Desulfovibrio species.

The central carbon/lactate utilization pathway in the model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, is encoded by the highly conserved operon DVU3025-3033. Our earlier in vitro genome-wide study had suggested a network of four two-component system regulators that target this large operon; however, how these four regulators control this operon was not known. Here, we probe the regulation of the lactate utilization operon with mutant strains and DNA-protein binding assays. We show that the LurR response regulator is required for optimal growth and complete lactate utilization, and that it activates the DVU3025-3033 lactate oxidation operon as well as DVU2451, a lactate permease gene, in the presence of lactate. We show by electrophoretic mobility shift assays that LurR binds to three sites in the upstream region of DVU3025, the first gene of the operon. NrfR, a response regulator that is activated under nitrite stress, and LurR share similar binding site motifs and bind the same sites upstream of DVU3025. The DVU3025 promoter also has a binding site motif (Pho box) that is bound by PhoB, a two-component response regulator activated under phosphate limitation. The lactate utilization operon, the regulator LurR, and LurR binding sites are conserved across the order Desulfovibrionales whereas possible modulation of the lactate utilization genes by additional regulators such as NrfR and PhoB appears to be limited to D. vulgaris.

Bacterial Interactomes: Interacting Protein Partners Share Similar Function and Are Validated in Independent Assays More Frequently Than Previously Reported.

Numerous affinity purification-mass spectrometry (AP-MS) and yeast two-hybrid screens have each defined thousands of pairwise protein-protein interactions (PPIs), most of which are between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here, we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial yeast two-hybrid and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli Compared with the nine published interactomes, our two networks are smaller, are much less highly connected, and have significantly lower false discovery rates. In addition, our interactomes are much more enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays than the pairs reported in prior studies. Our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.

Exploring the role of CheA3 in Desulfovibrio vulgaris Hildenborough motility.

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.

Membrane protein complex of APS reductase and Qmo is present in Desulfovibrio vulgaris and Desulfovibrio alaskensis.

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. Membrane proteins of Desulfovibrio vulgaris Hildenborough ΔqmoABCD JW9021, a deletion mutant, were compared to the parent strain using blue-native PAGE to determine whether Qmo formed a complex with Apr or other proteins. In the parent strain of D. vulgaris, a unique band was observed that contained all four Qmo subunits, and another band contained three subunits of Qmo, as well as subunits of AprA and AprB. Similar results were observed with bands excised from membrane preparations of Desulfovibrio alaskensis strain G20. These results are in support of the formation of a physical complex between the two proteins; a result that was further confirmed by the co-purification of QmoA/B and AprA/B from affinity-tagged D. vulgaris Hildenborough 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.

Effect of tobacco budworm (Lepidoptera: Noctuidae) infestation level on budworm-resistant and -susceptible varieties of flue-cured tobacco in North Carolina.

Field experiments were conducted from 1972 to 1978 and from 1998 to 1999 to evaluate tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), larval feeding on flue-cured tobacco, Nicotiana tabacum (L.), yield in eastern North Carolina. In the earlier studies, using variety Coker 319, treatment plots were evaluated when either 0 or 100% of plants in a plot were infested with H. virescens larvae. Treatment differences based on actual yield loss (kilograms per hectare) were compared with estimations of yield loss based on leaf consumption and leaf loss. Results indicate actual yield loss when 100% of plants were infested was less than the corresponding estimates of yield loss. In the later experiments, two tobacco budworm-resistant lines, 'CU 263' and 'CU 370', were compared with a commercial susceptible variety, K 326, when 0, 10, 20, or 40% of plants were infested (1998) and 0, 10, 40, 75, or 100% of plants were infested (1999). Although significant increases in leaf equivalents consumed were associated with infestations exceeding the recommended threshold, differences were not detected for yield (kilograms per hectare), quality (dollars per kilogram), and value (dollars per hectare) within each tobacco line. Additionally, there was not a significant correlation between value and infestations level for any of the tobacco lines. These results provide economic support for tolerance of a higher treatment threshold. Although K 326 sustained more leaf equivalent loss than CU 263 and CU 370, the value of K 326 harvested was higher than that of CU 263 and CU 370. To justify use of resistant varieties, the combination of pest pressure and the benefit of host plant resistance must be greater than the capacity of a susceptible variety to produce competitive yields, despite sustaining significantly higher loss.