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

2021 ◽  
Vol 10 (11) ◽  
Author(s):  
Judy D. Wall ◽  
Grant M. Zane ◽  
Thomas R. Juba ◽  
Jennifer V. Kuehl ◽  
Jayashree Ray ◽  
...  

ABSTRACT 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.

2004 ◽  
Vol 22 (5) ◽  
pp. 554-559 ◽  
Author(s):  
John F Heidelberg ◽  
Rekha Seshadri ◽  
Shelley A Haveman ◽  
Christopher L Hemme ◽  
Ian T Paulsen ◽  
...  

2009 ◽  
Vol 420 (2) ◽  
pp. 317-326 ◽  
Author(s):  
Susana A. L. Lobo ◽  
Amanda Brindley ◽  
Martin J. Warren ◽  
Lígia M. Saraiva

The biosynthesis of the tetrapyrrole framework has been investigated in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough by characterization of the enzymes required for the transformation of aminolaevulinic acid into sirohydrochlorin. PBG (porphobilinogen) synthase (HemB) was found to be a zinc-dependent enzyme that exists in its native state as a homohexamer. PBG deaminase (HemC) was shown to contain the dipyrromethane cofactor. Uroporphyrinogen III synthase is found fused with a uroporphyrinogen III methyltransferase (HemD-CobA). Both activities could be demonstrated in this amalgamated protein and the individual enzyme activities were separated by dissecting the relevant gene to allow the production of two distinct proteins. A gene annotated in the genome as a bifunctional precorrin-2 dehydrogenase/sirohydrochlorin ferrochelatase was in fact shown to act only as a dehydrogenase and is simply capable of synthesizing sirohydrochlorin rather than sirohaem. Genome analysis also reveals a lack of any uroporphyrinogen III decarboxylase, an enzyme necessary for the classical route to haem synthesis. However, the genome does encode some predicted haem d1 biosynthetic enzymes even though the bacterium does not contain the cd1 nitrite reductase. We suggest that sirohydrochlorin acts as a substrate for haem synthesis using a novel pathway that involves homologues of the d1 biogenesis system. This explains why the uroporphyrinogen III synthase is found fused with the methyltransferase, bypassing the need for uroporphyrinogen III decarboxylase activity.


2017 ◽  
Vol 199 (23) ◽  
Author(s):  
Lucy Zeng ◽  
Etsuko Wooton ◽  
David A. Stahl ◽  
Peter J. Walian

ABSTRACT Due in large part to their ability to facilitate the diffusion of a diverse range of solutes across the outer membrane (OM) of Gram-negative bacteria, the porins represent one of the most prominent and important bacterial membrane protein superfamilies. Notably, for the Gram-negative bacterium Desulfovibrio vulgaris Hildenborough, a model organism for studies of sulfate-reducing bacteria, no genes for porins have been identified or proposed in its annotated genome. Results from initial biochemical studies suggested that the product of the DVU0799 gene, which is one of the most abundant proteins of the D. vulgaris Hildenborough OM and purified as a homotrimeric complex, was a strong porin candidate. To investigate this possibility, this protein was further characterized biochemically and biophysically. Structural analyses via electron microscopy of negatively stained protein identified trimeric particles with stain-filled depressions and structural modeling suggested a β-barrel structure for the monomer, motifs common among the known porins. Functional studies were performed in which crude OM preparations or purified DVU0799 was reconstituted into proteoliposomes and the proteoliposomes were examined for permeability against a series of test solutes. The results obtained establish DVU0799 to be a pore-forming protein with permeability properties similar to those observed for classical bacterial porins, such as those of Escherichia coli. Taken together, these findings identify this highly abundant OM protein to be the major porin of D. vulgaris Hildenborough. Classification of DVU0799 in this model organism expands the database of functionally characterized porins and may also extend the range over which sequence analysis strategies can be used to identify porins in other bacterial genomes. IMPORTANCE Porins are membrane proteins that form transmembrane pores for the passive transport of small molecules across the outer membranes of Gram-negative bacteria. The present study identified and characterized the major porin of the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, observing its preference for anionic sugars over neutral ones. Its predicted architecture appears to be novel for a classical porin, as its core β-barrel structure is of a type typically found in solute-specific channels. Broader use of the methods employed here, such as assays for channel permeability and electron microscopy of purified samples, is expected to help expand the database of confirmed porin sequences and improve the range over which sequence analysis-based strategies can be used to identify porins in other Gram-negative bacteria. Functional characterization of these critical gatekeeping proteins from divergent Desulfovibrio species should offer an improved understanding of the physiological features that determine their habitat range and supporting activities.


2015 ◽  
Vol 197 (21) ◽  
pp. 3400-3408 ◽  
Author(s):  
Lara Rajeev ◽  
Amy Chen ◽  
Alexey E. Kazakov ◽  
Eric G. Luning ◽  
Grant M. Zane ◽  
...  

ABSTRACTSulfate-reducing bacteria (SRB) are sensitive to low concentrations of nitrite, and nitrite has been used to control SRB-related biofouling in oil fields.Desulfovibrio vulgarisHildenborough, a model SRB, carries a cytochromec-type nitrite reductase (nrfHA) that confers resistance to low concentrations of nitrite. The regulation of this nitrite reductase has not been directly examined to date. In this study, we show that DVU0621 (NrfR), a sigma54-dependent two-component system response regulator, is the positive regulator for this operon. NrfR activates the expression of thenrfHAoperon in response to nitrite stress. We also show thatnrfRis needed for fitness at low cell densities in the presence of nitrite because inactivation ofnrfRaffects the rate of nitrite reduction. We also predict and validate the binding sites for NrfR upstream of thenrfHAoperon using purified NrfR in gel shift assays. We discuss possible roles for NrfR in regulating nitrate reductase genes in nitrate-utilizingDesulfovibriospp.IMPORTANCEThe NrfA nitrite reductase is prevalent across several bacterial phyla and required for dissimilatory nitrite reduction. However, regulation of thenrfAgene has been studied in only a few nitrate-utilizing bacteria. Here, we show that inD. vulgaris, a bacterium that does not respire nitrate, the expression ofnrfHAis induced by NrfR upon nitrite stress. This is the first report of regulation ofnrfAby a sigma54-dependent two-component system. Our study increases our knowledge of nitrite stress responses and possibly of the regulation of nitrate reduction in SRB.


mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Megan L. Kempher ◽  
Xuanyu Tao ◽  
Rong Song ◽  
Bo Wu ◽  
David A. Stahl ◽  
...  

ABSTRACT Adaptation via natural selection is an important driver of evolution, and repeatable adaptations of replicate populations, under conditions of a constant environment, have been extensively reported. However, isolated groups of populations in nature tend to harbor both genetic and physiological divergence due to multiple selective pressures that they have encountered. How this divergence affects adaptation of these populations to a new common environment remains unclear. To determine the impact of prior genetic and physiological divergence in shaping adaptive evolution to accommodate a new common environment, an experimental evolution study with the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) was conducted. Two groups of replicate populations with genetic and physiological divergence, derived from a previous evolution study, were propagated in an elevated-temperature environment for 1,000 generations. Ancestor populations without prior experimental evolution were also propagated in the same environment as a control. After 1,000 generations, all the populations had increased growth rates and all but one had greater fitness in the new environment than the ancestor population. Moreover, improvements in growth rate were moderately affected by the divergence in the starting populations, while changes in fitness were not significantly affected. The mutations acquired at the gene level in each group of populations were quite different, indicating that the observed phenotypic changes were achieved by evolutionary responses that differed between the groups. Overall, our work demonstrated that the initial differences in fitness between the starting populations were eliminated by adaptation and that phenotypic convergence was achieved by acquisition of mutations in different genes. IMPORTANCE Improving our understanding of how previous adaptation influences evolution has been a long-standing goal in evolutionary biology. Natural selection tends to drive populations to find similar adaptive solutions for the same selective conditions. However, variations in historical environments can lead to both physiological and genetic divergence that can make evolution unpredictable. Here, we assessed the influence of divergence on the evolution of a model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, in response to elevated temperature and found a significant effect at the genetic but not the phenotypic level. Understanding how these influences drive evolution will allow us to better predict how bacteria will adapt to various ecological constraints.


2011 ◽  
Vol 193 (20) ◽  
pp. 5716-5727 ◽  
Author(s):  
M. N. Price ◽  
A. M. Deutschbauer ◽  
J. V. Kuehl ◽  
H. Liu ◽  
H. E. Witkowska ◽  
...  

2010 ◽  
Vol 87 (3) ◽  
pp. 1109-1118 ◽  
Author(s):  
Meng-Hsin Phoebe Lee ◽  
Sean M. Caffrey ◽  
Johanna K. Voordouw ◽  
Gerrit Voordouw

2015 ◽  
Author(s):  
◽  
Samuel R. Fels

Illumina sequencing and other massively-parallel, short read sequencing technologies have become prevalent methods for querying the genetic systems of organisms. This trend was initially driven by the demand for general de novo and resequencing applications in eukaryotes, but uses for these instruments have recently grown in scope to also include more specific assays and methods intended for use in prokaryotic systems. This thesis seeks to develop two such assays for use in the sulfate reducing bacteria type species Desulfovibrio vulgaris Hildenborough. These novel assays continue advances made in other bacteria, and the use of this environmentally relevant obligate anaerobe will ensure their extension to other bacteria outside the easily-manipulated groups studied previously. The first assay is a variation on existing transposon sequencing (Tn-seq) assays, which seek to determine gene fitness profiles and essential genes by simultaneous analyses of whether the absence of each gene product alters the growth kinetics of the bacterium. Here we modify the standard Tn-seq procedure by including delivery of the transposon through conjugation and liquid culture enrichment of the mutant pool, creating transposon liquid enrichment sequencing (TnLE-seq). This simplifies and shortens the process, and also reduces barriers to application of the technique in microbes lacking a facile genetic system. The second assay provides a means of mapping consensus 3' end sites of RNA transcripts across a reference genome. This method is known as 3' RNA-seq and compliments an established technique for determining 5' start sites of transcripts. Together these facilitate the application of current sequencing technology to a wider array of microbes and a new type of biologically relevant genetic data.


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