Development of a Markerless Genetic Exchange System for
            <i>Desulfovibrio vulgaris</i>
            Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency

Keller, Kimberly L.; Bender, Kelly S.; Wall, Judy D.

doi:10.1128/aem.01839-09

Cited by 81 publications

(111 citation statements)

References 32 publications

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“…For mutant complementation, the entire operons of DVU2097, DVU2547, and DVU3111 or a partial operon of DVU0379 as well as the upstream and downstream sequences were included in the complementation plasmids ( Table 1). The DNA fragments were amplified from wild-type (WT) D. vulgaris genomic DNA (gDNA) by PCR with Phusion high-fidelity DNA polymerase (Finnzymes Oy, Finland) and the primers listed in Table S1 in the supplemental material and were cloned into pMO719, which is stably replicated in D. vulgaris (22). The plasmids were propagated in Escherichia coli DH5␣, and the correctness of the construct was confirmed with Sanger sequencing.…”

Section: Methodsmentioning

confidence: 99%

Functional Characterization of Crp/Fnr-Type Global Transcriptional Regulators in Desulfovibrio vulgaris Hildenborough

Zhou

Chen

Zane

et al. 2012

Appl Environ Microbiol

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Crp/Fnr-type global transcriptional regulators regulate various metabolic pathways in bacteria and typically function in response to environmental changes. However, little is known about the function of four annotated Crp/Fnr homologs (DVU0379, DVU2097, DVU2547, and DVU3111) in Desulfovibrio vulgaris Hildenborough. A systematic study using bioinformatic, transcriptomic, genetic, and physiological approaches was conducted to characterize their roles in stress responses. Similar growth phenotypes were observed for the crp/fnr deletion mutants under multiple stress conditions. Nevertheless, the idea of distinct functions of Crp/Fnr-type regulators in stress responses was supported by phylogeny, gene transcription changes, fitness changes, and physiological differences. The four D. vulgaris Crp/Fnr homologs are localized in three subfamilies (HcpR, CooA, and cc). The crp/fnr knockout mutants were well separated by transcriptional profiling using detrended correspondence analysis (DCA), and more genes significantly changed in expression in a ⌬DVU3111 mutant (JW9013) than in the other three paralogs. In fitness studies, strain JW9013 showed the lowest fitness under standard growth conditions (i.e., sulfate reduction) and the highest fitness under NaCl or chromate stress conditions; better fitness was observed for a ⌬DVU2547 mutant (JW9011) under nitrite stress conditions and a ⌬DVU2097 mutant (JW9009) under air stress conditions. A higher Cr(VI) reduction rate was observed for strain JW9013 in experiments with washed cells. These results suggested that the four Crp/Fnr-type global regulators play distinct roles in stress responses of D. vulgaris. DVU3111 is implicated in responses to NaCl and chromate stresses, DVU2547 in nitrite stress responses, and DVU2097 in air stress responses.T ranscription factors, promoter sequences, and regulatory circuit architecture are often referred as "the regulatory genome" (27, 33), and transcription factors are central to the function of regulatory networks (5). Organisms rely on regulatory networks to orchestrate the transcription of genes in response to internal or external environmental changes in order to optimize metabolism and enhance survival. Crp/Fnr regulators are global transcriptional regulators widely distributed in bacteria. The characteristic structure of Crp/Fnr is a C-terminal helix-turn-helix (HTH) motif that fits the DNA major groove and an N-terminal nucleotide binding domain (34). This class of transcriptional factors is named after the first two representatives discovered in Escherichia coli, i.e., the Crp (cyclic AMP [cAMP] receptor protein) and Fnr (fumarate and nitrate reductase regulator protein) (4, 21, 24, 27). Crp/Fnr regulators are generally classified into different subfamilies (e.g., CooA, Crp, Dnr, FixK, Fnr, HbaR, NnrR, etc.) according to phylogenetic affiliations (50). The increasing number of sequenced whole genomes has added to a growing list of Crp/Fnr family regulators identified in bacteria (9, 26); however, functions for most are not well defin...

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Section: Methodsmentioning

confidence: 99%

Functional Characterization of Crp/Fnr-Type Global Transcriptional Regulators in Desulfovibrio vulgaris Hildenborough

Zhou

Chen

Zane

et al. 2012

Appl Environ Microbiol

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“…Incorporation of the pyrimidine analog 5-fluorouracil (5-FU) is lethal in D. vulgaris. Deletion of upp makes DvH resistant to 5-FU and re-introduction of upp restores sensitivity, which provides a selection marker for a two-step integration and excision in mutagenesis (Keller et al, 2009). SNPs were introduced into JW710 with a strategy similar to that of E. coli (Warming et al, 2005).…”

Section: Salt Tolerance Phenotype Testmentioning

confidence: 99%

“…Site-directed mutagenesis and growth phenotype test of SDMs DvH strain JW710 (Keller et al, 2009) with a deletion of the upp gene was used for generation of SDMs. upp encodes the pyrimidine salvage enzyme uracil phosphoribosyl transferase, which allows the recycling of free pyrimidines and the incorporation of base analogs into nucleoside monophosphates.…”

Section: Salt Tolerance Phenotype Testmentioning

confidence: 99%

Rapid selective sweep of pre-existing polymorphisms and slow fixation of new mutations in experimental evolution of Desulfovibrio vulgaris

Zhou

Hillesland

et al. 2015

The ISME Journal

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To investigate the genetic basis of microbial evolutionary adaptation to salt (NaCl) stress, populations of Desulfovibrio vulgaris Hildenborough (DvH), a sulfate-reducing bacterium important for the biogeochemical cycling of sulfur, carbon and nitrogen, and potentially the bioremediation of toxic heavy metals and radionuclides, were propagated under salt stress or non-stress conditions for 1200 generations. Whole-genome sequencing revealed 11 mutations in salt stress-evolved clone ES9-11 and 14 mutations in non-stress-evolved clone EC3-10. Whole-population sequencing data suggested the rapid selective sweep of the pre-existing polymorphisms under salt stress within the first 100 generations and the slow fixation of new mutations. Population genotyping data demonstrated that the rapid selective sweep of pre-existing polymorphisms was common in salt stress-evolved populations. In contrast, the selection of pre-existing polymorphisms was largely random in EC populations. Consistently, at 100 generations, stress-evolved population ES9 showed improved salt tolerance, namely increased growth rate (2.0-fold), higher biomass yield (1.8-fold) and shorter lag phase (0.7-fold) under higher salinity conditions. The beneficial nature of several mutations was confirmed by site-directed mutagenesis. All four tested mutations contributed to the shortened lag phases under higher salinity condition. In particular, compared with the salt tolerance improvement in ES9-11, a mutation in a histidine kinase protein gene lytS contributed 27% of the growth rate increase and 23% of the biomass yield increase while a mutation in hypothetical gene DVU2472 contributed 24% of the biomass yield increase. Our results suggested that a few beneficial mutations could lead to dramatic improvements in salt tolerance.

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“…Alternatively, a recipient strain can be constructed in which an organism is rendered sensitive to a substrate through mutagenesis. Frequently, the upp mutation is used to create sensitivity to 5-fluorouracil (10,12,20,24). Using S. mutans, we were able to create a galactose-sensitive recipient strain by mutagenizing the galKTE operon (33).…”

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confidence: 99%

Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans

Xie

Okinaga

et al. 2011

Appl Environ Microbiol

119

156

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Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed that utilize either temperature-sensitive plasmids or counterselectable suicide vectors containing both positive-and negative-selection markers. For many species, these techniques are not especially useful due to difficulties of cloning with Escherichia coli and/or a lack of functional negative-selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable to a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows extremely quick assembly and eliminates the requirement for either temperature-sensitive replicons or suicide vectors. As a proof of principle, we used Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild-type S. mutans strains, we further demonstrated that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches.Streptococcus mutans is a Gram-positive bacterial species that resides within multispecies oral biofilms formed on human tooth surfaces. It is also considered to be one of the principal species associated with dental caries initiation (6,7,34,35,44, 47,52). S. mutans genetic research has benefited tremendously from the many genetic tools that have been adapted for use in studies of the organism (4,5,13,15,22,25,26,29,33,45,51). For genetic studies of S. mutans, defined mutations are usually engineered in either of two ways: insertion duplication mutagenesis via single-crossover homologous recombination or marked allelic replacement mutagenesis using double-crossover homologous recombination (25,27,41). Both approaches are highly reliable strategies for mutagenesis and are simple to engineer, but they also have the potential to create unwanted artifacts that could influence the outcome of a genetic study. For example, insertion duplication mutations often result in the production of truncated proteins. Rarely is it known with certainty whether the protein fragments actually influence mutant phenotypes. Furthermore, due to significant polar effects downstream of the mutation site, both insertion duplication mutagenesis and allelic replacement mutagenesis are of limited utility within operons. In some cases, these issues have been addressed by creating allelic replacement mut...

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Development of a Markerless Genetic Exchange System for Desulfovibrio vulgaris Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency

Cited by 81 publications

References 32 publications

Functional Characterization of Crp/Fnr-Type Global Transcriptional Regulators in Desulfovibrio vulgaris Hildenborough

Functional Characterization of Crp/Fnr-Type Global Transcriptional Regulators in Desulfovibrio vulgaris Hildenborough

Rapid selective sweep of pre-existing polymorphisms and slow fixation of new mutations in experimental evolution of Desulfovibrio vulgaris

Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans

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