scholarly journals In Vitro Mutagenesis of Bacillus subtilis by Using a Modified Tn7 Transposon with an Outward-Facing Inducible Promoter

2008 ◽  
Vol 74 (11) ◽  
pp. 3419-3425 ◽  
Author(s):  
Christophe Bordi ◽  
Bronwyn G. Butcher ◽  
Qiaojuan Shi ◽  
Anna-Barbara Hachmann ◽  
Joseph E. Peters ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
High Frequency ◽  
Inducible Promoter ◽  
In Vitro Mutagenesis ◽  
Donor Plasmid ◽  
Positive Bacterium ◽  
Expression Of Genes ◽  
Regulated Expression ◽  
Selection Of

ABSTRACT A Tn7 donor plasmid, pTn7SX, was constructed for use with the model gram-positive bacterium Bacillus subtilis. This new mini-Tn7, mTn7SX, contains a spectinomycin resistance cassette and an outward-facing, xylose-inducible promoter, thereby allowing for the regulated expression of genes downstream of the transposon. We demonstrate that mTn7SX inserts are obtained at a high frequency and occur randomly throughout the B. subtilis genome. The utility of this system was demonstrated by the selection of mutants with increased resistance to the antibiotic fosfomycin or duramycin.

scholarly journals Biochemical Characterization of the C4-Dicarboxylate Transporter DctA from Bacillus subtilis

2010 ◽  
Vol 192 (11) ◽  
pp. 2900-2907 ◽  
Author(s):  
Maarten Groeneveld ◽  
Ruud G. J. Detert Oude Weme ◽  
Ria H. Duurkens ◽  
Dirk Jan Slotboom
Keyword(s):  
Bacillus Subtilis ◽  
Biochemical Characterization ◽  
Krebs Cycle ◽  
Membrane Vesicles ◽  
Positive Bacterium ◽  
Proton Symport ◽  
Negative Membrane Potential ◽  
Isolated Membrane Vesicles

ABSTRACT Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C4-dicarboxylates. Here, we have expressed the DctA homologue from Bacillus subtilis in the Gram-positive bacterium Lactococcus lactis. Transport of dicarboxylates in vitro in isolated membrane vesicles was assayed. We determined the substrate specificity, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns. DctA was found to catalyze proton-coupled symport of the four C4-dicarboxylates from the Krebs cycle (succinate, fumurate, malate, and oxaloacetate) but not of other mono- and dicarboxylates. Because (i) succinate-proton symport was electrogenic (stimulated by an internal negative membrane potential) and (ii) the divalent anionic form of succinate was recognized by DctA, at least three protons must be cotransported with succinate. The results were interpreted in the light of the crystal structure of the homologous aspartate transporter GltPh from Pyrococcus horikoshii.

scholarly journals Saccharomyces cerevisiae CYC1 mRNA 5'-end positioning: analysis by in vitro mutagenesis, using synthetic duplexes with random mismatch base pairs.

1985 ◽  
Vol 5 (12) ◽  
pp. 3545-3551 ◽  
Author(s):  
J B McNeil ◽  
M Smith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
In Vitro Mutagenesis ◽  
Base Pairs ◽  
Positioning Analysis ◽  
Dna Strand ◽  
Template Dna ◽  
Double Stranded Oligonucleotide ◽  
Selection Of

Expression of the Saccharomyces cerevisiae CYC1 gene produces mRNA with more than 20 different 5' ends. A derivative of the CYC1 gene (CYC1-157) was constructed with a deletion of a portion of the CYC1 5'-noncoding region, which includes the sites at which many of the CYC1 mRNAs 5' ends map. A 54-mer double-stranded oligonucleotide homologous with the deleted sequence of CYC1-157 and which included a low level of random base pair mismatches (an average of two mismatches per duplex) was used to construct mutants of the CYC1 gene and examine the role of the DNA sequence at and immediately adjacent to the mRNA 5' ends in specifying their locations. The effect of these mutations on the site selection of mRNA 5' ends was examined by primer extension. Results indicate that there is a strong preference for 5' ends which align with an A residue (T in the template DNA strand) preceded by a short tract of pyrimidine residues.

scholarly journals ModifiedmarinerTransposons for Random Inducible-Expression Insertions and Transcriptional Reporter Fusion Insertions in Bacillus subtilis

2011 ◽  
Vol 78 (3) ◽  
pp. 778-785 ◽  
Author(s):  
Eric R. Pozsgai ◽  
Kris M. Blair ◽  
Daniel B. Kearns
Keyword(s):  
Bacillus Subtilis ◽  
Insertional Mutagenesis ◽  
Inducible Promoter ◽  
Insertion Sequences ◽  
Content Type ◽  
Transcriptional Reporter ◽  
Species Specific ◽  
Genetically Manipulated

ABSTRACTTransposons are mobile genetic elements bounded by insertion sequences that are recognized by a specific mobilizing transposase enzyme. The transposase may mobilize not only the insertion sequences but also intervening DNA.marineris a particularly efficient transposon for the random chromosomal integration of genes and insertional mutagenesis. Here, we modify an existingmarinertransposon, TnYLB, such that it can easily be genetically manipulated and introduced intoBacillus subtilis. We generate a series of three newmarinerderivatives that mobilize spectinomycin, chloramphenicol, and kanamycin antibiotic resistance cassettes. Furthermore, we generate a series of transposons with a strong, outward-oriented, optionally isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter for the random overexpression of neighboring genes and a series of transposons with a promoterlesslacZgene for the random generation of transcriptional reporter fusions. We note that the modification of the base transposon is not restricted toB. subtilisand should be applicable to anymariner-compatible host organism, provided thatin vitromutagenesis or anin vivospecies-specific delivery vector is employed.

scholarly journals Functional analysis of subunits III and IV of Bacillus subtilis aa3-600 quinol oxidase by in vitro mutagenesis and gene replacement

1995 ◽  
Vol 1232 (1-2) ◽  
pp. 67-74 ◽  
Author(s):  
Gaetano Villani ◽  
Maria Tattoli ◽  
Nazzareno Capitanio ◽  
Philippe Glaser ◽  
Sergio Papa ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Bacillus Subtilis ◽  
Gene Replacement ◽  
In Vitro Mutagenesis ◽  
Quinol Oxidase

scholarly journals Subcellular Localization of TatAd of Bacillus subtilis Depends on the Presence of TatCd or TatCy

2009 ◽  
Vol 191 (13) ◽  
pp. 4410-4418 ◽  
Author(s):  
Anja N. J. A. Ridder ◽  
Esther J. de Jong ◽  
Jan D. H. Jongbloed ◽  
Oscar P. Kuipers
Keyword(s):  
Bacillus Subtilis ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Inducible Promoter ◽  
Specific Protein ◽  
Positive Bacterium ◽  
Protein Substrates ◽  
Gfp Fusion Protein ◽  
Dual Localization ◽  
Green Fluorescent

ABSTRACT The gram-positive bacterium Bacillus subtilis contains two minimal Tat translocases, TatAdCd and TatAyCy, which are each involved in the secretion of one or more specific protein substrates. We have investigated the subcellular localization of the TatA components by employing C-terminal green fluorescent protein (GFP) fusions and fluorescence microscopy. When expressed from a xylose-inducible promoter, the TatA-GFP fusion proteins displayed a dual localization pattern, being localized peripherally and showing bright foci which are predominantly located at the division sites and/or poles of the cells. Importantly, the localization of TatAd-GFP was similar when the protein was expressed from its own promoter under phosphate starvation conditions, indicating that these foci are not the result of artificial overexpression. Moreover, the TatAd-GFP fusion protein was shown to be functional in the translocation of its substrate PhoD, provided that TatCd is also present. Furthermore, we demonstrate that the localization of TatAd-GFP in foci depends on the presence of the TatCd component. Remarkably, however, the TatAd-GFP foci can also be observed in the presence of TatCy, indicating that TatAd can interact not only with TatCd but also with TatCy. These results suggest that the formation of TatAd complexes in B. subtilis is controlled by TatC.

scholarly journals The influence of Bacillus subtilis 87Y isolated from Eisenia fetida on the growth of pathogenic and probiotic microorganisms

2019 ◽  
Author(s):  
I. Szmigiel ◽  
J. Suchodolski ◽  
M. Łukaszewicz ◽  
A. Krasowska
Keyword(s):  
Bacillus Subtilis ◽  
Bile Salts ◽  
Eisenia Fetida ◽  
Animal Feed ◽  
Probiotic Strain ◽  
Rapeseed Meal ◽  
Positive Bacterium ◽  
Feed Supplements ◽  
Acidic Conditions

AbstractBacillus subtilis strain 87Y, isolated from the earthworm Eisenia fetida, decreases the growth of pathogenic Salmonella spp. and Staphylococcus aureus and promotes the growth of probiotic Lactococcus spp. Preserving viability in acidic conditions as well as in bile salts, B. subtilis 87Y meets two of the requirements of a probiotic strain. Thanks to the production of the biosurfactant surfactin, B. subtilis 87Y limits the growth of the Gram-positive bacterium S. aureus. In the presence of sucrose, B. subtilis produces levan, which contributes to promoting the growth of other probiotics. Our in vitro studies justify the continuation of enriching rapeseed meal waste from solid-state fermentation with B. subtilis 87Y, to produce high-value animal feed supplements.

scholarly journals Isolation of RNase H Genes That Are Essential for Growth of Bacillus subtilis 168

1999 ◽  
Vol 181 (7) ◽  
pp. 2118-2123 ◽  
Author(s):  
Mitsuhiro Itaya ◽  
Akira Omori ◽  
Shigenori Kanaya ◽  
Robert J. Crouch ◽  
Teruo Tanaka ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rnase H ◽  
Positive Bacterium ◽  
E Coli ◽  
Cellular Processes ◽  
Dna Library ◽  
Bacillus Subtilis 168 ◽  
Genes Encoding

ABSTRACT Two genes encoding functional RNase H (EC 3.1.26.4 ) were isolated from a gram-positive bacterium, Bacillus subtilis 168. Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a B. subtilis DNA library. One (28.2 kDa) revealed high similarity to Escherichia coli RNase HII, encoded by the rnhB gene. The other (33.9 kDa) was designated rnhC and encodes B. subtilisRNase HIII. The B. subtilis genome has anrnhA homologue, the product of which has not yet shown RNase H activity. Analyses of all three B. subtilis genes revealed that rnhB andrnhC cannot be simultaneously inactivated. This observation indicated that in B. subtilis both thernhB and rnhC products are involved in certain essential cellular processes that are different from those suggested by E. coli rnh mutation studies. Sequence conservation between the rnhB and rnhC genes implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the singlernhC homologue in the small genome ofMycoplasma species.

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