Chemical Synthesis and Cloning of a Gene Coding for Bacillus Subjilis Hbsu Protein

Groch, Nicolas; Quaas, Rainer; Hahn, Ulrich; Heinemann, Udo

doi:10.1080/07328318808056336

Cited by 7 publications

(5 citation statements)

References 9 publications

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“…The DNA sequence of the oligonucleotides used in the PCR is based on the amino acid sequence of HBsu protein (49). The oligomers represent two distinct parts of the chemically synthesized hbs gene (14). Both oligonucleotides were synthesized on an Applied Biosystems automatic DNA synthesizer and purified by hydrophobic interaction and anion-exchange fast protein liquid chromatography.…”

Section: Methodsmentioning

confidence: 99%

Molecular cloning, nucleotide sequence, and characterization of the Bacillus subtilis gene encoding the DNA-binding protein HBsu

et al. 1991

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A homologous class of histonelike proteins which are believed to wrap the DNA and to condense the chromosome into highly folded nucleoid structures has been identified in different bacterial species. Bacillus subtilis encodes a homodimeric DNA-binding protein called HBsu. We have cloned the corresponding gene (hbs) on a 3.8-kb fragment. The gene was subcloned to a 1-kb fragment, sequenced, and characterized. It encodes a 92-amino-acid protein with a predicted molecular mass of 9,884 Da. Fortunately, analysis of the DNA sequence downstream of the 3' end of hbs revealed the location of the first 19 amino acid residues of MtrA. This finding located the hbs gene unequivocally to the 5' end of the mtr operon at about 2040 on the standard genetic map of B. subtilis. Northern (RNA) blot analysis and primer extension studies indicated the presence of two distinct hbs transcripts, which were found to be initiated at two different sites located about 160 bases apart. Several attempts to replace the hbs gene in the B. subtilis chromosome with a cat-interrupted copy (hbs::cat) through marker replacement recombination were unsuccessful. In order to study whether hbs is an essential gene, we have constructed a strain containing a truncated copy of the gene behind its own promoter and another intact copy under control of the isopropyl-,-D-thiogalactopyranoside (IPTG)-inducible spac-1 promoter. In this strain (BM19), normal growth was found to depend on IPTG, whereas in the absence of IPTG, growth was severely affected. These results suggest an essential role for the hbs gene product for normal growth in B. subtilis.

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Section: Methodsmentioning

confidence: 99%

Molecular cloning, nucleotide sequence, and characterization of the Bacillus subtilis gene encoding the DNA-binding protein HBsu

et al. 1991

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“…The synthetic gene was cloned (Groch et al, 1988) into pUC120 as a construct having at the 5'-end an adapter cassette of two deoxyoligonucleotides linked to the coding part of the DNA sequence via a Sac1 restriction site. In addition to designed PstI and EcoRI sites, a BamHI site further downstream was gained from cloning experiments.…”

Section: Construction Of the Expression Vector Phbsulmentioning

confidence: 99%

“…We have recently described the synthesis and cloning of a gene coding for HBsu protein from B. subtilis (Groch et al, 1988) as well as the cloning and mapping of the chromosomal hbsu gene (Micka et al, 1991). The synthetic gene contains several unique restriction sites along the sequence for use in mutagenesis experiments.…”

mentioning

confidence: 99%

Determination of DNA‐binding parameters for the Bacillus subtilis histone‐like HBsu protein through introduction of fluorophores by site‐directed mutagenesis of a synthetic gene

Groch

Schindelin

Scholtz

et al. 1992

European Journal of Biochemistry

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A synthetic gene encoding the histone‐like DNA‐binding protein HBsu from Bacillus subtilis has been expressed in Escherichia coli. Yields of the purified protein are at least 20mg/l culture medium. The recombinant HBsu protein is chromatographically, immunologically and functionally identical with the authentic wild‐type protein. N‐terminal sequencing of the purified protein confirms the fidelity of expression of the synthetic gene in E. coli.. Site‐directed mutagenesis of the synthetic gene was employed to replace several amino acid residues of HBsu protein with tryptophan to facilitate the determination of DNA‐binding parameters by fluorescence spectroscopy. According to gel‐retardation experiments, the mutant protein [Phe47 → Trp]HBsu shows identical DNA binding to wild‐type HBsu protein. Analysis of fluorescence binding data reveals that [Phe47 → Trp]HBsu binds double‐stranded DNA with a dissociation constant in the micromolar range. Computer‐assisted fit of binding models to the experimental data renders positive cooperativity of binding unlikely. A dimer of [Phe47 → Trp]HBsu appears to contact three or four base pairs of DNA. These results are in pritial disagreement with earlier measurements on closely homologous proteins which tended to show cooperative binding and a longer DNA contact region.

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“…Site-directed mutagenesis of a synthetic gene coding for HBsu protein (8) was carried out as described earlier for [F47W] (10). Variant PCR fragments were synthesized with pHBsu 1 5' sequencing primer(introducing aXhol site) and mutagenic primers containing theXbal site ([F29W]) or the Bglll site ([F47W], [F50W]) of the synthetic gene in their hybridization region as well as with pHBsul 3' sequencing primer (introducing a Sail site) and a mutagenic primer containing the Kpnl site ([F79W]) of the gene in its hybridization region.…”

Section: Construction Of Synthetic Genes Of Hbsu Mutant Proteins [F29mentioning

confidence: 99%

“…The structure ofHBsu shows salt-dependent and protein concentration dependent changes in solution (7). Synthesis and cloning of a gene coding for HBsu protein from B. subtilis (8) and cloning and mapping of the chromosomal HBsu gene (9) were described recently. This allowed the introduction offluorophores by site-directed mutagenesis of a synthetic gene and the determination of DNA-binding parameters of HBsu (10).…”

Section: Introductionmentioning

confidence: 99%

Conformational stability of the DNA-binding histone-like protein, HBsu, fromBacillus subtilis, and of the four HBsu variants [F29W], [F47W], [F50W] and [F79W]

Welfle

Misselwitz

et al. 1993

Journal of Biomolecular Structure and Dynamics

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From denaturation studies with urea a free energy delta GuH2O of unfolding of 49.8 kJ.mol-1 at 25C was calculated for the histone-like DNA-binding protein HBsu from Bacillus subtilis. Unfolding was monitored by circular dichroism measurements observing the changes of the molar mean residue ellipticity [theta] at 222 nm. For the calculation of delta Gu a two-state model of unfolding, i.e. the unfolding of native dimers into unfolded monomers, was applied. The validity of this model in high ionic strength buffer was proven by measurements at different protein concentrations yielding the same delta Gu values. Four HBsu variants, each carrying one single point mutation ([F29W], [F47W], [F50W] and [F79W]) were analysed with respect to their stability against unfolding at increasing temperatures and urea concentrations. The delta Gu values of mutants were calculated using the two-state model and show a reduced stability of the variants [F29W], [F47W], [F50W] and [F79W] in comparison to the wild type HBsu with delta delta Gu values of -9.2 kJ.mol-1, -7.5 kJ.mol-1, -5.9 kJ.mol-1, and -7.5 kJ.mol-1, respectively. Similar delta delta Gu values were obtained for the HBsu mutant proteins by thermal unfolding experiments.

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Chemical Synthesis and Cloning of a Gene Coding for Bacillus Subjilis Hbsu Protein

Cited by 7 publications

References 9 publications

Molecular cloning, nucleotide sequence, and characterization of the Bacillus subtilis gene encoding the DNA-binding protein HBsu

Molecular cloning, nucleotide sequence, and characterization of the Bacillus subtilis gene encoding the DNA-binding protein HBsu

Determination of DNA‐binding parameters for the Bacillus subtilis histone‐like HBsu protein through introduction of fluorophores by site‐directed mutagenesis of a synthetic gene

Conformational stability of the DNA-binding histone-like protein, HBsu, fromBacillus subtilis, and of the four HBsu variants [F29W], [F47W], [F50W] and [F79W]

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