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H Bahl

Publications and source records attributed to H Bahl.

32 records · Page 2Linked to original sources
PubMed1993

Cloning, nucleotide sequence and structural analysis of the Clostridium acetobutylicum dnaJ gene.

The complete dnaJ gene of Clostridium acetobutylicum was isolated by chromosome walking using the previously cloned 5' end of the gene as a probe. Nucleotide sequencing of a positively reacting 2.2-kb HincII fragment, contained in the recombinant plasmid pKG4, revealed that the reading frame of the dnaJ gene of C. acetobutylicum consists of 1125 bp, encoding a protein of 374 amino acids with a calculated M(r) of 40376 and an isoelectric point of 9.54. The deduced amino acid sequence showed high similarity to the DnaJ proteins of other bacteria (e.g. Escherichia coli, Bacillus subtilis) as well as of an archaeon (Methanosarcina mazei) and to the corresponding proteins of eukaryotes (Saccharomyces cerevisiae, Homo sapiens). The areas of similarity included a conserved N-terminal domain of about 70 amino acids, a glycine-rich region of about 30 residues, and a central domain containing four repeats of a CXXCXGXG motif, whereas the C-terminal domain was less conserved. Northern (RNA) blot analysis indicated that dnaJ is induced by heat shock and that it is part of the dnaK operon of C. acetobutylicum. The 5' end (901 bp) of another gene (orfB), downstream of dnaJ and not heat-inducible, showed no significant similarity to other sequences available in EMBL and GenBank databases.

Amino Acid Sequence↗
PubMed1992

Parameters Affecting Solvent Production by Clostridium pasteurianum.

The effect of pH, growth rate, phosphate and iron limitation, carbon monoxide, and carbon source on product formation by Clostridium pasteurianum was determined. Under phosphate limitation, glucose was fermented almost exclusively to acetate and butyrate independently of the pH and growth rate. Iron limitation caused lactate production (38 mol/100 mol) from glucose in batch and continuous culture. At 15% (vol/vol) carbon monoxide in the atmosphere, glucose was fermented to ethanol (24 mol/100 mol), lactate (32 mol/100 mol), and butanol (36 mol/100 mol) in addition to the usual products, acetate (38 mol/100 mol) and butyrate (17 mol/100 mol). During glycerol fermentation, a completely different product pattern was found. In continuous culture under phosphate limitation, acetate and butyrate were produced only in trace amounts, whereas ethanol (30 mol/100 mol), butanol (18 mol/100 mol), and 1,3-propanediol (18 mol/100 mol) were the major products. Under iron limitation, the ratio of these products could be changed in favor of 1,3-propanediol (34 mol/100 mol). In addition, lactate was produced in significant amounts (25 mol/100 mol). The tolerance of C. pasteurianum to glycerol was remarkably high; growth was not inhibited by glycerol concentrations up to 17% (wt/vol). Increasing glycerol concentrations favored the production of 1,3-propanediol.

Journal Article↗
PubMed1992

Cloning, sequencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum.

The groESL operon of Clostridium acetobutylicum was cloned in Escherichia coli by using a gene probe of E. coli groESL. Sequencing of a positively reacting 2.2-kbp HindIII fragment contained in the recombinant plasmid pFN1 and a 2.5-kbp XbaI fragment present in pFN4 revealed that both fragments partially overlapped and together spanned 3,493 bp of the clostridial chromosome. Two complete open reading frames (288 and 1632 bp) were found and identified as the groES- and groEL-homologous genes of C. acetobutylicum, respectively. The 3' end of a third gene (orfZ), which was divergently transcribed, showed no significant homology to other sequences available in the EMBL and GenBank data bases. The length of the groESL-specific mRNA (2.2 kb), a transcription terminator downstream of groEL, and a transcription start site upstream of groES, identified by primer extension analysis, indicated that groES and groEL of C. acetobutylicum are organized in a bicistronic operon. From the transcription start site, the promoter structure 5'-TTGCTA (17 bp) TATTAT that shows high homology to the consensus promoter sequence of gram-positive bacteria as well as E. coli was deduced. Transcription of the groESL operon was strongly heat inducible, and maximum levels of mRNA were detected 15 min after heat shock from 30 to 42 degrees C. An 11-bp inverted repeat, located between promoter and translation start sites of groES and partially identical with similar structures in front of several heat shock genes of other bacteria, may play an important role in the regulation of heat shock gene expression in this organism.

Amino Acid Sequence↗
PubMed1992

Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene.

The dnaK gene region of Clostridium acetobutylicum was cloned in Escherichia coli by using the pBluescript SK+ and pUC18 vectors. By using the E. coli dnaK gene as a probe and by in vivo chromosome walking, three positive clones harboring the recombinant plasmids pKG1, pKG2, and pKG3 containing 1.2-kbp HindIII, 3.55-kbp EcoRV, and 1.2-kbp PstI fragments of the chromosome of C. acetobutylicum, respectively, were isolated. The cloned fragments partially overlapped, and together they spanned 4,083 bp of the clostridial genome that were completely sequenced. On one strand, four open reading frames of which the last was obviously truncated were identified. The last three genes showed high homology to the grpE, dnaK, and dnaJ heat shock genes of E. coli, respectively. They were preceded by an open reading frame (orfA) without any homology to sequences available in the EMBL or GenBank data bases. Typical translational start sites could be found in front of all four genes. Northern (RNA) blot analysis revealed transcripts of this region with a maximum length of 5.0 kb. Thus, these genes are probably organized in an operon. A transcription terminator could be found between the dnaK and dnaJ genes. By primer extension analysis, a major heat-inducible transcription start site was identified 49 bases upstream of orfA. This site was preceded by a region (5'-TTGACA[17 bp]TATTTT) that exhibited high homology to the consensus promoter sequences of gram-positive bacteria as well as sigma 70-dependent E. coli. Between this promoter and the initiation codon of orfA, a hairpin-loop structure with a possible regulatory role in the expression of these genes was found. Additional heat-inducible transcription start sites were located 69 bases upstream of orfA and 87 bases upstream of grpE; the corresponding promoter regions showed less similarity to other known promoter sequences. Maximum mRNA levels of this heat shock operon were found about 15 min after a heat shock from 30 to 42 degrees C. Our results indicate that orfA codes for an unknown heat shock protein.

Amino Acid Sequence↗
PubMed1991

Cloning and analysis of the beta-galactosidase-encoding gene from Clostridium thermosulfurogenes EM1.

Clostridium thermosulfurogenes EM1 produced a thermostable (up to 70 degrees C) beta-galactosidase (beta Gal) with a pH optimum of 7 during growth on lactose. The gene (lacZ) encoding this enzyme was cloned and expressed in Escherichia coli using pUC18 as a vector. The nucleotide sequence of a 2.7-kb PstI fragment carrying the lacZ gene was determined. The open reading frame for lacZ, which encoded a protein of 716 amino acids with a calculated Mr of 83,728, was confirmed by the identity of its deduced aa sequence with the chemically determined N-terminal aa sequence of the purified beta Gal of C. thermosulfurogenes EM1. The structural gene was preceded by a possible promoter sequence, 5'-TTGTAG (-35), 5'-TAATAT (-10); and a ribosome-binding site, 5'-AGGAGG. The cloned beta Gal was found to be indistinguishable from the native enzyme. The Mr of the active beta Gal was 170,000, as determined by Superose 12HR gel filtration and gradient gel electrophoresis. This indicated that this enzyme is composed of two identical subunits. Comparison of the aa sequences of different beta Gal revealed that five large regions of similarity with the enzymes from E. coli (lacZ, ebgA), Klebsiella pneumoniae (lacZ), and Lactobacillus bulgaricus are present in the beta Gal of C. thermosulfurogenes EM1 and that the putative active site residues (Glu461 and Tyr503 in the E. coli lacZ-encoded beta Gal) are conserved (Glu389 and Tyr429). Therefore, the thermostable beta Gal of C. thermosulfurogenes EM1 is more closely related to the enzyme of E. coli than to the likewise thermostable one of Bacillus stearothermophilus.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence of two Clostridium thermosulfurogenes EM1 genes homologous to Escherichia coli genes encoding integral membrane components of binding protein-dependent transport systems.

The complete nucleotide sequence of two genes from Clostridium thermosulfurogenes EM1 homologous to E. coli genes encoding transport proteins was determined by the dideoxy procedure. The genes were cloned from plasmid pCT4, which contains the alpha-amylase gene from C. thermosulfurogenes EM1 as a 2.9-kbp XbaI fragment, inserted into the XbaI site of pUC18, to yield plasmid pCT401. The proteins encoded by the two identified complete ORFs are very hydrophobic and thus are probably integral membrane proteins. They show over 50% similarity to the maltose transport proteins MalF and MalG and to the glycerol-3-phosphate uptake proteins UgpA and UgpE of Escherichia coli. Since these genes are located immediately upstream of the alpha-amylase gene (amyA) of C. thermosulfurogenes EM1, the encoded proteins might be involved in transport of starch degradation products. The genes were tentatively designated amyC and amyD.

ATP-Binding Cassette Transporters↗
PubMed1991

alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases.

The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of the DNA-dependent RNA polymerase from Clostridium acetobutylicum.

The DNA-dependent RNA polymerase (EC 2.7.7.6) from Clostridium acetobutylicum DSM 1731 has been purified to homogeneity and characterized. The purified enzyme was composed of four subunits and had a molecular mass of 370,000 Da. Western immunoblot analysis with polyclonal antibodies against the sigma 70 subunit of Escherichia coli RNA polymerase identified the 46,000-Da subunit as an immunologically and probably functionally related protein. The other three subunits of 128,000, 117,000, and 42,000 Da are tentatively analogous to the beta, beta', and alpha subunits, respectively, of other eubacterial RNA polymerases. The RNA polymerase activity was completely dependent on Mg2+, nucleoside triphosphates, and a DNA template. The presence of Mg2+ or Mn2+ in buffers used for purification or storage caused irreversible inactivation of the RNA polymerase.

Blotting, Western↗
PubMed1989

Cloning and expression of the thermostable alpha-amylase gene from Clostridium thermosulfurogenes (DSM 3896) in Escherichia coli.

The gene coding for a thermostable alpha-amylase from Clostridium thermosulfurogenes (DSM 3896) was cloned in Escherichia coli using pUC18 as a vector. The recombinant plasmid pCT2 of an amylolytic positive transformant of E. coli contained a 2.9 kbp fragment of chromosomal DNA of C. thermosulfurogenes carrying the alpha-amylase gene. In E. coli the gene was apparently transcribed by its own promoter. Comparative studies showed no difference between the original and the heterologously in E. coli expressed enzyme. The latter was not secreted into the medium.

Cloning, Molecular↗
PubMed1987

Induction of the heat shock response of E. coli through stabilization of sigma 32 by the phage lambda cIII protein.

The cIII protein of phage lambda favors the lysogenic response to infection by inhibiting the degradation of the lambda cII protein, which exerts the primary control on the developmental decision for lysis or lysogeny. To study the mechanism and scope of cIII-mediated regulation, we have used plasmid systems to examine the specific effect of cIII overproduction on the growth of Escherichia coli and the synthesis of bacterial proteins. We have found that maximal production of cIII prolongs the heat-induced synthesis of E. coli heat shock proteins and provokes elevated production of heat shock proteins even at low temperature. The overproduction of heat shock proteins is correlated with a rapid inhibition of cell growth, as judged by measurements of optical density. We suggest that an overactive heat shock response inhibits bacterial growth, either because excessive production of one or more of the proteins is highly deleterious or because only heat shock promoters are transcribed efficiently. To examine the effect of cIII on sigma 32, the specificity factor for the heat shock response, we have studied the stability of sigma 32 in cells carrying both cIII- and sigma 32-producing plasmids; the half-life of sigma 32 is increased fourfold in the presence of cIII. We conclude that overproduction of cIII provokes the heat shock response by increasing the steady-state level of active sigma 32. These studies also support the concept that the rate of expression of heat shock proteins is directly correlated with the amount of active sigma 32 and that regulation of the stability of sigma 32 may be an important factor for control of the heat shock response.

Bacteriophage lambda↗
PubMed1987

Changes in Photosynthetic Capacity and Photosynthetic Protein Pattern during Tomato Fruit Ripening.

Levels of polypeptides participating in the photosynthetic light and dark reactions have been measured during fruit ripening in tomato. Photosynthetic proteins were identified by Western blot analysis with heterologous antibodies. The concentrations of proteins of photosystem (PS) I (14 kilodaltons), of PSII (47-kilodalton reaction center protein, 32-kilodalton ;Q(B) binding' protein and light harvesting complex proteins), of the photosynthetic electron transport chain (ferredoxin-NADP-oxidoreductase and plastocyanin), and of the stroma (ribulose-1,5-bisphosphate carboxylase) decrease during the ripening process. The 32-kilodalton protein and plastocyanin were detectable in pericarp protein preparations of ripe tomato fruits. Absorbance difference spectrophotometry provided information on the relative concentrations of PSII and PSI reaction centers in leaf and green fruit tissue of tomato. These results indicate that green fruit pericarp of tomato is photosynthetically active. Photosynthetic activity decreases during chloroplast/chromoplast differentiation. This is consistent with changes that occur at the transcript level of photosynthesis-specific proteins during the differentiation process.

Journal Article↗
PubMed1986

Nutritional Factors Affecting the Ratio of Solvents Produced by Clostridium acetobutylicum.

Fermentation of whey by Clostridium acetobutylicum yielded butanol and acetone in a ratio of approximately 100:1. This ratio amounted to only 2:1 in synthetic media with glucose, lactose, or glucose plus galactose as substrates. Removal of citrate from whey and addition of minerals resulted in an increase in the amount of acetone produced. Experiments carried out in a chemostat with a low-phosphate synthetic medium revealed that the butanol/acetone ratio could be increased from 2:1 to 3.8:1 by cofermentation of l-lactate and from 2:1 to 8:1 by iron limitation. The performance of the fermentation in a low-iron glucose medium above pH 5.1 yielded l-lactate as the main product.

Journal Article↗