Biomedical subjects

B Henrich

Publications and source records attributed to B Henrich.

At least 37 records · Page 2Linked to original sources

PubMed1996

Characterization of pepR1, a gene coding for a potential transcriptional regulator of Lactobacillus delbrueckii subsp. lactis DSM7290.

The gene designated pepR1, encoding a potential transcription regulator of Lactobacillus delbrueckii subsp. lactis DSM7290, was identified by sequence similarity of an open reading frame located upstream of the prolidase pepQ orientated in opposite direction. pepQ and pepR1 coding regions are spaced by 152 nucleotides. Upstream of the -35 region of pepQ, a 14-bp palindromic sequence, homologous to the catabolite responsive element, could be identified. The pepRl gene has the potential to encode a protein of 333 amino acids with a calculated molecular mass of 36955 Da and a calculated pl of 5.5. The deduced protein sequence shows significant identity to the catabolite control protein of Bacillus. Co-expression in Escherichia coli was studied with the pepR1-pepQ intergenic region fused to the promoterless beta-galactosidase reporter gene. The pepQ-beta-galactosidase hybrid displayed an enhanced expression in the presence of cloned pepR1.

Amino Acid Sequence↗

PubMed1996

Biovar-specific epitopes of the urease enzyme of Ureaplasma urealyticum.

The importance of Ureaplasma urealyticum as a pathogen in premature neonates and patients with a profound defect in humoral immunity has, over the last few years, become well recognised. U. urealyticum is unique amongst the Mycoplasmataceae for its use of urea metabolism as an essential source of energy. The urease enzyme responsible for this is, therefore, of prime importance and any variability in expression of this enzyme may play a role in virulence of the organism. U. urealyticum is divided into 14 serovars comprising two biovars -- the parvo-biovar and T960-biovar. In this study monoclonal antibodies (MAbs) were produced against the urease enzyme. Two distinct epitopes of the 72-kDa alpha-subunit were recognised by three different MAbs. Under denaturing conditions both epitopes were shown to be specific for the parvo-biovar.

Amino Acid Sequence↗

PubMed1996

Repetitive elements of the Mycoplasma hominis adhesin p50 can be differentiated by monoclonal antibodies.

The gene encoding p50, an adhesin of Mycoplasma hominis, was identified, cloned, and sequenced. Comparison of the derived amino acid sequence with the N-terminal amino acids sequenced by the Edman reaction of the native protein revealed that p50 is expressed as a 467-amino-acid precursor. Posttranslational modification leads to a 441-amino-acid lipoprotein with an extended, predominantly helical structure and a leucine zipper. Computer analysis of the amino acid sequence identified a threefold-repetitive sequence motif comprising approximately three-quarters of the total protein. Different regions of the p50 polypeptide chain were expressed in Escherichia coli. Western blot (immunoblot) analysis of the E. coli lysates revealed that the epitopes of four p50-specific monoclonal antibodies were localized in the middle and C-terminal part of the protein. Epitope mapping by exonuclease III digestion showed that all of the four monoclonal antibodies bound within the same region of the threefold-repetitive amino acid sequence motif. The repeats, which were highly homologous but not identical in structure, could be differentiated by the monoclonal antibodies.

Adhesins, Bacterial↗

PubMed1995

Cloning and characterization of brnQ, a gene encoding a low-affinity, branched-chain amino acid carrier in Lactobacillus delbrückii subsp. lactis DSM7290.

A gene (brnQ), encoding a carrier for branched-chain amino acids in Lactobacillus delbrückii subsp. lactis DSM7290 was cloned in the low-copy-number vector pLG339 by complementation of a transport-deficient Escherichia coli strain. The plasmid carrying the cloned gene restored growth of an E. coli strain mutated in 4 different branched-chain amino acid transport genes at low concentrations of isoleucine, and increased its sensitivity to valine. Transport assays showed that leucine, isoleucine and valine are transported by this carrier and that transport is driven by the proton motive force. Nucleotide sequence analysis revealed an open reading frame of 1338 bp encoding a hydrophobic protein of 446 amino acids with a calculated molecular mass of 47864 Daltons. The start site of brnQ transcription was determined by primer extension analysis using mRNA from Lactobacillus delbrückii subsp. lactis DSM7290. The hydropathy profile suggests the existence of at least 12 hydrophobic domains that probably form membrane-associated alpha-helices. Comparisons of the nucleotide sequence of brnQ from Lactobacillus delbrückii subsp. lactis DSM7290, the amino acid sequence of its product and the topology of the hydrophobic domains with those of the respective carrier genes and proteins of Salmonella typhimurium and Pseudomonas aeruginosa revealed extensive homology.

Amino Acid Sequence↗

PubMed1995

Cloning and DNA sequence analysis of pepQ, a prolidase gene from Lactobacillus delbrueckii subsp. lactis DSM7290 and partial characterization of its product.

From a genomic library of Lactobacillus delbrueckii subsp. lactis (DSM7290) DNA, in the low-copy-number vector pLG339, a recombinant clone was selected, which complemented a mutation in the prolidase gene (pepQ) of Escherichia coli UK173. Nucleotide sequence analysis revealed an open reading frame of 1104 nucleotides corresponding to a protein of 368 amino acids with a calculated pI of 4.64 and a molecular mass of 41,087 Da. The start site of pepQ transcription was determined by primer extension analysis with mRNA prepared from L. delbrueckii. Based on homology of the gene product to various peptidases and on the substrate specificity determined, the peptidase was designated PepQ. The influence of various protease inhibitors and cations on peptidase activity indicated that PepQ is a metalloprotease. The absence of a membrane-spanning domain and a signal peptide sequence argues for a cytoplasmic localization of the enzyme.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and DNA sequence analysis of pepL, a leucyl aminopeptidase gene from Lactobacillus delbrueckii subsp. lactis DSM7290.

A genomic library of Lactobacillus delbrueckii subsp. lactis DSM7290 DNA fragments from a Sau3A partial digestion in the low-copy-number vector pLG339, was used to screen Escherichia coli for the presence of peptidases. Using the chromogenic substrate leucine-beta-naphthylamide (Leu-NH-Nap) and E. coli strain CM89 lacking the corresponding enzyme activity in an enzymic plate assay, allowed the isolation of two peptidase genes; the newly described pepL and the recently cloned and sequenced pepN. Clones could be distinguished not only by the restriction pattern of isolated plasmids but also by the rate and intensity of their colour reaction with Leu-NH-Nap. Three out of five clones were identified to express the Lactobacillus pepN gene; the others were shown to express a second aminopeptidase gene, designated pepL. This gene, together with 200 bp upstream of the proposed AUG initiation codon, was further subcloned and sequenced. The corresponding open reading frame of 897 nucleotides is predicted to encode a protein of 299 amino acids (34,541 Da). Searching the EMBL database revealed similarity to the prolinase of Lactobacillus helveticus (45.8% identity), to the iminopeptidases of Lb. delbrueckii subsp. lactis and Lb. delbrueckii subsp. bulgaricus (25.5%), and to the Bacillus coagulans prolinase (21.5%). Minor similarities were detected for hydrolytic enzymes with serine active sites. The product encoded by the pepL gene was functional but could not be visualized on Coomassie-blue-stained polyacrylamide gels. High level expression of peptidase L in E. coli was achieved by placing the gene under the control of the T7 promoter.

Amino Acid Sequence↗

PubMed1995

Positive-selection vector with enhanced lytic potential based on a variant of phi X174 phage gene E.

A cloning vector, pUH89, allowing positive selection of recombinant Escherichia coli clones by insertional inactivation of the modified lysis gene E of bacteriophage phi X174, was developed. Ten unique cloning sites were introduced into gene E by site-directed mutagenesis. To achieve efficient expression of the mutagenized gene, the combined lac and tac promoters were used. Additional restriction sites in the flanking sequences allow screening for transcription terminators and the excision of several cartridges suitable for vector construction.

Amino Acid Sequence↗

PubMed1995

Structure and expression of the gene (HNRPA2B1) encoding the human hnRNP protein A2/B1.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a major nuclear protein and one of the major components of the hnRNP core complex in mammalian cells. We first determined the complete sequence of the human gene for hnRNP protein A2 (HNRPA2B1). The human HNRPA2B1 gene exists in a single copy over 9 kb in length. The gene was split into 12 exons, including a 36-nucleotide mini-exon, which was specific to the hnRNP protein B1, providing genetic evidence that the B1 mRNA was generated from the primary HNRPA2B1 transcript by alternative splicing. The 5' region of HNRPA2B1 was GC-rich and contained several DNA motifs for the binding of several transcription factors, which included 2 CCAAT boxes and no TATA sequences. The 5' ends of the mRNA were mapped to multiple positions. These structural features are characteristic of promoter regions of housekeeping genes. Northern blot and RT-PCR analyses of the HNRPA2B1 transcripts revealed levels of B1 mRNA from 2 to 5% of total A2/B1 transcripts and showed that both A2 and B1 mRNAs were transcribed in all human cell lines and mouse tissues studied. The structural and evolutionary characteristics of the A2 and A1 proteins as they relate to each other are discussed.

Amino Acid Sequence↗

PubMed1995

Primary structure and functional analysis of the lysis genes of Lactobacillus gasseri bacteriophage phi adh.

The lysis genes of the Lactobacillus gasseri bacteriophage phi adh were isolated by complementation of a lambda Sam mutation in Escherichia coli. Nucleotide sequencing of a 1,735-bp DNA fragment revealed two adjacent coding regions of 342 bp (hol) and 951 bp (lys) in the same reading frame which appear to belong to a common transcriptional unit. Proteins corresponding to the predicted gene products, holin (12.9 kDa) and lysin (34.7 kDa), were identified by in vitro and in vivo expression of the cloned genes. The phi adh holin is a membrane-bound protein with structural similarity to lysis proteins of other phage, known to be required for the transit of murein hydrolases through the cytoplasmic membrane. The phi adh lysin shows homology with mureinolytic enzymes encoded by the Lactobacillus bulgaricus phage mv4, the Streptococcus pneumoniae phage Cp-1, Cp-7, and Cp-9, and the Lactococcus lactis phage phi LC3. Significant homology with the N termini of known muramidases suggests that phi adh lysin acts by a similar catalytic mechanism. In E. coli, the phi adh lysin seems to be associated with the total membrane fraction, from which it can be extracted with lauryl sarcosinate. Either one of the phi adh lysis proteins provoked lysis of E. coli when expressed along with holins or lysins of phage lambda or Bacillus subtilis phage phi 29. Concomitant expression of the combined holin and lysin functions of phi adh in E. coli, however, did not result in efficient cell lysis.

Amino Acid Sequence↗

PubMed1994

Cloning and nucleotide sequence analysis of the Lactobacillus delbrueckii ssp. lactis DSM7290 cysteine aminopeptidase gene pepC.

A genomic library of Lactobacillus delbrueckii ssp. lactis DSM7290 in the low copy number vector pLG339, was screened for the presence of peptidase genes. Using the chromogenic substrate gly-ala-beta-naphthylamide, which is not a substrate for any of the recently cloned peptidases of DSM7290, and the multiple peptidase deficient Escherichia coli strain CM89, allowed the isolation of clones, which contained the equivalent hydrolytic activity. To identify genes encoding the conserved catalytic active site of cysteine proteases, partial nucleotide sequencing with a degenerate oligonucleotide was performed on recombinant plasmids isolated from such clones. This allowed to identify two out of nine clones to carry the Lactobacillus pepC gene. A total of 2026 nucleotides were determined, and sequence analysis revealed a gene with strong homology to the recently cloned Lb. helveticus (73.2%) and Lactococcus lactis (51.03%) pepC genes, and the derived protein showed homology with the active site of a large number of cysteine proteases. The predicted open reading frame consists of 449 codons, coding for a protein of 50,909 Da. The enzyme is functional and extremely overexpressed in E. coli.

Amino Acid Sequence↗

PubMed1994

Peptidase D of Escherichia coli K-12, a metallopeptidase of low substrate specificity.

Peptidase D of Escherichia coli was overproduced from a multicopy plasmid and purified to electrophoretic homogeneity. The pure enzyme was stable at 4 degrees C or -20 degrees C and had a pH optimum at pH 9, and a pI of 4.7; the temperature optimum was at 37 degrees C. As the enzyme was activated by Co2+ and Zn2+, and deactivated by metal chelators, it appears to be a metallopeptidase. By activity staining of native gels, 11 dipeptides which are preferentially cleaved by peptidase D were identified. Peptidase D activity required dipeptide substrates with an unblocked amino terminus and the amino group in the alpha or beta position. Non-protein amino acids and proline were not accepted in the C-terminal position, whereas some dipeptide amides and formyl amino acids were hydrolyzed. Km values of 2 to 5 mM indicate a relatively poor interaction of the enzyme with its substrates.

Dipeptidases↗

PubMed1994

Determination and comparison of Lactobacillus delbrueckii ssp. lactis DSM7290 promoter sequences.

The transcriptional start points of ten Lactobacillus delbrückii ssp. lactis DSM7290 genes were determined by primer extension. The upstream located promoter regions, including potential -35 and -10 regions and the spacing between them were compared to the well-known Escherichia coli and Bacillus subtilis promoters. The Lb. delbrückii -35 consensus sequence (TTGACA) seems to be less conserved then the E. coli sequence. The nucleotides TGC were often found upstream of the -10 region (TATAAT). The most frequently observed spacing between the two core promoter regions was 17 nt and the main distance between the -10 region and the transcriptional start point was mostly determined to be 6 nt in contrast to 7 nt, as described for E. coli promoters. The preferred initiation nucleotides in Lb. delbrückii were shown to be definitely purines (A or G). The ribosome binding sites located downstream of the promoters revealed the consensus sequence 3'-UCCUCCU-5', being the predicted 3'-OH end of the Lactobacillus 16S rRNA with a high degree of homology to known 16S rRNAs.

Base Sequence↗

PubMed1993

Cytoadhesins of Mycoplasma hominis.

The mechanism of Mycoplasma hominis adherence to host cells of the urogenital tract was investigated with monoclonal antibodies (MAbs) directed against antigenic surface-localized polypeptides P50, P60, P80, and P100 of cytoadherent M. hominis FBG. A cell enzyme-linked immunosorbent assay was established allowing quantification of cytoadherent mycoplasmas detected by one of the following MAbs: four MAbs directed against P100 (molecular weight, about 100,000), three MAbs against P80, one MAb against P60, and three MAbs against P50. MAb binding to one of the surface proteins resulted in a decrease of mycoplasmal adherence to HeLa cells. To exclude the thesis that this is caused by nonspecific blocking of adherence, P100 and P50 were purified by affinity chromatography and tested instead of intact mycoplasmas in the cell enzyme-linked immunosorbent assay for cytoadherence. Both proteins bound to the surface of the eukaryotic cells. MAb binding to single epitopes of these proteins resulted in inhibition of protein adherence. These experiments strongly suggest that of the four surface-localized proteins at least P100 and P50 are adhesins of M. hominis FBG.

Animals↗

PubMed1993

dcp gene of Escherichia coli: cloning, sequencing, transcript mapping, and characterization of the gene product.

Dipeptidyl carboxypeptidase is a C-terminal exopeptidase of Escherichia coli. We have isolated the respective gene, dcp, from a low-copy-number plasmid library by its ability to complement a dcp mutation preventing the utilization of the unique substrate N-benzoyl-L-glycyl-L-histidyl-L-leucine. Sequence analysis of a 2.9-kb DNA fragment revealed an open reading frame of 2,043 nucleotides which was assigned to the dcp gene by N-terminal amino acid sequencing and electrophoretic molecular mass determination of the purified dcp product. Transcript mapping by primer extension and S1 protection experiments verified the physiological significance of potential initiation and termination signals for dcp transcription and allowed the identification of a single species of monocistronic dcp mRNA. The codon usage pattern and the effects of elevated gene copy number indicated a relatively low level of dcp expression. The predicted amino acid sequence of dipeptidyl carboxypeptidase, containing a potential zinc-binding site, is highly homologous (78.8%) to the corresponding enzyme from Salmonella typhimurium. It also displays significant homology to the products of the S. typhimurium opdA and the E. coli prlC genes and to some metalloproteases from rats and Saccharomyces cerevisiae. No potential export signals could be inferred from the amino acid sequence. Dipeptidyl carboxypeptidase was enriched 80-fold from crude extracts of E. coli and used to investigate some of its biochemical and biophysical properties.

Amino Acid Sequence↗

PubMed1992

Species differentiation of mycoplasmas by EF-Tu specific monoclonal antibodies.

Ten mouse hybridoma cell lines producing IgG monoclonal antibodies to mycoplasmal elongation factor Tu (EF-Tu) were established. These mAbs showed different degrees of cross-reactivity between mollicutes and even other bacteria. This finding, indicating protein structure diversities of pan bacterial EF-Tu should permit species differentiation of mycoplasmas by epitope pattern analysis of a single protein. Epitope patterns of 23 mollicute type strains and of 40 M. hominis isolates were determined by ELISA. All M. hominis patterns were found to be closely related whereas intrageneric patterns differed in a species specific manner.

Amino Acid Sequence↗

PubMed1992

The promoter region of the Escherichia coli pepD gene: deletion analysis and control by phosphate concentration.

A series of deletions removing progressively larger parts of the 5' flanking region of the Escherichia coli pepD gene was constructed. After fusing the resulting promoter fragments to the chromosomal malPQ operon, their activities were determined by assaying for amylomaltase, the product of the malQ gene. Transcription from the pepD promoter region in exponentially growing cells was estimated to be about 5 times less efficient than transcription from the induced lac promoter. Approximately 115 bp preceding the translation start site of the pepD gene are important for regular promoter functioning, whereas the more distal sequences could be deleted without any significant effects. In bacterial cultures containing limiting amounts of inorganic phosphate, the rate of de novo synthesis of peptidase D, simultaneously with the derepression of alkaline phosphatase, increased about fivefold as a consequence of phosphate starvation. This regulation was shown to occur at the transcriptional level by the use of chromosomal pepD promoter-malPQ fusions. The inducibility by phosphate limitation was conserved in all of the deletion clones in which the pepD promoter region was still functional. As demonstrated by the use of phoB, R, and M mutants, the modulation of pepD expression is independent of the genetic system controlling the pho regulon.

Base Sequence↗

PubMed1992

Decreased metabolism and viability of Mycoplasma hominis induced by monoclonal antibody-mediated agglutination.

Monoclonal antibodies (MAbs) were generated against lysates of clinical Mycoplasma hominis isolates. Three of these, designated BG2, BA10, and FE6, recognized an integral membrane protein of M. hominis with an apparent molecular weight of 50,000 (p50). Electron microscopy studies demonstrated that this protein is distributed evenly over the cell surface. These anti-p50 MAbs were species specific for M. hominis; they reacted with 42% of 126 tested clinical M. hominis isolates and showed no reactivity to heterologous mycoplasma species. Immunoblot analysis after limited proteolysis of purified p50 demonstrated that the three MAbs reacted with different epitopes of the protein. Unlike BA10 and FE6, MAb BG2 induced a decrease in arginine metabolism and a reduction of CFU in metabolic inhibition tests. F(ab)2 fragments of MAb BG2 showed the same inhibitory effect as the intact MAb molecule, while Fab and Fc fragments had no influence on vital functions. Preincubation of the mycoplasmas with MAb BG2 followed by trypsin treatment yielded the same amount of CFU as the control without antibodies. In conclusion, the cell aggregates were resolved by the trypsin treatment. These experiments and tests with the antibody fragments led to the conclusion that only the intact MAb structure or the F(ab)2 structure had metabolic inhibition potential and that the observed metabolism inhibition as well as the apparent decrease in viability were a result of agglutination by MAb BG2.

Agglutination↗

PubMed1991

Molecular analysis and nucleotide sequence of the envCD operon of Escherichia coli.

The chromosomal DNA insert in plasmid pJK131, which complements the phenotypic defects associated with a mutation in the envC gene of Escherichia coli strain PM61, was sequenced. The analysis of the nucleotide sequence revealed two open reading frames (ORFs) coding for the proteins EnvC (41,281 daltons) and EnvD (104,415 daltons). The envC gene product is synthesized as a pre-protein and, after cleavage of a signal peptide, the mature protein is incorporated into the cytoplasmic membrane. The detection of a common transcript for both ORFs indicated the existence of an envCD operon. Deletion analysis and the generation of frameshifts demonstrated that simultaneous expression of both genes is required to complement the defects in strain PM61. Overproduction of EnvC protein appears to be lethal to Escherichia coli. The envD gene, however, could be cloned and expressed at high levels under control of the tac promoter without deleterious effects on the host.

Amino Acid Sequence↗