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H Matzura

Publications and source records attributed to H Matzura.

At least 19 recordsLinked to original sources
PubMed1991

Nucleotide sequence analysis of a chloramphenicol-resistance determinant from Agrobacterium tumefaciens and identification of its gene product.

The nucleotide sequence of a chloramphenicol-resistance (CmR) determinant from the Gram- soil bacterium Agrobacterium tumefaciens was determined, and its gene product was identified as Cm acetyltransferase (CAT). Comparison of the amino acid sequences of the A. tumefaciens CAT and various CAT proteins of Gram+ and Gram- origin shows no homology between this and the other enzymes.

Amino Acid Sequence↗
PubMed1991

Chloramphenicol-induced stabilization of cat messenger RNA in Bacillus subtilis.

The expression of the chloramphenicol-inducible chloramphenicol-acetyltransferase gene (cat), encoded on Staphylococcus aureus plasmid pUB112, is regulated via a translational attenuation mechanism. Ribosomes, which are arrested by chloramphenicol during synthesis of a short leader peptide, activate catmRNA translation by opening a 5'-located stem-loop structure, thus setting free the cat ribosome-binding site. We have determined the 5' and 3' ends of catmRNA and analysed its stability in Bacillus subtilis. In the absence of the antibiotic, the half-life of catmRNA is shorter than 0.5 min; it is enhanced to about 8 min by sub-inhibitory concentrations of the drug. No decay intermediates of catmRNA could be detected, indicating a very fast degradation after an initial rate-limiting step. ochre nonsense mutations in the 5' region of the cat structural gene, which eliminate catmRNA translation, did not affect its chloramphenicol-induced stabilization. Mutations in the leader-peptide coding region, which abolish ribosome stalling and, therefore, cat gene induction, also eliminate catmRNA stabilization. We conclude that catmRNA is stabilized on induction by a chloramphenicol-arrested ribosome, which physically protects a nuclease-sensitive target site in the 5' region of catmRNA against exo- or endonucleolytic initiation of degradation. This protection is analogous to ermA and ermC mRNA and seems to reflect a general mechanism for stabilization of mRNA derived from inducible antibiotic resistance genes in B. subtilis.

Bacillus subtilis↗
PubMed1990

Chloramphenicol-induced translational activation of cat messenger RNA in vitro.

The expression of the chloramphenicol-inducible chloramphenicol acetyltransferase gene (cat) of the staphylococcal plasmid pUB112 is regulated at the post-transcriptional level. Previous in vivo analyses suggested that the antibiotic stalls ribosomes that are translating a regulatory leader peptide, and that a stalled ribosome activates the ribosome binding site of the acetyltransferase encoding sequence by opening an attenuating leader mRNA hairpin structure. To test this model, we used a Bacillus subtilis S-30 extract for an in vitro translation system and in vitro synthesized cat in RNAs. We showed that the leader portion of the cat transcript acts as a translational attenuator of cat gene expression in absence of chloramphenicol. The drug stimulates acetyltransferase synthesis by a leader mRNA-dependent activation of translation of the cat message. By using 5' end-labeled transcripts and employing the endogenous RNase activity of the S-30 extract we demonstrated that this activation is due to an antibiotic-induced stalling of a ribosome on cat leader mRNA.

Bacillus subtilis↗
PubMed1989

Nucleotide sequence analysis and expression studies of a chloramphenicol-acetyltransferase-coding gene from Clostridium perfringens.

The nucleotide sequence of a CmR determinant, located on the Clostridium perfringens plasmid pIP401, was determined and its gene product was identified as chloramphenicol acetyltransferase (CAT). The cat structural gene is preceded by transcription-initiation signals characteristic for Escherichia coli sigma 70 or Bacillus subtilis sigma 43 promoters. By promoter probing in the heterologous hosts the direction of transcription of the clostridial cat gene was analysed and the cat mRNA start point was determined in vitro using the RNA polymerases of E. coli and B. subtilis. Comparison of the amino acid sequences of C. perfringens CAT and other CAT proteins of Gram-positive and Gram-negative origin shows a remarkable degree of homology between the various enzymes.

Amino Acid Sequence↗
PubMed1988

Replication control of the Staphylococcus aureus chloramphenicol resistance plasmids pC223 and pUB112 in Bacillus subtilis.

A detailed physical and functional map of the chloramphenicol (Cm) resistance plasmid pC223 from Staphylococcus aureus was compiled. The plasmid's basic replicon and origin of replication were located and their nucleotide sequences determined. Two small RNAs of 92 and 155 nt, demonstrated by in vitro transcription with vegetative Bacillus subtilis RNA polymerase, were depicted as copy number regulating (cop) and incompatibility (inc) functions in Bacillus subtilis. pC223 and pUB112, another S. aureus Cm resistance plasmid, which exhibits marked sequence homology with pC223 and codes also for two small copRNAs, could be classified as members of the pT181-plasmid family (1). Copy numbers and segregational instability of pC223, pUB112 and deletion derivatives of both in B. subtilis showed great differences despite of their homologous basic replicons.

Amino Acid Sequence↗
PubMed1988

Positioning ribosomes on leader mRNA for translational activation of the message of an inducible Staphylococcus aureus cat gene.

The expression of the chloramphenicol (Cm) - inducible Cm acetyltransferase gene (cat) of the staphylococcal plasmid pUB112 is regulated at the translational level. The leader mRNA preceding the cat coding sequence can form a stable hairpin structure, in which the cat Shine-Dalgarno sequence is masked. Previous work showed that translation of a short leader peptide terminating within the stem of the inhibitory secondary structure is required for basal Cm acetyltransferase (CAT) synthesis and its inducibility. In the present study we shortened this leader peptide by introducing ochre codons in its coding sequence and found that synthesis of the N-terminal part of the leader peptide, terminating directly 5' to the stem, is sufficient to mediate basal and inducible CAT synthesis. Amino acid substitution in this region of the leader peptide abolished inducibility. We suggest that the 5' region of the leader peptide coding sequence specifies a particularly Cm-sensitive translation that represents the Cm-sensor mechanism for cat gene induction.

Amino Acid Sequence↗
PubMed1987

Dependence of expression of an inducible Staphylococcus aureus cat gene on the translation of its leader sequence.

The gene for chloramphenicol (Cm) acetyltransferase (CAT) carried by the staphylococcal plasmid pUB112, whose expression can be stimulated by Cm, is preceded by a regulatory region containing two control elements. One of these consists of a Shine-Dalgarno (SD) sequence followed by an open reading frame coding for a leader peptide of nine amino acids. Previous work has shown that the SD sequence is essential for inducibility of Cm resistance by the antibiotic (Brückner and Matzura 1985). Here we demonstrate that fusion of the leader peptide coding sequence to a truncated 'lacZ gene results in synthesis of a leader peptide-beta-galactosidase fusion protein. Introduction of an ochre nonsense codon into the reading frame of the leader peptide sequence leads to considerable reduction of the basal expression and loss of inducibility of the cat gene. These results reveal that synthesis of the leader peptide is required for the basal and inducible expression of the cat gene and support the model of translational attenuation for its regulation.

Acetyltransferases↗
PubMed1986

Characterization of signals promoting gene expression on the Staphylococcus aureus plasmid pUB110 and development of a gram-positive expression vector system.

The transcriptional and translational initiation signals of a portion of the Staphylococcus aureus plasmid pUB110 were analyzed. An Mbo I-Pvu II fragment was sequenced and the site of transcriptional initiation was determined by in vitro mapping. To convert the plasmid into a cloning vector, a multilinker was introduced in different positions relative to a detected reading frame. The Gram-negative beta-galactosidase gene and the Gram-positive chloramphenicol acetyltransferase (cat) gene were fused and the level of expression was determined in Bacillus subtilis. Hybrid proteins consisting of corresponding CAT polypeptides were produced in each translational reading frame. Therefore this vector system can be used to express cloned DNA in the Gram-positive host Bacillus subtilis. Furthermore a derived lacZ fusion plasmid may be used for rapid screening of inserts.

Acetyltransferases↗
PubMed1985

Regulation of the inducible chloramphenicol acetyltransferase gene of the Staphylococcus aureus plasmid pUB112.

Analyses of deletion mutants of the gene for chloramphenicol (Cm) acetyltransferase (CAT) carried by the staphylococcal plasmid pUB112 revealed a regulatory region, which is indispensable for Cm-inducible cat gene expression, located 70 bp in front of the CAT-coding sequence. This region consists of a possible ribosome binding site followed by an open reading frame coding for a peptide of nine amino acids and overlaps partially with an inverted repeat capable of forming a stem-loop structure. Deletion of the ribosome binding site and of parts of the open reading frame abolishes inducibility and results in a low-level cat gene expression, if the inverted repeat remains intact. Deletion of the 5' part of the possible stem leads to high-level constitutive CAT synthesis. The inverted repeat, therefore, exhibits negative control on cat gene expression whereas the preceding ribosome binding site is needed to enhance CAT synthesis in the presence of an inducer. These results suggest that translation of a leader peptide is a prerequisite for Cm-induced cat gene expression and that ribosome stalling on cat leader mRNA caused by Cm opens the stem-loop structure thereby releasing its negative effect on CAT synthesis.

Acetyltransferases↗
PubMed1984

Expression of a chloramphenicol-resistance determinant carried on hybrid plasmids in gram-positive and gram-negative bacteria.

To analyse the control of chloramphenicol (Cm) resistance conferred by the Staphylococcus aureus plasmid pUB112, a detailed restriction map of this plasmid has been constructed, and the position and orientation of the cat gene have been determined. An MboI restriction fragment carrying the entire cat gene of pUB112 was then cloned in another S. aureus plasmid, the kanamycin (Km) resistance vector pUB110. Depending on the orientation of the incorporated cat fragment, the level of Cm resistance varied dramatically in Bacillus subtilis cells. This effect could not be eliminated by deleting parts of the vector DNA, and only the introduction of a transcription termination signal led to orientation-independent Cm resistance. One such construct was further developed to yield a shuttle vector, replicating both in Escherichia coli and B. subtilis. Using this vector the expression of incorporated genes can be determined in both Gram-positive and Gram-negative bacteria. By in vitro transcription experiments using pUB110 DNA linearized with various restriction endonucleases as template, two pUB110 promoters could be localized and their orientations determined: one promoter controls a gene whose function is unknown, the other regulates the transcription of the KmR gene.

Acetyltransferases↗
PubMed1977

Transformation of Escherichia coli by a specific DNA restriction fragment.

Specific transformation of a rifampicin sensitive strain of Escherichia coli to rifampicin resistance has been performed by a single, defined DNA restriction fragment carrying the genetic information for the beta subunit of E. coli RNA polymerase. In this transformation the transforming genetic character has been substituted for the corresponding recipient gene locus by recombination. The value of the described transformation system for locating genetic markers on DNA restriction fragments is discussed in comparison to previously reported in vitro systems.

Coliphages↗
PubMed1976

The polypeptide chain growth rate in amino acid-starved Escherichia coli determined by a novel method.

The proteins synthesized by arginine-requiring Escherichia coli during growth or arginine starvation were characterized by polyacrylamide gel electrophoresis in sodium dodecyl sulfate to give size distributions. The proteins made during amino acid starvation were smaller than those made by growing cells. This was true for otherwise isogenic rel- ("relaxed") and rel+ ("stringent") bacteria. Also using electrophoretic profiles, the peptide chain growth rate was estimated by a novel method based on comparison of theoretically predicted and observed kinetics of pulse labeling protein chains of different sizes. During arginine starvation, the rate was 2--5 amino acids/s for both rel- and rel+ cells, compared to 20 amino acids/s for growing cells. The results rule out chain growth-rate differences as an aspect of the "relaxed" phenomenon.

Amino Acids↗