Hans Matzura

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.

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.

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.

In vivo synthesis of a polycistronic messenger RNA for the ribosomal proteins L11, L1, L10 and L7/12 in Escherichia coli

SummarySucrose density gradient centrifugation and DNA/RNA hybridization have been used to analyse the mRNA synthesized from the ribosomal protein—RNA polymerase subunits gene cluster rplKAJL-rpoBC in Escherichia coli. DNA/RNA hybrids obtained from total E. coli RNA and specific DNA restriction fragments from this chromosomal area were further subjected to endonuclease S1 digestion. This analysis permits the mapping of the ends of mRNA molecules for specific genes or operons by sizing the S1 resistant hybrids.Our results show that the predominant mRNA synthesized under conditions of balanced growth from the rplKAJL-rpoBC region codes for the four ribosomal proteins L11, L1, L10 and L7/12. This tetracistronic mRNA puts the transcription of the following rpoBC genes under the main control of the L11 promoter. Smaller distinct mRNA species could also be detected by this technique. They originate from intercistronic transcription termination and re-initiation as well as from processing of the larger polycistronic mRNA.

Binding of RNA polymerase and the catabolite gene activator protein within the cat promoter in Escherichia coli

Abstract The binding of RNA polymerase (nucleoside triphosphate: RNA nucleotidyl-transferase, EC 2.7.7.6) to the catabolite-sensitive cat promoter in Escherichia coli has been analysed in the absence and presence of the cAMP/catabolite gene activator protein complex. In its absence, weak binding was observed in both filter binding and DNAase protection experiments. Enhanced binding was achieved in its presence, and was shown to be most prominent within the Pribnow box. The region of the cat gene to which RNA polymerase was expected to bind has also been determined by elucidation of the nucleotide from which transcription starts. The resulting disposition of RNA polymerase and catabolite gene activator protein within the cat promoter could allow direct contact between the two proteins as a prerequisite for transcriptional activation.

Localisation of the transcription initiation site of the chloramphenicol resistance gene on plasmid pAC184

In Escherichia coli, R-factor carried resistance to chloramphenicol is exerted by acetylation of the antibiotic by the enzyme chloramphenicol acetyl transferase [l]. An unusual feature of the cat gene is its catabolite sensitivity, a feature more commonly associated with lac, gal and other sugar operons [2,3]. Catabolite sensitivity of the cat gene has been demonstrated both in vivo and in vitro [4]: however, since then there has been little biochemical or genetic analysis on such regulation of the cat operon. Here we have studied transcription of the cat gene of plasmid pAC184 151. These studies were suggested by and subsequently compared to nucleotide sequence studies of an analogous cat gene as well as electron microscopic analysis of transcription of pACl84, both carried out by independent research [6] (D. Stiiber and H. Bujard, personal communication). Our experiments have demonstrated that the predominant RNA species produced from a 1.5 kb HindIII/Eco RI restriction fragment of pAC184 is -270 nucleotides long. When this fragment is transcribed in presence of CAMP and CRP, the rate of synthesis of the 270 nucleotide transcript is markedly enhanced, an effect even more noticeable when Eco RI-cut, linearised pAC184 is used as template. The results of our transcription studies and a restriction analysis of the appropiate part of pACl84

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

SummaryThe 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.

Regulation of Biosynthesis of the DNA-Dependent RNA Polymerase in Escherichia coli

Publisher Summary DNA-dependent RNA polymerase is an enzyme that is vital for any living cell. It catalyzes the transcription of DNA into RNA, using the four 5’-nucleoside triphosphates—adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP) as substrates. In Escherichia coli ( E. c oli ), only one RNA polymerase synthesizes all RNA species—rRNA, tRNA, and mRNA, as well as the RNA primers for DNA chain initiation during replication. The complex composition of the RNA polymerase reflects the manifold control mechanisms that act on the enzyme during transcription rather than the complexity of the transcription process. E . coli RNA polymerase must transcribe a number of different reading units. It would interact with additional factors and effectors, would be subjected to structural modification, and would have to recognize modulation signals on the template. This chapter discusses the progress in the study of the regulation mechanisms that control the synthesis of the different RNA polymerase subunits and their assembly into the active enzyme under various conditions of bacterial growth. It presents the work regarding the localization of the genes for these subunits on the E . coli chromosome.

Transformation of Escherichia coli by a specific DNA restriction fragment

SummarySpecific 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 β 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.