Paul S. Lovett

Chloramphenicol-inducible gene expression in Bacillus subtilis

A cloned Bacillus pumilus cat gene expresses chloramphenicol-inducible chloramphenicol acetyltransferase activity in Bacillus subtilis. The chloramphenicol inducibility trait was shown to be determined by a 234-bp region of the cloned DNA. Nucleotide sequence analysis of this 234-bp segment indicated that the cat ribosome-binding site occurs within a 40-bp region containing 14-bp terminal inverted repeat sequences. Transcription of this region into RNA should sequester the cat ribosome-binding site in a stable stem-loop conformation. Chloramphenicol-mediated destabilization of the stem-loop is suggested as the basis for the chloramphenicol inducibility phenotype.

Nucleotide sequence of a Bacillus pumilus gene specifying chloramphenicol acetyltransferase.

Gene cat-86 of Bacillus pumilus, specifying chloramphenicol-inducible chloramphenicol acetyltransferase, was previously cloned in Bacillus subtilis on plasmid pUB110. The nucleotide sequence of cat-86 indicates that the gene encodes a protein of 220 amino acids and contains TTG as the translations-initiation codon. The proteins specified by cat-86 and the cat genes present on pC194, pC221 and Tn9 appear to share regions of amino acid sequence similarity. cat-86 is a structural gene on the B. subtilis expression plasmid pPL608. Restriction sites exist within the gene that should permit the product of inserted heterologous coding sequences to be synthesized in B. subtilis as fusion proteins.

Translation attenuation regulation of chloramphenicol resistance in bacteria--a review.

The chloramphenicol (Cm)-inducible cat and cmlA genes are regulated by translation attenuation, a regulatory device that modulates mRNA translation. In this form of gene regulation, translation of the CmR coding sequence is prevented by mRNA secondary structure that sequesters its ribosome-binding site (RBS). A translated leader of nine codons precedes the secondary structure, and induction results when a ribosome becomes stalled at a specific site in the leader. Here we demonstrate that the site of ribosome stalling in the leader is selected by a cis effect of the nascent leader peptide on its translating ribosome.

Enhanced expression of mouse dihydrofolate reductase in Bacillus subtilis.

pPL608-TR1 is a high-copy plasmid that permits phenotypic expression of the mouse dihydrofolate reductase (DHFR) gene in Bacillus subtilis. A plasmid mutation has been identified that increases expression of mouse DHFR more than ten-fold. The mutation is located in a 0.2-kb segment that intervenes between the DHFR gene and the 0.3-kb promoter fragment needed for transcriptional activation of DHFR. Nucleotide sequence analysis suggests that the effect of the mutation is to facilitate translation, initiated within the promoter fragment, through the 0.2-kb segment to the site of insertion of the DHFR-containing fragment. Additional promoter-containing fragments selected by their ability to promote expression of a plasmid gene located downstream from DHFR, the CAT gene, promote either high, intermediate or no phenotypic expression of DHFR. The results indicate that promoter fragments that allow phenotypic expression of the mouse DHFR gene contain two regulatory signals. One signal is essential to transcription of both DHFR and CAT and therefore functions as a promoter. The second signal may be necessary for translation of that portion of the mRNA specifying mouse DHFR.

PBP1: A flagella specific bacteriophage mediating transduction in Bacillus pumilus

Abstract Bacteriophage PBP1 was purified by differential and equilibrium centrifugation and characterized with respect to morphology, adsorption properties, and host range. PBP1 is a phage with a flexible noncontractile tail. The adsorption of PBP1 to cells of Bacillus pumilus NRRL B-3275 (BpB1) in penassay broth follows first-order kinetics with an average adsorption velocity constant at 37° of 6.1 × 10−9 ml−1 min−1. The activation energy for the adsorption reaction is approximately 24 kcal. On the basis of the following properties, it is concluded that PBP1 interacts with the bacterial flagellum during the adsorption process: (1) PBP1 does not adsorb to nonflagellated mutants derived from three phage-sensitive strains of B. pumilus. (2) Mechanical deflagellation of BpB1 renders the cells incapable of adsorbing PBP1 until the flagella have regenerated. (3) Nonflagellated mutants of B. pumilus ATCC 7065 can be isolated by selecting for resistance to PBP1. (4) Addition of a high multiplicity of PBP1 particles (>20) to motile BpB1 cells results in immobilization of the cells. (5) Electron microscopical examinations of mixtures of PBP1 and BpB1 cells demonstrate that the phage are capable of attaching to flagella. Comparison of the host ranges of PBP1 and PBS1 on thirty-two strains of B. pumilus divides the strains into three groups. One group is sensitive to PBP1 and PBS1, one group is sensitive to PBP1 but resistant to PBS1, and one group is resistant to both viruses. The possible use of PBP1 and PBS1 for distinguishing flagella types in B. pumilus strains is discussed. PBP1 mediates generalized transduction in BpB1 at a frequency on the order of 10−8 transductants per plaque-forming unit. Preliminary genetic studies indicate that contransducible markers in BpB1 are more weakly linked by PBP1 transduction than by PBS1 transduction.

CUG as a mutant start codon for cat-86 and xylE in Bacillus subtilis

The cat-86 gene specifies chloramphenicol acetyltransferase (CAT). The cat-86 start codon is UUG, although related genes have AUG as the start codon. Changing the start codon to AUG increased expression of cat-86 by 36% in Bacillus subtilis. Changing the start codon to GUG and CUG decreased expression to 65% and 30%, respectively, of the level obtained when AUG was the start codon. CUG has not been previously shown to function as a start codon in B. subtilis. N-terminal sequencing of purified CAT protein specified by the CUG mutant, revealed that CUG was indeed the start codon and specified methionine. The gene xylE, which specifies catechol 2,3-dioxygenase, has AUG as its start codon. Changing the start codon for xylE to CUG decreased expression by 98%. However, when the ribosome-binding site sequence for xylE was optimized and the spacing between it and the start codon was increased to 8 nucleotides, xylE activity increased to 13% of the activity observed for AUG. CUG did not function efficiently as a start codon for cat-86 in Escherichia coli. These data suggest conditions under which CUG can function, with modest efficiency, as a start codon in B. subtilis.

Constitutive variants of the pC194 cat gene exhibit DNA alterations in the vicinity of the ribosome binding site sequence.

The chloramphenicol-inducible regulation of the expression of cat genes from two Gram-positive bacteria, Staphylococcus aureus and Bacillus pumilus has been suggested to result from the presence of inverted repeat sequences that span the ribosome-binding site (RBS) for cat. In support of this hypothesis, we demonstrate that two derivatives of the pC194 cat gene which are constitutively expressed in Bacillus subtilis are deleted for all or a major portion of the inverted-repeat sequences.

The gene encoding glyoxalase I from Pseudomonas putida: cloning, overexpression, and sequence comparisons with human glyoxalase I

The gene encoding glyoxalase I (GlxI) from Pseudomonas putida has been cloned into the high-expression plasmid pBTacI. In the presence of IPTG, JM109 cells transformed with this vector give expression levels of GlxI 4000-fold higher than wild-type Escherichia coli. Contrary to a previous report, the nucleotide sequence of the gene encodes a 173-amino-acid polypeptide. Edman analysis indicates that the predicted N-terminal methionine is lost post-translationally to yield a 19407-Da protein. Mass spectrometry of the intact protein, and of the peptides generated from treatment with CNBr, does not indicate any additional post-translational modifications of the enzyme. Contrary to previous conclusions, there are no major regions of dissimilarity between the human and bacterial enzymes.

Nicotiana chloroplast genome : 7. Expression in E. coli and B. subtilis of tobacco and Chlamydomonas chloroplast DNA sequences coding for the large subunit of RuBP carboxylase.

SummaryRuBPCase, the enzyme responsible for carboxylation and oxidation of RuBP in a wide variety of photosynthetic organisms, is the major protein found in the chloroplast. Here we present the first evidence for direct expression in E. coli and B. subtilis of tobacco and Chlamydomonas ct-DNA sequences coding for the LS of RuBPCase as demonstrated by a simple in situ immunoassay.

The cis‐effect of a nascent peptide on its translating ribosome: influence of the cat‐86 leader pentapeptide on translation termination at leader codon 6

Inducible cat genes from Gram‐positive bacteria are regulated by translation attenuation. The inducer chloramphenicol stalls a ribosome at a specific site in the leader of cat transcripts; this destabillzes a downstream stem‐loop structure that normally sequesters the ribosome‐binding site for the cat structural gene. The five‐amino‐acid peptide MVKTD that is synthesized when a ribosome has translated to the leader induction site is an inhibitor of peptidyl transferase In vitro. Thus, the peptide may be the in vivo determinant of the site of ribosome stalling. Here we provide evidence that the leader pentapeptide can exert a cis‐effect on its translating ribosome In vivo. Converting leader codon 6 to the ochre codon results in expression of cat‐86 in the absence of Inducer. We term this autoinduction. Autoinduction is abolished by mutations that change the amino‐acid sequence of the leader peptide but have no, or little, effect on the sequence of nucleotides at the leader stall site. In contrast, four nucleotide changes within the leader site occupied by the stalled ribosome that result in synonymous codon replacements do not diminish autoinduction. Our evidence indicates that the cat‐86 leader pentapeptide can alter the function of its translating ribosome.