Cm Acetyltransferase Research Articles

Collective Resistance in Microbial Communities by Intracellular Antibiotic Deactivation.

The structure and composition of bacterial communities can compromise antibiotic efficacy. For example, the secretion of β-lactamase by individual bacteria provides passive resistance for all residents within a polymicrobial environment. Here, we uncover that collective resistance can also develop via intracellular antibiotic deactivation. Real-time luminescence measurements and single-cell analysis demonstrate that the opportunistic human pathogen Streptococcus pneumoniae grows in medium supplemented with chloramphenicol (Cm) when resistant bacteria expressing Cm acetyltransferase (CAT) are present. We show that CAT processes Cm intracellularly but not extracellularly. In a mouse pneumonia model, more susceptible pneumococci survive Cm treatment when coinfected with a CAT-expressing strain. Mathematical modeling predicts that stable coexistence is only possible when antibiotic resistance comes at a fitness cost. Strikingly, CAT-expressing pneumococci in mouse lungs were outcompeted by susceptible cells even during Cm treatment. Our results highlight the importance of the microbial context during infectious disease as a potential complicating factor to antibiotic therapy.

PRIBOTEX expression vector: a pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants

To improve the selection phenotype of the expression plasmid pTEX, a Trypanosoma cruzi rDNA (DNA coding for rRNA) gene spacer fragment (806 bp) containing a mapped transcription start point ( tsp) was cloned in the vectors pTEX and pTEX- cat, generating the plasmids pRIBOTEX and pRIBOTEX- cat. T. cruzi cultures transiently transfected with pRIBOTEX- cat expressed a chloramphenicol (Cm) acetyltransferase (CAT) activity 16 000-fold greater than the activity observed with the parental vector pTEX- cat. Moreover, T. cruzi cells transformed with pRIBOTEX and pRIBOTEX- cat exhibited logarithmic growth in the presence of Geneticin (G418) 2 weeks earlier than that observed with controls transformed with pTEX. The plasmid copy number in stably transformed trypanosomes was about 50-times higher in cultures transformed with pTEX- cat than in cells transformed with pRIBOTEX or pRIBOTEX- cat. However, the neo RNA steady-state level and the CAT activity observed among the stably transfected cultures showed only modest differences. Finally, it was found that the pRIBOTEX vector was not episomally maintained as pTEX, but integrated into a chromosome indistinguishable from the one encoding rRNA. These features make pRIBOTEX a useful tool for transfection and rapid expression of genes in T. cruzi.

Molecular Characterization of a Plasmid-Borne (pTC82) Chloramphenicol Resistance Determinant (cat-TC) fromLactobacillus reuteriG4

Lactobacillus reuteriG4 contains a 7.0-kb plasmid (pTC82) encoding resistance to chloramphenicol (Cmr). Determination of the nucleotide sequence of the genetic determinant (cat-TC) encoding resistance to Cm on pTC82 revealed an open reading frame for a 238-amino-acid Cm acetyltransferase (CAT) monomer. This structuralcatgene, 714 bp in length, was highly related (ca. 95% nucleotide and ca. 81% amino acid identity) to the 648-bpcatgene fromStaphylococcus aureusplasmid pC194. A total of 6 bp transversions and 4 bp deletions was observed along the whole DNA sequence ofcat-TC compared to that ofcat-pC194. To determine the activity of the putativecat-TC gene, recombinant plasmid pUC8217 containing thecatdeterminant from pTC82 was subjected to a maxicell analysis. The observed molecular mass of the synthesized protein, based on electrophoretic mobility, was in reasonable agreement with the 27.3 kDa predicted from the DNA sequence. This is the first reported nucleotide sequence of a Cm-resistance determinant fromL. reuteriand also the first evidence of addingLactobacillusto the list of versatile bacterial genera which naturally acquire thecat-pC194 gene in the microbial ecological system.

Functional analysis of transcription of the Mycobacterium tuberculosis 16S rDNA-encoding gene

A functional analysis of Mycobacterium tuberculosis 16S ribosomal RNA (rRNA) transcription and processing was undertaken in this study. RNA:DNA hybridizations indicated that the maximum transcriptional activity of rRNA-encoding genes (rDNA) corresponded to the earliest period of exponential growth. Transcription start points ( tsp) were mapped by primer extension analysis of RNA from M. tuberculosis H37Rv and M. tuberculosis H37Ra. An identical pattern of rRNA transcription and processing was exhibited in laboratory-grown cultures of M. tuberculosis H37Rv and H37Ra. One promoter represents the structural equivalent of the Escherichia coli rrn P2 promoter. The precursor transcripts are processed into mature 16S rRNA through a pathway that includes recognition of RNA secondary structure by ribonuclease III (RNase III) in the stem structure surrounding the 16S rRNA indicating that at least this RNA processing step is conserved in mycobacteria and E. coli. The 16S rDNA promoter region from H37Rv was cloned upstream from the promoterless chloramphenicol (Cm) acetyltransferase (CAT)-encoding gene ( cat) in a shuttle plasmid vector, pSD7. The promoter-fusion construct, pSD7.16S, was characterized by CAT assays, measurement of percent survival in Cm-containing medium and in vivo transcription analysis in M. smegmatis. The M. smegmatis transformant exhibited a CAT activity of 16669 nmol/min per mg protein, suggesting that the 16S promoter was of exceptionally high strength. Two tsp utilized in M. tuberculosis were also employed in M. smegmatis. The cat mRNA synthesized under the direction of the ribosomal promoter was less stable, as compared to genome-derived rRNA.

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

The nucleotide sequence of a chloramphenicol-resistance (Cm R) determinant from the Gram − soil bacterium Agro-bacterium 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.

Chloramphenicol resistance in Campylobacter coli: nucleotide sequence, expression, and cloning vector construction

A chloramphenicol-resistance determinant (Cm R), originally cloned from Campylobacter coli plasmid pNR9589 in Japan, was isolated and the nucleotide sequence determined, which contained an open reading frame of 621 bp. The gene product was identified as Cm acetyltransferase (CAT), which had a putative amino acid sequence that showed 43% to 57% identity with other CAT proteins of both Gram + and Gram − origin. Although expression of the cat gene was constitutive in both C. coli and Escherichia coli, results of primer extension experiments indicated that transcription was initiated at different sites in these two species. A kanamycin-resistance determinant, identified as the aphA-3 gene, was located downstream from the cat gene. The codon usage of the cat gene is very different from that used in E. coli, however, the CAT polypeptide was synthesized in large amounts in E. coli maxicells. Therefore, the codon usage bias is not one of the obstacles which affects Campylobacter spp. gene expression in E. coli. New Campylobacter cloning vectors were constructed in this study.

Cloning of two chloramphenicol acetyltransferase genes from Clostridium butyricum and their expression in Escherichia coli and Bacillus subtilis.

Two non-homologous chloramphenicol (Cm) acetyltransferase (CAT) genes, designated catA and catB, were cloned from Clostridium butyricum type strains and characterized by restriction mapping. Both genes are efficiently expressed in Escherichia coli and Bacillus subtilis. In contrast to analogous genes from staphylococci and bacilli, gene expression is not dependent on induction by Cm. The genes are considered as chromosomal, since no association with endogenous plasmids was detectable. Southern hybridization revealed a homology between catA and the staphylococcal Cm resistance plasmid, pC194. The subunit size of the clostridial CAT enzymes expressed in E. coli was determined as 22.5 kDa (catA) and 24 kDa (catB), respectively. The C. butyricum cat genes provide potentially useful selection markers for the construction of cloning vectors from cryptic clostridial plasmids.

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.

Improved cat gene cassette for promoter analysis and genetic constructions

A plasmid, pJS 133, was constructed which contains a promoterless cat gene encoding chloramphenicol (Cm) acetyltransferase (CAT) conferring Cm resistance. This improved cat cassette, derived from the cat gene in plasmid pK.K.232-8 [Brosius, Gene 27 (1984) 151–160], offers several advantages over currently available cat cassettes. Expression of cat from this cassette reflects only transcriptional activity because of translational stops in all three reading frames, and possesses facile insertion/excision properties employing the flanking Polylinker restriction sites. In plasmid pJS133, this cassette can be obtained by a single digestion with SmaI, HindIII, SalI, Bam HI or PstI restriction enzymes. Additionally, 28 different cat cassettes with unique flanking restriction sites can be created by the appropriate double digestions.

A simple phase-extraction assay for chloramphenicol acyltransferase activity

A simple and convenient phase extraction assay for chloramphenicol (Cm) acetyltransferase (CAT) activity has been developed, based on the enzymatic butyrylation of radiolabelled Cm. The assay is linear over two to three orders of magnitude of enzyme concentration, is highly sensitive, and substantially less expensive than all presently available alternatives. Methods for convenient CAT assay, adapted for mammalian cells, plant protoplasts, and mammalian cell culture supernatants, are described. These methods should also simplify measurement of CAT activity in other organisms, such as yeast and bacteria. In addition, a simple preextraction procedure is presented for purifying radiolabelled Cm which allows a 25-fold increase in sensitivity using tritiated substrates.

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.

Sequence and expression characteristics of a shuttle chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli

An efficiently transforming chloramphenicol-resistance (Cm r ) shuttle marker for Saccharomyces cerevisiae and Escherichia coli has been characterized in terms of its primary structure and expression characteristics. The complete nucleotide (nt) sequence of the Cm r marker is given, with details on restriction sites, apparent expression signals for both organisms, and translation of the Cm acetyltransferase (CAT)-coding sequence. SDS-polyacrylamide gel electrophoresis and Western blotting have confirmed that the marker produced an identical CAT protein in yeast and E. coli. Each copy of the marker, whether present in multiple copies or as a single copy, gave rise to approx. 0.1% of the total soluble protein as CAT in haploid yeast cells. When compared with homologous expression of alcohol dehydrogenase (ADH-I) by the same ADC1 promoter, this represents a 27-fold reduction for CAT expression, which is typical of heterologous gene expression in yeast. When the marker was on a multicopy plasmid in yeast, up to 2.1 % of the total soluble cell protein was produced as CAT, but this did not adversely affect the growth of host cells. Increase of the Cm concentration in the medium did not result in an increase in the number of plasmids nor the amount of CAT protein produced, showing that plasmid copy number and marker expression are regulated independently of the selection pressure. In E. coli, the ADC1 yeast-promoter DNA was found to contain both forwards and backwards promoter activity. The level of expression provided by these promoters was equivalent to that of an average E. coli gene.

Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids

A hybrid plasmid, pJS37, was made by combining pLSl, which confers tetracycline (Tc) resistance, and pC194, which confers chloramphenicol (Cm) resistance. Both pJS37 (7.3 kb) and its derivative pJ S 140 (6.0 kb), from which pC 194 replication genes were removed, were structurally and segregationally stable when introduced into Streptococcus pneumoniae and grown either in the presence of Tc or in the absence of drug. However, both hybrid plasmids underwent systematic deletion when grown in the presence of Cm. One of the deleted forms, pJS4(3.4 kb), could not be maintained in the absence of a helper plasmid; two others, pJS3 (4.1 kb) and pJS5 (3.8 kb), lost the tet gene but retained the replication functions of pLSl. They both expressed very high levels of Cm acetyltransferase (CAT), which, in the case of pJS5, were constitutive. Nucleotide sequence determination of the deletion junctions in pJS3 and pJS5 indicated that the deletions occurred, presumably by recombination, between short direct repeats of 6 and 9 bp, respectively. In both cases the tet promoter was juxtaposed to the cat gene. In the case of pJS5, the deletion removed a sequence that sequestered the ribosome-binding site (RBS) for cat, thereby rendering constitutive the production of CAT. The increased resistance to Cm afforded by the hyperexpression of the cat gene apparently provided a positive selective advantage for the accumulation of the deleted forms in the plasmid pool.

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.

Cloning of a chloramphenicol acetyltransferase gene of Streptomyces acrimycini and its expression in Streptomyces and Escherichia coli

A gene ( cat) for chloramphenicol (Cm) acetyltransferase (CAT) was cloned from Streptomyces acrimycini into S. lividans 66 on the plasmid vector pIJ61. The cat gene was localized on a 1.7-kb BclI fragment, which probably also carries the cat promoter. This DNA fragment conferred Cm resistance, through CAT activity, on S. lividans, S. coelicolor and S. parvulus, but not on Escherichia coli when inserted in the BamHI site of the tetracycline-resistance(Tc R) gene of pBR322. However, when inserted in a particular orientation in this site, spontaneous deletions of 0.7 kb led to CAT activity and Cm resistance. DNA homologous to the 1.7-kb BclI cat fragment was found in most, but not all, of a series of other streptomycetes that have CAT activity. The cat provides a potentially useful screening marker for Streptomyces cloning vectors.

Cloning and expression in streptomyces lividans of antibiotic resistance genes derived from Escherichia coli.

Hybrid plasmids that replicate in both Escherichia coli and Streptomyces lividans were constructed in vitro by joining the E. coli-derived plasmid pACYC184 or pACYC177, at their BamHI or PstI restriction site, respectively, to S. lividans plasmid pSLP111. After introduction of the composite replicons into S. lividans by transformation, chloramphenicol (Cm) resistance encoded by pACYC184 and kanamycin resistance encoded by pACYC177 were phenotypically expressed in the S. lividans host. A Sau3A restriction endonuclease-generated deoxyribonucleic acid fragment from pACYC184 containing the entire structural gene for the Cm acetyltransferase enzyme, but lacking the nucleotide sequence ordinarily serving as the Cm resistance gene promoter, also specified resistance to Cm when introduced in either orientation into the BamHI or BclI endonuclease cleavage site of pSLP111 or into corresponding sites of the analogous plasmid pSLP101. These findings make it unlikely that the biologically active CM acetyltransferase was being made in S. lividans as part of a fused protein, but instead indicate that the ATG start codon used for initiation of translation of the Cm resistance gene in E. coli was also utilized in S. lividans. In contrast, the synthesis of messenger ribonucleic acid that encodes the Cm acetyltransferase in S. lividans was, in at least one instance, apparently initiated at nucleotide sequences within the S. lividans plasmid vector, with resulting transcriptional read-through into the E. coli-derived deoxyribonucleic acid segment.