The galU gene expression in Streptococcus pneumoniae

Bacillus subtilis possesses carbon-flux regulating histidine protein (Crh), a paralog of the histidine protein (HPr) of the phosphotransferase system (PTS). Like HPr, Crh becomes (de)phosphorylated in vitro at residue Ser46 by the metabolite-controlled HPr kinase/phosphorylase HPrK/P. Depending on its phosphorylation state, Crh exerts regulatory functions in connection with carbohydrate metabolism. So far, knowledge on phosphorylation of Crh in vivo has been limited and derived from indirect evidence. Here, we studied the dynamics of Crh phosphorylation directly by non-denaturing gel electrophoresis followed by Western analysis. The results confirm that HPrK/P is the single kinase catalyzing phosphorylation of Crh in vivo. Accordingly, phosphorylation of Crh is triggered by the carbon source as observed previously for HPr, but with some differences. Phosphorylation of both proteins occurred during exponential growth and disappeared upon exhaustion of the carbon source. During exponential growth, ~80% of the Crh molecules were phosphorylated when cells utilized a preferred carbon source. The reverse distribution, i.e. around 20% of Crh molecules phosphorylated, was obtained upon utilization of less favorable substrates. This clear-cut classification of the substrates into two groups has not previously been observed for HPr(Ser)~P formation. The likely reason for this difference is the additional PTS-dependent phosphorylation of HPr at His15, which limits accumulation of HPr(Ser)~P.     

Transient expression of the chloramphenicol acetyltransferase gene in cultured mosquito cells

A recombinant plasmid in which the bacterial chloramphenicol acetyltransferase (CAT) gene is under the control of the Drosophila heat-shock protein (hsp) 70 promoter has been introduced into cultured mosquito (Aedes albopictus) cells using 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene) and dimethylsulfoxide (DMSO). CAT activity was induced by incubating transfected cells at 37 degrees C, and high levels of enzyme activity were maintained for more than 24 h after the temperature shock. Transfected DNA was maintained in the cells for at least 4 days. These experiments document an effective method for introducing purified DNA into cultured mosquito cells.     

Erythromycin induces expression of the chloramphenicol acetyltransferase gene cat-86.

The plasmid gene cat-86 specifies chloramphenicol-inducible chloramphenicol acetyltransferase in Bacillus subtilis. This gene, like the erythromycin-inducible erm genes, is regulated by translational attenuation. Here we show that cat-86 is also inducibly regulated by erythromycin. cat-86 does not confer resistance to erythromycin.     

A method to detect transfected chloramphenicol acetyltransferase gene expression in intact animals

A rapid procedure is described for assaying chloramphenicol acetyltransferase (CAT, EC 2.3.1.28) enzyme activity in intact animals following transfection of the RSV CAT plasmid into mouse bone marrow cells by electroporation. The reconstituted mice were injected with [14C]chloramphenicol and ethyl acetate extracts of 24-h urine samples were analyzed by TLC autoradiography for the excretion of 14C-labeled metabolites. CAT expression in vivo can be detected by the presence of acetylated 14C-labeled metabolites in the urine within 1 week after bone marrow transplantation and, under the conditions described, these metabolites can be detected for at least 3 months. CAT expression in intact mice as monitored by the urine assay correlates with the CAT expression in the hematopoietic tissues assayed in vitro. This method offers a quick mode of screening for introduced CAT gene expression in vivo without sacrificing the mice.     

Enhancing quantification of mammalian cell transfections with chloramphenicol acetyltransferase reporter plasmids

We modified existing techniques to optimize conditions for obtaining quantitative, highly replicable, and sensitive transfections. The processes described may serve as a model for investigators initiating transfection procedures who wish to obtain definitive and quantitative results quickly and efficiently. In our example, we compared specific gene expressions of plasmids with the chloramphenicol acetyltransferase (CAT) reporter. Techniques included measuring CAT activity in transfected mammalian cells, selecting a procedure for extracting plasmids from bacterial cells, evaluating the timing of the transfection, choosing a transfection reagent and the reagent: plasmid DNA ratio, and determining procedures for the extraction of cells.     

A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells

A rapid procedure is described for assaying chloramphenicol acetyltransferase (CAT) enzyme activity following transfection of the CAT gene into eucaryotic cells. CAT enzyme activity in cell extracts catalyzes the transfer of [14C]acetyl groups from labeled acetyl coenzyme A to unlabeled chloramphenicol. Labeled reaction product is quantitated by liquid scintillation counting after extraction into ethyl acetate. The method is valid for use with transfected cell extracts only if the extracts are first heated to 65 degrees C to remove a factor which degrades acetyl coenzyme A. The revised procedure offers considerable advantages in speed and ease of performance over the chromatographic assay in current use.     

Cloning and expression of a chloramphenicol acetyltransferase gene in cytosine-substituted T4 bacteriophage

We have developed an efficient method for transferring foreign genes into the T4 phage genome. Any foreign genes inserted into the T4 uvsY gene cloned on plasmids can be transferred into a cytosine-substituted T4dC(delta NB5060) phage genome by a replacement type of recombination. To achieve this, we constructed chimeric plasmids which had a chloramphenicol acetyltransferase gene (cat) derived from transposon Tn9 inserted into the Bg/II site within the T4 uvsY gene on pBR322. The cat gene was then transferred by in vivo recombination into the T4dC(delta NB5060) phage genome. Moreover, it was demonstrated that the cat gene in the hybrid T4dC phage was expressed upon phage infection and development.     

Resistance mechanism of chloramphenicol in Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis.

The chloramphenicol resistance of Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis isolated from clinical materials was proved to be due to an inactivating enzyme produced by these bacteria. The inactivated products of chloramphenicol were identified as 1-acetoxy, 3-acetoxy and 1,3-diacetoxy derivatives by thin-layer chromatography and infrared spectroscopy. The responsible enzyme was thus confirmed to be chloramphenicol acetyltransferase. The enzyme was inducible. It was partially purified by ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. The enzymes obtained from S. haemolyticus, S. pneumoniae and S. faecalis have been compared with the conclusion that they are identical with respect to molecular weight (approximately 75,000-80,000), optimum pH and heat stability.     

Physical and genetic characterization of deletions in Streptococcus pneumoniae.

Genetic properties of markers may discriminate between deletions and point mutations. We have designed a physical method for a direct characterization of deletions which also gives an estimate of their size.     

Chloramphenicol-inducible gene expression in Bacillus subtilis is independent of the chloramphenicol acetyltransferase structural gene and its promoter

Lactobacillus plantarum has an unusually high Mn(II) requirement for growth and accumulated over 30 mM intracellular Mn(II). The acquisition of Mn(II) by L. plantarum occurred via a specific active transport system powered by the transmembrane proton gradient. The Mn(II) uptake system has a Km of 0.2 microM and a Vmax of 24 nmol mg-1 of protein min-1. Above a medium Mn(II) concentration of 200 microM, the intracellular Mn(II) level was independent of the medium Mn(II) and unresponsive to oxygen stresses but was reduced by phosphate limitation. At a pH of 5.5, citrate, isocitrate, and cis-aconitate effectively promoted MN(II) uptake, although measurable levels of 1,5-[14C]citrate were not accumulated. When cells were presented with equimolar Mn(II) and Cd(II), Cd(II) was preferentially taken up by the Mn(II) transport system. Both Mn(II) and Cd(II) uptake were greatly increased by Mn(II) starvation. Mn(II) uptake by Mn(II)-starved cells was subject to a negative feedback regulatory mechanism functioning less than 1 min after exposure of the cells to Mn(II) and independent of protein synthesis. When presented with a relatively large amount of exogenous Mn(II), Mn(II)-starved cells exhibited a measurable efflux of their internal Mn(II), but the rate was only a small fraction of the maximal Mn(II) uptake rate.     

Purification and some properties of a chloramphenicol acetyltransferase mediated by plasmids from Vibrio anguillarum

Vibrio anguillarum strains were isolated from chloramphenicol-resistant bacteria in diseased fish. Plasmid Rms418, which confers chloramphenicol resistance, was transferred from V. anguillarum GN11379 to Escherichia coli K12 by conjugation. The Rms418-encoded chloramphenicol acetyltransferase (CAT) [EC 2.3.1.99] was isolated and purified to homogeneity using affinity chromatography on immobilized p-amino-chloramphenicol or ATP. The general CAT could be adsorbed by a matrix with a chloramphenicol base ligand (Zaidenzaig, Y. & Shaw, W.V. (1976) FEBS Lett. 62,266-271), but the Rms418-encoded CAT was not bound under these conditions. The specific activity of the enzyme, when measured by the spectrophotometric assay, was 71.4 units/mg protein at 37 degrees C. The molecular weight of the enzyme treated with SDS and 2-mercaptoethanol was shown to be approximately 22,000. The molecular weight of the native enzyme, as determined by gel filtration, was approximately 69,000, and the optimal pH was 7.8. The Km values for chloramphenicol and CoASAc were 34.5 and 150 microM, respectively. Enzyme activity was inhibited by HgCl2, p-chloromercuribenzoate (p-CMB), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and ethylendiaminotetraacetic acid (EDTA). The half life at 53 degrees C was approximately 100 min.     

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.     

A plasmid vector allowing positive selection of recombinant plasmids in Streptococcus pneumoniae

A new plasmid, pSP2, was constructed as a cloning vector for use in Streptococcus pneumoniae. It allows direct selection of recombinant plasmids, even for DNA fragments not homologous to the S. pneumoniae chromosome, as based on the failure to maintain long inverted repeats (LIRs) hyphen-free in bacterial plasmids. Plasmid pSP2 contains a 1.4-kb BamHI fragment ("hyphen") flanked by 1.9-kb LIRs. The removal of the 1.4-kb BamHI fragment followed by ligation creates a plasmid containing a 1.9-kb insert-free LIR; plasmids with such non-hyphenated LIRs were not established when transferred into S. pneumoniae. Replacement of the original 1.4-kb insert by other restriction fragments restored plasmid viability. Investigation of plasmid transfer by transformation suggests that intrastrand synapsis between the LIRs could occur, thus facilitating plasmid establishment (a process we call self-facilitation). Such an intrastrand synapsis could also account for rare occurrences of insert-inversion noticed upon transfer as well as for the formation of palindrome-deleted derivatives at low frequency. Plasmid pSP2 carries two selectable genes, tet and ermC, and can be used for cloning of fragments produced by a variety of restriction enzymes (BamHI, Bg/II, Bc/I or Sau3A, and Sa/I or XhoI).