Chloramphenicol-resistant Colonies Research Articles

Interaction of bacteriophage P1 with an epiphytic Pantoea agglomerans strain-the role of the interplay between various mobilome elements.

P1 is a model, temperate bacteriophage of the 94 kb genome. It can lysogenize representatives of the Enterobacterales order. In lysogens, it is maintained as a plasmid. We tested P1 interactions with the biocontrol P. agglomerans L15 strain to explore the utility of P1 in P. agglomerans genome engineering. A P1 derivative carrying the Tn9 (cmR) transposon could transfer a plasmid from Escherichia coli to the L15 cells. The L15 cells infected with this derivative formed chloramphenicol-resistant colonies. They could grow in a liquid medium with chloramphenicol after adaptation and did not contain prophage P1 but the chromosomally inserted cmR marker of P1 Tn9 (cat). The insertions were accompanied by various rearrangements upstream of the Tn9 cat gene promoter and the loss of IS1 (IS1L) from the corresponding region. Sequence analysis of the L15 strain genome revealed a chromosome and three plasmids of 0.58, 0.18, and 0.07 Mb. The largest and the smallest plasmid appeared to encode partition and replication incompatibility determinants similar to those of prophage P1, respectively. In the L15 derivatives cured of the largest plasmid, P1 with Tn9 could not replace the smallest plasmid even if selected. However, it could replace the smallest and the largest plasmid of L15 if its Tn9 IS1L sequence driving the Tn9 mobility was inactivated or if it was enriched with an immobile kanamycin resistance marker. Moreover, it could develop lytically in the L15 derivatives cured of both these plasmids. Clearly, under conditions of selection for P1, the mobility of the P1 selective marker determines whether or not the incoming P1 can outcompete the incompatible L15 resident plasmids. Our results demonstrate that P. agglomerans can serve as a host for bacteriophage P1 and can be engineered with the help of this phage. They also provide an example of how antibiotics can modify the outcome of horizontal gene transfer in natural environments. Numerous plasmids of Pantoea strains appear to contain determinants of replication or partition incompatibility with P1. Therefore, P1 with an immobile selective marker may be a tool of choice in curing these strains from the respective plasmids to facilitate their functional analysis.

Biochemical and Structural Analysis of Bacterial O-antigen Chain Length Regulator Proteins Reveals a Conserved Quaternary Structure

Lipopolysaccharide (LPS) is a major component of the Gram-negative outer membrane and is an important virulence determinant. The O-antigen polysaccharide of the LPS molecule provides protection from host defenses, and the length of O-antigen chains plays a pivotal role. In the Wzy-dependent O-antigen biosynthesis pathway, the integral inner membrane protein Wzz determines the O-antigen chain length. How these proteins function is currently unknown, but the hypothesis includes activities such as a "molecular ruler" or a "molecular stopwatch," and other possibilities may exist. Wzz homologs are membrane proteins with two transmembrane helices that flank a large periplasmic domain. Recent x-ray crystallographic studies of the periplasmic portions of Wzz proteins found multiple oligomeric forms, with quaternary structures favoring the "molecular ruler" interpretation. Here, we have studied full-length Wzz proteins with the transmembrane portions embedded in lipid membranes. Using electron microscopy and image analysis we find a unique hexameric state rather than differing oligomeric forms. The data suggest that in vivo Wzz proteins determine O-antigen chain length via subtle structure-function relationships at the level of primary, secondary, or tertiary structure within the context of a hexameric complex.

Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae.

The recent availability of bacterial genome sequence information permits the identification of conserved genes that are potential targets for novel antibiotic drug discovery. Using a coupled bioinformatic/experimental approach, a list of candidate conserved genes was generated using a Microbial Concordance bioinformatics tool followed by a targeted disruption campaign. Pneumococcal sequence data allowed for the design of precise PCR primers to clone the desired gene target fragments into the pEVP3 'suicide vector'. An insertion-duplication approach was employed that used the pEVP3 constructs and resulted in the introduction of a selectable chloramphenicol resistance marker into the chromosome. In the case of non-essential genes, cells can survive the disruption and form chloramphenicol-resistant colonies. A total of 347 candidate reading frames were subjected to disruption analysis, with 113 presumed to be essential due to lack of recovery of antibiotic-resistant colonies. In addition to essentiality determination, the same high-throughput methodology was used to overexpress gene products and to examine possible polarity effects for all essential genes.

A new transgenic mouse mutagenesis test system using Spi- and 6-thioguanine selections.

A new transgenic mouse mutagenesis test system has been developed for the efficient detection of point mutations and deletion mutations in vivo. The mice carry lambda EG10 DNA as a transgene. When the rescued phages are infected into Escherichia coli YG6020-expressing Cre recombinase, the phage DNA is converted into plasmid pYG142 carrying the chloramphenicol-resistance gene and the gpt gene of E. coli. The gpt mutants can be positively detected as colonies arising on plates containing chloramphenicol and 6-thioguanine. The EG10 DNA carries a chi site along with the red and gam genes so that the wild-type phages display Spi- (sensitive to P2 interference) phenotype. Mutant phages lacking both red and gam genes can be positively detected as plaques that grow in P2 lysogens of E. coli. These mutant phages are called lambda Spi-. The spontaneous gpt mutation frequencies of five independent transgenic lines were 1.7 to 3.3 x 10(-5) in bone marrow. When the mice were treated with ethylnitrosourea (single i.p. treatments with 150 mg/kg body weight; killed 7 days after the treatments), mutation frequencies were increased four- to sevenfold over the background in bone marrow. The average rescue efficiencies were more than 200,000 chloramphenicol-resistant colonies per 7.5 micrograms bone marrow DNA per packaging reaction. In contrast to gpt mutation frequencies, spontaneous Spi- mutation frequencies were 1.4 x 10(-6) and 1.1 x 10(-6) in bone marrow and sperm, respectively. No spontaneous Spi- mutants have been detected so far in spleen, although 930,000 phages rescued from untreated mice were screened. In gamma-ray-treated animals, however, induction of Spi- mutations was clearly observed in spleen, at frequencies of 1.4 x 10(-5) (5 Gy), 1.2 x 10(-5) (10 Gy), and 2.0 x 10(-5) (5O Gy). These results suggest that the new transgenic mouse "gpt delta" could be useful for the efficient detection of point mutations and deletion mutations in vivo.

Construction of a Synechocystic PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes

The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803 delta rbc, were detected within 8 days when grown under 5% CO2 in air. These transformants were unable to grow in air (0.03% CO2). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO2 in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803 delta rbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.

Initial studies on aBacillus subtilis mutant lacking the dnaK-homologue protein

A sequence of 412bp, spanning the terminal half of thegrpE and the proximal portion of thednaK-homologues inBacillus subtilis, was amplified with PCR technology. This fragment was cloned into pJH101, anEscherichia coli plasmid, and transformed intoB. subtilis strain YB886. Several chloramphenicol-resistant colonies were obtained from this transformation. The integration of the plasmid into theB. subtilis chromosome was verified by restriction endonuclease analysis and Southern hybridization. Strain BUL101, a chloramphenicol-resistant transformant, lacked the DnaK-homologue as demonstrated by two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. BUL101 grew at slower rates than parental cells at both 37°C and 48°C, produced abnormal cell shapes at 48°C, and was unable to grow at 51°C. The 412bp fragment did not exhibit detectable promoter activity.

Cysteinyl-tRNA synthetase is a direct descendant of the first aminoacyl-tRNA synthetase

The gene encoding the cysteinyl-tRNA synthetase of E. coli was cloned from an E. coli genomic library made in λ2761, a lambda vector which can integrate and which carries a chloramphenicol resistance gene. A thermosensitive cysS mutant of E. coli was lysogenised and chloramphenicol-resistant colonies able to grow at 42°C were selected to isolate phages containing the wild-type cysS gene. The sequence of the gene was determined. It codes for a 461 amino-acid protein and includes the sequences HIGH and KMSK known to be involved in the ATP and TRNA binding respectively of class I synthetases. The cysteinyl enzyme has segments in common with the cytoplasmic leucyl-tRNA synthetase of Neurospora crassa, the tryptophanyl-tRNA synthetase of Bacillus stearothermophilus, and the phenylalanyl-tRNA synthetase of Saccharomyces cerevisiae. Sequence comparisons show that the amino end of the cysteinyl-tRNA synthetase has similarities with prokaryotic elongation factors Tu; this region is close to the equivalent acceptor binding domain of the glutaminyl-tRNa synthetase of E. coli. There is a further similarity with the seryl enzyme (a class II enzyme) which has led us to propose that both classes had a common origin and that this was the ancestor of the cysteinyl-tRNA synthetase.

Transformation of Mycobacterium smegmatis with Escherichia coli plasmids carrying a selectable resistance marker.

One limiting factor in studies of tuberculosis and leprosy is the difficulty of genetic analysis and manipulation of mycobacteria. Two approaches were adopted for the construction of vectors, based on different Escherichia coli plasmids and using Mycobacterium smegmatis as the host. In both cases we found that the original E. coli plasmid is capable of being replicated in M. smegmatis, yielding chloramphenicol-resistant colonies. One such plasmid has been recovered from a M. smegmatis transformant and used to re-transform both M. smegmatis and E. coli to chloramphenicol resistance. This plasmid is indistinguishable from the original plasmid by restriction analysis, and can be used as a shuttle vector for the genetic manipulation of mycobacterial species.

Codon usage as a reason for unsuccessful search for amber-suppressor mutants inStreptomyces lividans?

SummaryAn amber mutation was created in the CAT gene of plasmid vector pACYC184 and this modified plasmid was fused with theStreptomycesvector pIJ702 for use as an indicator for the identification ofStreptomycesstrains carrying nonsense suppressor tRNA mutations. The resulting hybrid plasmid pGM1109 was introduced into the chloramphenicol-sensitive mutant M252 ofStreptomyces lividans. Chloramphenicol-resistant colonies were isolated and characterized. None of them was a nonsense suppressor mutant. The failure to obtain such mutants is discussed on the basis of codon usage in streptomycetes.

Resistance to chloramphenicol in Proteus mirabilis by expression of a chromosomal gene for chloramphenicol acetyltransferase.

Proteus mirabilis PM13 is a well-characterized chloramphenicol-sensitive isolate which spontaneously gives rise to resistant colonies on solid media containing chloramphenicol (50 micrograms ml-1) at a plating efficiency of 10(-4) to 10(-5). Such chloramphenicol-resistant colonies exhibit a novel phenotype with respect to chloramphenicol resistance. When a single colony grown on chloramphenicol agar is transferred to liquid medium and grown in the absence of antibiotic for 150 generations, a population of predominantly sensitive cells arises. This mutation-reversion phenomenon has been observed in other Proteus species and Providencia strains, wherein resistance has been shown to be mediated in each case by the enzyme chloramphenicol acetyltransferase. The cat gene responsible for the phenomenon is chromosomal and can be cloned from P. mirabilis PM13 with DNA prepared from cells grown in the absence or the presence of chloramphenicol. Recombinant plasmids which confer resistance to chloramphenicol carry an 8.5-kilobase PstI fragment irrespective of the source of host DNA. The location of the cat gene within the PstI fragment was determined by Southern blotting with a cat consensus oligonucleotide corresponding to the expected amino acid sequence of the active site region of chloramphenicol acetyltransferase, and the direction of transcription was deduced from homology with the type I cat variant.

Rosanilins: indicator dyes for chloramphenicol-resistant enterobacteria containing chloramphenicol acetyltransferase.

Rosanilin dyes such as crystal violet and basic fuchsin have been used as indicator dyes in solid growth medium for chloramphenicol-resistant enterobacterial colonies containing the enterobacterial resistance enzyme chloramphenicol acetyltransferase (CAT). On certain media containing rosanilins, cells containing CAT formed darker colonies than cells not containing CAT. Contrast was affected by the types and concentrations of complex nutrients, sugars salts, and rosanilin dyes present. When crystal violet was used as the indicator dye, contrast could not be obtained for strains whose growth was partially inhibited by crystal violet. Contrast could not be obtained between yeast colonies with and without the enterobacterial resistance enzyme, between Bacillus subtilis colonies with and without the staphylococcal resistance enzyme, or between enterobacterial colonies with and without the staphylococcal resistance enzyme. The darker coloration of enterobacterial colonies with the enterobacterial enzyme was due to the binding of dye to enzyme. Rosanilin dues have been used to score resistance phenotypes by colony color, to detect chloramphenicol-sensitive sectors in chloramphenicol-resistant colonies, and to screen for occasional chloramphenicol-sensitive cells in a resistant population during cloning by insertional inactivation of the chloramphenicol resistance gene.