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A family of cloning vectors derived from plasmid pACYC184 and, therefore, compatible with pBR322 and its derivatives (especially the pUC family of vectors), is described. They all contain a multiple cloning site (MCS) and the lacZ alpha reporter gene for easy cloning. They have been grouped in three sets: (i) six of the vectors contain a chloramphenicol-resistance (CmR)-encoding gene and each a different MCS with 16 unique restriction sites overall; (ii) another six vectors contain a kanamycin-resistance (KmR)-encoding gene and the same six MCS; and (iii) two CmR vectors that contain the SP6 and T7 promoters flanking the MCS and lacZ alpha reporter gene of pUC18/19.

A transformation system for Enterococcus faecalis was developed which uses untreated (i.e., non-protoplasted) cells and the electroporation technique. The optimized protocol resulted in transformation efficiencies of up to 4 x 10(6) transformants per microgram of plasmid DNA. All strains of E. faecalis tested could be transformed by this method, albeit with differing transformation efficiencies. Using the protocol optimized for E. faecalis we successfully transformed Enterococcus faecium, E. hirae, E. malodoratus and E. mundtii.

Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high level of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose to ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).

Twenty isolates of Salmonella typhi from cases of typhoid during the 1989-1990 epidemic in Calcutta were examined. Most isolates (84% of all isolates in the epidemic) were resistant to chloramphenicol, ampicillin, tetracycline and streptomycin but were sensitive to nalidixic acid and ciprofloxacin. Plasmids of 120 kb and 14 kb were identified amongst the multi-drug resistant isolates of S. typhi. However, there was no plasmid in the antibiotic-sensitive isolates. The 120-kb plasmid was transferable and transconjugants were resistant to chloramphenicol, ampicillin, tetracycline and streptomycin. Restriction endonuclease analysis patterns after EcoRI digestion of the 120-kb antibiotic-resistance plasmids from the S. typhi isolates and transconjugants were similar.

Replacement of cat-86 codon 7 or 144 with the UGA codon permitted the gene to confer chloramphenicol resistance in wild-type Bacillus subtilis. UAA replacements of the same codons resulted in a chloramphenicol-sensitive phenotype in wild-type B. subtilis and a chloramphenicol-resistant phenotype in suppressor-positive strains. N-terminal sequencing showed that UGA at codon 7 was decoded as tryptophan in wild-type cells, at an efficiency of about 6%.

Tn5253, carrying tetracycline and chloramphenicol resistance determinants, is a 65.5-kb conjugative transposon originally detected in the chromosome of Streptococcus pneumoniae BM6001. We have identified an 18-kb segment of DNA carrying the tet determinant within Tn5253 to be an independent conjugative transposon when removed from the context of the larger element. In vivo deletion of this DNA segment, now termed Tn5251, from within Tn5253 did not affect the conjugative transposition properties of the remaining sequences. Thus, Tn5253 is a composite element of two conjugative structures: Tn5252, constituting the sequences beyond Tn5251 within Tn5253, and Tn5251. The transfer properties of Tn5252 and Tn5251 suggest that these may belong to two different classes of mobile elements even though they were initially found associated. The notion that a tet-carrying transposon like Tn5251 may have been the ancestral element in the evolution of the larger streptococcal conjugative transposons must be reevaluated in the light of present observations.

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.

A series of controlled expression vectors was constructed based on the wide-host-range plasmid pMMB66EH. Some of these new vectors code for the alpha-peptide of beta-galactosidase and allow the direct screening of recombinant clones by inactivation of alpha-complementation. The bla gene was replaced in some plasmids by the cat gene of Tn9 coding for chloramphenicol resistance, extending the use into beta-lactam-resistant strains. They all feature either the tac or taclac (tac-lac UV5 in tandem) promoters in front of a polylinker followed by the rrnB transcriptional stop point. These vectors were tested by subcloning the xylE gene coding for the Pseudomonas putida catechol 2,3-oxygenase and the Escherichia coli lamB gene coding for the lambda receptor. The expression of these genes in E. coli indicated that the tac promoter is five times stronger than the taclac promoter and that both were tightly regulated. The tac promoter in Pseudomonas syringae pv glycinea and Xanthomonas campestris pv vesicatoria had a strength similar to that in E. coli, while the taclac promoter was much weaker, reaching only 6.5 and 3% of the level of expression of the tac promoter, respectively. The taclac promoter, however, proved to be useful for the cloning in E. coli of DNA fragments that were unstable in vectors with stronger promoters and higher copy number. Expression of the lamB gene in Vibrio cholerae strain TRH7000 was not sufficient to permit cosmid transduction. Two subunits of the E. coli mannose permease, coded by the ptsP and ptsM genes, are also required for cosmid DNA penetration into the recipient cells.

We replaced an Escherichia coli phr gene by a 1.4-kb fragment of DNA coding for resistance to chloramphenicol. Characterization of 2 deletions (phr-19 and phr-36) and 1 insertion (phr-34) in the phr gene revealed no photoreactivation. Photoreactivation-deficient strains of either recA56 or lexA1(ind-) were more sensitive to UV radiation in the dark than phr-proficient counterparts. The presence of the phr defect in uvrA6 strains increased by 1.5-2-fold his-4(Ochre) to His+ mutation induced by ultraviolet light compared to uvrA6 phr+ strains, although there was no difference in UV sensitivity between uvrA6 phr+ and uvrA6 phr- strains. 30-35% of the His+ mutations thus induced were suppressor mutations in uvrA6 phr+ and 49-55% in uvrA6 phr- strains. The UV mutagenesis results are consistent with the previous observations that suppressor mutations targeted by a thymine-cytosine pyrimidine dimer are reduced in the dark in cells with amplified DNA photolyase.

Seven (6.1%) of 115 strains of Salmonella typhi isolated from Malaysian patients harbored a single large plasmid of 71 to 166 mD. Two of the seven plasmid-bearing strains were resistant to chloramphenicol (Cm) and tetracycline (Tc) and they transferred Cm and Tc resistance traits to Escherichia coli K12 at frequencies from 1.6 x 10(-7) to 1.9 x 10(-6). Agarose gel electrophoresis provided evidence that the resistance traits were cotransferred on a conjugative plasmid. The significance and importance of these results are discussed.

The unstable feature of ristomycin resistance in S. coelicolor A3 (2) was studied. It was shown that the frequency of ristomycin-resistant derivatives was high in both chloramphenicol sensitive mutants and their resistant revertants. The 15- and 20-kb DNA sequences capable of amplifying were detected in the chloramphenicol resistant revertants. In the genomes of the studied strains they were represented by 50 and 40 copies, respectively.

We have examined in detail the effects of various induction temperatures on the expression of a heterologous fusion gene controlled by the bacteriophage lambda PL promoter in a heat-inducible Escherichia coli expression system which utilizes the CIts857 repressor. Experiments performed over a temperature range spanning 29-42 degrees C indicate that, under our conditions, temperatures as low as 29 degrees C may be required to fully repress the CI857-controlled transcription from PL, and that the highest protein yields are obtained after induction at 36 degrees C for 6 h. We cloned the cat reporter gene downstream from a heterologous gene controlled by PL and found that cat expression at a low induction temperature permits the monitoring of productive transcription through the heterologous gene and thus aids in selecting transformants that are capable of producing the heterologous protein in E. coli.

A small chloramphenicol resistance (Cm) plasmid of 4.65 kB could be detected in an "equine" Staphylococcus sciuri-culture. This plasmid, designated as pSC3, was identified by interspecific protoplast transformation. On the basis of restriction endonuclease analyses a detailed restriction map of pSC3 could be constructed. This allowed structural comparisons of pSC3 with Cm-plasmids of other staphylococcal species from infections of humans and animals and identification of pSC3 as a member of the pC 221-family of staphylococcal Cm-plasmids. The pSC3-plasmid encoded an inducible chloramphenicol acetyltransferase as confirmed by enzymatic assays. This enzyme could be demonstrated in cell-free lysates of Cm-induced pSC3-transformants.

A chloramphenicol-resistance determinant (CmR), 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.

We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B. subtilis chromosome. The procedure depends on the accuracy and high frequency of homologous recombination between the B. subtilis chromosome and the DNA taken up by the cell. The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst. The utility of the method has been demonstrated using leuB and pro of B. subtilis as target gene and catalyst, respectively, and mutations such as leuB::cat, leuB-, and pro::neo constructed in vitro on the cloned DNA fragments. Transformation in sequential steps as (leuB+ pro+)----(leuB::cat pro+)----(leuB- pro::neo)----(leuB- pro+) resulted in a leuB- single mutant without affecting other regions of the B. subtilis chromosome (gene-directed mutagenesis). We also demonstrate that other single mutations such as metD- and pro-, as well as the double mutation leuB- pro- can be introduced by the same procedure. In principle, true isogenies with multiple mutations can be constructed by the method described in this paper. Furthermore, the procedure should be generally applicable to any organisms in which homologous recombination is proficient.

A composite plasmid comprising the mini-F and pBR322 replicons was found to inhibit cell growth of a host with conditional mutations in dnaA and rnh under restrictive conditions, where the normal initiation of replication from oriC was inactivated, but the alternative replication initiation from oriK was active. It was further shown that the composite plasmid inhibited stable DNA replication (SDR) which occurs constitutively in cells mutant for rnh. Neither pBR322 nor mini-F alone produced these inhibitory effects. Deletion analyses revealed that the mini-F segment responsible for the inhibition of both processes was the promoter region of the sopA gene which had been cloned into a site upstream of the bla gene on pBR322 in such an orientation as to cause overexpression of bla. Inserting the promoter of the Escherichia coli lac gene into the same site had the same effect. Introduction of a deletion and a frameshift mutation into bla abolished the inhibition. Thus, the inhibition of growth and SDR appear to be due to overproduction of the bla gene product, beta-lactamase.

Chloramphenicol, thiamphenicol and three fluorinated derivatives, Sch 24893, Sch 25298 and Sch 25393, were studied with respect to inhibition of the growth of selected bacterial strains and cell-free translation systems. Thiamphenicol was the least potent inhibitor in the former experiments, but behaved similarly to chloramphenicol and Sch 25298 in the latter, thereby displaying selective inhibition of prokaryotic protein synthesis. Thiamphenicol and Sch 25298 were shown to be like chloramphenicol in inhibiting peptidyl transferase activity specifically on 70 S ribosomes, but the antibiotics bound to their common ribosomal-receptor site with different efficiencies in the order chloramphenicol greater than thiamphenicol greater than Sch 25298. Selected bacterial strains highly resistant to chloramphenicol and thiamphenicol because of chloramphenicol acetyltransferase production were, in contrast, highly sensitive to inhibition by the fluorinated antibiotics. Thus Sch 24893, Sch 25298 and Sch 25393 may have important uses in veterinary and clinical medicine.

We studied a clinical isolate of Salmonella typhi (strain 1895) characterized by resistance to 200 micrograms of chloramphenicol per ml despite the absence of chloramphenicol-inactivating activity. The outer membrane protein profile analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a deficiency of one of the major protein species which may serve as a porin for entry of chloramphenicol. When the strain was transformed with a plasmid encoding chloramphenicol acetyltransferase, chloramphenicol added to the culture was not inactivated, suggesting a drastic reduction of permeability towards the drug. Moreover, transformants bearing a plasmid coding for the Escherichia coli OmpF porin became considerably more susceptible to chloramphenicol (40 micrograms/ml). On the other hand, transformants carrying a plasmid encoding the Salmonella typhi ompC gene remained as resistant to the drug as the parental strain, even though they overexpressed OmpC. These findings indicate that the lack of OmpF plays a major role in the resistance to chloramphenicol in strain 1895.

The plasmids of a multiresistant "canine" Staphylococcus epidermidis-culture were investigated. Two small plasmids, the 4.55 kB chloramphenicol resistance (CmR-) plasmid pSC4 and the 4.45 kB tetracycline resistance (TetR-) plasmid pST 3 could be isolated. Detailed restriction maps of pSC 4 and pST 3 were constructed by double restriction endonuclease digests. The restriction maps revealed extensive structural homologies between pSC 4 from "canine" S. epidermidis and the CmR-plasmid pC 221 from "human" S. aureus as well as between pST 3 from "canine" S. epidermidis and the TetR-plasmid pT 181 from "human" S. aureus. These data suggested that an exchange of small plasmids between S. epidermidis and S. aureus might be possible.

Chloramphenicol resistance among Salmonella spp. has important public health and clinical implications, especially in areas of the world where these strains are endemic. The availability of rapid and sensitive screening methods for detection of antibiotic resistance is important. Therefore, we tested 33 strains of Salmonella for chloramphenicol acetyltransferase (CAT) activity using two rapid techniques. Evaluation of a 1-h tube method and a 30-min commercial disk procedure demonstrated that they are as accurate as standardized susceptibility techniques. Both the 1-h tube and 30-min disk methods detected CAT enzymatic activity produced by one CAT gene copy per cell.

The Bacillus subtilis small cytoplasmic RNA (scRNA) has an important, although not yet defined function in protein biosynthesis. Here we describe the mapping of the single copy scRNA gene and the flanking homolog to dnaZX of Escherichia coli, termed 'dnaX'. The scRNA gene region of a B. subtilis wild-type strain was marked with a cat gene and mapped by scoring chromosomal cotransformation rates of various mutant strains to chloramphenicol resistance and loss of the mutant phenotypes, respectively. This analysis, together with an EcoRI map comparison, places the scRNA gene and dnaX in the vicinity of recM near the replication origin region of B. subtilis.

A strain of Listeria monocytogenes, isolated from a patient with meningoencephalitis, was resistant to chloramphenicol, erythromycin, streptomycin, and tetracycline. The genes conferring resistance to these antibiotics were carried by a 37-kb plasmid, pIP811, that was self-transferable to other L monocytogenes cells, to enterococci-streptococci, and to Staphylococcus aureus. The efficacy of transfer and the stability of pIP811 were higher in enterococci-streptococci than in the other gram-positive bacteria. As indicated by nucleic acid hybridisation, the genes in pIP811 conferring resistance to chloramphenicol, erythromycin, and streptomycin were closely related to plasmid-borne determinants that are common in enterococci-streptococci. Plasmid pIP811 shared extensive sequence homology with pAM beta 1, the prototype broad host range resistance plasmid in these two groups of gram-positive cocci. These results suggest that emergence of multiple antibiotic resistance in Listeria spp is due to acquisition of a replicon originating in enterococci-streptococci. The dissemination of resistance to other strains of L monocytogenes is likely.

Several staphylococcal plasmids from different incompatibility (inc) groups which replicate by a rolling circle mechanism each specify a replication initiator protein (Rep) which is homologous with that of the inc3 tetracycline resistance plasmid pT181. The rep gene sequences of six pT181-like plasmids are known, each encoding proteins of molecular mass 38 kDa with 62% overall amino acid sequence identity. The initiation of replication in vivo by each of the Rep proteins is plasmid specific, acting in trans only at the cognate replication origin (ori) of the encoding plasmid. Previous studies in vitro of the RepC protein of pT181 demonstrated replication initiator, topoisomerase-like, and DNA binding activities, which appeared to be specific for the origin (oriC) of pT181 when compared with unrelated staphylococcal plasmids. Although RepD, specified by the inc4 chloramphenicol resistance plasmid pC221, has a range of activities similar to those noted previously for RepC, manipulation of in vitro conditions has revealed discrete steps in the overall reaction of RepD with oriD. In addition, factors have been identified which are necessary not only for sequence-dependent discrimination in vitro by Rep proteins for all pT181-like plasmids but also for the absolute specificity of RepD for its cognate pC221 replication origin (oriD), the latter occurring in vivo and a function of the topological state of the ori-containing target DNA. Here we also demonstrate the presence of a covalent phosphoryl-tyrosine linkage between the RepD protein of plasmid pC221 and an oligonucleotide substrate corresponding to its replication origin (oriD). The reactive tyrosine (Tyr-188) was identified from amino acid sequences of 32P-labeled peptide-oligonucleotide fragments. Substitution of Tyr-188 with phenylalanine confirms the importance of the tyrosyl hydroxyl group since the Y188F protein retains the sequence-specific DNA-binding capabilities of wild-type RepD but is unable to attach covalently to the replication origin or participate in the nicking-closing reaction in vitro.

We studied in vivo transcription and gene expression directed by a series of synthetic sequences, bearing the consensus hexamer (CH) pair of E.coli promoters in various contexts. The results demonstrate that, for the contexts tested, the CH pair supports transcription activity and gene expression, whether the spacer linking them is AT or GC rich, or is as short as 14 bp or as large as 26 bp (standard size 17 bp). However, we find that the context influences transcription efficiency by as much as an order of magnitude, and is able to scatter transcription start sites over a region of as much as 30 bp, including start sites within a CH or even between the two sequences of the CH pair. The results demonstrate that, although the CH pair can be sufficient for directing transcription by E.coli RNAP, important determinants for promoter activity are at least in part contained in the context of the consensus sequences; they advocate a synergic interplay of signals borne by the CH pair and its context, extending over all parts of the promoter sequence. A two-step model is proposed, in which properly located consensus sequences provide RNAP with facilities required for stereospecific docking along the promoter sequence; the result would be a sharp change in the local environment of the double helix inducing local isothermal unwinding. The size of the loop (related to the AT constraint in the promoter) and the extend of the environmental change required for unwinding would determine the rate of transcriptionally competent complex formation, positioning and grouping of start sites.

A 6.5 kb DNA fragment containing a chloramphenicol-resistance gene of Streptomyces venezuelae ISP5230 was cloned in Streptomyces lividans M252 using the high-copy-number plasmid vector pIJ702. The gene was located within a 2.4 kb KpnI-SstI fragment of the cloned DNA and encoded an enzyme (chloramphenicol hydrolase) that catalysed removal of the dichloroacetyl moiety from the antibiotic. The deacylated product, p-nitrophenylserinol, was metabolized to p-nitrobenzyl alcohol and other compounds by enzymes present in S. lividans M252. Examination of the genomic DNA from several sources using the cloned 6.5 kb SstI fragment from S. venezuelae ISP5230 as a probe showed a hybridizing region in the DNA from S. venezuelae 13s but none in the DNA from another chloramphenicol producer, Streptomyces phaeochromogenes NRRLB 3559. The resistance phenotype was not expressed when the 6.5 kb SstI fragment or a subfragment was subcloned behind the lac-promoter of plasmid pTZ18R in Escherichia coli.

Four plasmids encoding restriction and modification (R/M) systems are described that are different in the specificity of their restrictive activity toward the small isometric phage p2 and prolate phage c2. The R/M plasmids were cotransformed into Lactococcus lactis MG1363 with pVS2, encoding resistance to chloramphenicol and erythromycin, to indicate successful transformation events. Analysis of cotransformants showed that three different R/M plasmids could be combined in L. lactis MG1363. The efficiency at which phage plaqued on the transformants decreased as the number of R/M plasmids increased. Some plasmid combinations were unstable suggesting replicon incompatibility.

Transduction of Escherichia coli W3110(R702) and J53(RP4) (10(4) to 10(5) CFU/g of soil) by lysates of temperature-sensitive specialized transducing derivatives of bacteriophage P1 (10(4) to 10(5) PFU/g of soil) (P1 Cm cts, containing the resistance gene for chloramphenicol, or P1 Cm cts::Tn501, containing the resistance genes for chloramphenicol and mercury [Hg]) occurred in soil amended with montmorillonite or kaolinite and adjusted to a -33-kPa water tension. In nonsterile soil, survival of introduced E. coli and the numbers of E. coli transductants resistant to chloramphenicol or Hg were independent of the clay amendment. The numbers of added E. coli increased more when bacteria were added in Luria broth amended with Ca and Mg (LCB) than when they were added in saline, and E. coli transductants were approximately 1 order of magnitude higher in LCB; however, the same proportion of E. coli was transduced with both types of inoculum. In sterile soil, total and transduced E. coli and P1 increased by 3 to 4 logs, which was followed by a plateau when they were inoculated in LCB and a gradual decrease when they were inoculated in saline. Transduction appeared to occur primarily in the first few days after addition of P1 to soil. The transfer of Hg or chloramphenicol resistance from lysogenic to nonlysogenic E. coli by phage P1 occurred in both sterile and nonsterile soils. On the basis of heat-induced lysis and phenotype, as well as hybridization with a DNA probe in some studies, the transductants appeared to be the E. coli that was added. Transduction of indigenous soil bacteria was not unequivocally demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

To study recombination between short homologous sequences in Escherichia coli we constructed plasmids composed of the pBR322 replicon, M13 replication origin and a recombination unit inserted within and inactivating a gene encoding chloramphenicol resistance. The unit was composed of short direct repeats (9, 18 or 27 bp) which flanked inverted repeats (0, 8 or 308 bp) and a gene encoding kanamycin resistance. Recombination between direct repeats restored a functional chloramphenicol resistance gene, and could be detected by a simple phenotype test. The plasmids replicated in a double-stranded form, using the pBR322 replicon, and generated single-stranded DNA when the M13 replication origin was activated. The frequency of chloramphenicol-resistant cells was low (10(-8)-10(-4] when no single-stranded DNA was synthesized but increased greatly (to 100%) after induction of single-stranded DNA synthesis. Recombination between 9 bp direct repeats entailed no transfer of DNA from parental to recombinant plasmids, whereas recombination between 18 or 27 bp repeats entailed massive transfer. The presence or length of inverted repeats did not alter the pattern of DNA transfer. From these results we propose that direct repeats of 9 bp recombine by a copy choice process, while those greater than or equal to 18 bp can recombine by a breakage-reunion process. Genome rearrangements detected in many organisms often occur by recombination between sequences less than 18 bp, which suggests that they may result from copy choice recombination.

The involvement of GATC sites in directing mismatch correction for the elimination of replication errors in Escherichia coli was investigated in vivo by analyzing mutation rates for a gene carried on a series of related plasmids that contain 2, 1 and 0 such sites. This gene encoding chloramphenicol acetyl transferase (Cat protein) was inactivated by a point mutation. In vivo mutations restoring resistance to chloramphenicol were scored in mismatch repair proficient (mut+) and deficient (mutHLS-) strains. In mut+ cells, reduction of GATC sites from 2 to 0 increased mutation rates approximately 10-fold. Removal of the GATC site distal to the cat- mutation increased the rate of mutation less than 2-fold, indicating that mismatch repair can proceed normally with a single site. The mutation rate increased 3-fold after removal of the GATC site proximal to the mutation. In the absence of a GATC site, mutL- and mutS- strains exhibited a 2- to 3-fold increased mutation rate as compared to isogenic mutH- and mut+ strains. This indicates that 50%-70% of replication errors can be corrected in a mutLS-dependent way in the absence of any GATC site to target mismatch correction to newly synthesized DNA strands. Other strand targeting signals, possibly single strand discontinuities, might be used in mutLS-dependent repair.

A 4-month-old infant with congenital heart disease and sepsis and arthritis, and subsequently meningitis, caused by an antibiotic-resistant strain of Haemophilus influenzae type b, failed to respond to sequential therapy with ampicillin and trimethoprim/sulfamethoxazole. Following treatment with ceftizoxime, the infant was well for 42 days, until he returned to the hospital and died. A total of 10 Haemophilus influenzae type b isolates, all outer membrane protein subtype 51, was isolated from the pretreatment blood and synovium, cerebrospinal fluid and subdural fluids, and the petrous pyramids at autopsy. Pretreatment isolates had no detectable plasmid DNA, chloramphenicol acetyltransferase or beta-lactamase; the minimal inhibitory concentration for ampicillin (AM) and chloramphenicol (CM) was 0.2 and 0.8 microgram/ml, respectively. However, all cerebrospinal fluid isolates had a 42-44 mD plasmid and produced chloramphenicol acetyltransferase and beta-lactamase; the minimal inhibitory concentration of these isolates to AM and CM were 12.5 and 25 micrograms/ml, respectively, and were also resistant to tetracycline and sulfonamide. Resistance to AM and CM was cotransferred by filter-mating conjugation at a frequency of one to two transconjugants per 10(5) to an Rd haemophilus recipient. Posttreatment isolates from the petrous pyramids also were resistant to AM and CM and produced chloramphenicol acetyltransferase and beta-lactamase activity, but had no plasmid DNA. These findings and data from genetic studies suggested that plasmid-bearing antibiotic-resistant Haemophilus influenzae type b was selected from a heterogenous population, and that the AM/CM resistance transposons were incorporated into the bacterial chromosome.

A small plasmid of 3.95 kb, encoding resistance to chloramphenicol (Cm) was detected in three of 33 Staphylococcus hyicus strains. The plasmid in each of the three strains was indistinguishable by Southern-blot hybridization and restriction enzyme analysis. It was shown by curing and by transformation to specify resistance to Cm. A preliminary restriction map of the plasmid, designated pSC2, is presented. Chloramphenicol acetyltransferase was demonstrated by enzyme assay and by SDS-PAGE of cell-free lysates of pSC2 transformants.

The relationship between an unstable argG gene and a 5.7-kilobase (kb) amplifiable DNA sequence in Streptomyces lividans 66 was investigated. Spontaneous, high-frequency Arg mutants deleted for this gene typically contain 200 to 300 copies of the tandemly reiterated sequence. A library of S. lividans 66 (strain 1326) wild-type genomic DNA was prepared in the vector lambda Charon 35. Chromosome walking over 44 kb established that argG is located 25 kb distant from a duplicated amplifiable DNA structure. A sequence was characterized, located farther distal from the amplifiable structure, containing strong homology with an internal sequence of the amplifiable DNA, which may have a role in the deletion of argG. Genetic mapping showed that argG and the 5.7-kb amplifiable sequence are linked to another unstable gene, determining chloramphenicol resistance (Camr) and that together these genes may be located in a silent chromosomal arc.

We constructed a 7.9-kilobase-pair recombinant shuttle plasmid, designated pHR106, by combining desired segments of three plasmids: an Escherichia coli plasmid (pSL100) which provides a multiple cloning site, a Clostridium perfringens plasmid (pJU122) which provides a clostridial origin of replication, and an E. coli plasmid (pJIR62) which provides an E. coli origin of replication, an ampicillin resistance gene, and a chloramphenicol resistance gene of clostridial origin. The shuttle plasmid transformed E. coli HB101 with a frequency of 1 transformant per 10(4) viable cells and C. perfringens L-phase strain L-13 with a frequency of approximately 1 transformant per 10(6) viable cells. Because of the set of unique cloning sites and the chloramphenicol resistance marker, this shuttle plasmid should be particularly useful for studies of gene regulation and for enzyme production with C. perfringens.

The mitochondrial deoxyribonucleic acids (mtDNA's) from human HeLa and HT1080 cells differed in their restriction endonuclease cleavage patterns for HaeII, HaeIII, and HhaI. HaeII digestion yielded a 9-kilobase fragment in HT1080, which was replaced by 4.5-, 2.4-, and 2.1-kilobase fragments in HeLa. HaeIII and HhaI yielded distinctive 1.35- and 0.68-kilobase HeLa fragments. These restriction endonuclease polymorphisms were used as mtDNA markers in HeLa-HT1080 cybrid and hybrid crosses involving the cytoplasmic chloramphenicol resistance mutation was used. mtDNA's were purified and digested with the restriction endonucleases, the fragments were separated on agarose gels, and the bands were detected by ethidium bromide staining and Southern transfer analysis. Three cybrids and four hybrids (four expressing HeLa and three expressing HT1080 chloramphenicol resistance) contained 2- to 10-fold excesses of the mtDNA of the chloramphenicol-resistant parent. One cybrid, which was permitted to segregate chloramphenicol resistance and was then rechallenged with chloramphenicol, had approximately equal proportions of the two mtDNA's. Only one hybrid was discordant. These results indicated that chloramphenicol resistance is encoded in mtDNA and that expression of chloramphenicol resistance is related to the ratio of chloramphenicol-resistant and -sensitive genomes within cells.

The chloramphenicol resistance determinant from a nonconjugative strain of Clostridium perfringens was cloned and shown to be expressed in Escherichia coli. Subcloning and deletion analysis localized the resistance gene, catQ, to within a 1.25-kilobase (kb) partial Sau3A fragment. The catQ gene contained internal HindII, HaeIII, and DraI restriction sites and was distinct from the catP gene, which was originally cloned (L. J. Abraham, A. J. Wales, and J. I. Rood Plasmid 14:37-46, 1985) from the conjugative C. perfringens R plasmid, pIP401. Hybridization studies were carried out with a 0.35-kb DraI-P fragment of pJIR260 as an internal catQ-specific probe and a 0.38-kb EcoRV-HinfI fragment of pJIR62 as an internal catP-specific gene probe. The results showed that the catP and catQ genes were not similar and that neither probe hybridized with cat genes from other bacterial genera. However, the catP gene was similar to the cloned catD gene from Clostridium difficile. Comparative studies with both catP and catD probes showed that these genes had significant restriction identity. We therefore suggest that these genes were derived from a common source.

Southern blot hybridization and pneumococcal transformation were used to study the epidemiology at a molecular level of the genes for chloramphenicol resistance (cat) in streptococci and staphylococci. The cat gene of staphylococcal plasmid pC194 showed homology to the cat genes of the chromosomal elements of 5 different strains of Streptococcus pneumoniae and of Streptococcus agalactiae B109. DNA sequence homology was also detected between the cat gene of staphylococcal plasmid pC221 and the cat gene of broad host range conjugative plasmid pIP501, originally isolated from S. agalactiae. Two different cat genes appear to be present in clinical isolates of both streptococci and staphylococci.

The results indicative of chromosomal localization of the unstable chloramphenicol resistance determinant in Streptomyces coelicolor A3(2) have been obtained. Independent mutations specifying chloramphenicol sensitivity in different strains of S. coelicolor A3(2), S18 and A617M are localized in the same region flanked by markers argA1 and cysD18 on the genetic map. Mutations restoring chloramphenicol resistance are also localized in this region. Different locations of the genetically unstable determinant of chloramphenicol resistance detected in various laboratories are discussed, in relation to the results showing that transfer to chloramphenicol sensitivity is due to a set of various rearrangements (deletions, amplifications, deamplifications, etc.), differing in separate variants.

A deletion htpR mutant of Escherichia coli has been constructed on the basis of site-directed mutagenesis. To this end, the chromosomal allele of htpR gene was substituted by a mutant allele introduced into the cell with a recombinant plasmid. The htpR mutant is characterized by a reduced level of proteolysis and therefore by a decreased rate of proteolytic degradation of RNA polymerase of bacteriophage T7. The mutation in htpR is linked with chloramphenicol resistance.

Streptomyces lividans 66 exhibits genetic instability, involving sequential loss of resistance to chloramphenicol (Cams) and subsequent mutation of argG. Associated with this instability is the amplification of a 5.7-kilobase (kb) amplified DNA sequence (ADS). We have characterized a second, independent pathway of genetic instability, involving sequential loss of resistance to tetracycline (Tets) followed by mutation in nitrogen assimilation (Ntr). We detected DNA amplification in many of these mutant strains, as well as other reiterations coresident with the 5.7-kb ADS in Cams Arg mutants. However, in contrast to the 5.7-kb ADS, none of the novel elements were observed to amplify at high frequency. The mutation of argG is due to a deletion, one endpoint of which is defined by the 5.7-kb ADS. This amplification derives from a structure, the tandemly duplicated amplifiable unit of DNA (AUD), present in the wild-type genome. We found that progenitor strains containing just a single-copy AUD failed to reproducibly generate amplification of this element in Cams argG mutants, and DNA deletion endpoints proximal to the element were found to be unspecific. These results suggest that a duplicated AUD structure is required for high-frequency amplification and that this reiteration can subsequently buffer the extent of deletion formation in the relevant chromosomal region.

Expression of xylose isomerase was repressed in Bacillus subtilis strains W23, 168, and BR151 and could be induced in the presence of xylose. The expression was also glucose repressed in strains 168 and BR151, although this effect was not observed with W23. A xyl-cat fusion gene was constructed on a multicopy plasmid, from which the xyl promoter located on a 366-base-pair (bp) DNA fragment derived from W23 directed the expression of chloramphenicol resistance. The regulation of expression was not very pronounced in this multicopy situation. The xyl promoter is a strong signal for transcription initiation. The 5' sequence of the xyl mRNA was identified by nuclease S1 mapping. The promoter consisted of the -10 sequence TAAGAT, the -35 sequence TTGAAA spaced by 17 bp, and an upstream poly(A) block with 14 As out of 17 bp. To study the regulation, a xyl-lacZ fusion gene was constructed and integrated as a single copy into the amygene of B. subtilis 168. This strain grows blue on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) indicator plates in the presence of xylose and white in the presence of glucose. Quantitatively, the induction of beta-galactosidase by xylose was 100-fold. In the presence of xylose plus glucose, the expression of the indicator gene was repressed to 30% of the fully induced level. About 25 to 60% of the maximal lacZ expression was obtained with this strain when the 366-bp xyl DNA fragment was provided in trans on a multicopy plasmid. This result indicates that repression in the absence of xylose is mediated in trans by a soluble factor which is expressed at a low level in B. subtilis 168. The xylose effect depended on negative regulation. The estimations of mRNA amounts by dot blot analysis showed unambiguously that the induction by xylose occurs at the level of transcription. The possible molecular mechanisms are discussed with respect to the nucleotide sequence of the 366-bp xyl regulatory DNA.

An efficient method for DNA transfer is essential for the genetic manipulation of any organism. Such a capacity will be required for the genetic analysis of Actinobacillus pleuropneumoniae as a swine pathogen, as well as for its manipulation for vaccination purposes. For this reason, the use of electroporation as a means of plasmid DNA introduction into this species was examined. The multiple antibiotic-resistant strain 80-8141 of Actinobacillus pleuropneumoniae harbors 3 plasmids: pYG10, pYG15, and pYG12 of 5.0, 2.7, and 2.5 kb, respectively. Electroporation of A pleuropneumoniae strain 4074 with a plasmid extract of strain 80-8141 showed that pYG10 encodes chloramphenicol resistance and that pYG12 encodes ampicillin resistance. Electrical pulse conditions for efficient electroporation of strain 4074 were examined by use of pYG10 DNA isolated from a 4074 transformant. Efficiency, expressed as transformants per microgram of plasmid DNA, increased directly with pulse amplitude. However, high efficiencies were only observed in a narrow window of pulse duration (tau = 12 to 22 ms at 6.25 kV/cm). Longer pulse durations resulted in cell death. Electroporation efficiencies increased with cell density. Yield of transformants increased directly with DNA concentration. Results indicate that electroporation can be used to efficiently transform A pleuropneumoniae and that pYG10 and pYG12 are suitable plasmid vectors for use in the genetic manipulation of this organism.

We have constructed a small, transposition-defective derivative of the transposon Tn10 that carries the chloramphenicol acetyltransferase gene of pACYC184. This new genetic element, Tn10d-Cam, transposes when Tn10 transposase is provided from a multi-copy plasmid. Transposon insertion mutagenesis of Salmonella typhimurium was performed by using a strain carrying a Tn10d-Cam insertion in an Escherichia coli F' episome as the donor in transductional crosses into recipients that carried a plasmid expressing Tn10 transposase. Tn10d-Cam insertion mutations were also generated by complementation in cis of Tn10d-Cam by a cotransducible Tn10 element that overproduces transposase. Here, transposase was provided only transiently, and the Tn10d-Cam insertion mutations were recovered in a transposase-free strain. Cis complementation was used for mutagenesis of a plasmid target. The site specificity of insertion and the effect of insertions on expression of a downstream gene were investigated, using Tn10d-Cam insertions in a plasmid carrying a segment of the histidine operon.

The isopropylmalate isomerase of Salmonella typhimurium and Escherichia coli is a complex of the leuC and leuD gene products. The supQ/new D gene substitution system in S. typhimurium restores leucine prototrophy to leuD mutants of S. typhimurium. Previous genetic evidence supports a model that indicates the replacement of the missing LeuD polypeptide by the newD gene product. This model proposed that this gene substitution is possible when a mutation at the supQ locus (near newD) liberates unaltered newD polypeptide from its normal complex with the supQ protein product. In this study, recombinant plasmids carrying newD, supQ, or both were transformed into E. coli and S. typhimurium strains deleted for the leuD and supQ genes to test the supQ/newD gene substitution model for suppression of leucine auxotrophy. It was determined that the newD gene encodes a 22-kilodalton polypeptide which can restore leucine prototrophy to leuD deletion strains and that a functional supQ gene prevents this suppression. It was also determined that the supQ and newD genes are separated by a gene encoding a 50-kilodalton protein, pB. While there is extensive DNA sequence homology between the leucine operons of S. typhimurium and E. coli, DNA hybridization experiments did not indicate substantial homology between the newD and leuD genes. These data, taken together with previously obtained genetic data, eliminate the possibility that supQ and newD are recently translocated segments of the leucine operon.

The frequency of chloramphenicol-sensitive variants (Cmls) in Streptomyces lividans 66 is very high (0.57%). Correlation between chloramphenicol sensitivity and deamplification of PstI fragment with the length of 4.82 kb (RES1 genetic element) was shown. However, in some Cmls variants there was no RES1 deamplification. It was noted that in the cells of the Cmls variants isolated the levels of kanamycin and neomycin resistance determined by the Kanr determinant in the pSU17 plasmid were different. Expression of Kanr and Neor determinants inserted via pSU17 plasmid into the cells of Cmls variants was studied and three classes of chloramphenicol-sensitive variants were defined. After transformation of pSU17 plasmid into cells of Cmls variants of the class I, expression of Kanr and Neor genes, similar to that in S. lividans 66, was observed. The resistance level in Cmls variants of the class II was intermediate. In the cells of the class III no expression was noted. Cmls strains of classes I and II were unstable and those of the class III with impaired expression of Kanr and Neor genes were formed with high frequency. Cmlr variants formed from Cmls strain of the class III were studied. Two types of Cmlr variants were detected. Variants of the first type were identical to S. lividans 66 by their properties. The frequency of Cmls variants occurring in the cells of the first type was similar to that in S. lividans 66. The second type included pseudo-revertants. They were unstable and generated amplifications of the 5.7 kb fragment with high frequency.