Characterization of chloramphenicol resistance inLactobacillus plantarum caTC2

A strain ofLactobacillus plantarum caTC2R isolated from a meat source was resistant to chloramphenicol (30 g/ml). Resistance was mediated through an inducible chloramphenicol acetyltransferase. Plasmid analysis of this strain showed three plasmids, of which the 8.5-kb plasmid apparently encodes the gene for chloramphenicol resistance. This plasmid was lost at high frequency (25%) when theLactobacillus was subcultured at a higher than optimal temperature (40°C).     

Thermostability and superhelicity of plasmid DNA in Bacillus stearothermophilus

Summary The thermostability of the staphylococcal plasmids pC194 and pUB110 and their antibiotic-resistance determinants was examined upon transfer to Bacillus stearothermophilus CU21. Plasmid pGS13, a pUB110 derivative carrying the chloramphenicol acetyltransferase (CAT) gene of pC194, could be maintained up to the maximum growth temperature (68° C) by selection for chloramphenicol resistance. In the absence of selective pressure, pGS13 was lost at temperatures above 60° C. Segregational instability of pGS13 was accompanied by a progressive loss of negative superhelicity at elevated temperatures. Thermostable mutants of pGS13 were isolated by screening for expression of the antibiotic-resistance determinants after growth under non-selective conditions. These mutants were found to contain an insertion of a 1.7 kb DNA sequence derived from the cryptic B. stearothermophilus plasmid pBS02. Increased thermostability correlated with preservation of plasmid superhelicity at elevated temperatures.     

Effects of some curing agents on phenotypic stability in Pseudomonas putida degrading ε-caprolactam

A strain of Pseudomonas putida MCM B-408 capable of utilizing -caprolactam (monomer of nylon-6) as the sole source of carbon and nitrogen was found to harbour a single 32-kb plasmid with the same electrophoretic mobility as that of pARI180, a reference plasmid. Acridine orange, ethidium bromide, mitomycin C and SDS failed to cure the plasmid and the phenotype. Elevated temperature alone (40°C) was found to be ineffective in curing. Phenotype, but not the plasmid, was cured at a frequency of 2.63% when acridine orange and elevated temperature (40°C) were used together. The studies therefore indicated that the phenotypic expression of caprolactam degradative genes is quite stable and that Pseudomonas putida MCM B-408 may degrade -caprolactam from waste-water satisfactorily without spontaneous loss of the property under adverse environmental conditions.     

Improvement of plasmid encoded lactococcal bacteriophage resistance by mutator strain Epicurian coli XL1-Red

A random mutation strategy using mutator strain, Epicurian coli XL1-Red, was applied to a plasmid, pND018, constructed by inserting a Lactococcus lacis bacteriophage resistance gene (abiI) into a L. lactis/E. coli shuttle vector (pDL278), to introduce random mutations throughout the plasmid. Following transformation of the mutated plasmid library to a plasmid free and phage sensitive strain of L. lactis (LM0230), mutated plasmids were screened by cross-streaking and efficiency of plaquing (EOP) assays. Two strains with enhanced resistance were obtained, as well as several phage sensitive strains. Repeated transformation of the mutated plasmids to LM0230 confirmed that the observed phenotypes were caused by mutations located on the plasmids. The EOP values and plaque morphology of two enhanced phage resistance mutants were characterized at 30°C and 37°C. These results indicate that this simple procedure can be applied to generate modified plasmids with improved phage resistance, which may be of commercial value.     

Instability of plasmid-encoded citrate permease in Leuconostoc

M. KIHAL, H. PRÉVOST, M.E. LHOTTE, D.Q. HUANG AND C. DIVIÈS. 1996. The conversion from citrate positive (Cit⁺) to citrate negative (Cit^-) phenotype of six strains of Leuconostoc mesetiteroides was followed during growth in milk and buffered or unbuffered MRS medium at 30 or 37°C. High rate of loss of Cit⁺ phenotype was observed. The Cit^- phenotype was found to be linked to the loss of 22 to 23 kb plasmids. All Cit^- mutants isolated from Leuc. mesenteroides subsp. cremoris 195 reverted spontaneously to the Cit⁺ phenotype. Hybridization experiments using a 0.8 kb fragment of the citP gene of Leuc. mesenteroides showed that all the plasmids which were lost in Cit^- mutants encoded for a citrate permease. However, neither plasmid nor genomic DNA from Leuc. mesenteroides subsp. cremoris 195 hybridized with the citP probe.     

Cryptic plasmid pBf1 fromButyrivibrio fibrisolvens AR10: Its use as a replicon for recombinant plasmids

A cryptic 2.85 kb plasmid (pBf1) was isolated from the rumen bacteriumButyrivibrio fibrisolvens strain AR10, ampped with restriction endonucleases, and cleavage sites suitable for attachment toEscherichia coli plasmids were identified. AR10 was not able to be cured of pBf1 by growth at 42°C or in 0.25 g ampicillin/ml, but growth in 50 g acridine orange/ml for three culture passages produced cured colonies at a frequency of <1%. Chimeric plasmids were constructed by combining pBf1 with theE. coli plasmid pUC18, in addition to the clindamycin resistance gene fromBacteroides fragilis plasmid pDP1 (pCW2 and pCW3), or the CAT gene fromE. coli plasmid pKK232-8 (pCK1). For plasmid construction, pBf1 was cleaved at two alternative restriction sites to increase the likelihood that replication control sequences would remain functional in at least one of the plasmids. Electroporation of AR10 yielded transformant populations that clearly maintained the plasmids and that appeared to express the ampicillinase gene of pUC18, although transformants were not readily selectable with any of the three antibiotics. The suitability of pBf1 as a replicon on which to base the construction of shuttle vectors was demonstrated clearly, by persistence of plasmid pCW3 in the absence of selective pressure, and the addition of appropriate selection factors is expected to yield practical transformation vectors.     

Altered DNA-protein relaxation complex in a replication mutant of plasmid ColE1

Summary Approximately 200,000 clones of Escherichia coli carrying mutagen-treated colicinogenic plasmid E1 (ColE1) were examined for irreversible loss of the plasmid at 43°. Thirty of these clones that appeared to be most defective in plasmid DNA replication at the non-permissive temperature were selected for the study of: (a) the kinetics of plasmid and chromosomal DNA replication during a temperature shift in either the presence or absence of chloramphenicol; (b) the temperature stability of the plasmid DNA-protein relaxation complex; and (c) the temperature effect on F-promoted conjugal transfer. Two mutant plasmids, pJC307 and pJC301, showed defects in their relaxation complex. The relaxation complex of pJC307 exhibited an altered temperature stability in vitro. Reversion to temperature resistant replication resulted in four out of five cases in a concomitant change in the temperature stability of the relaxation complex. Conjugal mobility of this mutant was not markedly reduced at the permissive or non-permissive temperature. Plasmid pJC301 could not be isolated in the form of a relaxation complex and it was very poorly mobilized in an F-promoted conjugation. These results indicate that the ColE1 plasmid codes for at least one of the proteins of the relaxation complex and that the relaxation complex is involved in ColE1 DNA replication. In addition, the properties of the mutant plasmid pJC301 are consistent with a role for the complex in the mobilization of ColE1 during conjugation.     

Mutagenesis of plasmid DNA with hydroxylamine: Isolation of mutants of multi-copy plasmids

Summary An investigation of in vitro mutagenesis of plasmid DNA with hydroxylamine is described. The treated plasmid DNA was used to transform Escherichia coli K12. Mutants of the plasmid NTP3, which codes for resistance to ampicillin and sulphonamides, were isolated and characterised. They were classified according to the reduction in level of their -lactamase activity. Hydroxylamine-induced mutants of NTP14 were also isolated. This plasmid codes for ampicillin resistance, synthesis of colicin E1, and the EcoRI restriction and modification enzymes. One class of mutants is lethal to the host strain at temperatures above 33° C, but carrier strains grow well at 28° C. There is evidence that these mutants code for a temperature-sensitive EcoRI modification activity: the lethal effect probably results from the cleavage of the host-cell DNA by the restriction enzyme at non-permissive temperatures. The possible genetic uses of the mutant plasmids for the production of hybrid plasmids in the bacterial cell are discussed.     

The RAD3 gene of Saccharomyces cerevisiae: isolation and characterization of a temperature-sensitive mutant in the essential function and of extragenic suppressors of this mutant

Summary Mutations in the RAD3 gene of Saccharomyces cerevisiae were generated by integration of a mutagenized incomplete copy of the cloned gene into wild-type cells. Integrants were mass screened for colonies with abnormal growth characteristics at 37°C. A single temperature-sensitive mutant (rad3ts-1) was isolated and was shown to result from a missense mutation at codon 73 of the RAD3 gene. When shifted from 30° C to 37° C the strain undergoes only 2–4 cell doublings. This phenotype can be rescued by plasmids in which the essential function of the cloned RAD3 gene is intact, but not plasmids in which this function is inactivated. The mutant strain is weakly sensitive to ultraviolet (UV) radiation at restrictive temperatures. Measurement of RNA, DNA and protein synthesis at various times after shifting to restrictive temperatures does not show preferential inactivation of any one of these parameters and the temperature-sensitive mutation does not cause arrest at any specific phase of the cell cycle. The rad3ts-1 strain was transformed with multicopy plasmids from a normal yeast genomic library and two plasmids that partially suppress the temperature-sensitive phenotype were isolated. These suppressor genes (designated SRE1 and SRE2) are distinct from RAD3 and do not suppress the phenotype of several other temperature-sensitive mutants tested. Mutant strains carrying disruptions of the SRE1 gene are viable and are not sensitive to UV or radiation.     

Dispensability of either penicillin-binding protein-1a or -1b involved in the essential process for cell elongation in Escherichia coli

Summary A strain of Escherichia coli lacking the entire ponB gene and a strain lacking the proximal part of the ponA gene were constructed by substitution with a drug resistance gene. These strains lost either penicillin-binding protein(PBP) -1b or -1a totally and their growth was apparently normal at 30°C and 42°C except that growth of the ponB deletion strain was poor on a nutrient agar plate containing no NaCl at 30°C as well as at 42°C. Transductional experiments to introduce the ponB deletion into the ponA deletion strain, and vice versa, showed that the ponA ponB double deletion was lethal unless the deletion was functionally compensated, e.g., by the presence of a plasmid carrying either gene. Thus, either PBP-1b (ponB) or PBP-1a (ponA), but not both, is dispensable for cell viability, at least under ordinary culture conditions. Transductional experiments also suggested that the component of PBP-1b or the PBP-1b lacking the C-terminal portion encoded in the distal region to the SphI site on the ponB was sufficient for supporting growth of the E. coli cell.Abbreviations Ap ampicillin - Cm chloramphenicol - Km kanamycin - Tc tetracycline     

Comparative Genomics of the IncA/C Multidrug Resistance Plasmid Family

Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.Antimicrobial compounds have been used extensively in agriculture since the 1960s not only to treat and prevent disease in plants, fruits, vegetables, and animals but also to promote growth in fish, poultry, and other livestock (42). The risk of transferring antimicrobial drug resistance to nonresistant bacteria and the propagation of multidrug-resistant (MDR) bacteria from agricultural to clinical and/or community-associated settings are being debated by research, regulatory, and health authorities (27, 28). In this context, the recent discovery of a group of self-transferable IncA/C antimicrobial resistance plasmids, which are widely distributed among agricultural nontyphoidal Salmonella enterica isolates from the United States (24, 45) has caused considerable concern in the public health community. Similar IncA/C plasmids were identified in an MDR isolate from Madagascar of Yersinia pestis, the causative agent of the plague (16), and MDR strains of Vibrio cholerae O139 from China (34), as well as in MDR isolates of the fish pathogen Photobacterium damselae subsp. piscicida from the United States and Japan (21). While the IncA/C group of MDR plasmids seems to be efficient in collecting antimicrobial resistance traits and mobilizing them across geographical and taxonomical borders, little is known about the evolutionary origin of these plasmids or the genetic basis for their spread.The IncA/C reference plasmid, pRA1, was isolated in 1971 from the fish pathogen Aeromonas liquefaciens, later renamed Aeromonas hydrophila, as a transferable antimicrobial resistance plasmid conferring resistance to sulfonamides and tetracyclines (2). The repA gene of pRA1, located at the origin of replication and responsible for encoding the replication initiation protein A, has been sequenced (25) and is used for PCR-based replicon typing of IncA/C plasmids (7). repA genes from all sequenced IncA/C plasmids to date share at least 98% nucleotide sequence identity.To better understand the evolutionary origin of IncA/C plasmids, pRA1 was isolated, sequenced, and compared to all IncA/C plasmid sequences currently available. In addition to pRA1, a pRA1-derived cryptic IncA/C plasmid, designated pRAx, was also sequenced and included in the analysis. pRAx was isolated from Escherichia coli D7-3, a strain that was obtained through the conjugative transfer of pRA1 from A. hydrophila in 1980 (30). While the laboratory history of the pRAx-carrying strain E. coli D7-3 since the conjugative plasmid acquisition is unknown, pRAx was included in this study as it tested positive for the repA reference gene from pRA1 (100% nucleotide sequence identity) but negative for 11 out of 12 additional IncA/C marker genes that were shown to be part of a conserved plasmid backbone shared by recently isolated IncA/C plasmids (45).     

Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli

Summary The in vivo role of the Escherichia coli protein DnaA in the replication of plasmid pBR322 was investigated, using a plasmid derivative carrying an inducible dnaA ⁺ gene. In LB medium without inducer, the replication of this plasmid, like that of pBR322, was inhibited by heat inactivation of chromosomal DnaA46 protein so that plasmid accumulation ceased 1 to 2 h after the temperature shift. This inhibition did not occur when the plasmid dnaA ⁺ gene was expressed in the presence of the inducer isopropyl-1-thin--d-galactopyranoside (IPTG). Inhibition was also not observed in glycerol minimal medium or in the presence of low concentrations of rifampicin or chloramphenicol. Deletion of the DnaA binding site and the primosome assembly sites (pas, rri) downstream of the replication origin did not affect the plasmid copy number during exponential growth at 30° C, or after inactivation of DnaA by a shift to 42° C in a dnaA46 host, or after oversupply of DnaA, indicating that these sites are not involved in a rate-limiting step for replication in vivo. The accumulation of the replication inhibitor, RNAI, was independent of DnaA activity, ruling out the possibility that DnaA acts as a repressor of RNAI synthesis, as has been suggested in the literature. Changes in the rate of plasmid replication in response to changes in DnaA activity (in LB medium) could be resolved into an early, rom-dependent, and a late, rom-independent component. Rom ^– plasmids show only the late effect. After heat inactivation of DnaC, plasmid replication ceased immediately. These results, together with previously published reports, suggest that DnaA plays no specific role during in vivo replication of ColE1 plasmids and that the gradual cessation of plasmid replication after heat inactivation of DnaA in LB medium results from indirect effects of the inhibition of chromosome replication and the ensuing saturation of promoters with RNA polymerase under nonpermissive growth conditions.     

Repressor defective mutants, virulent mutants and adsorption resistance of lysogenic cells in case of phage kappa of Serration

Summary Thermoconditional clearplaque mutants (c_t) of phage have been isolated, which as prophages can be induced by exposure of the 30°C-grown lysogenic bacteria to 42°C. The thermoinduction of the lysogenic cells needs protein synthesis as shown by the inhibitory effect of chloramphenicol. The c_t-mutations are located in clearplaque region III of the genetic map of . Thus this region contains information for the prophage repressor.-lysogenic cells are adsorption resistant against -phages except the cells with mutant prophages of clearplaque region I (l_I mutants). Virulent (v) mutants which can grow in l_I-lysogenic cells appear after direct plating of extracellularly uv irradiated phage with l_I-lysogenic indicator bacteria. Surprisingly all these mutants show a more or less reduced plaque forming ability at 37°C compared with 30°C. For one of the v-mutants the maximum thermosensitive phase in the latent period was checked and found to be very shortly before lysis.     

Nitrogen excretion in Streptocephalus Dichotomus Baird (Crustacea: Anostraca)

Summary The total nitrogen excreted by Streptocephalus dichotomus is 1.5177 mg/g/day for males and 1.3875 mg/g/day for females at 30°C.The main excretory product of S. dichotomus is ammonia (75.3%). The rate of excretion of ammonia increased with increase in temperature up to 30°C and at 33°C it decreased.Analyses of covariance revealed that the F ratios are not significant in the case of rate of total nitrogen excretion between the two sexes at 30°C and in the case of rate of ammonia excretion between the two sexes or when comparison is made within the sexes at the different temperatures between 20° and 30°C.This work formed part of the thesis submitted to the University of Madras for the award of the Degree of Doctor of Philosophy in 1970.     

Operational and storage stability of neutral β-mannanase from Bacillus licheniformis

The stability of neutral -mannanase from Bacillus licheniformis during operation and storage was investigated. The enzyme activity decreased by 70% with a hydrolysate of glucomannan at 20 g l^–1 over 30 min at 25 °C. In an enzymatic membrane reactor operated at 50 °C after 24 h, the loss of enzyme activities were 23% and 9% in the absence/presence of the substrate. The residual activities of the enzyme were 21% and 90%, respectively, when stored in 30% (v/v) glycerol solution and in solid state at 4 °C after one year.     

Conjugal transfer of lactose-fermenting ability fromStreptococcus lactis C2 toLeuconostoc cremoris CAF7 yieldsLeuconostoc that ferment lactose and produce diacetyl

Summary Conjugation between lactose-fermenting (Lac⁺)Streptococcus lactis C2 and Lac^– Leuconostoc cremoris CAF7 was performed. The frequency of Lac⁺ transfer was 1.5 · 10^–2 per donor cell. Lac⁺ Leuconostoc transconjugants could ferment lactose significantly faster than wild-type cells. When grown in litmus milk fortified with 0.2% yeast extract, Lac⁺ transconjugants reached pH 4.68 within 24 h at 30°C and produced diacetyl. The identity of the transconjugants asLeuconostoc derivatives was confirmed by their resistance to phage c2 and to vancomycin (>500 g/ml), and by growth on selective medium containing azide. Plasmid profiles of 10 transconjugants showed two unique patterns. A novel enlarged plasmid was found. Southern blot hybridization revealed some homology with the 30 Md Lac⁺ plasmid of donor, recipient and the transconjugants, as well as with some of the remaining plasmids of the donor.Technical Paper No. 7953, Oregon Agricultural Experiment Station.     

Direct gene transfer to plant protoplasts by mild sonication

Summary A novel procedure employing mild sonication for transformation of plant protoplasts is described. Transient expression of a chloramphenicol acetyltransferase (CAT) gene in protoplasts of sugar beet (Beta vulgaris L.) and tobacco (Nicotiana tabacum L.) was obtained by a brief exposure of the protoplasts to 20 kHz ultrasound in the presence of plasmid DNA. Maximum levels of CAT activity were achieved by sonication for 500–900 ms at 30–70 W electric power (0.65–1.6 W/cm2 acoustic power). This reduced the viability to 15–20 % and 60 % for sugar beet and tobacco protoplasts, respectively. Up to 12 % (sugar beet) and 81 % (tobacco) of maximum transient expression could be achieved with no significant loss of viability. Protoplasts surviving exposure to ultrasound were found to have a similar long-term viability and to regenerate to micro-calli as untreated protoplasts. Plasmid DNA concentrations of 80–110 g/ml and sucrose concentrations of 21–28 % in the sonication medium were found to be optimal for transient expression.Abbreviations CAT chloramphenicol acetyltransferase     

Rapid enhancement of heterotrophic but not photosynthetic activities in Arctic microbial plankton at mesobiotic temperatures

Summary Marine microbial plankton from eastern Canadian Arctic waters have the remarkable ability of rapidly accumulating nutrients (amino acids, glucose, phosphate) at mesobiotic temperatures (30°C) after an acclimation period of a few hours. Curves of the nutrient uptake response to temperature established before and after an 8 h acclimation period at 30°C showed that maximum uptake rates attained much higher values and were established at much higher temperatures after the acclimation period. This enhancement was inhibited by chloramphenicol (but not cycloheximide) and occurred even when the possibility of bacterial enhancement due to animal and plant death was reduced by prior removal of organisms using a screen of 1 m nominal pore diameter. The enhancement at high temperatures was evidenced only in heterotrophic but not in photosynthetic activities and was concominant with large increases in the apparent activity of microbial DNA synthesis and bacterial cell density. The enhancement may have been due to a rapid and opportunistic growth of presumed non-psychrophilic members of the bacterioplankton.     

Conditional lysis ofEscherichia coli by the fusion of extracellular (STA) to periplasmic (LTB) enterotoxins: Apparent phenotypic suppression of lactose permease

The expression of a methanol-soluble, heat-stable enterotoxin (ST_A) fused to the B subunit of the heat-labile enterotoxin (LT_B) at 35°C or higher temperatures caused strains ofEscherichia coli deficient in lactose permease to behave on indicator media as Lac⁺; however, at 33°C or lower temperatures the original Lac^– phenotype of the host strains was maintained. The apparent phenotypic suppression oflacY was shown to be due to lysis of a fraction of the bacteria and the consequent release of active-galactosidase to the culture supernatant. After incubation at 37°C for 1 h, the cultures were committed to lyse. Plasmid and chromosomal mutants that do not show this phenotype were isolated by selecting Lac^– colonies at the unpermissive temperature. The mutations on the plasmids were localized in both the heat-stable and the heat-labile enterotoxin genes. Chromosomal mutants that show normal levels of-galactosidase and fused toxins have also been isolated.     

Direction of transcription affects the replication mode of lambda in an in vitro system

Summary pMV158 is a 5.4 kb broad host range multicopy plasmid specifying tetracycline resistance. This plasmid and two of its derivatives, pLS1 and pLS5, are stably mantained and express their genetic information in gram-positive and gram-negative hosts. The in vitro replication of plasmid pMV158 and its derivatives was studied in extracts prepared from plasmid-free Escherichia coli cells and the replicative characteristics of the streptococcal plasmids were compared to those of the E. coli replicons, ColE1 and the mini-R1 derivative pKN182. The optimal replicative activity of the E. coli extracts was found at a cellular phase of growth that corresponded to 2 g wet weight of cells per litre. Maximal synthesis of streptococcal plasmid DNA occurred after 90 min of incubation and at a temperature of 30° C. The optimal concentration of template DNA was 40 g/ml. Higher plasmid DNA concentrations resulted in a decrease in the incorporation of dTMP, indicating that competition of specific replication factor(s) for functional plasmid origins may occur. In vitro replication of plasmid pMV158 and its serivatives required the host RNA polymerase and de novo protein synthesis. The final products of the streptococcal plasmid DNAs replicated in the E. coli in vitro system were monomeric supercoiled DNA forms that had completed at least one round of replication, although a set of putative replicative intermediates could also be found. The results suggest that a specific plasmid-encoded factor is needed for the replication of the streptococcal plasmids.