Molecular Nature of Two Nonconjugative Plasmids Carrying Drug Resistance Genes

Two nonconjugative R-plasmids, N-SuSm and N-Tc, have been characterized. Both were of relatively small size (5 x 10(6) to 6 x 10(6) daltons) and present in multiple copies within their respective bacterial hosts. N-SuSm possessed a guanine plus cytosine content of 55%, whereas N-Tc was 49% guanine plus cytosine. Although these plasmids were inherently nontransmissible they could be mobilized by a large variety of transfer agents including Ent, Hly, and K88. The fi(-) transfer factors tested were far more likely (about 200x) to mobilize these nonconjugative plasmids than were the fi(+) transfer factors tested. Although the mobilization phenomenon was not found to be associated with a detectable level of direct stable recombinational union between N-SuSm or N-Tc with a transfer factor, we were able to demonstrate a low level of recombination between these replicons and a transfer factor by P1-mediated transduction. The isolation of recombinants between transfer factors and nonconjugative plasmids presumably represents one means by which unitary molecular types of R-plasmids arise and by which existing R-plasmids may acquire new resistance determinants.     

通过接合作用质粒从大肠杆菌到棒状杆菌的转移

构建了可接合转移的穿梭质粒pXZ911、pBZ51和pBZ52。这些质粒中含有具转移功能的Mob片段和在棒状杆菌中复制的复制区。以大肠杆菌S17—1为供体菌,通过接合转移作用,可将这些质粒转移到谷氨酸棒杆菌ATCCl3032、谷氨酸棒杆菌ATCC21543、北京棒杆菌B3、北京棒杆菌1．299、裂氏棒杆菌B43、黄色短杆菌ATCC 14067等棒状杆菌苗株,接合转移频率分别为：9X10^-5,1X10^-4,8．5x10,2．3X10^-4×10^-5,2．9X10^-5。本文还探索了大肠杆菌和几种革兰氏阳性棒状杆菌问基因转移的方法、转移频率、影响转移频率的因素、宿主范围等问题。     

Evolution of Plasmid Mobility: Origin and Fate of Conjugative and Nonconjugative Plasmids

Conjugation drives the horizontal transfer of adaptive traits across prokaryotes. One-fourth of the plasmids encode the functions necessary to conjugate autonomously, the others being eventually mobilizable by conjugation. To understand the evolution of plasmid mobility, we studied plasmid size, gene repertoires, and conjugation-related genes. Plasmid gene repertoires were found to vary rapidly in relation to the evolutionary rate of relaxases, for example, most pairs of plasmids with 95% identical relaxases have fewer than 50% of homologs. Among 249 recent transitions of mobility type, we observed a clear excess of plasmids losing the capacity to conjugate. These transitions are associated with even greater changes in gene repertoires, possibly mediated by transposable elements, including pseudogenization of the conjugation locus, exchange of replicases reducing the problem of incompatibility, and extensive loss of other genes. At the microevolutionary scale of plasmid taxonomy, transitions of mobility type sometimes result in the creation of novel taxonomic units. Interestingly, most transitions from conjugative to mobilizable plasmids seem to be lost in the long term. This suggests a source-sink dynamic, where conjugative plasmids generate nonconjugative plasmids that tend to be poorly adapted and are frequently lost. Still, in some cases, these relaxases seem to have evolved to become efficient at plasmid mobilization in trans, possibly by hijacking multiple conjugative systems. This resulted in specialized relaxases of mobilizable plasmids. In conclusion, the evolution of plasmid mobility is frequent, shapes the patterns of gene flow in bacteria, the dynamics of gene repertoires, and the ecology of plasmids.     

DNA Fragments Assembly Based on Nicking Enzyme System

A couple of DNA ligation-independent cloning (LIC) methods have been reported to meet various requirements in metabolic engineering and synthetic biology. The principle of LIC is the assembly of multiple overlapping DNA fragments by single-stranded (ss) DNA overlaps annealing. Here we present a method to generate single-stranded DNA overlaps based on Nicking Endonucleases (NEases) for LIC, the method was termed NE-LIC. Factors related to cloning efficiency were optimized in this study. This NE-LIC allows generating 3′-end or 5′-end ss DNA overlaps of various lengths for fragments assembly. We demonstrated that the 10 bp/15 bp overlaps had the highest DNA fragments assembling efficiency, while 5 bp/10 bp overlaps showed the highest efficiency when T4 DNA ligase was added. Its advantage over Sequence and Ligation Independent Cloning (SLIC) and Uracil-Specific Excision Reagent (USER) was obvious. The mechanism can be applied to many other LIC strategies. Finally, the NEases based LIC (NE-LIC) was successfully applied to assemble a pathway of six gene fragments responsible for synthesizing microbial poly-3-hydroxybutyrate (PHB).     

Drug Resistance of Staphylococci

Induction of chloramphenicol (CM) resistance in Staphylococcus aureus was investigated using 166 CM derivatives and analogues. It was found that 18 compounds of the samples used were able to induce resistance to CM in Staphylococcus aureus S1477 harboring an inducible CM-resistance determinant. Most of the samples which had a nitrophenyl moiety and a D- or DL-threo isomer in its steric configuration were found to have inducer activity for CM resistance. Competent inducers are D-isomer CM derivatives which have another substituent in place of the hydroxyl group at carbon atom 3 in the propanediol of the drug.     

Drug Resistance of Staphylococci

According to an epidemiological survey of drug resistance in Staphylococcus aureus, it was found that there are three types of cross-resistance to macrolide antibiotics (EM, erythromycin: OM, oleandomycin: LM, leucomycin: SP, spiramycin), i.e., resistance to EM, OM, LM, and SP, to EM and OM, and inducible resistance to “EM, OM”. In the inducible strains of S. aureus, EM and OM are active inducers, and the optimal concentrations of the inducers are 0.1 μg/ml and 1.0 μg/ml, respectively. The induction of high resistance (800 μg/ml or more) to both EM and OM occurred within 10 min exposure to 0.1 μg/ml of EM, and the resistance of induced cells was lost after overnight growth in the absence of inducer. After 1 to 3 hr exposure to 1.0 μg/ml of OM, the inducible strains acquired high resistance (100 μg/ml or more) to EM and to a lesser extent, resistance to OM, and the acquired resistance was lost when grown in antibiotic free media. When a known concentration of EM was mixed with the induced cells or with a crude extract from induced cells which had acquired high resistance to EM and OM, the antibiotic activity of EM was still retained in the mixture, indicating that the induced mixture or the extract from the induced cells was incapable of antibiotic (EM) inactivation under the test conditions.     

Drug Resistance of Staphylococci

It was reported that resistance to tetracycline (TC) in some staphylococcal strains was lost irreversibly without loss of resistance to other drugs when cultured at high temperature, and that the determinant for TC resistance exists on a plasmid. According to the transductional analysis of TC resistance in Staphylococcus aureus E169 it was found that the genes which govern resistance to tetracycline and streptomycin are located close together on a single genetic element. When TC resistance in MS146, one of our stock cultures, was transduced to E169S which had lost TC resistance, TC resistance in transductants was found to be unstable at elevated temperatures. By contrast, TC resistance in transductants MS353 TC^r, to which TC resistance was transduced from E169, was indicated to be stable even when cultured at high temperature. From these results, it is strongly suggested that instability of TC resistance in E169 is not accounted for by the genetic properties of the genes which govern TC resistance but those of host cell (E169) itself.     

Drug Resistance of Staphylococci

On the basis of resistance patterns to penicillins, strains of Staphylococcus aureus highly resistant to penicillin-G which were isolated from clinical sources, could be classified into 2 groups, A-1 and A-2. Group A-2 is a minor group which accounts for about 10% of these strains and exhibits lower degree of resistance to penicillin-G and phenethicillin than that exhibited by group A-1 strains. However, there was no marked difference between both groups in inducible production of penicillinase. The genetic determinants for penicillinase synthesis in strains of both groups were transduced into a recipient and the characteristics of the determinants were compared with each other under identical conditions. The results suggested that the genetic determinants for penicillinase synthesis in groups A-1 and A-2 strains differed from each other in both the structural gene for penicillinase production and the regulatory gene for the formation of the repressor.     

Drug Resistance of Staphylococci

Induction of chloramphenicol (CM) resistance in Staphylococcus aureus was investigated by using several CM derivatives. It was found that dl-threo-1-p-nitrophenyl-2-dichloroacetamino-1,3-dichloropropane has high antibacterial activity but low activity of induction for CM resistance. In spite of low antibacterial activity, induction of CM resistance occurred after prior treatment with dl-threo-1-p-nitrophenyl-2-dichloroacetamino-3-chloropropane-1-ol. It was found that dl-chloramphenicol di-acetate, dl-threo-1-p-nitrophenyl-2-dichloroacetamino-3-bromopropane-1-ol and dl-threo-1-phenyl-2-dichloroacetamino-1,3-propanediol have induction ability in spite of the absence of antibacterial activity. Other derivatives were classified into two groups; (1) low antibacterial activity and induction of CM resistance and (2) loss of both activities.     

Drug Resistance of Staphylococci

Examination of cross resistance to macrolide antibiotics in erythromycin resistant staphylococcal strains isolated from clinical sources in the United States showed that there were two types of cross resistance, group A (13.4%) and group C (86.6%). Group A possessed multiple resistance to the macrolide antibiotics, erythromycin, oleandomycin, leucomycin and spiramycin, and was also resistant to lincomycin. In group C, resistance to erythromycin alone or to both erythromycin and oleandomycin could be induced by exposure to erythromycin.     

The Population Biology of Bacterial Plasmids: A PRIORI Conditions for the Existence of Mobilizable Nonconjugative Factors

A mathematical model for the population dynamics of nonconjugative plasmids that can be mobilized by conjugative factors is presented. In the analysis of the properties of this model, primary consideration is given to the conditions under which these nonself-transmissible extrachromosomal elements could become established and would be maintained in bacterial populations. The results of this analysis demonstrate the existence of conditions where, as a consequence of infectious transmission via mobilization, nonconjugative plasmids could become established and be maintained even when the bacteria carrying them have lower reproductive fitnesses than plasmid-free members of the population. However, these existence conditions are stringent and suggest therefore, that it is highly unlikely that plasmids of this type would become established and maintained without some direct selection favoring their carriage. The general implications of these results and limitations of the model are discussed. Brief consideration is also given to the implications of these theoretical findings to the problems of the spread of multiple antibiotic resistance plasmids (R-factors) and the risk of contaminating natural populations of bacteria with chimeric plasmids produced by work with recombinant DNA.     

Conjugation in Archaea: Frequent Occurrence of Conjugative Plasmids inSulfolobus

We describe five novel conjugative plasmids (CPs) and two subfamilies, each comprising several closely related variants of CPs isolated from colony-cloned strains of the extremely thermophilic, heterotrophic archaeonSulfolobus islandicus,which were obtained by plating of samples from Icelandic solfataras after liquid enrichment. They are related to each other and to the previously described CP pNOB8 from a JapaneseSulfolobusstrain in that they share essential functions and limited similarity of genomes as demonstrated by DNA cross-hybridization and sequences. All these plasmids thus form a family of highly efficient self-spreading elements directly transferred from donor into recipient cells. Conjugation is initiated by pair formation, followed by selective transfer of the plasmids into the recipient and expression of transfer functions. Some of these CPs exclude superconjugation of the transcipients with closely related CPs. The novel CPs are stable upon conjugative transfer, but vary upon growth of transcipients. The stability of the CPs is higher in their original hosts or in relatedS. islandicusstrains, than inSulfolobus solfataricusstrain PH1 as recipient. The deletion variant pING3 has lost the ability to transfer itself but is still subject to being transferred by the transfer apparatus of its complete relative, pING6. The dissection of genes and functions has been initiated by characterizing this incomplete variant.     

Naphthalene and Donor Cell Density Influence Field Conjugation of Naphthalene Catabolism Plasmids

We examined transfer of naphthalene-catabolic genes from donor microorganisms native to a contaminated site to site-derived, rifampin-resistant recipient bacteria unable to grow on naphthalene. Horizontal gene transfer (HGT) was demonstrated in filter matings using groundwater microorganisms as donors. Two distinct but similar plasmid types, closely related to pDTG1, were retrieved. In laboratory-incubated sediment matings, the addition of naphthalene stimulated HGT. However, recipient bacteria deployed in recoverable vessels in the field site (in situ) did not retrieve plasmids from native donors. Only when plasmid-containing donor cells and naphthalene were added to the in situ mating experiments did HGT occur.     

Drug Resistance of Staphylococci VI. Genetic Determinant for Chloramphenicol Resistance

Naturally occurring strains of staphylococci which are resistant to chloramphenicol (CM) inactivate this antibiotic. One of the inactivation products of CM showed the chromatographic behavior of 3-acetoxychloramphenicol. Induction of resistance occurred after prior exposure to subinhibitory concentrations of the antibiotic. The resistance of induced populations, as well as CM-inactivation ability, was decreased when they were grown in CM-free medium. The CM-inactivation property was transduced together with CM resistance. Transductional analysis and CM-resistance elimination experiments indicated that CM resistance in naturally occurring strains of staphylococci is mainly accounted for by inactivation of the drug.     

Thermal Resistance of Salmonellae and Staphylococci in Foods

The heat-resistant Salmonella senftenberg 775W and two strains of Staphylococcus aureus were tested at temperatures up to 68.3 C (71.1 C for S. senftenberg) in four different media. From the survival data, decimal reduction times (D values) were calculated for each set of conditions, and decimal reduction time curves were constructed for each bacterial strain in each medium. Slopes of decimal reduction time curves (Z(D)) ranged from 4.52 to 6.38 C with a single exception. There was no statistical heterogeneity among the remaining values. Results were in close agreement with published results of similar studies conducted at somewhat lower temperatures and support the practice of using a slope value (Z(D)) of 5.56 C for establishing time-temperature relationships for food processing. It is recommended that such a decimal reduction time curve not be extrapolated to temperatures more than 5.56 C higher than those actually tested.     

Resistance of hospital Staphylococci to beta-lactam antibiotics]

Resistance of hospital Staphylococci to beta-lactam antibiotics was studied. The strains were isolated in two obstetric hospitals during an outbreak of purulent inflammatory infections in them. A modification of the "clover leaf" procedure providing elucidation of the resistance mechanism was used in the study. Development of the resistance to the beta-lactam antibiotics was shown to be mainly due the activity of beta-lactamases which makes it possible to discuss their role in the mechanism of resistance development.     

Novobiocin Resistance and the Classification of Staphylococci and Micrococci

S ummary . Isolates of staphylococci and micrococci, 105 in all, classified according to Baird-Parker's classification (Baird-Parker, 1963,1965) were examined for their sensitivity to novobiocin. All of the staphylococci were sensitive to novobiocin as also were strains of Micrococcus luteus and M. roseus. Almost all strains belonging to Micrococcus subgroups 1–6 were resistant to novobiocin.     

Phenotypic Suppression of Methicillin Resistance in Staphylococcus aureus by Mutant Noninducible Penicillinase Plasmids

Methicillin (intrinsic) resistance of Staphylococcus aureus was suppressed almost completely by regulatory gene (penI₁) mutations of penicillinase plasmids that made penicillinase production strictly noninducible. Methicillin resistance was restored by secondary regulatory gene mutations that altered the noninducible phenotype or by complementation with a compatible plasmid that did not bear the noninducible mutation. No evidence was obtained for genetic linkage between a penicillinase plasmid and the gene for methicillin resistance. We suggest, therefore, that the mutant noninducible repressor acted in trans by binding to a site on the methicillin resistance determinant. This hypothesis would imply an appreciable degree of homology between penicillinase plasmids and methicillin resistance genes.