Studies with RP1 deletion plasmids: Incompatibility properties,plasmid curing and the spontaneous loss of resistance

Four deletion plasmids, pHH301, pHH302, pHH303 and pHH401, obtained from RP1 DNA-transformed bacterial clones, were shown to be incompatible with three P plasmids inEscherichia coli K12 strains. Kinetic experiments and colony tests were used to verify the position of these R plasmids.Pseudomonas aeruginosa andE. coli strains, harbouring deletion plasmids, could be cured by using two mutagens, acriflavine and mitomycin C, which affect a percentage of the cell population. The deletion plasmid-positive strains could also be induced at an elevated temperature to spontaneously loose their plasmids.     

Novel mercury resistance determinants carried by IncJ plasmids pMERPH and R391

Summary HgCl₂ resistance (Hg^r) in a strain of Pseudomonas putrefaciens isolated from the River Mersey was identified as plasmid-borne by its transfer to Escherichia coli in conjugative matings. This plasmid, pMERPH, could not be isolated and was incompatible with the chromosomally integrated IncJ Hg^r plasmid R391. pMERPH and R391 both express inducible, narrow-spectrum mercury resistance and detoxify HgCl₂ by volatilization. The cloned mer determinants from pMERPH (pSP100) and R391 (pSP200) have very similar restriction maps and express identical polypeptide products. However, these features show distinct differences from those of the Tn501 family of mer determinants. pSP100 and pSP200 failed to hybridize at moderate stringency to merRTPA and merC probes from Tn501 and Tn21, respectively. We conclude that the IncJ mer determinants are only distantly related to that from Tn501 and its closely homologous relatives and that it identifies a novel sequence which is relatively rare in bacteria isolated from natural environments.     

Molecular characterization of antibiotic resistance in clinical Salmonella typhi isolated in Ghana

Fifty-eight clinical Salmonella typhi strains isolated from patients suspected of suffering from typhoid fever were obtained at the Korle-Bu Teaching Hospital and the Noguchi Memorial Institute for Medical Research, both located in Ghana, Africa. Each isolate was examined for susceptibility to ampicillin, chloramphenicol, streptomycin, tetracycline, and trimethoprim/sulfamethoxazole by the disk diffusion assay. Five of the isolates were resistant to all five antibiotics while 10 isolates were resistant to ampicillin, chloramphenicol, and trimethoprim/sulfamethoxazole, which are considered 'first line' antibiotics in the treatment of typhoid fever. Thirty-four isolates were resistant to at least one of the antibiotics tested and 62% of these isolates possessed conjugable plasmids belonging to incompatibility group IncHI. Ninety percent of the conjugable plasmids conferred a multiple drug-resistant phenotype on the strains harboring them. Additionally, 14 strains contained plasmids that were transformable and six of them encoded multiple drug resistance. Our findings indicate that multiple drug resistance to the 'first line' antibiotics in S. typhi may be more prevalent in Africa than previously thought.     

Plasmid-borne resistance to arsenate, arsenite, cadmium, and chloramphenicol in a Rhodococcus species

Summary A primarily genetic approach was employed to obtain plasmids in Rhodococcus erythropolis ATCC 12674 which carried genes conferring increased resistance to sodium arsenate and arsenite, cadmium chloride, and chloramphenicol. The plasmids were large, migrating more slowly than chromosomal DNA in agarose gels, and were made up of resistance determinants from the host organism together with part of the genome of nocardiophage Q4. Purified plasmid was used to transform a suitable recipient to increased resistance to sodium arsenate, sodium arsenite, and cadmium chloride.     

Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids

Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.     

Conjugative plasmids in multi-resistant bacterial isolates from Indian soil

Aims:  Determination of heavy metal and antibiotic resistance and presence of conjugative plasmids in bacteria isolated from soil irrigated with wastewater.
Methods and Results:  Composite soil samples were collected from Ghaziabad, Uttar Pradesh, India. Forty different bacteria were selected from nutrient agar and characterized by morphological, cultural and biochemical tests. All the isolates were tested for their resistance to different heavy metals and antibiotics. The DNA derived from multiple metal and antibiotic-resistant bacterial isolates was PCR amplified and plasmid-specific sequences (IncP, IncN, IncW, IncQ and pMV158-type) were analysed by dot blot hybridization. All isolates gave PCR products with trfA2 and oriT primers of the IncP group. These PCR products also hybridized with the RP4-derived probes. However, the samples were negative for all the other investigated plasmids as proved by PCR and dot blots.
Conclusions:  The presence of conjugative/mobilizable IncP plasmids in the isolates indicates that these bacteria have gene-mobilizing capacity with implications for potential dissemination of introduced recombinant DNA.
Significance and Impact of the Study:  The detection of IncP plasmids in all the bacterial isolates is another proof for the prevalence of these plasmids. We propose that IncP plasmids are mainly responsible for the spread of multi-resistant bacteria in these soils.     

Detection of conjugative plasmids and antibiotic resistance genes in anthropogenic soils from Germany and India

PCR typing methods were used to assess the presence of plasmids of the incompatibility (Inc) groups IncP, IncN, IncW, IncQ and rolling circle plasmids of the pMV158 type in total DNA extracts from anthropogenic soils from India and Germany. Ten different soils from two different locations in Germany, the urban park Berlin Tiergarten and the abandoned sewage field Berlin-Buch, and from four different locations in India were analysed. PCR amplification of the total DNA extracts revealed the prevalence of IncP-specific sequences in Berlin Buch and Indian soil samples. The detected IncP plasmids contained at least one transfer function, the origin of transfer, oriT. In contrast to IncP-specific sequences, IncQ, IncN, IncW and pMV158-specific sequences were never detected. The presence of ampC, tet (O), ermB, SHV-5, mecA, and vanA antibiotic resistance genes was also tested. Three Indian soil samples irrigated with wastewater contained the ampC gene, whereas the other resistance genes were not found in any of the samples. Detection of IncP trfA2 and oriT sequences by PCR amplification and hybridization is a clear indication that IncP plasmids are prevalent in these habitats. Exogenous plasmid isolation revealed conjugative plasmids belonging to the IncPbeta group encoding resistance to ampicillin.     

Comparison of the organisation of the genomes of phenotypically diverse plasmids of incompatibility group P: members of the IncP beta sub-group are closely related

Summary Comparison of physical maps of the broad host range plasmids R751, R906 and R772, belonging to the IncP sub-group of the Escherichia coli incompatibility group P, reveals two large regions of similarity, separated by dissimilar regions which contain the majority of the cleavage sites for restriction endonucleases with hexanucleotide recognition sites. Mapping of the regions of these plasmids which show homology to probes specific for genetically characterised segments of the distantly related IncP plasmid RK2, involved in plasmid maintenance or conjugal transfer, reveals that all four plasmids share a similar genetic organisation. In each case the homologous plasmid back-bone is interrupted by heterologous segments both between the essential replication loci oriV and trfA, and between the conjugal transfer regions tra1 and tra2, although in the case of R772 the segment of the backbone carrying the trfA and tra2 regions is inverted relative to that of the other plasmids. However, in the case of pJP4, shown to be a fourth member of the IncP sub-group, the back-bone is interrupted only by a single large segment adjacent to the trfA region. Mapping of the regions of the four IncP plasmids which show homology to Tn501 and nucleotide sequence determination at the ends of the homologous regions reveals that R906, R772 and pJP4 share a common mercury resistance region. This region, which appears to have been inactivated in R772, was probably inserted into a common ancestor of these plasmids by the transposition of an element related to an ancestor of Tn501. R751 shows no trace of the mercury resistance region, but contains a short relict of Tn501, derived from an independent insertion event.     

亚硝基胍消除链霉菌FR-008的线性质粒

【目的】利用亚硝基胍(NTG)消除链霉菌FR-008线性质粒以简化其基因组,获得背景清晰的菌株,作为抗生素异源生物合成的底盘细胞。【方法】NTG溶液处理链霉菌FR-008孢子悬液,从存活的诱变株中筛选砷敏感的突变株,再通过脉冲场凝胶电泳(PFGE)检测线性质粒是否被消除;用生测实验定性检测各个线性质粒消除突变株杀念菌素合成的能力,最后通过HPLC定量比较突变株和野生型产生杀念菌素的差异。【结果】从103个诱变株中筛选到3株砷敏感的突变株(10#、59#、115#)。PFGE检测发现它们均丢失了大线性质粒p SSFR1,此外,42#突变株的小线性质粒p SSFR2被消除,在此基础上,第二轮NTG诱变获得了双质粒消除的突变株。大线性质粒p SSFR1消除率约为3%,小线性质粒p SSFR2消除率约为1%。发酵结果显示:10#、115#突变株杀念菌素有效组分III产量分别提高了40%和30%。【结论】首次发现NTG是一种有效消除链霉菌线性质粒的诱变剂,2株大线性质粒消除的突变株杀念菌素的产量得到提高。此方法可以用来消除特定链霉菌菌株中的巨型线性质粒以高效简化其基因组,因而是一种有效的抗生素遗传育种的方法。     

Metal resistance and plasmid DNA in Thiobacillus ferrooxidans

The minimal inhibitory concentrations of copper and nickel were determined for each of fifteen isolates of T. ferrooxidans native to a Cu/Ni tailings environment. Ten isolates were inhibited by 160 mM Cu,²⁺ or less, and ten were inhibited by 160 mM Ni²⁺or less. The isolates were screened for plasmid DNA using an alkaline lysis method and CCC plasmid forms were confirmed using the Hintermann technique. Two isolates were found to be devoid of plasmid DNA, and only one isolate contained more than two plasmids. Variability existed in plasmid size, although the majority were larger than the standard pBR322 (4.3 kbp). One plasmid was selected for further analysis using restriction endonucleases. EcoRI, HindIII and KpnI all cleaved the plasmid in two locations, and PstI cleaved the plasmid in six locations. PstI-digested fragments of the plasmid were ligated into pBR322, and the recombinant plasmids were transformed into Escherichia coli ATCC 8739. Four genetically-different transformants resulted, and each was grown in media containing 2.0 mM Cu²⁺ and compared to the growth of a control under similar conditions. There was no conferred copper resistance in E. coli, although one recombinant plasmid appeared to decrease the tolerance for E. coli ATCC 8739 to Cu²⁺.     

Apparent incompatibility of plasmid pSfrYC4b of <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> with two different plasmids in another strain

Sinorhizobium fredii YC4B is a spontaneous mutant derivative of strain YC4 that is unable to nodulate soybeans. The second-largest plasmid of strain YC4B, termed pSfrYC4b (810 kb), was transferred to S. fredii HN01SR, a strain which contains three large indigenous plasmids (pSfrHN01a, pSfrHN01b and pSfrHN01c). Surprisingly, two stable indigenous plasmids (pSfrHN01a and pSfrHN01b) of strain HN01SR were cured simultaneously by the introduction of pSfrYC4b. Furthermore, a novel, unstable plasmid (pHY4) became visible in agarose gels. The electrophoretic mobility of plasmid pHY4 was slower than that shown by the cured plasmids, indicating that the molecular weight of the former is higher than that of plasmids pSfrYC4b and pSfrHN01b. Replication gene repC-like sequences were detected by polymerase chain reaction (PCR) on pSfrHN01a and pSfrYC4b, but not on pSfrHN01b. Sau3AI and PstI restriction patterns of the PCR-amplified repC-like sequences from HN01SR and YC4B were very similar.     

Persistence of pBR322-related plasmids in Escherichia coli grown in chemostat cultures

Abstract The stability of plasmid pBR322 and a number of close derivatives was examined by continuous culture of Escherichia coli . Cultures were subjected to either glucose, phosphate or magnesium limitation in non-selective medium at a dilution rate of 0.1/h. Under these conditions pBR322 was eventually lost from the population, but only after a distinct lag period. The closely related plasmids pBR325 and especially pBR327 and pBR328, but not pAT153, were lost more rapidly. Three cosmids pHC79, pSJ55 and pJB8 were generally found to be less stable than the pBR322-type plasmids from which they were derived. Chimaeric plasmids containing DNA from yeast and from a thermophilic bacillus were also unstable in E. coli .     

Diversity of tetracycline resistance genes in bacteria from aquaculture sources in Australia

AIMS: To determine the genetic determinants responsible for tetracycline resistance in oxytetracycline resistant bacteria from aquaculture sources in Australia. METHODS AND RESULTS: Twenty of 104 (19%) isolates tested were resistant to oxytetracycline (MIC > or = 16 microg ml(-1)). Using polymerase chain reaction (PCR) amplification, one or more tet genes were detected in 15/20 (75%) isolates tested, but none were found in 5/20 (25%). tetM (50%) was the most common determinant, followed by tetE (45%), tetA (35%) and tetD (15%). Five of 12 oxytetracycline resistant isolates studied were able to transfer their R-plasmid to Escherichia coli recipients of chicken, pig and human origin. tetA, tetD and tetM were found to be transferred while tetE was not transferred. Southern hybridization and PCR were used to confirm transfer of determinants. CONCLUSIONS: Bacterial isolates from aquaculture sources in Australia harbour a variety of tetracycline resistance genes, which can be transferred to other bacteria of different origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteria from aquaculture sources in Australia contribute to the resistance gene pool reservoir. The in vitro transfer of tetracycline R-plasmid from aquatic bacteria to E. coli isolates from various sources is an indication of the potential public health risk associated with these resistance determinants.     

Characterization of staphylococcal plasmids hybridizing with the fosfomycin resistance gene fosB

The distribution of the fosB gene, coding for fosfomycin resistance, in 105 fosfomycin-resistant isolates of Staphylococcus from various geographical areas, was studied by Southern blot hybridization. Nucleotide sequences related to fosB were detected in 36 strains belonging to five species. Plasmids bearing fosB were often of a size similar to that of pIP1842 (2.54 kb) in S. epidermidis, most often small (2.4 to 4.1 kb) in other species including S. aureus where a 2.7-kb plasmid was found in 16 out of the 18 strains studied. The fosB gene was geographically dispersed since it was present in six different locations in France and also in Japan. The weak hybridization observed with plasmid DNA of certain strains of S. aureus, S. epidermidis, S. haemolyticus, S. saprophyticus, and S. warneri may indicate gene heterogeneity for fosfomycin resistance in Staphylococcus spp.     

The dynamics of pAL2-1 homologous linear plasmids in Podospora anserina

A natural population of recently isolated Podospora anserina strains was screened for homologues of the linear longevity-inducing plasmid pAL2-1. Of the 78 wild-type isolates, 14 hybridised with a pAL2-1 specific probe, half of which contained a single plasmid and the other half multiple plasmid copies (plasmid family). All strains except one plasmid-containing strain, senesced normally. However, no inserted plasmid sequences were detected in the mitochondrial DNA, as was the case for the longevity-inducing pAL2-1 plasmid. Occasional loss of plasmids and of repeated plasmid sequences occurred during sexual transfer. Plasmid transmission was equally efficient for mono- and dikaryotic spores and was independent of the genetic background of the strains. Furthermore, horizontal transfer experiments showed that the linear plasmid could easily infect plasmid-free strains. Horizontal transfer was even observed between strains showing a clear vegetative incompatibility response (barrage). The linear plasmids are inherited maternally; however, paternal transmission was observed in crosses between confronted vegetative-incompatible strains. Paternal transmission of the plasmid was never observed using isolated spermatia for fertilisation, showing that mitochondrial plasmids can only gain access to maternal sexual reproductive structures following horizontal transfer. These findings have implications for both the function of vegetative incompatibility in fungi and for the mechanism of maintenance of linear plasmids. Received: 13 November 1997 / Accepted: 17 February 1998     

Isobutyrate as a precursor of n-butyrate in the biosynthesis of tylosine and fatty acids

Labelled sodium isobutyrate [(CD3)2-CHCOONa] was added to the culture medium of Streptomyces fradiae and up to 14 atoms of deuterium were found to be incorporated into a molecule of tylosin aglycone (tylactone). This observation is in accordance with the data in the literature. When fatty acids were analyzed, as much as 34% of the isobutyrate incorporated into the cell was formed to be transformed into butyrate that was used for the synthesis of even, straight-chain fatty acids; 57% of the labelled isobutyrate was incorporated into the even isoacids, whereas 9% was degraded to propionate and further used for the synthesis of the odd acids.     

Conserved regions in the T-DNA of different Agrobacterium rhizogenes root-inducing plasmids

The T-regions of the three so far identified types of Ri plasmids-corresponding to the synthesis of three different hairy root opines, agropine, mannopine and cucumopine-have been compared in detail by Southern blot cross hybridizations. Two distinct zones of very strong sequence homology, approximately 4 and 3 kilobases in length respectively, have been identified in all three T-regions. The highly conserved sequences, not present in Ti plasmid T-DNA, may encode essential rhizogenic functions common to all Agrobacterium rhizogenes T-DNAs.