Vegetative incompatibility in Neurospora: its effect on horizontal transfer of mitochondrial plasmids and senescence in natural populations

We have investigated the horizontal transfer of two mitochondrial plasmids and the Kalilo senescence phenotype in the fungus Neurospora without the use of heterokaryon-forcing markers. The Kalilo senescent state was only transferred between fully-compatible N. crassa strains, but not between strains differing at any of the loci het-c, het-d, het-e or mating-type. However, the linear plasmid kalDNA and the circular plasmid Han-2 were transferred following incompatible vegetative interactions. Our data suggest that vegetative incompatibility due to allelic differences at het-c is more effective in preventing transfer than that due to het-d, het-e or mating-type. Based on these observations we have developed a novel test for assessing vegetative incompatibility between Kalilo and non-Kalilo field isolates of N. intermedia. In this procedure combinations of Kalilo and non-Kalilo field isolates of N. intermedia were grown together and tested for senescence. Compatibility is inferred if the young non-Kalilo strain dies along with the senescent Kalilo strain, whereas incompatibility is inferred when the Kalilo strain dies without imposing its senescent state onto the non-Kalilo strain. Our results suggest that each of the nine Kalilo strains tested is incompatible with each of 20 non-Kalilo isolates from the same N. intermedia population of the Hawaiian island of Kauai. However, the observed incompatibility did not completely prevent cytoplasmic exchange, and in several cases plasmid transfer could be detected.     

A kalilo-like linear plasmid in Louisiana field isolates of the pseudohomothallic fungus Neurospora tetrasperma

Two Louisiana strains of Neurospora tetrasperma contain a linear plasmid (LA-kalDNA) with a restriction map identical to the Hawaiian Neurospora intermedia senescence plasmid, kalDNA, but with termini 100 nucleotide pairs shorter. One of these strains also bore a circular plasmid similar to the Hawaiian circular plasmid Hanalei-2. One species probably acquired both plasmids from the other by horizontal transfer, at a time sufficiently distant for sequence divergence to take place. Many LA-kalDNA-bearing derivative strains senesced, but this plasmid does not guarantee senescence. Furthermore, LA-kalDNA does not insert into mtDNA. One senescent strain showed no LA-kalDNA. The plasmids are effectively transmitted via the pseudohomothallic sexual cycle. Single mating-type derivatives transmit plasmids maternally.     

Suppression of cytoplasmic senescence in Neurospora

Summary We have shown that senescence in Kalilo strains of Neurospora, caused by a linear mitochondrial plasmid called kalDNA, is suppressible by existing variants of the nuclear genome. The suppressors are manifested by 4:4 segregation of senescence and immortality in asci from crosses between senescent female strains and males chosen from non-senescent candidate stocks. In one case of suppression, the asci also show segregation at the plasmid level. There is a reduction of kalDNA to barely detectable levels in the four ascospores showing immortality, so this suppressor evidently influences the maintenance of the plasmid itself. In the other case of suppression, the phenotypic segregation is not correlated with segregation at the plasmid level, and all eight ascospores in the asci show both free and inserted forms of kalDNA. This suggests that the suppression genotype provides a way of tolerating the presence of the plasmid rather than diminishing it. However, the allele f, which provides an analogous kind of suppression for the cytoplasmic mutation poky, does not suppress Kalilo or Maranhar senescence. Suppression is hence shown to be a possible option for host strains to combat the plasmid in nature, but no examples of suppressors were found in a limited survey of natural isolates. In addition, we have shown that long-lived, presumably non-senescent, strains do not arise by suppressor mutation, but lose senescence plasmid DNA by another mechanism.     

Distribution of seven homology groups of mitochondrial plasmids in Neurospora: evidence for widespread mobility between species in nature

A survey of mitochondrial DNAs from over 225 Neurospora and related fungal isolates from around the world uncovered three new homology groups of mitochondrial plasmids, two divergent subgroups of the Fiji plasmid family, and extended previous data about plasmid distribution patterns. Newly-discovered circular plasmids, Java and MB1, and the linear Moorea plasmids, were found in relatively-few isolates. A large proportion of isolates (51%) were found to have these or previously-discovered plasmids in the Varkud, kalilo, LaBelle, or Fiji families. Plasmids in most families were found in isolates world-wide and distributed nearly randomly with respect to species. As many as three types of plasmids were found in single isolates, and plasmids typically were found alone or in pairs in a random, independent pattern. The regional clustering of some plasmids was independent of species. providing a strong argument that horizontal transfer of plasmids occurs frequently in nature. Some plasmid families were much more diverse than others. The Fiji plasmids are a superfamily composed of distinct subgroups defined by degrees of cross-hybridization. Between some subgroups there were large regions of non-homology.     

Homologous linear plasmids in mitochondria of three species of wheat bunt fungi,Tilletia caries,T. laevis and T. controversa

Summary All isolates of Tilletia spp. investigated (five isolates of T. caries, including one from Japan, two isolates of T. laevis, and five isolates of T. controversa) contained a linear DNA plasmid ranging in size from 7.2 to 7.6 kb. All plasmids were highly homologous to each other as shown by DNA-DNA hybridization and comparison of restriction enzyme sites. Variability in the size of the plasmid was found to be due to differences within a central region of the plasmid. No homology between the plasmid and mitochondrial or nuclear DNA was found, but the mitochondrial origin of the plasmid was confirmed.     

Characterization and prevalence of a circular mitochondrial plasmid in senescence-prone isolates of Neurospora intermedia

Genetic and molecular analyses of the phenomenon of senescence—i.e., irreversible loss of growth and reproductive potential upon subculturing—in Neurospora intermedia strain M1991-60A, collected from Maddur in southern India, showed the presence of plasmid pMaddur1, which is homologous to the senescence-inducing circular mitochondrial plasmid, pVarkud. Maternal inheritance of senescence in M1991-60A correlated to the formation of variant pMaddur1, its subsequent insertion into mitochondrial (mt)DNA and the accumulation of defective mtDNA with the pMaddur1insert. PCR-based analyses for similar plasmids in 147 natural isolates of Neurospora from Maddur showed that nearly 40% of the strains had pMaddur1 or pMaddur2 that shared 97–98% sequence homology with pVarkud and pMauriceville. Nearly 50% of the strains that harbored either pMaddur1 or pMaddur2, also contained a circular Varkud satellite plasmid (pVS). Size polymorphism maps to the cluster of PstI sites in the non-coding region. Whereas senescence of nearly 40% of N. intermedia strains may be due to pMaddur, the presence in seven strains of pVS but not pMaddur and the absence of either of these two plasmids in other senescence-prone isolates suggests yet undiscovered mechanisms of senescence in the Maddur strains.     

Predominance of IncL/M and IncF plasmid types among CTX‐M‐ESBL‐producing Escherichia coli and Klebsiella pneumoniae in Bulgarian hospitals

Our objective was to investigate the plasmid replicon‐types involved in spread of ESBLs among Bulgarian Klebsiella pneumoniae and Escherichia coli. Sixty‐three isolates, with transferable beta‐lactam resistance determinants, collected between 2007 and 2009 in six medical institutions, were analysed with respect to their antimicrobial susceptibility, ESBL‐, RAPD‐, and plasmid replicon‐type. Phylogenetic typing and screening for the O25b‐ST131 lineage were carried out for E. coli. The predominant ESBLs were CTX‐M‐15 (81%) among E. coli and CTX‐M‐3 (58%) among K. pneumoniae. Other sporadically found ESBLs were SHV‐12 and TEM‐139, and for the first time in Bulgaria, CTX‐M‐1 and CTX‐M‐14. Replicon typing revealed that plasmids carrying bla_CTX‐M‐3 exclusively belonged to IncL/M‐type, while bla_CTX‐M‐15 was predominantly (94%) associated with IncF‐type plasmids. Among E. coli, 59% of the isolates were clonally related. Isolates of that cluster produced CTX‐M‐15, belonged to the O25b‐ST131 lineage, predominantly harboured plasmids with the FIA replicon, and were found in five centres. Among CTX‐M‐3‐producing K. pneumoniae, two prevailing RAPD‐types were found, one remained restricted to one centre and the second was found in three centres. The incompatibility groups IncN and IncA/C linked with bla_SHV‐12 respectively bla_TEM‐139 were found only once. To the best of our knowledge, this is the first detailed investigation of plasmids carrying ESBL genes among Bulgarian isolates demonstrating wide distribution of conjugative IncF plasmids among CTX‐M‐15‐producing E. coli and IncL/M plasmids among CTX‐M‐3 positive K. pneumoniae isolates.     

Plasmid profiles and restriction enzyme fragmentation patterns of plasmids of methicillin-sensitive and methicillin-resistant isolates of Staphylococcus aureus from hospital and the community

The number, frequency distribution and restriction enzyme fragmentation patterns of plasmids harboured by 163 methicillin-sensitive isolates of Staphylococcus aureus (MSSA) and 53 methicillin-resistant isolates (MRSA) were compared. Plasmids were demonstrated in less than half of the MSSA isolates; their frequency distribution did not differ from that predicted by a simple model of plasmid distributions. In contrast, all the MRSA isolates harboured plasmids, their distribution suggesting dissemination of a limited number of clones within the hospital. Among 72 MSSA isolates harbouring plasmids, 38 different restriction patterns were identified. There were fewer patterns among MRSA isolates; 11 were observed, and two predominant patterns accounted for 68% of those identified. These restriction patterns correlated with the presence or absence of aminoglycoside resistance. A multicopy plasmid of 2.6 kb was present in both MSSA and MRSA isolates that harboured more than one plasmid; it had the same restriction pattern irrespective of its source. The importance of these results in choosing a method of studying the spread of staphylococci is discussed.     

Molecular epidemiology of Salmonella gallinarum in chickens in Tanzania

A molecular epidemiological investigation of Salmonella gallinarum infection in scavenging local chickens and commercial layers in Tanzania was conducted between August 1997 and April 1998. A total of 1152 chickens were randomly selected from 10 villages and seven commercial farms. For serological and cultural prevalence studies, 1152 blood samples and 912 cloacal swabs were collected. In scavenging local chickens, the individual serological and cultural prevalences were 6.3 and 0%, while the prevalences were significantly higher in commercial layers at 18.4 and 2.6% (P < 0.001), respectively. The risk of infection in flocks of scavenging local chickens that had contact with commercial chickens was six times greater than the risk of infection in flocks of scavenging local chickens that had no contact with commercial chickens. Thirty-four S. gallinarum isolated from commercial chickens in this study, together with 29 Tanzanian historical isolates, were characterized using plasmid profiling and ribotyping. Fifty-one isolates contained both 85 and 2.5kb plasmids, five isolates contained only one plasmid of 85kb, and seven isolates had no plasmids. Ribotyping using HindIII restriction endonuclease demonstrated seven different ribotypes. Forty-seven isolates had similar results in both typing systems, suggesting they belonged to one clone. It is concluded that S. gallinarum infection in chickens in Tanzania is more prevalent in commercial layers than in scavenging local chickens. One strain of S. gallinarum from chickens first isolated in a Dar es Salaam hatchery was found to be common throughout the country.     

Detection of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium with pUO-StVR2-like virulence-resistance hybrid plasmids in the United Kingdom

The aim of this study was to investigate the presence in the United Kingdom (UK) of Salmonella enterica serovar Typhimurium isolates carrying pUO-StVR2-like virulence-resistance hybrid plasmids that originated from pSLT. One hundred and fifty ampicillin-resistant isolates of S. Typhimurium, collected in different regions of the UK during 2006, were screened for the presence of bla _OXA-1 carried by an InH-like integron (2000 bp/bla _OXA-1-aadA1) characteristic of pUO-StVR2. Positive isolates were tested for the presence of a large plasmid that hybridised with probes specific for the bla _OXA-1 and spvC genes, used as resistance and virulence markers of the hybrid plasmid, respectively. Eleven out of the 150 isolates fulfilled both criteria and were assigned to the S. Typhimurium pUO-StVR2 group. Nine were resistant to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfonamides and tetracycline, encoded by bla _OXA-1, catA1, aadA1-like, sul1 and tet(B), respectively, and carried a pUO-StVR2-like plasmid of ca. 130 kb. Two contained hybrid plasmids of smaller size and lacked resistance(s) to chloramphenicol or chloramphenicol and tetracycline. The eleven isolates, which showed five and six closely related XbaI and BlnI profiles, respectively, were resistant to nitrofurantoin. In conclusion, multidrug-resistant S. Typhimurium isolates of the pUO-StVR2 group, which are endemic in Spain, were also detected in the UK, albeit with a low frequency (7.3%).     

Molecular Characterization and Transfer Among Staphylococcus Strains of a Plasmid Conferring High-Level Resistance to Mupirocin

 In this work, mupirocin resistance was correlated with the presence of plasmids in methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in the Rio de Janeiro Federal University Hospital in Brazil, where topical mupirocin has been used extensively since 1990. Of 19 strains studied, those exhibiting high-level resistance carried a large and relaxable plasmid of about 35 kb. Mupirocin-sensitive derivatives, obtained by growth at 42  °C of a strain exhibiting high-level resistance, were devoid of the large plasmid, which was designated pMG1. Mupirocin resistance was transferred to strain RN8411 during overnight filter-matings at low frequencies (7.0×10^–9/donor). The pMG1 plasmid was shown to be responsible for high-level mupirocin resistance in our isolates and to be incompatible with pGO1. Hybridization experiments suggested that mupirocin resistance in pMG1 is due to the presence of the ileS-2 gene. The pMG1 plasmid was successfully and bidirectionally transferred from Staphylococcus aureus to Staphylococcus epidermidis, suggesting that the latter may be a reservoir of this resistance plasmid. No transfer was detected to Staphylococcus haemolyticus. The development of self-transferable high-level mupirocin resistance should be considered when using mupirocin to control the spread of MRSA in hospitals.     

Restriction endonuclease analysis ofStaphylococcus aureus plasmid DNA from three continents

Staphylococcus aureus isolates (n=1201) from 20 centers in Europe, the USA and Brazil were evaluated for the presence of epidemiologic markers. Plasmid typing and restriction endonuclease analysis of plasmid DNA confirmed the presence of an apparently identical plasmid in 13 % of clinical isolates. The plasmid was recovered from all 20 hospitals studied, with an overall frequency of >10 % on each of the three continents. Since relatively few staphylococcal plasmids may be shared by epidemiologically unrelated strains, there are inherent limitations to this otherwise useful technique. Additionally, these data demonstrate the importance of including unrelated strains ofStaphylococcus aureus from the local region as controls when molecular typing methods are performed.     

Kalilo plasmids are a family of four distinct members with individual global distributions across species

Kalilo is a linear 9-kb plasmid, isolated originally from Hawaiian strains of the heterothallic fungus Neurospora intermedia. Its properties include terminal inverted repeats, two ORFs coding for a presumptive DNA and an RNA polymerase, and the ability to cause senescence in its original host and in the closely related species Neurospora crassa. We have examined natural isolates alleged to contain plasmids homologous to kalilo. Most of these isolates do in fact contain plasmids with so close an identity to kalilo as to be certain relatives. We found a new case of kalilo in Neurospora tetrasperma from Moorea-Tahiti, and a new case of LA-kalilo (previously found only in N. tetrasperma) in N. crassa from Haiti. A previously unreported, substantially shorter, kalilo variant has been found in three geographically separate isolates of the heterothallic species Neurospora discreta. Therefore, if the previously reported kalilo variant from the genus Gelasinospora is included, in all there are four members of the kalilo plasmid family. The main differences between these plasmids are in the terminal inverted repeats (TIRs). The phylogeny of the TIR sequences is largely congruent with that of nuclear DNA in the species in which they are found, suggesting that the plasmids are related by vertical descent throughout the evolution of these species. However, there are two cases of a plasmid found in a heterothallic and a pseudohomothallic species in the same global area; these cases might have arisen from more recent horizontal transmission or introgression. Received: 14 July / 17 September 1999     

Protein patterns,serotyping and plasmid DNA profiles in the epidemiologic fingerprinting ofPseudomonas aeruginosa

The epidemiological typing schemes based on serotyping, antibiotic susceptibility pattern, plasmid DNA profile, and protein patterns determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were evaluated for their usefulness in typing clinical isolates ofPseudomonas aeruginosa. The serotypability was lower (45%) than reported in the literature (85–95%). The most commonly found serotypes were O:1 (19%), O:11 (25%), O:6 (35%). The electrophoretic analysis of plasmid DNAs showed plasmids (molecular weight=1 to >40 megadaltons). Two plasmids of M_r 2 and 38 megadaltons were found in various serotypes. The restriction enzyme analysis of plasmid DNA showed identical DNA fragment patterns among distinct serotypes. The SDS-PAGE protein banding patterns of whole-cell proteins showed homogeneity among the strains. However, analysis of the soluble protein patterns of the strains showed sufficiently distinct protein profiles that can be used to differentiate between various strains. The results of this study demonstrate that the electrophoretic patterns of soluble proteins, in combination with plasmid DNA profile or serotyping, can be of value in the epidemiologic fingerprinting of clinical isolates ofPseudomonas aeruginosa.     

Genetic characterization of plasmid-encoded multiple antibiotic resistance in a strain ofListeria monocytogenes causing endocarditis

One susceptible and two multiply resistant isolates ofListeria monocytogenes from a patient suffering from prosthetic valve endocarditis are described. They could not be distinguished by several typing methods. Two isolates were resistant to chloramphenicol, macrolide/lincosamide/streptogramin antibiotics and tetracycline. The resistance determinants were located on a 39 kb plasmid pWDB100 that was transferable by filter mating to several gram-positive bacteria. Evidence was obtained to support the hypothesis that the resistant variant had primarily infected the patient's blood and prosthetic valve, and later lost the resistance plasmid. The three resistance determinants showed homology to other known markers,cat221/cat223,ermB andtetM, which are frequently found in different gram-positive genera. Plasmid pWDB100 showed extensive homology to theStreptococcus agalactiae broad-host-range plasmid pIP501. It was also very similar to two listerial plasmids found in France. Thus, plasmid pWDB100 and the homologous plasmids from France, although isolated in geographically distant regions, may illustrate spread of a plasmid and its relatives.     

The mitochondrial plasmid of the true slime mold <Emphasis Type="Italic">Physarum polycephalum</Emphasis> bypasses uniparental inheritance by promoting mitochondrial fusion

Mitochondrial DNA (mtDNA) is inherited maternally in most eukaryotes. Linear mitochondrial plasmids in higher plants and fungi are also transmitted from the maternal parent to the progeny. However, mF, which is a mitochondrial linear plasmid of Physarum polycephalum, evades uniparental mitochondrial inheritance. We examined 36 myxamoebal strains of Physarum and isolated three novel mF⁺ strains (JE8, TU111, NG111) that harbored free mF plasmids. These strains were mated with the mF^– strain KM88. Of the three mF^– × mF⁺ crosses, only KM88 × JE8 displayed complete uniparental inheritance. However, in KM88 × TU111 and KM88 × NG111, the mtDNA of KM88 and mF of TU111 and NG111 were inherited by the plasmodia and showed recombination. For example, although the mtDNA of TU111 was eliminated, the mF of TU111 persisted and became inserted into the mtDNA of KM88, such that recombinant mtDNA represented 80% of the total mtDNA. The parental mitochondria fused to yield giant mitochondria with two or more mitochondrial nucleoids. The mF appears to exchange mitochondria from the recipient (paternal) to the donor (maternal) by promoting mitochondrial fusion.The first two authors have equally contributed to this work     

Characterization of IncA/C conjugative plasmid harboring blaTEM-52 and blaCTX-M-15 extended-spectrum β-lactamases in clinical isolates of Escherichia coli in Tunisia

To characterize the extended-spectrum β-lactamases (ESBLs) as well as their genetic environment in different isolates of Escherichia coli from patients with repeated urinary tract infections, large multidrug resistance (MDR) plasmids have been found. Definitive evidence for the presence of an A/C incompatibility complex (IncA/C) plasmid in the MDR isolates was provided by the probing of plasmids extracted from the clinical isolates. Conjugation experiments showed that bla genes were transferred by conjugation from the ten E. coli clinical isolates to E. coli XL1-Blue recipient. A comparative restriction fragment length polymorphism (RFLP) analysis of these plasmids showed that they are genetically similar, while the overall similarity of these plasmids supports the likelihood of recent movements among these E. coli isolates. Polymerase chain reaction (PCR) amplification and sequencing of the amplicons showed that the IncA/C plasmids harbor two ESBLs, identified as TEM-52 and CTX-M-15. Analysis of the plasmid DNA surrounding the bla _CTX-M-15 gene in the clinical isolates under study revealed a partially truncated fragment of ISEcp1 tnpA transposase. This result indicates the variety of genetic events that have enabled associations between ISEcp1 sequences and bla _CTX-M-15 genes in these clinical isolates.     

Heteroduplex analysis of Salmonella virulence plasmids and their prevalence in isolates of defined sources

Strains of the Salmonella serovars S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis harbour large plasmids which are required for extraintestinal colonization after oral infection of mice. Electron microscopic heteroduplex analysis showed that these virulence plasmids share large regions of homology. Nine hundred and eighty-six isolates of different origins were analysed for the presence of these plasmids by using a cloned fragment of a S. choleraesuis virulence plasmid as a gene probe. Virulence plasmids were detected in nearly 100% of strains isolated from animal organs or human blood. Frequencies of detection ranged from 48 to 87% in strains of faecal, food or environmental origin. These results suggest that Salmonella virulence plasmids are required for systemic infections in humans and livestock.     

Detection of LT and ST genes in the Escherichia coli isolated from the dogs, sheep and poultry

This study was designed to identify if the heat-labile enterotoxin (LT)-, heat-stable enterotoxin (ST)a- and STb-encoding genes are detectable in faecal samples from different healthy hosts and also on which part of the genome (chromosome or plasmid) they are located. Seventy-five samples of Escherichia coli were isolated from dog, sheep and poultry faecal samples (25 from each host). Plasmid and chromosomal DNA were extracted and polymerase chain reaction performed on all plasmid and chromosomal DNA using LT, STa and STb primers. Additionally, the plasmid profile of all E. coli isolates was defined using gel electrophoresis. The results showed that 36% of the E. coli isolates possessed genes for the production of LT toxin. All samples were negative for STa and STb genes. The plasmid profile of different hosts showed that all samples harboured plasmids. The results of this study indicate that enterotoxigenic E. coli are present in the faeces of different hosts. Since the genes encoding for LT and ST toxins reside on plasmids, or occasionally on transposons, they may transfer among the Gram-negative bacteria especially to the enterobacteriaceae family including E. coli which then may infect other hosts, for example, humans.     

Prevalence and profiles of plasmids inPseudomonas aeruginosa

The value of plasmid profile determination as an epidemiological tool inPseudomonas aeruginosa infections was investigated by determining the prevalence of plasmids in 450Pseudomonas aeruginosa strains and comparing the technique with other epidemiological tools. Since only 13.9% of these strains harbored plasmids and the majority of these plasmids were antibiotic resistant, the technique appeared to be less appropriate as an epidemiological tool in this organism than other techniques. Comparison of results obtained from plasmid profile determinations with those from antibiotyping, serotyping and pyocin typing in 50 non-epidemic strains showed the technique gave highly reproducible results and was sensitive; its ability to discriminate could be improved by additionally performing conjugation assays and hydrolysis of plasmidic DNA with restriction enzymes. It is concluded that plasmid profiles provide important epidemiological information onPseudomonas aeruginosa infections when performed in conjunction with either serotyping or, more importantly, pyocin typing.