Effect of the Prophage and Penicillinase Plasmid of the Recipient Strain Upon the Transduction and the Stability of Methicillin Resistance in Staphylococcus aureus

Transduction of a methicillin-resistance determinant (mec) in Staphylococcus aureus RN450 was dependent on its prior lysogenization with an appropriate temperate phage. In addition, an appropriate transduced penicillinase plasmid was usually required. Some phage 80-resistant variants of RN450 or of its parental lysogenic strain, NCTC 8325, were also effective recipients for transduction of mec. Elimination of prophage from RN450 abrogated its effectiveness as a transductional recipient of mec. Elimination of prophage from a methicillin-resistant transductant of RN450 reduced resistance to undetectable levels in six of seven phage-eliminated strains. In four of these a variable number of clones again became phenotypically resistant after lysogenization alone or lysogenization combined with reintroduction of a penicillinase plasmid. In two prophage-eliminated strains, no evidence of residual mec could be adduced. The establishment, expression, or stability of the transduced mec in strain RN450 appeared to depend on some function determined by a prophage or a prophage and a penicillinase plasmid.     

Transfer of plasmids pBR322 and pBR325 in wastewater from laboratory strains of Escherichia coli to bacteria indigenous to the waste disposal system.

Laboratory strains of Escherichia coli containing plasmid pBR325 (or pBR322) were coincubated with a mobilizer strain of E. coli (containing the conjugative plasmid R100-1) and a recipient strain of bacteria. Bacterial strains isolated from raw wastewater or a plasmid-free E. coli laboratory strain served as recipients. Transfer of the pBR plasmid into the recipient strain occurred during a 25-h coincubation in either L broth or sterilized wastewater; transfer frequencies were several orders of magnitude lower in wastewater. After the coincubation, recipients exhibited both plasmid-encoded phenotypic characteristics and an altered plasmid profile, as shown by agarose gel electrophoresis of purified plasmid DNA.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain.

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

Transfer of plasmids pBR322 and pBR325 in wastewater from laboratory strains of Escherichia coli to bacteria indigenous to the waste disposal system

Laboratory strains of Escherichia coli containing plasmid pBR325 (or pBR322) were coincubated with a mobilizer strain of E. coli (containing the conjugative plasmid R100-1) and a recipient strain of bacteria. Bacterial strains isolated from raw wastewater or a plasmid-free E. coli laboratory strain served as recipients. Transfer of the pBR plasmid into the recipient strain occurred during a 25-h coincubation in either L broth or sterilized wastewater; transfer frequencies were several orders of magnitude lower in wastewater. After the coincubation, recipients exhibited both plasmid-encoded phenotypic characteristics and an altered plasmid profile, as shown by agarose gel electrophoresis of purified plasmid DNA.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

Integration of plasmid DNA coding for beta-lactamase production in the Haemophilus influenzae chromosome.

Of beta-lactamase-producing strains of Haemophilus influenzae, 65% do not contain extrachromosomal plasmid DNA. These strains, however, conjugally transfer beta-lactamase production to a recipient strain from which a 30-megadalton plasmid can be isolated. Restriction enzyme analysis and Southern transfer of DNA from both donor and recipient strains revealed that chromosomal integration of plasmid sequences occurred in all donor strains examined.     

Lysogenic conversion for multiple characters in a strain of Staphylococcus aureus.

Lysogenization of nonlysogenic strains of Staphylococcus aureus was performed with two different bacteriophages, LS1 and LS2, that were unable to plaque on any of the strains of S. aureus tested. Infection of recipient strains was achieved when protoplasts were inoculated with LS1 or LS2 or when bacterial cultures were simultaneously inoculated with a virulent phage together with LS1 or LS2. Lysogenization was demonstrated by changes in phenotypic characters of the host strain and by liberation of bacteriophages from the modified strains as shown by electron microscopic examination. The lysogenic strains differed from the host strains by the following characters: they were coagulase, deoxyribonuclease, and lipase negative; they were untypable by the basic set of phages; they did not ferment mannitol under anaerobic conditions; and they produced only l-(+)-lactic acid by glucose fermentation. Their cell walls contained less glycine and concomitantly more serine than those of the host strains. Furthermore, they were devoid of protein A. Conversely, some antigenic factors as well as the presence of ribitol in the cell wall teichoic acid, indicated a parental relationship between the host strains and the derived lysogenic ones. Phages LS1 and LS2 could be excluded from the lysogenic strains by invading phages, and the revertant nonlysogenic strains recovered all of the characteristics of the initial host strains. It was thus concluded that the phenomenon described was due to lysogenic conversion. The origin of phages LS1 and LS2 is discussed.     

The Value of Plasmid Profiles for Strain Identification in Lactic Streptococci and the Relationship between Streptoccocus lactis 712, ML3 and C2

Plasmid DNA from lactic streptococci was subjected to electrophoresis on agarose gels. The plasmid profiles so obtained were strain specific and sufficiently stable to suggest their use in strain differentiation. A group of Streptococcus lactis strains, 712. 763 (ML3), 505 (C2) and 2031 (C2), found to have similar plasmid profiles, were shown to be closely related. Gene transfer by transduction and conjugation occurred between members of this group at frequencies comparable to those in homologous systems and temperate phages cross plated readily between their prophage cured derivatives.
Minor variations were, however, found between these four strains; slight differences in plasmid profiles, lysogenic status, prophage curability and temperate phage morphology were detected and it is suggested that these have evolved as a result of maintenance in different environments.     

Characterization of temperate bacteriophages of Leuconostoc oenos and evidence for two prophage attachment sites in the genome of starter strain PSU-1

The close relatedness between 17 Leuconostoc oenos bacteriophages, induced with mitomycin C from strains isolated in different geographic regions, was inferred from their morphology, DNA homology and protein composition. The genome of all the phages had cohesive end termini and ranged in size from 36.4 to 40.9 kb. According to the restriction patterns obtained by digestion with five enzymes, the phages were divided in six groups. Lysogenization of a spontaneous phage-cured derivative of Leuc. oenos strain PSU-1 was achieved with 16 phages and the analysis of the lysogens showed that the phage DNA integrates in the host chromosome in one or two sites. The att B loci were located on the macrorestriction Asc I and Not I fragments of the recipient strain. A survey of Leuc. oenos strains with a phage DNA probe confirmed the lysogenic nature of several, but not all of the original phage hosts. These results are discussed in the light of evidence for the instability of some lysogenic PSU-1 derivatives.     

Inter- and intraspecies conjugal transfer of different plasmids in bacilli

Conjugal transfer of plasmid pUB110 between different strains of bacilli was studied. The plasmid transfer was possible not only between various strains of B. subtilis, but also when many other species of bacilli served as recipients. Conjugation of a donor strain B. subtilis 19 (p19pUB110) was accompanied by a transfer of plasmid p19 along with plasmid pUB110 to the B. subtilis recipient strains lacking a large plasmid p19. If, like the donor cells, the recipient B. subtilis strain carried plasmid p19, the frequency of conjugation decreased. The small plasmid pBC16 was also capable of conjugative transfer. However, if this plasmid carried the mob gene with an inverted region, the frequency of its transmission dramatically decreased. If the donor strain contained another small plasmid, pV, which also carried the mob gene, the efficiency of transmission was partially restored.     

The identification, typing and fingerprinting of Salmonella: laboratory aspects and epidemiological applications

Since the 1930s, traditional methods of strain identification based on serotyping and phage typing have been the foundation of salmonella epidemiology. Although the incidence of diseases such as typhoid and paratyphoid has decreased in recent years, food-poisoning caused by non-typhoidal salmonella strains has now reached epidemic proportions in many countries, despite improvements in sanitation and hygiene. Precise strain identification is an essential prerequisite for epidemiological investigations aimed at combating the spread of these strains and eradicating the sources of infection. Modern methods of genotypic typing, particularly those based on physical characterization of the plasmid content of the organism have already proved invaluable for the identification and differentiation of strains in many outbreaks. These plasmid typing methods are now increasingly used with serotyping and phage typing for many epidemiological investigations. Other methods of genotypic typing, particularly those based on recognition of small differences in chromosome structure, are not yet practical for the examination of large numbers of strains. Nevertheless, improvements in small-scale methods for chromosomal DNA extraction coupled with the increasing use of non-isotopic labels for identification of restriction fragment length polymorphisms may provide a new dimension to Salmonella epidemiology.     

Fertility properties and regulation of antimicrobial substance production by plasmid SCP2 of Streptomyces coelicolor.

Streptomyces coelicolor A3(2) possesses two plasmids (SCP1 and SCP2) that act as sex factors. The plasmid deoxyribonucleic acid isolated from S. coelicolor A3(2) SCP1- strains A617 and A585 had the same molecular weight and endonuclease cleavage pattern as the SCP2 plasmid. The plasmidless strain S18 SCP2- was isolated from the A617 X A585 cross. SCP2 plasmid-containing strains acted as donors of chromosomal markers, whereas the plasmidless strain acted as recipient. The transfer of SCP2+ donor strain markers into the SCP2- recipient occurred at high frequencies (approximately 75%), was unidirectional, was initiated from a fixed region of the chromosome, and had the SCP2 fertility factor transferred first. The introduction of the SCP2 plasmid into a recipient strain greatly reduced the recombination frequency. These fertility properties differed from those previously reported, thereby suggesting that the SCP2 plasmid examined in this investigation may be an additional variant to those described in the literature. The SCP2 plasmid also regulated production of three antibacterial substances and conveyed resistance for S. coelicolor A3(2) strains against growth inhibition by one of them.     

Lysogeny in Leuconostoc oenos.

Thirty strains of Leuconostoc oenos were exposed to mitomycin C to induce lysogenic bacteriophages. Lysis curves typical for lysogenic strains were obtained with 19 strains. Indicator strans were found for 17 of these phages. Five were characterized by electron microscopy, lytic spectrum, molecular masses of the proteins, sequencing of five N-terminal amino acids of the two major proteins and DNA analysis (restriction patterns, cross hybridization). The results revealed a very close relationship between the phages. Hybridization experiments between the DNAs of the temperate phages and the appropriate lysogenic strains revealed phage-related sequences in the DNA of the lysogenic strain.     

Inter- and Intraspecies Conjugal Transfer of Different Plasmids in Bacilli

Conjugal transfer of plasmid pUB110 between different strains of bacilli was studied. The plasmid transfer was possible not only between various strains of B. subtilis, but also when many other species of bacilli served as recipients. Conjugation of a donor strain B. subtilis 19 (p19 pUB110) was accompanied by a transfer of plasmid p19 along with plasmid pUB110 to the B. subtilis recipient strains lacking a large plasmid p19. If, like the donor cells, the recipient B. subtilis strain carried plasmid p19, the frequency of conjugation decreased. The small plasmid pBC16 was also capable of conjugative transfer. However, if this plasmid carried the mob gene with an inverted region, the frequency of its transmission dramatically decreased. If the donor strain contained another small plasmid, pV, which also carried the mob gene, the efficiency of transmission was partially restored.     

Transfer of the pheromone-inducible plasmid pCF10 among Enterococcus faecalis microorganisms colonizing the intestine of mini-pigs.

A new animal model, the streptomycin-treated mini-pig, was developed in order to allow colonization of defined strains of Enterococcus faecalis in numbers sufficient to study plasmid transfer. Transfer of the pheromone-inducible pCF10 plasmid between streptomycin-resistant strains of E. faecalis OG1 was investigated in the model. The plasmid encodes resistance to tetracycline. Numbers of recipient, donor, and transconjugant bacteria were monitored by selective plating of fecal samples, and transconjugants were subsequently verified by PCR. After being ingested by the mini-pigs, the recipient strain persisted in the intestine at levels between 10(6) and 10(7) CFU per g of feces throughout the experiment. The donor strain, which carried different resistance markers but was otherwise chromosomally isogenic to the recipient strain, was given to the pigs 3 weeks after the recipient strain. The donor cells were initially present in high numbers (10(6) CFU per g) in feces, but they did not persist in the intestine at detectable levels. Immediately after introduction of the donor bacteria, transconjugant cells appeared and persisted in fecal samples at levels between 10(3) and 10(4) CFU per g until the end of the experiment. These observations showed that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from ingested E. faecalis cells to other E. faecalis organisms already present in the intestinal environment and that the plasmid subsequently persisted in the intestine.     

Effect of prophages on transfer frequency of conjugative plasmid G 873]

Transfer of the conjugative plasmid G873 on filters and mixed cultivation of the donor and recipient cells in liquid media is described. In the both systems the use of the lysogenic recipient cells (phages of serogroups B and F) in the crossings increased mor than 100-fold the frequency of plasmid transfer. The conjugative transfer of the plasmid in the mixed cultivation system was proved. The conjugative transfer required the presence (while not obligatory) of calcium chloride and was restricted by the serum factors.     

Effects of Mitomycin C and Thymidine Deprivation on Lysogenic and Sensitive Strains of Bacillus megaterium

A triple auxotroph of Bacillus megaterium strain KM was lysogenized with a phage suspension from B. megaterium 899a. The lysogenic and phage-sensitive derivatives of KM were found to die at the same exponential rate during thymineless incubation, despite the fact that the lysogenic strain became induced. The lysogenic strain was also induced by mitomycin C, and died at an exponential rate which was approximately twice that of the sensitive strain. With both strains, the lethality of mitomycin C was the same in the presence and absence of thymidine; thymidine was required for maximal phage production. Mitomycin C preferentially inhibited deoxyribonucleic acid (DNA) synthesis of both strains for the first 60 min. The (DNA) synthetic ability of the lysogenic strain was subsequently restored, due to phage production. Since there was no evidence that sensitive strains of KM contained other inducible elements (prophage or probacteriocins), it is concluded that both thymineless death and mitomycin C death can occur via mechanisms not involving induction.     

Cloning of genes from members of the family Enterobacteriaceae with mini-Mu bacteriophage containing plasmid replicons.

An in vivo cloning system that uses derivatives of the Escherichia coli bacteriophage Mu with plasmid replicons has been extended to five different species of the family Enterobacteriaceae. Mu and these mini-Mu replicon elements were introduced into strains of E. coli, Shigella flexneri, Salmonella typhimurium, Citrobacter freundii, and Proteus mirabilis by infection, by transformation, or by conjugation with newly constructed broad-host-range plasmids containing insertions of these elements. Lysates from these cells, lysogenic for Mu and mini-Mu elements, were used to infect sensitive recipient strains of E. coli, S. typhimurium, and C. freundii. Drug-resistant transductants had mini-Mu replicon elements with inserts of different DNA sequences. All of the lysogens made could be induced to yield high phage titers, including those coming from strains that were resistant to Mu and Mu derivatives. Clones of 10 particular genes were isolated by their ability to complement specific mutations in the recipient strains, even in the presence of the E. coli K-12 restriction system. Some of the mini-Mu replicon elements used contained lac gene fusing segments and resulted in fusions of the lac operon to control regions in the cloned sequences.     

Conjugation transfer of plasmid resistance to gentamycin and other antibiotics in clinical strains of Ps. aeruginosa]

A possibility of conjugation transfer of the markers of the plasmid resistance to gentamicin and other antibiotics from 10 clinical strains of Ps. aeruginosa, isolated from burn patients to the recipient strain of Ps. aeruginosa PTO 629 Rfr was shown. The marker of gentamicin resistance was transferred to 100 out of 110 of the exconjugants, i.e. 86.2 per cent. The rate of the conjugation transfer in the crosses between the clincal strains of Ps. aeruginosa and the recipient strain PTO 629 Rfr with respect to the gentamicin marker was about 10--7. The plasmid resistance markers in the clincal strains Ps. aeruginosa were transferred in various combinations. Transfer of the markers of resistance to streptomycin, carbenicillin, neomycin and combinations Sm, Nm and Sm, Nm, Cm was not achieved.