Characterization of mutations in the penicillinase operon of Staphylococcus aureus

Summary Mutant penicillinase plasmids, in which penicillinase synthesis is not inducible by penicillin or a penicillin analogue, were examined by biochemical and genetic analyses. In five of the six mutants tested, penicillinase synthesis could be induced by growth in the presence of 5-methyltryptophan. It is known that the tryptophan analogue 5-methyltryptophan is readily incorporated into protein by S. aureus and that staphylococcal penicillinase lacks tryptophan. 5-methyltryptophan seems to induce penicillinase synthesis in wild-type plasmids by becoming incorporated into the repressor and thereby inactivating the operator binding function of the penicillinase repressor. Therefore, induction of penicillinase synthesis in the mutant plasmids by 5-methyltryptophan strongly suggests that the noninducible phenotype of these five plasmids is due to a mutation that inactivates the effector binding site of the penicillinase repressor (i.e., the five mutant plasmids carry an i^s genotype for the penicillinase repressor). This conclusion was supported by heterodiploid analysis. The mutant plasmid that did not respond to 5-methyltryptophan either produces an exceedingly low basal level of penicillinase or does not produce active enzyme. This plasmid seems to carry a mutation in the penicillinase structural gene or in the promoter for the structural gene. Thus, a genetic characterization of many mutations in the penicillinase operon can be accomplished easily and rapidly by biochemical analysis.Journal Paper No. J-7994 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2029     

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.     

Nature of the plasmid-linked penicillinase regulatory region in Staphylococcus aureus

Summary Four noninducible staphylococcal penicillinase plasmids reported to produce a very low basal level of penicillinase were investigated. Incorporation of 5-methyltryptophan, which is known to inactivate the operator binding site of wild-type penicillinase repressor and thereby elicit penicillinase synthesis, did not induce penicillinase synthesis in any of these micro mutants. Therefore, these plasmids are not simply peni ^S mutants. Heterodiploid strains composed of a plasmid fully constitutive for penicillinase synthesis and one of the various micro penicillinase plasmids were constructed. Three of these heterodiploids produce a normal basal level of penicillinase and are inducible by 5-methyltryptophan but not by the standard gratuitous penicillinase inducer. Therefore, each of these three noninducible micro plasmids produces a peni ^S repressor, but in addition, each must bear a mutation in the penZ region. The fourth heterodiploid produces a fully constitutive level of penicillinase. The noninducible micro plasmid present in this heterodiploid must contain a penI ^– mutation and a mutation in the penZ region. Consequently, each of these four noninducible micro plasmids contains at least two mutants genes. Hence, the phenotype of noninducibility plus low basal penicillinase is not due to a point mutation in a second penicillinase regulatory region as has been proposed. Instead, these results strongly suggest that there is only one penicillinase regulatory gene located on the penicillinase plasmid and that this gene (penI) specifies the penicillinase repressor.Journal Paper No. J-8700 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa; Project No. 2029     

Constitutive penicillinase formation in Staphylococcus aureus owing to a mutation unlinked to the penicillinase plasmid

Previously described penicillinase-constitutive mutations in Staphylococcus aureus are caused by genetic lesions in a regulator gene (or genes) on the penicillinase plasmid in close linkage to the structural gene. This report describes a new class (R2(-)) of penicillinase-constitutive mutants of S. aureus unlinked to the plasmid. By transductional analysis, the penicillinase plasmids in these mutants were wild type. Wild-type plasmids transduced into penicillinase-negative (plasmid loss) derivatives of R2(-) mutants produced penicillinase constitutively in amounts comparable to a fully induced culture of the wild-type strain. Penicillinase production in R2(-) mutants was maximal at 30 to 32 C and was much reduced at 40 C.     

Conjugative plasmids in Neisseria gonorrhoeae.

A conjugation system initially discovered in beta-lactamase-producing gonococci mobilized small non-selftransmissible R plasmids encoding beta-lactamase (penicillinase) production into other gonococci, Neisseria, and Escherichia coli. This conjugation system was mediated by a separate selftransmissible plasmid of 23.9 X 10(6) daltons, pFA2. Conjugative plasmids capable of mobilizing R plasmids were also found in nearly 8% of the non-penicillinase-producing gonococci. These were similar to pFA2 in size, buoyant density, and restriction endonuclease digest patterns but were less efficient than pFA2 in mobilization of the penicillinase plasmid pFA3. The presence of conjugative plasmids in gonococci isolated before the appearance of penicillinase-producing strains indicates that a conjugation system for plasmid transfer predated the appearance of R plasmids in gonococci.     

Transformation-derived Neisseria gonorrhoeae plasmids with altered structure and function.

Plasmid deoxyribonucleic acid from Neisseria gonorrhoeae containing a 7.1-kilobase (kb) (4.7-megadalton) penicillinase (Pcr) plasmid transformed homogenic gonococci to penicillinase production at a low frequency. About 25% of the penicillinase-producing gonococcal transformants contained Pcr plasmids which were either larger or smaller than the 7.1 kb donor plasmid; these Pcr plasmids varied in size from 3.45 to 42 kb. Some of these altered plasmids differed from the donor plasmid in stability or in frequency of mobilization by a 36-kb (24-megadalton) conjugative plasmid. A restriction endonuclease cleavage map of the 7.1-kilobase Pcr plasmid and several of the smaller deleted plasmids was constructed. The most common size of altered Pcr plasmid was 5.1 kb (3.4 megadaltons). A Pcr plasmid isolated from a gonococcus in London, England, was identical with these 5.1-kb transformant plasmids in both size and restriction endonuclease cleavage profiles, suggesting that the 5.1-kb Pcr plasmid could have arisen from a 7.1-kb Pcr plasmid by a transformation-associated deletion in nature.     

Characteristics of penicillinase plasmids from Staphylococcus aureus strains

Penicillinase plasmids are present in most MRSA strains. They are very varying in their genotype and phenotype they confer. Penicillinase plasmids were transduced from 80 hospital MRSA strains to NCTC 8325 and the phenotype as well as the incompatibility group of plasmid were determined. Resistance to cadmium (high and low level), resistance to organic and nonorganic mercury compounds, arsenate/arsenite/antimonium resistance, resistance to bismuth and hypersensitivity to bismuth, resistance to macrolides as well as beta-lactamase production and its inductibility were checked. Among the examined strains 20 different phenotypes of penicillinase plasmids were found. Patterns of penicillinase plasmids were compared to DNA patterns of the investigated strains after digestion with SmaI and separation in pulsed field electrophoresis (PFGE). It was shown that strains with the same PFGE pattern often differ in the type of their penicillinase plasmid. Determining of penicillinase plasmid phenotype could be useful in differentiating S. aureus strains sharing the same pattern of PFGE.     

Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor.

Bacillus licheniformis penicillinase genes, penP and penI, are coded on a 4.2-kilobase EcoRI fragment of pTTE21 (T. Imanaka, T. Tanaka, H. Tsunekawa, and S. Aiba, J. Bacteriol. 147:776-186, 1981). The EcoRI fragment was subcloned in a low-copy-number plasmid pTB522 in Bacillus subtilis. B. subtilis carrying the recombinant plasmid pPTB60 (Tcr penP+ penI+) was chemically mutagenized. Of about 150,000 colonies, two penI(Ts) mutant plasmids, pPTB60D13 and pPTB60E24, were screened by the plate assay at 30 and 48 degrees C for penicillinase. By constructing recombinant plasmids between wild-type and mutant plasmids, the mutation points were shown to be located in a 1.7-kilobase EcoRI-PstI fragment. The EcoRI-PstI fragments of the wild-type plasmid and two mutant plasmids were sequenced. A large open reading frame, composed of 384 bases and 128 amino acid residues (molecular weight, 14,983), was found. Since the mutation points were located at different positions in the protein coding region (Ala to Val for pPTB60D13 and Pro to Leu for pPTB60E24), the coding region was concluded to be the penI gene. A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site (ATG). A probable promoter sequence which is very similar to the consensus sequence was also found upstream of the penP promoter, but in the opposite direction. A consensus twofold symmetric sequence (AAAGTATTA CATATGTAAGNTTT) which might have been used as a repressor binding region was found downstream and in the midst of the penP promoter and also downstream of the penI promoter. The regulation of penP and penI by the repressor is discussed.     

Cloning of the genes for penicillinase, penP and penI, of Bacillus licheniformis in some vector plasmids and their expression in Escherichia coli, Bacillus subtilis, and Bacillus licheniformis.

By using plasmid pMB9, penicillinase genes (penP and penI) from both the wild-type and constitutive strains of Bacillus licheniformis 9945A were cloned in EScherichia coli. When a low-copy-number plasmid was used, both wild-type and constitutive penicillinase genes could be transferred into Bacillus subtilis. However, when a high-copy-number plasmid was used, only the genes of the wild type could be transferred. These recombinant plasmids in B. subtilis could all be transferred by the protoplast transformation procedure into B. licheniformis. Transformants of E. coli were resistant to ampicillin (20 micrograms/ml) in spite of the low penicillinase activities (7 U/mg of cells). However, transformants of B. subtilis and B. licheniformis were sensitive to ampicillin (20 micrograms/ml) even in high penicillinase activities (more than 10,000 U/mg of cells). The secretion of penicillinase was rarely observed in E. coli. In contrast, penicillinases secreted from transformants of B. subtilis and B. licheniformis were around 30 and 60% of the total activities, respectively. We took advantage of the plasmids to permit the construction of hetero- and mero-polyploid structures in host cells, and we discuss a regulatory mechanism of penicillinase synthesis in B. licheniformis.     

The effect of membrane-bound beta-lactamase on linoleic acid sensitivity in Staphylococcus aureus

The presence of a plasmid conferring resistance to penicillin (PC plasmid, e.g. pI258blaI-) in Staphylococcus aureus NCTC 8325 increases the sensitivity of such a bacterium to the growth inhibitory effects of linoleic acid, whereas a plasmid conferring resistance to tetracycline does not affect linoleic acid sensitivity. The increased linoleic acid sensitivity of bacteria containing a PC plasmid may be related to the penicillinase protein itself since (i) strains having inducible penicillinase show increased sensitivity only after induction, (ii) strains in which penicillinase is directed from chromosomal or plasmid-borne genes show similar increased linoleic acid sensitivity and (iii) notwithstanding the above, the linoleic acid inhibitory effect is enhanced in a strain in which penicillinase activity is greatly reduced by a point mutation in the structural gene for penicillinase. The enhanced linoleic acid sensitivity seems to require the membrane-bound penicillinase since added extracellular penicillinase does not confer this sensitivity, and there appears to be a specific interaction between the membrane-bound penicillinase activity and linoleic acid.     

Genetic Studies on Plasmid-Linked Cadmium Resistance in Staphylococcus aureus

The genetic basis of cadmium resistance conferred by three penicillinase plasmids, PI(524), PI(258), and PII(147), of Staphylococcus aureus was examined by mutation, recombination, and deletion analysis. Three separate loci were identified: cadA, responsible for high-level resistance; cadB, giving a low-level resistance, nonadditive to cadA; and mad, a locus marginally decreasing the cadmium resistance of plasmid-positive staphylococci. The loci cadA and mad were present on all three plasmids, but cadB was only found on PII(147). Spontaneous deletions of mad involved up to three-fourths of the plasmid genome, which allowed derivation of a partial deletion map of PII(147), a plasmid with a contour length of 10.9 mum, corresponding to a molecular weight of 20.4 x 10(6).     

Transposition of penicillinase determinants in methicillin-resistant Staphylococcus aureus

Abstract A methicillin-resistant Staphylococcus aureus (MRSA) typical of those being isolated in Australian hospitals has been studied. It contains two plasmids, one of 1.4 megadalton (MDa) and one of 18 MDa. When selection is made for resistance to nucleic acid-binding (NAB) compounds in mixed-culture transfer, two types of transcipients are obtained; those containing an 18-MDa plasmid and resistant to NAB compounds, trimethoprim and aminoglycosides such as gentamicin and kanamycin and those having a 22 MDa plasmid and the additional phenotype of penicillinase production. The penicillinase determinants on the 22-MDa plasmid have been found to transpose to the chromosome and from the chromosome to an 18-MDa plasmid similar to that found in the original isolate. Restriction enzyme analysis has shown that a 7.3-kilobase pair (kb) element is involved. This has been designated Tn 3852 .     

Characteristics and distribution of plasmids in a clonally diverse set of methicillin-resistant Staphylococcus aureus strains

The aim of this study was to compare the plasmid contents of methicillin-resistant Staphylococcus aureus (MRSA) strains classified into different clonal clusters (CCs). The isolates were collected from 15 Czech hospitals in 2000-2008. Plasmid DNA was detected in 65 (89%) strains, and 33 of them harbored more than one plasmid type. Altogether 24 different types of plasmids were identified, ranging in size from 1.3 to 55 kb. Restriction endonuclease analysis, plasmid elimination, DNA hybridization, and sequencing were used for their further characterization. It has been found that the conjugative, erythromycin resistance and enterotoxin D encoding plasmids are harbored by strains from different CCs. On the other hand, chloramphenicol and tetracycline resistance plasmids, and most of the penicillinase and cryptic plasmids were only detected in certain CCs. Especially, the pUSA300-like plasmids were found exclusively in the USA300 clone strains. The high diversity in plasmid content detected in the study strains implies that plasmids play a major role in evolution of MRSA clonal lineages.     

Effects of the Recipient Strain and Ultraviolet Irradiation on Transduction Kinetics of the Penicillinase Plasmid of Staphylococcus aureus

When the penicillinase plasmid of Staphylococcus aureus PS 81(P(81))(T(81)) was transferred to its cured derivative of PS 81(N(P))(T(81)), there was a fivefold increase in the transduction frequency of penicillinase plasmid markers after ultraviolet (UV) irradiation of the phage instead of the expected decrease typical for plasmid-borne markers. These results were independent of the transducing phage, the donor, and the method of curing the recipient and were also obtained with a cured derivative of PS 80(PI(80)). With PS 52, a naturally occurring penicillin-sensitive strain, and a cured transductant of PS 52 as the recipients, typical plasmid kinetics were observed. The plasmid location of penicillinase plasmid markers in transductants was confirmed by their instability in ethidium bromide (EB). In a cross between isogenic plasmids (PI(258)penZ cad x PI(258)penI asa ero), transductants were doubly selected for cadmium and erythromycin resistances. There was a twofold increase in transduction frequency after UV irradiation of the transducing phage and an increase in the proportion of recombinant type transductants. CsCl-EB density centrifugation revealed that plasmid deoxyribonucleic acid (DNA) was present in PS 81(P(81))(N(T)) and its cured derivative [PS 81(N(P))(N(T))], but not in PS 52. Sucrose gradient analysis of plasmid DNA showed that the penicillinase plasmid of PS 81(P(81))(N(T)) was larger than the plasmid in its cured derivative. Thus, the cured derivative contains plasmid DNA which appears to recombine with the incoming plasmid, causing the rise in transduction frequency noted after UV irradiation of transducing phage.     

Plasmid-protein relaxation complexes in Staphylococcus aureus.

Protein-deoxyribonucleic acid relaxation complexes have been demonstrated for six Staphylococcus aureus plasmids out of sixteen examined. Four of these encode stretomycin resistence, have molecular weights of about 2.7 x 10(6), and are isolated as supercoiled molecules that are virtally 100% relaxable by treatment with sodium dodecyl sulfate. It is probable that these four isolates represent a single widely disseminated plasmid species. The other two plasmids showing relaxation complexes have molecular weights of about 3 x 10(6) and encode chloramphenicol resistance. The complexes in these cases are unstable, and it has not been possible to induce more than 50% relaxation by any of the standard treatments. Ten other plasmids do not show detectable complexes. These include three penicillinase plasmids, four tetracycline-resistance plasmids, one plasmid carrying kanamycin-neomycin resistance, and finally, two chloramphenicol-resistance plasmids.     

Naturally Occurring Penicillinase Plasmids in Staphylococcus aureus

A series of plasmids harbored by naturally occurring penicillin-resistant strains of Staphylococcus aureus were surveyed with a view toward exploring the variability in plasmid-linked marker patterns. Plasmids were transduced from their natural hosts to either of two plasmid-negative laboratory strains by selection for cadmium resistance, and the transductants were tested for all other markers previously found to be plasmid-linked. All of the strains that were able to serve as genetic donors to one of the two stock strains could donate cadmium and lead resistance as linked, plasmid-borne markers. Among the other plasmid markers, a wide variety of patterns was found, including four plasmids that did not carry the penicillinase determinant. Each of the 26 plasmids studied, including the latter 4, was found to belong to one of the two incompatibility sets of penicillinase plasmids previously identified. With the exception of the penicillinase-negative plasmids, which were found in both sets, all the plasmids of incompatibility set I directed the production of penicillinase type A; those belonging to set II directed either type A or type C. Those of set II without exception increased the sensitivity of their host strains to bismuth ion; those of set I carried determinants of bismuth resistance or did not affect the sensitivity of their host to this ion. No other perfect correlations between markers were encountered; in particular, there was no correlation between penicillinase serotype and the excretion of the enzyme. This finding allows the prediction that there is, in addition to all of the markers thus far identified, a plasmid-linked determinant of penicillinase excretion.     

Genetic transfer of methycilline resistance in Staphylococus epidermidis and Staphylococcus aureus strains in mixed cultures

In mixed cultures of staphylococci a transfer of the resistance to methicillin and penicillinase plasmids as well as tetracycline and chloramphenicol plasmids was investigated. It was shown that the resistance to methicillin was transferred in mixed cultures from one strain of S. aureus to another and from S. epidermidis to S. aureus. In both cases transfer of methicillin resistance required, the presence of penicillinase plasmid in recipient or donor strain. In the case of other markers transmission was independent. Moreover it was shown that the transfer of resistance genes in mixed cultures was mediated by bacteriophage of the serologic group A.     

Studies on plasmid replication. III. Isolation and characterization of replication-defective mutants

Some 85 Staphylococcus aureus mutants phenotypically thermosensitive for penicillinase plasmid segregation (Seg-) have been isolated and characterized. Some of the mutations were plasmid-linked and those studied in detail were found to be defective in plasmid replication, most problbly at the initiation stage. Analysis of the segregation behavior of these mutants suggested a figure of 2.7 for the average number of plasmid copies per cell in a random culture. Other mutations were host chromosome-linked and these could be divided into at least three classes on the basis of their ability to maintain plasmids of the two different incompatibility sets: some were defective for type I plasmids, some for type II, and some for both types. One host mutant, defective in segregation of type I but not type II plasmids, was defective in polymerization of both.     

Detection of penicillinase and bacteriocinogenicity plasmids in Staphylococcus aureus strain No. 580]

Staphylococcus aureus No. 580 strain contains simultaneously two plasmids: bacteriocinogenicity plasmid and penicillinase plasmid. Both plasmids are lost spontaneously with a high frequency, and also under the effect of acridine orange at temperatures of 37 degrees C and 44 degrees C, and in cultivation at a temperature of 44 degrees C for 5 days. Loss of one of the plasmids failed to lead to stabilization of another plasmid, and it was eliminated spontaneously with the same frequency as in the population of the initial strain. Plasmid loss did not lead to the changes in biochemical and pathogenic properties and also of the phagovar and bacteriogenovar. At the same time in elimination of one or both plasmids lag-phase diminished from 220 to 120 min.