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.     

In situ monitoring of IncF plasmid transfer on semi-solid agar surfaces reveals a limited invasion of plasmids in recipient colonies

Most natural conjugative IncF plasmids encode a fertility inhibition system that represses transfer gene expression in the majority of plasmid-carrying cells. The successful spread of these plasmids in clinically relevant bacteria has been suggested to be supported by a transitory derepression of transfer gene expression in newly formed transconjugants. In this study, we aimed to monitor the extent of transitory derepression during agar surface matings in situ by comparing plasmid spread of the IncF plasmid R1 and its derepressed mutant R1drd19 at low initial cell densities. A zygotic induction strategy was used to visualize the spatial distribution of fluorescent transconjugants within the heterogeneous environment. Epifluorescence and confocal microscopy revealed different transfer patterns for both plasmids, however, spread beyond the first five recipient cell layers adjacent to the donor cells was not observed. Similar results were observed for other prototypical conjugative plasmids. These results cannot rule out that transitory derepression contributes to the limited R1 plasmid invasion, but other factors like nutrient availability or spatial structure seem to limit plasmid spread.     

Genetic and physical studies of recombinant plasmids formed between an R plasmid of compatibility group FI and sex factor F of HfrH.

Recombinant plasmids between an R plasmid of the FI group (R162/3) and the sex factor F or HfrH were produced after the conjugal transfer of this R plasmid into HfrH. Three types of recombinant plasmids were identified after the mating of HfrH (R162/3) with recA and rec+ recipients. One specimen of each type (pIP218, pIP222, pIP226) was studied in this report. All three recombinant plasmids carry the same genetic information for resistance to antibiotics (CSSuT) retained from R162/3. pIP218 retained all the other properties from F of HfrH: derepression for pilus synthesis, mobilization of the chromosome for the proximally transferred HfrH genes (thr, leu, proA), interference with T7 propagation, and ability to be cured by acridine orange. pIP222 retained from F of HfrH the derepression for pilus synthesis and the same polarity of chromosome transfer (thr, leu, proA), while pIP226 retained the interference with T7 propagation and acridine orange curing. Physical studies revealed that replication control and/or recovery of F and pIP218 as covalent circles of deoxyribonucleic acid are similar, and are different from R162/3. The new plasmids are more likely the result of a substitutive recombination event than a fusion. We propose genetic maps of these recombinant plasmids, showing the unequal participation of the parental plasmids in their formation.     

Isolation of Saccharomyces cerevisiae mutants constitutive for invertase synthesis.

A new method for detecting invertase activity in Saccharomyces cerevisiae colonies was used to screen for mutants resistant to catabolite repression of invertase. Mutations causing the highest level of derepression were located in two previously identified genes, cyc8 and tup1. Several of the cyc8 mutations, notably cyc8-10 and cyc8-11, were temperature dependent, repressed at 23 degrees C, and derepressed at 37 degrees C. The kinetics of derepression of invertase mRNA in cyc8-10 cells shifted from 23 to 37 degrees C was determined by Northern blots. Invertase mRNA was detectable at 5 min after the shift, with kinetics of accumulation very similar to that of wild-type cells shifted from high-glucose to low-glucose medium. Assays of representative enzymes showed that many but not all glucose-repressible enzymes are derepressed in both cyc8 and tup1 mutants. cyc8 and tup1 appear to be the major negative regulatory genes controlling catabolite repression in yeasts.     

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.     

Genetic control of riboflavin biosynthesis in Pichia guilliermondii yeasts. The detection of a new regulator gene RIB81

The properties of mutants resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)-isoalloxazine (MTRY) were studied. The mutants were isolated from a genetic line of Pichia guilliermondii. Several of them were riboflavin overproducers and had derepressed flavinogenesis enzymes (GTP cyclohydrolase, 6.7-dimethyl-8-ribityllumazine synthase) in iron-rich medium. An additional derepression of these enzymes as well as derepression of riboflavin synthase occurred in iron-deficient medium. The characters "riboflavin oversynthesis" and "derepression of enzymes" were recessive in mutants of the 1st class, or dominant in those of the 2nd class. The hybrids of analogue-resistant strains of the 1st class with previously isolated regulatory mutants ribR (novel designation rib80) possessed the wild-type phenotype and were only capable of riboflavin overproduction under iron deficiency. Complementation analysis of the MTRY-resistant mutants showed that vitamin B2 oversynthesis and enzymes' derepression in these mutants are caused by impairment of a novel regulatory gene, RIB81. Thus, riboflavin biosynthesis in P. guilliermondii yeast is regulated at least by two genes of the negative action: RIB80 and RIB81. The meiotic segregants which contained rib80 and rib81 mutations did not show additivity in the action of the above regulatory genes. The hybrids of rib81 mutants with natural nonflavinogenic strain P. guilliermondii NF1453-1 were not capable of riboflavin oversythesis in the iron-rich medium. Apparently, the strain NF1453-1 contains an unaltered gene RIB81.     

Comparison of Proteins Involved in Pilus Synthesis and Mating Pair Stabilization from the Related Plasmids F and R100-1: Insights into the Mechanism of Conjugation

F and R100-1 are closely related, derepressed, conjugative plasmids from the IncFI and IncFII incompatibility groups, respectively. Heteroduplex mapping and genetic analyses have revealed that the transfer regions are extremely similar between the two plasmids. Plasmid specificity can occur at the level of relaxosome formation, regulation, and surface exclusion between the two transfer systems. There are also differences in pilus serology, pilus-specific phage sensitivity, and requirements for OmpA and lipopolysaccharide components in the recipient cell. These phenotypic differences were exploited in this study to yield new information about the mechanism of pilus synthesis, mating pair stabilization, and surface and/or entry exclusion, which are collectively involved in mating pair formation (Mpf). The sequence of the remainder of the transfer region of R100-1 (trbA to traS) has been completed, and the complete sequence is compared to that of F. The differences between the two transfer regions include insertions and deletions, gene duplications, and mosaicism within genes, although the genes essential for Mpf are conserved in both plasmids. F+ cells carrying defined mutations in each of the Mpf genes were complemented with the homologous genes from R100-1. Our results indicate that the specificity in recipient cell recognition and entry exclusion are mediated by TraN and TraG, respectively, and not by the pilus.     

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.     

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.     

Involvement of megaplasmids in heterotrophic derepression of the carbon-dioxide assimilating enzyme system in Alcaligenes spp.

The facultatively chemolithoautotrophic hydrogen-oxidizing bacteria Alcaligenes eutrophus and Alcaligenes hydrogenophilus partially derepressed the formation of phosphoribulokinase and ribulosebisphosphate carboxylase during heterotrophic growth on fructose or gluconate. We examined whether the indigenous magaplasmids in these bacteria that encode the ability to oxidize hydrogen affected this derepression. The results suggest an involvement of the plasmids in the derepression for the following reasons: (i) wild-type strains, except A. eutrophus TF93, exhibited the derepressible phenotype; (ii) plasmid-cured mutants formed the enzymes with formate as autotrophic growth substrate but did not derepress their formation during heterotrophic growth; (iii) the phenotype of the wild type was restored by transfer of the plasmids into plasmid-cured mutants. Plasmid pHG2 from strain TF93 differed from the other wild-type plasmids by conferring a non-derepressible phenotype onto the harboring strain. Mutants of A. eutrophus H16 carrying deletions in plasmid pHG1 showed a similar phenotype as that of the plasmid-cured mutants. We concluded that the plasmids from the various strains studied encode a regulatory ability to derepress phosphoribulokinase and ribulosebisphosphate carboxylase under heterotrophic growth conditions.Abbreviations PRK phosphoribulokinase - RuBPC ribulosebisphosphate carboxylase - Hox ability to oxidize hydrogen - Cfx ability to fix carbon dioxide autotrophically Dedicated to Prof. Dr. H. G. Schlegel on the occasion of his 60th birthday     

Physical map of the nrdA-nrdB-ftsB-glpT region of the chromosomal DNA of Escherichia coli

Seven pLC plasmids (pLC 3-46, 8-12, 8-24, 8-29, 14-12, 19-24 and 42-17) which complemented nrdA, nrdB, ftsB and/or glpT mutations of Escherichia coli were analyzed. A restriction map of each plasmid was constructed and restriction fragments were subcloned into pBR322. A physical map of approx. a 15 X 10(6) Mr segment of the chromosomal DNA was deduced from the overlapping region of the pLC plasmids. The pLC plasmids and newly constructed plasmids were examined for the ability to rescue the mutations. The complementation tests defined the location of the genes in the 15 X 10(6) Mr segment in the following order: nrdA-nrdB-ftsB-glpT. Functional nrdAB and ftsB genes were located in the 3.1 X 10(6) Mr EcoRI-PstI fragment.     

Genetic and nucleotide sequence analysis of the gene htdA, which regulates conjugal transfer of IncHI plasmids.

IncHI plasmids are naturally repressed for conjugative transfer and do not allow efficient propagation of the IncH pilus-specific phage Hgal. Transposons Tn7, Tn5, and TnlacZ were inserted into IncHI plasmids R478, R477-1, and R27, respectively, leading to the isolation of several plasmid mutants which exhibited increased levels of transfer and also permitted good lysis with phage Hgal. A 4.3-kb HindIII fragment from R478 reversed both phenotypic effects of derepression for the R477-1::Tn5 and the R478::Tn7 derivatives, pKFW99 and pKFW100, respectively. Exonuclease III deletions of this fragment and nucleotide sequence analysis indicated that the gene responsible for transfer repression, named here htdA, encoded a polypeptide of 150 amino acids. Cloning and sequence analysis of pDT2454 (R27::TnlacZ) revealed that the transposon had inserted into an open reading frame (ORF) which had an 83% amino acid identity with the R478 htdA gene. Maxicell analysis showed both the R27 and R478 HtdA products had molecular masses of 19.9 kDa. Conjugation experiments showed that the cloned htdA determinants caused a significant reduction of the transfer frequencies of wild-type R478 and R27 plasmids. Examination of both R478 derepressed mutants, pKFW100 and pKFW101, indicated that both transposon insertions occurred upstream of the htdA ORF. The results suggest that HtdA is a regulatory component of IncH plasmid transfer and also show that the region upstream of the htdA ORF is involved in transfer repression. The locations of the htdA determinants were identified on the plasmid maps of R27 and R478.     

Partial suppression of the phenotype of Escherichia coli K-12 dnaG mutants by some I-like conjugative plasmids.

Three I-like conjugative plasmids, ColIdrd1, R144drd3, and R64drd11, which are derepressed for functions involved in conjugation, were found to suppress at least partially the phenotype of temperature-sensitive dnaG mutants of Escherichia coli K-12, as judged from the kinetics of deoxyribonucleic acid synthesis at elevated temperature in newly formed and established plasmid-containing strains. In contrast, the corresponding wild-type plasmids and three F-like derepressed conjugative plasmids, F101, R100drd1, and R1drd16, all failed to suppress. Suppression is presumably caused by a different plasmid-determined function from that which promotes survival of ultraviolet-irradiated bacteria, because both the wild-type I-like plasmids and their drd mutants protected irradiated bacteria. One possible interpretation of these results is that the product of a gene carried by certain I-like plasmids can substitute for the bacterial dnaG gene product during ongoing deoxyribonucleic acid replication.     

Further characterization of the F fertility inhibition systems of "unusual" Fin+ plasmids.

Flac mutants insensitive to transfer inhibition by R factors. JR66a and R485 were isolated and characterized. Representative mutations were cis dominant and are therefore presumed to be at the sites of action, fisU and fisV, respectively, of the FinU and FinV transfer inhibition systems encoded by JR66a and R485. The mutants were used to confirm that the FinU and FinV fertility inhibition systems are different from each other and from the FinOP, FinQ, and FinW systems of R100, R62, and R455, respectively. Together with traO and fisQ mutants of Flac, the new mutants were also used to investigate the nature of the F fertility inhibition systems encoded by a further group of "unusual" Fin+ plasmids. Of these, two incompatibility group X plasmids were found to carry finO+ genes, and of five incompatibility group I plasmids, three encoded FinQ systems, one the FinU system, and one a new system (FinR). Transfer of a variety of derepressed F-like plasmids was inhibited by the FinQ, FinU, and FinV systems, but a quantitatively very different levels; this emphasizes the differences as well as the similarities between the conjugation systems of F-like plasmids.     

Multiple regulator gene control of the galactose operon in Escherichia coli K-12

Previous studies showed that nonsense mutations in either of two genes (capR or capS) or an undefined mutation in a third gene (capT) led to pleiotropic effects: (i) increased capsular polysaccharide synthesis (mucoid phenotype); (ii) increased synthesis of enzymes specified by at least four spatially separated operons involved in synthesis of capsular polysaccharide including the product of the galE gene, UDP-galactose-4-epimerase (EC 5.1.3.2) in capR mutants. The present study demonstrated that the entire galactose (gal) operon (galE, galT, and galK) is derepressed by mutations in either the capR or the capT genes, but not by mutation in capS. Double mutants (capR9 capT) were no more derepressed than the capR9 mutant, indicating that capR9 and capT regulate the gal operon via a common pathway. Isogenic double mutants containing either galR(+), galR(-), galR(s), or galO(c) in combination with either capR(+) or capR9 were prepared and analyzed for enzymes of the gal operon. The results demonstrated that capR9 caused derepression as compared to capR(+) in all of the combinations. Strains with a galR(s) mutation are not induced, for the gal operon, by any galactose compound including d-fucose, and this was confirmed in the present study using d-fucose. Nevertheless, the derepression of galR(s) capR9 compared to galR(s) capR(+) was four- to sixfold. The same derepression was observed when galR(+)capR9 was compared to galR(+)capR(+). The data eliminate the explanation that internal induction of the gal operon by a galactose derivative was causing increased gal operon enzyme synthesis in capR or capT mutants. Furthermore, the same data suggest that the galR and capR genes are acting independently to derepress the gal operon. A modified model for the structure of the gal operon is proposed to explain these results. The new feature of the model is that two operator sites are suggested, one to combine with the galR repressor and one to combine with the capR repressor.     

Interrelationship between metabolic and genetic regulation of alkaline and acid phosphatases in E. coli cells]

The effect of exogenous orthophosphate and mutations in regulatory genes of alkaline phosphatase on the level of nonspecific acid phosphatase was studied. The level of this enzyme as well as the level of alkaline phosphatase were shown to be regulated by exogenous orthophosphate being derepressed under phosphate starvation. The derepression of acid phosphatase is accompanied by more rapid secretion of enzyme from membranes to soluble fraction. Mutations in all the four regulatory genes decrease the level of enzyme in cells. Genes phoR and phoS, participating in regulation of alkaline phosphatase, are required for the derepression of acid phosphatase under the conditions of phosphate starvation.     

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.