Chromosomal mapping of a gene affecting enterotoxin A production in Staphylococcus aureus.

In a previous study, transformation demonstrated that a gene governing enterotoxin A production (entA+) in Staphylococcus aureus strain S-6 was located on the chromosome between the purB110 and ilv-129 markers; in contrast, the entA+ gene of strain FRI-196E was shown not to be located in the same position. In the current study, 54 enterotoxin A-producing strains of S. aureus were examined to locate the entA+ gene. Conventional transformation procedures and a series of multiply marked derivatives of NCTC 8325 were used as recipients for chromosomal mapping. Of the 54 strains tested, 23 were found to contain the entA+ gene at the original locus between the purB110 and ilv-129 markers. Twenty-seven strains could not be analyzed either because their DNA was genetically ineffective in transforming strain 8325 (23 strains), or Pur+ Ilv+ transformants could not be recovered (four strains). Four other strains contained an entA+ gene that could not be located in any of the chromosomal linkage groups. A new insertion site for Tn551 was located within the hla+ gene involved in alpha-toxin production. It eliminated alpha-toxin production and was used to separate the entA+ gene from the hla+ marker in the purB110-ilv-129 region. This segment of the chromosome is shown to consist of the purB110, entA+, hla+, and ilv-129 markers in that order.     

Localization of a chromosomal mutation affecting expression of extracellular lipase in Staphylococcus aureus.

We describe a Tn551 chromosomal insertion in Staphylococcus aureus S6C that results in sharply reduced expression of extracellular lipase. With Tn917 as a probe, the insertion in the original mutant (KSI905) was localized to a 12.6-kb EcoRI DNA fragment. The 12.6-kb fragment was cloned and used as a probe to identify a 26-kb EcoRI fragment containing the Tn551 insertion site in the S6C parent strain. Restriction endonuclease analysis of the 12.6- and 26-kb EcoRI fragments confirmed that the Tn551 insertion in KSI905 was accompanied by a deletion of 18.7 kb of chromosomal DNA. Tn551 was transduced from KSI905 back into the S6C parent strain. All transductants exhibited the same lipase-negative (Lip-) phenotype and contained the same mutation with respect to both the insertion and the 18.7-kb deletion. The inability to produce lipase was not caused by disruption of the lipase structural gene, since all Lip- mutants carried intact copies of geh. Moreover, the Tn551 insertion was localized to a region of the staphylococcal chromosome at least 650 kb from geh. Taken together, these results suggest that the Tn551 insertion occurred in a region of the chromosome encoding a trans-active element required for the expression of extracellular lipase. A 20-bp oligonucleotide corresponding to a sequence within the region encoding RNA II near the Tn551 insertion site in ISP546 (H.L. Peng, R.P. Novick, B. Kreiswirth, J. Kornblum, and P. Schlievert, J. Bacteriol. 170:4365-4372, 1988) and a 1.75-kb DNA fragment representing the region encoding RNA III were used as gene probes to show that the Tn551 insertion did not occur in the agr locus. We conclude that the genetic element functions independently of agr or as an unrecognized part of that regulatory system.     

Chromosomal mapping of a gene affecting enterotoxin A production in Staphylococcus aureus

In a previous study, transformation demonstrated that a gene governing enterotoxin A production (entA+) in Staphylococcus aureus strain S-6 was located on the chromosome between the purB110 and ilv-129 markers; in contrast, the entA+ gene of strain FRI-196E was shown not to be located in the same position. In the current study, 54 enterotoxin A-producing strains of S. aureus were examined to locate the entA+ gene. Conventional transformation procedures and a series of multiply marked derivatives of NCTC 8325 were used as recipients for chromosomal mapping. Of the 54 strains tested, 23 were found to contain the entA+ gene at the original locus between the purB110 and ilv-129 markers. Twenty-seven strains could not be analyzed either because their DNA was genetically ineffective in transforming strain 8325 (23 strains), or Pur+ Ilv+ transformants could not be recovered (four strains). Four other strains contained an entA+ gene that could not be located in any of the chromosomal linkage groups. A new insertion site for Tn551 was located within the hla+ gene involved in alpha-toxin production. It eliminated alpha-toxin production and was used to separate the entA+ gene from the hla+ marker in the purB110-ilv-129 region. This segment of the chromosome is shown to consist of the purB110, entA+, hla+, and ilv-129 markers in that order.     

A restriction endonuclease from Staphylococcus aureus.

A specific endonuclease, Sau 3AI, has been partially purified from Staphylococcus aureus strain 3A by DEAE-cellulose chromatography. The enzyme cleaves adenovirus type 5 DNA many times, SV40 DNA eight times but does not cleave double-stranded phi X174 DNA. It recognizes the sequence (see article) and cleaves as indicated by the arrows. Evidence is presented that this enzyme plays a role in the biological restriction-modification system of Staphylococcus aureus strain 3A.     

Characterization of the redox activity and disulfide bond formation in apurinic/apyrimidinic endonuclease

Apurinic/apyrimidinic endonuclease (APE1) is an unusual nuclear redox factor in which the redox-active cysteines identified to date, C65 and C93, are surface inaccessible residues whose activities may be influenced by partial unfolding of APE1. To assess the role of the five remaining cysteines in APE1's redox activity, double-cysteine mutants were analyzed, excluding C65A, which is redox-inactive as a single mutant. C93A/C99A APE1 was found to be redox-inactive, whereas other double-cysteine mutants retained the same redox activity as that observed for C93A APE1. To determine whether these three cysteines, C65, C93, and C99, were sufficient for redox activity, all other cysteines were substituted with alanine, and this protein was shown to be fully redox-active. Mutants with impaired redox activity failed to stimulate cell proliferation, establishing an important role for APE1's redox activity in cell growth. Disulfide bond formation upon oxidation of APE1 was analyzed by proteolysis of the protein followed by mass spectrometry analysis. Within 5 min of exposure to hydrogen peroxide, a single disulfide bond formed between C65 and C138 followed by the formation of three additional disulfide bonds within 15 min; 10 total disulfide bonds formed within 1 h. A single mixed-disulfide bond involving C99 of APE1 was observed for the reaction of oxidized APE1 with thioredoxin (TRX). Disulfide-bonded APE1 or APE1-TRX species were further characterized by size exclusion chromatography and found to form large complexes. Taken together, our data suggest that APE1 is a unique redox factor with properties distinct from those of other redox factors.     

Characterization of plasmids in aminocyclitol-resistant Staphylococcus aureus: electron microscopic and restriction endonuclease analysis

Plasmid species isolated from aminocyclitol-resistant Staphylococcus aureus have been analyzed by restriction endonuclease digestion and electron microscopy. These plasmids can be divided into two interrelated groups; intergroup variability is due to the gain or loss of defined DNA sequences. Plasmids pSJ1 and pSJ24 are related to staphylococcal penicillinase plasmid pI524 which was first described over 20 years ago. Both pSJ1 and pSJ24 differ from pI524 by the acquisition of 8 and 4 kbp, respectively, and encode additional resistance to the antibiotics erythromycin and kanamycin. The gain of these resistance determinants suggests that the evolution of staphylococcal resistance plasmids parallels that observed for plasmids of gram-negative bacteria and has serious implications for the spread of antibiotic resistance among the staphylococci.     

Characterization of a Staphylococcus aureus Bacteriocin

The bacteriocin produced by a strain of Staphylococcus aureus has been isolated and designated staphylococcin (414), and a study was made of its chemical, physical, and biological properties. The staphylococcin is released in appreciable quantities after breakage of the cells and can be purified through differential centrifugation and column chromatography. In the native state, it appears to be a lipoprotein-carbohydrate complex with a molecular weight in excess of 200,000. The complex can be dissociated by sodium dodecyl sulfate into smaller subunits which retain activity. The gross chemical and physical properties of the bacteriocin closely resemble those ascribed to certain preparations of cell membranes. Staphylococcin (414) is not a lytic enzyme like lysostaphin and does not have the same spectrum of activity. Like other bacteriocins from gram-positive microorganisms, it does not inhibit any gram-negative bacteria, but does inhibit several other genera.     

Drosophila ribosomal protein PO contains apurinic/apyrimidinic endonuclease activity.

Drosophila ribosomal protein PO was overexpressed in Escherichia coli to allow for its purification, biochemical characterization and to generate polyclonal antibodies for Western analysis. Biochemical tests were originally performed to see if overexpressed PO contained DNase activity similar to that recently reported for the apurinic/apyrimidinic (AP) lyase activity associated with Drosophila ribosomal protein S3. The overexpressed ribosomal protein was subsequently found to act on AP DNA, producing scissions that were in this case 5' of a baseless site instead of 3', as has been observed for S3. As a means of confirming that the source of AP endonuclease activity was in fact due to PO, glutathione S-transferase (GST) fusions containing a Factor Xa cleavage site between GST and PO were constructed, overexpressed in an E.coli strain defective for the major 5'-acting AP endonucleases and the fusions purified using glutathione-agarose affinity column chromatography. Isolated fractions containing purified GST-PO fusion proteins were subsequently found to have authentic AP endonuclease activity. Moreover, glutathione-agarose was able to deplete AP endonuclease activity from GST-PO fusion protein preparations, whereas the resin was ineffective in lowering DNA repair activity for PO that had been liberated from the fusion construct by Factor Xa cleavage. These results suggested that PO was a multifunctional protein with possible roles in DNA repair beyond its known participation in protein translation. In support of this notion, tests were performed that show that GST-PO, but not GST, was able to rescue an E.coli mutant lacking the major 5'-acting AP endonucleases from sensitivity to an alkylating agent. We furthermore show that GST-PO can be located in both the nucleus and ribosomes. Its nuclear location can be further traced to the nuclear matrix, thus placing PO in a subcellular location where it could act as a DNA repair protein. Other roles beyond DNA repair seem possible, however, since GST-PO also exhibited significant nuclease activity for both single- and double-stranded DNA.     

Staphylococcus aureus chromosomal mutation specifically affecting the copy number of Inc3 plasmids

A chromosomal mutation leading to an important increase in the copy number of plasmid pT181 and its derivatives has been isolated from Staphylococcus aureus strain 8325. The amplification effect in the mutant strain SA1350 was found to be specific for plasmids of the Inc3 group, to which belongs pT181. There are some other differences in the behavior of Inc3 plasmids between SA1350 and 8325, including stable maintenance in SA1350 at high copy number of temperature-sensitive replication mutants at restrictive temperatures, and altered incompatibility properties. Derivatives of SA1350 carrying only Inc3 plasmid mutants with high copy numbers (Cop mutants) could not be obtained, suggesting a lethal runaway plasmid replication in this situation. SA1350 expressed also a temperature-sensitive phenotype. The relationship of this character to the plaC1 mutation determining the amplification of Inc3 plasmids has not yet been elucidated.     

Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease

The apurinic/apyrimidinic endonucleases (APE) contain several highly conserved sequence motifs. The glutamic acid residue in a consensus motif, LQE96TK98 in human APE (hAPE-1), is crucial because of its role in coordinating Mg2+, an essential cofactor. Random mutagenesis of the inactive E96A mutant cDNA, followed by phenotypic screening in Escherichia coli, led to isolation of an intragenic suppressor with a second site mutation, K98R. Although the Km of the suppressor mutant was about sixfold higher than that of the wild-type enzyme, their kcat values were similar for AP endonuclease activity. These results suggest that the E96A mutation affects only the DNA-binding step, but not the catalytic step of the enzyme. The 3' DNA phosphoesterase activities of the wild-type and the suppressor mutant were also comparable. No global change of the protein conformation is induced by the single or double mutations, but a local perturbation in the structural environment of tryptophan residues may be induced by the K98R mutation. The wild-type and suppressor mutant proteins have similar Mg2+ requirement for activity. These results suggest a minor perturbation in conformation of the suppressor mutant enabling an unidentified Asp or Glu residue to substitute for Glu96 in positioning Mg2+ during catalysis. The possibility that Asp70 is such a residue, based on its observed proximity to the metal-binding site in the wild-type protein, was excluded by site-specific mutation studies. It thus appears that another acidic residue coordinates with Mg2+ in the mutant protein. These results suggest a rather flexible conformation of the region surrounding the metal binding site in hAPE-1 which is not obvious from the X-ray crystallographic structure.     

Characterization of Staphylococcus aureus in a Pediatric Burn Unit

A one-year study on an endemic strain of Staphylococcus aureus phage type 84/85 in a children's burn unit is described. The endemic strain rapidly colonized the burns and nares of acute patients after admission but was not isolated from a patient on admission. Nonendemic strains of S. aureus found on some new patients were mostly non-phage typable and did not prevail in burns. The endemic strain was rarely isolated from the nares and skin of reconstructive patients or from the nares of hospital personnel. The endemic strain did colonize the oral cavity, normal skin, and intestinal tract of some acute patients. Endemic and nonendemic strains of S. aureus from the burned children were compared in their biochemical activities and antibiotic sensitivities to two groups of S. aureus from one other local and one Danish burns unit. The latter groups of strains represented different combinations of staphylococcal phage group III strains. Each of the four groups of strains differed in production of hemolysins, Tween 80 hydrolysis, egg yolk reaction, and proteolysis of casein and gelatin. All of the strains were uniformly sensitive to gentamicin, oxacillin, and cephalothin. Only 4 of 162 strains tested were methicillin resistant. The endemic S. aureus strains of phage type 84/85 were uniformly resistant to eight other antibiotics including lincomycin and clindamycin. The endemic strain was not the known cause of a clinically documented infection in a group of 82 acute patients studied. The possible role of S. aureus strains of phage group III in burn grafting problems is discussed.     

Characterization of small plasmids from Staphylococcus aureus.

Small molecular weight plasmids from Staphylococcus aureus were characterized with respect to size, restriction enzyme cleavage pattern and transforming capacity. The plasmids pS194 and pC194 which encode streptomycin and chloramphenicol resistance respectively contained 3.0 and 2.0 megadaltons of DNA as determined by zonal rate centrifugation and electron-microscopy. Both plasmids transformed S. aureus with high efficiency. Plasmid pC194 contained only one cleavage site for endonuclease HindIII and pS194 contained single cleavage sites for HindIII and EcoRI. A natural recombinant between these two plasmids, pSC194, shared the high transforming capacity of the parental plasmids and contained one EcoRI site And two HindIII sites. pSC194 DNA also transformed B. subtilis with high efficiency. The recombinant plasmid pSC194 may be used as an EcoRI vector for construction and propagation of hybrid DNA in S. aureus as shown in the following paper (Löfdahl et al., 1978).     

Selective inhibition by harmane of the apurinic apyrimidinic endonuclease activity of phage T4-induced UV endonuclease.

1-Methyl-9H-pyrido-[3,4-b]indole (harmane) inhibits the apurinic/apyrimidinic (AP) endonuclease activity of the UV endonuclease induced by phage T4, whereas it stimulates the pyrimidine dimer-DNA glycosylase activity of that enzyme. E. coli endonuclease IV, E. coli endonuclease VI (the AP endonuclease activity associated with E. coli exonuclease III), and E. coli uracil-DNA glycosylase were not inhibited by harmane. Human fibroblast AP endonucleases I and II also were only slightly inhibited. Therefore, harmane is neither a general inhibitor of AP endonucleases, nor a general inhibitor of Class I AP endonucleases which incise DNA on the 3'-side of AP sites. However, E. coli endonuclease III and its associated dihydroxythymine-DNA glycosylase activity were both inhibited by harmane. This observation suggests that harmane may inhibit only AP endonucleases which have associated glycosylase activities.     

Purification and properties of a plant endonuclease specific for apurinic sites.

An endonuclease which hydrolyzes depurinated DNA has been isolated from Phaseolus multiflorus enbryos; it has a molecular weight around 40,000. The enzyme is specific for apurinic sites; it has no action on normal DNA strands or on alkylated sites, and is without exonulcease activity. The rate of phosphoester bond hydrolysis near apurinic sites is far greater in native than in denatured DNA. The endonuclease is not inactivated by 10 mM EDTA, but is activity is however stimulated by Mg2+ or Mn2+. Its optimum pH is 7.5 to 8.0, and its optimum temperature 40degrees although, at this temperature, it is rapidly denatured; even low NaCl concentrations inhibit the enzyme activity. The endonuclease for apurinic sites of P. multiflorus is a non-histone protein of chromatin; the properties (like thermosensitivity of susceptibility to ionic strength) of the enzyme in situ, working on chromatin DNA, might be different from those described for the isolated endonuclease in homogenous aqueous solution.     

Genetic mapping of a mutation affecting pyridine nucleotide transhydrogenase in Escherichia coli.

A mutation, pnt-1, causing loss of pyridine nucleotide transhydrogenase activity in Escherichia coli, was mapped by assaying for the enzyme in extracts of recombinant strains produced by conjugation, F-duction, and P1 transduction. The site of this mutation was near min 35, counterclockwise from man, and it co-transduced 59% with man. The mutation was associated with loss from the cell membrane fraction of energy-independent and adenosine 5'-triphosphate-dependent transhydrogenase activities, but reduced nicotinamide adenine dinucleotide dehydrogenase activity was not affected. Strains were constructed which lack phosphoglucoisomerase (pgi-2) and which carry either pnt+ or pnt-1. Although such strains, when grown on glucose, are expected to produce a large excess of reduced nicotinamide adenine dinucleotide phosphate, the growth rate was not affected by the pnt-1 allele.     

The exoA gene of Streptococcus pneumoniae and its product, a DNA exonuclease with apurinic endonuclease activity.

The gene encoding the major DNA exonuclease of Streptococcus pneumoniae, exoA, was cloned in a streptococcal host vector system. Its location was determined by subcloning and by insertion mutations. Transfer of a DNA segment containing the gene to an Escherichia coli expression vector showed that exoA was the structural gene for the enzyme and that it was adjacent to its promoter. DNA sequence determination indicated that the gene encoded a protein, ExoA, of molecular weight 31,263. Under hyperexpression conditions, the ExoA protein constituted 10% of total cellular protein. In addition to previously demonstrated 3' to 5' exonuclease and 3'-phosphatase activities, ExoA was shown to make single-strand breaks at apurinic sites in DNA. Its enzymatic activities are thus similar to those of exonuclease III of E. coli and other gram-negative bacteria. The nucleotide sequence of exoA revealed it to be homologous to xth of E. coli, with 26% identity of amino acid residues in the predicted proteins. So far, no null chromosomal mutants of exoA have been obtained, and the biological function of ExoA remains unknown.     

Characterization and genetic mapping of a mutation (ms35) which prevents anther dehiscence in Arabidopsis thaliana by affecting secondary wall thickening in the endothecium

The male sterile mutant, ms35 , of Arabidopsis thaliana was produced by X-irradiation of seeds. The mutant produces fertile pollen, but is male sterile because the anthers do not dehisce. Anther development in ms35 plants occurs as in wild-type Arabidopsis until shortly after microspores are released from meiotic tetrads. Thereafter, in the wild type, bands of lignified, cellulosic secondary wall thickenings are laid down around the cells of the anther endothecium. In contrast, wall thickenings are not formed in the endothecium of the ms35 mutant. Development of other lignified tissues, for example the vascular tissue of the stamen, occurs normally in ms35 plants. In mutant anthers, as pollen maturation is completed, the stomium is cleaved but the anther wall does not retract to release pollen. The block in anther dehiscence in ms35 plants is specifically correlated with the absence of endothecial wall thickenings. The ms35 mutation represents the first genetic evidence in support of the proposed role of the endothecium in anther dehiscence. The ms35 gene was mapped to the top arm of chromosome 3 ( hy2 -(4.17±2.31 cM)- ms35 -(32.14±5.45 cM)- gl1 ).