Thiaminokinase in parent and pyrithiamine mutant Staphylococcus aureus

1. As a result of the mutation of Staphylococcus aureus by pyrithiamine, deletion of the enzyme thiaminokinase in the system occurs. Some properties of thiaminokinase including the effects of pH, pyrophosphate donor nucleotides and metal ions on the enzyme in the parent S. aureus have been studied. Cell-free extract from mutant strain has been studied under similar conditions and thiaminokinase activity was found to be absent. Addition of thiamine (10μg./ml.) to the medium containing pyrithiamine (required for the growth of the mutant strain) did not give rise to thiaminokinase activity in the mutant bacteria. 2. The parent and the mutant strains of S. aureus have been studied for the fermentative production of acetylmethylcarbinol (3-hydroxybutan-2-one) and the mutant strain did not produce acetylmethylcarbinol under the conditions used.     

Metabolism of pyrithiamine by the pyrithiamine-requiring mutant of Staphylococcus aureus

1. A mutant strain of Staphylococcus aureus that requires pyrithiamine for its optimum growth was found to utilize pyrithiamine during the exponential phase of growth. 2. Pyrithiamine was deaminated by the organism to form oxypyrithiamine, the reaction being enzymic with no cofactor requirement. 3. On prolonged incubation of S. aureus A cultures, the concentration of deaminating enzyme increased in the culture broth, from which pyrithiamine-deaminating enzyme could be isolated by solvent fractionation. 4. Oxypyrithiamine is not a competitive analogue of thiamine although it inhibited the growth of the parent strain of S. aureus; the inhibition index of this compound, however, was lower than that of pyrithiamine.     

Isolation and characterization of a mutant of Staphylococcus aureus deficient in autolytic activity.

A mutant of Staphylococcus aureus H (RUS3) uas isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The rate of autolysis of whole cells and isolated cell walls of RUS3 was less than 10% of the parent strain. In addition, the ability of the crude soluble enzyme isolated from RUS3 to degrade cell walls was negligible compared with the parent strain. The cell wall composition and the generation time of RUS3 were comparable to the parent strain. Unlike S. aureus H, RUS3 grew in clumps and did not undergo cell wall turnover. Both strains exhibited identical kinetics of killing by penicillin G. This may indicate that autolytic enzymes play a role in cell wall turnover and cell separation, but in S. aureus most of the autolytic activity is unrelated to the lethal effect of cell wall antibiotics.     

Infection in Mouse Peritoneal Cavity with a Pyrimidine-requiring Mutant and Naturally Occurring Staphylococcus aureus Strains

The lethal activity of a thymineless mutant of Staphylococcus aureus Wood 46 strain has been compared with that of three naturally occurring strains: parent Wood 46, Smith, and coagulase-negative SA-13. The thymineless mutant and the parent Wood 46 strain showed a sharp decline in culturable units from the peritoneal cavity in the first 4 hr after their injection. After 6 hr, that is, 2 hr before the mice began to die, the number of culturable units of the thymineless mutant was still declining, whereas that of the parent strain increased; for both strains, the number of units was still lower than that of the inoculum. Although the thymineless mutant, unlike the parent strain, was apparently unable to multiply in mouse peritoneal cavity, it killed mice at a similar rate. The highly virulent Smith strain known to multiply rapidly and the avirulent coagulase-negative SA-13 strain were used as additional controls. Under our experimental conditions, death of mice after the injection of the thymineless mutant in the peritoneal cavity did not seem to be due to bacterial multiplication but to toxicity, death being delayed by antitoxin. The pyrimidine-requiring auxotroph we used could be better material than killed bacteria to study some aspects of the lethal activity of S. aureus.     

Two thermostable nucleases coexisted in Staphylococcus aureus: evidence from mutagenesis and in vitro expression

Thermostable nuclease is known to be an important pathogenic factor unique to Staphylococcus aureus and it is commonly presumed to have had the same genetic origin. However, two ORFs in S. aureus genomes were predicted to encode nucleases. One encoded an unnamed nuclease A (SNase) (termed nuc1 ), and the other encoded a thermonuclease (TNase) named nuc (termed nuc2 ). In order to verify whether the two thermostable nuclease proteins are coexpressed in S. aureus , the nuc1 and nuc2 genes were cloned and expressed in Escherichia coli , and both of the recombinant proteins showed thermostable nuclease activity in a toluidine blue-DNA assay. Furthermore, a nuc1 -deleted mutant of S. aureus strain RN4220 (termed RNΔ nuc1 ) was successfully constructed by homologous recombination. Selection and characterization of this mutant strain revealed that it still exhibited thermostable nuclease activity, but at a relative lower level than that of the parent strain. The nucleases secreted by the parent strain and nuc1 -deleted strain still showed functional activity after 30 min at 121 °C. The findings indicated that two types of thermostable nucleases, encoded by two different genes, coexisted in S. aureus .     

Regulation of bacterial cell walls: correlation between autolytic activity and cell wall turnover in Staphylococcus aureus.

Cell wall turnover was examined in parent and mutant strains of Staphylococcus aureus. Peptidoglycan and teichoic acid were observed to undergo turnover in the wild-type strain during exponential growth; however, the rate of turnover did not decrease when the growth rate slowed, as the culture entered stationary phase. Isolated native cell walls and crude soluble autolytic enzyme were prepared from cells harvested during exponential and postexponential phases of growth. Native cell walls from both phases of growth autolyzed in buffer at identical rates; similarily, crude soluble enzyme from both preparations degraded radioactive cell walls at the same rate. Therefore, the activity of the autolysin in both exponential and postexponential cells was similar. The autolysis of whole cells of a mutant tar-1 was enhanced by 1.0 M NaCl. When 1.0 M NaCl was present under growing conditions, the rate of cell wall turnover was greatly increased. The presence of chloramphenicol, which inhibits whole-cell autolysis, also inhibited turnover. Analysis of the cell wall material recovered from spent medium revealed products consistent with the known mode of action of the endogenous autolysin. It is concluded that cell wall turnover in S. aureus is independent of the stage of culture growth but is dependent instead on the activity of the autolysin.     

Mutant strains of Rhodopseudomonas spheroides which form photosynthetic pigments aerobically in the dark. Growth characteristics and enzymic activities

Mutant strains of Rhodopseudomonas spheroides have been isolated which contain 5–50 times more bacteriochlorophyll and carotenoids than the wild type when grown under highly aerobic conditions in the dark. Their pigment content is similar to the wild type when grown in the light. One of the mutants (TA-R) grew more slowly than its parent strain under aerobic conditions but formed pigments at about 60% of the rate observed under photosynthetic conditions. The other mutants grew at rates similar to the wild type under all conditions. Synthesis of bacteriochlorophyll by suspensions of the mutants began without delay upon transfer from conditions of high to low aeration. In contrast to the wild type, magnesium protoporphyrin-S-adenosylmethionine methyltransferase (EC 2.1.1.11) activity in particulate preparations from the mutants was not repressed by growth under aerobic conditions in the light or dark. Ribulose diphosphate carboxylase (EC 4.1.1.39) activity was repressed by O₂ in the mutants as in the wild type. Other enzyme activities were compared in mutant TA-R and its parent strain grown under the same conditions. NADH oxidase activity in particles from aerobically grown TA-R was about one third that found in the parent strain. However, the respiration rates of the intact cells did not differ. Light inhibited the respiration of aerobically grown TA-R, indicating that the bacteriochlorophyll formed under these conditions had photochemical activity. It is concluded that the insensitivity of the mutants to O₂ repression is due to defects in the regulatory system which controls formation of the enzymes concerned in pigment synthesis.     

Isolation of a peptide transport-deficient mutant of yeast.

A peptide transport mutant of a leucine-lysine auxotroph of Saccharomyces cerevisiae (strain Z1-2D) was isolated on the basis of its resistance to L-ethionyl-L-alanine. The mutant, designated Z1-2D Etar, did not utilize di- and tripeptides containing leucine or lysine although it contained peptidases which released the required amino acids from these substrates. S. cerevisiae Z1-2D Etar did not accumulate radioactivity from [14C]glycyl-L-leucine under conditions identical to those in which the parent took up the label from this dipeptide. These results indicate that the mutant lacks the cellular mechanism to transport peptides to the site of the peptidase activity and that di- and tripeptides share a common mode of entry into yeast.     

Properties of a Novel Pleiotropic Bacteriophage-Resistant Mutant of Staphylococcus aureus H

A phage-resistant mutant of Staphylococcus aureus H (Sm(R)), S. aureus 52A5, was previously shown to lack polymeric teichoic acid. This paper characterizes other phenotypic differences between the strains. In broth cultures the mutant cells grew more slowly, were larger, and formed much larger clumps than the parent strain. The clumps of cells appeared to be covalently linked and could only be separated by mild sonic energy-a process which yielded viable cells. Mutant and parent cells autolyzed at equal rates, whereas isolated cell walls of the mutant strain autolyzed faster than the wild type. Nevertheless, the specific activity of the autolytic enzyme in the wild type soluble fraction was much higher than in the mutant. In contrast to the parent, strain 52A5 failed to accumulate nucleotide-bound murein precursors when treated with penicillin. Mutant strains with these characteristics were repeatedly isolated both spontaneously and by chemical mutagenesis. Strain 52A5 was shown to be fully revertible. Thus, it appears to be a pleiotropic mutation, and the possible nature of the defect which causes these varied effects is discussed.     

The regulation of synthesis of iron and magnesium tetrapyrroles. Observations with mutant strains of Rhodopseudomonas spheroides

1. Cell suspensions of mutant strains of Rhodopseudomonas spheroides, which cannot form bacteriochlorophyll, have been examined for their ability to form other tetrapyrroles under conditions of low aeration. With the exception of strain L-57, the mutants could form carotenoids. 2. All strains, like the parent organism, formed iron protoporphyrin when incubated with delta-aminolaevulate, showing that the iron branch of the biosynthetic pathway operated. 3. Magnesium protoporphyrin or its monomethyl ester was also formed from delta-aminolaevulate by all strains with the exception of L-57. 4. Coproporphyrin and coproporphyrinogen were accumulated by the parent and by strains 2/73 and 2/21 when incubated with glycine and succinate in the presence of ethionine. Strain 2/33, which required methionine for growth, accumulated these compounds in the presence and absence of methionine. 5. Strain L-57 did not accumulate porphyrins from glycine and succinate under any conditions. However, the delta-aminolaevulate synthase of this mutant showed the same rise in activity in response to reduced aeration as did that of the parent organism. 6. Ethionine inhibited production of protoporphyrin and its derivatives from delta-aminolaevulate by the parent strain. 7. The accumulation of coproporphyrin(ogen) under conditions of methionine deficiency may reflect the presence of enzymes of the magnesium branch of the biosynthetic pathway. Strain L-57 may lack a genetic element which determines the development of the entire photosynthetic apparatus. Since this strain did not accumulate coproporphyrin(ogen), the possibility of a specific delta-aminolaevulate synthase, directed towards bacteriochlorophyll synthesis, should be considered.     

Inhibition of citric acid accumulation by manganese ions in Aspergillus niger mutants with reduced citrate control of phosphofructokinase.

Mutant strains of Aspergillus niger with reduced citrate control of carbohydrate catabolism (cic mutants) grow faster than the parent strain on media containing 5% (wt/vol) citrate. The mutants tolerated a higher intracellular citrate concentration than the parent strain. One mutant (cic-7/3) contained phosphofructokinase activity significantly less sensitive towards citrate than the enzyme from the parent strain. When this mutant was grown under citrate-accumulating conditions, acidogenesis was far less sensitive to inhibition by Mn2+ than in the parent strain. Some of the cic mutants also showed altered citrate inhibition of NADP-specific isocitrate dehydrogenase.     

Structure of the S. aureus PI-specific phospholipase C reveals modulation of active site access by a titratable π-cation latched loop

Staphylococcus aureus secretes a phosphatidylinositol-specific phospholipase C (PI-PLC) as a virulence factor that is unusual in exhibiting higher activity at acidic pH values than other enzymes in this class. We have determined the crystal structure of this enzyme at pH 4.6 and pH 7.5. Under slightly basic conditions, the S. aureus PI-PLC structure closely follows the conformation of other bacterial PI-PLCs. However, when crystallized under acidic conditions, a large section of mobile loop at the αβ-barrel rim in the vicinity of the active site shows ~10 ? shift. This loop displacement at acidic pH is the result of a titratable intramolecular π-cation interaction between His258 and Phe249. This was verified by a structure of the mutant protein H258Y crystallized at pH 4.6, which does not exhibit the large loop shift. The intramolecular π-cation interaction for S. aureus PI-PLC provides an explanation for the activity of the enzyme at acid pH and also suggests how phosphatidylcholine, as a competitor for Phe249, may kinetically activate this enzyme.     

Isolation and characterization of autolysis-defective mutants of Staphylococcus aureus created by Tn917-lacZ mutagenesis.

Two autolysis-defective mutants (Lyt-1 and Lyt-2) of Staphylococcus aureus have been isolated by transposon Tn917-lacZ mutagenesis. The mutants exhibited normal growth rate, cell division, cell size, and adaptive responses to environmental changes. No autolytic activities were detected in a crude autolytic enzyme preparation from the Lyt- mutants. The rate of autolysis of whole cells and cell walls in the mutants were negligible, but mutant cell wall preparations were degraded by crude enzyme preparations from the wild-type strain. Zymographic analyses of enzyme extracts from the mutants showed a single autolytic enzyme band, compared with more than 10 autolytic enzyme bands from the parent strain. Analyses of intracellular and exoprotein fractions gave results similar to those in experiments with total-cell extracts. Southern blot analysis indicated the insertion of a single copy of the transposon into the chromosome of Lyt mutants. Isogenic Lyt mutants constructed by phage phi 11 transduction showed similar phenotypes. Because both Lyt- mutants had Tn917-lacZ inserted in the appropriate orientation, it was possible to determine gene activity under various conditions by measuring beta-galactosidase activity. The gene activity was found to be induced by low pH, low temperature, and high sucrose and high sodium chloride concentrations. From these data, we propose that the mutation lies in either a master regulatory gene or a structural gene which is responsible for the synthesis or processing of a majority of the autolytic enzyme bands.     

sn-Glycerol-3-phosphate dehydrogenase and its interaction with nitrate reductase in wild-type and hem mutant strains of Staphylococcus aureus

Staphylococcus aureus has membrane-associated sn-glycerol-3-phosphate dehydrogenase activity that is strongly activated by detergents. The enzyme can be measured spectrophotometrically in intact cells in assay systems containing lauryldimethylamine oxide (Ammonyx LO). The dehydrogenase activity was located exclusively in the membrane fraction of cells grown with glycerol under aerobic conditions or under anaerobic conditions with the addition of nitrate; there was no evidence of multiple forms. Development of sn-glycerol-3-phosphate dehydrogenase activity was studied with suspensions of cells grown previously under semianaerobic conditions with glucose and nitrate. The wild-type strain rapidly formed the enzyme when incubated with glycerol under aerobic conditions or under semianaerobic conditions in the presence of nitrate. Under similar conditions, suspensions of hem mutant H-14 required the addition of hemin. Induction of the enzyme was strongly repressed by glucose with both organisms. A procedure was established to obtain cells of mutant H-14 with sn-glycerol-3-phosphate dehydrogenase and nitrate reductase activities, but which could not link the systems unless supplemented with hemin. The coupled activity could also be reconstructed in vitro by the addition of hemin to the depleted membranes.     

A new two-component regulatory system involved in adhesion, autolysis, and extracellular proteolytic activity of Staphylococcus aureus

A transposition mutant of Staphylococcus aureus was selected from the parent strain MT23142, a derivative of strain 8325. The site of transposition was near the 5' terminus of the gene arlS. ArlS exhibits strong similarities with histidine protein kinases. Sequence analysis suggested that arlS forms an operon with upstream gene arlR. The predicted product of arlR is a member of the OmpR-PhoB family of response regulators. The arlS mutant formed a biofilm on a polystyrene surface unlike the parent strain and the complemented mutant. Biofilm formation was associated with increased primary adherence to polystyrene, whereas cellular adhesion was only slightly decreased. In addition, the arlS mutant exhibited increased autolysis and altered peptidoglycan hydrolase activity compared to the parental strain and to the complemented mutant. As it has been shown for coagulase-negative staphylococci that some autolysins are able to bind polymer surfaces, these data suggest that the two-component regulatory system ArlS-ArlR may control attachment to polymer surfaces by affecting secreted peptidoglycan hydrolase activity. Finally, the arlS mutant showed a dramatic decrease of extracellular proteolytic activity, including serine protease activity, in comparison to the wild-type strain and the complemented mutant, and cells grown in the presence of phenylmethylsulfonyl fluoride (a serine protease inhibitor) showed an increased autolysin activity. Since the locus arlR-arlS strikingly modifies extracellular proteolytic activity, this locus might also be involved in the virulence of S. aureus.     

The iron-regulated surface proteins IsdA, IsdB, and IsdH are not required for heme iron utilization in Staphylococcus aureus

The iron-regulated surface determinant proteins (Isd) of Staphylococcus aureus are expressed during iron limitation and have been proposed to be involved in the scavenging of iron from heme. In this study, the genes encoding the surface proteins IsdA, IsdB, and IsdH were inactivated in order to determine their combined role. The triple mutant was found to have no defect in growth under any conditions of iron limitation tested. Also using a mouse septic arthritis model of S.?aureus systemic disease, no significant difference in bacterial load was observed for the triple mutant, compared with its otherwise isogenic parent.     

The expression of alpha-haemolysin is required for Staphylococcus aureus phagosomal escape after internalization in CFT-1 cells

Staphylococcus aureus colonizes the lungs of cystic fibrosis patients and treatment with antibiotics usually results in recurrent and relapsing infections. We have shown that S. aureus can invade and replicate within a cystic fibrosis epithelial cell line (CFT-1), and that these internalized bacteria subsequently escape from the endocytic vesicle. The a ccessory g ene r egulator, agr , in S. aureus has been shown to control the expression of a large number of secreted toxins involved in virulence. Here we show that an agr mutant of S. aureus strain RN6390 was unable to escape from the endocytic vesicle after invasion of the CFT-1 cells using markers of vesicular trafficking (LAMP-1 and 2, LysoTracker and Vacuolar-ATPase). Trafficking analysis of live S. aureus which did not express alpha-haemolysin, a specific agr regulated toxin, revealed a defect in vesicular escape that was undistinguishable from the trafficking defect exhibited by the agr mutant. Furthermore, overexpression of alpha-haemolysin under an inducible promoter in an agr mutant of S. aureus partially restored the phagosome-escaping phenotype of an agr mutant. These results demonstrate that the expression of agr is required for vesicular escape, and that biologically active alpha-haemolysin is required for S. aureus escape from the endocytic vesicle into the cytosol of CFT-1 cells.     

Aerobic and anaerobic alcohol dehydrogenases in Escherichia coli

Abstract Mutants unable to use ethanol for carbon and energy were counterselected from an ethanolutilizing mutant of Escherichia coli K12 derepressed for alcohol dehydrogenase (ADH). Mutants of one class were devoid of ADH activity under anaerobic conditions but exhibited aerobic activities comparable to those of wild-type E. coli. Mutants of a second class exhibited ADH activity levels intermediate between those of the wild-type and derepressed parent. Immunological studies showed that mutants of the former class synthesized far less ADH protein than did the derepressed parent while mutants of the latter class synthesized about the same amount. The ADH mutations in both classes were located within the previously described adh region which contains the structural gene for the activity that is derepressed in the parent. An Eth⁻ adh-lac fusion mutant with an insertion in the structural gene was also isolated and characterized. It exhibited no ADH activity under anaerobic conditions and wild-type levels under aerobic conditions. These data are consistent with the existence in E. coli of distinct aerobic and anaerobic ADH enzymes and a derepression of the anaerobic but not the aerobic enzyme in the ethanol utilizing strain.     

Development of a mutant of Trichoderma citrinoviride for enhanced production of cellulases

Considering importance of a microbial strain capable of increased cellulases production and insensitive to catabolite repression for industrial use, we have developed a mutant strain of Trichoderma citrinoviride by multiple exposures to EMS and ethidium bromide. The mutant produced 0.63, 3.12, 8.22 and 1.94 IU ml(-1) FPase, endoglucanase, beta-glucosidase and cellobiase, respectively. These levels were, respectively, 2.14, 2.10, 4.09 and 1.73 fold higher than those in parent strain. Glucose (upto 20 mM) did not repress enzyme production by the mutant under submerged fermentation conditions. In vitro activity assay with partially purified cellulase showed lack of inhibition by glucose. Interestingly, the partially purified endoglucanase and beta-glucosidase were activated by 2.0 fold and 2.6 fold, respectively, by 20 mM and 30 mM ethanol in the assay mixture. Genetic distinction of the mutant was revealed by the presence of two unique amplicans in comparative DNA fingerprinting performed using 20 random primers.