Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus

We isolated and characterized temperature-sensitive mutants for two genes, dnaE and polC, that are essential for DNA replication in Staphylococcus aureus. DNA replication in these mutants had a slow-stop phenotype when the temperature was shifted to a non-permissive level. The dnaE gene encodes a homolog of the alpha-subunit of the DNA polymerase III holoenzyme, the replicase essential for chromosomal DNA replication in Escherichia coli. The polC gene encodes PolC, another catalytic subunit of DNA polymerase, which is specifically found in gram-positive bacteria. The wild-type dnaE or polC gene complemented the temperature-sensitive phenotypes of cell growth and DNA replication in the corresponding mutant. Single mutations resulting in amino-acid exchanges were identified in the dnaE and polC genes of the temperature-sensitive mutants. The results indicate that these genes encode two distinct DNA polymerases which are both essential for chromosomal DNA replication in S. aureus. The number of viable mutant cells decreased at non-permissive temperature, suggesting that inactivation of DnaE and PolC has a bactericidal effect and that these enzymes are potential targets of antibiotics.     

Synthesis of catalase in Staphylococcus aureus MF-31

During the growth of Staphylococcus aureus MF-31, initial catalase activity dropped to a reduced level at the onset of exponential phase before increasing. When S. aureus was grown at 25, 32, or 37 degrees C, catalase activity was found to decrease by 80 to 90% within 1 h of inoculation. Two catalase-negative mutants and wild-type S. aureus MF-31 cells were exposed to exogenous 20 mM H2O2 for 15 min. For wild-type S. aureus, there was no effect from H2O2 until min 15, at which time a 10% decrease in CFU was observed. Both mutants showed increased sensitivity to the H2O2, with 56 and 71% reductions in the CFU for mutants C3 and C4, respectively, after a 15-min exposure. Cells of mutant and wild-type S. aureus were subjected to sublethal heating at 52 degrees C for 20 min. The lack of catalase activity in the mutants resulted in large decreases in enumeration.     

Synthesis of catalase in Staphylococcus aureus MF-31.

During the growth of Staphylococcus aureus MF-31, initial catalase activity dropped to a reduced level at the onset of exponential phase before increasing. When S. aureus was grown at 25, 32, or 37 degrees C, catalase activity was found to decrease by 80 to 90% within 1 h of inoculation. Two catalase-negative mutants and wild-type S. aureus MF-31 cells were exposed to exogenous 20 mM H2O2 for 15 min. For wild-type S. aureus, there was no effect from H2O2 until min 15, at which time a 10% decrease in CFU was observed. Both mutants showed increased sensitivity to the H2O2, with 56 and 71% reductions in the CFU for mutants C3 and C4, respectively, after a 15-min exposure. Cells of mutant and wild-type S. aureus were subjected to sublethal heating at 52 degrees C for 20 min. The lack of catalase activity in the mutants resulted in large decreases in enumeration.     

Remodeling of arachidonate-containing phosphoglycerides within the human neutrophil

Initial incorporation and subsequent remodeling of 16 phosphoglyceride molecular species containing arachidonate in the human neutrophil have been studied. Neutrophils were pulse-labeled with [3H]arachidonic acid (AA) for 5 min, then phospholipids were analyzed either at this time point or after a subsequent 120-min incubation. [3H]AA was found to be incorporated into phosphoglycerides phosphatidylinositol (PI) greater than phosphatidylcholine (PC) greater than phosphatidylethanolamine (PE) by 5 min. Incorporation of [3H]AA was not related to pool size, but reflected an increase in phosphoglyceride turnover. Following the 120-min incubation, only PE gained a significant amount of labeled arachidonate. Specific activity analysis revealed that PI contained the highest labeled/unlabeled ratio at both 5 min and 120 min. After the initial 5-min pulse, the majority of [3H]arachidonate was incorporated into 1-acyl-2-[3H]arachidonoyl-sn-glycero-3-PC, -PE, and -PI showing no preference for fatty acyl chains at the sn-1 position. However, [3H]AA was remodeled into 1-alkyl-acyl-and 1-alk-1-enyl-acyl-sn-glycero-3-PC and -PE molecular species in those neutrophils incubated for the additional 120 min. Specific activities of [3H]AA within all diacyl molecular species were initially higher relative to those alkyl-acyl and alk-1-enyl-acyl molecular species, but for PC and PE became more uniform as label shifted into ether and plasmalogen pools during the additional 120-min incubation. In contrast, the specific activity of 1-stearoyl-2-arachidonoyl-sn-glycero-3-PI remained constant throughout the 120-min incubation.     

Cardiac glucose uptake and suppressed expression/translocation of myocardium glucose transport-4 in dogs undergoing ischemia-reperfusion

Impaired glucose metabolism is implicated in cardiac failure during ischemia-reperfusion. This study examined cardiac glucose uptake and expression of glucose transport-4 (GLUT-4) in dogs undergoing ischemia-reperfusion. Cardiac ischemia was induced by cardiopulmonary bypass for 30 min or 120 min in dogs. Plasma insulin and glucose concentrations were measured at pre-bypass (control), and aortic cross-clamp off (ischemia-reperfusion) at 15, 45, and 75 min. At the same time, the left ventricle biopsies were taken for GLUT-4 immunohistochemistry and glycogen content analysis. In dogs receiving 120-min ischemia, coronary arterial and venous glucose concentrations were increased, but the net glucose uptake in ischemia-reperfusion heart were significantly decreased from 25% (control) to zero at 15 and 45 min of reperfusion, and recovered to only 7% after 75 min reperfusion. Myocardium glycogen contents were decreased by 65%. Plasma insulin levels and Insulin Resistant Index were markedly increased in dogs undergoing 120-min ischemia and reperfusion. These changes were relatively mild and reversible in dogs receiving only 30-min ischemia followed by reperfusion. Expression of total GLUT-4 in myocardium was decreased 40% and translocation of GLUT-4 from cytoplasm to surface membrane was decreased 90% in dogs receiving 120-min ischemia followed by 15-min reperfusion. Suppressed translocation of GLUT-4 was also evident in dogs receiving 30-min ischemia, but to a lesser extent. Reduced myocardium glucose uptake, utilization, and glycogen content are clearly associated with ischemia-reperfusion heart injury. This appears to be due, at least in part, to suppressed expression and translocation of myocardium GLUT-4.     

Involvement of a urethane-sensitive system in timing the onset of gastrulation in Xenopus laevis embryos

This paper describes success in delaying the onset of gastrulation in Xenopus laevis embryos without damage to their subsequent development by temporarily arresting cleavage with urethane. Exposure of X. laevis embryos to 150 mM urethane before gastrulation resulted in cleavage arrest and its removal led to cleavage resumption. During cleavage arrest, cyclic activities including nuclear replication and the M-phase-promoting factor cycle continued, although their duration was lengthened to nearly 1.8-fold that of the controls. Because of a 30-min time lag from removal of urethane to resumption of cleavage, as well as the retardation of cyclic activities during cleavage arrest, the development of embryos after a 60-min exposure to urethane lagged two cell cycles behind that of control embryos. Here, the two cell cycle delay is equivalent to 50 min at 22-23 degrees C. The start of gastrulation in exposed embryos was accordingly delayed about 50 min, although the delay in mid-blastula transition was as little as 20-25 min. Consistent results were obtained in embryos exposed to urethane for 90 or 120 min and those exposed to procaine or NH4Cl for 60 min. Although these results imply that delay in the start of gastrulation in exposed embryos is ascribed simply to delay in their development raised by cleavage arrest, at the same time they suggest that the onset of gastrulation is timed by systems sensitive to urethane, procaine and NH4Cl in X. laevis embryos.     

Mouse sperm capacitation assessed by kinetics and morphology of fertilization in vitro

Epididymal mouse spermatozoa were preincubated for periods of 5-120 min and then tested for their ability to penetrate freshly ovulated eggs synchronously and rapidly. When zona-intact eggs were used, only suspensions preincubated for 120 min gave consistently high rates of fertilization, but suspensions preincubated for 30 min were functionally equivalent to those incubated for 120 min when used with zona-free eggs; the only major observable differences were a 15-min lag in sperm-egg interaction and an increased incidence of asynchrony with multiple sperm penetrations. A morphological study of sperm-egg interactions using zona-intact eggs indicated that, within 35 min of gamete mixing, egg microvilli could be detected by SEM in association with the fertilizing sperm head. Using conventional light microscopic examination of fixed and stained preparations, initial stages of sperm head decondensation could be detected in the majority of eggs after 45-60 min and the process was essentially completed, with the egg at the telophase-second polar body stage of meiosis II, after 75 min. Similar kinetics were observed with sperm concentrations of 10(5) and 10(6)/ml. The time required for penetration by capacitated sperm suspensions is therefore relatively short and the most accurate information regarding state of capacitation and rate of sperm penetration can be obtained by choosing an appropriately short interval for sperm-egg interaction before assessment.     

Deoxyribonucleic Acid Replication and Cell Division in Escherichia coli at 33 C

At 33 C (60-min generation time) the time required to replicate the chromosome is C = 60 min. The time between the end of a round of replication and cell division is D = 20 min, as at 37 C. Nalidixic acid and a temperature shift in a dnaB mutant give identical results for the determination of the end of a round of replication.     

Understanding the enzymology of archaeal DNA replication: progress in form and function

The analysis of completed archaeal genome sequences led to the identification of a set of approximately 10-20 genes whose protein products were inferred to be involved in chromosomal DNA replication. Until recently, however, little was known of the biochemical properties of these proteins. Here, I review recent progress in this area brought about by biochemical and structural analysis. Aside from shedding considerable new light on the molecular machinery of DNA replication in the archaea, the results of these studies also present new opportunities for understanding the molecular events of chromosomal DNA replication in eukaryotic cells.     

Chromosomal gene transfer during conjugation by Staphylococcus aureus is mediated by transposon-facilitated mobilization.

A chromosomal copy of the transposon Tn551 and a copy coresident on a gentamicin-resistant conjugative plasmid of Staphylococcus aureus resulted in the mobilization of chromosomal genes during filter mating. Gene mobilization was recA dependent and was not restricted to any specific region of the chromosome. Both essential and nonessential genes were transferred.     

Fine structural in situ analysis of nascent DNA movement following DNA replication

Nascent DNA (newly replicated DNA) was visualized in situ with regard to the position of the previously replicated DNA and to chromatin structure. Localization of nascent DNA at the replication sites can be achieved through pulse labeling of cells with labeled DNA precursors during very short periods of time. We were able to label V79 Chinese Hamster cells for as shortly as 2 min with BrdU; Br-DNA, detected by immunoelectron microscopy, occurs at the periphery of dense chromatin, at individual dispersed chromatin fibers, and within dispersed chromatin areas. In these regions DNA polymerase alpha was also visualized. After a 5-min BrdU pulse, condensed chromatin also became labeled. When the pulse was followed by a chase, a larger number of gold particles occurred on condensed chromatin. Double-labeling experiments, consisting in first incubating cells with IdU for 20 min, chased for 10 min and then labeled for 5 min with CldU, reveal CldU-labeled nascent DNA on the periphery of condensed chromatin, while previously replicated IdU-labeled DNA has been internalized into condensed chromatin. Altogether, these results show that the sites of DNA replication correspond essentially to perichromatin regions and that the newly replicated DNA moves rapidly from replication sites toward the interior of condensed chromatin areas.     

Isolation of a temperature-sensitive dnaA mutant of Staphylococcus aureus

Of 750 temperature-sensitive mutants of Gram-positive Staphylococcus aureus, one was complemented by the dnaA gene. This mutant had a single base transition in the dnaA gene causing the amino-acid substitution mutation, Ala40Thr. Phage transduction experiments showed that this temperature-sensitive phenotype was linked with a drug-resistant marker inserted near the dnaA gene, suggesting the dnaA mutation is responsible for the phenotype. Flow cytometric analysis revealed that the dnaA mutant was unable to initiate DNA replication at a restrictive temperature and exhibited asynchrony in the replication initiation at a permissive temperature. This is the first report of a temperature-sensitive dnaA mutant in S. aureus, and the results show that DnaA is required for the initiation of chromosomal replication and for the regulation of synchrony in the bacterial cells.     

Control of F′lac Replication in Escherichia coli B/r

The timing of replication of an F'lac plasmid during the division cycle of Escherichia coli B/r lac(-)/F'lac was examined in relation to the timing of initiation of chromosome replication. This was accomplished by measuring the induction of beta-galactosidase and the incorporation of radioactive thymidine into cells at different ages in cultures growing exponentially at various rates. In cells growing with interdivision times of 27, 36, and 55 min, the F'lac replicated at various stages in the division cycle but always at approximately the same time as initiation of chromosome replication. In cells growing with an interdivision time of 85 min, the F'lac episome replicated midway through the division cycle, whereas chromosome replication initiated at the start of the cycle. Measurements of absorbance at 450 nm per cell suggested that the F'lac replicated when the cells reached a mass which was a constant multiple of the number of episomes per cell at each growth rate. In contrast, the mass per cell at initiation of chromosome replication in cells with an 85-min interdivision time was significantly lower than this constant value. A possible explanation for the apparent coupling between F'lac replication and initiation of chromosome replication at the higher growth rates, and the lack of coupling at the lowest growth rate, is discussed.     

Transfection of Lysostaphin-treated Cells of Staphylococcus aureus

After treatment with 1 unit of lysostaphin per ml for 3 min, two strains of Staphylococcus aureus, 233 and PS 44A HJD, were transfected with phenol-extracted deoxyribonucleic acid (DNA) from the staphylococcal bacteriophages, 53 and 44A HJD, respectively. The number of transfected cells was low in both systems, approximately two in 10(7) enzyme-treated cells. There was a saturation effect at high concentrations of DNA; optimal results were obtained at concentrations between 10 to 25 mug/ml. Growth curves and fluctuation tests indicated that cells of strain 44A HJD infected with phage, then converted to protoplasts by a 10-min treatment with lysostaphin, produce only one phage particle and lose their ability to lyse spontaneously in hypertonic media.     

Superoxide dismutase activity in thermally stressed Staphylococcus aureus.

The effects of heat and NaCl on the activity of superoxide dismutase from Staphylococcus aureus were examined. A linear decrease in superoxide dismutase activity occurred when S. aureus MF-31 cells were thermally stressed for 90 min at 52% C in 100 mM potassium phosphate buffer (pH 7.2). After 20 min of heating, only 5% of the superoxide dismutase activity was lost. Heating for 60, 90 and 120 min resulted in decreases of approximately 10, 22, and 68%, respectively. The rates of thermal inactivation of superoxide dismutase from S. aureus strains 196E and 210 were similar and slightly greater than those of strains MF-31, S-6, and 181. The addition of NaCl before or after heating resulted in increased losses of superoxide dismutase activity.     

Erythropoietin response to acute normobaric hypoxia in humans.

Hypoxia causes an increased production of erythropoietin (EPO), but the time course of the EPO response in humans has not been well characterized. This study examines the relationship between the duration of normobaric hypoxic exposure and plasma EPO levels in healthy human subjects. Six volunteers breathed a gas mixture of 10.5% O2-89.5% N2 continuously for 5, 60, and 120 or intermittently for 240 min. O2 saturations were maintained between 75 and 85% during the exposure. Arterial pH was 7.467 +/- 0.019, PO2 37.05 +/- 2.43 Torr, and PCO2 36.69 +/- 2.05 Torr. O2 half-saturation pressures of hemoglobin were normal for all subjects. Plasma EPO was measured every 30 min for 360 min by radioimmunoassay. No increase in EPO was seen after the 5- and 60-min exposures. However, a 50% increase was seen 240 min after the initiation of the 120-min hypoxic exposure (P less than 0.01). Intermittent exposure resulted in an increase of EPO by 52% 360 min after the onset of exposure (P less than 0.05). Therefore, exposing humans continuously to an inspiratory O2 fraction of 0.105 for 120 min or intermittently for 240 min provides a sufficient stimulus to increase production of EPO.     

DNA replication in methotrexate-treated human lymphoblasts.

The rate of DNA synthesis in cultures of human lymphoblasts decreased more than 80% within 30 min after the cells were exposed to methotrexate, a potent inhibitor of dihydrofolate reductase. Despite this rapid initial inhibition, DNA continued to be synthesized for at least an additional 6 h. The mode of this subsequent replication appeared to be semiconservative, as indicated by the buoyant density of 5-bromodeoxyuridine-substituted DNA in alkaline CsCl gradients. The growth rates of DNA chains in cells exposed to methotrexate were determined by sedimentation rate analysis in alkaline sucrose gradients. DNA synthesized during 2-min or 10-min pulses with labeled deoxycitidine in the presence of methotrexate had about the same sedimentation coefficient, 35 S, as controls. When methotrexate-treated cultures were pulse-labeled for 10 min and then chased for various times, DNA fragments of about 80 S accumulated. DNA synthesized in the presence of methotrexate was stable and elongated to bulk-size DNA after methotrexate inhibition of growth was removed by addition of thymidine and deoxycytidine. The data suggest that methotrexate reduces the rate of DNA replication by inhibiting chain initiation independently of chain elongation.