Structure of the nucleoid in cells of Streptococcus faecalis

The structure of the nucleoid of Streptococcus faecalis (ATCC 9790) was examined and compared in the unfixed and fixed states by immersive refractometry and electron microscopy. It appears from these studies that the nucleoid structure is much more centralized in unfixed chloramphenicol-treated (stationary-phase) cells than it is in cells in the exponential phase of growth. The more dispersed configuration of the exponential-phase nucleoid could be preserved by fixation in glutaraldehyde, but not in Formalin or in osmium tetroxide. One important factor in explaining these differences in preservation is that glutaraldehyde (but not Formalin or osmium tetroxide) can rapidly cross-link the amino groups of macromolecules in cells. It was also observed that osmium tetroxide resulted in a preferential breakdown of nascent ribonucleic acid. These results are interpreted as indicating that glutaraldehyde is able to stabilize the exponential-phase nucleoid before it assumes the more central appearance seen in osmium tetroxide- and Formalin-fixed cells. These results are discussed in terms of the proposed organization of the exponential-phase nucleoid in unfixed cells.     

Folate metabolism in Streptococcus faecalis

The possibility that the inability of Streptococcus faecalis to utilize 5-methyltetrahydropteroylglutamate or pteroyltriglutamate might be due to permeability was investigated. Whereas the former was taken up by S. faecalis cells growing on pteroylglutamic acid, the latter was not. No subsequent conversion of the 5-methyltetrahydropteroylglutamate took place and accumulation, which was against a considerable concentration gradient, was inhibited by fluoride. It would thus appear to be an active process.     

Turnover rates at regulatory phosphorylation sites on myosin II in endothelial cells

Assembly and motor activity of non-muscle myosin II can be regulated by phosphorylation. Because myosin II-containing structures undergo continuous assembly, disassembly, and remodeling in living cells, especially during cell migration, myosin II should undergo frequent phosphorylation and dephosphorylation. This study examines the turnover of phosphate on myosin II in stationary and migrating endothelial cells. Cultured bovine aortic endothelial cells were metabolically labeled with (32)P-phosphate, and the incorporation of phosphate into myosin II was assessed by quantitative phosphor imaging of electrophoretic gels of myosin II immunoadsorbed from cell lysates. Likewise, phosphate turnover was measured upon chasing the (32)P with unlabeled phosphate. Phosphate incorporated very slowly into heavy chains, taking >8 h to plateau, and turned over at 相似文献   

Sodium-stimulated ATPase in Streptococcus faecalis.

We measured Na+-stimulated ATPase activity in a mutant of Streptococcus faecalis defective in the generation of proton motive force. The activity in membrane vesicles was 62.1 +/- 5.9 nmol of phosphate produced per min per mg of protein when cells were grown on medium containing 0.12 M Na+. Activity decreased as the concentration of Na+ in the growth medium decreased. The decrease in enzyme activity corresponded to the decrease in transport activity for Na+ in both whole cells and membrane vesicles. The effects of pH on both activities were identical. Thus, it is suggested that Na+ movement is mediated by this enzyme. Sodium extrusion and ATPase activity in the wild-type strain were markedly lower than those observed in the mutant strain. Elevated activities of both Na+ extrusion and Na+-stimulated ATPase could be detected in the wild-type strain when cells were grown in the absence of proton motive force. Thus, we propose that the level of ATPase is increased by dissipation of the proton motive force.     

The virulence of Streptococcus pneumoniae strains--the causative agents of pneumococcal infection at different sites]

A total of 256 S. pneumoniae strains, the causative agents of infectious processes with different localization, were studied for their virulence (in experiments on mice), neuraminidase and aldolase-protease activity (APA). In pneumococcal strains isolated 18-20 hours after intraperitoneal infection their virulence for mice increased, on the average, 1,000-fold and the average level of extracellular and cellular neuraminidase and APA increased 2- to 5-fold in comparison with the initial values. Pneumococcal strains causing acute pneumococcal infections with different localization, or the aggravation of such infections, exhibited higher virulence for mice and higher levels of neuraminidase and APA, while the inflammatory process at the period of clinical remissions was mainly maintained by S. pneumoniae cultures with low virulence.     

Three-dimensional reconstruction of whole cells of Streptococcus faecalis from thin sections of cells.

A new ultrastructural technique has been developed to study the geometry of cell wall assembly in Streptococcus faecalis, which is believed to occur between pairs of raised bands located on the organism's surface. Three-dimensional reconstructions of these new regions of envelope growth are produced from the mathematical rotation (around a central axis) of various measurements taken from central, longitudinal thin sections of cells. These reconstructions can be used to calculate the surface area and volume of the septal and peripheral walls that were supposedly present in any given cell before sectioning. In an accompanying paper, it is shown how such surface and volume estimations, coupled with other measurements of length, thickness, and curvature, can be used to characterize a cycle of envelope growth in this organism. The validity of the assumptions used to reconstruct cells by rotation and the possible sources of error in using this technique are discussed.     

Protein:DNA interactions at chromosomal loop attachment sites

We have recently identified an evolutionarily conserved class of sequences that organize chromosomal loops in the interphase nucleus, which we have termed "matrix association regions" (MARs). MARs are about 200 bp long, AT-rich, contain topoisomerase II consensus sequences and other AT-rich sequence motifs, often reside near cis-acting regulatory sequences, and their binding sites are abundant (greater than 10,000 per mammalian nucleus). Here we demonstrate that the interactions between the mouse kappa immunoglobulin gene MAR and topoisomerase II or the "nuclear matrix" occur between multiple and sometimes overlapping binding sites. Interestingly, the sites most susceptible to topoisomerase II cleavage are localized near the breakpoints of a previously described illegitimate recombination event. The presence of multiple binding sites within single MARs may allow DNA and RNA polymerase passage without disrupting primary loop organization.     

The structure of NADH peroxidase from Streptococcus faecalis at 3.3 A resolution

NADH peroxidase (EC 1.11.1.1) previously isolated from Streptococcus faecalis 10C1 has been crystallized. The crystal structure has been solved by multiple isomorphous replacement and solvent-flattening at 3.3 A (1 A = 0.1 nm) resolution. The enzyme forms a tetramer consisting of 4 crystallographically related subunits. The monomer chain fold is in general similar to those of glutathione reductase and lipoamide dehydrogenase. FAD binds in the same region and in a similar conformation as in glutathione reductase. The unusual cysteine-sulfenic acid participating in catalysis is located at the isoalloxazine of FAD.     

Amplification of the Na+-ATPase of Streptococcus faecalis at alkaline pH

The Na+-ATPase activity of Streptococcus faecalis was influenced by the medium pH. Activities of the protonophore-resistant Na+ extrusion and the KtrII (active K+ uptake by the Na+-ATPase) were maximal in the cells grown at pH 9.5, and were minimal in those grown at pH 6.0. In the cells grown at pH 7.5, they were moderately observed. The Na+-stimulated ATPase activity of the cells grown at pH 9.5 was about 4-fold higher than that of the cells grown at pH 6.0. Thus, amplification of the Na+-ATPase is remarkable at alkaline pH in this organism, possibly by an increase of the cytoplasmic Na+ level as a signal.     

Fatty Acid Composition of L-Forms of Streptococcus faecalis Cultured at Different Osmolalities

The fatty acid composition of the membranes of three different penicillin-produced L-forms of Streptococcus faecalis was determined: (i) a stable (nonreverting) L-form (T(53)) cultured in brain heart infusion (BHI) with 0.5 M sucrose; (ii) a stable L-form (T(531)) cultured in BHI without sucrose; and (iii) an unstable L-form (T(9)) cultured in BHI with 0.5 M sucrose and 1,000 U of penicillin per ml. L-forms were obtained by centrifugation and lysed by washing in 1 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer. The parent S. faecalis was also cultured in BHI and BHI containing 0.5 M sucrose, and washed with buffer. The fatty acid composition of L-forms of S. faecalis cultured in BHI without sucrose (370 mosmol) had higher C(18:1) and lower C(18) than L-forms cultured in the same media with added 0.5 M sucrose (950 mosmol) in both exponential and stationary cultures. In the stationary phase of growth, C(19) was reduced in the L-forms cultured without sucrose. Similar changes were seen in the parent S. faecalis cultured in the two types of media. These changes in membrane fatty acids may relate to osmo-regulation of the L-forms.     

Identification and characterization of a small sequence located at two sites on the amplifiable tetracycline resistance plasmid pAMalpha1 in Streptococcus faecalis.

Streptococcus faecalis DT-11 harbors the 6.0-megadalton plasmid pAMalpha1, which determines resistance to tetracycline (TC). When this strain is grown in the presence of TC for a number of generations, a reversible gene amplification occurs, generating tandem repeats of a 2.65-megadalton segment of the plasmid. On the basis of heteroduplex studies between various forms of pAMalpha1 and fragments generated by the Escherichia coli restriction endonuclease EcoRI, we have obtained direct evidence for the presence of a small sequence designated RS1 (for recombination sequence) located on both sides of the TC resistance determinant. Corresponding points on the two sequences are separated by 2.65 megadaltons of deoxyribonucleic acid. The two RS1 sequences have the same polarity and are of a size corresponding to about 380 nucleotide base pairs. The data presented serve as strong support for amplification models that are based on recombinational events involving the RS1 sequences.