Effect of Inhibition of Deoxyribonucleic Acid and Protein Synthesis on the Direction of Cell Wall Growth in Streptococcus faecalis

Selective inhibition of protein synthesis in Streptococcus faecalis (ATCC 9790) was accompanied by a rapid and severe inhibition of cell division and a reduction of enlargement of cellular surface area. Continued synthesis of cell wall polymers resulted in rapid thickening of the wall to an extent not seen in exponential-phase populations. Thus, the normal direction of wall growth was changed from a preferential feeding out of new wall surface to that of thickening existing cell surfaces. However, the overall manner in which the wall thickened, from nascent septa toward polar regions, was the same in both exponential-phase and inhibited populations. In contrast, selective inhibition of deoxyribonucleic acid (DNA) synthesis using mitomycin C was accompanied by an increase in cellular surface area and by division of about 80% of the cells in random populations. Little or no wall thickening was observed until the synthesis of macromolecules other than DNA was impaired and further cell division ceased. Concomitant inhibition of both DNA and protein synthesis inhibited cell division but permitted an increase in average cell volume. In such doubly inhibited cells, walls thickened less than in cells inhibited for protein synthesis only. On the basis of the results obtained, a model for cell surface enlargement and cell division is presented. The model proposes that: (i) each wall enlargement site is influenced by an individual chromosome replication cycle; (ii) during chromosome replication peripheral surface enlargement would be favored over thickening (or septation); (iii) a signal associated with chromosome termination would favor thickening (and septation) at the expense of surface enlargement; and (iv) a factor or signal related to protein synthesis would be required for one or more of the near terminal stages of cell division or cell separation, or both.     

Autolytic Enzyme System of Streptococcus faecalis III. Localization of the Autolysin at the Sites of Cell Wall Synthesis

Cell walls from exponential-phase cultures of Streptococcus faecalis ATCC 9790 autolyzed in dilute buffers. Walls were isolated from cultures grown in the presence of (14)C-lysine for about 10 generations and then on (12)C-lysine for 0.1 to 0.8 of a generation (prelabeled). These walls released (14)C to the soluble fraction more slowly than they lost turbidity during the initial stages of autolysis. Walls isolated from cultures grown in the presence of (14)C-lysine for only the last 0.1 to 0.4 of a generation (postlabeled) released (14)C to the supernatant fluid more rapidly than they lost turbidity. Autolysin in both pre- and postlabeled walls was inactivated, and such walls were then incubated in the presence of unlabeled walls containing active autolysin. The inactivated walls lost their (14)C label only very slowly until autolysis of the unlabeled walls was virtually complete and release of soluble autolysin was expected. When this experiment was done in the presence of trypsin, a fourfold increase in the autolysis rate resulted, but the same pattern of (14)C release was observed. A parallel release of (14)C and loss of turbidity from pre- or postlabeled walls was observed upon trypsin "activation" and by addition of isolated soluble autolysin to inactivated walls. We conclude that the wall-bound autolysin acts first on the more recently synthesized portion of the wall. Trypsin appears to speed wall autolysis by activating additional latent autolysin in situ at sites in the older portion of the wall.     

Influence of Macromolecular Biosynthesis on Cellular Autolysis in Streptococcus faecalis

The addition of several different antibiotics to growing cultures of Streptococcus faecalis, ATCC 9790, was found to inhibit autolysis of cells in sodium phosphate buffer. When added to exponential-phase cultures, mitomycin C (0.4 mug/ml) or phenethyl alcohol (3 mg/ml) inhibited deoxyribonucleic acid synthesis, but did not appreciably affect the rate of cellular autolysis. Addition of chloramphenicol (10 mug/ml), tetracycline (0.5 mug/ml), puromycin (25 mug/ml), or 5-azacytidine (5 mug/ml) to exponential-phase cultures inhibited protein synthesis and profoundly decreased the rate of cellular autolysis. Actinomycin D (0.075 mug/ml) and rifampin (0.01 mug/ml), both inhibitors of ribonucleic acid (RNA) synthesis, also reduced the rate of cellular autolysis. However, the inhibitory effect of actinomycin D and rifampin on cellular autolysis was more closely correlated with their concomitant secondary inhibition of protein synthesis than with the more severe inhibition of RNA synthesis. The dose-dependent inhibition of protein synthesis by 5-azacytidine was quickly diluted out of a growing culture. Reversal of inhibition was accompanied by a disproportionately rapid increase in the ability of cells to autolyze. Thus, inhibition of the ability of cells to autolyze can be most closely related to inhibition of protein synthesis. Furthermore, the rapidity of the response of cellular autolysis to inhibitors of protein synthesis suggests that regulation is exerted at the level of autolytic enzyme activity and not enzyme synthesis.     

Reinitiation of Cell Wall Growth After Threonine Starvation of Streptococcus faecalis

Cultures of Streptococcus faecalis ATCC 9790 were starved of threonine for 10 hr and then allowed to reinitiate growth in a fresh complete medium. On regrowth, culture turbidity began to increase within 10 min, but the ability of cells to autolyze did not begin to increase until after 30 min. Ultrastructural studies of regrowth of the initially thick-walled cells showed, at about 30 min, centripetal linear extension of new thin cross wall. This was followed, at about 40 min, by a notching, splitting, and peeling apart of the base of the cross wall. After this, extension of new thin peripheral wall from the nascent cross wall appeared to push old thick wall toward the poles. After the first cell division, asymmetric cells with one initial generation thick-walled pole and one second generation thin-walled pole were seen. After two divisions, thick-walled hemispheres were still seen, suggesting conservation of old wall during this time. A small fraction of the initial cell population exhibited aberrations and difficulties in reinitiating linear wall extension and were useful in the establishment of a model for the reinitiation of linear wall extension.     

AcmD,a Homolog of the Major Autolysin AcmA of Lactococcus lactis,Binds to the Cell Wall and Contributes to Cell Separation and Autolysis

Lactococcus lactis expresses the homologous glucosaminidases AcmB, AcmC, AcmA and AcmD. The latter two have three C-terminal LysM repeats for peptidoglycan binding. AcmD has much shorter intervening sequences separating the LysM repeats and a lower iso-electric point (4.3) than AcmA (10.3). Under standard laboratory conditions AcmD was mainly secreted into the culture supernatant. An L. lactis acmAacmD double mutant formed longer chains than the acmA single mutant, indicating that AcmD contributes to cell separation. This phenotype could be complemented by plasmid-encoded expression of AcmD in the double mutant. No clear difference in cellular lysis and protein secretion was observed between both mutants. Nevertheless, overexpression of AcmD resulted in increased autolysis when AcmA was present (as in the wild type strain) or when AcmA was added to the culture medium of an AcmA-minus strain. Possibly, AcmD is mainly active within the cell wall, at places where proper conditions are present for its binding and catalytic activity. Various fusion proteins carrying either the three LysM repeats of AcmA or AcmD were used to study and compare their cell wall binding characteristics. Whereas binding of the LysM domain of AcmA took place at pHs ranging from 4 to 8, LysM domain of AcmD seems to bind strongest at pH 4.     

Autolytic Formation of Protoplasts (Autoplasts) of Streptococcus faecalis 9790: Release of Cell Wall, Autolysin, and Formation of Stable Autoplasts

A system for the formation of apparently wall-free protoplasts from exponential-phase cells of Streptococcus faecalis ATCC 9790 in the absence of added lytic enzymes was developed. Exponential-phase cells suspended in 0.04 M ammonium acetate, pH 6.7, 1 mM magnesium acetate, and 0.5 M sucrose become osmotically fragile within 1 to 1.5 h due to the action of the native, autolytic enzyme on the cell wall peptidoglycan. However, maximal cell wall loss occurred much more slowly, being complete only after 3 to 6 h. Under these conditions, the autolytically formed protoplasts (autoplasts) remained intact for prolonged periods (up to 24 h) with less than 5% of their deoxyribonucleic acid, ribonucleic acid, and protein lost during the first 6 h. During dissolution of the cell wall, release of autolytic enzyme to the supernatant fluid began after 60% of the wall was lost. The addition of trypsin to the incubation mixture increased the rate of attainment of osmotic fragility and cell wall loss two- to threefold, apparently due to the activation of the latent form of the autolysin. Electron microscopy was used to confirm cell wall loss and the presence of intact protoplasts at the end of the incubation periods.     

Site of Initiation of Cellular Autolysis in Streptococcus faecalis as Seen by Electron Microscopy

Low concentrations of glutaraldehyde (0.1% or higher) blocked cellular and wall autolysis. The site of autolytic activity was studied by allowing cell autolysis to proceed for very short periods (0 to 15 min) before addition of glutaraldehyde. Electron microscopy of ultrathin sections showed that the primary site of autolytic activity was the leading edge of the nascent cross wall. The base of the cross wall seemed more resistant than the tip. Evidence supporting the involvement of autolysin activity in continued wall extension and in cell separation as well as in the initiation of new sites of wall extension was obtained. In cells exposed for 10 min to chloramphenicol, wall dissolution was very much slower but occurred at the same cross wall site.     

Protein Synthesis in Cell-Free Extracts of Streptococcus faecalis

If a cell-free extract of Streptococcus faecalis is fractionated by way of a 10 to 70% sucrose gradient, at least three areas are found capable of protein synthesis having a density greater than can be accounted for by association of individual ribosomes. These areas represent distinct “polysome” peaks rather than random distribution of polymers of varied length. They appear to be membrane subunits. In addition there is a further particle with a density of about 150S, incapable of protein synthesis but which is capable of stimulating protein synthesis in some of the larger fractions found by gradient centrifugation.     

Relationship of Cellular Organelles to the Formation and Development of the Plant Cell Wall

It has been shown that Golgi bodies, endoplasmic reticulum,and microtubules are concerned with the organization and synthesisof materials which are incorporated into the wall of the manycells making up the various tissues of a young plant. Preformedmaterial is added to the wall from vesicles which in some cellscan be inferred to be derived from the Golgi bodies. The materialis passed to the wall by a process of pinocytosis. In othercells although the same process is apparent the origin of thevesicles cannot at present be ascertained. The organization of the growth and development of the wall iscontrolled to some extent by the endoplasmic reticulum whichcan be seen to be situated in the cell at positions relativeto particular regions of cell-wall development. This is veryapparent in the formation of pit fields, sieve plates, and thesecondary thickenings of the xylem. The microtubules are organized in the cytoplasm relative towall growth and can be seen in cells in which growth is eitheroccurring uniformly along the wall or as organized annular orspiral thickenings. In the former case the microtubules arealso present all along the length of the wall whereas in thelatter cells they are found grouped in relation to the developingthickenings.     

Resistance mechanism of chloramphenicol in Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis.

The chloramphenicol resistance of Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis isolated from clinical materials was proved to be due to an inactivating enzyme produced by these bacteria. The inactivated products of chloramphenicol were identified as 1-acetoxy, 3-acetoxy and 1,3-diacetoxy derivatives by thin-layer chromatography and infrared spectroscopy. The responsible enzyme was thus confirmed to be chloramphenicol acetyltransferase. The enzyme was inducible. It was partially purified by ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. The enzymes obtained from S. haemolyticus, S. pneumoniae and S. faecalis have been compared with the conclusion that they are identical with respect to molecular weight (approximately 75,000-80,000), optimum pH and heat stability.     

Inhibition of the Initiation of Cellular DNA Synthesis After Reovirus Infection

The synthesis of cellular DNA was measured in synchronized L cells after reovirus infection. Initiation of the synthetic phase of the cell cycle was completely inhibited in cells infected 8 h before the beginning of DNA synthesis.     

Cell Wall Stress Stimulates the Activity of the Protein Kinase StkP of Streptococcus pneumoniae,Leading to Multiple Phosphorylation

Streptococcus pneumoniae is an opportunistic human pathogen that encodes a single eukaryotic-type Ser/Thr protein kinase StkP and its functional counterpart, the protein phosphatase PhpP. These signaling enzymes play critical roles in coordinating cell division and growth in pneumococci. In this study, we determined the proteome and phosphoproteome profiles of relevant mutants. Comparison of those with the wild-type provided a representative dataset of novel phosphoacceptor sites and StkP-dependent substrates. StkP phosphorylates key proteins involved in cell division and cell wall biosynthesis in both the unencapsulated laboratory strain Rx1 and the encapsulated virulent strain D39. Furthermore, we show that StkP plays an important role in triggering an adaptive response induced by a cell wall-directed antibiotic. Phosphorylation of the sensor histidine kinase WalK and downregulation of proteins of the WalRK core regulon suggest crosstalk between StkP and the WalRK two-component system. Analysis of proteomic profiles led to the identification of gene clusters regulated by catabolite control mechanisms, indicating a tight coupling of carbon metabolism and cell wall homeostasis. The imbalance of steady-state protein phosphorylation in the mutants as well as after antibiotic treatment is accompanied by an accumulation of the global Spx regulator, indicating a Spx-mediated envelope stress response. In summary, StkP relays the perceived signal of cell wall status to key cell division and regulatory proteins, controlling the cell cycle and cell wall homeostasis.     

Cellular Immune Responses to Cell Wall Peptidoglycan Associated Protein Antigens in Tuberculosis Patients and Healthy Subjects

We have isolated cell wall peptidoglycan associated proteins (CW-Pr) of Mycobacterium tuberculosis H₃₇Ra by chemical treatment with trifluoromethanesulfonic acid:anisole (2:1), which further resolved into 71, 60 and 45 kDa proteins on SDS-PAGE. A study was carried out to investigate the immunoreactivity of these proteins with blood samples from 4 categories, including 15 tuberculous patients (TB), 5 tuberculous patients on ATT (TBT), 10 PPD non-reactive healthy controls (HPPD^?) and 11 PPD reactive healthy controls (HPPD⁺). Comparing the proliferative responses to cell wall protein antigens, it was observed that the 71 kDa protein gave maximum stimulation with PBMCs from the TB and HPPD⁺ groups. The adherent PBMCs from the TB group also demonstrated enhanced phagocytosis, particularly in the presence of 71 and 45 kDa proteins, and the phagocytic index was significantly higher (P < 0.05) than the TBT group. However, PBMCs from of the groups recognized the 60 kDa cell wall antigen. Our results suggest that the 71 kDa protein from the cell wall of M. tuberculosis is highly immunogenic.     

Ethylene-induced Pea Internode Swelling: Its Relation to Ribonucleic Acid Metabolism, Wall Protein Synthesis, and Cell Wall Structure

Exposure of etiolated pea (Pisum sativum L. var. Alaska) subapical sections to ethylene disrupts normal polar cell expansion, but fresh weight increase is little affected and the tissue expands radially, swelling. Ethylene has no effect on gross ribonucleic acid metabolism before or during the period when swelling occurs, but incorporation of ¹⁴C-proline and leucine into wall-associated protein is markedly inhibited after an initial 3-hour lag period which precedes swelling. Ethylene affects the composition of this protein, altering the proline-hydroxyproline ratio. The gas also alters the optical birefringence pattern of the cell wall, indicating that the cellulose microfibrillar orientation has been changed.     

Cell Wall Synthesis in Staphylococcus aureus in the Presence of Protein Synthesis Inhibitory Agents

Electron-microscopic and biochemical studies on morphological changes in Staphylococcus aureus following exposure to protein synthesis inhibitory agents such as lincomycin (LCM), clindamycin (CLM), erythromycin (EM), and spiramycin (SP) are presented in this paper. It was demonstrated that bacterial cell walls became extremely thickened usually with the formation of multilayers, when exposed to each of the above-mentioned antibiotics. Furthermore, electron density of the cytoplasm was higher in those cells exposed to drugs than in intact control cells. Incorporations of ¹⁴C-labeled l-lysine into the cell-wall fraction and the protein fraction were measured for biochemical elucidation of these phenomena. Labeled lysine was selectively incorporated into the cell-wall fraction when the test organism was exposed to the respective antibiotics. Uptake at 15 min after exposure was about twice as large as that of intact control cells. SP and CLM inhibited protein synthesis while they stimulated cell-wall synthesis. The evidences for thickening of, and formation of multilayers in the bacterial cell walls following exposure to drugs were closely related to the stimulating action of these antibiotics on the cell-wall synthesizing system. Morphology of resistant clinical isolates following such antibiotic exposure was also investigated using two staphylococcal strains, one resistant to EM alone and the other completely cross-resistant to all the macrolides.     

Secondary Cell Wall Polymers of Enterococcus faecalis Are Critical for Resistance to Complement Activation via Mannose-binding Lectin

The complement system is part of our first line of defense against invading pathogens. The strategies used by Enterococcus faecalis to evade recognition by human complement are incompletely understood. In this study, we identified an insertional mutant of the wall teichoic acid (WTA) synthesis gene tagB in E. faecalis V583 that exhibited an increased susceptibility to complement-mediated killing by neutrophils. Further analysis revealed that increased killing of the mutant was due to a higher rate of phagocytosis by neutrophils, which correlated with higher C3b deposition on the bacterial surface. Our studies indicated that complement activation via the lectin pathway was much stronger on the tagB mutant compared with wild type. In concordance, we found an increased binding of the key lectin pathway components mannose-binding lectin and mannose-binding lectin-associated serine protease-2 (MASP-2) on the mutant. To understand the mechanism of lectin pathway inhibition by E. faecalis, we purified and characterized cell wall carbohydrates of E. faecalis wild type and V583ΔtagB. NMR analysis revealed that the mutant strain lacked two WTAs with a repeating unit of →6)[α-l-Rhap-(1→3)]β-d-GalpNAc-(1→5)-Rbo-1-P and →6) β-d-Glcp-(1→3) [α-d-Glcp-(1→4)]-β-d-GalpNAc-(1→5)-Rbo-1-P→, respectively (Rbo, ribitol). In addition, compositional changes in the enterococcal rhamnopolysaccharide were noticed. Our study indicates that in E. faecalis, modification of peptidoglycan by secondary cell wall polymers is critical to evade recognition by the complement system.