Role of murein lipoprotein in morphogenesis of the bacterial division septum: phenotypic similarity of lkyD and lpo mutants.

Phenotypes were compared in two different classes of mutants with defects in murein-lipoprotein (lkyD mutants of Salmonella typhimurium and an lpo mutant of Escherichia coli). Both mutations are associated with the same triad of phenotypic abnormalities, consisting of defective formation of the division septum, leakage of periplasmic proteins during growth, and increased sensitivity to several unrelated external toxic agents. The abnormality in septum formation consists of a defect in invagination of the outer membrane during formation of the nascent septum. The results suggest that formation of the murein-lipoprotein link plays an important role in differentiation of the division septum and perhaps also in maintaining the normal barrier function of the outer membrane.     

Interference with murein turnover has no effect on growth but reduces beta-lactamase induction in Escherichia coli

Physiological studies of a mutant of Escherichia coli lacking the three lytic transglycosylases Slt70, MltA, and MltB revealed that interference with murein turnover can prevent AmpC beta-lactamase induction. The triple mutant, although growing normally, shows a dramatically reduced rate of murein turnover. Despite the reduction in the formation of low-molecular-weight murein turnover products, neither the rate of murein synthesis nor the amount of murein per cell was increased. This might be explained by assuming that during growth in the absence of the major lytic transglycosylases native murein strands are excised by the action of endopeptidases and directly reused without further breakdown to muropeptides. The reduced rate of murein turnover could be correlated with lowered cefoxitin-induced expression of beta-lactamase, present on a plasmid carrying the ampC and ampR genes from Enterobacter cloacae. Overproduction of MltB stimulated beta-lactamase induction, whereas specific inhibition of Slt70 by bulgecin repressed ampC expression. Thus, specific inhibitors of lytic transglycosylases can increase the potency of penicillins and cephalosporins against bacteria inducing AmpC-like beta-lactamases.     

The induction of protein X in DNA repair and cell division mutants of Escherichia coli

Certain temperature-sensitive Escherichia coli cell division mutants and DNA repair mutants were treated in several ways to alter DNA synthesis or cell division. The bacteria were pulsed with [³⁵S]methionine; then membrane proteins were prepared and examined using sodium dodecyl sulfate/polyacrylamide slab gels. Autoradiography was performed on the slab gels so that the rate of synthesis of protein X could be determined by microdensitometry.Several changes in the rate of synthesis of the 40,000 molecular weight protein X were found in the different mutants. The wild-type (rec⁺ and lex⁺) strains synthesized protein X in response to DNA synthesis inhibition. However, neither recA^? strains nor lex^? strains synthesized protein X.Both the filament forming, temperature-sensitive mutants tif^? and tsl^? (which was derived from lex^?) synthesized protein X when DNA synthesis was inhibited, but at rates different from the wild-type strains. Moreover, these strains also produced protein X at their non-permissive temperature, even though DNA synthesis was not inhibited. In the tif^? mutant, the rate of synthesis of protein X was influenced by the addition of nucleic acid precursors.A double mutant tsl^?recA^? produced protein X when DNA synthesis was inhibited, or at the non-permissive temperature (although DNA synthesis was normal). This was the only strain carrying a recA^? mutation capable of synthesizing protein X.From these results it is suggested that the genes lex, recA and tif comprise a system that controls DNA repair and limits DNA degradation by the recBC nuclease. The inducer of this control system might be a DNA degradation product.     

Role of precursor translocation in coordination of murein and phospholipid synthesis in Escherichia coli.

Inhibition of phospholipid synthesis in Escherichia coli by either cerulenin treatment or glycerol starvation of a glycerol-auxotrophic mutant resulted in a concomitant block of murein synthesis. The intracellular pool of cytoplasmic and lipid-linked murein precursors was not affected by an inhibition of phospholipid synthesis, nor was the activity of the penicillin-binding proteins. In addition, a decrease in the activity of the two lipoprotein murein hydrolases, the lytic transglycosylases A and B, could not be demonstrated. The indirect inhibition of murein synthesis by cerulenin resulted in a 68% decrease of trimeric muropeptide structures, proposed to represent the attachment points of newly added murein. Importantly, inhibition of phospholipid synthesis also inhibited O-antigen synthesis with a sensitivity and kinetics similar to those of murein synthesis. It is concluded that the step common for murein and O-antigen synthesis, the translocation of the respective bactoprenolphosphate-linked precursor molecules, is affected by an inhibition of phospholipid synthesis. Consistent with this assumption, it was shown that murein synthesis no longer depends on ongoing phospholipid synthesis in ether-permeabilized cells. We propose that the assembly of a murein-synthesizing machinery, a multienzyme complex consisting of murein hydrolases and synthases, at specific sites of the membrane, where integral membrane proteins such as RodA and FtsW facilitate the translocation of the lipid-linked murein precursors to the periplasm, depends on ongoing phospholipid synthesis. This would explain the well-known phenomenon that both murein synthesis and antibiotic-induced autolysis depend on phospholipid synthesis and thereby indirectly on the stringent control.     

Escherichia coli mutants altered in murein lipoprotein.

Mutants with alterations in the structure, biosynthesis, or assembly of murein lipoprotein were selected by a procedure based on radiation suicide of wild-type organisms by [3H]arginine under conditions where the radioactive arginine was preferentially incorporated into lipoprotein. Further screening for the potential mutants among the survivors of [3H]arginine suicide was carried out by using a sensitive immunodiffusion test, followed by radioactive double-labeling experiments. Three mutants were obtained and partially characterized.     

Defective cell division in thermosensitive mutants of Salmonella typhimurium

Summary Two temperature-sensitive mutants ofSalmonella typhimurium defective in cell division (divA anddivC) have been isolated. Cell division in these mutants is arrested at elevated temperature while DNA and protein synthesis are unaffected. This results in formation of long filaments. Filaments returned to permissive temperature divide after a short lag. Inhibition of DNA synthesis by nalidixic acid does not block these divisions. This suggests that the thermosensitive step is required late in the division cycle. Chloramphenicol prevents the division of filaments shifted back to permissive temperature in one of these mutants (divA) and allows limited division to take place in the other mutant (divC). The arrest of cell division at elevated temperature may be phenotypically cured by high osmolarity of the medium. The mutationdivA has been mapped betweenrha andmetB and the mutationdivC betweenleu andaziA.If the filaments ofdivA are starved for thymine and then returned to permissive temperature with the simultaneous restoration of thymine the start of their division is delayed in comparison with the division of the control (unstarved) filaments. The argument is raised that a proper ratio of terminated chromosomes to cell mass must be attained to allow division.     

Signal sequence mutants of beta-lactamase

The function of the NH2-terminal signal peptide in the translocation of beta-lactamase across the inner membrane of Escherichia coli has been studied by characterization of 15 signal sequence mutants. Three amino acid substitutions (Pro 20 to Ser, Pro 20 to Phe, and Cys 18 to Tyr) in the 23-amino acid signal sequence each cause, to varying degrees, a defect in the proteolytic processing of pre-beta-lactamase, abnormal growth of the host strain, and a severe reduction in the expression of beta-lactamase in vivo but not in vitro. The results are consistent with a model for protein secretion in E. coli that parallels the pathway proposed for translocation across the endoplasmic reticulum in eucaryotic cells.     

Fluphenazine-resistant Saccharomyces cerevisiae mutants defective in the cell division cycle.

An fls1 mutant of Saccharomyces cerevisiae, which did not grow in the presence of 30 micrograms of fluphenazine per ml, was isolated. Mutants that were resistant to 90 micrograms of fluphenazine per ml and temperature sensitive for growth were obtained from the fls1 mutant. One fluphenazine-resistance mutation, fsr1, was located near the his7 locus on chromosome II. Growth of the fsr1 mutants at 35 degrees C was arrested after nuclear division. The other group of fluphenazine-resistant mutants, carrying fsr2 mutations, showed Ca2+-dependent growth at 35 degrees C. Growth of the fsr2 mutants at 35 degrees C was arrested at the G2 stage of the cell cycle in Ca2+-poor medium.     

Low-temperature conditional cell division mutants of Escherichia coli.

Fifteen low-temperature conditional division mutants of Escherichia coli K-12 was isolated. They grew normally at 39 degrees C but formed filaments at 30 degrees C. All exhibited a coordinated burst of cell division when the filaments were shifted to the permissive temperature (39 degrees C). None of the various agents that stimulate cell division in other mutant systems (salt, sucrose, ethanol, and chloramphenicol) was very effective in restoring colony-forming ability at 25 degrees C or in stimulating cell division in broth. One of these mutants, strain JS10, was found to have an altered cell envelope as evidenced by increased sensitivity to deoxycholate and antibiotics, as well as leakage of ribonulcease I, a periplasmic enzyme. This mutant had normal rates of DNA synthesis, RNA synthesis, and phospholipid synthesis at both the nonpermissive and permissive temperatures. However, strain JS10 required new protein synthesis in the apparent absence of new RNA synthesis for division of filaments at the permissive temperature. The division of lesion in strain JS10 is cotransducible with malA, aroB, and glpD and maps within min 72 to 75 on the E. coli chromosome.     

Ribosomal precursor RNA metabolism and cell division in the yeast Saccharomyces cerevisiae

Summary In a mating mixture of Hfr and F ^- bacteria the gene for alkaline phosphatase undergoes a transient derepression at the time of transfer. It is shown that this escape from repression occurs in the donor cells and is probably connected with the synchronous duplication of the transferred genome.     

Volume growth, murein synthesis, and murein cross-linkage during the division cycle of Escherichia coli PA3092.

Cells of Escherichia coli PA3092 were synchronized by centrifugal elutriation. The synchronously growing cells were double labeled with -3H or DL-[meso-2,6-14C]diaminopimelic acid (DAP) at different times. Cells incorporated [3H]DAP at a continuously increasing rate during their cycle, with a maximum occurring at about 30 min before division for trichloroacetic acid-precipitated cells (whole cells) and about 10 min before division for sodium dodecyl sulfate-treated cells (sacculi). This was in good agreement with the observed kinetics of volume growth under these conditions. Furazlocillin, which preferentially interacts with penicillin-binding protein 3, modified the pattern of incorporation of [3H]DAP. Electron microscopy indicated that furazlocillin did not inhibit the initiation of division but rather its completion. In addition, we measured the cross-linking of the murein inserted at different times during synchronous growth. The highest percentages were found to occur around division. At this same time, the cross-linking of old peptidoglycan was found to be decreased.     

Role of specific cell surface receptors in thrombin-stimulated cell division

Previous studies have shown that thrombin action at the cell surface is sufficient to bring about division of cultured fibroblast-like cells (Carney and Cunningham, 1978). This prompted the present binding experiments with ¹²⁵I-thrombin which led to the Identification of a thrombin receptor on the surface of mouse embryo cells. Scatchard plots of binding data at 4, 22 and 37°C were linear over a broad range of thrombin concentrations, indicating a single affinity class of receptors. The association constant was about 1 × 10⁹ M^?1 and there were approximately 2 × 10⁵ receptors per cell. Neither insulin, epidermal growth factor nor prothrombin competed for thrombin binding to its receptor, indicating that It was unique for thrombin. Comparisons of thrombin binding and the amount of cell division produced by various concentrations of thrombin indicated that there was a relationship between receptor occupancy and increase in cell number. Low concentrations of serum (0.1%) inhibited both the mitogenic action of thrombin and the specific binding of thrombin to its receptor. It did not, however, inhibit nonspecific association of ¹²⁵I-thrombin with the cells. Experiments showed that this inhibition by serum resulted from a masking of thrombin receptors on the cells and not from binding of thrombin by serum factors. Together these studies suggest that thrombin must bind to Its surface receptor to stimulate cell division.     

Catabolite repression mutants of yeast

The mechaṅism of catabolite repression in yeast is not well understood, although it has been established that cAMP does not play a role similar to that found in Escherichia coli. To identify the elements implicated in catabolite repression in yeast, a variety of mutants affected in this process have been isolated by different research groups. A systematic review of the results reported in the literature is presented. The conclusion that can be drawn is that the mechanism of catabolite repression is a complex one, with no single gene controlling all the genes subject to repression. The expression of a given gene or set of genes is controlled by several regulatory genes, but it is not yet known whether these genes act cooperatively or sequentially.     

Methionine and vitamin B 12 repression and precursor induction in the regulation of homocysteine methylation in Salmonella typhimurium

Summary 5-methyltetrahydrofolate, the product of a reaction catalysed by 5-methyltetrahydrofolate: FAD oxidoreductase (metF), is the methyl donor in the transmethylation of homocysteine in Salmonella typhimurium either via a vitamin B₁₂ dependent (metH) or independent (metE) pathway. Both the metF and H enzymes were shown to be repressible by methionine.B₁₂ was found to repress synthesis of the metF enzyme in some metH mutants but not in others although all lacked B₁₂-dependent 5-methyltetrahydrofolate homocysteine transmethylase. This suggested a dual enzymatic and regulatory role for the metH gene but no complementation was detected between any metH mutants.The levels of metF and H enzymes were elevated in mutants blocked at early stages in methionine synthesis. Also the metH enzyme level in a metF mutant was increased by the addition to the medium of known precursors unable to support its growth, suggesting precursor induction of the enzymes. This increase did not occur in the presence of chloramphenicol.The different regulatory systems involved in the methylation of homocysteine could reflect the importance of this step in the inter-relationship of different metabolic pathways.