Mapping of the mecillinam-resistant, round morphological mutants of Escherichia coli.

Genes responsible for round morphology in mecillinam-resistant, round morphological mutants of Escherichia coli have been mapped. Three mutants, called rodX, mapped at around 14 min, and two, called rodY, mapped at around 70 min by P1 transduction. These are either the same or very close to the loci reported, respectively, for the rodA (H. Matsuzawa, K. Hayakawa, T. Sato, and K. Imahori, J. Bacteriol. 115:436-442, 1973) and envB genes (B. Westling-H?ggstr?m and S. Normark, J. Bacteriol. 123:75-82, 1975). This suggests that mecillinam can be used very efficiently to select for found morphological mutants of rodA and envB after nitrosoguanidine treatment.     

Accumulation of a murein-membrane attachment site fraction when cell division is blocked in lkyD and cha mutants of Salmonella typhimurium and Escherichia coli.

Membrane fractionation studies were performed on Salmonella typhimurium lkyD(Ts) and E. coli cha(Ts) mutants that appeared to be blocked at a late stage of the cell division cycle. In both cases growth of the mutant strains at nonpermissive temperatures was associated with accumulation of a characteristic cell envelope fraction (fraction OML) that contained inner membrane, murein, and outer membrane components. The isolated fraction corresponded in composition and bouyant density to a fraction from wild-type strains that had previously been suggested (M. H. Bayer, G. P. Costello, and M. E. Bayer, J. Bacteriol. 149:758-767, 1982; K. Ishidate, E. S. Creeger, J. Zrike, S. Deb, B. Glauner, T. J. MacAlister, and L. I. Rothfield, J. Biol. Chem. 261:428-443, 1986) to contain adhesion sites between inner membrane, murein, and outer membrane. The accumulation of OML in LkyD- and Cha- cells was prevented by treatments that blocked DNA synthesis. The effects of interference with DNA synthesis did not appear to involve the SOS response.     

Murein segregation in Escherichia coli.

Peptidoglycan (murein) segregation has been studied by means of a new labeling method. The method relies on the ability of Escherichia coli cells to incorporate D-Cys into macromolecular murein. The incorporation depends on a periplasmic amino acid exchange reaction. At low concentrations, D-Cys is innocuous to the cell. The distribution of modified murein in purified sacculi can be traced and visualized by immunodetection of the -SH groups by fluorescence and electron microscopy techniques. Analysis of murein segregation in wild-type and cell division mutant strains revealed that murein in polar caps is metabolically inert and is segregated in a conservative fashion. Elongation of the sacculus apparently occurs by diffuse insertion of precursors over the cylindrical part of the cell surface. At the initiation of cell division, there is a FtsZ-dependent localized activation of murein synthesis at the potential division sites. Penicillin-binding protein 3 and the products of the division genes ftsA and ftsQ are dispensable for the activation of division sites. As a consequence, under restrictive conditions ftsA,ftsI,or ftsQ mutants generate filamentous sacculi with rings of all-new murein at the positions where septa would otherwise develop.     

A major autolysin of Pseudomonas aeruginosa: subcellular distribution, potential role in cell growth and division and secretion in surface membrane vesicles.

A 26-kDa murein hydrolase is the major autolysin of Pseudomonas aeruginosa PAO1, and its expression can be correlated with the growth and division of cells in both batch and synchronously growing cultures. In batch cultures, it is detected primarily during the mid-exponential growth phase, and in synchronous cultures, it is detected primarily during the cell elongation and division phases. Immunogold labeling of thin sections of P. aeruginosa using antibodies raised against the 26-kDa autolysin revealed that it is associated mainly with the cell envelope and in particular within the periplasm. It is also tightly bound to the peptidoglycan layer, since murein sacculi, isolated by boiling 4% sodium dodecyl sulfate treatment, could also be immunogold labeled. Since division is due to cell constriction in this P. aeruginosa strain (septa are rarely seen), we cannot comment on the autolysin's contribution to septation, although constriction sites were always heavily labeled. Some labeling was also found in the cytoplasm, and this was thought to be due to the de novo synthesis of the enzyme before translocation to the periplasm. Interestingly, the autolysin was also found to be associated with natural membrane vesicles which blebbed from the surface during cell growth; the enzyme is therefore part of the complex makeup of these membrane packages of secreted materials (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). The expression of these membrane vesicles was correlated with the expression of B-band lipopolysaccharide.     

Alignment of multiple chromosomes along helical ParA scaffolding in sporulating Streptomyces hyphae

The dynamic, mitosis-like segregation of bacterial chromosomes and plasmids often involves proteins of the ParA (ATPase) and ParB (DNA-binding protein) families. The conversion of multigenomic aerial hyphae of the mycelial organism Streptomyces coelicolor into chains of unigenomic spores requires the synchronous segregation of multiple chromosomes, providing an unusual context for chromosome segregation. Correct spatial organization of the oriC-proximal region prior to septum formation is achieved by the assembly of ParB into segregation complexes (Jakimowicz et al., 2005; J Bacteriol 187: 3572-3580). Here, we focus on the contribution of ParA to sporulation-associated chromosome segregation. Elimination of ParA strongly affects not only chromosome segregation but also septation. In wild type hyphae about to undergo sporulation, immunostained ParA was observed as a stretched double-helical filament, which accompanies the formation of ParB foci. We show that ParA mediates efficient assembly of ParB complexes in vivo and in vitro, and that ATP binding is crucial for ParA dimerization and interaction with ParB but not for ParA localization in vivo. We suggest that S. coelicolor ParA provides scaffolding for proper distribution of ParB complexes and consequently controls synchronized segregation of several dozens of chromosomes, possibly mediating a segregation and septation checkpoint.     

Evidence for diffuse growth of the cylindrical portion of the Escherichia coli murein sacculus.

High-resolution autoradiography of thin sections of Escherichia coli cells whose murein was pulse-labeled with [3H]diaminopimelic acid after a period of diaminopimelic acid deprivation indicated that elongation of the murein sacculus occurs by a multisite (diffuse) process. Upon chasing, radioactivity in polar murein was stable, whereas radioactivity in cylindrical murein was reduced, indicating that diffuse intercalation of new murein occurred during cell elongation. Elongation and septation were shown to be overlapping processes.     

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.     

Cell cycle-independent lysis of Escherichia coli by cefsulodin, an inhibitor of penicillin-binding proteins 1a and 1b.

Cefsulodin lyses actively growing Escherichia coli by binding specifically to penicillin-binding proteins (PBPs) 1a and 1b. Recent findings (F. García del Portillo, M. A. de Pedro, D. Joseleau-Petit, and R. D'Ari, J. Bacteriol. 171:4217-4221, 1989) have linked cefsulodin-induced lysis to septation during the first division cycle after a nutritional shift-up or chromosome replication realignment. We synchronized cells by membrane filtration to determine whether cefsulodin-induced lysis depended on septation in normally growing cells. Populations of newly divided cells were allowed to grow for variable lengths of time. Cefsulodin was added to these synchronous cultures, which represented points in two to three rounds of the cell cycle. Since the cell numbers were small, a new lysis assay was developed that was based on the release of DNA measured by fluorometry. Lysis occurred at a constant time after addition of the antibiotic, regardless of the time in the cell cycle at which the addition was made. Thus, cefsulodin-induced lysis is not linked to septation or to any other cell cycle-related event.     

On the control of septation in Escherichia coli.

Mutants of E. coli defective in cell septation (ftsA to ftsG, conditional thermosensitive mutants isolated by Ricard and Hirota) were studied with respect to their membrane protein composition, murein hydrolase activities and rates of synthesis of murein and phospholipids. Three classes of mutants have been distinguished: 1) those affected in both murein and phospholipid synthesis; 2) those affected in either murein or phospholipid synthesis and 3) those affected in neither of these parameters. Overall murein hydrolase activities, after activation, is of the same order in all the mutants screened. In addition to soluble products of murein splitting, we have found insoluble products that appear to be in dynamic equilibrium with the murein of the sacculus. Endogenous levels of cyclic adenosine 3',5'-monophosphate measured after blocking septation showed no variation. This suggests that the cyclic nucleotide is not involved in the metabolic control of septation.     

A simple method for following the fate of alanine-containing components in murein synthesis inEscherichia coli

The fate of the alanine-containing components in murein synthesis was followed by incorporation of¹⁴C-l-alanine inE. coli under conditions allowing cell-wall synthesis while preventing protein synthesis. The components were separated by chromatography and detected by autoradiography.Spots containing murein, cell-wall precursors, alanine andd-alanyl-d-alanine were identified. A further component was probably identical to pyruvic acid. Two unidentified spots were found in the region where lipid-intermediates in cell-wall synthesis are usually found. However, the absence of turnover of these two components was at variance with the proposed properties of the lipidintermediates. d-Alanyl-d-alanine and the component which is probably identical to pyruvic acid were excreted into the medium, whereas murein and cell-wall precursors were found in the cellular fraction.The influence of the concentration of alanine, and of the number of cells per ml, on the acid-precipitable activity were studied. The latter increased during, at least, the first two hours and represented mainly lysozyme-degradable material.Significant turnover of murein could be detected neither in the presence nor in the absence of protein synthesis.A time course of the activity of the radioactive components is provided. The influence of a number of antibiotics inhibiting cell-wall synthesis on the acid-precipitable activity and on the activity of the main intermediates in murein synthesis was studied.We thank Mrs. Arna van Schijndel-van Dam and Mr. A. A. G. Verweij for excellent assistance. We thank Dr. P. E. Reynolds (University of Cambridge) for teaching one of us (E J. J. L.) several techniques in the field of bacterial cell walls, and Dr. H. J. W. Wijsman for stimulating discussions.     

Murein biosynthesis during a synchromous cell cycle of Escherichia coli B.

The last stages of murein biosynthesis were studied in relation to the division cycle of Escherichia coli in cells synchronized by amino acid starvation (Ron et al., J. Bacteriol. 123:374--376, 1975). Murein synthesis and the activities of the D-alanine carboxypeptidase and transpeptidase were found to vary significantly during the cell cycle. Maximal synthesis and transpeptidation were observed immediately after cell division, whereas maximal D-alanine carboxypeptidase activity was detected before cell division. These results are in agreement with our earlier findings that before cell division there is a stage of increased hydrolysis of the C-terminal D-alanine moiety of newly synthesized murein strands.     

Membrane proteins correlated with expression of the polysialic acid capsule in Escherichia coli K1.

Growth of Escherichia coli K1 strains at 15 degrees C results in a defect in the synthesis or assembly of the K1 polysialic acid capsule. Synthesis is reactivated in cells grown at 15 degrees C after upshift to 37 degrees C, and activation requires protein synthesis (Whitfield et al., J. Bacteriol. 159:321-328, 1984). Using this temperature-induced defect, we determined the molecular weights and locations of membrane proteins correlated with the expression of K1 (polysialosyl) capsular antigen. Pulse-labeling experiments demonstrated the presence of 11 proteins whose synthesis was correlated with capsule appearance at the cell surface. Using the differential solubility of inner and outer membranes in the detergent Sarkosyl, we localized five of the proteins in the outer membrane and four in the inner membrane. The subcellular location of two of the proteins was not determined. Five proteins appeared in the membrane simultaneously with the initial expression of the K1 capsule at the cell surface. One of these proteins, a 40,000-dalton protein localized in the outer membrane, was identified as porin protein K, which previously has been shown to be present in the outer membrane of encapsulated E. coli. The possible role of these proteins in the synthesis of the polysialosyl capsule is discussed.     

Branching of Escherichia coli cells arises from multiple sites of inert peptidoglycan

Some strains of Escherichia coli defective for dacA, the gene coding for penicillin-binding protein 5, exhibit a strong branching phenotype when cell division is blocked. Since such branch formation implies a differentiation of polar caps at ectopic locations in the cell envelope, we analyzed murein segregation and observed a strong correlation between areas of inert murein and these morphological anomalies. In particular, the tips of branches exhibited the same properties as those described for polar caps of wild-type cells, i.e., the synthesis and turnover of murein were inhibited. Also, the mobility of cell envelope proteins was apparently constrained in areas with morphological defects. Polar regions of branching cells and sacculi had aberrant morphologies with a very high frequency. Of special interest was that areas of inert murein at polar caps were often split by areas of active synthesis, a situation unlike that observed in wild-type cells. These observations suggest that in dacA mutants, branches and other morphological anomalies may arise from split polar caps or by de novo generation of new poles built around inert peptidoglycan patches in the side walls of the cell.     

Penicillin-binding proteins 4 and 5 of Haemophilus influenzae are involved in cell wall incorporation

Abstract Permeabilized cells of Haemophilus influenzae incorporate wall precursors into murein material in an ampicillin-sensitive reaction. In resistant transformants that contain the low antibiotic affinity penicillin-binding proteins (PBPs) 4 and 5, the sensitivity of this incorporation reaction to ampicillin is proportionally lower, suggesting a catalytic role for these proteins in wall synthesis. We conclude that, analogous to the reaction in Escherichia coli , PBPs 4 and 5 of H. influenzae have transpeptidase activity.     

Peptidoglycan biosynthesis in stationary-phase cells of Escherichia coli.

The ability of stationary-phase cells of Escherichia coli W7 to incorporate radioactive precursors into macromolecular murein has been studied. During the initial 6 h of the stationary phase, resting cells incorporated meso-[3H]diaminopimelic acid at a rate corresponding to the insertion of 1.3 X 10(4) disaccharide units min-1 cell-1. Afterwards, the rate of incorporation dropped drastically (90%) to a low but still detectable level. Incorporation during stationary phase did not result in an increased amount of total murein in the culture, suggesting that it was related to a turnover process. Analysis of the effects of a number of beta-lactam antibiotics indicated that incorporation of murein precursors in stationary-phase cells was mediated by penicillin-binding proteins, suggesting that the activity of penicillin-binding protein 2 was particularly relevant to this process.     

Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli

The murein (peptidoglycan) sacculus is an essential polymer embedded in the bacterial envelope. The Escherichia coli class B penicillin-binding protein (PBP) 3 is a murein transpeptidase and essential for cell division. In an affinity chromatography experiment, the bifunctional transglycosylase-transpeptidase murein synthase PBP1B was retained by PBP3-sepharose when a membrane fraction of E. coli was applied. The direct protein-protein interaction between purified PBP3 and PBP1B was characterized in vitro by surface plasmon resonance. The interaction was confirmed in vivo employing two different methods: by a bacterial two-hybrid system, and by cross-linking/co-immunoprecipitation. In the bacterial two-hybrid system, a truncated PBP3 comprising the N-terminal 56 amino acids interacted with PBP1B. Both synthases could be cross-linked in vivo in wild-type cells and in cells lacking FtsW or FtsN. PBP1B localized diffusely and in foci at the septation site and also at the side wall. Statistical analysis of the immunofluorescence signals revealed that the localization of PBP1B at the septation site depended on the physical presence of PBP3, but not on the activity of PBP3. These studies have demonstrated, for the first time, a direct interaction between a class B PBP (PBP3) and a class A PBP (PBP1B) in vitro and in vivo, indicating that different murein synthases might act in concert to enlarge the murein sacculus during cell division.     

A New Fungal Metabolite and Root Growth-stimulating Activity of Its Methyl Ester

Escherichia coli rodA mutant AOS151 grows as round cells at 30 and 42°C (H. Matsuzawa, K. Hayakawa, T. Sato, and K. Imahori, J. Bacteriol., 115, 436–442 (1973)). The mutant was found to be resistant to mecillinam at both temperatures. lip⁺ transductants were prepared by Pl phage transduction via strain AOS151, the cotransduction frequency of round morphology (Rod^?) at 42°C with the lip gene being about 90%. At 42°C all 54 Rod^? transductants tested were resistant to mecillinam. At 30°C all but two of these Rod^? (at 42°C)-type transductants were rod-shaped, and all were sensitive to mecillinam; the two strains grew as ovoid cells. The original rodA mutant AOS151 probably involves an additional mutation(s), that expresses the round cell shape at lower temperature, whereas the rodA51 mutation alone seems to result in temperature-sensitive expression of round cell morphology and mecillinam resistance. rodA mutant cells cultured at either 30 or 42°C had wild-type penicillin-binding protein 2, judging from penicillin-binding activity, electrophoretic mobility, and thermosensitivity.     

High resolution autoradiography of Escherichia coli cells infected with bacteriophage R17

Granboulan, Nicole (Institute de Recherches sur le Cancer, Villejuif, Seine, France), and Richard M. Franklin. High-resolution autoradiography of Escherichia coli cells infected with bacteriophage R17. J. Bacteriol. 91:849-857. 1966.-The ultrastructural alterations in Escherichia coli infected with the RNA bacteriophage R17 were further investigated by means of the technique of high-resolution autoradiography. Tritiated precursors to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein were employed in separate experiments. A striking inhibition of cellular RNA, DNA, and protein synthesis was noted. Whereas normal RNA synthesis occurs in the nucleoid, in infected cells RNA synthesis is predominantly cytoplasmic, but later in the latent period, and during the stage of active viral growth, the label is localized in a polar region. In the late stages of viral growth, RNA synthesis occurs only around the crystals. Protein synthesis also becomes localized in a polar region, but DNA synthesis remains confined to the nucleoid. Under conditions of chloramphenicol inhibition of viral-coat protein synthesis, RNA label is localized in the paranuclear lesion, providing further indication that RNA forms this fibrillar structure.     

Effects of furazlocillin, a beta-lactam antibiotic which binds selectively to penicillin-binding protein 3, on Escherichia coli mutants deficient in other penicillin-binding proteins.

Furazlocillin binds selectively to penicillin-binding protein 3 (PBP-3), prevents septation of Escherichia coli, and allows the cells to form long filaments without lysis. The effect of furazlocillin on the morphology, autolysis, and murein synthesis of E. coli mutants deficient in either PBP-1A, PBP-1Bs, or PBP-2 was studied. The results reveal that PBP-1A and PBP-1Bs functions are not equivalent since furazlocillin affects the morphology, autolysis, and murein synthesis of PBP1A- mutants quite differently from that of PBP-1Bs mutants. Different "PBP-2-" mutants were found to respond to furazlocillin in dramatically different ways: strain LS-1 cells formed elongated rods with a central bulge which eventually lysed, whereas SP6 cells formed stable "barbells" in which the two daughter cells were well separated but remained connected by a thick central region.