Overproduction of outer membrane protein suppresses envA-induced hyperpermeability.

A quantitative study on outer membrane components was performed in a number of envelope mutants of Escherichia coli K-12 exhibition different permeability properties for antimicrobial agents. The envA1 allele causing an increased influx for both hydrophobic and hydrophilic drugs was found to be associated with a deficiency in the amount of lipopolysaccharides. The sefA1 envA1 double mutant was found to have a higher outer membrane buoyant density, apparently due to an increase in protein content. This double mutant was still low in lipopolysaccharide content.     

Phenethyl Alcohol as a Suppressor of the EnvA Phenotype Associated with the envA Gene in Escherichia coli K-12

In Escherichia coli K-12 the envA gene was previously shown to mediate chain formation and a decreased tolerance to several antibacterial agents. Phenethyl alcohol at low concentrations has now been found to increase the tolerance to actinomycin D, ampicillin, rifampin, and gentian violet in strains containing envA. The increased tolerance to gentian violet was correlated to a decreased uptake of the dye. A phenotype suppression of chain formation and colony morphology in envA mutants was also obtained. Except for an increase in palmitic acid, chemical analysis revealed no differences between an envA and its wild-type strain in the lipopolysaccharide part of the envelope. However, a decrease in the amount of phosphatidylglycerol and a C18: 1 fatty acid was observed in the extractable lipids of a strain containing envA. Growth in the presence of phenethyl alcohol reversed the changes in fatty acid and the phospholipid composition. Phenethyl alcohol was found to cause an immediate but transient inhibition of ribonucleic acid synthesis. It is suggested that this inhibition affects the penetrability barrier of the outer cell envelope layers in strains containing envA.     

Dual function of the germinal centre kinase Don3 during mitosis and cytokinesis in Ustilago maydis

Septum formation is a crucial step of cytokinesis in fungi. In the basidiomycete Ustilago maydis, the germinal centre kinase Don3 triggers initiation of a secondary septum necessary for cell separation after cytokinesis. Here we show that oligomerization of Don3 via a putative coiled-coil domain is critical for secondary septum formation. Within the Don3 sequence we detected a characteristic C-terminal sequence motif (T-motif), which determines the subcellular localization of Don3 but is not required for regulation of cell separation. This motif defines a novel family of fungal protein kinases including Sid1p, an essential component of the septation initiation network (SIN) in Schizosaccharomyces pombe. Using the yeast two-hybrid system we isolated the Don3-interacting protein Dip1, which is similar to S. pombe Cdc14p, another member of the SIN. Remarkably, deletion of dip1 did not interfere with cytokinesis in U. maydis, but both dip1 and don3 mutants were affected in nuclear envelope breakdown (NEBD) during mitosis. This phenotype has already been described for mutants, which lack the small GTPase Ras3, the U. maydis homologue of the SIN component Spg1p. We propose that the Don3 kinase exerts a dual function in the regulation of cell separation and NEBD.     

Evidence for a role of N-acetylmuramyl-L-alanine amidase in septum separation in Escherichia coli.

Septum formation and septum separation have been studied in a chain-forming mutant of Escherichia coli K-12 bearing the envA mutation and its parental strain. In comparison to the wild type, the mutant showed a sixfold reduction in the specific activity of the enzyme, N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28), part of which was associated to the outer membrane. Genetic as well as physiological suppression of chain formation resulted in an increase in amidase activity. The addition of N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid to growing wild-type cells and to cells bearing the envA mutation caused an inhibition of cell separation and an increased frequency of visible septa. The kinetics of septum formation and separation was followed in chains by the use of ampicillin and nalidixic acid. The latter drug inhibited initiation of new septa but allowed preformed ones to go to cell separation at a rate corresponding to that of steady-state growing cells. Ampicillin treatment, on the other hand, resulted in a more rapid decrease in the frequency of septa. The disparate effects of ampicillin and nalidixic acid were not explained by a difference in amidase activity but could be due to an inhibitory effect of ampicillin on a septal peptidoglycan fusing activity.     

Mutant of Escherichia coli with Anomalous Cell Division and Ability to Decrease Episomally and Chromosomally Mediated Resistance to Ampicillin and Several Other Antibiotics

In a mutation experiment with a rough, ampicillin-resistant strain, we isolated two smooth mutants which were both sensitive to ampicillin and carried defects in the cell envelope. One of the strains (with the envA gene) is hindered in its completion of septa and forms chains of cells. The envA gene has been mapped to a position between leu and proB, at 2 to 4 min. The envA gene decreased the resistance mediated by both episomal and chromosomal genes for resistance to several antibiotics. During growth the envA mutant was characterized by abnormal ratios between viable count or cell count and optical density. The ratio between viable count and optical density was affected during shift-up and shift-down experiments. When compared to the parent strain, the envA mutant was found to be more resistant to ultraviolet irradiation on plates. Prestarvation for tryptophan had a protective effect against irradiation both on the parent strain and the envA mutant.     

Morphogenesis of the bacterial division septum: identification of potential sites of division in lkyD mutants of Salmonella typhimurium.

It previously has been shown that lkyD mutants of Salmonella typhimurium form large blebs of outer membrane over the septal and polar regions of dividing cells. To determine whether the outer membrane blebs are formed over potential sites of division even in the absence of septal ingrowth, lkyD strains were studied under conditions in which ingrowth of inner membrane and murein was prevented by inactivation of the envA gene product. In aseptate filaments of the LkyD EnvA strain, outer membrane blebs occurred with the usual frequency and were preferentially located over regions where new septa were formed when cell division was subsequently permitted to resume. The results indicate that the outer membrane blebs of the LkyD strain are markers for potential sites of cell division, implying that an alteration in association of outer membrane and murein exists in these sites before the initiation of septal ingrowth. This localized change in cell envelope organization is independent of the septation-inducing effects of the envA gene product.     

Rapid Method for Isolation of Large Quantities of Outer Membrane from Escherichia coli K-12 and Its Application to the Study of Envelope Mutants

A rapid method for the isolation of large quantities of bacterial outer membrane is described. This cell envelope component was removed from plasmolyzed cells of Escherichia coli K-12 by lysozyme-ethylenediaminetetra-acetic acid treatment, aggregated by lowering the pH to 5.0, and recovered by centrifugation. Aggregates of membrane fragments were clearly identified in an electron microscope. A criterion of homogeneity of the preparation was obtained by isopycnic sucrose gradient centrifugation. A single band appeared at a density of 1.24 g/cc. The cytoplasmic membrane marker, succinate dehydrogenase activity, was 40 times lower in the outer membrane preparation than in complete cell envelope preparations. A rich activity was, however, found for the outer membrane marker, phospholipase A. The compositions of outer membranes from a transductant pair were compared. One transductant was a chain-forming, antibiotic-supersensitive envA strain, whereas the other contained the envA(+) allele. The envA strain showed a slightly modified protein pattern and a lower relative content of phosphatidylglycerol.     

Inhibition of cell division in Escherichia coli K-12 by the R-factor R1 and copy mutants of R1.

The effect of the copy number of plasmid R1drd-19 on cell division of Escherichia coli K-12 was studied in populations growing as steady-state cultures at different growth rates, the growth rate being varied by use of different carbon sources. The plasmid copy number was also varied by using copy mutants of the R-factor. The mean cell size was larger in populations carrying an R-factor than in R-factorless populations, an effect that was more pronounced at low growth rates and in populations carrying R-factor copy mutants. The increased cell size was due to formation of elongated cells in a fraction of the population and to an increase in the diameter of all cells. The majority of the cells divided at a normal cell length, but the presence of an R-factor caused some cells to elongate, probably by the uncoupling of chromosome replication and cell division. This can be explained as a competition between the chromosome and plasmid replicons for some replication factor(s), presumably acting on both initiation and elongation of replication. The formation of elongated cells was a reversible process, but occasionally some of the elongated cells reached lengths 20 times that of newborn cells. If cell division did not occur at the normal cell size, the septum was not formed until the cell size was four times that of a newborn cell. When an elongated cell divided, it usually formed a polar septum, thus producing a newborn cell of normal cell length. The ability of plasmid-containing cells to omit one cell division but to retain the capacity of dividing one mass doubling later is compatible with a mechanical model for septum formation and cell division.     

Chs1 of Candida albicans is an essential chitin synthase required for synthesis of the septum and for cell integrity

CaCHS1 of the fungal pathogen Candida albicans encodes an essential chitin synthase that is required for septum formation, viability, cell shape and integrity. The CaCHS1 gene was inactivated by first disrupting one allele using the ura-blaster protocol, then placing the remaining allele under the control of the maltose-inducible, glucose-repressible MRP1 promoter. Under repressing conditions, yeast cell growth continued temporarily, but daughter buds failed to detach from parents, resulting in septumless chains of cells with constrictions defining contiguous compartments. After several generations, a proportion of the distal compartments lysed. The conditional Deltachs1 mutant also failed to form primary septa in hyphae; after several generations, growth stopped, and hyphae developed swollen balloon-like features or lysed at one of a number of sites including the hyphal apex and other locations that would not normally be associated with septum formation. CHS1 therefore synthesizes the septum of both yeast and hyphae and also maintains the integrity of the lateral cell wall. The conditional mutant was avirulent under repressing conditions in an experimental model of systemic infection. Because this gene is essential in vitro and in vivo and is not present in humans, it represents an attractive target for the development of antifungal compounds.     

The eli1 mutation reveals a link between cell expansion and secondary cell wall formation in Arabidopsis thaliana

Mutants with altered patterns of lignification have been identified in a population of mutagenised Arabidopsis seedlings. One of the mutants exhibited ectopic lignification (eli) of cells throughout the plant that never normally lignify. The reduced expansion of eli1 cells resulted in a stunted phenotype, and xylem cells were misshapen and failed to differentiate into continuous strands, causing a disorganized xylem. Analysis of phenotypes associated with double mutants of eli1 lit (lion's tail), a cell expansion mutant, indicated that the primary defect in eli1 plants may be inappropriate initiation of secondary wall formation and subsequent aberrant lignification of cells caused by altered cell expansion. Related ectopic lignification phenotypes were also observed in other cell expansion mutants, suggesting a mechanism that senses cell size and controls subsequent secondary wall formation. Interactions between eli1 and wol (woodenleg), a mutant altering xylem cell specification, revealed a role for ELI1 in promoting formation of continuous xylem strands, and demonstrated that ELI1 functions during cell elongation zone in the primary root and other tissues.     

Leakage of periplasmic enzymes from envA1 strains of Escherichia coli

Previous work ascribed antibiotic hypersensitivity of the envA1 mutant to lowered lipopolysaccharide levels and exposure of the lipid bilayer. In the detailed characterization of the EnvA permeability phenotype presented here, the envA1 mutation was shown to confer leakage of the periplasmic enzymes beta-lactamase and RNase I. Leakage was observed in three different genetic backgrounds, including the original envA1 strain and its parent. In contrast, no detectable leakage of the cytoplasmic enzyme beta-galactosidase was observed. Sensitivity of envA1 strains to a range of antibiotics not previously reported was tested, and lipophilicity (partition coefficient) of a number of antibiotics was determined. On the basis of observations of periplasmic leakage and sensitivity to large hydrophilic antibiotics and lysozyme, part of the permeability phenotype of the envA1 mutant is proposed to be due to transient rupture and resealing of the EDTA-sensitive outer membrane layer. In this regard, the EnvA permeability phenotype falls into a general class of permeability/leaky mutants of both Escherichia coli and Salmonella typhimurium.     

Cross Talk between NDR Kinase Pathways Coordinates Cytokinesis with Cell Separation in Schizosaccharomyces pombe

NDR (nuclear Dbf-2-related) kinases constitute key regulatory nodes in signaling networks that control multiple biological processes such as growth, proliferation, mitotic exit, morphogenesis, and apoptosis. Two NDR pathways called the septation initiation network (SIN) and the morphogenesis Orb6 network (MOR) exist in the fission yeast Schizosaccharomyces pombe. The SIN promotes cytokinesis, and the MOR drives cell separation at the end of cytokinesis and polarized growth during interphase. We showed previously that cross talk exists between these two pathways, with the SIN inhibiting the MOR during cytokinesis through phosphorylation of the MOR component Nak1 by the SIN Sid2 kinase. The reason for this inhibition remained uncertain. We show here that failure to inhibit MOR signaling during cytokinesis results in cell lysis at the site of septum formation. Time-lapse analysis revealed that MOR signaling during cytokinesis causes cells to prematurely initiate septum degradation/cell separation. The cell lysis phenotype is due to premature initiation of cell separation because it can be rescued by mutations in genes required for cell separation/septum degradation. We also shed further light on how the SIN inhibits the MOR. Sid2 phosphorylation of the MOR proteins Sog2 and Nak1 is required to prevent cell lysis during cytokinesis. Together, these results show that SIN inhibition of the MOR enforces proper temporal ordering of cytokinetic events.     

LytR-CpsA-Psr proteins in Staphylococcus aureus display partial functional redundancy and the deletion of all three severely impairs septum placement and cell separation

Staphylococcus aureus contains three members of the LytR-CpsA-Psr (LCP) family of membrane proteins: MsrR, SA0908 and SA2103. The characterization of single-, double- and triple-deletion mutants revealed distinct phenotypes for each of the three proteins. MsrR was involved in cell separation and septum formation and influenced β-lactam resistance; SA0908 protected cells from autolysis; and SA2103, although displaying no apparent phenotype by itself, enhanced the properties of msrR and sa0908 mutants when deleted. The deletion of sa0908 and sa2103 also further attenuated the virulence of msrR mutants in a nematode-killing assay. The severely defective growth phenotype of the triple mutant revealed that LytR-CpsA-Psr proteins are essential for optimal cell division in S. aureus. Growth could be rescued to varying degrees by any one of the three proteins, indicating some functional redundancy within members of this protein family. However, differing phenotypic characteristics of all single and double mutants and complemented triple mutants indicated that each protein played a distinct role(s) and contributed differently to phenotypes influencing cell separation, autolysis, cell surface properties and virulence.     

Antibacterial activity of rifampicin against the causative agents of suppurative, septic diseases]

The antibacterial activity of rifampicin was studied in comparison with other antibiotics with respect to clinical strains isolated from cases with various purulent inflammatory processes caused by Staphylococcus, E. coli, Ps. aeruginose, Proteus. The aim of the study was to define the role of rifampicin in the treatment of the above infections. No rifampicin resistant strains were found among staphylococci belonging to the phenotype carrying the determinants of resistance to 2-8 antibiotics. Rifampicin was less active against gramnegative organisms. High heterogeneity of the microbial population of rifampicin was shown with respect to all microbial strains tested. The rate of the spontaneous mutants was high. The average rate of the mutants was 1-7.7-10-8. The studies on the dynamics of the rifampicin resistance increase in the strains of Staphylococci, E. Coli, Ps. aeruginosa and Proteus showed that the resistance increased after 1-2 passages, which means that one-stage mutation was characteristic rifampicin.     

Candida albicans Rho-type GTPase-encoding genes required for polarized cell growth and cell separation

Rho proteins are essential regulators of morphogenesis in eukaryotic cells. In this report, we investigate the role of two previously uncharacterized Rho proteins, encoded by the Candida albicans RHO3 (CaRHO3) and CaCRL1/CaRHO4 genes. The CaRHO3 gene was found to contain one intron. Promoter shutdown experiments using a MET3 promoter-controlled RHO3 revealed a strong cell polarity defect and a partially depolarized actin cytoskeleton. Hyphal growth after promoter shutdown was abolished in rho3 mutants even in the presence of a constitutively active ras1(G13V) allele, and existing germ tubes became swollen. Deletion of C. albicans RHO4 indicated that it is a nonessential gene and that rho4 mutants were phenotypically different from rho3. Two distinct phenotypes of rho4 cells were elongated cell morphology and an unexpected cell separation defect generating chains of cells. Colony morphology of crl1/rho4 resulted in a growth-dependent smooth (long cell cycle length) or wrinkled (short cell cycle length) phenotype. This phenotype was additionally dependent on the rho4 cell separation defect and was also found in a Cacht3 chitinase mutant that shows a strong cytokinesis defect. The overexpression of the endoglucanase encoding the ENG1 gene, but not CHT3, suppressed the cell separation defect of crl1/rho4 but could not suppress the cell elongation phenotype. C. albicans Crl1/Rho4 and Bnr1 both localize to septal sites in yeast and hyphal cells but not to the hyphal tip. Deletion of RHO4 and BNR1 produced similar morphological phenotypes. Based on the localization of Rho4 and on the rho4 mutant phenotype, we propose a model in which Rho4p may function as a regulator of cell polarity, breaking the initial axis of polarity found during early bud growth to promote the construction of a septum.     

Recombination between a temperature-sensitive mutant and a deletion mutant of Rous sarcoma virus.

Cells doubly infected with two mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), ts68, which is temperature sensitive for cell transformation (srcts), and a deletion mutant, N8, which is deficient in the envelope glycoprotein (env-), produced a recombinant which carried the defects of both parents. The frequency of formation of such a recombinant was exceptionally high and made up 45 to 55% of the progeny carrying the srcts marker. By contrast, the reciprocal recombinant, which is wild type in transformation (srcts) and contains the subgroup A envelope glycoprotein (envA), was almost undetectable. This remarkable difference in the frequency of the formation of the two possible recombinants suggests that a unique mechanism may be involved in the genetic interaction of the two virus genomes, one of which has a large deletion. When an RNA-dependent DNA polymerase-negative variant of the N8 (N8alpha) was crinants also became deficient in the polymerase. Cells infected by the srctsenv- recombinant were morphologically normal at the nonpermissive temperature (41 degrees C) and susceptible to all subgroups of RSV. The rate by which the wild-type RSV transformed the recombinant-preinfected cells was indistinguishable from that of transformation of uninfected chicken cells by the same wild-type virus. This indicates that no detectable interference exists at postpenetration stages between the preinfected and superinfecting virus genomes and confirms that the expression of the transformed state is dominant over the suppressed state.     

Identification and Characterization of Aspergillus Nidulans Mutants Defective in Cytokinesis

Filamentous fungi undergo cytokinesis by forming crosswalls termed septa. Here, we describe the genetic and physiological controls governing septation in Aspergillus nidulans. Germinating conidia do not form septa until the completion of their third nuclear division. The first septum is invariantly positioned at the basal end of the germ tube. Block-and-release experiments of nuclear division with benomyl or hydroxyurea, and analysis of various nuclear division mutants demonstrated that septum formation is dependent upon the third mitotic division. Block-and-release experiments with cytochalasin A and the localization of actin in germlings by indirect immunofluorescence showed that actin participated in septum formation. In addition to being concentrated at the growing hyphal tips, a band of actin was also apparent at the site of septum formation. Previous genetic analysis in A. nidulans identified four genes involved in septation (sepA-D). We have screened a new collection of temperature sensitive (ts) mutants of A. nidulans for strains that failed to form septa at the restrictive temperature but were able to complete early nuclear divisions. We identified five new genes designated sepE, G, H, I and J, along with one additional allele of a previously identified septation gene. On the basis of temperature shift experiments, nuclear counts and cell morphology, we sorted these cytokinesis mutants into three phenotypic classes. Interestingly, one class of mutants fails to form septa and fails to progress past the third nuclear division. This class of mutants suggests the existence of a regulatory mechanism in A. nidulans that ensures the continuation of nuclear division following the initiation of cytokinesis.     

Division behavior and shape changes in isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli during temperature shift experiments.

Isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli were compared with their parent strain in temperature shift experiments. To improve detection of phenotypic differences in division behavior and cell shape, the strains were grown in glucose-minimal medium with a decreased osmolality (about 100 mosM). Already at the premissive temperature, all mutants, particularly the pbpB and ftsQ mutants, showed an increased average cell length and cell mass. The pbpB and ftsQ mutants also exhibited a prolonged duration of the constriction period. All strains, except ftsZ, continued to initiate new constrictions at 42 degrees C, suggesting the involvement of FtsZ in an early step of the constriction process. The new constrictions were blunt in ftsQ and more pronounced in ftsA and pbpB filaments, which also had elongated median constrictions. Whereas the latter strains showed a slow recovery of cell division after a shift back to the permissive temperature, ftsZ and ftsQ filaments recovered quickly. Recovery of filaments occurred in all strains by the separation of newborn cells with an average length of two times LO, the length of newborn cells at the permissive temperature. The increased size of the newborn cells could indicate that the cell division machinery recovers too slowly to create normal-sized cells. Our results indicate a phenotypic resemblance between ftsA and pbpB mutants and suggest that the cell division gene products function in the order FtsZ-FtsQ-FtsA, PBP3. The ftsE mutant continued to constrict and divide at 42 degrees C, forming short filaments, which recovered quickly after a shift back to the permissive temperature. After prolonged growth at 42 degree C, chains of cells, which eventually swelled up, were formed. Although the ftsE mutant produced filaments in broth medium at the restrictive temperature, it cannot be considered a cell division mutant under the presently applied conditions.