Ethanol sensitivity of sporulation in Bacillus subtilis: a new tool for the analysis of the sporulation process.

The growth rate of Bacillus subtilis is lowered but the final cell yield is unchanged when certain concentrations of ethanol are present in the culture medium. At the concentration allowing growth at half-maximal rate, practically no spores are formed. Blockage of spore formation generally occurs at stage 0-I. Sensitivity to ethanol of the capacity to form spores is limited, in a nonsynchronized culture, to a period of at most 45 min around t1. Postexponential events such as excretion of certain enzymes and modification of ribonucleic acid polymerase are altered or suppressed in the presence of ethanol, possibly as the results of a physical change upon the cell membrane. In effect, ethanol is turning wild-type cells into phenocopies of spoO mutants.     

Yeast sphingolipid bypass mutants as indicators of antifungal agents selectively targeting sphingolipid synthesis

Standard methods for evaluating the target specificity of antimicrobial agents often involve the use of microorganisms with altered expression of selected targets and thus either more resistant or more susceptible to target specific inhibitors. In this study we present an alternative approach that utilizes physiological bypass mutants. The Saccharomyces cerevisiae sphingolipid bypass mutant strain AGD is able to grow without making sphingolipids and importantly, tolerates loss-of-function mutations in the otherwise essential genes for both serine palmitoyltransferase (SPT) and inositol phosphorylceramide (IPC) synthase. We found that strain AGD was >1000-fold more resistant than the wild-type strain to selective inhibitors of SPT and IPC synthase. In contrast, strain AGD, which due to abnormal composition of the plasma membrane is sensitive to a variety of environmental stresses, was more susceptible than the wild-type to amphotericin B, voriconazole, and to cycloheximide. We show that in a simple growth assay the AGD strain is an appropriate and useful indicator for inhibitors of IPC synthase, a selective antifungal target.     

Isolation and characterization of mutants of Rhizobium leguminosarum bv. viciae 248 with altered lipopolysaccharides: possible role of surface charge or hydrophobicity in bacterial release from the infection thread

Effects of alterations in lipopolysaccharide (LPS) structure of Rhizobium leguminosarum bv. viciae on effective symbiosis and on a number of cell surface characteristics were studied. Tn5 mutants with altered LPSs were screened for their inability to bind monoclonal antibody 3, one of three monoclonal antibodies to the tentative O-antigenic part of the wild-type LPS of strain 248. Ten class I LPS mutants completely lacked the O-antigen-containing LPS species. The class II LPS mutant had a severely diminished amount of an antigenically altered O-antigen-containing LPS. The class III LPS mutant had normal amounts of an altered, O-antigen-containing LPS. Class I and II mutants, but not the class III mutant, showed abnormal nodule development (i.e., blocked in the stage of bacterial release from the infection thread) resulting in nodules in which very few, at the most, plant cells contained bacteroids and which were unable to fix nitrogen. Class I and II mutants were nonmotile and were more sensitive to hydrophobic compounds than the parent strain. The most striking difference between the symbiotically defective class I and II LPS mutants on one hand and the wild-type strain and the class III mutant on the other hand was that the class I and II mutants have a more hydrophobic cell surface and a higher electrophoretic mobility. A role for an O-antigen-containing LPS in bacterial release from the infection thread, through its effects on general physicochemical cell surface characteristics, is proposed.     

Survival of rifampin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soil systems

The fate of spontaneous chromosomal rifampin-resistant (Rifr) mutants of Pseudomonas putida and Pseudomonas fluorescens in sterile and live organic soil from which they were isolated was studied. In sterile native-soil assays, a Rifr mutant of P. putida showed no decrease in competitive fitness when compared with the wild-type parent. However, mutants of P. fluorescens were of two general categories. Group 1 showed no difference from the wild type in terms of growth rate, competitive fitness, and membrane protein composition. Group 2 showed a slower growth rate in both minimal and enriched media and an altered membrane protein profile. These mutants also demonstrated decreased competitive fitness compared with the wild-type strain. In live soil, the Rifr P. putida strain persisted throughout the 38-day test period with a decay rate of 0.7 log10 CFU/g of soil per 10 days. A group 1 Rifr P. fluorescens mutant maintained its inoculated titer for 7 to 10 days and then decayed at a rate of 0.2 to 0.4 log10 CFU/g of soil per 10 days. A group 2 Rifr P. fluorescens mutant remained at its titer for 1 to 5 days before decaying at a two- to threefold-faster rate. These findings indicate that rifampin resistance may not be an innocuous mutation in some pseudomonads and that marked strains should be compared with wild-type parents before being used as monitors of parental strain survival. Colonization of sterile soil with either the wild-type or mutant strain precluded normal colonization of the second added strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival of rifampin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soil systems.

The fate of spontaneous chromosomal rifampin-resistant (Rifr) mutants of Pseudomonas putida and Pseudomonas fluorescens in sterile and live organic soil from which they were isolated was studied. In sterile native-soil assays, a Rifr mutant of P. putida showed no decrease in competitive fitness when compared with the wild-type parent. However, mutants of P. fluorescens were of two general categories. Group 1 showed no difference from the wild type in terms of growth rate, competitive fitness, and membrane protein composition. Group 2 showed a slower growth rate in both minimal and enriched media and an altered membrane protein profile. These mutants also demonstrated decreased competitive fitness compared with the wild-type strain. In live soil, the Rifr P. putida strain persisted throughout the 38-day test period with a decay rate of 0.7 log10 CFU/g of soil per 10 days. A group 1 Rifr P. fluorescens mutant maintained its inoculated titer for 7 to 10 days and then decayed at a rate of 0.2 to 0.4 log10 CFU/g of soil per 10 days. A group 2 Rifr P. fluorescens mutant remained at its titer for 1 to 5 days before decaying at a two- to threefold-faster rate. These findings indicate that rifampin resistance may not be an innocuous mutation in some pseudomonads and that marked strains should be compared with wild-type parents before being used as monitors of parental strain survival. Colonization of sterile soil with either the wild-type or mutant strain precluded normal colonization of the second added strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetyl phosphate and the phosphorylation of OmpR are involved in the regulation of the cell division rate in Escherichia coli

Carbon sources that can be converted to acetate were added to the growth medium of Escherichia coli wild-type cells. Cells responded with an increased cell division rate. The addition of acetate also caused a decreased synthesis of flagella. Mutants in phosphotransacetylase, which are incapable of synthesizing acetyl phosphate, and mutants in the osmoregulator OmpR divided at a lower rate than did wild-type cells. The mutants did not increase their cell division rate upon the addition of serine, as observed for wild-type cells. These data are consistent with the idea that the previously described effect of serine upon the cell division rate is mediated by acetyl phosphate and phosphorylation of OmpR. Received: 23 March 1998 / Accepted: 8 May 1998     

Cytokinesis is defective in Dictyostelium mutants with altered phagocytic recognition, adhesion, and vegetative cell cohesion properties

Mutants that have been selected for defects in phagocytic recognition, adhesion, and vegetative cell-cell cohesion were found to be larger and more highly multinucleate than their parent strain. This defect is associated with the complex mutant phenotype of these mutants since revertants of the mutants coordinately acquire the wild-type phenotype for all of the defects. The larger size and multinuclearity were due to a high frequency of failure of cytokinesis in cells of wild-type size. This was shown by purifying the small cells in mutant populations and observing their growth and cell division. The mutant phenotype is more penetrant during axenic growth. Most of the mutants are not multinucleate when grown on bacteria. Recently, new mutants have been isolated that are also multinucleate when grown on bacteria by a strong selection procedure for non-adhesion to tissue culture dishes. The pleiotropic mutant phenotype and the greater penetrance of the mutant phenotype in axenic culture can be explained by hypothesizing a deficiency in a membrane component of the actomyosin motor that is involved in all of the processes defective in the mutants.     

Gap junction distribution in the Drosophila wing disc mutants vg,l(2)gd,l(3)c43hs1, and l(2)gl4

The density of gap junctions in four Drosophila melanogaster mutants with abnormal wing disc development has been determined using quantitative electron microscopy and compared with the gap junction density in wild-type wing discs. No appreciable differences relative to wild-type controls were found in the cell death mutant vestigial or in the mildly hyperplastic mutant lethal giant disc which could not be accounted for in terms of altered lateral plasma membrane surface density or as an extension of the gap junction growth which normally occurs during the third larval stage of development in wild-type wing discs. However, both the severely hyperplastic mutant l₍₃₎c43^hs1 and the neoplastic mutant lethal giant larva have significant reductions in the gap junction surface density, the number of gap junctions, and the gap junction areal fraction of the lateral plasma membrane compared with wild-type controls. These differences cannot be attributed to altered lateral plasma membrane surface densities which are not significantly different from wild-type control wing discs. The reduced gap junction density in severely hyperplastic and neoplastic wing discs suggests that alterations in the number or distribution of gap junctions may be as disruptive to normal growth and development as their complete absence.     

Temperature sensitivity and cell division defects in an Escherichia coli strain with mutations in yghB and yqjA, encoding related and conserved inner membrane proteins

Ludox density gradients were used to enrich for Escherichia coli mutants with conditional growth defects and alterations in membrane composition. A temperature-sensitive mutant named Lud135 was isolated with mutations in two related, nonessential genes: yghB and yqjA. yghB harbors a single missense mutation (G203D) and yqjA contains a nonsense mutation (W92TGA) in Lud135. Both mutations are required for the temperature-sensitive phenotype: targeted deletion of both genes in a wild-type background results in a strain with a similar phenotype and expression of either gene from a plasmid restores growth at elevated temperatures. The mutant has altered membrane phospholipid levels, with elevated levels of acidic phospholipids, when grown under permissive conditions. Growth of Lud135 under nonpermissive conditions is restored by the presence of millimolar concentrations of divalent cations Ca(2+), Ba(2+), Sr(2+), or Mg(2+) or 300 to 500 mM NaCl but not 400 mM sucrose. Microscopic analysis of Lud135 demonstrates a dramatic defect at a late stage of cell division when cells are grown under permissive conditions. yghB and yqjA belong to the conserved and widely distributed dedA gene family, for which no function has been reported. The two open reading frames encode predicted polytopic inner membrane proteins with 61% amino acid identity. It is likely that YghB and YqjA play redundant but critical roles in membrane biology that are essential for completion of cell division in E. coli.     

Altered biological properties of cell membranes in Escherichia coli dnaA and seqA mutants.

We present evidence that biological properties of cell membranes are altered in dnaA and seqA mutants of Escherichia coli relative to wild-type bacteria. We found that bacteriophage lambda forms extremely large plaques on the dnaA seqA double mutants. On the single mutants, dnaA and seqA, the plaques are also bigger than those formed on the wild-type host. However, no significant differences in intracellular phage lambda development were observed between wild-type and mutant hosts, indicating that differences in burst size do not account for the observed differences in plaque size. On the other hand, more efficient release of the phage lytic proteins and/or higher sensitivity of the cell membranes to these proteins may result in more efficient cell lysis. We found that the efficiency of adsorption of bacteriophage lambda to the dnaA seqA mutant cells is decreased at 0 degrees C , but not at 30 degrees C, relative to the wild-type strain. A considerable increase in the permeability of membranes of the mutant cells for beta-galactosidase is demonstrated. The dnaA and seqA mutants are more sensitive to ethanol (an organic solvent) than wild-type bacteria, and the seqA strain and the double mutant dnaA seqA are very sensitive to deoxycholate (a detergent). We conclude that lesions in the genes dnaA and seqA result in alterations in cell membranes, such that the permeability and possibly also other properties of the membranes are significantly altered relative to wild-type bacteria.     

Regulatory mutants of Streptomyces clavuligerus affected in free diaminopimelic acid content and antibiotic biosynthesis

The composition of intracellular free amino acid pools was determined in Streptomyces clavuligerus mutants possessing an altered aspartokinase which is insensitive to concerted feedback inhibition by threonine and lysine. These mutants contained total free amino acid pool contents that were considerably higher than those found in the wild-type strain. Diaminopimelic acid accounted for 10 to 20% of the total free amino acid pools, depending on the individual mutant and its culture growth phase, whereas diaminopimelic acid contained in the wild-type strain accounted for only 0.5% of the total free amino acid pool.     

Mutation in the structural gene for release factor 1 (RF-1) of Salmonella typhimurium inhibits cell division.

A temperature-sensitive mutant of Salmonella typhimurium LT2 was isolated. At the nonpermissive temperature cell division stopped and multinucleated filaments were formed. DNA, RNA, or protein synthesis was not affected until after about two generations. Different physiological conditions, such as anaerobiosis and different growth media, suppress the division deficiency at high temperatures. Certain mutations causing a reduced polypeptide chain elongation rate also suppress the division deficiency. The mutation is recessive and shown to be in the structural gene for release factor I (prfA). DNA sequencing of both the wild-type (prfA+) and mutant (prfA101) allele revealed a GC-to-AT transition in codon 168. Like other known prfA mutants, prfA101 can suppress amber mutations. The division defect in the prfA101 mutant strain could not be suppressed by overexpression of the ftsQAZ operon. Moreover, at the nonpermissive temperature the mutant shows a normal heat shock and SOS response and has a normal ppGpp level. We conclude that the prfA101-mediated defect in cell division is not directed through any of these metabolic pathways, which are all known to affect cell division. We speculate that the altered release factor I induces aberrant synthesis of an unidentified protein(s) involved in the elaborate process of septation.     

Cell division in Escherichia coli minB mutants

In Escherichia coli minB mutants, cell division can take place at the cell poles as well as non-polarly in the cell. We have examined growth, division patterns, and nucleoid distribution in individual cells of a minC point mutant and a minB deletion mutant, and compared them to the corresponding wild-type strain and an intR1 strain in which the chromosome is over-replicated. The main findings were as follows. In the minB mutants, polar and non-polar divisions appeared to occur independently of each other. Furthermore, the timing of cell division in the cell cycle was found to be severely affected. In addition, nucleoid conformation and distribution were considerably disturbed. The results obtained call for a re-evaluation of the role of the MinB system in the E. coli cell cycle, and of the concept that limiting quanta of cell division factors are regularly produced during the cell cycle.     

Ethanol and glycerol production under aerobic conditions by wild-type,respiratory-deficient mutants and a fusion product of Saccharomyces cerevisiae

Summary Experiments were performed to investigate growth, ethanol and glycerol production by wild-type strains (RHO) and respiratory-deficient (rho) mutants of Saccharomyces cerevisiae. Furthermore protoplasts were fused in order to enhance the fermentation capacity of a flocculent strain. At high substrate conditions, 150 g/l of saccharose, there is no difference in cell growth. However, at a glucose concentration of 10–20 g/l the mutants grow much slower. After 3 days of incubation at 28° C in a complete medium the viability of the two strains is the same. In minimal medium on the other hand the number of viable cells of the mutant is 100-fold reduced. All mutants tested showed a higher specific activity of alcohol dehydrogenase (ADH I) and an enhanced production of glycerol compared with the wild-type strain. By protoplast fusion a modified flocculent strain was obtained with higher specific activity of ADH I and a reduced biosynthesis of glycerol. However, the yields of ethanol (75–78%) are about the same for the wild-type strain and the rho mutants under aerobic conditions in absence of catabolite repression.     

Plate ethanol-screening assay for selection of the Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability for xylose alcoholic fermentation

A new method for the selection of Pichia stipitis and Hansenula polymorpha yeast mutants with altered capability to ferment xylose to ethanol was developed. The method is based on the ability of P. stipitis and H. polymorpha colonies to grow and produce ethanol on agar plates with xylose as the sole carbon and energy source. Secreted ethanol, in contrast to xylose, supports growth of cells of the indicator xylose-negative strains (the wild-type strain of Saccharomyces cerevisiae or Δxyl1 mutant of H. polymorpha) mixed with agar medium. The size of the tester culture-growth zone around xylose-grown colonies appeared to be dependent on the amount of secreted ethanol. Mutants with altered (decreased or elevated) ethanol production in xylose medium have been isolated using this method. The mutants exhibited pleiotropic alterations in enzymatic activities of the intermediary xylose metabolism.     

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.     

Salt stress affects cortical microtubule organization and helical growth in Arabidopsis

Cortical microtubule arrays are critical in determining the growth axis of diffusely growing plant cells, and various environmental and physiological factors are known to affect the array organization. Microtubule organization is partly disrupted in the spiral1 mutant of Arabidopsis thaliana, which displays a right-handed helical growth phenotype in rapidly elongating epidermal cells. We show here that mutations in the plasma membrane Na(+)/H(+) antiporter SOS1 and its regulatory kinase SOS2 efficiently suppressed both microtubule disruption and helical growth phenotypes of spiral1, and that sos1 and sos2 roots in the absence of salt stress exhibited altered helical growth response to microtubule-interacting drugs at low doses. Salt stress also altered root growth response to the drugs in wild-type roots. Suppression of helical growth appeared to be specific to spiral1 since other helical growth mutants were not rescued. The effects of sos1 in suppressing spiral1 defects and in causing abnormal drug responses were nullified in the presence of the hkt1 Na(+) influx carrier mutation in roots but not in hypocotyls. These results suggest that cytoplasmic salt imbalance caused by insufficient SOS1 activity compromises cortical microtubule functions in which microtubule-localized SPIRAL1 is specifically involved.     

The effect of ethanol on the phase behavior of membrane lipids extracted from Clostridium thermocellum strains

The phase behavior of aqueous dispersions of extracted lipids from Clostridium thermocellum wild-type and ethanol-tolerant C919 cells has been examined by DSC. The optimum growth temperature of this anaerobe is 60°C. The wild-type lipids exhibit a broad phase transition centered at 30°C; the C919 mutant lipids show a 10°C lower T_m. The direct addition of growth inhibiting concentrations of ethanol has no significant effect on T_m or headgroup mobility (monitored by ²H-NMR) of either set of lipids. In contrast, wild-type cells adapted to growth in ethanol exhibit a broadened and lower T_m (15–25°C plateau); C919 membrane lipids do not exhibit significantly altered phase behavior when adapted to growth in ethanol. Both wild-type and mutant membranes have fatty acid composition changes upon growth in ethanol, which increases lower-melting components. It is concluded that fatty acid changes which occur upon adaptation of the organism to growth in ethanol are secondary responses and not necessarily direct responses to alter membrane fluidity.