Correlation Between Reduced Nicotinamide Adenine Dinucleotide Phosphate Levels and Morphological Changes in Neurospora crassa

A colonial mutant of Neurospora crassa, previously shown to be altered in the structure of glucose-6-P dehydrogenase [a reduced nicotinamide adenine dinucleotide phosphate (NADPH) producing reaction], contained only 40% as much NADPH in extracts as did the wild type. A partial revertant strain, when grown at 23 C, had the same total NADPH content as the wild type, but, at 34 C, had lower levels of NADPH as well as a colonial morphology. A revertant with complete wild-type morphology had wild-type levels of NADPH. Two different colonial mutants, which have also been reported to be altered in NADPH-generating reactions, were found to have a lower content of NADPH, whereas other colonial mutants had wild-type levels. The wild-type strain, when grown under conditions in which it contained a lower total content of NADPH, had a morphology similar to that of a colonial mutant. The evidence indicates that lowered NADPH content leads to a dramatic alteration in the morphology of Neurospora, but not necessarily vice versa. The possible pleiotropic effects of the NADPH deficiency are discussed.     

Specific Lysogenicity in Streptomyces azureus

Slope (or plate) cultures of thiostrepton-producing Streptomyces azureus (ATCC 14921) often showed spontaneously developing plaques. Plaques increased in number during serial subcultures. The production of aerial mycelia and sporulating aerial hyphae was interrupted by the overlapping plaques, whereas the growth of substrate mycelia continued in the plaques. These abnormal (eroded) cultures were easily restored to their normal conditions once they were passed through liquid cultures under shaking conditions. A few phage particles were found in the plaques, together with some headless tails and numerous tail tips which formed a hexagonal crystal or a large crystal mass when viewed in an electron microscope. No lytic phenomenon and no phage production were found in the liquid cultures, although all mycelia and spores harbored phage-producing abilities. It was also found that the propagation of phages was successful in solid culture, but not in liquid culture. The whole phage was named SAt2, which belongs to group B of Bradley's morphological classification. From these results, it is considered that S. azureus is lysogenic with temperate phage SAt2, of which virulent mutants are able to infect the aerial mycelia and sporulating hyphae of their lysogenic host.     

Point mutations in Aβ result in the formation of distinct polymorphic aggregates in the presence of lipid bilayers

A hallmark of Alzheimer's disease (AD) is the rearrangement of the β-amyloid (Aβ) peptide to a non-native conformation that promotes the formation of toxic, nanoscale aggregates. Recent studies have pointed to the role of sample preparation in creating polymorphic fibrillar species. One of many potential pathways for Aβ toxicity may be modulation of lipid membrane function on cellular surfaces. There are several mutations clustered around the central hydrophobic core of Aβ near the α-secretase cleavage site (E22G Arctic mutation, E22K Italian mutation, D23N Iowa mutation, and A21G Flemish mutation). These point mutations are associated with hereditary diseases ranging from almost pure cerebral amyloid angiopathy (CAA) to typical Alzheimer's disease pathology with plaques and tangles. We investigated how these point mutations alter Aβ aggregation in the presence of supported lipid membranes comprised of total brain lipid extract. Brain lipid extract bilayers were used as a physiologically relevant model of a neuronal cell surface. Intact lipid bilayers were exposed to predominantly monomeric preparations of Wild Type or different mutant forms of Aβ, and atomic force microscopy was used to monitor aggregate formation and morphology as well as bilayer integrity over a 12 hour period. The goal of this study was to determine how point mutations in Aβ, which alter peptide charge and hydrophobic character, influence interactions between Aβ and the lipid surface. While fibril morphology did not appear to be significantly altered when mutants were prepped similarly and incubated under free solution conditions, aggregation in the lipid membranes resulted in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also had a variable ability to disrupt bilayer integrity.     

Supramolecular dynamics of gap junctions

During the life cycle of a membrane protein its molecular structure may change and for aggregated proteins this process may be observed on the supramolecular level. Here we demonstrate that this is the case for gap junction channels which maintain cell-cell communication. Freshly synthesized connexins are integrated as hexamers (connexons) into the plasma membrane where they form plaques after pairing with connexons of an attached cell. We inhibited protein trafficking by applying the fungal metabolite brefeldin A (BFA), quantified cell-cell coupling by calcein transfer and fluorescence-activated flow cytometry, and examined the degradation and formation of gap junction plaques by indirect immunofluorescence and immunogold labeling. Under control conditions 50% of the detected plaques were ubiquitylated and less than 10% showed a two-dimensional crystalline packing. One hour after BFA reversal about 60% of the plaques were crystalline and ubiquitylation dropped to 14%. Label for ubiquitin was predominantly found on non-crystalline plaques. We, therefore, conclude that newly formed gap junction plaques are of crystalline morphology which changes to a pleomorphic structure when individual channels are modified during their aging process. This dynamic in plaque morphology correlates with channel inactivation and plaque ubiquitylation.     

Ecological strategies and fitness tradeoffs inEscherichia coli mutants adapted to prolonged starvation

Many bacteria in nature are nutritionally deprived, and there has been heightened interest during the past decade in the properties of these bacteria. We subjected five populations ofEscherichia coli to prolonged starvation in a minimal salts medium, during which time the density of viable cells declined by several orders of magnitude. From each one, we isolated a surviving clone that showed some heritable difference in colony morphology. We then characterized these mutants in two ecologically relevant respects. First, we determined the nature of their selective advantage, if any, during prolonged starvation. (i) Three of the five mutants had significantly lower net death rate when progenitor and mutant clones were starved separately. (ii) Three mutants showed a significant reduction in death rate in mixed culture that was frequency dependent and manifest when the mutant clone was initially rare. This pattern suggests that these mutants fed on some byproduct of progenitor cells (living or dead). (iii) Two mutants caused the death rate of their progenitors to increase significantly relative to the rate measured in the absence of the mutant. This pattern suggests that these mutants had become allelopathic to their progenitors. Thus, three distinct ecological adaptations to prolonged starvation are evident. No advantage was detected for one mutant, whereas two mutants exhibited multiple advantages. Second, we asked whether the starvation-selected mutants were as fit in growth-supporting conditions as their progenitors. All five mutants were inferior to their progenitor during competition in fresh medium. Evidently, there is an evolutionary tradeoff between performance under growth and starvation conditions.     

Nodulation of soybean byRhizobium japonicum mutants with altered capsule synthesis

Spontaneous mutants with altered capsule synthesis were isolated from a marked strain of the symbiont,Rhizobium japonicum. Differential centrifugation was used to enrich serially for mutants incapable of forming capsules. The desired mutants were detected by altered colony morphology and altered ability to bind host plant lectin. Three mutants failed to form detectable capsules at any growth phase when cultured in vitro or in association with the host (soybean,Glycine max (L.) Merr.) roots. These mutants were all capable of nodulating and attaching to soybean roots, indicating that the presence of a capsule physically surrounding the bacterium is not required for attachment or for infection and nodulation. Nodulation by several of the mutants was linearly proportional to the amount of acidic exopolysaccharide that they released into the culture medium during the exponential growth phase, indicating that such polysaccharide synthesis is important and perhaps required for nodulation. Two of the mutants appeared to synthesize normal lectin-binding capsules when cultured in association with host roots, but not when cultured in vitro. Nodulation by these mutants appeared to depend on how rapidly after inoculation they synthesized capsular polysaccharide.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - FITC fluorescein isothiocyanate Contribution No. 719 of the C.F. Kettering Research Laboratory     

Effect of defined lipopolysaccharide core defects on resistance of Salmonella typhimurium to freezing and thawing and other stresses

A family of mutants of Salmonella typhimurium with altered lipopolysaccharide (LPS) core chain lengths were assessed for sensitivity to freeze-thaw and other stresses. Deep rough strains with decreased chain length in the LPS core were more susceptible to novobiocin, polymyxin B, bacitracin, and sodium lauryl sulfate during growth, to ethylenediaminetetraacetic acid and sodium lauryl sulfate in resting suspension, and to slow and rapid freeze-thaw in water and saline, and these strains exhibited more outer membrane damage than the wild type or less rough strains. Variations in the LPS chain length did not dramatically affect the sensitivity of the strains to tetracycline, neomycin, or NaCl in growth conditions or the degree of freeze-thaw-induced cytoplasmic membrane damage. The deeper rough isogenic strains incorporated larger quantities of less-stable LPS and less protein into the outer membrane than did the wild type or less rough mutants, indicating that the mutations affected outer membrane synthesis or organization or both. Nikaido's model of the role of LPS and protein in determining the resistance of gram-negative bacteria to low-molecular-weight hydrophobic antibiotics is discussed in relation to the stress of freeze-thaw.     

Effect of defined lipopolysaccharide core defects on resistance of Salmonella typhimurium to freezing and thawing and other stresses.

A family of mutants of Salmonella typhimurium with altered lipopolysaccharide (LPS) core chain lengths were assessed for sensitivity to freeze-thaw and other stresses. Deep rough strains with decreased chain length in the LPS core were more susceptible to novobiocin, polymyxin B, bacitracin, and sodium lauryl sulfate during growth, to ethylenediaminetetraacetic acid and sodium lauryl sulfate in resting suspension, and to slow and rapid freeze-thaw in water and saline, and these strains exhibited more outer membrane damage than the wild type or less rough strains. Variations in the LPS chain length did not dramatically affect the sensitivity of the strains to tetracycline, neomycin, or NaCl in growth conditions or the degree of freeze-thaw-induced cytoplasmic membrane damage. The deeper rough isogenic strains incorporated larger quantities of less-stable LPS and less protein into the outer membrane than did the wild type or less rough mutants, indicating that the mutations affected outer membrane synthesis or organization or both. Nikaido's model of the role of LPS and protein in determining the resistance of gram-negative bacteria to low-molecular-weight hydrophobic antibiotics is discussed in relation to the stress of freeze-thaw.     

Circadian rhythms in Neurospora crassa: a clock mutant, prd-1, is altered in membrane fatty acid composition

The fatty acid compositions of the phospholipids of Neurospora crassa mutants with altered periods were determined to test the possibility that some of these mutants might have altered membrane composition. In liquid shaker culture in constant light the bd (band) strain, which has a normal period (21.6 h), exhibited a growth-dependent increase in linoleic acid content and a decrease in linolenic acid content during early log phase growth. By late log phase, fatty acid composition was essentially constant. The phospholipid fatty acid compositions of bd strains containing mutations at the frq (frequency) and chr (chrono) loci were indistinguishable from that of the bd strain under the conditions used. However, a bd strain containing a mutation at the prd-1 (period) locus, as well as prd-1 segregants from a cross of this strain to a bd strain, had altered patterns of growth-dependent fatty acid composition; linoleic and linolenic acid contents changed more slow than in the bd strain and continued to change throughout growth. In addition, the fatty acid composition of a bd prd-1 strain on solid medium differed from that of the bd strain. It is proposed that the prd-1 mutation leads to altered membrane homeostasis, which in turn affects circadian rhythmicity because some or all components of the rhythm-generating system are membrane-localized.     

Farnesol restores wild-type colony morphology to 96% of Candida albicans colony morphology variants recovered following treatment with mutagens.

Candida albicans is a diploid fungus that undergoes a morphological transition between budding yeast, hyphal, and pseudohyphal forms. The morphological transition is strongly correlated with virulence and is regulated in part by quorum sensing. Candida albicans produces and secretes farnesol that regulates the yeast to mycelia morphological transition. Mutants that fail to synthesize or respond to farnesol could be locked in the filamentous mode. To test this hypothesis, a collection of C. albicans mutants were isolated that have altered colony morphologies indicative of the presence of hyphal cells under environmental conditions where C. albicans normally grows only as yeasts. All mutants were characterized for their ability to respond to farnesol. Of these, 95.9% fully or partially reverted to wild-type morphology on yeast malt (YM) agar plates supplemented with farnesol. All mutants that respond to farnesol regained their hyphal morphology when restreaked on YM plates without farnesol. The observation that farnesol remedial mutants are so common (95.9%) relative to mutants that fail to respond to farnesol (4.1%) suggests that farnesol activates and (or) induces a pathway that can override many of the morphogenesis defects in these mutants. Additionally, 9 mutants chosen at random were screened for farnesol production. Two mutants failed to produce detectable levels of farnesol.     

A mutant of Escherichia coli capable of utilizing 3-deoxy-d-manno-2-octulosonic acid as sole carbon source

Abstract The hypothesis that rifampicin resistance mutations (possibly leading to altered RNA polymerases) have a pleiotropic effect on symbiotic nitrogen fixation was tested using the Rhizobium japonicum -soybean symbiosis. A total of 20 spontaneous rifampicin-resistant mutants of R. japonicum strain 110 were analyzed biochemically. RNA polymerase assays revealed that the enzyme from 15 mutants was indeed rifampicin-insensitive. Two of these mutants were found to possess an enzyme with an electrophoretically altered β subunit. All rifampicin-resistant mutants were able to form nodules on soybeans and fix nitrogen symbiotically; free-living nitrogen fixation under microaerophilic culture conditions was also unaffected.     

Thermolysin activation mutants with changes in the fusogenic region of an influenza virus hemagglutinin.

Influenza virus A/seal/Mass/1/80 (H7N7) mutants were obtained; the hemagglutinins (HAs) of the mutants were not activated by trypsin, as in the wild-type virus, but by thermolysin. The mutants grew efficiently under multiple replication cycle conditions and formed plaques in chicken embryo cells only when thermolysin was added to the culture medium. They exhibited hemolytic activity and induced protective immunity in chickens after an asymptomatic course of infection. Nucleotide sequencing of the HA gene and direct amino acid sequencing showed that insertion of a single leucine into the fusion peptide of the HA2 chain close to the cleavage site and a shift of the cleavage site toward the C terminus by one amino acid were responsible for the changes in the biological properties of the thermolysin activation mutants. Revertants could be obtained when trypsin or trypsin-like endoproteases were present in the virus-producing system.     

The longevity of Caenorhabditis elegans in soil

Relatively simple model organisms such as yeast, fruit-flies and the nematode, Caenorhabditis elegans, have proven to be invaluable resources in biological studies. An example is the widespread use of C. elegans to investigate the complex process of ageing. An important issue when interpreting results from these studies is the similarity of the observed C. elegans mortality pattern in the laboratory to that expected in its natural environment. We found that the longevity of C. elegans under more natural conditions is reduced up to 10-fold compared with standard laboratory culture conditions. Additionally, C. elegans mutants that live twice as long as wild-type worms in laboratory conditions typically die sooner than wild-type worms in a natural soil. These results indicate that conclusions regarding extended longevity drawn from standard laboratory assays may not extend to animals in their native environment.     

Organic Solvents as Probes for the Structure and Function of the Bacterial Membrane: Effects of Ethanol on the Wild Type and an Ethanol-Resistant Mutant of Escherichia coli K-12

The effects of ethanol on the growth of a wild-type Escherichia coli K-12 are described. These effects include a reduction of the steady-state growth rate and an interference with the division process. They appear as an immediate response to the addition of ethanol and are rapidly reversed by removal of ethanol. Mutants were selected that could grow at a concentration of ethanol that stopped wild-type growth. The growth of one of the mutants we studied (strain S9L100) is stimulated by the presence of ethanol, methanol, or dimethyl sulfoxide. This strain exhibits pleiotropic growth defects including abnormal cell division and morphology. It also appears to have an altered lac permease function which is not due to a mutation in the Y gene itself. We conclude that this mutant has an altered membrane and that the membrane defect may be the cause of the abnormal growth properties. The use of compounds which serve as general membrane perturbants and mutants resistant to these perturbants form a system accessible to both genetic and physical-biochemical techniques.     

Regulation of toxinogenesis in Corynebacterium diphtheriae: mutations in the bacterial genome that alter the effects of iron on toxin production

Mutants of Corynebacterium diphtheriae C7(beta) that are resistant to the inhibitory effects of iron on toxinogenesis were identified by their ability to form colonies surrounded by toxin-antitoxin halos on agar medium containing both antitoxin and a high concentration of iron. Chromosomal mutations were essential for the altered phenotypes of four independently isolated mutant strains. During growth in deferrated liquid medium containing various amounts of added iron, these mutants differed from wild-type C. diphtheriae C7(beta) in several ways. Their growth rates were slower under low-iron conditions and were stimulated to various degrees under high-iron conditions. The concentrations of iron at which optimal toxin production occurred were higher for the mutants than for wild-type C. diphtheriae C7(beta). Toxin production by the mutants during growth in low-iron medium occurred throughout the period of exponential growth at nearly constant rates that were proportional to the bacterial growth rates. In contrast, toxin production by wild-type C. diphtheriae C7(beta) in similar low-iron cultures occurred predominantly during the late exponential phase, when iron was a growth-limiting nutrient. Additional studies demonstrated that these mutants had severe defects in their transport systems for ferric iron. We propose that the altered regulation of toxinogenesis by iron in our mutants was caused by the severe defects in their iron transport systems. As a consequence, the mutants exhibited a low-iron phenotype during growth under conditions that permitted wild-type C. diphtheriae C7(beta) to exhibit a high-iron phenotype.     

Isolation and characterization of heat-resistant (HR) mutants of Newcastle disease virus

Heat-resistant (HR) mutants (MR 70 and HR 74) of Newcastle disease virus (NDV) which exhibited significantly higher thermostability in their infectivity than wild-type virus were isolated and characterized. They differ from each other in their plaque morphology; HR 70 produces small turbid plaques, whereas those of HR 74 are large and clear. Cytopathogenicity of these mutants is much lower than that of the wild-type virus in cultured cells such as CEF, LLCMK2 and HeLa cells. Moreover, these HR mutants exhibited extended mean embryo survival times. Synthesis of cellular RNA's and proteins in cells infected with HR mutants was not significantly reduced under conditions in which synthesis of these macromolecules was strongly reduced in cells infected with wild-type virus. No significant differences were observed between HR mutants and wild-type virus in their other phenotypic characteristics such as the capacity for interferon production, growth characteristics at a low multiplicity of infection, and cleavage of viral glycoproteins in infected cells. From these findings, it was suggested that the inhibitory effect of virus infection on cellular macromolecular synthesis is a possible determinant of cytopathogenicity of NDV.     

Cardiolipin Accumulation in the Inner and Outer Membranes of Escherichia coli Mutants Defective in Phosphatidylserine Synthetase

Mutants of Escherichia coli defective in phosphatidylserine synthetase (pss) make less phosphatidylethanolamine than normal cells, and they are temperature sensitive for growth. We have isolated a new mutant, designated RA2021, which is better than previously available strains in that the residual phosphatidylethanolamine level approaches 25% after 4 h at 42 degrees C. The total amount of phospholipid normalized to the density of the culture is about the same in RA2021 (pss-21) as in the isogenic wild-type RA2000 (pss(+)). Consequently, there is a net accumulation of polyglycerophosphatides in the mutant, particularly of cardiolipin. The addition of 10 to 20 mM MgCl(2) to a culture of RA2021 prolongs growth under nonpermissive conditions and prevents loss of cell viability, but it does not eliminate the temperature-sensitive phenotype. Divalent cations, like Mg(2+), do not correct the phospholipid composition of the mutant, but may act indirectly by balancing the negative charges of phosphatidylglycerol and cardiolipin. To determine the effects of the pss mutation on membrane composition, we have examined the subcellular distribution of the polyglycerophosphatides that accumulate in these strains. All of the excess anionic lipids of RA2021 are associated with the envelope fraction and are distributed equally between the inner and outer membranes. The protein compositions of the isolated membranes do not differ significantly in the mutant and wild type. The fatty acid composition of RA2021 is almost the same as wild type at 30 degrees C, but there is more palmitic and cyclopropane fatty acid at 42 degrees C. These results demonstrate that the modification of the polar lipid composition observed in pss mutants affects both membranes and that cardiolipin, which is not ordinarily present in large quantities, can accumulate in the outer membrane when it is overproduced by the cell. The altered polar headgroup composition of the outer membrane in pss mutants may account, in part, for their hypersensitivity to the aminoglycoside antibiotics.     

Host range mutants of bacteriophage Ox2 can use two different outer membrane proteins of Escherichia coli K-12 as receptors.

The Escherichia coli K-12 outer membrane protein OmpA functions as the receptor for bacteriophage Ox2. We isolated a host range mutant of this phage which was able to grow on an Ox2-resistant ompA mutant producing an altered OmpA protein. From this mutant, Ox2h5, a second-step host range mutant was recovered which formed turbid plaques on a strain completely lacking the OmpA protein. From one of these mutants, Ox2h10, a third-step host range mutant, Ox2h12, was isolated which formed clear plaques on a strain missing the OmpA protein. Ox2h10 and Ox2h12 apparently were able to use both outer membrane proteins OmpA and OmpC as receptors. Whereas there two proteins are very different with respect to primary structures and functions, the OmpC protein is very closely related to another outer membrane protein, OmpF, which was not recognized by Ox2h10 or Ox2h12. An examination of the OmpC amino acid sequence, in the regions where it differs from that of OmpF, revealed that one region shares considerable homology with a region of the OmpA protein which most likely is required for phage Ox2 receptor activity.     

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.