Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis.

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.     

Role of protein O-mannosyltransferase Pmt4 in the morphogenesis and virulence of Cryptococcus neoformans

Protein O mannosylation is initiated in the endoplasmic reticulum by protein O-mannosyltransferases (Pmt proteins) and plays an important role in the secretion, localization, and function of many proteins, as well as in cell wall integrity and morphogenesis in fungi. Three Pmt proteins, each belonging to one of the three respective Pmt subfamilies, are encoded in the genome of the human fungal pathogen Cryptococcus neoformans. Disruption of the C. neoformans PMT4 gene resulted in abnormal growth morphology and defective cell separation. Transmission electron microscopy revealed defective cell wall septum degradation during mother-daughter cell separation in the pmt4 mutant compared to wild-type cells. The pmt4 mutant also demonstrated sensitivity to elevated temperature, sodium dodecyl sulfate, and amphotericin B, suggesting cell wall defects. Further analysis of cell wall protein composition revealed a cell wall proteome defect in the pmt4 mutant, as well as a global decrease in protein mannosylation. Heterologous expression of C. neoformans PMT4 in a Saccharomyces cerevisiae pmt1pmt4 mutant strain functionally complemented the deficient Pmt activity. Furthermore, Pmt4 activity in C. neoformans was required for full virulence in two murine models of disseminated cryptococcal infection. Taken together, these results indicate a central role for Pmt4-mediated protein O mannosylation in growth, cell wall integrity, and virulence of C. neoformans.     

Atmospheric and room temperature plasma mutagenesis and astaxanthin production from sugarcane bagasse hydrolysate by Phaffia rhodozyma mutant Y1

In this study, atmospheric and room temperature plasma and ultraviolet mutagenesis was studied for astaxanthin overproducing mutant. Phaffia rhodozyma mutant Y1 was obtained from the selection plate with 120 μmol/L diphenylamine as selection agent, and its carotenoid concentration and content were 54.38 mg/L and 5.38 mg/g, which were 19.02 % and 21.20 % higher than that of the original strain, respectively. Sugarcane bagasse hydrolysate was used for astaxanthin production by mutant Y1 at 22 °C and 220 rpm for 96 h, and the biomass and carotenoid concentration reached 12.65 g/L and 88.57 mg/L, respectively. Ultrasonication and cellulase were used to break cell wall and the parameters were optimized, achieving an astaxanthin extraction rate of 96.01 %. The present work provided a novel combined mutagenesis method for astaxanthin overproducing mutant and a green cell wall disruption process for astaxanthin extraction, which would play a solid foundation on the development of natural astaxanthin.     

Disruption of Botrytis cinerea class I chitin synthase gene Bcchs1 results in cell wall weakening and reduced virulence

To get a better insight into the relationship between cell wall integrity and pathogenicity of the fungus Botrytis cinerea, we have constructed chitin synthase mutants. A 620 bp class I chitin synthase gene fragment (Bcchs1) obtained by PCR amplification was used to disrupt the corresponding gene in the genome. Disruption of Bcchs1 occurred at a frequency of 8%. Nine independent mutants were obtained and the Bcchs1 mutant phenotype compared to that of transformants in which the gene was not disrupted. These disruption mutants were dramatically reduced in their in vitro Mg2+, Mn2+, and Co2+-dependent chitin synthase activity. Chitin content was reduced by 30%, indicating that Bcchs1p contributes substantially to cell wall composition. Enzymatic degradation by a cocktail of glucanases revealed cell wall weakening in the mutant. Bcchs1 was transcribed at a constant level during vegetative exponential growth, suggesting that it was necessary throughout hyphal development. Bcchs1 mutant growth was identical to undisrupted control transformant growth, however, the mutant exhibited reduced pathogenicity on vine leaves. It can be assumed that disruption of Bcchs1 leads to cell wall weakening which might slow down in planta fungal progression.     

Absence of fks1p in lager brewing yeast results in aberrant cell wall composition and improved beer flavor stability

The flavor stability during storage is very important to the freshness and shelf life of beer. However, beer fermented with a yeast strain which is prone to autolyze will significantly affect the flavor of product. In this study, the gene encoding β-1,3-glucan synthetase catalytic subunit (fks1) of the lager yeast was destroyed via self-clone strategy. β-1,3-glucan is the principle cell wall component, so fks1 disruption caused a decrease in β-1,3-glucan level and increase in chitin level in cell wall, resulting in the increased cell wall thickness. Comparing with wild-type strain, the mutant strain had 39.9 and 63.41 % less leakage of octanoic acid and decanoic acid which would significantly affect the flavor of beer during storage. Moreover, the results of European Brewery Convention tube fermentation test showed that the genetic manipulation to the industrial brewing yeast helped with the anti-staling ability, rather than affecting the fermentation ability. The thiobarbituric acid value reduced by 65.59 %, and the resistant staling value increased by 26.56 %. Moreover, the anti-staling index of the beer fermented with mutant strain increased by 2.64-fold than that from wild-type strain respectively. China has the most production and consumption of beer around the world, so the quality of beer has a significant impact on Chinese beer industry. The result of this study could help with the improvement of the quality of beer in China as well as around the world.     

Isolation, and morphological and chemical properties of an autolysis-deficient mutant of Clostridium botulinum type A.

An autolysis-deficient mutant was isolated from Clostridium botulinum type A 190L by treatment with ethyl methanesulfonate. The cell wall prepared from the mutant autolyzed at much slower rate than that from the parent strain, accompanying with much less liberation of both amino terminals and reducing groups. Electron microscopic observation revealed that the mutant strain was converted to short rod or curved spherical form with thickened cell walls when the growth temperature was shifted from 37 to 45 C. The mutant had a significantly larger amount of non-peptidoglycan-carbohydrate complexes than did the parent strain and became markedly resistant to the autolysin partially purified from the parent, compared with the parent strain. Furthermore, the mutant was fairly tolerant to killing by penicillin. These results suggest that the autolysis deficiency of the mutant was due not only to the deficient production of autolysin but also to the excess accumulation of carbohydrate in the cell wall.     

Evidence for vital role of endo-β-N-acetylglucosaminidase in the resistance of Arthrobacter protophormiae RKJ100 towards elevated concentrations of o-nitrobenzoate

Arthrobacter protophormiae RKJ100 was previously characterized for its ability to tolerate extremely high concentrations of o-nitrobenzoate (ONB), a toxic xenobiotic environmental pollutant. The physiological responses of strain RKJ100 to ≥30 mM ONB indicated towards a resistance mechanism manifested via alteration of cell morphology and cell wall structure. In this study, we aim to characterize gene(s) involved in the resistance of strain RKJ100 towards extreme concentrations (i.e. 150 mM) of ONB. Transposon mutagenesis was carried out to generate a mutant library of strain RKJ100, which was then screened for ONB-sensitive mutants. A sensitive mutant was defined and selected as one that could not tolerate ≥30 mM ONB. Molecular and biochemical characterization of this mutant showed that the disruption of endo-β-N-acetylglucosaminidase (ENGase) gene caused the sensitivity. ENGase is an important enzyme for oligosaccharide processing and cell wall recycling in bacteria, fungi, plants and animals. Previous reports have already indicated several possible roles of this enzyme in cellular homeostasis. Results presented here provide the first evidence for its involvement in bacterial resistance towards extreme concentrations of a toxic xenobiotic compound and also suggest that strain RKJ100 employs ENGase as an important component in osmotic shock response for resisting extreme concentrations of ONB.     

Osmotic-Sensitive Mutant of Salmonella typhimurium

A strain (DA82) having peculiar osmotic properties was isolated in Salmonella typhimurium. The mutant shows increased elasticity of its cell wall and makes spherical instead of elongated cells, regardless of the osmolality of the medium. The strain withstands dilution in distilled water without disruption or death and grows normally in 0.1 molal NaCl broth (240 milliosmol), but it dies exponentially in low-osmolality broth (40 milliosmol). Addition of salts or sucrose instantly stops death and allows growth and cell division to proceed. Death is not due to lysis because this appears at later times and at a much lower rate. Osmotic inactivation is temperature-dependent: higher death rates occur at higher incubation temperatures. Inhibition of protein synthesis by chloramphenicol (20 mug/ml) prevents osmotic death. At 37 C and at lower temperatures, the phenomenon of osmotic death is transient. After a variable interval, growth of the osmotic-sensitive strain resumes. It is assumed that the strain's osmotic behavior is due to membrane defectiveness. The membrane disfunction and the wall defect shown by the strain may be consequences of a single genetic alteration or the results of independent mutations.     

Abnormal Cell Envelope Ultrastructure of a Saccharomyces Mutant with Invertase Formation Resistant to Hexoses

The most obvious morphological characteristic of Saccharomyces mutant FH4C cells is the tendency to form clumps (production of invertase and alpha-glucosidase by this mutant is highly resistant to repression by hexoses). This peculiar feature arises from the abnormal cell envelope ultrastructure of the mutant. Clumps are formed as a result of the failure of the cell wall of the bud to separate from that of the mother cell. The cell wall also shows irregular thickening. There are many cells with a doughnut shape and with small budlike protrusions. Abnormal septation and wall invagination into vacuoles give rise to cells of differing sizes and irregular profiles. Many vesicles, tubules, and coiled membranous bodies originate from invaginations of the plasmalemma. These structures are frequently observed in the cell wall or the periplasmic space. The cells of mutant FH4C growing in 0.2 M glucose, unlike parent strain 303-67, contain many mitochondria. Large numbers of glycogen deposits are also found in many cells of FH4C.     

Cell wall-linked cryptococcal phospholipase B1 is a source of secreted enzyme and a determinant of cell wall integrity

Phospholipase B (Plb1) is secreted by pathogenic fungi and is a proven virulence determinant in Cryptococcus neoformans. Cell-associated Plb1 is presumptively involved in fungal membrane biogenesis and remodelling. We have also identified it in cryptococcal cell walls. Motif scanning programs predict that Plb1 is attached to cryptococcal membranes via a glycosylphosphatidylinositol (GPI) anchor, which could regulate Plb1 export and secretion. A functional GPI anchor was identified in cell-associated Plb1 by (G)PI-specific phospholipase C (PLC)-induced release of Plb1 from strain H99 membrane rafts and inhibition of GPI anchor synthesis by YW3548, which prevented Plb1 secretion and transport to membranes and cell walls. Plb1 containing beta-1,6-linked glucan was released from H99 (wild-type strain) cell walls by beta-1,3 glucanase, consistent with covalent attachment of Plb1 via beta-1,6-linked glucans to beta-1,3-linked glucan in the central scaffold of the wall. Naturally secreted Plb1 also contained beta-1,6-linked glucan, confirming that it originated from the cell wall. Plb1 maintains cell wall integrity because a H99 deletion mutant, DeltaPLB1, exhibited a morphological defect and was more susceptible than H99 to cell wall disruption by SDS and Congo red. Growth of DeltaPLB1 was unaffected by caffeine, excluding an effect of Plb1 on cell wall biogenesis-related signaling pathways. Environmental (heat) stress caused Plb1 accumulation in cell walls, with loss from membranes and reduced secretion, further supporting the importance of Plb1 in cell wall integrity. This is the first demonstration that Plb1 contributes to fungal survival by maintaining cell wall integrity and that the cell wall is a source of secreted enzyme.     

Cell wall structure of a mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids

A mutant strain of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids was recently isolated (Liu, J., and Nikaido, H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4011-4016). This mutant failed to synthesize full-length mycolic acids and accumulated a series of long chain beta-hydroxymeromycolates. In this work, we provide a detailed characterization of the localization of meromycolates and of the cell wall structure of the mutant. Thin layer chromatography showed that the insoluble cell wall matrix remaining after extraction with chloroform/methanol and SDS still contained a large portion of the total meromycolates. Matrix-assisted laser desorption/ionization and electrospray ionization mass spectroscopy analysis of fragments arising from Smith degradation of the insoluble cell wall matrix revealed that the meromycolates were covalently attached to arabinogalactan at the 5-OH positions of the terminal arabinofuranosyl residues. The arabinogalactan appeared to be normal in the mutant strain, as analyzed by NMR. Analysis of organic phase lipids showed that the mutant cell wall contained some of the extractable lipids but lacked glycopeptidolipids and lipooligosaccharides. Differential scanning calorimetry of the mutant cell wall failed to show the large cooperative thermal transitions typical of intact mycobacterial cell walls. Transmission electron microscopy showed that the mutant cell wall had an abnormal ultrastructure (without the electron-transparent zone associated with the asymmetric mycolate lipid layer). Taken together, these results demonstrate the importance of mycolic acids for the structural and functional integrity of the mycobacterial cell wall. The lack of highly organized lipid domains in the mutant cell wall explains the drug-sensitive and temperature-sensitive phenotypes of the mutant.     

Involvement of the cell envelope of Listeria monocytogenes in the acquisition of nisin resistance

The involvement of the cell wall in the acquisition of nisin resistance by Listeria monocytogenes F6861 and its nisin-resistant mutant was investigated. Results indicated that without a cell wall, the acquired nisin resistance of the mutant was lost. Cell surface hydrophobicity was shown to correlate with nisin sensitivity; the wild type strain being more hydrophobic than its mutant. The possible role of S-layer proteins in nisin resistance was investigated. Examination of strains by freeze-etching and atomic force microscopy did not demonstrate the presence of S-layers in either strain while SDS-PAGE following S-layer extraction procedures revealed no major protein bands. Chloramphenicol did not adversely affect the frequency of isolation of nisin-resistant mutants, indicating that de novo protein synthesis was not involved. The involvement of other cell surface components, teichoic and lipoteichoic acids, was also examined. In contrast with other reports, comparison of the total phospholipid content of the mutant with its parental strain showed no significant difference ( P > 0.05).     

A Recombinant Saccharomyces cerevisiae Strain Overproducing Mannoproteins Stabilizes Wine against Protein Haze

Stabilization against protein haze was one of the first positive properties attributed to yeast mannoproteins in winemaking. In previous work we demonstrated that deletion of KNR4 leads to increased mannoprotein release in laboratory Saccharomyces cerevisiae strains. We have now constructed strains with KNR4 deleted in two different industrial wine yeast backgrounds. This required replacement of two and three alleles of KNR4 for the EC1118 and T73-4 backgrounds, respectively, and the use of three different selection markers for yeast genetic transformation. The actual effect of the genetic modification was dependent on both the genetic background and the culture conditions. The fermentation performance of T73-4 derivatives was clearly impaired, and these derivatives did not contribute to the protein stability of the wine, even though they showed increased mannoprotein release in vitro. In contrast, the EC1118 derivative with both alleles of KNR4 deleted released increased amounts of mannoproteins both in vitro and during wine fermentation assays, and the resulting wines were consistently less susceptible to protein haze. The fermentation performance of this strain was slightly impaired, but only with must with a very high sugar content. These results pave the way for the development of new commercial strains with the potential to improve several mannoprotein-related quality and technological parameters of wine.     

Role of penicillin-binding protein 2 (PBP2) in the antibiotic susceptibility and cell wall cross-linking of Staphylococcus aureus: evidence for the cooperative functioning of PBP2, PBP4, and PBP2A

Ceftizoxime, a beta-lactam antibiotic with high selective affinity for penicillin-binding protein 2 (PBP2) of Staphylococcus aureus, was used to select a spontaneous resistant mutant of S. aureus strain 27s. The stable resistant mutant ZOX3 had an increased ceftizoxime MIC and a decreased affinity of its PBP2 for ceftizoxime and produced peptidoglycan in which the proportion of highly cross-linked muropeptides was reduced. The pbpB gene of ZOX3 carried a single C-to-T nucleotide substitution at nucleotide 1373, causing replacement of a proline with a leucine at amino acid residue 458 of the transpeptidase domain of the protein, close to the SFN conserved motif. Experimental proof that this point mutation was responsible for the drug-resistant phenotype, and also for the decreased PBP2 affinity and reduced cell wall cross-linking, was provided by allelic replacement experiments and site-directed mutagenesis. Disruption of pbpD, the structural gene of PBP4, in either the parental strain or the mutant caused a large decrease in the highly cross-linked muropeptide components of the cell wall and in the mutant caused a massive accumulation of muropeptide monomers as well. Disruption of pbpD also caused increased sensitivity to ceftizoxime in both the parental cells and the ZOX3 mutant, while introduction of the plasmid-borne mecA gene, the genetic determinant of the beta-lactam resistance protein PBP2A, had the opposite effects. The findings provide evidence for the cooperative functioning of two native S. aureus transpeptidases (PBP2 and PBP4) and an acquired transpeptidase (PBP2A) in staphylococcal cell wall biosynthesis and susceptibility to antimicrobial agents.     

Stabilization of cell wall proteins in Bacillus subtilis: a proteomic approach

Even though cell wall proteins of Bacillus subtilis are characterized by specific cell wall retention signals, some of these are also components of the extracellular proteome. In contrast to the majority of extracellular proteins, wall binding proteins disappeared from the extracellular proteome during the stationary phase and are subjected to proteolysis. Thus, the extracellular proteome of the multiple protease-deficient strain WB700 was analyzed which showed an increased stability of secreted WapA processing products during the stationary phase. In addition, stabilization of the WapA processing products was observed also in a sigD mutant strain which is impaired in motility and cell wall turnover. Next, we analyzed if proteins that can be extracted from B. subtilis cell walls are stabilized in the WB700 strain as well as in the sigD mutant. Thus, the cell wall proteome of B. subtilis wild type was defined showing most abundantly cell wall binding proteins (CWBPs) resulting from the WapA and WprA precursor processing. The inactivation of extracellular proteases as well as SigmaD caused an increase of CWBP105 and a decrease of CWBP62 in the cell wall proteome. We conclude that WapA processing products are substrates for the extracellular proteases which are stabilized in the absence of sigD due to an impaired cell wall turnover.     

Autolytic defective mutant of Streptococcus faecalis.

Properties of a variant of Streptococcus faecalis ATCC 9790 with defective cellular autolysis are described. The mutant strain was selected as a survivor from a mutagenized cell population simultaneously challenged with two antibiotics which inhibit cell wall biosynthesis, penicillin G and cycloserine. Compared to the parental strain, the mutant strain exhibited: (i) a thermosensitive pattern of cellular autolysis; (ii) an autolytic enzyme activity that had only a slightly increased thermolability when tested in solution in the absence of wall substrate; and (iii) an isolated autolysin that had hydrolytic activity on isolated S. faecalis wall substrate indistinguishable from that of the parental strain, but that was inactive when tested on walls of Micrococcus lysodeikticus as a substrate. These data indicate an alteration in the substrate specificity of the autolytic enzyme of the mutant which appears to result from the synthesis of an altered form of autolytic enzyme.