Structure peculiarities of cell walls of <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis>—an autotroph of cephalosporin C

Alterations of cell walls of Acremonium chrysogenum occurring at intensive synthesis of cephalosporin C has been studied. It is shown, using electron microscopy, that the cell wall of the cells of ATCC 11550 strain (“wild” type) became looser and thicker during growth. The cell wall of the cells of strain 26/8 (hyperautotroph of cephalosporin C) considerably degraded by the end of the stationary phase. Biochemical analysis has shown that these alterations entailed decrease of the proteins’ content covalently or noncovalently linked with the polysaccharides of cell walls of both strains. An increase of sensitivity of cell walls of the strain-hyperautotroph to an activity of lytic enzymes of chitinase, laminarinase, proteinase K, and lyticase preparation has been observed during the growth, but this increase has not been found in the case of “wild” type strain. The obtained results evidence to the structure failure of the cell wall of A. chrysogenum entailing the intensive creation of antibiotic.     

Aspergillus nidulans RhoA is involved in polar growth, branching, and cell wall synthesis

Growth of the filamentous fungus Aspergillus nidulans begins when the conidium breaks dormancy and grows isotropically. Eventually a germ tube emerges and the axis of growth remains fixed in the primary hypha while new growth axes are established basally to form secondary germ tubes and lateral branches. Rho1 is a Rho family GTPase that has been shown to be involved in polarity establishment and cell wall deposition in Saccharomyces cerevisiae. A gene predicted to encode a Rho1 homolog was cloned from A. nidulans and named rhoA. Strains carrying ectopic copies of the constitutively active rhoA(G14V) allele or the dominant rhoA(E40I) allele were created and characterized. The constitutively active rhoA(G14V) strain grew slowly relative to wild type and showed an abnormal clustered pattern of branch emergence. The rhoA(G14V) strain also labeled intensely with calcofluor, showed elevated levels of cell wall N-acetylglucosamine and had unusually thick cell walls. The dominant rhoA(E40I)strain was accelerated in the emergence of secondary and tertiary germ tubes, and lateral branches relative to wild type and showed lysis with prolonged incubation. The rhoA(E40I) strain also was hypersensitive to the cell wall disrupting agents calcofluor and caspofungin acetate and showed an increase in cell wall N-acetylglucosamine levels. Our results suggest that rhoA plays a role in polarity, proper branching pattern, and cell wall deposition.     

Correlation of cephalosporin C synthesis and proteolytic enzymes in a differentiating culture of Acremonium chrysogenum (Cephalosporum acremonium) mutants

The effect of the growth conditions and composition of the nutrient medium on the synthesis of cephalosporin C, alkaline exoproteases and cell differentiation was studied in various strains of Acremonium chrysogenum. It was observed that the changes in the above processes occurred simultaneously and depended on the aeration rate, temperature, illumination level and concentrations of methionine and soybean meal. Close correlation between the synthesis of alkaline exoproteases, cephalosporin C and formation of secondary structures in the strains of A. chrysogenum was shown.     

The Cytotoxic Effects of Camptothecin and Mastoparan on the Unicellular Green Alga Chlamydomonas reinhardtii

We have recently reported that protease inhibitors affecting the activity of the proteasome cause necrotic cell death in Chlamydomonas reinhardtii instead of inducing apoptosis as shown for some mammalian cell lines. Therefore, we have studied other well‐known inducers of apoptosis in mammalian cells for their effects on C. reinhardtii cells. Mastoparan caused rapid cell death without a prominent lag‐phase under all growth conditions, whereas the cytotoxic effect of the topoisomerase I inhibitor camptothecin exclusively occurred during the cell‐division phase. Essentially no differences between wall‐deficient and wild‐type cells were observed with respect to dose‐response and time‐course of camptothecin and mastoparan. In cultures of the wall‐deficient strain, cell death was accompanied by swelling and subsequent disruption of the cells, established markers of necrosis. In case of the wild‐type strain, camptothecin and mastoparan caused accumulation of apparently intact, but dead cells instead of cell debris due to the presence of the wall. Both in cultures of the wall‐deficient and the wild‐type strains, cell death was accompanied by an increase of the protein concentration in the culture medium indicating a lytic process like necrosis. Taking together, we have severe doubts on the existence of an apoptotic program in case of C. reinhardtii.     

Coordinate increase of isopenicillin N synthetase, isopenicillin N epimerase and deacetoxycephalosporin C synthetase in a high cephalosporin-producing mutant of Acremonium chrysogenum and simultaneous loss of the three enzymes in a non-producing mutant

Abstract An early blocked mutant in cephalosporin biosynthesis ( Acremonium chrysogenum N₂) had simultaneously lost 3 enzymes of the cephalosporin biosynthetic pathway (isopenicillin N synthetase, isopenicillin N epimerase and deacetoxycephalosporin C synthetase) and accumulated the tripeptide α-aminoadipyl-cysteinyl-valine. An overproducing mutant ( A. chrysogenum C-10) showed a 2-fold increase in the same 3 enzymes throughout fermentation, with respect to the low-producing strain A. chrysogenum CW-19. These results suggest that expression of the genes coding for cephalosporin biosynthetic enzymes is altered in a coordinate form in these mutants.     

Expression of the transporter encoded by the cefT gene of Acremonium chrysogenum increases cephalosporin production in Penicillium chrysogenum

By introduction of the cefEF genes of Acremonium chrysogenum and the cmcH gene of Streptomyces clavuligerus, Penicillium chrysogenum can be reprogrammed to form adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (ad7-ACCCA), a carbamoylated derivate of adipoyl-7-aminodeacetoxy-cephalosporanic acid. The cefT gene of A. chrysogenum encodes a cephalosporin C transporter that belongs to the Major Facilitator Superfamily. Introduction of cefT into an ad7-ACCCA-producing P. chrysogenum strain results in an almost 2-fold increase in cephalosporin production with a concomitant decrease in penicillin by-product formation. These data suggest that cephalosporin production by recombinant P. chrysogenum strains is limited by the ability of the fungus to secrete these compounds.     

Effect of carbohydrates in the culture medium on amino acid composition of a cephalosporin C producer

The intracellular and extracellular amino acid composition of an auxotrophic methionine-deficient strain of Acremonium chrysogenum was studied in respect of the content of various carbohydrates in the fermentation medium. In the presence of glucose, an intensive involvement of exogenous DL-methionine into the cell metabolism was observed at earlier stages than in the presence of dextran or succrose. The total content of intracellular amino acids was lower in the cells grown on the medium with glucose. The production of cephalosporin C depended on the intracellular content of methionine, glutamic acid and lysine.     

Gene organization and plasticity of the β-lactam genes in different filamentous fungi

The genes pcbAB, pcbC and penDE encoding enzymes that catalyze the three steps of the penicillin biosynthesis have been cloned from Penicillium chrysogenum and Aspergillus nidulans. They are located in a cluster in Penicillium chrysogenum, Penicillium notatum, Aspergillus nidulans and Penicillium nalgiovense. The three genes are clustered in chromosome I (10.4 Mb) of P. chrysogenum, in chromosome II of P. notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. The cluster of the penicillin biosynthetic genes is amplified in strains with high level of antibiotic production. About five to six copies of the cluster are present in the AS-P-78 strain and 11 to 14 copies in the E1 strain (an industrial isolate), whereas only one copy is present in the wild type (NRRL 1951) strain and in the low producer Wis 54-1255 strain. The amplified region in strains AS-P-78 and E1 is arranged in tandem repeats of 106.5 or 57.6-kb units, respectively. In Acremonium chrysogenum the genes involved in cephalosporin biosynthesis are separated in at least two clusters. The pcbAB and pcbC genes are linked in the so-called early cluster of genes involved in the cephalosporin biosynthesis. The late cluster, which includes the cefEF and cefG genes, is involved in the last steps of cephalosporin biosynthesis. The early cluster was located in chromosome VII (4.6 Mb) in the C10 strain and the late cluster in chromosome I (2.2 Mb). Both clusters are present in a single copy in the A. chrysogenum genome, in the wild-type and in the high cephalosporin-producing C10 strains.     

Quantifying the importance of galactofuranose in Aspergillus nidulans hyphal wall surface organization by atomic force microscopy

The fungal wall mediates cell-environment interactions. Galactofuranose (Galf), the five-member ring form of galactose, has a relatively low abundance in Aspergillus walls yet is important for fungal growth and fitness. Aspergillus nidulans strains deleted for Galf biosynthesis enzymes UgeA (UDP-glucose-4-epimerase) and UgmA (UDP-galactopyranose mutase) lacked immunolocalizable Galf, had growth and sporulation defects, and had abnormal wall architecture. We used atomic force microscopy and force spectroscopy to image and quantify cell wall viscoelasticity and surface adhesion of ugeAΔ and ugmAΔ strains. We compared the results for ugeAΔ and ugmAΔ strains with the results for a wild-type strain (AAE1) and the ugeB deletion strain, which has wild-type growth and sporulation. Our results suggest that UgeA and UgmA are important for cell wall surface subunit organization and wall viscoelasticity. The ugeAΔ and ugmAΔ strains had significantly larger surface subunits and lower cell wall viscoelastic moduli than those of AAE1 or ugeBΔ hyphae. Double deletion strains (ugeAΔ ugeBΔ and ugeAΔ ugmAΔ) had more-disorganized surface subunits than single deletion strains. Changes in wall surface structure correlated with changes in its viscoelastic modulus for both fixed and living hyphae. Wild-type walls had the largest viscoelastic modulus, while the walls of the double deletion strains had the smallest. The ugmAΔ strain and particularly the ugeAΔ ugmAΔ double deletion strain were more adhesive to hydrophilic surfaces than the wild type, consistent with changes in wall viscoelasticity and surface organization. We propose that Galf is necessary for full maturation of A. nidulans walls during hyphal extension.     

Genetic alteration of pore size and other properties of the Neurospora cell wall

Trevithick, John R. (University of Wisconsin Medical School, Madison), Robert L. Metzenberg and Donald F. Costello. Genetic alteration of pore size and other properties of the Neurospora cell wall. J. Bacteriol. 92:1016-1020. 1966.-Several properties of the cell walls of wild type and the osmotic mutant of Neurospora crassa have been examined. The peameability of the isolated cell walls to polyethylene glycol and dextran polymers of different molecular weights was investigated by the volume of distribution technique. The exclusion thresholds were evaluated by a statistical treatment. The molecular weights corresponding to these thresholds for wild type and osmotic were approximately 4,750 and 18,500, respectively; these values are significantly different. The cell walls of osmotic appeared to be thinner, more easily broken, and more easily compressed to ribbonlike shapes, whereas those of wild type were tubular and strong. Chemical analysis showed that osmotic walls had roughly a 30-fold higher galactosamine-glucosamine ratio than did wild type. It is proposed that the osmotic mutant has a cell wall with abnormally large pores, and that this may account for the increased rate of egress of invertase and the decreased fractionation of light from heavy invertase in this strain.     

Two separate zones of helicoidally orientated microfibrils are present in the walls ofNitella internodes during growth

Summary The dynamic changes in microfibril architecture in the internode cell walls of the giant unicellular algaNitella translucens were studied during cell expansion. Thin section electron microscopy in conjunction with mild matrix polysaccharide extraction techniques revealed three distinct architectural zones in the walls of fully grown cells. These zones were related to distinct phases of growth by monitoring changes in cell wall architecture of internodes during active cell expansion. The initial microfibril deposition before the onset of active cell growth is helicoidal. A helicoid is a structurally complex but ordered arrangement of microfibrils that has been detected increasingly often in higher plant cell walls. During active cell elongation microfibrils are deposited transversely to the direction of cell elongation as shown in earlier studies by birefringence measurements in the polarizing microscope. The gradual decline in cell elongation corresponds with a final helicoidal deposition which continues after cell expansion ceases entirely.The continual presence of the initial helicoidal zone in the outer wall region during the whole growth process suggests that these microfibrils do not experience strain reorientation and are continually reorganized, or maintained, in a well ordered helicoidal arrangement.     

Pathways of the synthesis of glutamic acid by cephalosporin C-producing Acremonium chrysogenum

There were observed two pathways of glutamic acid formation in two strains of Acremonium chrysogenum differing in the production levels of cephalosporin C. The pathway involving glutamate dehydrogenase is known. The other pathway involved amination catalyzed by glutamine synthetase. Activity of both the enzymes during intensive synthesis of the antibiotic was higher in the highly productive strain. Under conditions of limited nitrogen content in the medium production of glutamate during the antibiotic biosynthesis depended on glutamine synthetase. When there was an excess of nitrogen in the medium the main role in production of glutamic acid at the phase of cephalosporin synthesis was played by the other enzyme i. e. glutamate dehydrogenase. By the dynamics the curve of the glutamate dehydrogenase activity correlated with that of the antibiotic production.     

Autolytic enzyme-deficient mutants of Bacillus subtilis 168.

Mutants of Bacillus subtilis strain 168 have been isolated that are at least 90 to 95% deficient in the autolytic enzymes N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase. These mutants grow at normal rates as very long chains of unseparated cells. The length of the chains is directly related to the growth rates. They are nonmotile and have no flagella, but otherwise appear to have normal cell morphology. Their walls are fully sysceptible to enzymes formed by the wild type and have the same chemical composition as the latter. Cell wall preparations from the mutants lyse at about 10% of the rate of those from the isogenic wild type, with the correspondingly small liberation of both the amino groups of alanine at pH 8.0 and of reducing groups at pH 5.6. Likewise, Microcococcus luteus walls at pH 5.6 and B. subtilis walls at pH 8 are lysed only very slowly by LiCl extracts made from the mutants as compared with rates obtained with wild-type extracts. Thus, the activity of both autolytic enzymes in the mutants is depressed. The frequencies of transformation, the isolation of revertants, and observations with a temperature-sensitive mutant all point to the likelihood that the pleiotropic, phenotypic properties of the strains are due to a single mutation. The mutants did not produce more protease or amylase than did the wild type. They sporulate and the spores germinate normally. The addition of antibiotics to exponentially growing cultures prevents wall synthesis but leads to less lysis than is obtained with the wild type. The bacteriophage PBSX can be induced in the mutants by treatment with mitomycin C.     

ISOLATION AND CHARACTERIZATION OF A CELL WALL‐DEFECTIVE MUTANT OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1

Cell wall–defective strains of Chlamydomonas have played an important role in the development of transformation protocols for introducing exogenous DNA (foreign genes or cloned Chlamydomonas genes) into C. reinhardtii. To promote the development of similar protocols for transformation of the distantly related homothallic species, C. monoica, we used UV mutagenesis to obtain a mutant strain with a defective cell wall. The mutant, cw‐1, was first identified on the basis of irregular colony shape and was subsequently shown to have reduced plating efficiency and increased sensitivity to lysis by a non‐ionic detergent as compared with wild‐type cells. Tetrad analysis of crosses involving the cw‐1 mutant confirmed 2:2 segregation of the cw:cw⁺ phenotypes, indicating that the wall defect resulted from mutation of a single nuclear gene. The phenotype showed incomplete penetrance and variable expressivity. Although some cells had apparently normal cell walls as viewed by TEM, many cells of the cw‐1 strain had broken cell walls and others were protoplasts completely devoid of a cell wall. Several cw‐1 isolates obtained from crosses involving the original mutant strain showed a marked enhancement of the mutant phenotype and may prove especially useful for future work involving somatic cell fusions or development of transformation protocols.     

Targeted inactivation of the mecB gene, encoding cystathionine-gamma-lyase, shows that the reverse transsulfuration pathway is required for high-level cephalosporin biosynthesis in Acremonium chrysogenum C10 but not for methionine induction of the cephalosporin genes

Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of the mecB gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysis and no cystathionine-gamma-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH(2)) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-gamma-lyase.