The patterns of extracellular protein formation by spontaneously-occurring rifampicin-resistant mutants of Staphylococcus aureus

Spontaneously-occurring rifampicin-resistant mutants of Staphylococcus aureus were isolated on 4% (w/v) Tryptone Soya Agar containing 4 and 40 times the m.i.c. for rifampicin. A number of colonies were selected at each rifampicin concentration and were grown aerobically in 3% (w/v) Tryptone Soya Broth for 24 h at 37 degrees C. In the case of S. aureus RN4220 all the mutants grew to bacterial densities up to approximately 1.7 times more than the parent organism. The corresponding levels of extracellular protein secretion varied over a 5-fold range, all the mutants being less productive than the parent. By contrast, mutants of the wild-type Wood 46 strain achieved bacterial densities of only 45-83% that of the parent whilst exoprotein secretion showed a smaller 1.7-fold variation. However, widely-differing patterns of exoproteins were revealed by SDS-polyacrylamide gel electrophoresis of the parent and mutant organisms of both strains.     

The use of cellulases from a beta-glucosidase-hyperproducing mutant of Trichoderma reesei in simultaneous saccharification and fermentation of wheat straw

Conidia of the cellulolytic strain Trichoderma reesei F522 were mutagenized with UV irradiation and N-methyl|-N'-nitro-N-nitrosoguanidine (NTG). A visual agar plate detection system was developed, using esculin and ferric ions, to identify mutants of T. reesei with increased beta-glucosidase activity. Selected mutants were tested for production of extracellular cellulases in shake flasks on autohydrolyzed wheat straw as carbon source. The most active mutant V-7 showed about 6-times higher activity of beta-glucosidase than the parent strain F-522, whereas the filter paper degrading and endo-1,4-beta-D-glucanase activities increased by 45% and by almost 31%, respectively. Cellulase preparations obtained from the parent and mutant strains were then used along with Kluyveromyces fragilis cells for ethanol production from ethanol-alkali pulped straw in the simultaneous saccharification and fermentation (SSF) process. From 10% (w/v) of straw pulp (dry matter), 2.5% (w/v) ethanol was obtained at 43 degrees C after 48 h using cellulase derived from the parent strain of T. reesei. When the beta-glucosidase-hyperproducing mutant V-7 was employed, the ethanol yield in the SSF process increased to 3.4% (w/v), the reaction time was shortened to 24 h and no cellobiose was detected in straw hydrolyzates.     

Elevated 3-hydroxypropionaldehyde production from glycerol using a Citrobacter freundii mutant

A mutant strain of Citrobacter freundii capable of elevated 3-hydroxypropionaldehyde production from glycerol was isolated using chemical mutagenesis and a screening protocol. The protocol involved screening mutagenized bacterial cells on solid minimal medium containing 5 % (v/v) glycerol. Colonies were picked onto duplicate solid minimal medium plates and one plate was stained with 1 % (w/v) phloroglucinol. Those colonies staining red were further screened and a mutant, HPAO-1, was identified. The mutant strain produced a several-fold higher 3-hydroxypropionaldehyde concentration than did the parent strain when grown on 5 % (v/v) glycerol. The ratio of culture volume to flask volume influenced 3-hydroxypropionaldehyde production by the mutant cells compared to the parent cells. Aldehyde production was highest when the mutant strain was grown on 5 % (v/v) glycerol at a ratio of culture volume to flask volume of 1:3 or 1:12.5.     

Yeast mutants with enhanced ability to secrete human lysozyme: Isolation and identification of a protease-deficient mutant

Summary Yeast mutant strains which secrete large amounts of human lysozyme were screened using an agar medium containing bacterial cells. Nine mutants secreted over 10 times more lysozyme than the wild-type parent strain. The mRNA levels for lysozyme in the mutants were not higher than that of the wild-type strain. Three of the mutant strains were deficient in carboxypeptidase Y activity. It was found that the protease deficiency was caused by a deficiency in conversion of proenzyme to mature enzyme in ssl1 mutant cells. The ssl1 gene was found to be closely linked to the centromere and determine both the efficiency of secretion of lysozyme and the processing of carboxypeptidase Y.Abbreviations CPY carboxypeptidase Y (yscY) - HLY a synthetic gene for human lysozyme     

The effect of glucose on the expression of extracellular protein genes by Staphylococcus aureus strain V8

The bacteria from overnight cultures (20 h) of S. aureus V8 and exp negative mutant K6812-1, grown, aerobically, in 3% (w/v) Tryptone Soya Broth, at 37 degrees C, were resuspended in fresh medium, in the case of the parent strain +/- 1% (w/v) glucose, without change in bacterial density. During a 6 h incubation period there was an approximate doubling of bacterial density, to the same level, in each case. However, exoprotein production by the mutant was only 20% that of the parent whilst the addition of glucose to the V8 strain resulted in a tenfold reduction in the exoprotein formed. SDS-polyacrylamide gel electrophoresis showed that the exoprotein patterns of both organisms after 6 h incubation were the same as those observed in the overnight cultures whilst the presence of 1% (w/v) extra glucose changed the pattern produced by the parent to one similar to that of the mutant. The results showed that conditions which lead to the rapid formation of glucose catabolites produced an effect consistent with the inhibition of the activity of the exp gene product.     

Localization of proteolytic enzymes in cells of Bacillus intermedius

The activities of proteinases in the culture fluid and cellular fractions of Bacillus intermedius 3-19 grown under various conditions were studied. Thiol-dependent serine proteinase was the prevalent enzyme in the total pool of extracellular proteinases (70%); its catalytically active form was also detected in the cell membrane and, during active enzyme production, in the cell wall. Another enzyme, glutamyl endopeptidase (10% of the total pool), was detected in the cell membrane; it was also found in the cell wall and cytoplasm during active enzyme secretion into the growth medium. Production of these enzymes was maximal on medium containing inorganic phosphate and gelatin and decreased 2- to 4-fold on medium with glucose and lactate. The level of activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of CoCl2 (2 mM) into the medium caused an essential increase in extracellular glutamyl endopeptidase activity and promoted the release of the membrane-bound enzyme into the culture fluid. Proteolytic activity towards casein was also detected in the cytoplasm. The proteinases localized in the cytoplasm were shown to differ in their properties from those secreted.     

Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004

An extracellular protease was produced under stress conditions of high temperature and high salinity by a newly isolated moderate halophile, Salinivibrio sp. strain AF-2004 in a basal medium containing peptone, beef extract, glucose and NaCl. A modification of Kunitz method was used for protease assay. The isolate was capable of producing protease in the presence of sodium chloride, sodium sulfate, sodium nitrate, sodium nitrite, potassium chloride, sodium acetate and sodium citrate. The maximum protease was secreted in the presence of 7.5 to 10% (w/v) sodium sulfate or 3% (w/v) sodium acetate (4.6 U ml⁻¹). Various carbon sources including glucose, lactose, casein and peptone were capable of inducing enzyme production. The optimum pH, temperature and aeration for enzyme production were 9.0, 32 °C and 220 rpm, respectively. The enzyme production corresponded with growth and reached a maximum level during the mid-stationary phase. Maximum protease activity was exhibited in the medium containing 1% (w/v) NaCl at 60 °C, with 18% and 41% activity reductions at temperature 50 and 70 °C, respectively. The optimum pH for enzyme activity was 8.5, with 86% and 75% residual activities at pH 10 and 6, respectively. The activity of enzyme was inhibited by EDTA. These results suggest that the protease secreted by Salinivibrio sp. strain AF-2004 is industrially important from the perspectives of its activity at a broad pH ranges (5.0–10.0), its moderate thermoactivity in addition to its high tolerance to a wide range of salt concentration (0–10% NaCl).     

Comparison of Alkaline Proteinase from Hyperproductive Mutants and Parent Strain of Aspergillus sojae

Physico-chemical properties of alkaline proteinase from the parent strain were compared with those from hyperproductive mutants of Aspergillus sojae. All the results on behavior of enzyme protein to ion exchange resin and celluloses, gel filtration, ultracentrifugal sedimentation, disc electrophoresis and isoelectrofocusing on polyacrylamide gel column, specific activity, substrate specificity, and kinetic constants provided evidence in favor of the conclusion that the parent and mutant strains produced the chemically identical enzymes and that superactivity of alkaline proteinase in culture extracts or filtrates of mutant strains was not attributed to alteration of catalytic property of the enzyme, but to hyperproduction of the identical enzyme resulting from the genetic change in the regulatory mechanism of enzyme synthesis.     

Culture Conditions for the Production of Alkaline Protease from n-Paraffins by a Kabicidin Resistant Mutant No. 5–128B

A novel process for the microbial production of alkaline protease on an industrial scale was successfully established by using a kabicidin resistant mutant, No. 5–128B, derived from Fusarium sp. S–19–5. The most suitable carbon source for producing alkaline protease was n-paraffins (C₁₀~C₁₄) and the effective nitrogen source was dried-yeast cells containing no nucleic acid, the optimum concentrations being 12.5% (w/v) and 7.0% (w/v), respectively. The optimal temperature and initial pH for protease production were 24°C and 6.0, respectively. Under the optimal conditions using a shaker flask mutant No. 5–128B produced 41000 PU/ml of alkaline protease, which corresponded to about 10 times the amount produced by the parent strain. The relation between the high ability to produce alkaline protease and the resistance to kabicidin, a polyene antibiotic, is discussed.     

The cellular location of proteolytic enzymes ofBacillus intermedius

The activities of proteinases in the culture fluid and cellular fractions ofBacillus intermedius 3–19 grown under various conditions were studied. Thiol-dependent serine proteinase was the prevalent enzyme in the total pool of extracellular proteinases (70%); its catalytically active form was also detected in the cell membrane and, during active enzyme production, in the cell wall. Another enzyme, glutamyl endopeptidase (10% of the total pool), was detected in the cell membrane; it was also found in the cell wall and cytoplasm during active enzyme secretion into the growth medium. The production of these enzymes was maximal on medium containing inorganic phosphate and gelatin and decreased 2-to 4-fold on medium with glucose and lactate. The level of activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of C0Cl₂ (2 mM) into the medium caused an essential increase in extracellular glutamyl endopeptidase activity and promoted the release of the membrane-bound enzyme into the culture fluid. Proteolytic activity towards casein was also detected in the cytoplasm. The proteinases localized in the cytoplasm were shown to differ in their properties from those secreted.     

Identification of an endo-beta-1,4-D-xylanase from Magnaporthe grisea by gene knockout analysis, purification, and heterologous expression

Magnaporthe grisea, a destructive ascomycetous pathogen of rice, secretes cell wall-degrading enzymes into a culture medium containing purified rice cell walls as the sole carbon source. From M. grisea grown under the culture conditions described here, we have identified an expressed sequenced tag, XYL-6, a gene that is also expressed in M. grisea-infected rice leaves 24 h postinoculation with conidia. This gene encodes a protein about 65% similar to endo-beta-1,4-D-glycanases within glycoside hydrolase family GH10. A M. grisea knockout mutant for XYL-6 was created, and it was shown to be as virulent as the parent strain in infecting the rice host. The proteins secreted by the parent strain and by the xyl-6Delta mutant were each fractionated by liquid chromatography, and the collected fractions were assayed for endo-beta-1,4-D-glucanase or endo-beta-1,4-D-xylanase activities. Two protein-containing peaks with endo-beta-1,4-D-xylanase activity secreted by the parent strain are not detectable in the column eluant of the proteins secreted by the mutant. The two endoxylanases (XYL-6alpha and XYL-6beta) from the parent were each purified to homogeneity. N-terminal amino acid sequencing indicated that XYL-6alpha is a fragment of XYL-6beta and that XYL-6beta is identical to the deduced protein sequence encoded by the XYL-6 gene. Finally, XYL-6 was introduced into Pichia pastoris for heterologous expression, which resulted in the purification of a fusion protein, XYL-6H, from the Pichia pastoris culture filtrate. XYL-6H is active in cleaving arabinoxylan. These experiments unequivocally established that the XYL-6 gene encodes a secreted endo-beta-1,4-D-xylanase.     

Improved ethanol tolerance and production in strains of Clostridium thermocellum

Two Clostridium thermocellum strains were improved for ethanol tolerance, to 5% (v/v), by gradual adaptation and mutation. The best mutant gave an ethanol yield of 0.37 g/g substrate, with a growth yield 1.5 times more than its parent. Accumulation of acids and reducing sugars by the mutant strain with 5% (v/v) ethanol was lower than that of the parent strain with 1.5% (v/v) ethanol.     

YscP of Yersinia pestis is a secreted component of the Yop secretion system

The Yersinia pestis low-Ca2+ response stimulon is responsible for the environmentally regulated expression and secretion of antihost proteins (V antigen and Yops). We have previously shown that yscO encodes a secreted core component of the Yop secretion (Ysc) mechanism. In this study, we constructed and characterized in-frame deletions in the adjacent gene, yscP, in the yscN-yscU operon. The DeltaP1 mutation, which removed amino acids 246 to 333 of YscP, had no effect on Yop expression or secretion, and the mutant protein, YscP1, was secreted, as was YscP in the parent. In contrast, the DeltaP2 strain expressed and secreted less of each Yop than did the parent under the inductive conditions of 37 degrees C and the absence of Ca2+, with an exception being YopE, which was only minimally affected by the mutation. The YscP2 protein, missing amino acids 57 to 324 of YscP, was expressed but not secreted by the DeltaP2 mutant. The effect of the DeltaP2 mutation was at the level of Yop secretion because YopM and V antigen still showed limited secretion when overproduced in trans. Excess YscP also affected secretion: overexpression of YscP in the parent, in either yscP mutant, or in an lcrG mutant effectively shut off secretion. However, co-overexpression of YscO and YscP had no effect on secretion, and YscP overexpression in an lcrE mutant had little effect on Yop secretion, suggesting that YscP acts, in conjunction with YscO, at the level of secretion control of LcrE at the bacterial surface. These findings place YscP among the growing family of mobile Ysc components that both affect secretion and themselves are secreted by the Ysc.     

Biochemical and mutational analysis of a gingipain-like peptidase activity from Prevotella ruminicola B(1)4 and its role in ammonia production by ruminal bacteria.

A chemical mutagenesis protocol was used with the ruminal bacterium Prevotella ruminicola strain B(1)4 to generate mutant strains defective in peptidase activity. Compared with the wild-type parent strain, the isolated mutants possessed 1/10 of the enzyme activity responsible for cleavage of glycine-arginine-4-methoxy-beta-naphthylamide (Gly-Arg-MNA). A concomitant loss in activity against arginine-arginine-4-methoxy-beta-naphthylamide (Arg-Arg-MNA) was also observed. Both activities were similarly affected by various proteinase inhibitors, suggesting that the same enzyme is responsible for the Arg-Arg-MNA peptidase and Gly-Arg-MNA peptidase activities. Growth rates of wild-type and mutant strains grown in batch culture with various nitrogen sources did not differ. However, a role for the Gly-Arg-MNA peptidase activity was demonstrated in coculture experiments with gram-positive, ammonia-producing ruminal bacteria. The rate and extent of ammonia production were reduced by approximately 25% in cocultures containing the mutants when compared with that of wild-type-containing cultures. These reductions could not be accounted for simply by the decrease in ammonia production by the mutant strain alone. To our knowledge, this paper reports the first successful use of chemical mutagenesis with ruminal microorganisms.     

Characterization of a pyoverdine-deficient mutant of Pseudomonas fluorescens impaired in the secretion of extracellular lipase

A mutant of Pseudomonas fluorescens strain B52 deficient in the synthesis of the fluorescent pigment, pyoverdine, was isolated. Absence of pyoverdine and other siderophores was confirmed by gel filtration, a specific siderophore assay, and inhibition studies with the iron chelator EDDA. Both parent and mutant synthesized additional outer membrane proteins in response to iron-limitation. Mutant cells cultured in the absence of iron(III) accumulated ⁵⁵Fe-labeled pyoverdine. The mutant produced extracellular proteinase normally on various media, but was deficient in lipase secretion. Growth of the mutant with partially-purified pyoverdine resulted in a 2.5-fold stimulation of lipase secretion. The mutant grew poorly in deferrated medium; however, the addition of iron(III) stimulated growth. Proteinase secretion in deferrated medium was stimulated over a narrow range of iron(III) concentration, while lipase secretion was only slightly affected. The data suggest that separate regulatory mechanisms exist for the control of proteinase and lipase secretion by iron(III).Contribution No. 768 from the Food Research Centre     

Efficient recovery of secretory recombinant proteins from protease negative mutant Escherichia coli strains

Osmotic shock and lysozyme/EDTA methods were used to recover secreted recombinant proteins from protease negative mutant strains of E. coli. Up to 80% of protein A--lactamase fusion protein was recovered from protease negative mutants by simple osmotic shock. Fractionation by lysozyme/EDTA treatment, increased the recovery of protein A--lactamase fusion protein from the mutant strain up to 93%. Mild fractionation condition allowed efficient recovery of secreted protein from protease negative mutant strains, but not from the parent strain possessing proteases. © Rapid Science Ltd. 1998     

Isolation and characterization of a mutant of Neisseria gonorrhoeae that is defective in the uptake of iron from transferrin and haemoglobin and is avirulent in mouse subcutaneous chambers

Iron-uptake mutants of Neisseria gonorrhoeae strain 340 were obtained following treatment with streptonigrin, and one such mutant (Fud14) was characterized. N. gonorrhoeae strain Fud14 was unable to grow with human transferrin or haemoglobin as the sole source of iron, but grew normally with heat-inactivated normal human serum or haemin. Internalization of 55Fe from transferrin by strain Fud14 was only 25% of the parent level. Strain Fud14 (less than or equal to 1 x 10(8) c.f.u.) did not grow in subcutaneous chambers implanted in mice, whereas the parent strain was infective at an ID50 of 4.3 x 10(1) c.f.u. Supplementation of chambers with either normal human serum or haemin resulted in the establishment of strain Fud14 in vivo for at least 240 h post-inoculation. Electroporation of Fud14 with wild-type DNA and selection for growth on medium containing human transferrin resulted in a recombinant (Fud15) that was capable of utilizing haemoglobin, and was virulent in mice. These results suggest that a gonococcal strain defective in the ability to utilize in vivo iron sources is not capable of survival in vivo.     

A Mucor pusillus mutant defective in asparagine-linked glycosylation.

A Mucor pusillus mutant defective in asparagine-linked glycosylation was found in our stock cultures. This mutant, designated 1116, secreted aspartic proteinase (MPP) in a less-glycosylated form than that secreted by the wild-type strain. Analysis of enzyme susceptibility, lectin binding, and carbohydrate composition indicated that this mutant secreted three glycoforms of MPPs, one of which contained no carbohydrate; the other two had truncated asparagine-linked oligosaccharide chains such as Man0-1GlcNAc2. Further analysis using oligosaccharide processing inhibitors, such as castanospermine, 1-deoxynojirimycin and N-methyldeoxynojirimycin, suggested that MPPs in the mutant were glycosylated through a transfer of the truncated lipid-linked oligosaccharides, Man0-1GlcNAc2, to the MPP protein but not through an aberrant processing. In addition, genetic studies with forced primary heterokaryons indicated that the mutation in strain 1116 was recessive.     

Butanol production by hypersolvent-producing mutant Clostridium beijerinckii BA101 in corn steep water medium containing maltodextrin

Clostridium beijerinckii NCIMB 8052 parent strain and BA101, a hypersolvent-producing mutant, fermented 6% (w/v) glucose, maltodextrin, maltose or xylose in a medium containing corn steep water (CSW) to produce butanol. Batch fermentation in an unoptimized 6% (w/v) maltodextrin plus 1.6% solids CSW medium demonstrated that C. beijerinckii NCIMB 8052 and BA101 produced 10.7 g butanol/L and 14.5 g butanol/L, respectively.     

The epiphytic lichen Evernia prunastri synthesizes a secretable cellulase system that degrades crystalline cellulose

Evernia prunastri (L.) Ach. produces a cellulase system capable of solubilizing crystalline cellulose. The enzyme is secreted into the incubation medium in response to the presence of 0.5% (w/v) cellobiose. Cellulase secretion is pH dependent, the maximum occurring at an initial pH of 9.0, which is rapidly decreased by the action of the lichen. The effect of cellobiose on the cellulase system is inhibited by cycloheximide and 8-azaguanine. The cellulasc activity increases with the metabolic reactivation that is initiated by thallus hydration. Several functions of cellulase in this lichen are discussed.