Control of phosphatidylserine synthase II activity in Chinese hamster ovary cells.

Phosphatidylserine (PtdSer) in Chinese hamster ovary (CHO) cells is synthesized through the action of PtdSer synthase (PSS) I and II, which catalyzes the exchange of L-serine with the base moiety of phosphatidylcholine and phosphatidylethanolamine, respectively. The PtdSer synthesis in a CHO cell mutant, PSA-3, which lacks PSS I but has normal PSS II activity, was almost completely inhibited by the addition of PtdSer to the culture medium, like that in the wild-type CHO-K1 cells. In contrast, the PtdSer synthesis in a PSS II-overproducing stable transformant of CHO-K1, K1/wt-pssB, was reduced by only 35% upon addition of PtdSer. The serine exchange activity in a membrane fraction of K1/wt-pssB cells was not inhibited by PtdSer at all, whereas those of PSA-3 and CHO-K1 cells were inhibited by >95%. These results indicated that PSS II activity in PSA-3 and CHO-K1 cells is inhibited by exogenous PtdSer and that overproduction of PSS II leads to the loss of normal control of PSS II activity by exogenous PtdSer. Although overproduced PSS II in K1/wt-pssB cells was not normally controlled by exogenous PtdSer, K1/wt-pssB cells cultivated without exogenous PtdSer exhibited a normal PtdSer biosynthetic rate similar to that in CHO-K1 cells. In contrast to K1/wt-pssB cells, another stable transformant of CHO-K1, K1/R97K-pssB, which overproduces R97K mutant PSS II, exhibited a approximately 4-fold higher PtdSer biosynthetic rate compared with that in CHO-K1 cells. These results suggested that for maintenance of a normal PtdSer biosynthetic rate, the activity of overproduced wild-type PSS II in K1/wt-pssB cells is depressed by an as yet unknown post-translational mechanisms other than those for the exogenous PtdSer-mediated inhibition and that Arg-97 of PSS II is critical for this depression of overproduced PSS II activity. When the cDNA-directed wild-type and R97K mutant PSS II activities were expressed at nonoverproduction levels in a PSS I- and PSS II-defective mutant of CHO-K1 cells, expression of the mutant PSS II activity but not that of the wild-type PSS II activity induced the PtdSer-resistant PtdSer biosynthesis. This suggested that Arg-97 of PSS II is critical also for the exogenous PtdSer-mediated inhibition of PSS II.     

Properties of a Tn5 insertion mutant defective in the structural gene (fruA) of the fructose-specific phosphotransferase system of Rhodobacter capsulatus and cloning of the fru regulon.

In photosynthetic bacteria such as members of the genera Rhodospirillum, Rhodopseudomonas, and Rhodobacter a single sugar, fructose, is transported by the phosphotransferase system-catalyzed group translocation mechanism. Previous studies indicated that syntheses of the three fructose catabolic enzymes, the integral membrane enzyme II, the peripheral membrane enzyme I, and the soluble fructose-1-phosphate kinase, are coordinately induced. To characterize the genetic apparatus encoding these enzymes, a Tn5 insertion mutation specifically resulting in a fructose-negative, glucose-positive phenotype was isolated in Rhodobacter capsulatus. The mutant was totally lacking in fructose fermentation, fructose uptake in vivo, phosphoenolpyruvate-dependent fructose phosphorylation in vitro, and fructose 1-phosphate-dependent fructose transphosphorylation in vitro. Extraction of the membrane fraction of wild-type cells with butanol and urea resulted in the preparation of active enzyme II free of contaminating enzyme I activity. This preparation was used to show that the activity of enzyme I was entirely membrane associated in the parent but largely soluble in the mutant, suggesting the presence of an enzyme I-enzyme II complex in the membranes of wild-type cells. The uninduced mutant exhibited measurable activities of both enzyme I and fructose-1-phosphate kinase, which were increased threefold when it was grown in the presence of fructose. Both activities were about 100-fold inducible in the parental strain. Although the Tn5 insertion mutation was polar on enzyme I expression, fructose-1-phosphate kinase activity was enhanced, relative to the parental strain. ATP-dependent fructokinase activity was low, but twofold inducible and comparable in the two strains. A second fru::Tn5 mutant and a chemically induced mutant selected on the basis of xylitol resistance showed pleiotropic loss of enzyme I, enzyme II, and fructose-1-phosphate kinase. These mutants were used to clone the fru regulon by complementing the negative phenotype with a wild-type cosmid bank.     

Isolation and properties of Bacillus brevis mutants unable to produce tyrocidine.

Mutants of Bacillus brevis ATCC 10068 were isolated which produced less than 1/100 of the amount of tyrocidine produced by the parent strain. These mutants produced spores at the same frequency and which were as resistant to heating at 80 degrees C for up to 3 h as were those produced by the parent strain. A partially purified tyrocidine synthetase from strain ATCC 10068 catalyzed [32P]PPi-ATP exchange reactions dependent on added tyrocidine-constituent amino acids. These activities were separated into three groups (I, II, and III) by fractionation on an Ultrogel AcA34 column. Each group was similar to one of the three components (heavy, intermediate, and light, respectively) found previously for strain ATCC 8185 except that glutamate-dependent activity was not detected in the group I activities and some amino acyl-tRNA synthetase activities were associated with the group III activities. Some of the mutants were shown to have defective tyrocidine synthetase enzymes. Mutant BH30 was defective in two of the group II amino acid-dependent [32P]PPi-ATP exchange reactions, mutant BH16 was defective in one of the group I and one of the group II reactions, and mutant BH34 had alterations to activities in all of the groups. It is unlikely that any of these mutants could synthesise tyrocidine. We conclude that tyrocidine is not involved in either the sporulation process or the resistance of spores of B. brevis ATCC 10068 to heating at 80 degrees C for up to 3 h.     

RecBC enzyme activity is required for far-UV induced respiration shutoff in Escherichia coli K12

Shutoff of respiration is one of a number of recA+ lexA+ dependent (SOS) responses caused by far ultraviolet (245 nm) radiation (UV) damage of DNA in Escherichia coli cells. Thus far no rec/lex response has been shown to require the recB recC gene product, the RecBC enzyme. We report in this paper that UV-induced respiration shutoff did not occur in either of these radiation-sensitive derivatives of K12 strain AB1157 nor in the recB recC double mutant. The sbcB gene product is exonuclease I and it has been reported that the triple mutant strain recB recC sbcB has near normal recombination efficiency and resistance to UV. The sbcB strain shut off its respiration after UV but the triple mutant did not show UV-induced respiration shutoff; the shutoff and death responses were uncoupled. We concluded that respiration shutoff requires RecBC enzyme activity. The RecBC enzyme has ATP-dependent double-strand exonuclease activity, helicase activity and several other activities. We tested a recBC+ (double dagger) mutant strain (recC 1010) that had normal recombination efficiency and resistance to UV but which possessed no ATP-dependent double-strand exonuclease activity. This strain did not shut off its respiration. The presence or absence of other RecBC enzyme activities in this mutant is not known. These results support the hypothesis that ATP-dependent double-strand exonuclease activity is necessary for UV-induced respiration shutoff.     

The respiratory chain complex thresholds in mitochondria of a Drosophila subobscura mutant strain

Analysis of a mutant strain of Drosophila subobscura revealed that most (80%) mitochondrial genomes have undergone a large scale deletion (5 kb) in the coding region. Compared with the wild-type strain, complex I and III activities are, respectively, reduced by 50% and 30% in the mutant. However, the ATP synthesis capacities remain unchanged. In order to elucidate how the ATP synthesis is maintained at a normal level, despite a significant decrease in complex I and III activities, we progressively inhibited respiratory chain complex activities, respiration rate and ATP synthesis. Complex I, III and IV activities were inhibited by rotenone, antimycin and KCN, respectively. Threshold curves were thus determined for each complex. Our results demonstrated that in the mutant strain, both mitochondrial respiration and ATP synthesis had decreased when complex I activity was inhibited by more than 20%, whereas 70% inhibition is required to induce similar changes in the wild-type. The complex I inhibition pattern of the wild-type was restored by a backcross (mutant female/wild-type male). The complex III activity threshold is below 20% in both strains, and we observed some difference in antimycin sensitivity, suggesting a modification of the complex enzymatic properties in the mutant. In contrast, threshold values of 70% were measured for complex IV inhibition. Our data suggest that the difference in the complex I threshold curves between the wild-type and mutant strains could partially account for the absence of pathological phenotype in the mutant.     

ilvB-encoded acetolactate synthase is resistant to the herbicide sulfometuron methyl.

The herbicide sulfometuron methyl is a potent inhibitor of the branched-chain amino acid biosynthetic enzyme acetolactate synthase (ALS) isolated from bacteria, fungi, and plants. However, it did not prevent growth of wild-type Salmonella typhimurium LT2 or Escherichia coli K-12. These species each contain two acetolactate synthase isozymes. Growth of S. typhimurium and E. coli mutants lacking ALS I was prevented by the herbicide, suggesting that activity of the remaining ALS isoenzyme (II or III, respectively) was stopped by sulfometuron methyl. Synthesis of ALS I requires either an relA function or an elevated cyclic AMP level. A relA mutant of S. typhimurium was inhibited by sulfometuron methyl on rich carbon sources that display a basal cyclic AMP level but not on poor carbon sources where the cyclic AMP concentration is elevated. When L-valine, which allosterically inhibits ALS I activity, was added, growth retardation of the relA- strain by sulfometuron methyl was observed on both poor and rich carbon sources. Enzymological analyses indicated that ALS I activities derived from both species were resistant to the herbicide. In contrast, activities of S. typhimurium ALS II and E. coli ALS III were abolished by sulfometuron methyl.     

Human ATP:Cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase

The final step in the conversion of vitamin B(12) into coenzyme B(12) (adenosylcobalamin, AdoCbl) is catalyzed by ATP:cob(I)alamin adenosyltransferase (ATR). Prior studies identified the human ATR and showed that defects in its encoding gene underlie cblB methylmalonic aciduria. Here two common polymorphic variants of the ATR that are found in normal individuals are expressed in Escherichia coli, purified, and partially characterized. The specific activities of ATR variants 239K and 239M were 220 and 190 nmol min(-1) mg(-1), and their K(m) values were 6.3 and 6.9 mum for ATP and 1.2 and 1.6 mum for cob(I)alamin, respectively. These values are similar to those obtained for previously studied bacterial ATRs indicating that both human variants have sufficient activity to mediate AdoCbl synthesis in vivo. Investigations also showed that purified recombinant human methionine synthase reductase (MSR) in combination with purified ATR can convert cob(II)alamin to AdoCbl in vitro. In this system, MSR reduced cob(II)alamin to cob(I)alamin that was adenosylated to AdoCbl by ATR. The optimal stoichiometry for this reaction was approximately 4 MSR/ATR and results indicated that MSR and ATR physically interacted in such a way that the highly reactive reaction intermediate [cob(I)alamin] was sequestered. The finding that MSR reduced cob(II)alamin to cob(I)alamin for AdoCbl synthesis (in conjunction with the prior finding that MSR reduced cob(II)alamin for the activation of methionine synthase) indicates a dual physiological role for MSR.     

ATPase activity of RecD is essential for growth of the Antarctic Pseudomonas syringae Lz4W at low temperature

RecD is essential for growth at low temperature in the Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W. To examine the essential nature of its activity, we analyzed wild-type and mutant RecD proteins with substitutions of important residues in each of the seven conserved helicase motifs. The wild-type RecD displayed DNA-dependent ATPase and helicase activity in vitro, with the ability to unwind short DNA duplexes containing only 5' overhangs or forked ends. Five of the mutant proteins, K229Q (in motif I), D323N and E324Q (in motif II), Q354E (in motif III) and R660A (in motif VI) completely lost both ATPase and helicase activities. Three other mutants, T259A in motif Ia, R419A in motif IV and E633Q in motif V exhibited various degrees of reduction in ATPase activity, but had no helicase activity. While all RecD proteins had DNA-binding activity, the mutants of motifs IV and V displayed reduced binding, and the motif II mutant showed a higher degree of binding to ssDNA. Significantly, only RecD variants with in vitro ATPase activity could complement the cold-sensitive growth of a recD-inactivated strain of P. syringae at 4 degrees C. These results suggest that the requirement for RecD at lower temperatures lies in its ATP-hydrolyzing activity.     

Pleiotropic hydrogenase mutants of Escherichia coli K12: growth in the presence of nickel can restore hydrogenase activity

Anaerobic growth in the presence of 0.6 mM NiCl2 was able to restore hydrogenase and benzyl-viologen-linked formate dehydrogenase activities to a mutant (FD12), which is normally defective in these activities. This mutant carries a mutation located near minute 58 in the genome. Hydrogenase isoenzyme I and II activities were restored along with the hydrogenase activity that forms part of the formate hydrogen lyase system. A plasmid (pRW1) was constructed, containing a 4.8 kb chromosomal DNA insert, which was able to complement the lesion in mutant FD12. Further mutants with mutations near 58 minutes on the chromosome, and which lacked hydrogenase and formate dehydrogenase activities were isolated. These mutants were divided into three groups. Class I mutants were restored to the wild-type phenotype either by growth with 0.6 mM NiCl2 or following transformation with pRW1. Class II mutants were also complemented by pRW1 but were unaffected by growth with NiCl2. Class III mutants were unaffected by both pRW1 and growth with NiCl2. The cloned 4.8 kb fragment of chromosomal DNA therefore encodes two genes essential for hydrogenase activity. Restriction analysis indicates that the cloned DNA is the same as a fragment that has previously been cloned and which complements the hydB locus (Sankar et al. (1985) J. Bacteriol., 162, 353-360). None of the three classes of mutants possess mutations in hydrogenase structural genes.     

Insertional inactivation of the gene encoding subunit II of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 carries out oxygenic photosynthesis analogous to higher plants. Its photosystem I contains seven different polypeptide subunits. The cartridge mutagenesis technique was used to inactivate the psaD gene which encodes subunit II of photosystem I. A mutant strain lacking subunit II was generated by transforming wild type cells with cloned DNA in which psaD gene was interrupted by a gene conferring kanamycin resistance. The photoautotrophic growth of mutant strain is much slower than that of wild type cells. The membranes prepared from mutant cells lack subunit II of photosystem I. Studies on the purified photosystem I reaction center revealed that the complex lacking subunit II is assembled and is functional in P700 photooxidation but at much reduced rate. Therefore, subunit II of photosystem I is required for efficient function of photosystem I.     

Tryptophan Analog Resistance Mutations in Chlamydomonas Reinhardtii

Forty single gene mutations in Chlamydomonas reinhardtii were isolated based on resistance to the compound 5'-methyl anthranilic acid (5-MAA). In other organisms, 5-MAA is converted to 5'-methyltryptophan (5-MT) and 5-MT is a potent inhibitor of anthranilate synthase, which catalyzes the first committed step in tryptophan biosynthesis. The mutant strains fall into two phenotypic classes based on the rate of cell division in the absence of 5-MAA. Strains with class I mutations divide more slowly than wild-type cells. These 17 mutations map to seven loci, which are designated MAA1 to MAA7. Strains with class II mutations have generation times indistinguishable from wild-type cells, and 7 of these 23 mutations map to loci defined by class I mutations. The remainder of the class II mutations map to 9 other loci, which are designated MAA8-MAA16. The maa5-1 mutant strain excretes high levels of anthranilate and phenylalanine into the medium. In this strain, four enzymatic activities in the tryptophan biosynthetic pathway are increased at least twofold. These include the combined activities of anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, indoleglycerol phosphate synthetase and anthranilate synthase. The slow growth phenotypes of strains with class I mutations are not rescued by the addition of tryptophan, but the slow growth phenotype of the maa6-1 mutant strain is partially rescued by the addition of indole. The maa6-1 mutant strain excretes a fluorescent compound into the medium, and cell extracts have no combined anthranilate phosphoribosyl transferase, phosphoribosyl anthranilate isomerase and indoleglycerol phosphate synthetase activity. The MAA6 locus is likely to encode a tryptophan biosynthetic enzyme. None of the other class I mutations affected these enzyme activities. Based on the phenotypes of double mutant strains, epistatic relationships among the class I mutations have been determined.     

Developmental Changes in Lactate Dehydrogenase and Aldolase Activity of the A2G-adr Mouse with Abnormal Muscle Function: Further Comparison with the 129Re-dy Mutant

Abstract: Lactate dehydrogenase and aldolase activity were reduced in lateral gastrocnemius muscle from two mouse mutants, A2G- adr and 129Re- dy , with abnormal muscle function. The activities of both of these enzymes were significantly reduced in the lateral gastrocnemius muscle from the A2G- adr mice at ages varying from 2 weeks to 32 weeks, whereas the activities in the soleus, heart, liver, and brain were the same as in the control animals. The lactate dehydrogenase isoenzymes in the lateral gastrocnemius and soleus muscles from the A2G mice were quantified, and although those of the soleus were comparable in mutant and control muscle, the lateral gastrocnemius from the adr mutant had reduced activity of LDH 5 and increased activities of the other four isoenzymes. The findings suggest that the adr mutation is expressed in the white (Type II) muscle fibres and not in the red (Type I) fibres or in any of the organs studied. It is suggested that the initiation of differentiation into Type II fibres from the embryonic form is absent or delayed in the A2G mutant. The reduced activities of lactate dehydrogenase and aldolase in 129Re- dy muscle confirm the findings of other workers.     

Organization of chlorophyll-protein complexes of Photosystem I in Chlamydomonas reinhardii

The polypeptide pattern, chlorophyll-protein complexes, fluorescence emission spectra and light intensity required for saturation of electron flow via Photosystem (PS) II and PS I in a pale-green photoautotrophic mutant, y-lp, were compared to those of the parent strain, Chlamydomonas reinhardii y-1 cells. The mutant exhibits a 686 nm fluorescence yield at 25°C and 77 K 2–6-fold higher than that of the parent strain cells, and is deficient in thylakoid polypeptides 14, 17.2, 18 and 22 according to the nomenclature of Chua (Chua, N.-H. (1980) Methods Enzymol. 60C, 434–446). All chlorophyll-protein complexes ascribed to PS II and the CP I complex were present in both type of cells. However, a chlorophyll-protein complex CP Ia containing — in the parent strain — the 66–68 kDa polypeptides of CP I and the four above-mentioned polypeptides, was absent in the mutant. It was previously reported that a chlorophyll-protein complex, CP O, obtained from C. reinhardii contains five polypeptides, namely, 14, 15, 17.2, 18 and 22 (Wollman, F.A. and Bennoun, P. (1982) Biochim. Biophys. Acta 680, 352–360). A CP O-like complex was present also in the mutant y-lp cells but it contains only one polypeptide, 15. Energy transfer from PS II to PS I was not impaired in the mutant, although a 4-fold higher light intensity was required for the saturation of PS I electron flow in the y-lp cells as compared with the parent strain. No difference was found in the light saturation curves for PS II activity between the mutant and parent strain cells. Based on these and additional data (Gershoni, J.M., Shochat, S., Malkin, S. and Ohad, I. (1982) Plant Physiol. 70, 637–644), it is concluded that the chlorophyll-protein complexes of PS I in Chlamydomonas comprise a reaction center-core antenna complex containing the 66–68 kDa polypeptides (CP I), a connecting antenna consisting of four polypeptides (14, 17.2, 18 and 22), and a light-harvesting antenna containing one polypeptide, 15. These appear to be organized as a complex, CP Ia. The interconnecting antenna is deficient in the y-lp mutant and thus the CP Ia complex is unstable and energy is not transferred from CP O to CP I. The effective cross-section of PS I antenna is thus reduced and a high fluorescence is emitted at 686 nm.     

The nrfI gene is essential for the attachment of the active site haem group of Wolinella succinogenes cytochrome c nitrite reductase

The cytochrome c nitrite reductase complex (NrfHA) is the terminal enzyme in the electron transport chain catalysing nitrite respiration of Wolinella succinogenes. The catalytic subunit NrfA is a pentahaem cytochrome c containing an active site haem group which is covalently bound via the cysteine residues of a unique CWTCK motif. The lysine residue serves as the axial ligand of the haem iron. The other four haem groups of NrfA are bound by conventional haem-binding motifs (CXXCH). The nrfHAIJ locus was restored on the genome of the W. succinogenes DeltanrfAIJ deletion mutant by integration of a plasmid, thus enabling the expression of modified alleles of nrfA and nrfI. A mutant (K134H) was constructed which contained a nrfA gene encoding a CWTCH motif instead of CWTCK. NrfA of strain K134H was found to be synthesized with five bound haem groups, as judged by matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry. Its nitrite reduction activity with reduced benzyl viologen was 40% of the wild-type activity. Ammonia was formed as the only product of nitrite reduction. The mutant did not grow by nitrite respiration and its electron transport activity from formate to nitrite was 5% of that of the wild-type strain. The predicted nrfI gene product is similar to proteins involved in system II cytochrome c biogenesis. A mutant of W. succinogenes (stopI) was constructed that contained a nrfHAIJ gene cluster with the nrfI codons 47 and 48 altered to stop codons. The NrfA protein of this mutant did not catalyse nitrite reduction and lacked the active site haem group, whereas the other four haem groups were present. Mutant (K134H/stopI) which contained the K134H modification in NrfA in addition to the inactivated nrfI gene had essentially the same properties as strain K134H. NrfA from strain K134H/stopI contained five haem groups. It is concluded that NrfI is involved in haem attachment to the CWTCK motif in NrfA but not to any of the CXXCH motifs. The nrfI gene is obviously dispensable for maturation of a modified NrfA protein containing a CWTCH motif instead of CWTCK. Therefore, NrfI might function as a specific haem lyase that recognizes the active site lysine residue of NrfA. A similar role has been proposed for NrfE, F and G of Escherichia coli, although these proteins share no overall sequence similarity to NrfI and belong to system I cytochrome c biogenesis, which differs fundamentally from system II.     

Biochemical characterization of mutant forms of DNA polymerase I from Escherichia coli. I. The polA12 mutation.

DNA polymerase I has been purified to greater than 90% homogeneity from a strain of Escherichia coli K12 that bears the temperature-sensitive DNA polymerase I mutatation, polA12. The mutant enzyme has a reduced electrophoretic mobility and sedimentation rate. It is abnormally thermolabile and is rapidly inactivated at low salt concentrations. Its polymerase and 5' leads to 3' exonuclease activities are not grossly defective at 30 degrees, yet its capacity to promote the concerted 5' leads to 3' polymerization and the 5' leads to 3' exonucleolytic hydrolysis of nucleotides at a nick ("nick translation") is decreased 10-fold. These effects are probably the result of a significant alteration in the tertiary structure of the enzyme.     

Reductive pentose phosphate-independent CO2 fixation in Rhodobacter sphaeroides and evidence that ribulose bisphosphate carboxylase/oxygenase activity serves to maintain the redox balance of the cell.

Whole-cell CO2 fixation and ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity were determined in Rhodobacter sphaeroides wild-type and mutant strains. There is no obvious difference in the levels of whole-cell CO2 fixation for the wild type, a form I RubisCO deletion mutant, and a form II RubisCO deletion mutant. No ribulose 1,5-bisphosphate-dependent CO2 fixation was detected in a form I-form II RubisCO double-deletion mutant (strain 16) or strain 16PHC, a derivative from strain 16 which was selected for the ability to grow photoheterotrophically with CO2 as an electron acceptor. However, significant levels of whole-cell CO2 fixation were detected in both strains 16 and 16PHC. Strain 16PHC exhibited CO2 fixation rates significantly higher than those of strain 16; the rates found for strain 16PHC were 30% of the level found in photoheterotrophically grown wild-type strain HR containing both form I and form II RubisCO and 10% of the level of the wild-type strain grown photolithoautotrophically. Strain 16PHC could not grow photolithoautotrophically in a CO2-H2 atmosphere; however, CO2 fixation catalyzed by photoheterotrophically grown strain 16PHC was repressed by addition of the alternate electron acceptor dimethyl sulfoxide. Dimethyl sulfoxide addition also influenced RubisCO activity under photolithoautotrophic conditions; 40 to 70% of the RubisCO activity was reduced without significantly influencing growth. Strain 16PHC and strain 16 contain nearly equivalent but low levels of pyruvate carboxylase, indicating that CO2 fixation enzymes other than pyruvate carboxylase contribute to the ability of strain 16PHC to grow with CO2 as an electron acceptor.     

Glucose-induced secretion of Trichoderma reesei xylanases.

To produce two xylanases with Trichoderma reesei grown on glucose, recombinant strains which carry either the xyn1 or the xyn2 (xylanase I and II [XYN I and XYN II]-encoding) structural genes under the expression signals of the homologous pki1 (pyruvate kinase-encoding) gene were constructed. The two types of transformants secreted XYN I or II, respectively, during growth on glucose, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostaining. The corresponding specific xylanase activities of the best transformants on glucose were 76 and 145 U/mg of protein for XYN I and XYN II, respectively, as opposed to that obtained by the parent strain (26 U/mg of protein). When related to the amount of biomass formed, however, they produced only about 4 to 5 U/g, in contrast to much higher activities (10 to 12 U/g) during growth on xylan. The ultrastructural location of XYN II in the transformant strain producing the highest constitutive XYN II formation (ATX2-12) was investigated by immunoelectron microscopy and compared with that in the wild-type strain growing on xylan. Cell extracts from both types of transformants grown on glucose exhibited a higher intracellular xylanase activity than did the parent strain grown on xylan. By using electron microscopy and immunogold labelling, XYN II was detected in the endoplasmic reticulum, Golgi-like vesicles, secretory vesicles, vacuoles, and cell walls. The immunolabel in the vacuoles was detected preferentially in subapical cells. When a recombinant strain which expressed xyn2 from the pki1 promoter was compared with the parent strain during growth on xylan, the former exhibited a less proliferated endoplasmic reticulum and a smaller number of secretory vesicles; however, a higher density of labelling was observed. The relationship of these findings to the efficacy of protein secretion during growth on glucose is discussed.     

Isolation and characterization of a mutant of Escherichia coli K12 synthesizing DNA polymerase I and endonuclease I constitutively

A mutant of Escherichia coli K12, highly resistant to ultraviolet radiation, has been isolated. Preliminary tests show that this mutant is also resistant to mitomycin C, nalidixic acid, fluorouracil and thymineless death. The mutant strain apparently repairs its damaged DNA more efficiently than wild-type E. coli K12 strains and, to do so, constitutively produces 35 times more DNA polymerase I and 12 times more endonuclease I than the wild-type strain.