Wild-type Cell Extracts Research Articles

The lithium-chloride-soluble cell-wall layers of Chlamydomonas reinhardii contain several immunologically related glycoproteins

Cell-wall glycoproteins of the unicellular green alga Chlamydomonas reinhardii have been purified from LiCl extracts of intact cells by gel exclusion chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies were raised against several polypeptide components isolated from the LiCl extracts. All these antibodies specifically reacted with the cell surface of formaldehyde-fixed cells. They showed cross-reactivity with the different antigens and were also reactive against some other polypeptides present in the LiCl extracts of intact wild-type cells as shown by double-diffusion assays and immunoblot analyses. These antigens were largely missing in LiCl extracts from the cell-wall-deficient mutant CW-15. The pattern of immunologically related cell-wall polypeptides of C. reinhardii varied during the vegetative cell cycle and was found to be also dependent on the growth conditions. Dot-immunobinding assays on chemically modified cell-wall glycoproteins demonstrated differences between the various antibodies with respect to their specificities. Differences were observed especially with respect to their reactivities against chemically deglycosylated cell-wall polypeptides. Chemical deglycosylation generally reduced the binding of the different antibodies indicating that all these antibodies recognize carbohydrate side chains. Only two of these antibody preparations, raised against cell-wall glycoproteins of relative molecular mass 35 and 150 kilodaltons, were found to be strongly reactive against deglycosylated cell-wall polypeptides. When these antibodies were saturated with cell-wall-derived glycopeptides in order to abolish the binding to carbohydrate side chains, they still recognized the same cell-wall polypeptides as did the untreated antibodies. These findings indicate that the cross-reactivity of the different cell-wall polypeptides with the antibodies is not exclusively the consequence of similar glycosylation patterns but is also the result of the presence of similar structures within the non-glycosylated stretches of the polypeptide backbones. Cell walls isolated from growing tobacco pollen tubes contained a single polypeptide component which showed crossreactivity with the antibodies to the cell-wall glycoproteins of C. reinhardii.

Allyl Alcohol Selection for Lower Alcohol Dehydrogenase Activity in Nicotiana plumbaginifolia Cultured Cells

One cell strain with stable tolerance to allyl alcohol (AA(r)) was selected from 6 x 10(8) suspension cultured Nicotiana plumbaginifolia Viviani cells. The selected strain contained one-half the alcohol dehydrogenase (ADH) activity of the wild type (NP) due to the loss of two of three bands of ADH activity seen on starch gels following electrophoresis of wild-type cell extracts. Anaerobic conditions, simulated by not shaking the suspension cultures, increased the ADH specific activity to more than 3-fold the initial level in both strains but did not change the number of activity bands or the relative levels of activity. The cell strain with decreased ADH activity lost viability more rapidly than the wild type under the anaerobic conditions. The AA(r) cells were 10 times more tolerant to ethanol than the NP cells and were also somewhat more tolerant to acetaldehyde and antimycin A. The substrate specificities of the ADH enzymes from both strains were very similar. Further selection of AA(r) cells with allyl alcohol produced strains with even lower ADH activity and selection under anaerobic conditions produced strains with increased ADH activity. Genetic studies indicate that the N. plumbaginifolia ADH activity bands arise from subunits produced by two nonallelic genes. This is the first example of the use of allyl alcohol to select for decreased ADH using cultured plant cells.

The absence of γ‐glutamyltransferase activity in transport‐dependent methotrexate‐resistant hepatoma cells

A cell line derived from H35 hepatoma cells resistant to methotrexate (MTX) as a result of a defective transport system for MTX has been examined to determine how closely the variant resembles the parent cells with regard to other biochemical properties. The capacity of extracts of resistant cells to catalyze the poly-gamma-glutamylation of MTX was approximately twice as great as that of wild-type cell extracts. Evidence of similarity between wild-type and H35 R0.3 cells was derived from the equitoxic activity to both cell lines of nonclassical antifolates and other miscellaneous antineoplastics which act by a variety of mechanisms. Two phenotypic markers of hepatic cell function, alpha-aminoisobutyric acid (AIB) transport and tyrosine aminotransferase (TAT) activity inducibility, were present in both cell types, demonstrating the maintenance of these phenotypic properties in the H35 R0.3 cells. gamma-Glutamyltransferase (GGT, EC 2.3.2.2) activity differed in that it was present in wild-type cells and barely detectable in H35 R0.3 cells. The GGT activity reappeared in the H35 cells when they regained MTX sensitivity after incubation for 14-20 weeks in MTX-free media. Although defective MTX transport appeared to be correlated with the disappearance of GGT activity in an H35 variant cell line, no functional relationship between them is apparent at this time. It is possible that a lack of GGT activity may be evidence of a more differentiated phenotype in the transport-resistant cell line.

Complementation of Bacillus subtilis polA mutants by DNA polymerase I from Streptococcus pneumoniae.

The polA gene of Streptococcus pneumoniae cloned in the recombinant plasmid pSM22 is expressed in Bacillus subtilis. Extracts of B. subtilis polA mutants containing pSM22 showed 6 times more DNA polymerase activity than extracts of wild-type cells without the plasmid. Complete complementation of the B. subtilis polA5 and polA59 mutations with respect to in vivo resistance to UV irradiation and methyl methanesulfonate was observed when four copies of the pneumococcal polA gene were present in each cell. Ectopic integration of the polA gene together with a cat marker into the chromosome of B. subtilis gave chromosomal insertions containing single and double doses of the pneumococcal polA gene. Correlation with gene dosage was observed for both chloramphenicol acetyltransferase and DNA polymerase activities measured in vitro. Depending on the number of copies of the S. pneumoniae polA gene present, restoration of DNA repair functions in polA mutants of B. subtilis was either partial or complete.

Adenosine kinase deficiency in tritiated deoxyadenosine-resistant mouse S49 lymphoma cell lines.

Mutant sublines were derived of S49 mouse T-lymphoma cells that were resistant to tritiated deoxyadenosine. Twenty-five isolates that were selected in 1 microCi/ml of the nucleoside were cross-resistant to 6-thioguanine, were sensitive to HAT (hypoxanthine, aminopterin, and thymidine), and contained less than 1% of hypoxanthine phosphoribosyltransferase activity in wild-type cells. One of the mutant clones, S49-dA2, was further subjected to selection in a medium containing 2 microCi/ml tritiated deoxyadenosine and 1 microgram/ml deoxycoformycin, an inhibitor of adenosine deaminase. All resistant subclones were cross-resistant to tubercidin, 6-methylmercaptopurine riboside, and arabinosyladenine. One of the subclones, S49-12, was completely devoid of adenosine kinase and was partially deficient in deoxyadenosine kinase. This subclone, however, contained wild-type levels of deoxycytidine kinase. DEAE chromatography of the wild-type cell extracts revealed two deoxyadenosine phosphorylating activities, one of which coeluted with adenosine kinase and was the enzyme missing in S49-12. The other species phosphorylated both deoxyadenosine and deoxycytidine, of which deoxycytidine was the preferred substrate.

Enzymatic conversion of glutamate to delta-aminolevulinic acid in soluble extracts of Euglena gracilis.

Glutamate was converted to the chlorophyll and heme precursor delta-aminolevulinic acid in soluble extracts of Euglena gracilis. delta-Aminolevulinic acid-forming activity depended on the presence of native enzyme, glutamate, ATP, Mg2+, NADPH or NADH, and RNA. The requirement for reduced pyridine nucleotide was observed only if, prior to incubation, the enzyme extract was filtered through activated carbon to remove firmly bound reductant. Dithiothreitol was also required for activity after carbon treatment. delta-Aminolevulinic acid formation was stimulated by RNA from various plant tissues and algal cells, including greening barley leaves and members of the algal groups Chlorophyta (Chlorella vulgaris, Chlamydomonas reinhardtii), Rhodophyta (Cyanidium caldarium), Cyanophyta (Anacystis nidulans, Synechocystis sp. PCC 6803), and Prochlorophyta (Prochlorothrix hollandica), but not by RNA derived from Escherichia coli, yeast, wheat germ, bovine liver, and Methanobacterium thermoautotrophicum. E. coli glutamate-specific tRNA was inhibitory. Several of the RNAs that did not stimulate delta-aminolevulinic acid formation nevertheless became acylated when incubated with glutamate in the presence of Euglena enzyme extract. RNA extracted from nongreen dark-grown wild-type Euglena cells was about half as stimulatory as that from chlorophyllous light-grown cells, and RNA from aplastidic mutant cells stimulated only slightly. delta-Aminolevulinic acid-forming enzyme activity was present in extracts of light-grown wild-type cells, but undetectable in extracts of aplastidic mutant and dark-grown wild-type cells. Gabaculine inhibited delta-aminolevulinic acid formation at submicromolar concentration. Heme inhibited 50% at 25 microM, but protoporphyrin IX, Mg-protoporphyrin IX, and protochlorophyllide inhibited only slightly at this concentration.

Inhibition of chondroitin and heparan sulfate biosynthesis in Chinese hamster ovary cell mutants defective in galactosyltransferase I.

We have isolated five Chinese hamster ovary cell mutants defective in galactosyltransferase I (UDP-D-galactose:xylose beta-1,4-D-galactosyltransferase) and studied the effect of p-nitrophenyl-beta-D-xyloside supplementation on glycosaminoglycan biosynthesis in the mutant cells. Assays of galactosyltransferase I showed that the mutants contained less than 2% of the enzyme activity present in wild-type cells, and enzyme activity was additive in mixtures of mutant and wild-type cell extracts, suggesting that the mutations most likely defined the structural gene encoding the enzyme. Cell hybridization studies showed that the mutations in all five strains were recessive and that the mutants belonged to the same complementation group. The mutants contained wild-type levels of xylosyltransferase (UDP-D-xylose:core protein (serine) beta-D-xylosyltransferase), lactose synthase (UDP-D-galactose:N-acetyl-glucosaminide beta-1,4-D-galactosyltransferase), and lactosylceramide synthase (UDP-D-galactose:glucosylceramide beta-1,4-D-galactosyltransferase). Their sensitivity to lectin-mediated cytotoxicity was virtually identical to that of the wild-type, indicating that there were no gross alterations in glycoprotein or glycolipid compositions. Anion-exchange high performance liquid chromatography of 35S-glycosaminoglycans from one of the galactosyltransferase I-deficient mutants showed a dramatic reduction in both heparan sulfate and chondroitin sulfate, demonstrating that galactosyltransferase I is responsible for the formation of both glycosaminoglycans in intact cells. Surprisingly, the addition of 1 mM-p-nitrophenyl-beta-D-xyloside, a substrate for galactosyltransferase I, restored glycosaminoglycan synthesis in mutant cells. This finding suggested that another galactosyltransferase, possibly lactose synthase, can transfer galactose to xylose in intact cells.

Isolation and characterization of novobiocin-resistant BHK cells

We isolated two novobiocin-resistant mutants which were stable and approximately three and four times more resistant than the parent cells to novobiocin. Both mutants (Novr A2, Novr A41) were more sensitive than the wild-type cells to nalidixic acid, and cold sensitive for cell growth. When we isolated derivatives of Novr A2 and Novr A41 cells which are resistant to nalidixic acid, those are found to be phenotypically reverted to novobiocin sensitivity like wild-type cells, thereby suggesting the relationship between the targets for novobiocin and for nalidixic acid. But the cold sensitivity did not always revert to wild type, with accompanying resistance to nalidixic acid. The DNA and RNA syntheses of Novr mutants were more resistant to novobiocin but more sensitive to nalidixic acid, than those of wild-type cells. However, in vitro assays of wild-type and Novr cell extracts were unable to demonstrate any differences in the sensitivity of topoisomerase II activity to inhibition by novobiocin. While the targets of novobiocin and nalidixic acid show a mutual interaction in vivo and play a role in DNA replication and transcription, our results suggest that these targets are probably not topoisomerase II.

Probing the structural domains and function in vivo of Escherichia coli DNA topoisomerase I by mutagenesis

Insertion and deletion mutagenesis within the gene topA of Escherichia coli encoding DNA topoisomerase I was carried out to test the existence of subdomains in the enzyme and the relationship between the slow-growth topA − phenotype and the known DNA relaxation activity of the enzyme. All mutants that show no detectable DNA relaxation activity in cell extracts fail to complement the temperature-sensitive growth defect of strain AS17 topA am harboring a plasmid-borne temperature-sensitive suppressor tRNA. All mutants that show partial or full levels of DNA relaxation activity in cell extracts (relative to activity in extracts of wild-type cells) can complement this defect. The carboxyl-proximal 25% of the enzyme appears to be in a domain that is dispensable both in terms of the catalytic function of the enzyme and its biological role. Analysis of the mutant enzyme also indicates that the formation of the covalent topoisomerase-DNA complex is correlated with the DNA relaxation activity, which supports the notion that the covalent complex is an obligatory intermediate in the catalysis of DNA topoisomerization.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and lipid metabolism in a concanavalin A-resistant chinese hamster ovary cell line

Lipid metabolism in a concanavalin A-resistant, glycosylation-defective mutant cell line was investigated by comparing growth properties, lipid composition, and lipid biosynthesis in wild-type (WT), mutant (C R-7), and revertant (RC R-7) cells. In contrast to WT and RC R-7, the mutant was auxotrophic for cholesterol, but mevalonolactone did not restore growth on lipoprotein-deficient medium. The use of R-[2- 14C]mevalonolactone revealed that C R-7 was deficient in the conversion of lanosterol to cholesterol. Total lipid and phospholipid content and composition were similar in all three cell lines, but C R-7 displayed subnormal content and biosynthesis of cholesterol and unsaturated fatty acids. The mutant was hypersensitive to compactin and was unable to upregulate either 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase activity or the binding and internalization of 125I-labeled low-density lipoprotein (LDL) in response to lipoprotein deprivation. HMG-CoA reductase activity in all three cell lines showed similar kinetics and phosphorylation status, and the binding kinetics and degradation of 125I-LDL were also similar, suggesting that C R-7 possesses kinetically normal reductase and LDL binding sites, but is deficient in their coordinate regulation. Tunicamycin (1–2 μg/ml) strongly and reversibly suppressed reductase activity in WT and RC R-7. C R-7 was resistant to this inhibitor. In WT cells this suppressive effect was accompanied by inhibition of 3H-labeled mannose incorporation into cellular protein, but 3H-labeled leucine incorporation was unaffected. Immunotitration of HMG-CoA reductase activity in extracts of WT cells, cultured in the presence and absence of tunicamycin, showed that suppression of reductase activity reflected the presence of reduced amounts of reductase protein, implying that glycosylation plays an important role in the coordinate regulation of HMG-CoA reductase activity and LDL binding.

Characterization of a cyclic AMP-resistant Chinese hamster ovary cell mutant containing both wild-type and mutant species of type I regulatory subunit of cyclic AMP-dependent protein kinase.

We have characterized a cyclic AMP-resistant Chinese hamster ovary (CHO) cell mutant in which one of two major species of type I regulatory subunit (RI) of cyclic AMP-dependent protein kinase is altered. Wild-type CHO cell extracts contain two cyclic AMP-dependent protein kinase activities. As shown by DEAE-cellulose chromatography, there is a peak of type I protein kinase activity in mutant extracts, but the type II protein kinase activity is considerably reduced even though free type II regulatory subunit (RII) is present. The type I kinase from the mutant has an altered RI (RI*) whose KD for the binding of 8-N3[32P] cAMP (KD = 1.3 X 10(-5) M) is increased by more than 200-fold compared to RI from the wild-type enzyme (KD = 5.5 X 10(-8) M). No differences were found between the catalytic subunits from the wild-type and mutant type I kinases. A large portion of RI in mutant and wild-type extracts is present in the free form. The RI* derived from mutant type I protein kinase shows altered labeling by 8-N3[32P]cAMP (KD = 1.3 X 10(-5) M) whereas the free RI from the mutant is labeled normally by the photoaffinity label (KD = 7.2 X 10(-8) M), suggesting that the RI* which binds to the catalytic subunit is functionally different from the free form of RI. The decreased amount of type II kinase activity in the mutant appears to be due to competition of RI* with RII for binding to the catalytic subunit. Translation of mRNA from wild-type CHO cells results in the synthesis of two different charge forms of RI, providing biochemical confirmation of two different species of RI in CHO cells. Additional biochemical evidence based on isoelectric focusing behavior of 8-N3[32P]cAMP-labeled RI species and [35S]methionine-labeled RI from mutant and wild-type extracts confirms the charge heterogeneity of RI species in CHO cells. These genetic and biochemical data taken together are consistent with the conclusion that there are at least two different species of RI present in CHO cells and that one of these species is altered in the mutant analyzed in this work.

Mechanisms determining aerobic or anaerobic growth in the facultative anaerobe Salmonella typhimurium.

We isolated mutant strains of the facultative anaerobe Salmonella typhimurium that grow either aerobically or anaerobically. Strict anaerobic mutants contained a defective DNA topoisomerase I gene (topI), while strict aerobic mutants contained a defective DNA gyrase subunit A gene (gyrA, also nalA). Topoisomerase I activity was detected in cell-free extracts of strict aerobic mutants but not of strict anaerobic mutant strains, whereas gyrase activity was detected in extracts of strict anaerobic mutants but not of strict aerobic mutants. Furthermore, extracts of wild-type cells, cultured under vigorous aerobic condition, contain topoisomerase I activity but no significant gyrase activity. In contrast, the extracts of anaerobically cultured wild-type cells contain gyrase activity but no significant topoisomerase I activity. Sucrose gradient centrifugation with ethidium bromide showed that chromosomal DNA in strict aerobic mutants and aerobically grown wild-type cells was relaxed, while the chromosomal DNA of strict anaerobic mutants and anaerobically grown wild-type cells was more supercoiled. Aerobic cultures of wild type and strict aerobic mutants produced both superoxide dismutase and catalase, whereas anaerobic cultures of wild type and strict anaerobic mutants did not. These results lead us to conclude that activity of topoisomerase I, associated with relaxation of chromosomal DNA, is necessary for expression of genes required for aerobic growth, whereas activity of gyrase, associated with supercoiling of chromosomal DNA, is necessary for expression of genes required for anaerobic growth.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells.

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells.

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

A complex from cultured Chinese hamster ovary cells containing nine aminoacyl-tRNA synthetases. Thermolabile leucyl-tRNA synthetase from the tsH1 mutant cell line is an integral component of this complex.

The size distribution of the 20 aminoacyl-tRNA synthetases from wild-type Chinese hamster ovary (CHO) cells and from the mutant cell line tsH1, containing a temperature-sensitive leucyl-tRNA synthetase, was determined by gel filtration. Nine aminoacyl-tRNA synthetases, specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine and proline, which coeluted as high-Mr entities (Mr approximately 1.2 X 10(6)), were further co-purified to yield a multienzyme complex, the polypeptide composition of which was identical to that previously determined for the complex from rabbit liver. Immunoprecipitates obtained from crude extracts of wild-type and tsH1 mutant cells, using specific antibodies directed to the lysyl-tRNA or methionyl-tRNA synthetase components of the complex, displayed the same polypeptide compositions as that of the purified complex, thereby establishing the heterotypic nature of this complex. Although the activity of leucyl-tRNA synthetase from the mutant cells, grown at a permissive temperature, was low compared to that from the wild-type, the polypeptide of Mr 129 000, corresponding to this enzyme, was present in similar amounts and occurred exclusively as a component of the high-Mr complex. Finally, we report that attempts to demonstrate phosphorylation of the components of the complex from cultured CHO, HeLa and C3 cells were unsuccessful.

Growth, enzyme levels, and some metabolic properties of an Escherichia coli mutant grown on L-threonine as the sole carbon source.

A mutant of Escherichia coli (designated E. coli SBD-76) that utilizes L-threonine as the sole carbon source was isolated. In contrast with levels in extracts of wild-type cells, the levels of threonine dehydrogenase in extracts of this mutant were 100-fold higher than levels of threonine aldolase or degradative threonine dehydratase. Catabolite repression of threonine dehydrogenase was manifested in wild-type, but not SBD-76, cells. For purposes of isolating enzymes, large quantities of SBD-76 cells with the elevated threonine dehydrogenase level could be grown in a fermentor in modified Fraser medium containing 1% glycerol, rather than in the 0.2% L-threonine minimal medium used to isolate the mutant. SBD-76 cells grown on L-threonine excreted glycine and aminoacetone into the medium, and extracts of the mutant strain catalyzed a quantitative conversion of L-threonine to glycine and aminoacetone.

Adenosine metabolism in wild‐type and enzyme‐deficient variants of Chinese hamster ovary and Novikoff rat hepatoma cells

Variants of Chinese hamster ovary and Novikoff rat hepatoma cells resistant to tubercidin and 2,5-diaminopurine, or to both drugs, were isolated, and their ability to convert adenosine and various adenosine analogs to nucleotides was compared to that of wild-type cells, both in intact cells and cell-free extracts. Adenosine deamination, and thus its conversion to nucleotides via inosine-hypoxanthine-inosine monophosphate, was inhibited by pretreatment of the cells or cell extracts with 2-deoxycoformycin. Cell-free extracts of the tubercidin-resistant variants, as well as of two adenosine-resistant mutants of Chinese hamster ovary cells, phosphorylated adenosine, tubercidin, pyrazofurin, or tricyclic nucleoside in the presence of ATP at less than 1% of the rate of extracts of wild-type cells. However, addition of phosphoribosyl pyrophosphate stimulated the conversion of adenosine to nucleotides 40-fold. Similarly, intact adenosine kinase-deficient cells failed to phosphorylate the adenosine analogs, but still converted adenosine to nucleotides at 5-10% the rate observed with wild-type cells. Phosphorylation of adenosine and tubercidin in wild-type cells was inhibited by substrate at concentration above 5-10 microM. In contrast, the rate of conversion of adenosine to nucleotides by adenosine kinase-deficient cells increased linearly up to a concentration of 400 microM adenosine, with the consequence that, at this concentration, these cells took up adenosine almost as rapidly as wild-type cells. Adenosine uptake by these kinase-deficient cells was inhibited by adenine and 5'-deoxyadenosine, and was largely abolished in mutants devoid also of adenine phosphoribosyltransferase. We conclude that adenosine is converted to nucleotides in adenosine kinase-deficient cells via adenine. Indirect evidence implicates 5'-methylthioadenosine phosphorylase as the enzyme responsible for the degradation of adenosine to adenine.

A Mutant of Bacillus subtilis Secreting a DNAase Inhibitor During Sporulation

Under sporulation conditions, Bacillus subtilis mutant SE63 secreted into the medium an inhibitor of the extracellular DNAase that is specifically associated with stage II to III of sporulation. The inhibitor was a heat-labile protein with a molecular weight of 18000–20000. A cell-bound, presumably intracellular, inhibitor with the same properties was found in extracts of wild-type cells and of the mutant. The mechanisms by which the mutation, designated din, might cause the secretion of a protein that is normally intracellular are discussed. The mutation was mapped by transduction with phage PBS1 and it lies between met and fru. It was also introduced into a variety of sporulation mutants including some blocked at the earliest known stages of the process. In all of these it caused secretion of the inhibitor. It is concluded that the secretion is therefore not integrally associated with sporulation but is, instead, part of the response of the cells to the nutritional step-down conditions in which spore formation is induced.

Expression of an alternative nitrogen fixation system in Azotobacter vinelandii.

Nitrogenase activities were determined from maximum acetylene reduction rates for mutant strains of Azotobacter vinelandii which are unable to fix N2 in the presence of molybdenum (Nif-) but undergo phenotypic reversal to Nif+ under conditions of Mo deficiency. The system responsible for N2 fixation under these conditions is thought to be an alternative N2 fixation system (Bishop et al., Proc. Natl. Acad. Sci. U.S.A. 77:7342-7346, 1980). Phenotypic reversal of Nif- strains to Nif+ strains was also observed in N-free medium without Mo but with either V or Re. Two protein patterns were found on two-dimensional gels of proteins from the extracts of wild-type cells cultured in N-free medium without Mo and with or without V or Re. The expression of each protein pattern in the wild-type strain of A. vinelandii seemed to depend upon the physiological state of the N2-fixing culture. Electron paramagnetic resonance experiments were conducted on whole cells of A. vinelandii grown under conditions of Mo deprivation in the absence of fixed N. No g = 3.65 signal (an electron paramagnetic resonance signal characteristic of the Mo-containing component of nitrogenase) was detectable in these cells, regardless of whether V or Re was present during growth of these cells, These results are discussed from the perspective that the well-known effect of V on N2 fixation by A. vinelandii may involve an alternative N2 fixation system.

A mutant of the cyanobacterium Plectonema boryanum resistant to photooxidation

Using a novel method for selecting mutants resistant to photooxidation a highly resistant mutant of the blue-green alga (cyanobacterium) Plectonema boryanum was obtained and its behaviour under photooxidative conditions was studied in comparison with sensitive strains. This mutant, designated PBstr LR-1, grown organotrophically, contained 40–50% less phycocyanin and chlorophyll per mg protein than the light-grown wild type cells. A pigment absorbing between 455 nm and 490 nm was detected in an aqueous extract of the mutant cells which was absent in extracts of wild type cells. An analysis of the ratio of superoxide dismutase (SOD) isoenzymes revealed that in the photooxidation resistant mutant, the major component of SOD is due to a hydrogen peroxide insensitive isoenzyme, while in the wild type and other sensitive strains, the majority of the SOD activity was due to a hydrogen peroxide sensitive isoenzyme. The significance of these finding, in terms of mechanisms protecting against photodynamic action is discussed.