Isolation and Characterization of Chlamydomonas reinhardtii Mutants with Defects in the Induction of Flagellar Protein Synthesis after Deflagellation1,2

Amputating the flagella of Chlamydomonas reinhardtii stimulates increased synthesis of many flagellar proteins within 30 min. We have isolated a series of mutants which are defective in this stimulation, taking advantage of the fact that cells which cannot stimulate flagellar protein synthesis cannot regenerate flagella. More than a dozen mutants which have flagella, but cannot regenerate them after amputation, were isolated and studied by in vivo labeling to identify those non-regenerator mutants which were specifically defective in the induction of flagellar protein synthesis. Ten such mutants have been identified, and in each of them flagellar amputation does not stimulate the synthesis of any of the major flagellar proteins. At least four of the mutants display an interesting conditional phenotype. The synthesis of flagellar proteins after deflagellation is defective only in gametic cells; vegetative cells of these mutants are capable of flagellar protein synthesis after flagellar amputation.     

Temperature sensitive mutations affecting RNA synthesis in Escherichia coli

Summary A streptomycin method has been used for the isolation of mutants with RNA synthesis inhibited at elevated temperature. The method is based on the observation that streptomycin kills bacteria with normal RNA synthesis and does not affect the cells with inhibited synthesis of RNA. This selection method increases the yield of temperature sensitive mutants by a factor 10–20, the amount of mutants with disturbed RNA synthesis is increased 3–5 fold as compared with the method of replicas.Several types of mutants were found among the temperature sensitive strains: those possessing temperature sensitivity of one, two or three types of cellular macromolecules DNA, RNA and protein. The screening among the mutants with affected RNA synthesis revealed a strain ts-19 showing low RNA polymerase activity in cell extracts and partially purified RNA polymerase preparations. The presented evidence suggests that ts-19 mutation affects the structural gene of one of the RNA polymerase subunits.The mapping of the corresponding locus indicated that it was located between the str and thy loci in E. coli K 12 chromosome at a distance of about 20 recombination units from the first locus.     

Screening for mutants temperature sensitive in protein synthesis by using methionine analogues

Summary Two new mutants are described which are temperature sensitive in protein synthesis. The mutants were obtained by a screening procedure using methionine analogues. The method is based on two findings: a) that in E. coli, and in other members of the Enterobacteriaceae, sensitivity to methionine analogues increases with the growth temperature, and b) that cells which are not synthesizing proteins during treatment with methionine analogues have a shorter lag in resuming growth subsequent to removal of the analogue.     

Reversibility of DnaA protein activity in the'irreversible'dnaA204 mutant of Escherichia coli

The dnaA204 mutant, one of the so-called irreversible dnaA mutants which cannot reinitiate chromosome replication upon a shift from non-permissive to permissive growth temperature in the absence of protein synthesis, was reinvestigated using flow cytometry and marker frequency analysis. In a temperature downshift experiment and in the presence of protein synthesis the dnaA204 mutant reinitiates chromosome replication very fast. Using a lac promoter-controlled wild type or a dnaA204 mutant gene carried on a plasmid, we have observed instantaneous initiation of replication when synthesis of DnaA protein is induced in the dnaA204 mutant at 42δC. The data indicate that the dnaA204 mutant after a shift to 42δC still contains functional DnaA protein, but that the activity level is below the initiation threshold. Thus, after synthesis of very small amounts of additional DnaA protein, initiation occurs very fast both after a shift to 30δC, and after induction of DnaA protein synthesis at 42 C. A model describing the processing of DnaA protein in mutants and in the wild type Is presented.     

Escherichia coli mutants with temperature-sensitive synthesis of DNA

Summary Seven mutants of E. coli with temperature-sensitive synthesis of DNA have been isolated. Synthesis of RNA, protein and DNA precursors does not appear to be directly affected. The mutants can be divided into at least two groups on the basis of their pattern of DNA synthesis, their ability to support phage growth at 41° and their genetic mapping.Mutants of the first group are heterogeneous in their pattern of DNA synthesis at 40°. Some mutants cease DNA synthesis abruptly upon transfer to 40° and any residual DNA synthesis is barely detectable. In others there is substantial residual synthesis at 40°. All these Group 1 mutants are alike, however, in that they support the growth of phage T4 but not Lambda at 41°. Two mutants with barely detectable residual DNA synthesis carry DNA mutations which have been mapped by P1 transduction and show about 72% linkage to the malB locus. It has not yet proved possible to map accurately the mutants showing substantial residual synthesis, and the possibility that these mutations are in a different gene(s) has not been excluded.A single mutant has been placed in a second group. Like some Group 1 mutants it synthesizes substantial amounts of DNA at 40° before synthesis stops. However, unlike them it supports the growth of T4 and Lambda at 41°. The DNA mutation maps near the leu locus. Certain properties of this mutant are consistent with the idea that initiation of DNA synthesis is temperature-sensitive in this strain.Adapted from a dissertation presented in partial fulfillment of the degree of Doctor of Philosophy. This investigation was supported in part by U.S. Public Health Services Grant 5-TO1-GM00829 from the National Institute of General Medical Sciences and in part by U.S.P.H.S. research grant GM12524.     

Reinitiation kinetics in eight dnaA(Ts) mutants of Escherichia coli: rifampicin-resistant Initiation of chromosome replication

The kinetics of reinitiation of chromosome replication of eight dnaA(Ts) mutants was investigated in an isogenic set of strains. Five mutants (167, 46, 601, 606 and 5) are classified as reversible, since they can reinitiate at 30 C without protein synthesis, whereas the other three (508, 205, 204) require protein synthesis. In the presence of protein synthesis, reversible mutants initiate one round of replication rapidly after a shift to 30δC, indicating that they contain active or renaturable DnaA protein. The dnaA508 and dnaA204 mutants also reinitiate chromosome replication rapidly, whereas reinitiation is delayed 15–20min in dnaA205. The dnaA508 and dnaA204 mutants might contain active DnaA protein just below the threshold level at 42δC and only require synthesis of small amounts of new DnaA protein before initiation at 30δC, whereas dnaA205 accumulates DnaA protein for some time at 30δC before reaching the initiation threshold. Three of the reversible mutants (5, 601, and 606) exhibited, in addition to the protein synthesis-independent initiation capacity, an RNA synthesis-independent initiation capacity. The thermal stability of these initiation capacities is the same as for mutant DnaA protein, strongly suggesting that mutant DnaA protein is responsible for both.     

Streptomycin-Suppressible Lethal Mutations in Escherichia coli

Forty-one mutants have been isolated which require streptomycin for growth on complete medium. These streptomycin-suppressible lethal mutations are located randomly around the Escherichia coli genetic map; during growth in liquid culture, they exhibit a variety of responses to the removal of streptomycin as judged by turbidity, cell morphology, and macromolecular synthesis. In particular, some mutants are primarily affected in protein or ribonucleic acid (RNA) synthesis (or both), one in deoxyribonucleic acid synthesis, and two in lipid synthesis. Ten mutants affected in protein synthesis were examined for the activities of all twenty aminoacyl-transfer RNA synthetases, and three were found to have altered glutamyl-transfer RNA synthetase activities. The advantages of this method for isolating a wide variety of conditional lethal mutants are discussed.     

Derepressed leucine transport activity in Escherichia coli

Transport activity and synthesis of binding protein for the amino acids leucine, isoleucine and valine in E. coli are coordinately controlled by the level of leucine in the growth medium. Spontaneous mutants (dlu) which can utilize D-leucine as a source of L-leucine show derepressed transport activity for the three-branched chain amino acids. The increased transport activity is a result of an increase in the binding protein for these amino acids. Azaleucine-resistant mutants have been isolated which have a defect in leucine transport but normal levels of the binding protein for leucine.     

Mutants of yeast with depressed DNA synthesis

Summary Seven temperature-sensitive mutants have been isolated in Saccharomyces cerevisiae which show a reproducible defect in DNA synthesis at the restrictive temperature. One of these is allelic with rnal1 (Hartwell et al., 1970) but the remaining mutants define six complementation groups and probably represent six different genes. The gene symbol dds (for depressed DNA synthesis) is proposed.At the restrictive temperature, rnal1-2, dds2-1 and dds6-1 show a rapid and almost total cessation of DNA and RNA synthesis, whilst protein synthesis continues for several hours. The remaining dds mutants show a reduced rate of DNA synthesis from the time of temperature shift (dds1, dds3, dds4) or a cessation of DNA synthesis at a later time (dds5). In some cases, RNA synthesis is affected concomitantly with, or soon after, the depression in DNA synthesis. Possible reasons for the phenotypes of these mutants, and for the relative absence of yeast mutants which are unambiguously and specifically affected in DNA synthesis, are discussed.In addition, we report the isolation of seven new alleles of known cdc genes and ten new mutants with a cell cycle phenotype that complement those already known.     

Isolation of metabolically active Petite mutants of Kluyveromyces lactis, a petite-negative yeast

Summary Conditions under which complete cultures of the petite-negative yeast Kluyveromyces lactis can be converted to metabolically active petite mutants have been found. These mutants, which lack mitochondrial protein synthesis have been shown to be metabolically active by their ability to exclude the dye trypan blue. They appear to possess a functional protein synthesising system, which is sensitive to the inhibitor trichodermin. However, on transfer to solid nutrient medium, these mutants fail to grow normally, and give rise to microcolonies composed of up to a thousand cells. These colonies autolyse after several days.     

Mitochondrial ribosome assembly in Neurospora crassa: mutants with defects in mitochondrial ribosome assembly.

Summary We have examined mitochondrial (mt) ribosome assembly and-function in five nuclear and six extranuclear mutants of Neurospora crassa which had previously been characterized as deficient in cytochromes b and aa ₃. All six extranuclear mutants showed phenotypes similar to that previously described for the extranuclear [poky] mutant: small subunit-deficient with 19 S rRNA rapidly degraded. The nuclear mutants have the following phenotypes: 297-24 is mt small subunit deficient with 19 S RNA rapidly degraded. 289-56 is mt small subunit deficient but contains normal ratios of 19 S to 25 S RNA in whole mitochondria. 289-67 and 299-9 show defects in the processing of 25 S RNA leading to accumulation of a large precursor RNA. 289-4 is deficient in large subunits although a substantial, but less than normal, amount of 25 S RNA is present in the mitochondria.The present work provides new insight into the phenotypes of mt small subunit-deficient mutants. Previous studies using chloramphenicol suggest that some defects in the assembly of mt small subunits may arise secondarily as a result of inhibition of mt protein synthesis (LaPolla and Lambowitz, 1977; Lambowitz et al., 1979). Three mutants (289-56, 289-67 and 299-9) appear to show such defects. These strains contain incomplete mt small subunits which sediment more slowly than normal and are deficient in at least two proteins, S-5 and S-9. Correlation of mutant phenotypes with rates of mt protein synthesis in the different strains suggests that mt protein synthesis must be decreased to less than one half of the wild-type rate before secondary defects in mt small subunit assembly are observed. This threshold value is much lower than that which leads to gross deficiencies of cytochromes b and aa ₃. Although several mutants have phenotypes suggestive of alterations in mt ribosomal proteins, no such alterations could be identified by two dimensional gel electrophoresis.     

The synthesis of S-adenosylmethionine by mutants with defects in S-adenosylmethionine synthetase

Summary Some metK mutants of Salmonella typhimurium with constitutive methionine biosynthesis have no detectable S-adenosylmethionine (SAM) synthetase, the enzyme which converts methionine to SAM, the postulated corepressor of the methionine pathway. However these mutants are not auxotrophic for SAM, an essential compound for many reactions. Here it is shown that these mutants have normal functioning of pathways involving SAM and do in fact produce SAM at as high levels as wild-type. Also, SAM synthetase is shown to be dispensible for growth but not for methionine regulation. These results indicate that there is another route of SAM synthesis independent of SAM synthetase. This route probably also uses methionine as substrate as metK mutants are shown to convert methionine to SAM as efficiently as analogous non-metK strains. The existence of a second route of SAM synthesis makes it necessary to postulate a compartmentalization of SAM made via the SAM synthetase reaction from SAM made in any other way to explain the reduced ability of metK mutants to repress methionine biosynthesis.     

Replication of small plasmids in extracts of Escherichia coli: involvement of the dnaB and dnaC protein in the replication of early replicative intermediates.

Summary The role of E. coli dnaB and dnaC protein in the replication of plasmid ColE1 and RSF1030 DNA was investigated in a soluble in vitro system (Staudenbauer, 1976a). Extracts from dnaB and dnaC mutants which are phenotypically DNA initiationor DNA elongation-defective were examined for their replicative capacity. It was found that all mutants tested are deficient in the synthesis of supercoiled plasmid DNA. Deficient extracts of dnaB mutants could be partially complemented by purified dnaB wild type protein but required for full complementation dnaC wild type protein as well. The dnaB wild type protein could be replaced by a P1dnaB analog (ban) protein complexed with a dnaB ts protein. Deficient extracts of dnaC mutants were complemented by purified dnaC wild type protein alone.The in vitro plasmid replication cycle had been separated into an early and late stage (Staudenbauer, 1977). Analysis by CsCl velocity centrifugation of the plasmid DNA synthesized in mutant extracts indicates that the early stage, namely the synthesis of early replicative intermediates, proceeds in all dnaB and dnaC mutants tested. However, replication of the early intermediates during the late stage depends on both the dnaB and dnaC protein. These conclusions were confirmed using inhibitors of DNA synthesis.     

Temperature sensitive mutants affecting the activity and regulation of the acetamidase of Aspergillus nidulans

Summary Mutants of Aspergillus nidulans with temperature sensitive growth on various amides have been isolated. Three of these mutants have a lesion in the amdS gene and their properties indicate that this is the structural gene for the acetamidase enzyme. In addition one of these mutants appears to be temperature sensitive for assembly of enzyme sub-units. The fourth mutant has a lesion in the amdR gene and, while producing a normal enzyme, is temperature sensitive for synthesis of the acetamidase. The properties of these mutants provide support for a model in which amdR codes for a protein which acts positively to activate synthesis of the acetamidase. A discussion of the present knowledge concerning acetamidase regulation is presented.     

Outer membrane protein a and other polypeptides regulate capsular polysaccharide synthesis in E. coli K-12.

Summary capR (lon) mutants of Escherichia coli K-12 are mucoid on minimal agar because they produce large quantities of capsular polysaccharide. When such mutants are transformed to tetracycline resistance by plasmid pMC44, a hybrid plasmid that contains a 2 megadalton (Mdal) endonuclease EcoR1 fragment of E. coli K-12 DNA joined to the cloning vehicle-pSC101, capsular polysaccharide synthesis is inhibited and the transformed colonies exhibit a nonmucoid phenotype. Re-cloning of the 2 Mdal EcoR1 fragment onto plasmid pHA105, a min-colE1 plasmid, yielded plasmid pFM100 which also inhibited capsular polysaccharide synthesis in the capR mutants. A comparison of the polypeptides specified by both plasmids pFM100 and pMC44 in minicells demonstrated that seven polypeptide bands were specified by the 2 Mdal DNA, one of which was previously demonstrated to be outer membrane protein a; also known as 3b or M2 (40 kilodaltons, Kdal). Plasmid mutants no longer repressing capsular polysaccharide synthesis were either unable to specify the 40 K dal outer membrane protein a or were deficient in synthesis of 25 K dal and 14.5 K dal polypeptides specified by the 2 Mdal DNA fragment. Studies with a minicell-producing strain that also contained a capR mutation indicated that the capR gene product regulated processing of at least one normal protein, the precursor of outer membrane protein a.     

Synthesis of flagellin and hook subunit protein in flagellar mutants of Escherichia coli K12

Summary We have examined Escherichia coli K12 flagellar mutants affected in each of 29 different loci for the synthesis of flagellin and hook subunit protein. Immune precipitation experiments were employed by treating cell extracts with antiserum against each protein. Flagellin was synthesized in mutants defective in genes flaS, flaT, flaU and flbC. The flaE and flaZ mutants produced small amounts of flagellin, while all the other mutants failed to produce any detectable amount of flagellin.Hook subunit protein was found in most mutants including those defective in genes flaA, flaB, flaC, flaD, flaE, flaG, flaH, flaL, flaM, flaN, flaO, flaP, flaQ, flaR, flaS, flaT, flaU, flaV, flaW, flaX, flaY, flaZ, flbA, flbC, flbD, and hag but not in mutants of flaK, flaI, and flbB. The results conform to the predictions made by our previous indirect gene fusion study (Komeda 1982).     

Cellular compartmentation of aromatic amino acids in Neurospora crassa. II. Synthesis and misplaced accumulation of phenylalanine in Phen-2 auxotrophs

Two mutants which require phenylalanine for normal growth and which show no prephenate dehydratase activity in vitro have been found to accumulate and excrete phenylalanine when incubated on minimal medium or grown on low concentrations of phenylalanine. The high levels of phenylalanine accumulated in these mutants apparently cannot be used for protein synthesis or for the regulation of the biosynthetic enzymes in the aromatic pathway. Mutant mycelia grown in high phenylalanine maintain a much lower level of free phenylalanine in the cells than do those grown on low phenylalanine or those which eventually grow on minimal. If all the phenylalanine required for the protein in a 3-day mycelial pad is supplied, little or no phenylalanine can be found in the medium after 3 days: if only a fraction of the total protein phenylalanine is supplied, the concentration of phenylalanine in the medium after 3 days is actually higher than the initial concentration. It is proposed that the mutation in these organisms has resulted in abnormal compartmentation of the phenylalanine produced so it cannot be utilized by the cells until it has been excreted and transported back into the normal pool channels. In this case, the transport (exogenous) and protein synthesis pools would be involved. The abnormal mislocation of the phenylalanine in the cell might be a result of the diffusion of free prephenate to low pH regions in the cell where it is nonenzymatically converted to phenylpyruvate. If, however, the mutant prephenate dehydratase is active in vivo, the mutation must somehow affect the activity or stability of the enzyme in vitro and also cause the release of the end product in the wrong place in the cell. This might be expected if the normal wild-type prephenate dehydratase is directionally oriented, e.g., as a result of membrane association, to release the product into normal cell channels (protein synthesis pool) while such oriented release might not occur in the mutants.This work was supported, in part, by an Atomic Energy Commission grant to the Institute of Molecular Biophysics, The Florida State University, and by the Genetics Training Grant, funded by the National Institutes of Health. It contains, in part, data from the doctoral thesis of the senior author, who was supported by a Florida State University Nuclear Fellowship and by a Public Health Service Fellowship.     

Isolation and characterization of Chlamydomonas reinhardtii mutants with defects in the induction of flagellar protein synthesis after deflagellation

Amputating the flagella of Chlamydomonas reinhardtii stimulates increased synthesis of many flagellar proteins within 30 min. We have isolated a series of mutants which are defective in this stimulation, taking advantage of the fact that cells which cannot stimulate flagellar protein synthesis cannot regenerate flagella. More than a dozen mutants which have flagella, but cannot regenerate them after amputation, were isolated and studied by in vivo labeling to identify those non-regenerator mutants which were specifically defective in the induction of flagellar protein synthesis. Ten such mutants have been identified, and in each of them flagellar amputation does not stimulate the synthesis of any of the major flagellar proteins. At least four of the mutants display an interesting conditional phenotype. The synthesis of flagellar proteins after deflagellation is defective only in gametic cells; vegetative cells of these mutants are capable of flagellar protein synthesis after flagellar amputation.     

Homogeneous incorporation of secondary cell wall polysaccharides to the cell wall of <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB27

Regular surface protein layers (S-layers) from most Gram-positive bacteria and from the ancestral bacterium Thermus thermophilus attach to pyruvylated polysaccharides (SCWP) covalently bound to the peptidoglycan through their SLH domain. However, it is not known whether the synthesis of SCWP and S-layer is coordinated enough as to follow a similar pattern of incorporation to the cell wall during growth. In this work we analyse the localization of newly synthesized SCWP on the cell wall of T. thermophilus by immunoelectron microscopy. For this, we obtained mutants with a reduced amount of pyruvylated SCWP through mutation of the csaB gene encoding the SCWP-pyruvylating activity, and its upstream gene csaA, a putative sugar transporter. We hypothesized that CsaA would be required for the synthesis of the SCWP. However, we found that csaA mutants showed only a minor decrease in the amount of SCWP immunodetected on the cell walls in comparison with csaB mutants, revealing its irrelevance in the process. Complementation experiments of csaB mutants with CsaB expressed from inducible promoters revealed that newly synthesized SCWP was homogeneously distributed along the cell wall. Fusions with thermostable fluorescent protein revealed that CsaB was distributed also in homogeneous pattern associated with the membrane. These data support that synthesis of SCWP takes place in disperse and homogeneous form all over the cell surface, in contrast to the zonal incorporation at the cell centre recently demonstrated for SlpA.