Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus

Aspergillus fumigatus secretes a serine alkaline protease (ALP) and a metalloprotease (MEP) when the fungus is cultivated in the presence of collagen as sole nitrogen and carbon source. The gene encoding ALP was isolated and characterized previously. We report here the cloning and the sequencing of the gene encoding MEP. Genomic and cDNA clones were isolated from A. fumigatus libraries using synthetic oligonucleotides as probes. Stretches of the deduced amino acid sequence were found to be in agreement with the N-terminal amino acid sequence of MEP and with internal peptide sequences. The amino acid sequence of the enzyme contains a putative active-site sequence HEYTH homologous to the active site of other bacterial and eukaryotic zinc metalloproteases. Sequence analysis reveals that MEP has a pre-proregion consisting of 245 amino acid residues preceding the 388 amino acid residues of the mature region (molecular mass of 42 kDa). An alp mep mutant, deficient in proteolytic activity at neutral pH in vitro, was constructed and tested for pathogenicity in a murine model. No difference in pathogenicity was observed between the wild-type strain and the alp mep double mutant, suggesting that ALP and MEP are not essential for the invasion of the lung tissues by A. fumigatus.     

Temperature-sensitive mutants of B. subtilis defective in deoxyribonucleotide synthesis

Summary Cessation of DNA synthesis in the temperature sensitive mutant 167 tsA 13 of Bacillus subtilis is correlated with the disappearance of dCTP and dATP pools at the nonpermissive temperature; dGTP and dTTP residual pools are stable. In the presence of AdR and CdR at 45°C, the dCTP and dATP pools remain normal and the cells continue to synthesise DNA and grow. It is inferred that in 167 tsA 13 AdR and CdR kinases exist, that the deoxynucleotide kinases function normally and the ribonucleotide reduction is deficient. B. subtilis strains have a hydroxyurea sensitive reductase and the drug inhibition can be reversed by exogenous deoxynucleosides. Evidence that the tsA 13 mutation is in the structural gene of the ribonucleotide reductase is discussed.     

The gene for ribosomal protein L25 (rplY) maps at 47.3 min near nalA in Escherichia coli K-12.

Summary A temperature sensitive mutant, termed JE1306, derived from Escherichia coli strain PA3092 was found to have an alteration in the ribosomal protein L25. Crosses with various Hfr strains and transductions with P1kc phage have revealed that the mutation maps at 47.3 min between nalA and fpk, in a region where no ribosomal protein gene has so far been located. The gene affected by this mutation is most probably the structural gene for protein L25 (rplY), because a strain heteromerozygous for the region shows both wild type and mutant forms of protein L25.     

Functional characteristics of green alga Scenedesmus obliquus (Chlorophyceae): 276–6 wild type and its two photosystems deficient mutants cultured under photoautotrophic,mixotrophic and heterotrophic conditions

Functional features of Scenedesmus obliquus: wild type 276–6 strain (WT) and its two mutants reported as photosystem I‐deficient (mutant 56.80) and photosystem II‐deficient (mutant 57.80) were characterized. Algae were cultured aseptically under continuous light or in darkness on mineral bold basal medium (BBM), yeast extract‐enriched BBM and yeast extract to evaluate the physiology of algal cells under photoautotrophic, mixotrophic and heterotrophic conditions. Growth, superoxide dismutase activity and photosynthetic parameters, including polyphasic fluorescence rise during the first seconds of chlorophyll a illumination (OJIP), were analyzed to find relationships between the photosynthetic/respiratory activity of the cells, occurrence of oxidative stress and trophic conditions applied to PSs‐deficient algae. Despite the highest superoxide dismutase activity, indicating the presence of oxidative stress, mixotrophic conditions appeared to be optimal for S. obliquus WT and mutant strains kept in non‐aerated cultures. OJIP analysis indicated that in mutant 56.80 part of photosystem (PS) I was functional and in mutant 57.80 residual PS II activity was found.     

Mutants of Salmonella typhimurium deficient in DNA polymerase. I. Detection by their failure to produce colicin E1

Summary A colicinogenic strain of Salmonella typhimurium was treated with nitrosoguanidine, and the survivors were tested for spontaneous production of colicin E1. Among about 10000 clones tested, two were found which appeared to have lost the ColE1 factor and had become sensitive to methyl methanesulphonate (MMS). These two isolates also proved to be more sensitive to ultraviolet (UV) irradiation and ionizing () radiation than their parent strain, and to be at least partly deficient in ability to host-cell reactivate bacteriophages damaged by UV-irradiation, -irradiation or MMS treatment. A third mutant with these properties has previously been described. Revertants of all three mutants selected on the basis of resistance to MMS were found to have regained wild-type resistance to UV, , or MMS treatment, suggesting that each of the original mutants carries a single mutation responsible for increased radiation sensitivity and reduced HCR capacity. All three mutants were of approximately normal fertility in transduction, and released temperate phages spontaneously at a significantly higher frequency than did their parent strain. Assays performed on crude extracts obtained by ultrasonic treatment established that the various mutants were deficient in an enzyme with DNA polymerase activity, and that their MMS-resistant derivates had regained almost 100% of the enzyme activity found in extracts of the wild-type parent strain. Preliminary mapping by conjugation indicated that the mutation conferring radiation sensitivity in one of the three strains lies between cysI and rha on the S. typhimurium chromosome, but attempts to determine its location more precisely by P22-mediated transduction were unsuccessful.     

Ribosomal protein modification in Escherichia coli. I. A mutant lacking the N-terminal acetylation of protein S5 exhibits thermosensitivity.

A thermosensitive mutant (JE386) of Escherichia coli which harbours an alteration in protein S5 of the smaller ribosomal subunit has been isolated. Genetic studies have shown that the lesion causing thermosensitivity also causes the alteration in protein S5, and that this mutation is not in the structural gene for S5 (rpsE). Hence the mutation has been termed rimJ (ribosomal modification). Protein-chemical studies of protein S5 purified from JE386 and its wild-type parent indicated an alteration in the N-terminal tryptic peptide. Amino acid sequence analysis of the N-terminal peptides showed complete homology between wild-type and mutant, suggesting that the N-terminal modification (acetylation) of the parent was absent in the mutant. Gradient transmission mapping has located the rimJ mutation at 31 minutes on the current E. coli genetic map. By constructing a derivative of the mutant heterozygous for rimJ, it has been found that the wild-type allele is dominant over the mutant one. Ts⁺ revertants of JE386 have been isolated which show either a wild-type ribosomal protein electrophoresis pattern, or an additional alteration in either protein S4 or S5. The mutations in S4 and S5 may compensate the lesion caused by the rimJ mutation of JE386, that is even though the N-terminus of S5 remains unacetylated, bacteria can grow at 42 °C. Furthermore, a mutation near or at strA carried by JE386 has been found to be involved in the phenotypic expression of the rimJ mutation. This mutation was also found to be present in four other strA mutants. Possible implications of the modification of ribosomal proteins in vivo are discussed.     

Escherichia coli uvrD mutants with thermosensitive DNA-dependent adenosine triphosphatase I (helicase II)

Three mutants producing thermosensitive DNA-dependent Adenosine triphosphatase (ATPase) I were screened from a collection of temperature-sensitive mutants of Escherichia coli K12. ATPase I purified to near homogeneity from one of the mutants (JE11000) possesses both thermosensitive DNA-dependent ATPase and DNA helicase activities. We have shown that ATPase I is encoded by the uvrD gene as first suggested by Oeda et al. (1982): (i) the thermosensitive ATPase I mutation present in JE11040 lies in or very close to the uvrD gene, (ii) ATPase I activity is absent in uvrD210, uvrD156, and uvrD252 mutants. Thus the thermosensitive mutations correspond to new uvrD mutations. However, the mutation present in JE11040 confers neither UV sensitivity nor mutator phenotype at high temperature. Evidence is presented that the mutant ATPase I is stabilized in vivo at 42 degrees C.     

Preparation and analysis of isogenic mutants in the transferrin receptor protein genes, tbpA and tbpB, from Neisseria meningitidis

Isogenic mutants were constructed in the tbpA and tbpB genes from Neisseria meningitidis strain B16B6, which code for the transferrin receptor proteins, Tbp1 and Tbp2. Insertion mutants of the tbpA and tbpB genes were obtained by shuttle mutagenesis and by in vitro cassette mutagenesis, respectively. The Isogenic mutants were verified by Southern blot and Western blot analysis. Isogenic mutants deficient in Tbp1 or Tbp2 demonstrated a reduced transferrin binding activity in intact cells and total membranes but were incapable of utilizing transferrin iron for growth. Tbp1 could be isolated by affinity methods from the mutant lacking Tbp2 but isolation of Tbp2 from the mutant lacking Tbp1 required the presence of exogenous Tbp1.     

Bacillus subtilis mutants dependent on streptomycin

Summary Six streptomycin-dependent mutants of Bacillus subtilis, two of which were asporogenous, were isolated. All six mutants, SD1, SD2, SD6, SD7, SD9 and SD10, contained a single mutation causing streptomycin dependence and asporogeny, but four of these mutants (SD6, SD7, SD9, SD10) contained a second mutation which phenotypically suppressed the asporogenous character of the streptomycin dependence mutation. All six mutants grew more slowly than the wild type strain BR151, but those defective in sporulation grew the slowest. The streptomycin dependence mutations of SD9 and SD10B (a sporeplus transformant from SD10 carrying both the dependence mutation and the phenotypic suppressor) lie near or possibly within the strA locus. Ribosomes from SD9, SD10A (a spore-minus transformant from SD10 carrying only the dependence mutation), and SD10B were stimulated in vitro by concentrations of streptomycin that inhibit the activity of wild type strain BR151 ribosomes. The level of misreading as measured by poly(U)-directed isoleucine incorporation was greatly enhanced by streptomycin in wild type strain BR151 ribosomes, but misreading of mutant SD9, SD10A, and SD10B ribosomes, irrespective of the sporulation phenotype, was little affected by streptomycin. There were no apparent differences in the patterns obtained by two-dimensional polyacrylamide gel electrophoresis of the 70S ribosomal proteins of the mutants SD9, SD10A, SD10B, and wild type strain BS151.     

Early blocked asporogenous mutants of Bacillus subtilis 168. I. Isolation and characterization of mutants resistant to antibiotic(s) produced by sporulating Bacillus subtilis 168

Summary A number of mutants (abs)-resistant to antibiotic(s) produced by sporulating Bacillus subtilis 168 have been isolated from an early blocked asporogenous mutant (spoA12). At least four classes were recognized according to their phenotypic properties. Genetic analysis has shown that these mutants were neither partial revertants nor suppressor mutants of the spoA gene. Both nonsense and missense mutants of the spoA gene are reverted partially by a secondary mutation which is resistant to antibiotic of B. subtilis 168. Another asporogenous mutant, spoB, whose locus is closely linked to pheA, is also affected by the same abs mutation. The nature of abs mutants is discussed.     

Isolation of a Tn501 insertion mutant lacking porin protein P of Pseudomonas aeruginosa

Summary In order to demonstrate a role for anion-specific protein P channels in phosphate transport in Pseudomonas aeruginosa PAO, we wished to isolate a transposon insertion mutant deficient in protein P. A number of transposon delivery systems were tested which yielded, for the most part, whole plasmid inserts. Plasmid pMT1000 (Tsuda et al. 1984), a temperature-sensitive R68 plasmid carrying the transposon Tn501, was successfully employed in the isolation of a Tn501 insertion mutant lacking protein P under normally inducing conditions. To identify the mutant deficient in protein P, a protein P-specific polyclonal antiserum was used. This mutant, strain H576, was deficient in high-affinity phosphate transport exhibiting a Km for uptake (3.60±0.64 M) almost ten times greater than that of the wild type strain (Km=0.39 M). There was, however, no change in the V_max for high-affinity phosphate transport as a result of the loss of protein P in this mutant. The protein P-deficiency of the mutant correlated with a growth defect in a phosphate-limited medium resulting in an 18%–35% decrease in growth when compared with the wild type.     

Mutations affecting growth of the Escherichia coli cell under a condition of DNA polymerase I-deficiency

Summary We constructed a strain of E. coli K12 carrying polA1 (an amber mutation of the DNA polymerase I gene; De Lucia and Cairns, 1969), and sup-126 (a temperature-sensitive amber suppressor; Nagata and Horiuchi, 1973). DNA polymerizing activity of the enzyme in this strain is virtually undetectable if the cells are grown at 42°C, but if grown at 30°C it is sufficiently present. By mutagenizing this strain, and after appropriate screening, we obtained mutants no longer able to grow at 42°, but able to do so when the normally functioning polA gene is present. One of them, called TS41, was most extensively studied. It acquired a mutation named pdeB41 which was found to be located between ilv and metE on the E. coli linkage map. Its phenotype is pleiotropic. The mutation by itself, i.e., if present in a polA ⁺ cell, does not kill the cell at 42°, but does so as in TS41 when it is reconstructed into a pdeB41 polA1 sup-126 triple mutant by P1 transduction. The mutation by itself renders the cell sensitive to UV, and tolerant to phage deficient in recombination. It is also a mutator.     

Prophage induction in Escherichia coli K12 cells deficient in DNA polymerase I.

Summary The induction of prophage by ultraviolet light has been measured inE. coli K12 lysogenic cells deficient in DNA polymerase I. The efficiency of the induction process was greater inpolA1 polC(dnaE) double mutants incubated at the temperature that blocks DNA replication than inpolA ⁺ polC single mutants. Similarly, thepolA1 mutation sensitizedtif-promoted lysogenic induction in apolA1 tif strain at 42°. In strains bearing thepolA12 mutation, which growth normally at 30°, induction of the prophage occured after the shift to 42°. It is concluded that dissapearance of the DNA polymerase I activity leads to changes in DNA replication that are able, per se, to trigger the prophage induction process.     

Extrachromosomal inheritance in Schizosaccharomyces pombe. II. Evidence for extrakaryotically inherited respiratory deficient mutants.

Summary In contrast to the wild-type, mutant [ANT ^R8] is able spontaneously to throw off stable respiratory deficient mutants. The frequency of these mutants is considerably enhanced by treatment with ethidium bromide (EB) or the azo-dye Janus green (JG).An unstable cell state with a petite-like phenotype is found in both mutant [ANT ^R8] and wild-type after EB-treatment. However, only in the mutant is this unstable cell state followed by the appearance of stable respiratory deficient (RD) mutants. Formation of microcolonies is observed both in [ANT ^R8] and wild-type.RD mutants were isolated after EB treatment. Three of them (mit-12, mit-25, and mit-30) were analyzed and mit-25 characterized in more detail.Mutant mit-25 shows mitotic segregation in the diploid state, indicating non-Mendelian mode of inheritance.The results of haploidization experiments also indicate extrachromosomal inheritance. Mit-25 shows a spontaneous rate of reversion of 10^-6, which can considerably increased by EB. Mit-25 possesses enzymatically active complexes I, II, and III of the respiratory chain (the latter without cytochrome b-566), lacks complex IV and binds antimycin, showing that mitochondrial protein synthesis is functional. Several lines of evidence presented in this paper make it very likely that the lesion in the mutant mit-25 is a point mutation in mitochondrial DNA.     

Male sterility associated with APRT deficiency in Arabidopsis thaliana results from a mutation in the gene APT1

Four mutants of Arabidopsis thaliana that are deficient in adenine phosphoribosyl transferase (APRT) activity have been isolated by selecting for germination of seeds and growth of the plantlets on a medium containing 2,6-diaminopurine (DAP), a toxic analog of adenine. In all mutants, DAP resistance is due to a recessive nuclear mutation at a locus designated apt. The mutants are male sterile due to pollen abortion after meiosis. Furthermore, it has been shown that metabolism of cytokinins is impaired in the mutant BM3, which has the lowest level of APRT activity among the mutants tested. However, three different cDNAs encoding APRT have been isolated in A. thaliana and this raised the question of the nature of the mutation which results in low APRT activity. The mutation was genetically mapped to chromosome I and lies within 6 cM of the phenotypic marker dis2, indicating that the mutation affects the APT1 gene, a result confirmed by sequencing of mutant alleles. The mutation in the allele apt1-3 is located at the 5′ splicing site of the third intron, and eliminates a BstNI restriction site, as verified by Southern blotting and PCR fragment length analysis. Received: 20 February 1997 / Accepted: 28 August 1997     

Construction of recA mutants of Acholeplasma laidlawii by insertional inactivation with a homologous DNA fragment

Mycoplasmas (class Mollicutes) are wall-less prokaryotes phylogenetically related to gram-positive bacteria. This study describes the construction of recA mutants of the mycoplasma Acholeplasma laidlawii. An internal fragment of the recA gene from A. laidlawii was cloned into a plasmid that does not replicate in this organism. When this plasmid construct was used to transform A. laidlawii, it inserted into the chromosome, disrupting the recA gene. The pheno-type of the resulting recA mutant was compared to that of wild-type cells and to that of a strain that has a naturally occurring ochre mutation in its recA gene. As found in other bacterial systems, loss of RecA activity resulted in cells deficient in DNA repair.     

Gene affecting longevity of messenger RNA: a mutant of Escherichia coli with altered mRNA stability.

Summary we have screened 897 temperature sensitive growth mutants ofE. coli for mutant strains showing longer mRNA half-life. The fate of pulse-labelled RNA was examined at 42° C after cessation of RNA synthesis and with prior exposure to nonpermissive temperature (42° C). Eight stains showed altered turn-over of RNA (presumably mRNA), and further analysis on mutant strain JE15144 indicated that the stability of pulse-labeled RNA as well as of tryptophan (trp) mRNA increased four to seven fold over its parental strain at 42° C. At 4 min or 10 min after addition of rifampicin, some 70 to 80% of polyribosome in the growing cells could still be conserved in JE15144 cultured at the nonpermissive temperature while little, if any, polyribosomes remained in its parental strain (PA3092) under the same condition. Two generation times were required for complete stoppage of growth of this mutant strain after shifting to 42° C, and protein synthesis continued at a significant, but slightly reduced, rate at 42° C. However, functional decay of mRNA in the mutant strain, with respect to the capacity for producing peptides, appeared to be similar to the parent strain, with half-lives of 3.5 min in PA3092 and 4.7 min in JE15144.     

Photocontrol of anthocyanin biosynthesis in tomato

Juvenile anthocyanin biosynthesis has been studied in dark-grown seedlings of tomato (Lycopersicon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. During a subsequent 24-hr period of monochromatic irradiation at different fluence rates of red light (R) the fluence-rate response relationships for induction of anthocyanin in all the WTs are similar, yet complex, showing a response at low fluence rates (LFRR) followed by a fluence rate-dependent high irradiance response (HIR). In the hypocotyl this response is restricted to the sub-epidermal layer of cells. The high-pigment-1 (hp-1) mutant exhibits a strong amplification of both response components. Theatroviolacea (atv) mutant shows strongest amplification of the HIR component. In contrast, a transgenic line overexpressing an oat phytochrome A gene (PHYA3 ⁺) shows a most dramatic amplification of the LFRR component. The far-red light (FR)-insensitive (fri) mutant, deficient in phytochrome A (phyA), lacks the LFRR component whilst retaining a normal HIR. The temporarily R-insensitive (tri) mutant, deficient in phytochrome B1 (phyB1) retains the LFRR, but lacks the HIR. Thehp-1,fri andhp-1,tri double mutant, exhibit amplified, yet qualitatively similar responses to the monogenicfri andtri mutants. Thefri,tri double mutant lacks both response components in R, but a residual response to blue light (B) remains. Similarly, theaurea (au) mutant deficient in phytochrome chromophore biosynthesis and presumably all phytochromes, lacks both response components in the R and FR regions of the spectrum. Experiments at other wavelengths demonstrate that while there is only a small response in the FR spectral region (729 nm) in tomato, there is an appreciable HIR response in the near FR at 704 nm, which is retained in thetri mutant. This suggests that the labile phyA pool participates in the HIR at this wavelength. The intense pigmentation (Ip) mutant appears to be specifically deficient in the B1 induced anthocyanin biosynthesis. Adult plants, grown under fluorescent light/dark cycles, show a reduction of anthocyanin content of young developing leaves upon application of supplemtary or end-of-day FR. The involvement of different phytochrome species in anthocyanin biosynthesis based on micro-injection studies into theau mutant and studies using type specific phytochrome mutants is discussed.     

Cold-sensitive growth of a mutant of Escherichia coli with an altered ribosomal protein S8: analysis of revertants.

Summary 26 cold-resistant revertants of a cold-sensitiveEscherichia coli mutant with an altered ribosomal protein S8 were analyzed for their ribosomal protein pattern by two-dimensional polyacrylamide gel electrophoresis. It was found that 16 of them had acquired the apparent wild-type form of protein S8, one exhibits a more strongly altered S8 than the original mutant and two revertants regained the wild-type form of S8 and, in addition, possess alterations in protein L30. The ribosomes of the residual revertants showed no detectable difference from those of the parental S8 mutant.The mutation leading to the more strongly altered S8 was genetically not separable from the primary S8 mutation; this indicates that both mutations are very close to each other or at the same site. The structural gene for ribosomal protein L30 was mapped relative to two other ribosomal protein genes (for proteins S5 and S8) by the aid of one of the L30 mutants: The relative order obtained is:aroE....rpmD(L30)....rpsE(S5)....rpsH(S8)....THe L30 mutation impairs growth and ribosomal assembly at 20°C and is therefore the first example of a mutant with a defined 50S alteration that has (partial) cold-sensitive ribosome assembly. A double mutant was constructed which possesses both the S8 and the L30 mutations. It was found that the L30 mutation had a slight antagonistic effect on the growth inhibition caused by the S8 mutation. Thus the L30 mutants might have possibly arisen from the original S8 mutants first as S8/L30 double mutants which was followed by the loss of the original S8 lesion.