Nikkomycin-resistant mutants ofMucor rouxii: physiological and biochemical properties

We isolated three nikkomycin-resistant mutants of the dimorphic fungusM. rouxii which were physiologically characterized regarding their response to yeast-phase inducing conditions and their sensitivity to bacilysin. Mutant strains G21 and G23, showed a qualitatively normal, though delayed, dimorphic transition and partial cross-resistance to bacilysin. Mutant strain G27 showed an altered dimorphism, producing a high proportion (50%) of hyphal cells, and a wild-type sensitivity to bacilysin. Cell-free extracts from this mutant exhibited an activity of both basal and protease-activated chitin synthetase which was overexpressed as compared with the parental strain and mutants G21 and G23. Results are discussed in terms of the different genetic background of the mutants.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - UDP-GlcNAc uridine 5-diphospho-N-acetylglucosamine - GlcNAc N-acetylglucosamine     

Mycolytic effect of extracellular enzymes of antagonistic microbes to Colletotrichum falcatum,red rot pathogen of sugarcane

Strains of selected bacteria and Trichoderma harzianum isolated from sugarcane rhizosphere and endosphere regions were tested for the production of chitinolytic enzymes and their involvement in the suppression of Colletotrichum falcatum, red rot pathogen of sugarcane. Among several strains tested for chitinolytic activity, 12 strains showed a clearing zone on chitin-amended agar medium. Among these, bacterial strains AFG2, AFG 4, AFG 10, FP7 and VPT4 and all the tested T. harzianum strains produced clearing zones of a size larger than 10 mm. The antifungal activity of these strains increased when chitin was incorporated into the medium. Trichoderma harzianum strain T5 showed increased levels of activity of N-acetylglucosaminidase and -1,3-glucanase when grown on minimal medium containing chitin or cell wall of the pathogen. Lytic enzymes of bacterial strains AFG2, AFG4, VPT4 and FP7 and T. harzianum T5 inhibited conidial germination and mycelial growth of the pathogen. Enzymes from T. harzianum T5 were found to be the most effective in inhibiting the fungus. When mycelial discs of the pathogen were treated with the enzymes, electrolytes were released from fungal mycelia. The results indicated that antagonistic T. harzianum T5 caused a higher level of lysis of the pathogen mycelium, and the inhibitory effect was more pronounced when the lytic enzymes were produced using chitin or cell wall of the pathogen as carbon source.     

Molecular characterization of thirteen gyrA mutations conferring nalidixic acid resistance in Bacillus subtilis

We isolated 607 independent nalidixic acid-resistant mutants from Bacillus subtilis. A 163 by DNA segment from a 5 portion of the gyrA gene was amplified from the DNA of each mutant strain. After heat denaturation, the product was subjected to gel electrophoresis to detect conformational polymorphism of single-strand DNA (PCR-SSCP analysis). Mobility patterns of the two DNA strands from all the mutant strains examined differed from those of the parental wild-type strains. The patterns were classified into 13 types, and the DNA sequence of each type was determined. A unique sequence alteration was found in mutants belonging to each of the 13 types, defining 13 gyrA alleles. Eight were single base pair substitutions, four were substitutions of two consecutive base pairs, and one was a substitution of three consecutive base pairs. Only three amino acid residues (Ser-84, Ala-85, and Glu-88) were altered in the deduced amino acid sequences of the mutated genes. We conclude that molecular typing based on the PCR-SSCP method is a powerful technique for the exhaustive identification of allelic variants among mutants selected for a phenotypic trait.     

Transmission of yeast mitochondrial loci to progeny is reduced when nearby intergenic regions containing ori sequences are deleted

Summary Mitochondrial DNAs (mtDNA) from four stable revertant strains generated from high frequency petite forming strains of Saccharomyces cerevisiae have been shown to contain deletions which have eliminated intergenic sequences encompassing ori1, ori2 and ori7. The deleted sequences are dispensable for expression of the respiratory phenotype and mutant strains exhibit the same relative amount of mtDNA per cell as the wild-type (wt) parental strain. These deletion mutants were also used to study the influence of particular intergenic sequences on the transmission of closely linked mitochondrial loci. When the mutant strains were crossed with the parental wt strains, there was a strong bias towards the transmission into the progeny of mitochondrial genomes lacking the intergenic deletions. The deficiency in the transmission of the mutant regions was not a simple function of deletion length and varied between different loci. In crosses between mutant strains which had non-overlapping deletions, wt mtDNA molecules were formed by recombination. The wt recombinants were present at high frequencies among the progeny of such crosses, but recombinants containing both deletions were not detected at all. The results indicate that mitochondrial genomes can be selectively transmitted to progeny and that two particular intergenic regions positively influence transmission. Within these regions other sequences in addition to ori/rep affect transmission.This paper is dedicated to colleagues J. Jana, D. Tasi, I. Bortner, and F. Zavrl     

Temperature sensitivity as a general phenomenon in a collection of chlorophyll-deficient mutants of sweetclover (Melilotus alba)

A collection of chlorophyll (Chl)-deficient mutants of sweetclover (Melilotus alba) with defects in eight nuclear loci were grown at 17 or 26° C. Plants grown at either temperature were examined for Chl content, Chla/b ratio, expression of the light-harvesting complex II (LHC-II) apoproteins, and protochlorophyllide (Pchlide) biosynthetic capacity. Except for thech4 mutant, the parental strain and all mutants accumulate more Chl when grown at 26° C than at 17° C. Thech5 mutants, lacking Chl b under any growth condition, and thech12 mutant showed little temperature-dependent phenotypic plasticity, whereas this was a marked phenomenon in the other mutants. Thech10 andch11 mutants demonstrated extreme temperature sensitivity with regard to the production of Chlb and the Chlb-binding LHC-II apoproteins. When excised trifoliolates were supplemented with exogenously supplied -aminolevulinic acid, only thech4 mutant was markedly impaired in the ability to produce Pchlide. These data indicate that temperature-sensitive phenotypic plasticity is a common phenomenon of chlorophyll-deficient mutants and substantiate that only a minority of Chl-deficient mutants is impaired in the biosynthesis of Chl.This research was supported by Grants GM84-CRCR-1-1479 (J.C.O.) and 89-00641 (J.M.) of the United States Department of Agriculture and by National Science Foundation Grant DMB87-03100 (J.M.). This is paper No. 8971, Nebraska Agricultural Research Division.     

chs-4, a class IV chitin synthase gene fromNeurospora crassa

InSaccharomyces cerevisiae, most of the cellular chitin is produced by chitin synthase III, which requires the product encoded by theCSD2/CAL1/DIT101/KT12 gene. We have identified, isolated and structurally characterized aCSD2/CAL1/DIT101/KT12 homologue in the filamentous ascomyceteNeurospora crassa and have used a reverse genetics approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of aN. crassa gene (designatedchs-4) encoding a polypeptide belonging to a class of chitin synthases which we have designated class IV. The predicted polypeptide encoded by this gene is highly similar to those ofS. cerevisiae andCandida albicans. N. crassa strains in whichchs-4 had been inactivated by the Repeat-Induced Point mutation (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in thechs-4 ^RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme inN. crassa and that, in contrast to yeasts, it is possible that filamentous fungi may have more than one class IV chitin synthase.A. Beth Din and C. A. Specht contributed equally to this work     

Chitin synthetase mutants of Phycomyces blakesleeanus

Mutants resistant to nikkomycin, an inhibitor of chitin biosynthesis, were isolated after exposure of wild-type spores of the fungus Phycomyces blakesleeanus to N-methyl-N-nitro-N-nitrosoguanidine. Genetic analysis revealed that nikkomycin resistance was due to mutations in a single gene, chsA. Mutants and wild type grew equally well in the absence of nikkomycin. In contrast to the wild type, whose spore germination and mycelial growth were inhibited by 5 M nikkomycin, chsA mutants grew reasonably well in the presence of 50 M nikkomycin. Chitin synthesis in vivo was much less affected by the drug in the mutants than in the wild type. Resistance was not due to impaired uptake or detoxification of the drug. Analysis of the kinetics of chitin synthesis in vitro showed that the mutants had a decreased K_a for the allosteric activator, N-acetylglucosamine, and gross alterations in nikkomycin inhibition kinetics. These results indicate that chsA is the structural gene for chitin synthetase, or at least for the polypeptide that bears the catalytic and allosteric sites.     

Citric acid production from cellobiose by 2-deoxyglucose-resistant mutant strains of Aspergillus niger in semi-solid culture

Citric acid production from cellobiose by Aspergillus niger was studied by a semi-solid culture method using bagasse as a carrier. From the parental strain Yang no. 2, mutant strains showing resistance to 2-deoxy-d-glucose (DG) on minimal medium containing glucose as a carbon source were induced. The representative mutant strain M155 was selected and subjected to further mutation. The new series of mutant strains showing resistance to DG on minimal medium containing cellobiose as a carbon source was induced, and among them the best mutant strain C192 showed higher citric acid productivity than Yang no. 2 in semi-solid culture when glucose was used as a carbon source. Moreover, in semi-solid culture, the strain C192 produced 49.6 g/l of citric acid, 1.6 times as much citric acid as Yang no. 2 produced, from 100 g cellobiose/l and showed enhanced -glucosidase production. In shake culture, the extracellular -glucosidase activity of C192 was higher than that of Yang no. 2 when not only cellobiose but also glucose and glycerol, catabolite repressors, were used as a carbon source. These results indicate that mutant strains such as C192 are insensitive to catabolite repression. Correspondence to: S. Usami     

Protection of apple against fire blight induced by an hrpL mutant of Erwinia amylovora

A regulatory hrpL non-virulent mutant of Erwinia amylovora is effective in controlling fire blight disease when inoculated on apple seedlings simultaneously with the pathogenic parental strain. Mechanisms involved in this protective effect were investigated. The use of two marker genes, uidA and lacZ, expressed in the hrpL mutant and the pathogenic strain, respectively, allowed to localize simultaneously the two inoculated strains in plant tissue. An anti-β-glucuronidase antibody was also used to detect the hrpL mutant. Both techniques indicated that the two strains localized mainly in separate areas of the leaf tissue. In addition, leaves infiltrated with the hrpL mutant exhibited a significant increase in peroxidase activity in contrast to a hrp secretion mutant known to be less effective in the protection. It is suggested that protection obtained with the hrpL mutant relies on the physical separation between the mutant and the parental strain after co-inoculation and the rapid and sustained activation of plant defense mechanisms in reactive tissue, i.e. not invaded by the virulent strain.     

Mycelial growth of strains of the genera <Emphasis Type="Italic">Suillus</Emphasis> and <Emphasis Type="Italic">Boletinus</Emphasis> in media with a wide range of concentrations of carbon and nitrogen sources

Suillus and Boletinus were studied using Ohta medium. In media with glucose or trehalose, all tested strains grew well. With mannose and cellobiose, strains generally grew well, except for one strain of Suillus. Utilization of dextrin and soluble starch differed with each strain, and that of sucrose and glycerol was low for all strains. Utilization of four amino acids, arginine, glutamic acid, aspartic acid, and alanine, was similar to that of ammonium tartrate for Suillus strains, but mycelial growth with amino acids was clearly lower than with ammonium tartrate for the Boletinus strain. The effect of glucose and ammonium tartrate concentrations for nine strains of the genera Suillus and Boletinus was studied with ranges for glucose of 1–100 and 200g/l, respectively, and for ammonium tartrate of 0.2–5 and 20g/l, respectively. Six strains showed maximal growth at a glucose concentration greater than 25g/l, and one strain showed maximal growth at 70g/l. The results indicate that these fungi are adapted to relatively high concentrations of carbon sources. In general, glucose concentration at mycelial growth maximum decreased as ammonium tartrate concentration increased, and at higher concentrations of glucose, mycelial growth decreased more rapidly in higher concentrations of ammonium tartrate.     

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.     

A novel eye morphology induced by a P element in somatic tissue of Drosophila melanogaster.

Summary We found a specific eye morphology designated as Square, which is induced when some Drosophila melanogaster strains harboring P elements are crossed with the 2–3 strain carrying a modified P element, P[ry ⁺, 2–3], which produces transposase in somatic tissue. This phenotype was dominant and also induced in the reciprocal crosses. Square was induced when the 2–3 strain was crossed with Q and M strains such as the sn^w (M) strain carrying three small P elements but not with P strains. Inheritance of Square was also tested and its phenotype was not transmitted to the next generation. These results suggest that Square is caused by the transposition of P elements in somatic cells.     

Strain improvement ofArthrobacter simplex by protoplast fusion

Anl-tryptophan auxotroph and milky mutants were derived from an inducible cholesterol oxidase-producing bacterium,Arthrobacter simplex USA18, via UV-mutagenesis. Protoplasts of these mutants and a constitutive cholesterol oxidase producer, strain US3011, were prepared by growing cells in the presence of ampicillin (20g ml^–1) followed by digestion with lysozyme. Protoplast fusion between tested strains with complementary characteristics was achieved in the presence of 20–40% polyethylene glycol 6000. The fusion frequency was about 1.5–1.7×10^–3. The cholesterol oxidase activity of four fusants in a cholesterol-containing medium was 20–60% higher than that of parental strains. This study demonstrated that protoplast fusion is applicable to strain improvement ofArthrobacter strains for enzyme production.     

Bacteriophage λ-mediated transposon mutagenesis of phytopathogenic and epiphytic Erwinia species is strain dependent

Summary Using transformation and conjugal mobilization, plasmids carrying the lamB gene of Escherichia coli were transferred to a range of Erwinia strains. The resultant strains were infected with 467, and kanamycin resistant transductants were screened for various mutant phenotypes including auxotrophy and altered extracellular enzyme activities. Reversion analysis suggested that most mutant phenotypes were due to Tn5 insertion. The applicability of the techniques was highly strain dependent. However a rapid and simple route to mutant isolation was obtained, which could allow the use of other -related genetic techniques in several important species which, to date, have not been genetically manipulated.     

Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains

Summary A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD ^- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.     

Introduction of a carboxyl group in the loop of the F0 c-subunit affects the H+/ATP coupling ratio of the ATP synthase from Synechocystis 6803

The proton translocation stoichiometry (H⁺/ATP ratio) was investigated in membrane vesicles from a Synechocystis 6803 mutant in which the serine at position 37 in the hydrophilic loop of the c-subunit from the wild type was replaced by a negatively charged glutamic acid residue (strain plc37). At this position the c-subunit of chloroplasts and the cyanobacterium Synechococcus 6716 already contains glutamic acid. H⁺/ATP ratios were determined with active ATP synthase in thermodynamic equilibrium between phosphate potential (G _p ) and the proton gradient ( _H ⁺) induced by acid–base transition. The mutant displayed a significantly higher H⁺/ATP ratio than the control strain (wild type with kanamycin resistance) at pH 8 (4.3 vs. 3.3); the higher ratio also being observed in chloroplasts and Synechococcus 6716. Furthermore, the pH dependence of the H⁺/ATP of strain plc37 resembles that of Synechococcus 6716. When the pH was increased from 7.6 to 8.4, the H⁺/ATP of the mutant increased from 4.2 to 4.6 whereas in the control strain the ratio decreased from 3.8 to 2.8. Differences in H⁺/ATP between the mutant and the control strain were confirmed by measuring the light-induced phosphorylation efficiency (P/2e), which changed as expected, i.e., the P/2e ratio in the mutant was significantly less than that in the wild type. The need for more H⁺ ions used per ATP in the mutant was also reflected by the significantly lower growth rate of the mutant strain. The results are discussed against the background of the present structural and functional models of proton translocation coupled to catalytic activity of the ATP synthase.     

Response to carbon-starvation in Pseudomonas aeruginosa strain IE-6S+: analysis of general cross protection,production of some nematocidal compounds in vitro,and the biological control of Meloidogyne javanica in tomato

Adaptation to nutrient-limited conditions by repeated culture on soil agar media was found to induce resistance to osmotic, oxidation, thermal and pH stress as well as carbon-limited culture conditions in Pseudomonas aeruginosa strain IE-6S⁺. Culture filtrate of the resistant strains obtained from 10% strength King's medium B (KMB) caused greater (32–54%) mortality of Meloidogyne javanica juveniles compared with their parental strain. When 10% strength KMB was amended with 1% (w/v) glucose, the ability to cause nematode mortality was substantially enhanced by adapted strains, while activity of the parental strain was repressed. Two of the four starved bacteria IE-6S⁺PBK1 and IE-6S⁺KUC2 grown in KMB liquid medium amended with glucose synthesized salicylic acid (5.1 and 5.8 g ml^–1, respectively) and hydrogen cyanide (picrate paper turned yellow to brownish red for both strains) in greater quantities compared to wild type strain (SA = 4.4 g ml^–1, picrate paper turned orange-yellow). Neither wild type strain IE-6S⁺ nor its adapted strains were capable of utilizing tomato root exudates as a sole carbon source. Strains adapted to carbon-limiting conditions exhibited enhanced colonization in the rhizosphere and inner root tissues of tomato compared to their exponentially growing counterpart. Pre-adapted bacterial inoculants applied in the soil also caused greater (15%) reduction in nematode penetration compared to the parental strain or controls.     

Cloning and expression of the α-amylase gene and oxytetracycline production in Streptomyces rimosus

An 8.4 kb Sau3AI DNA fragment containing the Streptomyces rimosus TM-55 -amylase gene (amy) was ligated to a vector pIJ702, named pCYL01, and cloned into amylase deficient mutant S. lividans M2 (amy ^–). Subcloning study showed that the amy gene was localized in 3.3 kbKpnI-PstI fragment. The molecular weight of the purified -amylases of S. lividans M2/pCYL01 and S. rimosus TM-55 were estimated to be 65.7 kDa. Different sizes of recombinant plasmids carrying the amy gene had been retransferred into the parental strain of S. rimosus TM-55. Among these S. rimosus transformants, TM-55/pCYL01, TM-55/pCYL12 and TM-55/pCYL36 showed amylase activity 1.36- to 2.05-fold at the seventh day (1.61 to 2.42 units vs 1.18 units), and oxytetracycline (OTC) production 2.00- to 2.50-fold at the ninth day (approximate 140 to 170 g ml^–1 vs 72 g ml^–1), higher than that of S. rimosus TM-55 alone, respectively. These results showed that industrial microorganisms could be improved by genetic and metabolic engineering.     

Characterization of an exopolysaccharide mutant of Nostoc spongiaeforme: Zn2+-sorption and uptake

Exposure of the exopolysaccharide (EPS)-synthesizing cyanobacterium Nostoc spongiaeforme to Zn²⁺ (20 M) transformed the biomass into white debris. However, a few blue–green pin-heads emerged after 2 weeks in the same Zn²⁺-containing medium and formed less mucoid microcolonies (1–2 mm) relative to the protruding colonies (2–4 mm) of the parent strain on nutrient agar. One of such survivors (designated as Zn₂₀) that was stable through 10 successive transfers in Zn²⁺-lacking medium has been adopted for further characterization. The parent strain retained almost 88% of the total EPS synthesized, the rest being released into the ambient medium, while for Zn₂₀, the EPS retained approximated to 74%. Although the Zn²⁺-sensitivity of the mutant was comparable with that of the parent (LD₅₀, 7 M), Zn²⁺ uptake was still 5-fold higher in the former (2 g mg^–1 biomass dry wt., 20 M, external concentration). Also, both the strains showed insignificant difference in Zn²⁺-sorption onto their isolated EPS. The mutant was characterized by having higher cell carbohydrate content (642.8 g mg^–1 dry wt.) than its parent (513.6 g). The X-ray diffraction pattern revealed Zn²⁺ deposition on EPS from the parent mainly as zinc hypophosphite monohydrate [Zn(H₂PO₂)₂·H₂O], whereas there was a lack of distinct peaks in similar samples from Zn₂₀, thus confirming the amorphous nature. There was participation in Zn²⁺ binding of only COO^–, N=O, NO₂, SO₂ groups in the parent while participation of P—O and C=O groups in mutant EPS was evident in IR spectra. The observations suggest that the mutant could be deployed to achieve sustained EPS synthesis, its release and metal sorption/desorption in repeated cycles.     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.