The development of tolerance to dodine in Venturia inaequalis

The screening in vitro of large numbers of conidia of Venturia inaequalis against the selection pressure of dodine revealed a spontaneous mutation rate to dodine tolerance of about 1 in 106 conidia screened. Mutants cultured as mycelial disks grew 53–92% as well on a medium containing 0.5 ppm dodine as on a dodine-free medium. A comparable level of tolerance was developed by the wild type after a period of continuous vegetative growth on the dodine medium, but, unlike the mutants, the wild type lost its tolerance when cultured briefly on medium containing no fungicide. One of the seven mutants exhibited the capacity to adapt to increasingly higher concentrations of dodine. It is postulated that the events leading to the development of tolerance to dodine in V. inaequalis may be not only mutational and stable, but also adaptive and reversible, or a combination of both mechanisms.

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Potyviral 6K2 Protein Long-Distance Movement and Symptom-Induction Functions Are Independent and Host-Specific

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2004.17.5.502 ◽

2004 ◽

Vol 17 (5) ◽

pp. 502-510 ◽

Cited By ~ 28

Author(s):

Carl Spetz ◽

Jari P. T. Valkonen

Keyword(s):

Spontaneous Mutation ◽

Systemic Infection ◽

Wild Type ◽

Virus Propagation ◽

Long Distance ◽

Potato Virus A ◽

Viral Mutant ◽

Membrane Bound ◽

Long Distance Movement

Deletion of various portions, or insertion of six histidine residues (6×His) into various positions of the membrane-bound 6K2 protein (53 amino acids) of Potato virus A (PVA, genus Potyvirus), inhibited systemic infection in Nicotiana tabacum and N. benthamiana plants. However, a spontaneous mutation (Gly2Cys) that occurred in 6K2 adjacent to the 6×His insert placed between Ser1 and Gly2 enabled systemic infection in a single N. benthamiana plant. No symptoms were observed, but virus titers were similar to the symptom-inducing wild-type (wt) PVA. N. tabacum plants were not systemically infected, albeit virus propagation was observed in inoculated protoplasts. The 6×His/Gly2Cys mutant was reconstructed in vitro and serially propagated by mechanical inoculation in N. benthamiana. Following the third passage, a novel viral mutant appeared, lacking the last four His residues of the insert, as well as the Gly2 and Thr3 of 6K2. It infected N. tabacum plants systemically, and in the systemically infected N. benthamiana leaves, vein chlorosis and mild yellowing symptoms were observed, typical of wt PVA infection. The mutant virus accumulated to titers similar to wt PVA in both hosts. These results show that the PVA 6K2 protein affects viral long-distance movement and symptom induction independently and in a host-specific manner.

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Functional Analysis of the NucS/EndoMS of the Hyperthermophilic Archaeon Sulfolobus islandicus REY15A

Frontiers in Microbiology ◽

10.3389/fmicb.2020.607431 ◽

2020 ◽

Vol 11 ◽

Author(s):

Sohail Ahmad ◽

Qihong Huang ◽

Jinfeng Ni ◽

Yuanxi Xiao ◽

Yunfeng Yang ◽

...

Keyword(s):

Mismatch Repair ◽

Mutation Rate ◽

Spontaneous Mutation ◽

Repair Pathway ◽

Wild Type ◽

Spontaneous Mutation Rate ◽

Hyperthermophilic Archaeon ◽

Cleavage Activity ◽

Sulfolobus Islandicus ◽

Mismatch Repair Pathway

EndoMS is a recently identified mismatch specific endonuclease in Thermococcales of Archaea and Mycobacteria of Bacteria. The homologs of EndoMS are conserved in Archaea and Actinobacteria, where classic MutS-MutL-mediated DNA mismatch repair pathway is absent or non-functional. Here, we report a study on the in vitro mismatch cleavage activity and in vivo function of an EndoMS homolog (SisEndoMS) from Sulfolobus islandicus REY15A, the model archaeon belonging to Crenarchaeota. SisEndoMS is highly active on duplex DNA containing G/T, G/G, and T/T mismatches. Interestingly, the cleavage activity of SisEndoMS is stimulated by the heterotrimeric PCNAs, and when Mn2+ was used as the co-factor instead of Mg2+, SisEndoMS was also active on DNA substrates containing C/T or A/G mismatches, suggesting that the endonuclease activity can be regulated by ion co-factors and accessory proteins. We compared the spontaneous mutation rate of the wild type strain REY15A and ∆endoMS by counter selection against 5-fluoroorotic acid (5-FOA). The endoMS knockout mutant had much higher spontaneous mutation rate (5.06 × 10−3) than that of the wild type (4.6 × 10−6). A mutation accumulation analysis also showed that the deletion mutant had a higher mutation occurrence than the wild type, with transition mutation being the dominant, suggesting that SisEndoMS is responsible for mutation avoidance in this hyperthermophilic archaeon. Overexpression of the wild type SisEndoMS in S. islandicus resulted in retarded growth and abnormal cell morphology, similar to strains overexpressing Hje and Hjc, the Holliday junction endonucleases. Transcriptomic analysis revealed that SisEndoMS overexpression led to upregulation of distinct gene including the CRISPR-Cas IIIB system, methyltransferases, and glycosyltransferases, which are mainly localized to specific regions in the chromosome. Collectively, our results support that EndoMS proteins represent a noncanonical DNA repair pathway in Archaea. The mechanism of the mismatch repair pathway in Sulfolobus which have a single chromosome is discussed.

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Allorestricted cytotoxic T cells. Large numbers of allo-H-2Kb-restricted antihapten and antiviral cytotoxic T cell populations clonally develop in vitro from murine splenic precursor T cells.

Journal of Experimental Medicine ◽

10.1084/jem.162.2.592 ◽

1985 ◽

Vol 162 (2) ◽

pp. 592-606 ◽

Cited By ~ 12

Author(s):

J Reimann ◽

D Kabelitz ◽

K Heeg ◽

H Wagner

Keyword(s):

T Cells ◽

High Probability ◽

Cytotoxic T Lymphocyte ◽

Cytotoxic T Cells ◽

Large Fraction ◽

Cytotoxic T Cell ◽

Wild Type ◽

Large Numbers ◽

Simplex Virus

Cytotoxic T lymphocyte (CTL) responses of splenic T cells from C57BL/6 B6) mice and mutant H-2Kbm1 (bm1) mice to haptenic (trinitrophenyl [TNP] ) and herpes simplex virus (HSV) determinants in the context of an allogenic (wild-type or mutant) H-2Kb molecule were analyzed in a modified limiting dilution system. In the B6-anti-bm1TNP mixed leukocyte reaction (MLR), estimated frequencies for precursors of CTL clones that lysed bm1TNP targets ranged from 1/120 to 1/400; in the bm1-anti-B6TNP MLR, estimated frequencies of precursors of CTL clones that lysed B6TNP targets ranged from 1/500 to 1/1,300. Estimated frequencies for precursors of CTL clones that lysed the respective unmodified and TNP-modified allogeneic targets were two- to three-fold lower. Lytic specificity patterns determined by split-well analysis showed that at least 20-30% of the generated CTL populations (selected for a high probability of clonality) in both MLR displayed allorestricted lysis of TNP-modified concanavalin A blast targets. In the B6-anti-bm1HSV MLR, estimated frequencies for precursors of CTL clones that lysed bm1HSV targets ranged from 1/70 to 1/300; in the bm1-anti-B6HSV MLR, estimated frequencies for precursors of CTL clones that lysed B6HSV targets ranged from 1/300 to 1/1,200. Again, estimated frequencies for precursors of CTL clones that lysed the respective noninfected and virus-infected allogeneic targets were two- to fourfold lower. Of the CTL populations selected for a high probability of clonality at least 30-60% displayed allorestricted lysis of virus-infected lipopolysaccharide blast targets in both MLR. It is concluded that a large fraction of clonally developing CTL populations stimulated with TNP-modified or HSV-infected allo-H-2Kb-bearing cells displayed an allorestricted pattern of recognition. It was further evident that the estimated frequencies of splenic precursors that generated allorestricted CTL clones was two- to threefold higher than the estimated frequencies of precursors that gave rise to the respective alloreactive CTL populations.

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The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana regulates formation of a symmetric lamina, establishment of venation and repression of meristem-related homeobox genes in leaves

Development ◽

10.1242/dev.128.10.1771 ◽

2001 ◽

Vol 128 (10) ◽

pp. 1771-1783 ◽

Cited By ~ 13

Author(s):

E. Semiarti ◽

Y. Ueno ◽

H. Tsukaya ◽

H. Iwakawa ◽

C. Machida ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Apical Meristem ◽

Homeobox Genes ◽

Free Medium ◽

Wild Type ◽

Leaf Lamina ◽

Chain Reaction ◽

Class 1 ◽

Polymerase Chain

The asymmetric leaves2 (as2) mutant of Arabidopsis thaliana generated leaf lobes and leaflet-like structures from the petioles of leaves in a bilaterally asymmetric manner. Both the delayed formation of the primary vein and the asymmetric formation of secondary veins were apparent in leaf primordia of as2 plants. A distinct midvein, which is the thickest vein and is located in the longitudinal center of the leaf lamina of wild-type plants, was often rudimentary even in mature as2 leaves. However, several parallel veins of very similar thickness were evident in such leaves. The complexity of venation patterns in all leaf-like organs of as2 plants was reduced. The malformed veins were visible before the development of asymmetry of the leaf lamina and were maintained in mature as2 leaves. In vitro culture on phytohormone-free medium of leaf sections from as2 mutants and from the asymmetric leaves1 (as1) mutant, which has a phenotype similar to that of as2, revealed an elevated potential in both cases for regeneration of shoots from leaf cells. Analysis by the reverse transcription-polymerase chain reaction showed that transcripts of the KNAT1, KNAT2 and KNAT6 (a recently identified member of the class 1 knox family) genes accumulated in the leaves of both as2 and as1 plants but not of wild type. Transcripts of the STM gene also accumulated in as1 leaves. These findings suggest that, in leaves, the AS2 and AS1 genes repress the expression of these homeobox genes, which are thought to maintain the indeterminate cell state in the shoot apical meristem. Taken together, our results suggest that AS2 and AS1 might be involved in establishment of a prominent midvein and of networks of other veins as well as in the formation of the symmetric leaf lamina, which might be related to repression of class 1 knox homeobox genes in leaves.

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Emergence of Secretion-Defective Sublines of Pseudomonas aeruginosa PAO1 Resulting from Spontaneous Mutations in the vfr Global Regulatory Gene

Applied and Environmental Microbiology ◽

10.1128/aem.02539-07 ◽

2008 ◽

Vol 74 (6) ◽

pp. 1902-1908 ◽

Cited By ~ 16

Author(s):

Áine Fox ◽

Dieter Haas ◽

Cornelia Reimmann ◽

Stephan Heeb ◽

Alain Filloux ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Extracellular Enzymes ◽

Spontaneous Mutation ◽

Regulatory Gene ◽

Selective Advantage ◽

Wild Type ◽

Strain Pao1 ◽

Cultivation In Vitro

ABSTRACT Pseudomonas aeruginosa undergoes spontaneous mutation that impairs secretion of several extracellular enzymes during extended cultivation in vitro in rich media, as well as during long-term colonization of the cystic fibrosis lung. A frequent type of strong secretion deficiency is caused by inactivation of the quorum-sensing regulatory gene lasR. Here we analyzed a spontaneously emerging subline of strain PAO1 that exhibited moderate secretion deficiency and partial loss of quorum-sensing control. Using generalized transduction, we mapped the secretion defect to the vfr gene, which is known to control positively the expression of the lasR gene and type II secretion of several proteases. We confirmed this secretion defect by sequencing and complementation of the vfr mutation. In a reconstruction experiment conducted with a 1:1 mixture of wild-type strain PAO1 and a vfr mutant of PAO1, we observed that the vfr mutant had a selective advantage over the wild type after growth in static culture for 4 days. Under these conditions, spontaneous vfr emerged in a strain PAO1 population after four growth cycles, and these mutants accounted for more than 40% of the population after seven cycles. These results suggest that partial or complete loss of quorum sensing and secretion can be beneficial to P. aeruginosa under certain environmental conditions.

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A DNA adenine methylase mutant of Shigella flexneri shows no significant attenuation of virulence

Microbiology ◽

10.1099/mic.0.26781-0 ◽

2004 ◽

Vol 150 (4) ◽

pp. 1073-1078 ◽

Cited By ~ 12

Author(s):

Yasuko Honma ◽

Reinaldo E. Fernández ◽

Anthony T. Maurelli

Keyword(s):

Shigella Flexneri ◽

Spontaneous Mutation ◽

Bacterial Pathogen ◽

Wild Type ◽

Vaccine Strategy ◽

Cell Monolayers ◽

Dna Adenine Methylase ◽

Significant Attenuation

Mutants of Salmonella defective in DNA adenine methylase (dam) have been reported to be attenuated for virulence and to provide protective immunity when used as vaccine strains. To determine whether these observations could be extended to Shigella, a dam mutant of Shigella flexneri 2a was characterized and examined for the role of dam in pathogenesis. The Shigella dam mutant showed some unique characteristics; however, it retained virulence in vivo as well as in vitro. The mutant invaded cultured L2 monolayer cells as efficiently as the wild-type parent, but its intracellular growth was suppressed up to 7 h post-invasion. Furthermore, the invading dam mutant formed smaller plaques in cell monolayers compared to the parent strain. However, the mutant produced keratoconjunctivitis in the Sereny test in guinea pigs only slightly more slowly than the wild-type. While the effect of the dam mutation on virulence was modest, the rate of spontaneous mutation in the dam mutant was 1000-fold greater compared with the wild-type. The virulence and high mutability displayed by the dam mutant of Sh. flexneri suggest that a general anti-bacterial pathogen vaccine strategy based on mutations in dam needs to be re-evaluated.

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THE ORIGIN OF SPONTANEOUS MUTATION IN SACCHAROMYCES CEREVISIAE

Genetics ◽

10.1093/genetics/96.4.819 ◽

1980 ◽

Vol 96 (4) ◽

pp. 819-839 ◽

Cited By ~ 1

Author(s):

Siew-Keen Quah ◽

R C von Borstel ◽

P J Hastings

Keyword(s):

Mutation Rate ◽

Spontaneous Mutation ◽

Repair System ◽

Wild Type ◽

Spontaneous Mutation Rate ◽

Double Mutant Strain ◽

In The Wild ◽

Radiation Resistant ◽

Almost All

ABSTRACT Characterization of two antimutator loci in yeast shows that both are members of the same mutagenic repair system known to be responsible for almost all induced mutation (Lawrence and Christensen 1976, 1979a,b; Prakash 1976). One of the these newly isolated antimutator mutations is an allele of rev3 (Lemontt 1971b). Two other alleles of rev3 were tested and were also found to be antimutators. Double mutants carrying rev3 and mutator mutations of rad3, rad51 or rad18 are like rev3 single mutants with respect to spontaneous mutation rate, supporting the hypothesis (Hastings, Quah and von Borstel 1976) that many mutators in yeast act by channelling spontaneous lesions from accurate to mutagenic repair. However, the enhanced mutation rate seen in a radiation-resistant mutator mutant mut1 is not dependent on REV3, but is dependent on another gene designated ANT1. An additive effect on the reduction in spontaneous mutation, seen in the ant1 rev3 double-mutant strain, leads to the conclusion that at least 90% of spontaneous mutations seen in the wild type are caused by mutagenic repair of spontaneous lesions.

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Bioluminescence screening in vitro (Bio-Siv) assays for high-volume antimycobacterial drug discovery.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.6.1536 ◽

1996 ◽

Vol 40 (6) ◽

pp. 1536-1541 ◽

Cited By ~ 40

Author(s):

T M Arain ◽

A E Resconi ◽

M J Hickey ◽

C K Stover

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Discovery ◽

High Volume ◽

Luciferase Gene ◽

Direct Identification ◽

Mycobacterium Intracellulare ◽

Large Numbers ◽

Antimycobacterial Agents ◽

Screening In Vitro

Bioluminescence-based assays to indicate antimicrobial susceptibility have been developed and validated for recombinant strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium avium, and Mycobacterium intracellulare expressing an integrated eukaryotic luciferase gene. MICs determined with these bioluminescence assays for several antimycobacterial agents, including isoniazid, ethambutol, rifampin, amikacin, streptomycin, ciprofloxacin, and clarithromycin, compared favorably with traditional BACTEC methods and visual estimations of the inhibitory end point. Assay methodology has been optimized for the analysis of large numbers of novel compounds and is simple, inexpensive, and labor efficient. The availability of these four recombinant mycobacteria has permitted a strategy for drug discovery employing the nonpathogenic BCG strain for mass screening purposes with subsequent confirmation of activity against the pathogenic mycobacteria. Furthermore, evidence suggests that the BCG-based screen may allow the direct identification of bactericidal agents.

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Analysis of the roles of cysteine proteinases of Leishmania mexicana in the host–parasite interaction

Parasitology ◽

10.1017/s0031182099006435 ◽

2000 ◽

Vol 121 (4) ◽

pp. 367-377 ◽

Cited By ~ 41

Author(s):

M. J. FRAME ◽

J. C. MOTTRAM ◽

G. H. COOMBS

Keyword(s):

Time Course ◽

Cysteine Proteinase ◽

Mammalian Host ◽

Leishmania Mexicana ◽

Wild Type ◽

Large Numbers ◽

Host Parasite

Promastigotes of Leishmania mexicana mutants lacking the multicopy CPB cysteine proteinase genes (ΔCPB) are markedly less able than wild-type parasites to infect macrophages in vitro. ΔCPB promastigotes invade macrophages in large numbers but are unable to survive in the majority of the cells. In contrast, ΔCPB amastigotes invade and survive within macrophages in vitro. This extreme in vitro stage-specific difference was not mimicked in vivo; both promastigotes and amastigotes of ΔCPB produced lesions in BALB/c mice, but in each case the lesions grew considerably more slowly than those caused by wild-type parasites and only small lesions resulted. Inhibition of CPB in situ using cell-permeant peptidyldiazomethylketones had no measurable effect on parasite growth or differentiation axenically in vitro. In contrast, N-benzoyloxycarbonyl-phe-ala-diazomethylketone reduced the infectivity of wild-type parasites to macrophages by 80%. Time-course experiments demonstrated that application of the inhibitor caused effects not seen with ΔCPB, suggesting that CPB may not be the prime target of this inhibitor. The data show that the CPB genes of L. mexicana encode enzymes that have important roles in intracellular survival of the parasite and more generally in its interaction with its mammalian host.

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Participation of RecJ in the base excision repair pathway of Deinococcus radiodurans

Nucleic Acids Research ◽

10.1093/nar/gkaa714 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9859-9871

Author(s):

Kaiying Cheng ◽

Ying Xu ◽

Xuanyi Chen ◽

Huizhi Lu ◽

Yuan He ◽

...

Keyword(s):

Base Excision Repair ◽

Deinococcus Radiodurans ◽

Excision Repair ◽

Spontaneous Mutation ◽

Nuclease Activity ◽

Binding Pocket ◽

Spontaneous Mutation Rate ◽

Base Excision

Abstract RecJ reportedly participates in the base excision repair (BER) pathway, but structural and functional data are scarce. Herein, the Deinococcus radiodurans RecJ (drRecJ) deletion strain exhibited extreme sensitivity to hydrogen peroxide and methyl-methanesulphonate, as well as a high spontaneous mutation rate and an accumulation of unrepaired abasic sites in vivo, indicating the involvement of drRecJ in the BER pathway. The binding affinity and nuclease activity preference of drRecJ toward DNA substrates containing a 5′-P-dSpacer group, a 5′-deoxyribose-phosphate (dRP) mimic, were established. A 1.9 Å structure of drRecJ in complex with 5′-P-dSpacer-modified single-stranded DNA (ssDNA) revealed a 5′-monophosphate binding pocket and occupancy of 5′-dRP in the drRecJ nuclease core. The mechanism for RecJ 5′-dRP catalysis was explored using structural and biochemical data, and the results implied that drRecJ is not a canonical 5′-dRP lyase. Furthermore, in vitro reconstitution assays indicated that drRecJ tends to participate in the long-patch BER pathway rather than the short-patch BER pathway.

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