Extragenic Suppressors of a Dynein Mutation That Blocks Nuclear Migration in Aspergillus Nidulans

Cytoplasmic dynein is a large molecular weight protein complex that functions as a microtubule-dependent, negative, end-directed ``motor.' Mutations in nudA, which encodes the heavy chain of cytoplasmic dynein, inhibit nuclear migration in Aspergillus nidulans. This paper describes the selection and characterization of extragenic suppressors of the nudA1 mutation preparatory to the identification of other proteins that interact directly or indirectly with the cytoplasmic dynein heavy chain. To facilitate future cloning of the suppressor genes, we have searched particularly for extragenic suppressor mutations that also convey a selectable phenotype, such as cold or dimethyl sulfoxide sensitivity. Genetic analysis of 16 revertants has defined at least five extragenic suppressors of nudA1 (snaA-E). All the sna mutations except one were recessive in diploids homozygous for nudA1 and heterozygous for sna mutations. To characterize the nuclear migration phenotype in the sna mutants, conidia of one representative of each complementation group were germinated, fixed and nuclei stained. The sna mutants display partial suppression of the nudA1 nuclear migration defect. Although conidiophores were produced in the sna mutants, they failed to develop normally and to produce spores. Examination of the nudA1,sna conidiophores under the microscope showed that nuclear migration into the metulae and phialides was defective.     

Suppressors of Recb Mutations in Salmonella Typhimurium

Using a screen that directly assesses transductional proficiency, we have isolated suppressors of recB mutations in Salmonella typhimurium. The alleles of sbcB reported here are phenotypically distinct from those isolated in Escherichia coli in that they restore recombination proficiency (Rec(+)), resistance to ultraviolet light (UV(R)), and mitomycin C resistance (MC(R)) in the absence of an accompanying sbcCD mutation. In addition the sbcB alleles reported here are co-dominant to sbcB(+). We have also isolated insertion and deletion mutants of the sbcB locus. These null mutations suppress only the UV(S) phenotype of recB mutants. We have also isolated sbcCD mutations, which map near proC. These sbcCD mutations increase the viability, recombination proficiency and MC(R) of both the transductional recombination suppressors (sbcB1 & sbcB6) and the sbcB null mutations. S. typhimurium recB sbcB1 sbcCD8 strains are 15-fold more recombination proficient than wild-type strains. The increase in transductants in these strains is accompanied by a loss of abortive transductants suggesting that these fragments are accessible to the mutant recombination apparatus. Using tandem duplications, we have constructed sbcB merodiploids and found that, in a recB mutant sbcCD(+) genetic background, the sbcB(+) allele is dominant to sbcB1 for transductional recombination but co-dominant for UV(R) and MC(R). However, in a recB sbcCD8 genetic background, the sbcB1 mutation is co-dominant to sbcB(+) for all phenotypes. Our results lead us to suggest that the SbcB and SbcCD proteins have roles in RecBCD-dependent recombination.     

Extragenic Suppressors of Nudc3, a Mutation That Blocks Nuclear Migration in Aspergillus Nidulans

Nuclear migration plays an important role in the growth and development of many organisms including the filamentous fungus Aspergillus nidulans. We have cloned three genes from A. nidulans, nudA, nudC, and nudF, in which mutations affect nuclear migration. The nudA gene encodes the heavy chain of cytoplasmic dynein. The nudC gene encodes a 22-kD protein. The nudF gene was identified as an extracopy suppressor of the temperature sensitive (ts(-)) nudC3 mutation. The nudC3 mutation substantially decreases the intracellular concentration of the nudF protein at restrictive temperature. This is restored toward the normal level by an extra copy of nudF. To identify other genes whose products interact directly or indirectly with the NUDC protein, we have isolated a set of extragenic suppressors of the nudC3 temperature-sensitive mutation. Genetic analysis of 16 such extragenic suppressors showed them to represent nine different genes, designated sncA-sncI (for suppressor of nudC). sncA-sncH were either dominant or semidominant in diploids homozygous for nudC3 and heterozygous for the snc mutations. All of the suppressors reversed the ts(-) phenotype of nudC3 by restoring the intracellular concentration of the NUDF protein.     

Extragenic Suppressors of Saccharomyces Cerevisiae Prp4 Mutations Identify a Negative Regulator of Prp Genes

The PRP4 gene encodes a protein that is a component of the U4/U6 small nuclear ribonucleoprotein particle and is necessary for both spliceosome assembly and pre-mRNA splicing. To identify genes whose products interact with the PRP4 gene or gene product, we isolated second-site suppressors of temperature-sensitive prp4 mutations. We limited ourselves to suppressors with a distinct phenotype, cold sensitivity, to facilitate analysis of mutants. Ten independent recessive suppressors were obtained that identified four complementation groups, spp41, spp42, spp43 and spp44 (suppressor of prp4, numbers 1-4). spp41-spp44 suppress the pre-mRNA splicing defect as well as the temperature-sensitive phenotype of prp4 strains. Each of these spp mutations also suppresses prp3; spp41 and spp42 suppress prp11 as well. Neither spp41 nor spp42 suppresses null alleles of prp3 or prp4, indicating that the suppression does not occur via a bypass mechanism. The spp41 and spp42 mutations are neither allele- nor gene-specific in their pattern of suppression and do not result in a defect in pre-mRNA splicing. Thus the SPP41 and SPP42 gene products are unlikely to participate directly in mRNA splicing or interact directly with Prp3p or Prp4p. Expression of PRP3-lacZ and PRP4-lacZ gene fusions is increased in spp41 strains, suggesting that wild-type Spp41p represses expression of PRP3 and PRP4. SPP41 was cloned and sequenced and found to be essential. spp43 is allelic to the previously identified suppressor srn1, which encodes a negative regulator of gene expression.     

Extragenic Suppressors of the Arabidopsis Det1 Mutant Identify Elements of Flowering-Time and Light-Response Regulatory Pathways

Light regulation of seedling morphogenesis is mediated by photoreceptors that perceive red, far-red, blue and UV light. Photomorphogenetic mutants of Arabidopsis have identified several of the primary photoreceptors, as well as a set of negative regulators of seedling photomorphogenesis, including DET1, that appear to act downstream of the photoreceptors. To study the regulatory context in which DET1 acts to repress photomorphogenesis, we used a simple morphological screen to isolate extragenic mutations in six loci, designated ted (for reversal of the det phenotype), that partially or fully suppress the seedling morphological phenotype of det1-1. Genetic analyses indicate that mutations in the ted4 and ted5 loci identify new alleles of the previously described photomorphogenetic loci hy1 and hy5, respectively. Molecular analyses indicate that the ted mutations partially suppress the dark-grown gene expression phenotype of det1-1, and that the mechanism of suppression does not involve direct remediation of the splicing defect caused by the det1-1 mutation. The ted mutations also partially suppress the light-grown morphological phenotype of mature det1-1 plants, and ted1 and ted2 suppress a daylength insensitivity phenotype of det1. TED1, TED2 and TED3 are newly described genes, whose function appears closely associated with that of DET1. In addition, alleles of ted1 are associated with a moderate late-flowering phenotype, suggesting that TED1 plays a role in the pathways that regulate both seedling morphogenesis and the initiation of flowering.     

Extragenic Suppressors of Mutations in the Cytoplasmic C Terminus of Sec63 Define Five Genes in Saccharomyces Cerevisae

Mutations in the SEC63 gene of Saccharomyces cerevisiae affect both nuclear protein localization and translocation of proteins into the endoplasmic reticulum. We now report the isolation of suppressors of sec63-101 (formerly npl1-1), a temperature-sensitive allele of SEC63. Five complementation groups of extragenic mutations, son1-son5 (suppressor of npl1-1), were identified among the recessive suppressors. The son mutations are specific to SEC63, are not bypass suppressors, and are not new alleles of previously identified secretory (SEC61, SEC62, KAR2) or nuclear protein localization genes (NPL3, NPL4, NPL6). son1 mutations show regional specificity of suppression of sec63 alleles. At low temperatures, son1 mutants grow slowly and show partial mislocalization of nuclear antigens. The SON1 gene maps to chromosome IV and encodes a nuclear protein of 531 amino acids that contains two acidic stretches and a putative nuclear localization sequence. We show that son1 mutations suppress sec63-101 by elimination of Son1p function.     

Intragenic Suppressors of Folding Defects in the P22 Tailspike Protein

Within the amino acid sequences of polypeptide chains little is known of the distribution of sites and sequences critical for directing chain folding and assembly. Temperature-sensitive folding (tsf) mutations identifying such sites have been previously isolated and characterized in gene 9 of phage P22 encoding the tailspike endorhamnosidase. We report here the isolation of a set of second-site conformational suppressors which alleviate the defect in such folding mutants. The suppressors were selected for their ability to correct the defects of missense tailspike polypeptide chains, generated by growth of gene 9 amber mutants on Salmonella host strains inserting either tyrosine, serine, glutamine or leucine at the nonsense codons. Second-site suppressors were recovered for 13 of 22 starting sites. The suppressors of defects at six sites mapped within gene 9. (Suppressors for seven other sites were extragenic and distant from gene 9.) The missense polypeptide chains generated from all six suppressible sites displayed ts phenotypes. Temperature-sensitive alleles were isolated at these amber sites by pseudoreversion. The intragenic suppressors restored growth at the restrictive temperature of these presumptive tsf alleles. Characterization of protein maturation in cells infected with mutant phages carrying the intragenic suppressors indicates that the suppression is acting at the level of polypeptide chain folding and assembly.     

Inhibition of Growth by Amber Suppressors in Yeast

Strains of the yeast Saccharomyces cerevisiae that contain highly efficient amber (UAG) suppressors grow poorly on nutrient medium, while normal or nearly normal growth rates are observed when these strains lose the supressors or when the suppressors are mutated to lower efficiencies. The different growth rates account for the accumulation of mutants with lowered efficiencies in cultures of strains with highly efficient amber suppressors. Genetic analyses indicate that one of the mutations with a lowered efficiency of suppression is caused by an intragenic mutation of the amber supressor. The inhibition of growth caused by excessive suppression is expected to be exacerbated when appropriate suppressors are combined together in haploid cells if two suppressors act with a greater efficiency than a single suppressor. Such retardation of growth is observed with combinations of two UAA (ochre) suppressors (Gilmore 1967) and with combinations of two UAG suppressors when the efficiencies of each of the suppressors are within a critical range. In contrast, combinations of a UAA suppressor and a UAG suppressor do not affect growth rate. Apparently while either excessive UAA or excessive UAG suppression is deleterious to yeast, a moderate level of simultaneous UAA and UAG suppression is not.     

Extragenic Suppressors of Schizosaccharomyces Pombe Rad9 Mutations Uncouple Radioresistance and Hydroxyurea Sensitivity from Cell Cycle Checkpoint Control

Schizosaccharomyces pombe cells that contain a mutation within rad9 are sensitive to ionizing radiation, UV light and hydroxyurea, relative to wild-type strains. In addition, the mutants are moderately hypomutable by UV and unable to delay initiation of mitosis after treatment with radiation or hydroxyurea. Three radioresistant derivatives of rad9::ura4 cells were isolated, and each contained a single unique extragenic suppressor responsible for the acquired resistance. The suppressor loci also conferred radioresistance upon cells containing rad9-192, which differs from rad9(+) by a single base pair change. The suppressors additionally enhanced the radioresistance of cells containing rad3-136, a mutation that leads to phenotypes similar to those mediated by rad9::ura4. None of the derivatives of rad9::ura4 cells recovered the ability to delay cycling in G2 after exposure to ionizing radiation or UV light. All three suppressor derivatives, relative to the parental rad9::ura4 strain, also exhibited a moderate increase in resistance to the DNA replication inhibitor hydroxyurea without gaining the ability to stop progression into mitosis despite the inhibition of DNA synthesis. Results are discussed in terms of models to explain the putative role of rad9 and the suppressor genes in promoting radioresistance and mediating checkpoint controls responsive to DNA damage or incomplete DNA replication.     

Intragenic and Extragenic Suppressors of Temperature Sensitive Mutations in the Replication Initiation Genes dnaD and dnaB of Bacillus subtilis

Background

The Bacillus subtilis genes dnaD and dnaB are essential for the initiation of DNA replication and are required for loading of the replicative helicase at the chromosomal origin of replication oriC. Wild type DnaD and DnaB interact weakly in vitro and this interaction has not been detected in vivo or in yeast two-hybrid assays.

Methodology/Principal Findings

We isolated second site suppressors of the temperature sensitive phenotypes caused by one dnaD mutation and two different dnaB mutations. Five different intragenic suppressors of the dnaD23ts mutation were identified. One intragenic suppressor was a deletion of two amino acids in DnaD. This deletion caused increased and detectable interaction between the mutant DnaD and wild type DnaB in a yeast two-hybrid assay, similar to the increased interaction caused by a missense mutation in dnaB that is an extragenic suppressor of dnaD23ts. We isolated both intragenic and extragenic suppressors of the two dnaBts alleles. Some of the extragenic suppressors were informational suppressors (missense suppressors) in tRNA genes. These suppressor mutations caused a change in the anticodon of an alanine tRNA so that it would recognize the mutant codon (threonine) in dnaB and likely insert the wild type amino acid (alanine).

Conclusions/Significance

The intragenic suppressors should provide insights into structure-function relationships in DnaD and DnaB, and interactions between DnaD and DnaB. The extragenic suppressors in the tRNA genes have important implications regarding the amount of wild type DnaB needed in the cell. Since missense suppressors are typically inefficient, these findings indicate that production of a small amount of wild type DnaB, in combination with the mutant protein, is sufficient to restore some DnaB function.     

Oxidoreductases that Act as Conditional Virulence Suppressors in Salmonella enterica Serovar Typhimurium

In Salmonella enterica serovar Typhimurium, oxidoreductases of the thioredoxin superfamily contribute to bacterial invasiveness, intracellular replication and to the virulence in BALB/c mice as well as in the soil nematode Caenorhabditis elegans. The scsABCD gene cluster, present in many but not all enteric bacteria, codes for four putative oxidoreductases of the thioredoxin superfamily. Here we have analyzed the potential role of the scs genes in oxidative stress tolerance and virulence in S. Typhimurium. An scsABCD deletion mutant showed moderate sensitization to the redox-active transition metal ion copper and increased protein carbonylation upon exposure to hydrogen peroxide. Still, the scsABCD mutant was not significantly affected for invasiveness or intracellular replication in respectively cultured epithelial or macrophage-like cells. However, we noted a significant copper chloride sensitivity of SPI1 T3SS mediated invasiveness that strongly depended on the presence of the scs genes. The scsABCD deletion mutant was not attenuated in animal infection models. In contrast, the mutant showed a moderate increase in its competitive index upon intraperitoneal challenge and enhanced invasiveness in small intestinal ileal loops of BALB/c mice. Moreover, deletion of the scsABCD genes restored the invasiveness of a trxA mutant in epithelial cells and its virulence in C. elegans. Our findings thus demonstrate that the scs gene cluster conditionally affects virulence and underscore the complex interactions between oxidoreductases of the thioredoxin superfamily in maintaining host adaptation of S. Typhimurium.     

pmrA(Con) confers pmrHFIJKL-dependent EGTA and polymyxin resistance on msbB Salmonella by decorating lipid A with phosphoethanolamine

Mutations in pmrA were recombined into Salmonella strain ATCC 14028 msbB to determine if pmrA-regulated modifications of lipopolysaccharide could suppress msbB growth defects. A mutation that functions to constitutively activate pmrA [pmrA(Con)] suppresses msbB growth defects on EGTA-containing media. Lipid A structural analysis showed that Salmonella msbB pmrA(Con) strains, compared to Salmonella msbB strains, have increased amounts of palmitate and phosphoethanolamine but no aminoarabinose addition, suggesting that aminoarabinose is not incorporated into msbB lipid A. Surprisingly, loss-of-function mutations in the aminoarabinose biosynthetic genes restored EGTA and polymyxin sensitivity to Salmonella msbB pmrA(Con) strains. These blocks in aminoarabinose biosynthesis also prevented lipid A phosphoethanolamine incorporation and reduced the levels of palmitate addition, indicating previously unknown roles for the aminoarabinose biosynthetic enzymes. Lipid A structural analysis of the EGTA- and polymyxin-resistant triple mutant msbB pmrA(Con) pagP::Tn10, which contains phosphoethanolamine but no palmitoylated lipid A, suggests that phosphoethanolamine addition is sufficient to confer EGTA and polymyxin resistance on Salmonella msbB strains. Additionally, palmitoylated lipid A was observed only in wild-type Salmonella grown in the presence of salt in rich media. Thus, we correlate EGTA resistance and polymyxin resistance with phosphoethanolamine-decorated lipid A and demonstrate that the aminoarabinose biosynthetic proteins play an essential role in lipid A phosphoethanolamine addition and affect lipid A palmitate addition in Salmonella msbB strains.     

msbB deletion confers acute sensitivity to CO2 in Salmonella enterica serovar Typhimurium that can be suppressed by a loss-of-function mutation in zwf

Background  

Pathogens tolerate stress conditions that include low pH, oxidative stress, high salt and high temperature in order to survive inside and outside their hosts. Lipopolysaccharide (LPS), which forms the outer-leaflet of the outer membrane in Gram-negative bacteria, acts as a permeability barrier. The lipid A moiety of LPS anchors it to the outer membrane bilayer. The MsbB enzyme myristoylates the lipid A precursor and loss of this enzyme, in Salmonella, is correlated with reduced virulence and severe growth defects that can both be compensated with extragenic suppressor mutations.