The Candida albicans histidine kinase Chk1p: Signaling and cell wall mannan

Several published functions associated with the CHK1 histidine kinase of Candida albicans resemble those of the MAPK Cek1p and its cognate receptor Sho1p (SSU81). To explore this further, we have compared mutants lacking the proteins mentioned above and have constructed a double sho1/chk1Δ null mutant to determine relationships among these proteins. We observed that the sensitivity to Congo red (CR), calcofluor white (CW), as well as clumping of cells, was slightly increased in the double mutant compared to the single chk1Δ or sho1Δ mutants. However, Cek1p phosphorylation via Sho1p, which occurs during log phase growth in the presence or absence of CR in Wt cells, does not require Chk1p. These data suggest that Chk1p and Sho1p are components of parallel but independent signal pathways. In addition, bulk mannan of strains was analyzed by GLC/MS and GPC MALLS and NMR. Compared to Wt and a CHK1 gene-reconstituted strain (CHK23) that contained high, intermediate and low Mw mannan species, we found that the mannan of strains CHK21 (chk1Δ null), the cek1Δ null, and the double mutant consisted only of low Mw mannan. The sho1Δ null mutant only demonstrated a reduced intermediate type of mannan. Alcian blue binding was lower in cek1Δ, chk1Δ, and the double sho1/chk1Δ null mutant lacking high and intermediate Mw mannan than in the sho1Δ null which had a partial loss of intermediate Mw mannan only. We conclude that the Chk1p HK is part of a functionally similar but parallel pathway to the Sho1p-Cek1p pathway that confers resistance to the cell wall inhibitors CR and CW. However, a functional relationship in mannan biosynthesis of Chk1p and Cek1p exists that only partially requires Sho1p.     

The RCK1 and RCK2 protein kinase genes from Saccharomyces cerevisiae suppress cell cycle checkpoint mutations in Schizosaccharomyces pombe

The protein kinase-encoding genes RCK1 and RCK2 from Saccharomyces cerevisiae have been identified as suppressors of Schizosaccharomyces pombe cell cycle checkpoint mutations. Upon expression of these genes, radiation resistance is partially restored in S. pombe mutants with checkpoint deficiencies, but not in mutants with DNA repair defects. Some checkpoint mutants are sensitive to the DNA synthesis inhibitor hydroxyurea, and this sensitivity is also suppressed by RCK1 and RCK2. The degree of suppression can be modulated by varying expression levels. Expression of RCK1 or RCK2 in S. pombe causes cell elongation and decelerated growth. Cells expressing these genes have a single nucleus and a 2n DNA content. We conclude that these genes act in S. pombe to prolong the G2 phase of the cell cycle.     

Adaptative response to enhanced basal oxidative damage in sod mutants from Saccharomyces cerevisiae

We investigated the adaptative response of S. cerevisiae in sod mutants (sod1Δ, sod2Δ and sod1Δsod2Δ) after H₂O₂ treatment in the stationary phase. sod2Δ and sod1Δsod2Δ demonstrated the highest levels of GSH in the control, suggesting that pathways which include GSH protect these double mutants against oxidative stress. In addition, sod1Δ and sod1Δsod2Δ had higher iron levels than the wild-type, independently of H₂O₂ stress. Fe levels were increased in sod2Δ following H₂O₂ In addition, the sod2Δ mutant was more sensitive to H₂O₂ treatment than the wild-type. These results suggest that sod2Δ sensibility may be associated with •OH production by the Fenton reaction. This increased iron demand in the sod2Δ mutant may be a reflection of the cells’ efforts to reconstitute proteins that are inactivated in conditions of excess superoxide. MDA levels were assayed by HPLC in these mutants. The highest MDA levels could be observed after 10mM H₂O₂ treatment in the sod1Δsod2Δ double mutant. After treatment with a GSH inhibitor, the MDA level was still higher in the same strain. Thus, both direct and indirect GSH pathways are involved in the protection of lipid membranes and proteins in these mutants and may constitute an adaptative response to enhanced basal oxidative damage produced by superoxide.     

The HOG1-like MAP kinase Sak1 of Botrytis cinerea is negatively regulated by the upstream histidine kinase Bos1 and is not involved in dicarboximide- and phenylpyrrole-resistance

In filamentous ascomycetes, HOG-like signal transduction cascades are involved in the resistance to hyper-osmotic conditions and to dicarboximides and phenylpyrroles. The histidine kinase (HK) Bos1 and the mitogen-activated protein kinase (MAPK) Sak1 are important for the adaptation to hyper-osmotic and oxidative stress, development, and pathogenicity in the phytopathogenic fungus Botrytis cinerea. However, bos1Δ and sak1Δ mutants created previously, also presented different phenotypes, especially the sak1Δ mutants were not resistant to high fungicide concentrations. Since both single mutants were constructed in different parental strains, phenotypic variations due to the genetic background might be suspected. In order to establish the relationship between both protein kinases, we analyzed Sak1 phosphorylation under the control of the Bos1 HK and we realized epistasis analysis between bos1Δ and sak1Δ mutations through the construction of isogenic single and double mutants. Our results show that Bos1 negatively regulates Sak1 phosphorylation and that Bos1 regulates certain phenotypes independently of Sak1. They include fungicide susceptibility, adaptation and conidiation on high neutral osmolarity.     

AtBS14a and AtBS14b, two Bet1/Sft1-like SNAREs from Arabidopsis thaliana that complement mutations in the yeast SFT1 gene

SNAREs are membrane-associated proteins that play a central role in vesicle targeting and intra-cellular membrane fusion reactions in eukaryotic cells. Here we describe the identification of AtBS14a and AtBS14b, putative SNAREs from Arabidopsis thaliana that share 60% amino acid sequence identity. Both AtBS14a and BS14b are dosage suppressors of the temperature-sensitive growth defect in sft1-1 cells and over-expression of either AtBS14a or AtBS14b can support the growth of sft1Δ cells but not bet1Δ cells. These data together with structure–function and biochemical studies presented herein suggest that AtBS14a and AtBS14b share properties that are consistent with them being members of the Bet1/Sft1 SNARE protein family.     

A role for yeast glutaredoxin genes in selenite-mediated oxidative stress

Since the double Δgrx1Δgrx2 mutant is hypersensitive to selenite we decided to evaluate mechanisms underlying this phenomenon and establish the roles of other components of yeast glutaredoxin system, in particular glutaredoxin 5 in the selenite resistance. We found elevation in the intracellular and mitochondrial superoxide production in the Δgrx1Δgrx2 and Δgrx5 mutants after Se(IV) treatment. The last effect was more pronounced for cells lacking the mitochondrial Grx5 protein. We also recorded selenite-induced increase in the peroxide production in all strains tested. Nonfermentable carbon sources, glycerol and ethanol, augmented selenite toxicity. Hypo- and anoxia protected against the harmful effects of Se(VI). Augmentation of the intracellular levels of two endogenous antioxidants, erythroascorbic acid and glutathione confers resistance to selenite. We recorded a strain-unspecific, selenite-mediated decrease in the level of acid-soluble thiols. Collectively, our data demonstrate that hypersensitivity to the Δgrx1Δgrx2 and Δgrx5 disruptants to selenite is mediated by altered intracellular redox equilibrium.     

Phenotypic analysis of the ccp1Δ and ccp1Δ-ccp1 mutant strains of Saccharomyces cerevisiae indicates that cytochrome c peroxidase functions in oxidative-stress signaling

Yeast cytochrome c peroxidase (CCP) efficiently catalyzes the reduction of H₂O₂ to H₂O by ferrocytochrome c in vitro. The physiological function of CCP, a heme peroxidase that is targeted to the mitochondrial intermembrane space of Saccharomyces cerevisiae, is not known. CCP1-null-mutant cells in the W303-1B genetic background (ccp1Δ) grew as well as wild-type cells with glucose, ethanol, glycerol or lactate as carbon sources but with a shorter initial doubling time. Monitoring growth over 10 days demonstrated that CCP1 does not enhance mitochondrial function in unstressed cells. No role for CCP1 was apparent in cells exposed to heat stress under aerobic or anaerobic conditions. However, the detoxification function of CCP protected respiring mitochondria when cells were challenged with H₂O₂. Transformation of ccp1Δ with ccp1^W191F, which encodes the CCP^W191F mutant enzyme lacking CCP activity, significantly increased the sensitivity to H₂O₂ of exponential-phase fermenting cells. In contrast, stationary-phase (7-day) ccp1Δ-ccp1^W191F exhibited wild-type tolerance to H₂O₂, which exceeded that of ccp1Δ. Challenge with H₂O₂ caused increased CCP, superoxide dismutase and catalase antioxidant enzyme activities (but not glutathione reductase activity) in exponentially growing cells and decreased antioxidant activities in stationary-phase cells. Although unstressed stationary-phase ccp1Δ exhibited the highest catalase and glutathione reductase activities, a greater loss of these antioxidant activities was observed on H₂O₂ exposure in ccp1Δ than in ccp1Δ-ccp1^W191F and wild-type cells. The phenotypic differences reported here between the ccp1Δ and ccp1Δ-ccp1^W191F strains lacking CCP activity provide strong evidence that CCP has separate antioxidant and signaling functions in yeast.     

Efficient, galactose-free production of Candida antarctica lipase B by GAL10 promoter in Δgal80 mutant of Saccharomyces cerevisiae

An efficient yeast gene expression system with GAL10 promoter that does not require galactose as an inducer was developed using Δgal80 mutant strain of Saccharomyces cerevisiae. We constructed several combinations of gal mutations (Δgal1, Δgal80, Δmig1, Δmig2, and Δgal6) of S. cerevisiae and tested for their effect on efficiency of recombinant protein production by GAL10 promoter using a lipase, Candida antarctica lipase B (CalB), as a reporter. While the use of Δgal1 mutant strain required the addition of a certain amount of galactose to the medium, Δgal80 mutant strain did not require galactose. Furthermore, it was found that the recombinant CalB could be produced more efficiently (1.6-fold at 5 L-scale fermentation) in Δgal80 mutant strain than in the Δgal1 mutant. The Δgal80 mutant strain showed glucose repressible mode of expression of GAL10 promoter. Using Δgal80 mutant strain of S. cerevisiae, CalB was efficiently produced in a glucose-only fermentation at volumes up to 500 L.     

Identification of mucAB-like homologs on two IncT plasmids, R394 and Rts-1

Recent phylogenetic analysis of the superfamily of lesion-replicating DNA polymerases suggest that they can be broadly divided into four sub-groups comprised of UmuC-like, DinB-like, Rev1-like and Rad30-like proteins. The UmuC-like sub-family is best characterized at the genetic level and sequence analysis of eleven umu orthologs, residing on bacterial chromosomes or on self-transmissible R-plasmids allows further subdivision into five sub-groups (UmuDC, MucAB, ImpAB, RumAB and RulAB) based on amino acid sequence conservation. Some of these orthologs are apparently inactive in situ, but may promote increased mutagenesis and survival when subcloned and expressed from high-copy number plasmids. We were, therefore, interested in devising an assay that would identify umuC-like genes in situ in the absence of a functional assay. To this end, degenerate primers directed towards conserved amino acid regions within the UmuC-like sub-family of DNA polymerases were designed and used to identify mucAB-like operons on the IncT plasmids, R394 and Rts-1.Interestingly, DNA sequence analysis of an 7 kb region of R394 identified two LexA-regulated genes immediately downstream of mucAB_(R394) that are similar to the chromosomally-encoded Escherichia coli tus gene and the IncI plasmid-encoded impC gene, respectively. Analysis of the R394 and Rts-1 mucB genes revealed that both contain insertions which result in the expression of a truncated inactive MucB protein. While R394 was unable to restore mutagenesis functions to a ΔumuDC E. coli strain, Rts-1 surprisingly promoted significant levels of MMS-induced SOS mutagenesis, raising the possibility that Rts-1 encodes another, yet unidentified, umu-like homolog.     

在钙离子敏感性方面与RCH1遗传互作的酿酒酵母基因的筛选

酿酒酵母ScRCH1是白念珠菌CaRCH1的同功基因,作为人体溶质转运蛋白SLC10A7的同源蛋白,两者都是细胞质膜上钙离子内流的抑制因子。为了研究酿酒酵母RCH1与基因组中其他基因之间的遗传互作,利用合成遗传阵列(Synthetic Genetic Array,SGA)方法构建了RCH1分别与其他非必需基因之间的双基因缺失株文库。钙离子表型筛选表明RCH1与17个基因之间存在遗传互作,其中4个基因BUD9、THR1、RAS2和CPR7在钙离子敏感性方面的功能以前没有报道过。这些结果为深入研究Rch1对钙离子稳态的调控提供了参考。     

Nuclear protein import, but not mRNA export, is defective in all Saccharomyces cerevisiae mutants that produce temperature-sensitive forms of the Ran GTPase homologue Gsp1p

A series of ts mutations in the GSP1 gene of Saccharomyces cerevisiae was isolated by error-prone PCR. A total of 25 ts gsp1 strains was obtained. Each of these mutants showed between one and seven different amino acid alterations. In several of these ts gsp1 strains, the same amino acid residues in Gsp1p were repeatedly mutated, indicating that our screen for ts gsp1 mutations was saturating. All of the ts gsp1 strains isolated had a defect in nuclear protein import, but only 16 of the 25 ts gsp1 strains had a defect in mRNA export. Thus, Gsp1p is suggested to be directly involved in nuclear protein import, but not in mRNA export. Following release from α-factor arrest, 11 of the ts gsp1 mutants arrested in G1; the remainder did not show any specific cell-cycle arrest, at 37° C, the nonpermissive temperature. While the mutants that are defective in both mRNA export and protein import have a tendency to arrest in G1, there was no clear correlation between the cell cycle phenotype and the defects in mRNA export and nuclear protein import. Based on this, we assume that Ran/Gsp1p GTPase regulates the cell cycle and the nucleus/cytosol exchange of macromolecules through interactions with effectors that were independent of each other, and are differentially affected by mutation. Received: 30 June 1997 / Accepted: 23 October 1997     

Synergistic effect of TRM2/RNC1 and EXO1 in DNA double-strand break repair in Saccharomyces cerevisiae

In our recently published study, we provided in vitro as well as in vivo data demonstrating the involvement of TRM2/RNC1 in homologous recombination based repair (HRR) of DNA double strand breaks (DSBs), in support of such claims reported earlier. To further validate its role in DNA DSB processing, our present study revealed that the trm2 single mutant displays higher sensitivity to persistent induction of specific DSBs at the MAT locus by HO-endonuclease with higher sterility rate among the survivors compared to wild type (wt) or exo1 single mutants. Intriguingly, both sensitivity and sterility rate increased dramatically in trm2exo1 double mutants lacking both endo-exonucleases with a progressively increased sterility rate in trm2exo1 double mutants with short-induction periods, reaching a very high level of sterility with persistent DSB inductions. Mutation analysis of the mating type (MAT) locus among the sterile survivors with persistent HO-induction in trm2 and exo1 single mutants as well as in trm2exo1 double mutants revealed a similar small insertions and deletions events, characteristic of non-homologous end joining (NHEJ) that might have occurred due to the lack of proper processing function in these mutants. In addition, trm2ku80 and trm2rad52 double mutants also displayed significantly higher sterility with persistent DSB induction compared to ku80 and rad52 single mutants, respectively, exhibiting a mutation spectra that shifted from base substitution (in ku80 and rad52 single mutants) to small insertions and deletions in the double mutants (in trm2ku80 and trm2rad52 mutants). These data indicate a defective processing in absence of TRM2, with a synergistic effect of TRM2, and EXO1 in such processing.     

A putative pheromone signaling pathway is dispensable for self-fertility in the homothallic ascomycete Gibberella zeae

Gibberella zeae, a homothallic ascomycetous fungus, does not seek a partner for mating. Here, we focused on the role(s) of putative pheromone and receptor genes during sexual development in G. zeae. Orthologs of two pheromone precursor genes (GzPPG1 and GzPPG2), and their cognate receptor genes (GzPRE2 and GzPRE1) were transcribed during sexual development. The expression of these genes was controlled by the mating-type (MAT) locus and a MAP kinase gene, but not in a MAT-specific manner. Targeted gene deletion and subsequent outcrosses generated G. zeae strains lacking these putative pheromone/receptor genes in various combinations (from single to quadruple deletions). All G. zeae deletion strains were similar to the self-fertile progenitor in both male- and female fertility and other traits. Sometimes, the deletions including ΔGzPPG1;ΔGzPRE2 caused increased numbers of immature perithecia. Taken together, it is clear that these putative pheromones/receptors play a non-essential role in the sexual development of G. zeae.     

Regulation of the Saccharomyces cerevisiae Srs2 helicase during the mitotic cell cycle, meiosis and after irradiation

The expression of theSRS2 gene, which encodes a DNA helicase involved in DNA repair inSaccharomyces cerevisiae, was studied using anSRS2-lacZ fusion integrated at the chromosomalSRS2 locus. It is shown here that this gene is expressed at a low level and is tightly regulated. It is cell-cycle regulated, with induction probably being coordinated with that of the DNA-synthesis genes, which are transcribed at the G1-S boundary. It is also induced by DNA-damaging agents, but only during the G2 phase of the cell cycle; this distinguishes it from a number of other repair genes, which are inducible throughout the cycle. During meiosis, the expression ofSRS2 rises at a time nearly coincident with commitment to recombination. Sincesrs2 null mutants are radiation sensitive essentially when treated in G1, the mitotic regulation pattern described here leads us to postulate that either secondary regulatory events limit Srs2 activity to G1 cells or Srs2 functions in a repair mechanism associated with replication.     

Storage of light-driven transthylakoid proton motive force as an electric field (Δψ) under steady-state conditions in intact cells of <Emphasis Type="Italic">Chlamydomonasreinhardtii</Emphasis>

Proton motive force (pmf) is physiologically stored as either a ΔpH or a membrane potential (Δψ) across bacterial and mitochondrial energetic membranes. In the case of chloroplasts, previous work (Cruz et al. 2001, Biochemistry 40: 1226–1237) indicates that Δψ is a significant fraction of pmf, in vivo, and in vitro as long as the activities of counterions are relatively low. Kinetic analysis of light-induced changes in the electrochromic shift (ECS) in intact leaves was consistent with these observations. In this work, we took advantage of the spectroscopic properties of the green alga, Chlamydomonas reinhardtii, to demonstrate that light-driven Δψ was stored in vivo over the hours time scale. Analysis of the light-induced ECS kinetics suggested that the steady-state Δψ in 400 μmol photons m⁻² s⁻¹ red light was between 20 and 90 mV and that this represented about 60% of the light-induced increase in pmf. By extrapolation, it was surmised that about half of total (basal and light-induced) pmf is held as Δψ. It is hypothesized that Δψ is stabilized either by maintaining low chloroplast ionic strength or by active membrane ion transporters. In addition to the strong implications for regulation of photosynthesis by the xanthophyll cycle, these results imply that pmf partitioning is important across a wide range of species.     

Properties of γ-aminobutyraldehyde dehydrogenase from Escherichia coli

γ-Aminobutyraldehyde dehydrogenase from Escherichia coli K-12 has been purified and characterized from cell mutants able to grow in putrescine as the sole carbon and nitrogen source. The enzyme has an M_r of 195 000±10 000 in its dimeric form with an M_r of 95 000±1000 for each subunit, a pH optimum at 5.4 in sodium citrate buffer, and does not require bivalent cations for its activity. K_m values are 31.3±6.8 μM and 53.8±7.4 μM for Δ-1-pyrroline and NAD⁺, respectively. An inhibitory capacity for NADH is also shown using the purified enzyme.     

Investigation of a requirement for the PsbP-like protein in Synechocystis sp. PCC 6803

The sll1418 gene encodes a PsbP-like protein in Synechocystis sp. PCC 6803. Expression of sll1418 was similar in BG-11 and in Cl⁻- or Ca²⁺-limiting media, and inactivation of sll1418 did not prevent photoautotrophic growth in normal or nutrient-limiting conditions. Also the wild-type and ΔPsbP strains exhibited similar oxygen evolution and assembly of Photosystem II (PS II) centers. Inactivation of sll1418 in the ΔPsbO: ΔPsbP, ΔPsbQ:ΔPsbP, ΔPsbU:ΔPsbP and ΔPsbV:ΔPsbP mutants did not prevent photoautotrophy or alter PS II assembly and oxygen evolution in these strains. Moreover, the absence of PsbP did not affect the ability of alkaline pH to restore photoautotrophic growth in the ΔPsbO:ΔPsbU strain. The PsbO, PsbU and PsbV proteins are required for thermostability of PS II and thermal acclimation in Synechocystis sp. PCC 6803 [Kimura et al. (2002) Plant Cell Physiol 43: 932–938]. However, thermostability and thermal acclimation in ΔPsbP cells were similar to wild type. These results are consistent with the conclusion that PsbP is associated with ∼3 of PS II centers, and may play a regulatory role in PS II [Thornton et al. (2004) Plant Cell 16: 2164–2175].     

Isolation and characterization of temperature-sensitiveplc1 mutants of the yeastSaccharomyces cerevisiae

ThePLC1 gene of the yeastSaccharomyces cerevisiae has been discovered to encode a homolog of mammalian phosphoinositide-specific phospholipase C (PLC). Five temperature-sensitiveplc1 mutants were isolated by in vitro mutagenesis with subsequent plasmid shuffling. All of the amino acid substitutions that caused a temperature-sensitive growth phenotype were located in the X or the Y region, both of which are conserved among PLC isoenzymes. The PLC activity of all products of mutantplc1 genes was dramatically lower than that of the wild-type product, indicating that PLC activity itself is important for cell growth. At the restrictive temperature,plc1 mutant cells ceased growth at random times during the cell cycle, a result that suggests thatPLC1 is required at several or all stages of the cell cycle.