RIM101-dependent and-independent pathways govern pH responses in Candida albicans

Growth and differentiation of Candida albicans over a broad pH range underlie its ability to infect an array of tissues in susceptible hosts. We identified C. albicans RIM101, RIM20, and RIM8 based on their homology to components of the one known fungal pH response pathway. PCR product-disruption mutations in each gene cause defects in three responses to alkaline pH: filamentation, induction of PRA1 and PHR1, and repression of PHR2. We find that RIM101 itself is an alkaline-induced gene that also depends on Rim20p and Rim8p for induction. Two observations indicate that a novel pH response pathway also exists. First, PHR2 becomes an alkaline-induced gene in the absence of Rim101p, Rim20p, or Rim8p. Second, we created strains in which Rim101p activity is independent of Rim20p and Rim8p; in these strains, filamentation remains pH dependent. Thus, pH governs gene expression and cellular differentiation in C. albicans through both RIM101-dependent and RIM101-independent pathways.     

Candida albicans Rim13p, a protease required for Rim101p processing at acidic and alkaline pHs

Candida albicans is an important commensal of mucosal surfaces that is also an opportunistic pathogen. This organism colonizes a wide range of host sites that differ in pH; thus, it must respond appropriately to this environmental stress to survive. The ability to respond to neutral-to-alkaline pHs is governed in part by the RIM101 signal transduction pathway. Here we describe the analysis of C. albicans Rim13p, a homolog of the Rim13p/PalB calpain-like protease member of the RIM101/pacC pathway from Saccharomyces cerevisiae and Aspergillus nidulans, respectively. RIM13, like other members of the RIM101 pathway, is required for alkaline pH-induced filamentation and growth under extreme alkaline conditions. Further, our studies suggest that the RIM101 pathway promotes pH-independent responses, including resistance to high concentrations of lithium and to the drug hygromycin B. RIM13 encodes a calpain-like protease, and we found that Rim101p undergoes a Rim13p-dependent C-terminal proteolytic processing event at neutral-to-alkaline pHs, similar to that reported for S. cerevisiae Rim101p and A. nidulans PacC. However, we present evidence that suggests that C. albicans Rim101p undergoes a novel processing event at acidic pHs that has not been reported in either S. cerevisiae or A. nidulans. Thus, our results provide a framework to understand how the C. albicans Rim101p processing pathway promotes alkaline pH-independent processes.     

PRR1, a homolog of Aspergillus nidulans palF, controls pH-dependent gene expression and filamentation in Candida albicans

The pH of the environment has been implicated in controlling the yeast-hypha transition and pathogenesis of Candida albicans. Several C. albicans genes, including PHR1 and PHR2, are pH dependent in their expression. To investigate the mechanism of pH-dependent expression, we have cloned and characterized PRR1 (for pH response regulator). PRR1 is homologous to palF, a component of the pH response pathway in Aspergillus nidulans. Expression of PRR1 was itself pH dependent, being maximal at acid pH but reduced severalfold at alkaline pH. In a prr1 null mutant the alkaline-induced expression of PHR1 was completely abolished. Conversely, expression of PHR2 was no longer repressed at alkaline pH. A prr1 null mutant exhibited no morphological abnormalities at either pH; however, it lost the ability to form hyphae on medium 199 and on 10% serum plates. The ability to filament on serum was not restored by forced expression of PHR1, indicating that additional PRR1-dependent genes are required for hyphal development. These developmental genes appear to be distinct from those controlled by the developmental regulator EFG1, since the EFG1-dependent gene HWP1 was expressed normally in the prr1 null mutant. We conclude that PRR1 encodes a component of the pH-dependent response pathway in C. albicans and that this pathway regulates the expression of multiple components of hyphal development.     

Developmental bypass suppression of Myxococcus xanthus csgA mutations.

The csgA mutations of Myxococcus xanthus (formerly known as spoC) inhibit sporulation as well as rippling, which involves ridges of cells moving in waves. Sporulating revertants of CsgA cells were isolated by direct selection, since spores are much more resistant to heat and ultrasonic treatment than are vegetative cells. The revertants fell into seven groups on the basis of phenotype and the chromosomal location of the suppressor alleles. Group 1 contained one allele that was a back mutation of the original csgA mutation. Group 2 contained two linked alleles that were unlinked to the csgA locus and restored fruiting-body formation, sporulation, and rippling. Group 3 revertants regained the ability to sporulate in fruiting bodies but not the ability to ripple. Revertants in groups 4 to 7 were able to sporulate but unable to form fruiting bodies or ripples. The suppressors were all found to be bypass suppressors even though they were not selected as such in most cases. The csgA mutation prevented expression of several developmentally regulated promoters, each fused to a lacZ reporter gene and assayed by beta-galactosidase production. In four of five suppressor groups (groups 4 to 7), expression of each of these csgA-dependent fusions was restored, which suggests that bypass suppression restores developmental gene expression near the point at which expression is disrupted in CsgA mutants. Bypass suppression did not restore production of C factor, and morphological manifestations of development such as rippling and fruiting-body formation were usually abnormal. One interpretation of these results is that C factor has multiple functions and few suppressors can compensate for all of them.     

PHR2 of Candida albicans encodes a functional homolog of the pH-regulated gene PHR1 with an inverted pattern of pH-dependent expression.

Deletion of PHR1, a pH-regulated gene of Candida albicans, results in pH-conditional defects in growth, morphogenesis, and virulence evident at neutral to alkaline pH but absent at acidic pH. Consequently, we searched for a functional homolog of PHR1 active at low pH. This resulted in the isolation of a second pH-regulated gene, designated PHR2. The expression of PHR2 was inversely related to that of PHR1, being repressed at pH values above 6 and progressively induced at more acidic pH values. The predicted amino acid sequence of the PHR2 protein, Phr2p, was 54% identical to that of Phr1p. A PHR2 null mutant exhibited pH-conditional defects in growth and morphogenesis analogous to those of PHR1 mutants but manifest at acid rather than alkaline pH values. Engineered expression of PHR1 at acid pH in a PHR2 mutant strain and PHR2 at alkaline pH in a PHR1 mutant strain complemented the defects in the opposing mutant. Deletion of both PHR1 and PHR2 resulted in a strain with pH-independent, constitutive growth and morphological defects. These results indicate that PHR1 and PHR2 represent a novel pH-balanced system of functional homologs required for C. albicans to adapt to environments of diverse pH.     

Adaptation to environmental pH: integrating the Rim101 and calcineurin signal transduction pathways

The ability to appropriately respond to environmental conditions is critical for the survival of simple microbes and for development of complex multicellular organisms. Sensing and responding to a given environmental condition requires the integration of numerous signals through one or more signal transduction pathways. This leads to changes in gene expression, and potentially post-translational modifications, that favour growth in the given environment. In the fungus Candida albicans, an important opportunistic pathogen, environmental pH has profound effects on morphology and proper adaptation to extracellular pH is critical for pathogenesis. Here, we demonstrate that the Rim101/PacC pH-sensing pathway acts in parallel to Crz1, via calcineurin, to adapt to alkaline pH. We also show that the Rim101 pathway acts in parallel to Crz2, independent of calcineurin, to adapt to high lithium concentrations and to repress filamentation at acidic pH. Our studies also revealed a novel requirement for Crz1, Crz2 and calcineurin for growth at acidic pH. From these studies, we propose that the Crz1 homologue Crz2 is calcineurin-independent, but like Crz1, acts in parallel to promote specific Rim101-dependent processes. These results establish and begin to dissect the complex interactions between important signal transduction pathways in C. albicans, which are critical for virulence.     

Dissociation of tsl-tif-Induced Filamentation and recA Protein Synthesis in Escherichia coli K-12

In Escherichia coli, expression of the tif-1 mutation (in the recA gene) induces the "SOS response" at 40 degrees C, including massive synthesis of the recA(tif) protein, cell filamentation, appearance of new repair and mutagenic activities, and prophage induction. Expression of the tsl-1 mutation (in the lexA gene) induces massive synthesis of the recA protein and cell filamentation at 42 degrees C, although other SOS functions are not induced. In this paper we show that the septation inhibition induced in tif and tsl strains at 42 degrees C is not due to the presence of a high concentration of recA protein since (i) no recA mutants (相似文献   

Thermoresistant revertants of an Escherichia coli strain carrying tif-1 and ruv mutations: non-suppressibility of ruv by sfi.

Spontaneous thermoresistant revertants were isolated from Tif1 Ruv- and Tif1 Ruv+ strains of Escherichia coli K-12. They were divided into five groups; backmutants to tif+ and recA structural gene mutants accounted for at least two of these groups. Mutations with an unconditional RecA- phenyotype were detected at a higher frequency in the Tif1 Ruv- strains (65%) than in the Tif1 Ruv+ strains (25%). A third group consisted of revertants exhibiting a RecA- phenotype at low temperature. Revertants with normal recombination ability and UV resistance, but with a thermosensitive defect in propagating lambda bio11 phage, were also isolated (group 4). The alleles responsible for this property were cotransducible with the srl gene, suggesting that they are located at the recA locus. Other revertants, which might carry lex, LEXB, or zab mutations, were UV sensitive and were able to propagate lambda bio11 phage (group 5). The sfi mutation, which suppresses filamentation in the Tif1 and UV-sensitive Lon- strains, does not restore UV resistance of the Ruv- mutant.     

Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions

Disruption of newly identified genes in the pathogen Candida albicans is a vital step in determination of gene function. Several gene disruption methods described previously employ long regions of homology flanking a selectable marker. Here, we describe disruption of C. albicans genes with PCR products that have 50 to 60 bp of homology to a genomic sequence on each end of a selectable marker. We used the method to disrupt two known genes, ARG5 and ADE2, and two sequences newly identified through the Candida genome project, HRM101 and ENX3. HRM101 and ENX3 are homologous to genes in the conserved RIM101 (previously called RIM1) and PacC pathways of Saccharomyces cerevisiae and Aspergillus nidulans. We show that three independent hrm101/hrm101 mutants and two independent enx3/enx3 mutants are defective in filamentation on Spider medium. These observations argue that HRM101 and ENX3 sequences are indeed portions of genes and that the respective gene products have related functions.     

Suppressors of mutations in the bacteriophage T4 gene coding for both RNA ligase and tail fiber attachment activities.

The protein product of T4 gene 63 catalyzes both the attachment of tail fibers to fiberless phage particles and the ligation of single-stranded RNA (Snopek at al., Proc. Natl. Acad. Sci. U.S.A. 74:3355-3359, 1977). To investigate whether the gene 63 product has a role in nucleotide metabolism, we isolated false revertants of amM69 in gene 63. We screened for revertants that could grow at 30 degrees C but not at 43 degrees C on Escherichia coli OK305 when nucleotides were limiting. These false revertants contained the original mutation in gene 63 and new suppressor mutations. Some of these suppressor mutations caused temperature sensitivity by themselves, allowing single mutants carrying the suppressor to be recognized and isolated. The results of mapping and complementation studies indicated that most of these ts suppressors were in the t gene (lysis), one was in gene 5 (baseplate), and one was in gene 18 (sheath). The mutation in gene 18, tsDH638, suppressed three different amber mutations in gene 63 but did not suppress amber mutations in several other genes. None of the suppressors that were characterized were in genes with known functions in nucleotide metabolism. However, an intriguing property of these false revertants was that they were very sensitive to hydroxyurea, an inhibitor of nucleotide metabolism.     

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.     

Isolation of cell size mutants of a fission yeast by a new selective method: characterization of mutants and implications for division control mechanisms.

Previously known cell size (wee) mutations of fission yeast suppress the mitotic block caused by a defective cdc25 allele. Some 700 revertants of cdc25-22 were obtained after ultraviolet mutagenesis and selection at the restrictive temperature. Most revertants carried the original cdc25 lesion plus a mutation in or very close to the wee1 gene. Two partial wee1 mutations of a new type were found among the revertants. Two new wee mutations mapping at the cdc2 gene (cdc2-w mutants) were also obtained. The various mutations were examined for their effects on cell division size, their efficiency as cdc25 suppressors, and their dominance relations. Full wee1 mutations were found to suppress cdc25 lesions very efficiently, whereas partial wee1 mutations were poor suppressors. The cdc25 suppression ability of cdc2-w mutations was allele specific for cdc2, suggesting bifunctionality of the gene product. The wee1 mutations were recessive for cdc25 suppression; cdc2-w mutations were dominant. A model is proposed for the genetic control of mitotic timing and cell division size, in which the cdc2+ product is needed and is rate limiting for mitosis. The cdc2+ activity is inhibited by the wee1+ product, whereas the cdc25+ product relieves this inhibition.     

Suppressors of snf2 Mutations Restore Invertase Derepression and Cause Temperature-Sensitive Lethality in Yeast

Mutations in the SNF2 gene of Saccharomyces cerevisiae prevent derepression of the SUC2 (invertase) gene, and other glucose-repressible genes, in response to glucose deprivation. We have isolated 25 partial phenotypic revertants of a snf2 mutant that are able to derepress secreted invertase. These revertants all carried suppressor mutations at a single locus, designated SSN20 (suppressor of snf2). Alleles with dominant, partially dominant and recessive suppressor phenotypes were recovered, but all were only partial suppressors of snf2, reversing the defect in invertase synthesis but not other defects. All alleles also caused recessive, temperature-sensitive lethality and a recessive defect in galactose utilization, regardless of the SNF2 genotype. No significant effect on SUC2 expression was detected in a wild-type (SNF2) genetic background. The ssn20 mutations also suppressed the defects in invertase derepression caused by snf5 and snf6 mutations, and selection for invertase-producing revertants of snf5 mutants yielded only additional ssn20 alleles. These findings suggest that the roles of the SNF2, SNF5 and SNF6 genes in regulation of SUC2 are functionally related and that SSN20 plays a role in expression of a variety of yeast genes.     

Genetic Analysis of Bacteriophage P22 Lysozyme Structure

The suppression patterns of 11 phage P22 mutants bearing different amber mutations in the gene encoding lysozyme (19) were determined on six different amber suppressor strains. Of the 60 resulting single amino acid substitutions, 18 resulted in defects in lysozyme activity at 30 degrees; an additional seven were defective at 40 degrees. Revertants were isolated on the "missuppressing" hosts following UV mutagenesis; they were screened to distinguish primary- from second-site revertants. It was found that second-site revertants were recovered with greater efficiency if the UV-irradiated phage stocks were passaged through an intermediate host in liquid culture rather than plated directly on the nonpermissive host. Eleven second-site revertants (isolated as suppressors of five deleterious substitutions) were sequenced: four were intragenic, five extragenic; three of the extragenic revertants were found to have alterations near and upstream from gene 19, in gene 13. Lysozyme genes from the intragenic revertant phages were introduced into unmutagenized P22, and found to confer the revertant plating phenotype.