CHS5, a gene involved in chitin synthesis and mating in Saccharomyces cerevisiae.

The CHS5 locus of Saccharomyces cerevisiae is important for wild-type levels of chitin synthase III activity. chs5 cells have reduced levels of this activity. To further understand the role of CHS5 in yeast, the CHS5 gene was cloned by complementation of the Calcofluor resistance phenotype of a chs5 mutant. Transformation of the mutant with a plasmid carrying CHS5 restored Calcofluor sensitivity, wild-type cell wall chitin levels, and chitin synthase III activity levels. DNA sequence analysis reveals that CHS5 encodes a unique polypeptide of 671 amino acids with a molecular mass of 73,642 Da. The predicted sequence shows a heptapeptide repeated 10 times, a carboxy-terminal lysine-rich tail, and some similarity to neurofilament proteins. The effects of deletion of CHS5 indicate that it is not essential for yeast cell growth; however, it is important for mating. Deletion of CHS3, the presumptive structural gene for chitin synthase III activity, results in a modest decrease in mating efficiency, whereas chs5delta cells exhibit a much stronger mating defect. However, chs5 cells produce more chitin than chs3 mutants, indicating that CHS5 plays a role in other processes besides chitin synthesis. Analysis of mating mixtures of chs5 cells reveals that cells agglutinate and make contact but fail to undergo cell fusion. The chs5 mating defect can be partially rescued by FUS1 and/or FUS2, two genes which have been implicated previously in cell fusion, but not by FUS3. In addition, mating efficiency is much lower in fus1 fus2 x chs5 than in fus1 fus2 x wild type crosses. Our results indicate that Chs5p plays an important role in the cell fusion step of mating.     

Chitin synthesis in a gas1 mutant of Saccharomyces cerevisiae

The existence of a compensatory mechanism in response to cell wall damage has been proposed in yeast cells. The increase of chitin accumulation is part of this response. In order to study the mechanism of the stress-related chitin synthesis, we tested chitin synthase I (CSI), CSII, and CSIII in vitro activities in the cell-wall-defective mutant gas1 delta. CSI activity increased twofold with respect to the control, a finding in agreement with an increase in the expression of the CHS1 gene. However, deletion of the CHS1 gene did not affect the phenotype of the gas1 delta mutant and only slightly reduced the chitin content. Interestingly, in chs1 gas1 double mutants the lysed-bud phenotype, typical of chs1 null mutant, was suppressed, although in gas1 cells there was no reduction in chitinase activity. CHS3 expression was not affected in the gas1 mutant. Deletion of the CHS3 gene severely compromised the phenotype of gas1 cells, despite the fact that CSIII activity, assayed in membrane fractions, did not change. Furthermore, in chs3 gas1 cells the chitin level was about 10% that of gas1 cells. Thus, CSIII is the enzyme responsible for the hyperaccumulation of chitin in response to cell wall stress. However, the level of enzyme or the in vitro CSIII activity does not change. This result suggests that an interaction with a regulatory molecule or a posttranslational modification, which is not preserved during membrane fractionation, could be essential in vivo for the stress-induced synthesis of chitin.     

Individual chitin synthase enzymes synthesize microfibrils of differing structure at specific locations in the Candida albicans cell wall

The shape and integrity of fungal cells is dependent on the skeletal polysaccharides in their cell walls of which beta(1,3)-glucan and chitin are of principle importance. The human pathogenic fungus Candida albicans has four genes, CHS1, CHS2, CHS3 and CHS8, which encode chitin synthase isoenzymes with different biochemical properties and physiological functions. Analysis of the morphology of chitin in cell wall ghosts revealed two distinct forms of chitin microfibrils: short microcrystalline rodlets that comprised the bulk of the cell wall; and a network of longer interlaced microfibrils in the bud scars and primary septa. Analysis of chitin ghosts of chs mutant strains by shadow-cast transmission electron microscopy showed that the long-chitin microfibrils were absent in chs8 mutants and the short-chitin rodlets were absent in chs3 mutants. The inferred site of chitin microfibril synthesis of these Chs enzymes was corroborated by their localization determined in Chsp-YFP-expressing strains. These results suggest that Chs8p synthesizes the long-chitin microfibrils, and Chs3p synthesizes the short-chitin rodlets at the same cellular location. Therefore the architecture of the chitin skeleton of C. albicans is shaped by the action of more than one chitin synthase at the site of cell wall synthesis.     

Different Chitin Synthase Genes Are Required for Various Developmental and Plant Infection Processes in the Rice Blast Fungus Magnaporthe oryzae

Chitin is a major component of fungal cell wall and is synthesized by chitin synthases (Chs). Plant pathogenic fungi normally have multiple chitin synthase genes. To determine their roles in development and pathogenesis, we functionally characterized all seven CHS genes in Magnaporthe oryzae. Three of them, CHS1, CHS6, and CHS7, were found to be important for plant infection. While the chs6 mutant was non-pathogenic, the chs1 and chs7 mutants were significantly reduced in virulence. CHS1 plays a specific role in conidiogenesis, an essential step for natural infection cycle. Most of chs1 conidia had no septum and spore tip mucilage. The chs6 mutant was reduced in hyphal growth and conidiation. It failed to penetrate and grow invasively in plant cells. The two MMD-containing chitin synthase genes, CHS5 and CHS6, have a similar expression pattern. Although deletion of CHS5 had no detectable phenotype, the chs5 chs6 double mutant had more severe defects than the chs6 mutant, indicating that they may have overlapping functions in maintaining polarized growth in vegetative and invasive hyphae. Unlike the other CHS genes, CHS7 has a unique function in appressorium formation. Although it was blocked in appressorium formation by germ tubes on artificial hydrophobic surfaces, the chs7 mutant still produced melanized appressoria by hyphal tips or on plant surfaces, indicating that chitin synthase genes have distinct impacts on appressorium formation by hyphal tip and germ tube. The chs7 mutant also was defective in appressorium penetration and invasive growth. Overall, our results indicate that individual CHS genes play diverse roles in hyphal growth, conidiogenesis, appressorium development, and pathogenesis in M. oryzae, and provided potential new leads in the control of this devastating pathogen by targeting specific chitin synthases.     

Double chitin synthetase mutants from the corn smut fungus Ustilago maydis

Using genetic crosses between single chs mutants of Ustilago maydis inoculated into maize ( Zea mays ) seedlings, two classes of double mutants affected in genes coding for chitin synthetases were isolated: chs3 / chs4 , and chs4 / chs5 . Analysis of the mutants showed almost no change in their phenotype compared with wild-type strains. Growth rate, effect of stress conditions, dimorphic transition and mating were not affected. The only salient differences were increased sensitivity to osmotics at acid pH, and decrease in chitin synthetase activity, especially when measured with CO²⁺, and in chitin content. Most significant was a decrease in virulence, although this appeared to be due a factor unrelated to CHS genes. These data can be taken as further evidence that multigenic control of chitin synthetase in fungi operates as a safety mechanism to guarantee fungal viability in changing and hostile environmental conditions.     

CHS8-a fourth chitin synthase gene of Candida albicans contributes to in vitro chitin synthase activity, but is dispensable for growth

In silico analysis of the genome sequence of the human pathogenic fungus Candida albicans identified an open reading frame encoding a putative fourth member of the chitin synthase gene family. This gene, named CaCHS8, encodes an 1105 amino acid open reading frame with the conserved motifs characteristic of class I zymogenic chitin synthases with closest sequence similarity to the non-essential C. albicans class I CHS2 gene. Although the CaCHS8 gene was expressed in both yeast and hyphal cells, homozygous chs8 Delta null mutants had normal growth rates, cellular morphologies and chitin contents. The null mutant strains had a 25% reduction in chitin synthase activity and were hypersensitive to Calcofluor White. A chs2 Delta chs8 Delta double mutant had less than 3% of normal chitin synthase activity and had increased wall glucan and decreased mannan but was unaffected in growth or cell morphology. The C. albicans class I double mutant did not exhibit a bud-lysis phenotype as found in the class I chs1 Delta mutant of Saccharomyces cerevisiae. Therefore, C. albicans has four chitin synthases with two non-essential class I Chs isoenzymes that contribute collectively to more than 97% of the in vitro chitin synthase activity.     

Survey of the Botrytis cinerea chitin synthase multigenic family through the analysis of six euascomycetes genomes.

We describe a strategy for systematic amplification of chitin synthase genes (chs) in the filamentous ascomycetes plant-pathogen Botrytis cinerea using PCR with multiple degenerate primers designed on specific and conserved sequence motifs. Eight distinct chs genes were isolated, named Bcchs I, II, IIIa, IIIb, IV, V, VI and VII. They probably constitute the entire chs multigenic family of this fungus, as revealed by careful analysis of six euascomycetes genomes. Bcchs I, IIIa, IIIb, IV and VI genes were subjected to DNA walking and their deduced amino acid sequences were compared by hydrophobic cluster analysis (HCA) to localize putative residues critical for CHS activity. HCA also enabled us to highlight three different transmembrane topologies of the CHS membranous isoenzymes. We found that the N-terminal region of the BcCHSI isoenzyme, and its orthologues in other euascomycetes, probably contain folded peptide motifs with conserved tyrosine residues. Their putative role is discussed. The BcCHSVII isoenzyme appeared to belong to a new class of CHS orthologues that was demonstrated by phylogenetic study to branch apart from division 1 and 2 of CHS.     

The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo

The chitin synthase of Saccharomyces is a plasma membrane-bound zymogen. Following proteolytic activation, the enzyme synthesizes insoluble chitin that has chain length and other physical properties similar to chitin found in bud scars. We isolated mutants lacking chitin synthase activity (chs1) and used these to clone CHS1. The gene has an open reading frame of 3400 bases and encodes a protein of 130 kd. The fission yeast S. pombe lacks chitin synthase and chitin. When a plasmid encoding a CHS1-lacZ fusion protein is introduced into S. pombe, both enzymatic activities are expressed in the same ratio as in S. cerevisiae, demonstrating that CHS1 encodes the structural gene of chitin synthase. Three CHS1 gene disruption experiments were performed. In all cases, strains with the disrupted gene have a recognizable phenotype, lack measurable chitin synthase activity in vitro but are viable, contain normal levels of chitin in vivo, and mate and sporulate efficiently.     

Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae.

The GGP1/GAS1 gene codes for a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae. The ggp1delta mutant shows morphogenetic defects which suggest changes in the cell wall matrix. In this work, we have investigated cell wall glucan levels and the increase of chitin in ggp1delta mutant cells. In these cells, the level of alkali-insoluble 1,6-beta-D-glucan was found to be 50% of that of wild-type cells and was responsible for the observed decrease in the total alkali-insoluble glucan. Moreover, the ratio of alkali-soluble to alkali-insoluble glucan almost doubled, suggesting a change in glucan solubility. The increase of chitin in ggp1delta cells was found to be essential since the chs3delta ggp1delta mutations determined a severe reduction in the growth rate and in cell viability. Electron microscopy analysis showed the loss of the typical structure of yeast cell walls. Furthermore, in the chs3delta ggp1delta cells, the level of alkali-insoluble glucan was 57% of that of wild-type cells and the alkali-soluble/alkali-insoluble glucan ratio was doubled. We tested the effect of inhibition of chitin synthesis also by a different approach. The ggp1delta cells were treated with nikkomycin Z, a well-known inhibitor of chitin synthesis, and showed a hypersensitivity to this drug. In addition, studies of genetic interactions with genes related to the construction of the cell wall indicate a synthetic lethal effect of the ggp1delta kre6delta and the ggp1delta pkc1delta combined mutations. Our data point to an involvement of the GGP1 gene product in the cross-links between cell wall glucans (1,3-beta-D-glucans with 1,6-beta-D-glucans and with chitin). Chitin is essential to compensate for the defects due to the lack of Ggp1p. Moreover, the activities of Ggp1p and Chs3p are essential to the formation of the organized structure of the cell wall in vegetative cells.     

Disruption of the chitin synthase gene CHS1 from Fusarium asiaticum results in an altered structure of cell walls and reduced virulence

Natural resistance of wheat against Fusarium head blight (FHB) is inadequate and new strategies for controlling the disease are required. Chitin synthases that catalyze chitin biosynthesis would be an ideal target for antifungal agents. In this study, a class I chitin synthase gene (CHS1) from Fusarium asiaticum, the predominant species of FHB pathogens on wheat in China, was functionally disrupted via Agrobacterium tumefaciens-mediated transformation. Specific disruption of the CHS1 gene resulted in a 58% reduction of chitin synthase activity, accompanied by decreases of 35% in chitin content, 22% in conidiation, and 16% in macroconidium length. The Δchs1 mutant strain had a growth rate comparable to that of the wild-type on PDA medium but had a 35% increase in the number of nuclear cellulae and exhibited a remarkably increased sensitivity to osmosis stresses. Electron microscopy revealed substantial changes occurring in cell wall structures of the macroconidium, ascospore, and mycelium, with the most profound changes in the mycelium. Furthermore, the Δchs1 mutant displayed significantly reduced pathogenicity on wheat spikes and seedlings. Re-introduction of a functional CHS1 gene into the Δchs1 mutant strain restored the wild-type phenotype. These results reveal an important in vivo role played by a CHS1 gene in a FHB pathogen whose mycelial chitin could serve as a target for controlling the disease.     

Morphological changes induced by class III chitin synthase gene silencing could enhance penicillin production of Penicillium chrysogenum

Chitin synthases catalyze the formation of β-(1,4)-glycosidic bonds between N-acetylglucosamine residues to form the unbranched polysaccharide chitin, which is the major component of cell walls in most filamentous fungi. Several studies have shown that chitin synthases are structurally and functionally divergent and play crucial roles in the growth and morphogenesis of the genus Aspergillus although little research on this topic has been done in Penicillium chrysogenum. We used BLAST to find the genes encoding chitin synthases in P. chrysogenum related to chitin synthase genes in Aspergillus nidulans. Three homologous sequences coding for a class III chitin synthase CHS4 and two hypothetical proteins in P. chrysogenum were found. The gene which product showed the highest identity and encoded the class III chitin synthase CHS4 was studied in detail. To investigate the role of CHS4 in P. chrysogenum morphogenesis, we developed an RNA interference system to silence the class III chitin synthase gene chs4. After transformation, mutants exhibited a slow growth rate and shorter and more branched hyphae, which were distinct from those of the original strain. The results also showed that the conidiation efficiency of all transformants was reduced sharply and indicated that chs4 is essential in conidia development. The morphologies of all transformants and the original strain in penicillin production were investigated by light microscopy, which showed that changes in chs4 expression led to a completely different morphology during fermentation and eventually caused distinct penicillin yields, especially in the transformants PcRNAi1-17 and PcRNAi2-1 where penicillin production rose by 27 % and 41 %, respectively.     

Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae

Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH-activity profiles showed that yeast and hyphae contain a protease-dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin synthases in C. albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane-bound zymogen capable of chitin biosynthesis in vitro was based on several criteria. (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]-GlcNAc from the substrate, UDP[U-14C]-GlcNAc, into alkali-insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH-activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 8.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88,281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).     

Differential cell wall remodeling of two chitin synthase deletants Δchs3A and Δchs3B in the pathogenic yeast Candida glabrata

It is known that cell wall remodeling and the salvaging pathway act to compensate for an impaired or a damaged cell wall. Lately, it has been indicated that this mechanism is partly required for resistance to the glucan synthesis inhibitor echinocandin. While cell wall remodeling has been described in mutants of glucan or mannan synthesis, it has not yet been reported in a chitin synthesis mutant. Here, we describe a novel cell wall remodeling and salvaging pathway in chitin synthesis mutants, Δchs3A and Δchs3B, of the pathogenic yeast Candida glabrata. Electron microscopic analysis revealed a thickened mannoprotein layer in Δchs3A cells and a thickened chitin-glucan layer of Δchs3B cells, and it indicated the hypothesis that mannan synthase and chitin-glucan synthase indemnify Δchs3A and Δchs3B cells, respectively. The double-mutant CHS3A and MNN10, encoding α-1,6-mannosyltransferase, showed synergistic stress sensitization, and the Δchs3B strain showed supersensitivity to echinocandins. Hence, these findings support the above hypothesis of remodeling. Furthermore, unlike Δchs3A cells, Δchs3B cells showed supersensitivity to calcineurin inhibitor FK506 and Tor1p kinase inhibitor rapamycin, indicating that the Δchs3B strain uses the calcineurin pathway and a Tor1p kinase for cell wall remodeling.     

Genetic analysis of chs1+ and chs2+ encoding chitin synthases from Schizosaccharomyces pombe

To explore the function of chitin in Schizosaccharomyces pombe, we have cloned chs1+ and chs2+, encoding putative chitin synthases, based on sequences in the Sanger Centre database. The synthetic lethal phenotype of the S. cerevisiae chs1 chs2 chs3 mutant was complemented by expression of S. pombe chs1+ or chs1+, indicating that both chs1+ and chs2+ in fact encode chitin synthase. The homothallic Deltachs1 strain formed abnormal asci that contained 1, 2, or 3 spores, while the Deltachs2 strain had no noticeable phenotype. The chs1 chs2 double disruptant looked similar phenotypically to the Deltachs1 strain. The Chs2-GFP fusion protein predominantly localized at the septum after the septum was formed during vegetative growth. The level of chs2+ mRNA increased just before the septum was formed. Levels of Chs2-13Myc synthesis also changed during the cell cycle. Thus, chs1+ is required for proper spore formation, and chs2+ is perhaps involved in septum formation.     

Chs6p-dependent Anterograde Transport of Chs3p from the Chitosome to the Plasma Membrane in Saccharomyces cerevisiae

Chitin synthase III (CSIII), an enzyme required to form a chitin ring in the nascent division septum of Saccharomyces cerevisiae, may be transported to the cell surface in a regulated manner. Chs3p, the catalytic subunit of CSIII, requires the product of CHS6 to be transported to or activated at the cell surface. We find that chs6Δ strains have morphological abnormalities similar to those of chs3 mutants. Subcellular fractionation and indirect immunofluorescence indicate that Chs3p distribution is altered in chs6 mutant cells. Order-of-function experiments using end4–1 (endocytosis-defective) and chs6 mutants indicate that Chs6p is required for anterograde transport of Chs3p from an internal endosome-like membrane compartment, the chitosome, to the plasma membrane. As a result, chs6 strains accumulate Chs3p in chitosomes. Chs1p, a distinct chitin synthase that acts during or after cell separation, is transported normally in chs6 mutants, suggesting that Chs1p and Chs3p are independently packaged during protein transport through the late secretory pathway.     

Isolation of a class IV chitin synthase gene from a zygomycete fungus, Rhizopus oligosporus

We found the presence of DNA sequence which shows sequence similarity to the class IV chitin synthase gene (CHS3) of Saccharomyces cerevisiae in the genome of 14 Rhizopus species which belong to zygomycetes. We cloned a gene (chs3), which might correspond to one of these homologous sequences, from Rhizopus oligosporus by low stringency plaque hybridization probed with CHS3. The deduced amino acid sequence of this gene showed highest similarity to the class IV chitin synthase of Neurospora crassa (46.7% identity over 1087 amino acids), showing that this gene encodes a class IV chitin synthase. Northern analysis revealed the differential expression pattern of this gene in the asexual life cycle with highest expression in the early stage of asexual spore formation. This is the first report of the isolation and analysis of a class IV chitin synthase gene from zygomycete fungi.     

Proper ascospore maturation requires the chs1+ chitin synthase gene in Schizosaccharomyces pombe

We have cloned chs1+, a Schizosaccharomyces pombe gene with similarity to class II chitin synthases, and have shown that it is responsible for chitin synthase activity present in cell extracts from this organism. Analysis of this activity reveals that it behaves like chitin synthases from other fungi, although with specific biochemical characteristics. Deletion or overexpression of this gene does not lead to any apparent defect during vegetative growth. In contrast, chs1+ expression increases significantly during sporulation, and this is accompanied by an increase in chitin synthase activity. In addition, spore formation is severely affected when both parental strains carry a chs1 deletion, as a result of a defect in the synthesis of the ascospore cell wall. Finally, we show that wild-type, but not chs1-/chs1-, ascospore cell walls bind wheatgerm agglutinin. Our results clearly suggest the existence of a relationship between chs1+, chitin synthesis and ascospore maturation in S. pombe.     

Two chitin synthases in Saccharomyces cerevisiae

Disruption of the yeast CHS1 gene, which encodes trypsin-activable chitin synthase I, yielded strains that apparently lacked chitin synthase activity in vitro, yet contained normal levels of chitin (Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L., and Robbins, P. W. (1986) Cell 46, 213-225). It is shown here that disrupted (chs1 :: URA3) strains have a particulate chitin synthetic activity, chitin synthase II, and that wild type strains, in addition to chitin synthase I, have this second activity. Chitin synthase II is measured in wild type strains without preincubation with trypsin, the condition under which highest chitin synthase II activities are obtained in extracts from the chs1 :: URA3 strain. Chitin synthase II, like chitin synthase I, uses UDP-GlcNAc as substrate and synthesizes alkali-insoluble chitin (with a chain length of about 170 residues). The enzymes are equally sensitive to the competitive inhibitor Polyoxin D. The two chitin synthases are distinct in their pH and temperature optima, and in their responses to trypsin, digitonin, N-acetyl-D-glucosamine, and Co2+. In contrast to the report by Sburlati and Cabib (Sburlati, A., and Cabib, E. (1986) Fed. Proc. 45, 1909), chitin synthase II activity in vitro is usually lowered on treatment with trypsin, indicating that chitin synthase II is not activated by proteolysis. Chitin synthase II shows highest specific activities in extracts from logarithmically growing cultures, whereas chitin synthase I, whether from growing or stationary phase cultures, is only measurable after trypsin treatment, and levels of the zymogen do not change. Chitin synthase I is not required for alpha-mating pheromone-induced chitin synthesis in MATa cells, yet levels of chitin synthase I zymogen double in alpha factor-treated cultures. Specific chitin synthase II activities do not change in pheromone-treated cultures. It is proposed that of yeast's two chitin synthases, chitin synthase II is responsible for chitin synthesis in vivo, whereas nonessential chitin synthase I, detectable in vitro only after trypsin treatment, may not normally be active in vivo.     

A chitin synthase and its regulator protein are critical for chitosan production and growth of the fungal pathogen Cryptococcus neoformans

Chitin is an essential component of the cell wall of many fungi. Chitin also can be enzymatically deacetylated to chitosan, a more flexible and soluble polymer. Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. In this work, we show that both chitin and chitosan are present in the cell wall of vegetatively growing C. neoformans yeast cells and that the levels of both rise dramatically as cells grow to higher density in liquid culture. C. neoformans has eight putative chitin synthases, and strains with any one chitin synthase deleted are viable at 30 degrees C. In addition, C. neoformans genes encode three putative regulator proteins, which are homologs of Saccharomyces cerevisiae Skt5p. None of these three is essential for viability. However, one of the chitin synthases (Chs3) and one of the regulators (Csr2) are important for growth. Cells with deletions in either CHS3 or CSR2 have several shared phenotypes, including sensitivity to growth at 37 degrees C. The similarity of their phenotypes also suggests that Csr2 specifically regulates chitin synthesis by Chs3. Lastly, both chs3Delta and the csr2Delta mutants are defective in chitosan production, predicting that Chs3-Csr2 complex with chitin deacetylases for conversion of chitin to chitosan. These data suggest that chitin synthesis could be an excellent antifungal target.