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The invasive species Aedes albopictus is a major vector of several arboviruses. The global spread of this species seriously threatens human health. Insecticide resistance is an increasing problem worldwide that limits the efficacy of mosquito control. As the major structural component of cuticles, chitin is indispensable to insects. Chitin synthase (CHS) is the enzyme that catalyzes the biosynthesis of chitin at the final step. In this study, two CHS genes of Aedes albopictus (AaCHS1 and AaCHS2) were identified and their basic characteristics were evaluated via bioinformatics analysis. The highest abundance of AaCHS1 transcripts was detected in pupae, whereas that of AaCHS2 transcripts was detected in females; the highest expression levels of AaCHS1 and AaCHS2 were found in the epidermis and the midgut of pupae, respectively. The survival and emergence rates of pupae were significantly reduced after the injection of double-stranded RNA of AaCHS1 or AaCHS2, indicating that both AaCHS1 and AaCHS2 play crucial roles in the pupal development. In addition, the chitin content of pupae was obviously decreased after the suppression of AaCHS1 expression by RNA interference (RNAi) treatment. This influence of the RNAi treatment was further supported by the reduced chitin thickness and weakened chitin fluorescence signal in the new cuticle. The midgut of pupae presented a reduced intensity of the chitin fluorescence signal along with RNAi treatment specific to AaCHS2 expression. The results of this study indicate that CHS genes may be suitable as molecular targets used for controlling mosquitoes., (© 2024 Wiley Periodicals LLC.)

Chitin is of great importance in the cuticle and inner cuticular linings of insects. Chitin synthases (CHSs), chitin deacetylases (CDAs), chitinases (CHTs), and β-N-acetylhexosaminidases (HEXs) are important enzymes required for chitin metabolism, and play essential roles in development and metamorphosis. Although chitin metabolism genes have been well characterized in limited insects, the information in the yellow mealworm, Tenebrio molitor, a model insect, is presently still unavailable. With the help of bioinformatics, we identified 54 genes that encode putative chitin metabolism enzymes, including 2 CHSs, 10 CDAs, 32 CHTs, and 10 HEXs in the genome of T. molitor. All these genes have the conserved domains and motifs of their corresponding protein family. Phylogenetic analyses indicated that CHS genes were divided into two groups. CDA genes were clustered into five groups. CHT genes were phylogenetically grouped into 11 clades, among which 1 in the endo-β-N-acetylglucosaminidases group and the others were classified in the glycoside hydrolase family 18 groups. HEX genes were assorted into six groups. Developmental and tissue-specific expression profiling indicated that the identified chitin metabolism genes showed dynamical expression patterns concurrent with specific instar during molting period, suggesting their significant roles in molting and development. They were predominantly expressed in different tissues or body parts, implying their functional specialization and diversity. The results provide important information for further clarifying their biological functions using the yellow mealworm as an ideal experimental insect., (© 2022 Wiley Periodicals LLC.)

The polytopic yeast protein Chs3 (chitin synthase III) relies on a dedicated membrane-localized chaperone, Chs7, for its folding and expression at the cell surface. In the absence of Chs7, Chs3 forms high molecular weight aggregates and is retained in the endoplasmic reticulum (ER). Chs7 was reported to be an ER resident protein, but its role in Chs3 folding and transport was not well characterized. Here, we show that Chs7 itself exits the ER and localizes with Chs3 at the bud neck and intracellular compartments. We identified mutations in the Chs7 C-terminal cytosolic domain that do not affect its chaperone function, but cause it to dissociate from Chs3 at a post-ER transport step. Mutations that prevent the continued association of Chs7 with Chs3 do not block delivery of Chs3 to the cell surface, but dramatically reduce its catalytic activity. This suggests that Chs7 engages in functionally distinct interactions with Chs3 to first promote its folding and ER exit, and subsequently to regulate its activity at the plasma membrane., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The yeast spore wall is an excellent model to study the assembly of an extracellular macromolecule structure. In the present study, mutants defective in β-1,6-glucan synthesis, including kre1∆, kre6∆, kre9∆ and big1∆, were sporulated to analyse the effect of β-1,6-glucan defects on the spore wall. Except for kre6∆, these mutant spores were sensitive to treatment with ether, suggesting that the mutations perturb the integrity of the spore wall. Morphologically, the mutant spores were indistinguishable from wild-type spores. They lacked significant sporulation defects partly because the chitosan layer, which covers the glucan layer, compensated for the damage. The proof for this model was obtained from the effect of the additional deletion of CHS3 that resulted in the absence of the chitosan layer. Among the double mutants, the most severe spore wall deficiency was observed in big1∆ spores. The majority of the big1∆chs3∆ mutants failed to form visible spores at a higher temperature. Given that the big1∆ mutation caused a failure to attach a GPI-anchored reporter, Cwp2-GFP, to the spore wall, β-1,6-glucan is involved in tethering of GPI-anchored proteins in the spore wall as well as in the vegetative cell wall. Thus, β-1,6-glucan is required for proper organization of the spore wall. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Chitin synthase (CHS) is an important enzymatic component, which is required for chitin formation in the cuticles and cuticular linings of other tissues in insects. CHSs have been divided into two classes, classes A and B, based on their amino acid sequence similarities and functions. Class A CHS (CHS-A) is specifically expressed in the epidermis and related ectodermal cells such as tracheal cells, while class B CHS (CHS-B) is expressed in gut epithelial cells that produce peritrophic matrices. In this study, we cloned the CHS-A gene from the beet armyworm, Spodoptera exigua (SeCHS-A). The SeCHS-A contains an open reading frame of 4,698 nucleotides, encoding a protein of 1,565 amino acids with a predicted molecular mass of approximately 177.8 kDa. The SeCHS-A mRNA was expressed in all developmental stages and specifically in the epidermis and tracheae tissue by quantitative real-time-PCR analysis. Expression of SeCHS-A gene was suppressed by feeding double-stranded RNA (dsCHS-A, 400 ng/larva) in the third instar larvae of S. exigua. Suppression of the SeCHS-A gene expression significantly increased 35% of mortality on pupation of S. exigua. Also, the third instar larvae fed with dsCHS-A significantly increased susceptibility to entomopathogenic fungi, Beauveria bassiana ANU1 at 3 days after treatment. These results suggest that the SeCHS-A gene plays an important role in development of S. exigua and RNA interference may apply to effective pest control with B. bassiana., (© 2017 Wiley Periodicals, Inc.)

Chitin synthase (ChS) plays a critical role in chitin synthesis and excretion. In this study, two ChS genes (LdChSA and LdChSB) were identified in Leptinotarsa decemlineata. LdChSA contains two splicing variants, LdChSAa and LdChSAb. Within the first, second, and third larval instars, the mRNA levels of LdChSAa, LdChSAb, and LdChSB coincide with the peaks of circulating 20-hydroxyecdysone (20E) and juvenile hormone (JH). In vitro culture of midguts and an in vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide stimulated the expression of the three LdChSs. Conversely, a reduction of 20E by RNA interference (RNAi) of an ecdysteroidogenesis gene LdSHD repressed the expression of these LdChSs, and ingestion of halofenozide by LdSHD RNAi larvae rescued the repression. Moreover, disruption of 20E signaling by RNAi of LdEcR, LdE75, LdHR3, and LdFTZ-F1 reduced the expression levels of these genes. Similarly, in vitro culture and an in vivo bioassay showed that exogenous JH and a JH analog methoprene activated the expression of the three LdChSs, whereas a decrease in JH by RNAi of a JH biosynthesis gene LdJHAMT downregulated these LdChSs. It seems that JH upregulates LdChSs at the early stage of each instar, whereas a 20E pulse triggers the transcription of LdChSs during molting in L. decemlineata., (© 2016 Wiley Periodicals, Inc.)

The peritrophic matrix (PM) is an acellular structure that lines the gut of most insects. It is an attractive target for pest management strategies because of its close involvement in digestive processes and role as a barrier against pathogens and toxins. The purpose of this study was to identify and characterize the genes that translate for principal components of the Reticulitermes flavipes PM. Genes encoding a gut chitin synthase (CHS), two proteins with peritrophin-A domains, and a chitin deacetylase were identified from an R. flavipes symbiont-free gut cDNA library, a pyrosequencing study of termite lignocellulose digestion, and a metatranscriptomic analysis of R. flavipes fed on agricultural biomass. Quantitative expression analysis of the identified genes, in the termite digestive tract, revealed that the transcripts coding for a CHS (RfCHSB) and the proteins with peritrophin-A domains (RfPPAD1 and RfPPAD2) were predominantly expressed in the midgut, suggesting an association with the PM. The peritrophin identity of the RfPPAD2 gene was confirmed by immunodetection of its translated peptide in the midgut and PM. The discovery and characterization of PM components of R. flavipes provides a basis for further investigation of the viability of this structure as a target for candidate termiticides., (© 2016 Wiley Periodicals, Inc.)

Although chitin is an essential component of the fungal cell wall (CW), its biosynthesis and role in virulence is poorly understood. In Aspergillus fumigatus, there are eight chitin synthase (CHS) genes belonging to two families CHSA-C, CHSG in family 1 and CHSF, CHSD, CSMA, CSMB in family 2). To understand the function of these CHS genes, their single and multiple deletions were performed using β-rec/six system to be able to delete all genes within each family (up to a quadruple ΔchsA/C/B/G mutant in family 1 and a quadruple ΔcsmA/csmB/F/D mutant in family 2). Radial growth, conidiation, mycelial/conidial morphology, CW polysaccharide content, Chs-activity, susceptibility to antifungal molecules and pathogenicity in experimental animal aspergillosis were analysed for all the mutants. Among the family 1 CHS, ΔchsA, ΔchsB and ΔchsC mutants showed limited impact on chitin synthesis. In contrast, there was reduced conidiation, altered mycelial morphotype and reduced growth and Chs-activity in the ΔchsG and ΔchsA/C/B/G mutants. In spite of this altered phenotype, these two mutants were as virulent as the parental strain in the experimental aspergillosis models. Among family 2 CHS, phenotypic defects mainly resulted from the CSMA deletion. Despite significant morphological mycelial and conidial growth phenotypes in the quadruple ΔcsmA/csmB/F/D mutant, the chitin content was poorly affected by gene deletions in this family. However, the entire mycelial cell wall structure was disorganized in the family 2 mutants that may be related to the reduced pathogenicity of the quadruple ΔcsmA/csmB/F/D mutant strain compared to the parental strain, in vivo. Deletion of the genes encompassing the two families (ΔcsmA/csmB/F/G) showed that in spite of being originated from an ancient divergence of fungi, these two families work cooperatively to synthesize chitin in A. fumigatus and demonstrate the essentiality of chitin biosynthesis for vegetative growth, resistance to antifungal drugs, and virulence of this filamentous fungus., (© 2014 John Wiley & Sons Ltd.)

Forkhead (Fox) transcription factors display functional diversity and are involved in various metabolic and developmental processes. The Spodoptera exigua Fox (SeFox) encodes a protein of 353 amino acids with a theoretical molecular mass of approximately 38.99 kDa and an isoelectric point of 8.86. qPCR results revealed that SeFox was expressed mainly in the brain, fat body, epidermis, midgut, Malpighian tubules, and testis. SeFox was expressed, with some changes, throughout development in the fat body and whole body. Injection of dsSeFox (SeFox dsRNA) into larvae resulted in incidences of albino plus molting deformity (4.8%), molting deformity (26.2%), and albino phenotypes (69.1%). dsSeFox injection resulted in approximately 50% knockdown of transcript levels at 36 h. Compared with control groups, hexokinase (HK) expression was reduced to approximately 40% at 48 h postinjection. Chitin synthase A (CHSA) expression was reduced to two-thirds at 24 h, but increased at 72 h. Compared with untreated control and green fluorescent protein-treated groups, Chitin synthase B (CHSB) expression decreased to 33% following dsSeFox injection by 36 h. We infer from our results that forkhead transcription factors act in chitin synthesis in S. exigua., (© 2014 Wiley Periodicals, Inc.)

Chitin synthase (CHS) is the key regulatory enzyme in chitin synthesis and excretion in insects, and a specific target of insecticides. We cloned a CHS B gene of Bombyx mori (BmChsB) and showed it to be midgut specific, highly expressed during the feeding process in the larva. Knockdown of BmChsB expression in the third-instar larvae increased the number of nonmolting and abnormally molting larvae. Exposure to nikkomycin Z, a CHS inhibitor, reduced the amount of chitin in the peritrophic membrane of molted larvae, whereas abnormally elevated BmChsB mRNA levels were readily detected from the end of molting and in the newly molted larvae. Exogenous 20-hydroxyecdysone (20E) and methoprene, a juvenile hormone analogue, significantly upregulated the expression of BmChsB when the levels of endogenous molting hormone (MH) were low and the levels of endogenous juvenile hormone (JH) were high immediately after molting. When levels of endogenous MH were high and those of endogenous JH were low during the molting stage, exogenous 20E did not upregulate BmChsB expression and exogenous methoprene upregulated it negligibly. When the endogenous hormone levels were low during the mulberry-leaf intake process, BmChsB expression was upregulated by exogenous methoprene. We conclude that the expression of BmChsB is regulated by insect hormones, and directly affects the chitin-synthesis-dependent form of the peritrophic membrane and protects the food intake and molting process of silkworm larvae., (© 2013 Wiley Periodicals, Inc.)

Analyses of environmental DNAs have provided tantalizing evidence for "rozellida" or "cryptomycota", a clade of mostly undescribed and deeply diverging aquatic fungi. Here, we put cryptomycota into perspective through consideration of Rozella, the only clade member growing in culture. This is timely on account of the publication in Nature of the first images of uncultured cryptomycota from environmental filtrates, where molecular probes revealed non-motile cyst-like structures and motile spores, all lacking typical fungal chitinous cell walls. Current studies of Rozella can complement these fragmentary observations from environmental samples. Rozella has a fungal-specific chitin synthase and its resting sporangia have walls that appear to contain chitin. Cryptomycota, including Rozella, lack a cell wall when absorbing food but like some other fungi, they may have lost their "dinner jacket" through convergence. Rather than evolutionary intermediates, the cryptomycota may be strange, divergent fungi that evolved from an ancestor with a nearly complete suite of classical fungal-specific characters., (Copyright © 2012 WILEY Periodicals, Inc.)

Chitin synthase catalyzes chitin synthesis in the exoskeleton, tracheal system and gut during insect development. A chitin synthase 1 (CfCHS1) cDNA was identified and cloned from the spruce budworm, Choristoneura fumiferana. The CfCHS1 cDNA is 5,300 bp in length and codes a 1,564-amino acid protein with a molecular mass of 178 kDa. The deduced protein contains 16 transmembrane helixes in its domains A and C. The single copy CfCHS1 gene expressed during each of the larval molts from the 3rd to the 6th instar. The gene expressed highly and periodically in the epidermis during each of molts, whereas no transcripts were detected in the midgut and fat body. 20-hydroxyecdysone and the ecdysone agonist RH5992 suppressed CfCHS1 expression, whereas the juvenile hormone analog methoprene induced CfCHS1 expression. These results implicate that CfCHS1 is involved in the chitin synthase and new chitin formation during molting in the insect., (© 2010 Wiley Periodicals, Inc.)

We report the isolation and sequencing of CHS3, a gene that encodes one of several chitin synthases in Paracoccidioides brasiliensis, a medically important fungus restricted geographically to Latin America. The gene contains a single open reading frame of 3817 bp with two introns (71 and 86 bp) and encodes a 1220 amino acid polypeptide with high similarity to other fungal chitin synthases. Northern analysis reveals a high expression of CHS3 in the pathogenic yeast-like phase of the fungus and at the end of the mycelium-yeast transition. Expression of P. brasiliensis CHS3 in a Saccharomyces cerevisiae chs3 null mutant enhanced calcofluor white staining in parallel to an increase in total chitin synthase activity and chitin content in its cell wall., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Dihydromaltophilin (heat-stable antifungal factor [HSAF]) is an antifungal metabolite produced in Lysobacter enzymogenes biocontrol strain C3. This compound induces cell wall thickening in Aspergillus nidulans. Here we show that the cell wall thickening is a general response to HSAF in diverse fungal species. In the A. nidulans model, the thickened cell wall negatively affects hyphal growth. Growth of HSAF-pre-treated hyphae failed to resume at hyphal tips with thick cell wall and the actin cable could not re-polarize at the thickened region of the cell wall, even after the treated hyphae were transferred to drug-free medium. Moreover, HSAF-induced cell wall thickening is mediated by sphingolipid synthesis: HSAF failed to induce cell wall thickening in the absence of ceramide synthase BarA and the sphingolipid synthesis inhibitor myriocin was able to suppress HSAF-induced cell wall thickening. The thickened cell wall could be digested by chitinase suggesting that chitin contributes to the HSAF-induced thickening. Furthermore, HSAF treatment activated the transcription of two chitin synthase encoding genes chsB and chsC.

Botrytis cinerea is an important phytopathogenic fungus requiring new methods of control. Chitin biosynthesis, which involves seven classes of chitin synthases, could be an attractive target. A fragment encoding one of the class III enzymes was used to disrupt the corresponding Bcchs3a gene in the B. cinerea genome. The resulting mutant exhibited a 39% reduction in its chitin content and an 89% reduction in its in vitro chitin synthase activity, compared with the wild-type strain. Bcchs3a mutant was not affected in its growth in liquid medium, neither in its production of sclerotia, micro- and macroconidia. In contrast, the mutant Bcchs3a was severely impaired in its growth on solid medium. Counterbalancing this defect in radial growth, Bcchs3a mutant presented a large increase in hyphal ramification, resulting in an enhanced aerial growth. Observations by different techniques of microscopy revealed a thick extracellular matrix around the hyphal tips. Moreover, Bcchs3a mutant had a largely reduced virulence on Vitis vinifera and Arabidopsis thaliana leaves.

Until recently the only intein known to be encoded by the nuclear genome of a eukaryote was the VMA intein in the vacuolar ATPase precursor of several species of saccharomycete yeast. This intein has been intensively studied and much information has been gained about its structure, mode of action and evolutionary history. We recently reported a second nuclear intein, Cne PRP8, encoded within the PRP8 gene of the basidiomycete Cryptococcus neoformans. Subsequent studies have found allelic PRP8 inteins in several species of yeast and filamentous ascomycetes. Here we report two further, non-allelic, inteins from ascomycete species. The yeast Debaryomyces hansenii (which also has a VMA intein) has an intein encoded within the sequence of the glutamate synthase gene (GLT1). There are also inteins encoded in the homologous GLT1 genes of the yeast Candida (Pichia) guilliermondii and the filamentous fungus Podospora anserina. These allelic GLT1 inteins occupy exactly the same site in the glutamate synthase and all contain domains that indicate the presence of a homing endonuclease (HEG). Podospora anserina, in addition, contains a second, non-allelic, intein encoded in the chitin synthase gene (CHS2); this intein also contains a HEG domain. We describe the phylogenetic relationships among the four eukaryote nuclear encoded inteins (VMA, PRP8, GLT1 and CHS2). We also consider this phylogeny in the broader context of eubacterial, archaeal and eukaryote viral and organelle inteins.

Exoskeletons stabilize cell, tissue, and body morphology in many living organisms including fungi, plants, and arthropods. In insects, the exoskeleton, the cuticle, is produced by epidermal cells as a protein extracellular matrix containing lipids and the polysaccharide chitin, and its formation requires coordinated synthesis, distribution, and modification of these components. Eventually, the stepwise secretion and sorting of the cuticle material results in a layered structure comprising the envelope, the proteinaceous epicuticle, and the chitinous procuticle. To study the role of chitin during cuticle development, we analyzed the consequences of chitin absence in the embryo of Drosophila melanogaster caused by mutations in the Chitin Synthase-1 (CS-1) gene, called krotzkopf verkehrt (kkv). Our histological data confirm that chitin is essential for procuticle integrity and further demonstrate that an intact procuticle is important to assemble and to stabilize the chitin-less epicuticle. Moreover, the phenotype of CS-1/kkv mutant embryos indicates that chitin is required to attach the cuticle to the epidermal cells, thereby maintaining epidermal morphology. Finally, sclerotization and pigmentation, which are the last steps in cuticle differentiation, are impaired in tissues lacking CS-1/kkv function, suggesting that proper cuticle structure is crucial for the activity of the underlying enzymes., (Copyright 2005 Wiley-Liss, Inc.)

The cellular response of Saccharomyces cerevisiae to a linear plasmid encoded killer toxin from Pichia acaciae was analysed. As for the Kluyveromyces lactis zymocin, such toxin was recently shown to bind to the target cell's chitin and probably acts by facilitating the import of a toxin subunit. However, as distinct from zymocin, which arrests cells in G1, it provokes S-phase arrest and concomitant DNA damage checkpoint activation. Here, we report that such novel toxin type causes cell death in a two-step process. Within 4 h in toxin, viability of cells is immediately reduced to approximately 30%. Elevated mutation rates at the CAN1 locus prove DNA damaging mediated by the toxin. Cells arrested artificially in G1 or G2/M are very rapidly affected, while cells arrested in S loose their viability at a slower rate. S-phase arrest is, thus, a response of target cells to cope with DNA damage induced by the toxin. A second decline in viability requiring metabolically active target cells emerges upon toxin exposure over 10 h. During this phase, toxin treated cells develop abnormal nuclear morphology and react positive to terminal deoxynucleotidyl transferase-mediated nick end-labelling (TUNEL), indicative of DNA fragmentation. Furthermore, as judged from staining with fluorescein conjugated annexinV, cells expose phosphatidylserine at the outer membrane face and the formation of reactive oxygen species (ROS) is increased. ROS formation and concomitant cell death was heavily suppressed in a rho- derivative of the tester strain, while immediate reduction of viability was indistinguishable from the wild type. As a strain lacking the cellular target because of defects in the major chitinsynthase (Chs3) did not display such characteristic changes, the chitin binding and DNA-damaging P. acaciae toxin constitutes an apoptosis inducing protein. Both, DNA-damaging and apoptosis induction are unique features of this novel toxin type.

In Saccharomyces cerevisiae, Chs4p directly interacts with chitin synthase III (Chs3p) to act as a post-translational regulator of the Chs3p complex. We identified four Chs4p homologous proteins in Schizosaccharomyces pombe which we named Chr1, Chr2, Chr3 and Chr4 (putative chitin synthase regulatory factor). We assessed the functions of these proteins and found that while overproduction of Chr1, Chr2 or Chr3 did not affect the cellular morphology of wild-type Sz. pombe cells, overproduction of Chr4 caused the cells to form multi-septa and delayed their growth. All multiple disruptants of chr1, chr2, chr3 and chr4 grew normally under a variety of growth conditions. However, while chitin synthase II (Chs2) normally localizes exclusively at the septum, in many chr4-disrupted cells it was found in the cytoplasm and the septa. Chs2 did localize at the abnormal septa caused by the overproduction of chr4+. Chr4-13Myc expression was unaffected by the different media or growth conditions in both wild-type and the chs2 disruptant. Chs2 expression was also unaltered by the absence of Chr4. Moreover, Chr4-13Myc localized mostly at the tips and the septum during vegetative growth in chs2, chr1, chr2 and chr3 disruptants as well as in wild-type. Thus, chr4+ is involved in septum formation and is required for the proper localization of Chs2 at the septum in Sz. pombe., (Copyright (c) 2004 John Wiley & Sons, Ltd.)

The objective of this study was to quantify the effect of disrupting two chitin synthases, chsB and csmA, on the morphology and rheology during batch cultivation of Aspergillus oryzae. The rheological properties were characterized in batch cultivations at different biomass concentrations (from 3.4-22.5 g kg(-1) biomass) and the power-law model adequately described the rheological properties. In the cultivations there were pellets, clumps, and freely dispersed hyphal elements. The different morphological fractions were quantified using image analysis. The apparent viscosity of the fermentation broth was significantly affected by the biomass concentration, the morphology, and also by pH. The chsB disruption strain had lower consistency index K values for all biomass concentrations investigated, which is a desirable trait for industrial Aspergillus fermentations., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 525-534, 2003.)

Chitin synthase III is essential for the increase in chitin level and for cell integrity in cells lacking Gas1p, a beta(1,3)-glucanosyltransferase. In order to discover whether the upregulation of chitin synthesis proceeds through the canonical transport and activation pathway of Chs3p or through an alternative one, here we studied the effects of the inactivation of the GAS1 and CHS4-5-6-7 genes. All the double-null mutants showed a temperature-sensitive cell lysis phenotype that could be suppressed by the presence of an osmotic stabilizer. In liquid YEPD at 30 degrees C, chs4 delta gas1 delta, chs5 delta gas1 delta, chs6 deltagas1 delta and chs7 delta gas1 delta mutants were unable to grow, whereas they grew very slowly in minimal medium and showed low viability. High osmolarity suppressed the defective phenotype and restored growth. In chs4 gas1, chs5 gas1 and chs7 gas1, chitin levels did not increase and were reduced to only 10%, while in chs6 gas1 the value of gas1 was reduced to 20-40%. To investigate at which level the upregulation of chitin synthesis could occur, mRNA levels were monitored. The expression of CHS4-5-6-7 did not change significantly in gas1 delta. In strains expressing HA-tagged forms, the localization of Chs3p and Chs5p was examined. In the gas1 mutant the fluorescence pattern was affected and the proteins appeared abnormally present in the bud. The results indicate that: (a) the function of the CHS4-7 genes is required for chitin hyperaccumulation in gas1 mutant and for cell integrity; (b) homologous genes do not replace their function; (c) the regulation of CHS4-7 genes does not occur at transcriptional level. Control of the position of chitin synthesis could be important in protecting the bud from lysis., (Copyright 2002 John Wiley & Sons, Ltd.)

The exozymocin secreted by Kluyveromyces lactis causes sensitive yeast cells, including Saccharomyces cerevisiae, to arrest growth in the G(1) phase of the cell cycle. Despite its heterotrimeric (alpha beta gamma) structure, intracellular expression of its smallest subunit, the gamma-toxin, is alone responsible for the G(1) arrest. The alpha subunit, however, has a chitinase activity that is essential for holozymocin action from the cell exterior. Here we show that sensitive yeast cells can be rescued from zymocin treatment by exogenously applying crude chitin preparations, supporting the idea that chitin polymers can compete for binding to zymocin with chitin present on the surface of sensitive yeast cells. Consistent with this, holozymocin can be purified by way of affinity chromatography using an immobilized chitin matrix. PCR-mediated deletions of chitin synthesis (CHS) genes show that most, if not all, genetic scenarios that lead to complete loss (chs3 Delta), blocked export (chs7 Delta) or reduced activation (chs4 Delta), combined with mislocalization (chs4 Delta chs5 Delta; chs4 Delta chs6 Delta; chs4 Delta chs5 Delta chs6 Delta) of chitin synthase III activity (CSIII), render cells refractory to the inhibitory effects of exozymocin. In contrast, deletions in CHS1 and CHS2, which code for CSI and CSII, respectively, have no effect on zymocin sensitivity. Thus, CSIII-polymerized chitin, which amounts to almost 90% of the cell's chitin resources, appears to be the carbohydrate receptor required for the initial interaction of zymocin with sensitive cells., (Copyright 2001 John Wiley & Sons, Ltd.)

The nucleotide sequence of a chitin synthase gene (CHS2) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined. The deduced amino acid sequence of Chs2p consists of 1043 residues and is highly homologous to other class II fungal chitin synthases. Computational structural analyses suggest very high similarity to other fungal chitin synthases with a highly variable region at the cytosolic amino-terminal region which may be related to its possible zymogenic nature, and the putative catalytic region close to seven membrane-spanning regions at the carboxyl terminus.

A mutant screen has been designed to isolate mutants in Saccharomyces cerevisiae deficient in spore wall dityrosine. As shown by electron microscopy, most of the mutant spores lacked only the outermost, dityrosine-rich layer of the spore wall. Mutant dit101, however, was additionally lacking the chitosan layer of the spore wall. Chemical measurements showed that this mutant does not synthesize chitosan during sporulation. The mutant spores were viable but sensitive to lytic enzymes (glusulase or zymolyase). Unlike most of the dit-mutants, dit101 did show a distinctive phenotype in vegetative cells: they grew normally but contained very little chitin and were therefore resistant to the toxic chitin-binding dye, Calcofluor White. The cells showed barely detectable staining of the walls with Calcofluor White or primulin. The decrease in the amount of chitin in vegetative cells and the absence of chitosan in spores suggested that the mutant dit101 could be defective in a chitin synthase. Indeed, a genomic yeast clone harboring the gene, CSD2, sharing significant sequence similarity with yeast chitin synthases I and II (C. E. Bulawa (1992), Mol. Cell. Biol. 12, 1764-1776), complemented our mutant and was shown to correspond to the chromosomal locus of dit101. Thus, the mutations dit101 and csd2 (and probably also call; M. H. Valdivieso et al., (1991), J. Cell Biol. 114, 101-109) were shown to be allelic. The gene was mapped to chromosome II and was located about 3 kb distal of GAL1. Using this DNA clone, a transcript of about 3500-4000 nucleotides was detected. Comparing RNA isolated from vegetative cells and from sporulating cells at different times throughout the sporulation process, no significant differences in DIT101 transcript levels could be detected indicating absence of sporulation-specific transcriptional regulation. However, the amount of DIT101 transcript changed significantly at different stages of the mitotic cell cycle, peaking after septum formation, but before cytokinesis. As most of the chitin synthesis of vegetative cells occurs at this stage of the cell division cycle, chitin synthesis mediated by DIT101 could be primarily regulated at the level of transcription in vegetatively growing cells.

Saccharomyces cerevisiae produces two chitin synthases (Chs1 and Chs2) encoded by separate genes. Although these enzymes catalyze the same reaction, Chs2 is essential for septum formation whereas Chs1 has a repair function. To determine if these physiological differences are reflected in the enzyme structures, the CHS2 gene was sequenced and compared to that of CHS1. The predicted amino acid sequence of Chs2 shares substantial similarity with that of Chs1 in the carboxyl two-thirds of the protein. The amino one-third segments differ in predicted isoelectric point by almost 5 pH units. It is suggested that the similar regions are related to common catalytic function. The unrelated regions may be involved in regulation or localization of the respective enzymes. CHS1 and CHS2 are unlinked but may have arisen from the duplication of an ancestral gene.