The family of amphioxus chitin synthases offers insight into the evolution of chitin formation in chordates.

Chitin is a very important and widely-used biopolymer in fungi and lower metazoans, but mysteriously disappears in mammals. Recent studies reveal that at least lower vertebrates have chitin synthases (CS) and use them to synthesize endogenous chitin. Amphioxus, a basal chordate, therefore becomes critical to understand the evolution of CS, as it occupies the transitional position from invertebrates to vertebrates, and is considered as a good proxy to the chordate ancestor. Here, by exploiting multiple genome assemblies, high-depth RNA-seq data and synteny relations, we identify 11-12 CS genes for each amphioxus species. It represents the largest CS gene pool ever found in eukaryotes so far. As comparison, most metazoans have one or two CSs. Amphioxus is the only chordate that has both the very ancient type-I CS family and the more broadly distributed type-II CS family. Specifically, amphioxus has only one type-II CS but 10-11 type-I CSs, which means that amphioxus is the only metazoan with a greatly expanded type-I CS family. Further analysis suggests that the chordate ancestor have at least one type-II CS and an expanded of type-I CS family. We hypothesize that: these ancient CSs are mostly retained in amphioxus; but the whole type-I CS family was lost in urochordates and vertebrates; the type-II CS was later duplicated into two lineages in vertebrates and followed by stochastic losses, till all type-II CSs were eventually lost in birds and mammals. Finally, our expression profiling and preliminary gene knockout analysis suggest that amphioxus CSs could have highly diverse but mildly overlapping functions in various tissues and organs. Taken together, these findings not only provide insights into the evolution of chordate CSs, lay a foundation for further functional study of the chordate CSs. After all, it is mysterious that our chordate ancestor needed so many isoenzymes for chitin formation.

Genome-wide analyses of chitin synthases identify horizontal gene transfers towards bacteria and allow a robust and unifying classification into fungi.

BACKGROUND: Chitin, the second most abundant biopolymer on earth after cellulose, is found in probably all fungi, many animals (mainly invertebrates), several protists and a few algae, playing an essential role in the development of many of them. This polysaccharide is produced by type 2 glycosyltransferases, called chitin synthases (CHS). There are several contradictory classifications of CHS isoenzymes and, as regards their evolutionary history, their origin and diversity is still a matter of debate. RESULTS: A genome-wide analysis resulted in the detection of more than eight hundred putative chitin synthases in proteomes associated with about 130 genomes. Phylogenetic analyses were performed with special care to avoid any pitfalls associated with the peculiarities of these sequences (e.g. highly variable regions, truncated or recombined sequences, long-branch attraction). This allowed us to revise and unify the fungal CHS classification and to study the evolutionary history of the CHS multigenic family. This update has the advantage of being user-friendly due to the development of a dedicated website ( http://wwwabi.snv.jussieu.fr/public/CHSdb ), and it includes any correspondences with previously published classifications and mutants. Concerning the evolutionary history of CHS, this family has mainly evolved via duplications and losses. However, it is likely that several horizontal gene transfers (HGT) also occurred in eukaryotic microorganisms and, even more surprisingly, in bacteria. CONCLUSIONS: This comprehensive multi-species analysis contributes to the classification of fungal CHS, in particular by optimizing its robustness, consensuality and accessibility. It also highlights the importance of HGT in the evolutionary history of CHS and describes bacterial chs genes for the first time. Many of the bacteria that have acquired a chitin synthase are plant pathogens (e.g. Dickeya spp; Pectobacterium spp; Brenneria spp; Agrobacterium vitis and Pseudomonas cichorii). Whether they are able to produce a chitin exopolysaccharide or secrete chitooligosaccharides requires further investigation.

Large-scale phylogenetic classification of fungal chitin synthases and identification of a putative cell-wall metabolism gene cluster in Aspergillus genomes.

The cell wall is a protective and versatile structure distributed in all fungi. The component responsible for its rigidity is chitin, a product of chitin synthase (Chsp) enzymes. There are seven classes of chitin synthase genes (CHS) and the amount and type encoded in fungal genomes varies considerably from one species to another. Previous Chsp sequence analyses focused on their study as individual units, regardless of genomic context. The identification of blocks of conserved genes between genomes can provide important clues about the interactions and localization of chitin synthases. On the present study, we carried out an in silico search of all putative Chsp encoded in 54 full fungal genomes, encompassing 21 orders from five phyla. Phylogenetic studies of these Chsp were able to confidently classify 347 out of the 369 Chsp identified (94%). Patterns in the distribution of Chsp related to taxonomy were identified, the most prominent being related to the type of fungal growth. More importantly, a synteny analysis for genomic blocks centered on class IV Chsp (the most abundant and widely distributed Chsp class) identified a putative cell wall metabolism gene cluster in members of the genus Aspergillus, the first such association reported for any fungal genome.

Exposure to diflubenzuron results in an up-regulation of a chitin synthase 1 gene in citrus red mite, Panonychus citri (Acari: Tetranychidae).

Chitin synthase synthesizes chitin, which is critical for the arthropod exoskeleton. In this study, we cloned the cDNA sequences of a chitin synthase 1 gene, PcCHS1, in the citrus red mite, Panonychus citri (McGregor), which is one of the most economically important pests of citrus worldwide. The full-length cDNA of PcCHS1 contains an open reading frame of 4605 bp of nucleotides, which encodes a protein of 1535 amino acid residues with a predicted molecular mass of 175.0 kDa. A phylogenetic analysis showed that PcCHS1 was most closely related to CHS1 from Tetranychus urticae. During P. citri development, PcCHS1 was constantly expressed in all stages but highly expressed in the egg stage (114.8-fold higher than in the adult). When larvae were exposed to diflubenzuron (DFB) for 6 h, the mite had a significantly high mortality rate, and the mRNA expression levels of PcCHS1 were significantly enhanced. These results indicate a promising use of DFB to control P. citri, by possibly acting as an inhibitor in chitin synthesis as indicated by the up-regulation of PcCHS1 after exposure to DFB.

Functional differentiation of chitin synthases in Yarrowia lipolytica.

In this study, we identified seven chitin synthase-encoding genes in the genome of the dimorphic yeast Yarrowia lipolytica. Three encoded chitin synthases with myosin motor-like domains at their N-termini, and we designated these CSM1 to CSM3, whereas four were identified as CHS1 to CHS4. To investigate the functions of these seven genes, we constructed and characterized their deletion mutants. The chs2Δ mutant formed chained cells in which daughter cells were connected with mother cells and had abnormally thick septa at the bud neck. The chs4Δ mutant showed remarkably reduced chitin content in its cell wall. The chs2Δ, csm1Δ, and csm2Δ mutants were found to be highly sensitive to chitin binding dyes, calcofluor white (CFW) and Congo red, whereas the chs4Δ mutant was resistant to CFW. These results suggest that Chs2 and Chs4 play major roles in septum formation and cell wall chitin synthesis respectively, whereas Csm1 and Csm2 are involved in the maintenance of cell wall architecture and/or cell wall integrity. The populations of filamentous cells, a type of cell population that are defined by the lengths of the cellular long and short axes, decreased in the chs3Δ mutant, suggesting that Chs3 is involved in cellular morphogenesis.

Fusarium verticillioides chitin synthases CHS5 and CHS7 are required for normal growth and pathogenicity.

Fusarium verticillioides is both an endophyte and a pathogen of maize and is a health threat in many areas of the world because it can contaminate maize with fumonisins, a toxic secondary metabolite. We identified eight putative chitin synthase (CHS) genes in F. verticillioides genomic sequence, and phylogenetic evidence shows that they group into seven established CHS gene classes. We targeted two CHSs (CHS5 and CHS7) for deletion analysis and found that both are required for normal hyphal growth and maximal disease of maize seedlings and ears. CHS5 and CHS7 encode a putative class V and class VII fungal chitin synthase, respectively; they are located adjacent to each other and are divergently transcribed. Fluorescent microscopy found that both CHS deficient strains produce balloon-shaped hyphae, while growth assays indicated that they were more sensitive to cell wall stressing compounds (e.g., the antifungal compound Nikkomycin Z) than wild type. Pathogenicity assays on maize seedlings and ears indicated that both strains were significantly reduced in their ability to cause disease. Our results demonstrate that both CHS5 and CHS7 are necessary for proper hyphal growth and pathogenicity of F. verticillioides on maize.

Cloning and characterization of a Lentinula edodes class II chitin synthase gene, LeChs2.

A genomic DNA sequence and cDNA encoding a putative chitin synthase were isolated from the white rot basidiomycete Lentinula edodes. The gene, named LeChs2, consists of a 2,598-bp open reading frame interrupted by 14 introns and encodes a putative protein of 866 amino acid residues. The data obtained in this study suggest that LeChs2 belongs to the class II chitin synthases.

Chitin in diatoms and its association with the cell wall.

Chitin is a globally abundant polymer widely distributed throughout eukaryotes that has been well characterized in only a few lineages. Diatoms are members of the eukaryotic lineage of stramenopiles. Of the hundreds of diatom genera, two produce long fibers of chitin that extrude through their cell walls of silica. We identify and describe here genes encoding putative chitin synthases in a variety of additional diatom genera, indicating that the ability to produce chitin is more widespread and likely plays a more central role in diatom biology than previously considered. Diatom chitin synthases fall into four phylogenetic clades. Protein domain predictions and differential gene expression patterns provide evidence that chitin synthases have multiple functions within a diatom cell. Thalassiosira pseudonana possesses six genes encoding three types of chitin synthases. Transcript abundance of the gene encoding one of these chitin synthase types increases when cells resume division after short-term silicic acid starvation and during short-term limitation by silicic acid or iron, two nutrient conditions connected in the environment and known to affect the cell wall. During long-term silicic acid starvation transcript abundance of this gene and one additional chitin synthase gene increased at the same time a chitin-binding lectin localized to the girdle band region of the cell wall. Together, these results suggest that the ability to produce chitin is more widespread in diatoms than previously thought and that a subset of the chitin produced by diatoms is associated with the cell wall.

Dissection of the Candida albicans class I chitin synthase promoters.

Maintenance of the integrity of the cell wall in fungi is essential. One mechanism that cells use to maintain cell wall integrity in response to cell wall damage is to up-regulate chitin synthesis. In Candida albicans, the PKC cell wall integrity, Ca(2+)/calcineurin and high osmolarity glycerol (HOG) signalling pathways co-ordinately regulate chitin synthesis in response to cell wall stress. The transcription factors downstream of these pathways and their DNA binding sites within the promoters of target genes are well characterised in Saccharomyces cerevisiae, but not in C. albicans. The promoters of the C. albicans class I CHS genes (CaCHS2 and CaCHS8) were functionally dissected with the aim of identifying and characterising the transcription factors and promoter elements that mediate the transcriptional up-regulation of CaCHS2 and CaCHS8 in response to cell wall stress. This analysis provided evidence that the PKC cell wall integrity pathway may operate through RLM1-elements in the CaCHS2 and CaCHS8 promoters, but that promoter sequences that respond to the Ca(2+)/calcineurin and HOG signalling pathways in S. cerevisiae did not directly regulate chitin synthase 2 and 8 gene transcription in C. albicans.

The class A chitin synthase gene of Spodoptera exigua: molecular cloning and expression patterns.

Chitin synthase (CHS) is an important enzymatic component required for chitin formation in the cuticles and cuticular linings of other tissues. In the present study, a new CHS gene was characterized from the beet army worm Spodoptera exigua (Hübner) (Se). Homologous alignment and phylogenetic analysis of S. exigua CHS (SeCHS) with other related proteins suggest that SeCHS belongs to the class A CHS family (SeCHSA). Northern blot analysis revealed that SeCHSA is transcribed preferentially in the cuticle and tracheae. Further investigation indicated that SeCHSA mRNA is highly expressed in the early and late stages of each larval instar, and consistently expressed in high level during the pupal stage. Using antibody specific for CHS, SeCHS was further localized in the underlying epidermal cells of the integument and tracheal cells, but not in the fat body or Malpighian tubules. These data suggest that SeCHS plays an important role in cuticle formation and development of S. exigua.

Coccidioides posadasii contains single chitin synthase genes corresponding to classes I to VII.

Coccidioides posadasii is a dimorphic fungal pathogen of humans and other mammals. The switch between saprobic and parasitic growth involves synthesis of new cell walls of which chitin is a significant component. To determine whether particular subsets of chitin synthases (CHSes) are responsible for production of chitin at different stages of differentiation, we have isolated six CHS genes from this fungus. They correspond, together with another reported CHS gene, to single members of the seven defined classes of chitin synthases (classes I-VII). Using Real-Time RT-PCR we show their pattern of expression during morphogenesis. CpCHS2, CpCHS3, and CpCHS6 are preferentially expressed during the saprobic phase, while CpCHS1 and CpCHS4 are more highly expressed during the parasitic phase. CpCHS5 and CpCHS7 expression is similar in both saprobic and parasitic phases. Because C. posadasii contains single members of the seven classes of CHSes found in fungi, it is a good model to investigate the putatively different roles of these genes in fungal growth and differentiation.

WdChs1p, a class II chitin synthase, is more responsible than WdChs2p (Class I) for normal yeast reproductive growth in the polymorphic, pathogenic fungus Wangiella (Exophiala) dermatitidis.

The chitin synthase gene WdCHS1 was isolated from a partial genomic DNA library of the pathogenic polymorphic fungus Wangiella dermatitidis. Sequencing showed that WdCHS1 encoded a class II chitin synthase composed of 988 amino acids. Disruption of WdCHS1 produced strains that were hyperpigmented in rich media, grew as yeast at wild-type rates at both 25 and 37 degrees C and were as virulent as the wild type in a mouse model. However, detailed morphological and cytological studies of the wdchs1Delta mutants showed that yeast cells often failed to separate, tended to be enriched with chitin in septal regions and, sometimes, were enlarged with multiple nuclei, had broader mother cell-daughter bud regions and had other cell wall defects seen considerably less often than in the wild type or wdchs2 Delta strains. Disruption of WdCHS1 and WdCHS2 in the same background revealed that WdChs1p had functions synergistic to those of WdChs2p, because mutants devoid of both isozymes produced growth that was very abnormal at 25 degrees C and was not viable at 37 degrees C unless osmotically stabilized. These results suggested that WdChs1p was more responsible than WdChs2p for normal yeast cell reproductive growth because strains with defects in the latter exhibited no morphological abnormalities, whereas those with defects in WdChs1p were frequently impaired in one or more yeast developmental processes.

Polar localizing class V myosin chitin synthases are essential during early plant infection in the plant pathogenic fungus Ustilago maydis.

Fungal chitin synthases (CHSs) form fibers of the cell wall and are crucial for substrate invasion and pathogenicity. Filamentous fungi contain up to 10 CHSs, which might reflect redundant functions or the complex biology of these fungi. Here, we investigate the complete repertoire of eight CHSs in the dimorphic plant pathogen Ustilago maydis. We demonstrate that all CHSs are expressed in yeast cells and hyphae. Green fluorescent protein (GFP) fusions to all CHSs localize to septa, whereas Chs5-GFP, Chs6-GFP, Chs7-yellow fluorescent protein (YFP), and Myosin chitin synthase1 (Mcs1)-YFP were found at growth regions of yeast-like cells and hyphae, indicating that they participate in tip growth. However, only the class IV CHS genes chs7 and chs5 are crucial for shaping yeast cells and hyphae ex planta. Although most CHS mutants were attenuated in plant pathogenicity, Deltachs6, Deltachs7, and Deltamcs1 mutants were drastically reduced in virulence. Deltamcs1 showed no morphological defects in hyphae, but Mcs1 became essential during invasion of the plant epidermis. Deltamcs1 hyphae entered the plant but immediately lost growth polarity and formed large aggregates of spherical cells. Our data show that the polar class IV CHSs are essential for morphogenesis ex planta, whereas the class V myosin-CHS is essential during plant infection.

Class I and class II chitin synthases are involved in septum formation in the filamentous fungus Aspergillus nidulans.

The class II and class I chitin synthases of the filamentous fungus Aspergillus nidulans are encoded by chsA and chsC, respectively. Previously, we presented several lines of evidence suggesting that ChsA and ChsC have overlapping functions in maintaining cell wall integrity. In order to determine the functions of these chitin synthases, we employed electron and fluorescence microscopy and investigated in detail the cell wall of a DeltachsA DeltachsC double mutant (DeltaAC mutant) along with the localization of ChsA and ChsC. In the lateral cell wall of the DeltaAC mutant, electron-transparent regions were thickened. Septa of the DeltaAC mutant were aberrantly thick and had a large pore. Some septa were located abnormally close to adjacent septa. A functional hemagglutinin (HA)-tagged ChsA (HA-ChsA) and a functional FLAG-tagged ChsC (FLAG-ChsC) were each localized to a subset of septation sites. Comparison with the localization pattern of actin, which is known to localize at forming septa, suggested that ChsA and ChsC transiently exist at the septation sites during and shortly after septum formation. Double staining of HA-ChsA and FLAG-ChsC indicated that their localizations were not identical but partly overlapped at the septation sites. Fluorescence of FLAG-ChsC, but not of HA-ChsA, was also observed at hyphal tips. These data indicate that ChsA and ChsC share overlapping roles in septum formation.

CsmA, a class V chitin synthase with a myosin motor-like domain, is localized through direct interaction with the actin cytoskeleton in Aspergillus nidulans.

One of the essential features of fungal morphogenesis is the polarized synthesis of cell wall components such as chitin. The actin cytoskeleton provides the structural basis for cell polarity in Aspergillus nidulans, as well as in most other eukaryotes. A class V chitin synthase, CsmA, which contains a myosin motor-like domain (MMD), is conserved among most filamentous fungi. The DeltacsmA null mutant showed remarkable abnormalities with respect to cell wall integrity and the establishment of polarity. In this study, we demonstrated that CsmA tagged with 9x HA epitopes localized near actin structures at the hyphal tips and septation sites and that its MMD was able to bind to actin. Characterization of mutants bearing a point mutation or deletion in the MMD suggests that the interaction between the MMD and actin is not only necessary for the proper localization of CsmA, but also for CsmA function. Thus, the finding of a direct interaction between the chitin synthase and the actin cytoskeleton provides new insight into the mechanisms of polarized cell wall synthesis and fungal morphogenesis.

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.

Disruption of Botrytis cinerea class I chitin synthase gene Bcchs1 results in cell wall weakening and reduced virulence.

To get a better insight into the relationship between cell wall integrity and pathogenicity of the fungus Botrytis cinerea, we have constructed chitin synthase mutants. A 620 bp class I chitin synthase gene fragment (Bcchs1) obtained by PCR amplification was used to disrupt the corresponding gene in the genome. Disruption of Bcchs1 occurred at a frequency of 8%. Nine independent mutants were obtained and the Bcchs1 mutant phenotype compared to that of transformants in which the gene was not disrupted. These disruption mutants were dramatically reduced in their in vitro Mg2+, Mn2+, and Co2+-dependent chitin synthase activity. Chitin content was reduced by 30%, indicating that Bcchs1p contributes substantially to cell wall composition. Enzymatic degradation by a cocktail of glucanases revealed cell wall weakening in the mutant. Bcchs1 was transcribed at a constant level during vegetative exponential growth, suggesting that it was necessary throughout hyphal development. Bcchs1 mutant growth was identical to undisrupted control transformant growth, however, the mutant exhibited reduced pathogenicity on vine leaves. It can be assumed that disruption of Bcchs1 leads to cell wall weakening which might slow down in planta fungal progression.

Activation of chsC transcription by AbaA during asexual development of Aspergillus nidulans.

The temporal and spatial regulation of chitin synthesis plays an important role in morphogenesis during fungal growth and development. Northern blot analysis showed that the transcription level of chsA, chsC, and chsD was significantly decreased in an Aspergillus nidulans abaA mutant. Electrophoretic mobility shift assays revealed that AbaA bound tightly to all three AREs (AbaA response elements) in the chsC promoter region. Experiments with the Saccharomyces cerevisiae heterologous expression system confirmed AbaA-dependent transcriptional activation of chsC. Taken together, these data suggest that AbaA plays an important role in chitin biosynthesis during conidiophore development by controlling the transcription level of certain chitin synthase genes.

csmA, a gene encoding a class V chitin synthase with a myosin motor-like domain of Aspergillus nidulans, is translated as a single polypeptide and regulated in response to osmotic conditions.

The csmA gene of Aspergillus nidulans encodes a polypeptide that consists of an N-terminal myosin motor-like domain and a C-terminal chitin synthase domain. csmA null mutants showed marked abnormalities in polarized growth, hyphal wall integrity, and conidiophore development. Furthermore, the growth of the csmA null mutants was sensitive to low osmotic conditions. In an effort to investigate the intracellular behavior of the csmA product (CsmA) and the regulation of its production, we constructed strains that produced CsmA tagged with nine repeats of the hemagglutinin A (HA) epitope at its COOH terminus (CsmA-HA) instead of CsmA. Western blot analysis with anti-HA antibody showed that the entire coding region of csmA was translated as a single polypeptide with an approximate molecular mass of 210kDa. CsmA-HA was produced during vegetative growth; however, its yield was significantly reduced under high osmotic conditions, suggesting that the role of CsmA in growth and morphogenesis is particularly important under low osmotic conditions.