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The genetics and molecular biology of marine fungi
mycologist 20 (2006) 144–151
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/mycol
The genetics and molecular biology of marine fungi Paul HOOLEY*, Michael WHITEHEAD School of Applied Sciences, University of Wolverhampton, WV1 1SB, UK
abstract Keywords: Climate change
Interest in the genetics of marine fungi has focused upon the basis of stress adaptation and
Cloning
the control of the production of secondary metabolites and enzymes. Analysis by molecu-
Debaryomyces
lar genetics has been applied to marine fungal taxonomy, phylogeny and species identifi-
Genomics
cation. The advent of the Debaryomyces hansenii genome project and the influence of
Marine fungi
climate change on this research are discussed. ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Mutants
1.
Introduction
Fungal genetics was first studied in detail in the 1940’s as fungi appeared to present ideal models to study the nature of the gene. Beadle and Tatum’s classic work on Neurospora crassa Shear and Dodge laid the foundations for the ‘‘one gene – one enzyme hypothesis’’. Nutritionally deficient mutants (auxotrophs) allowed the dissection of biochemical pathways that illustrated the stepwise conversion of precursor molecules to products, each step under the control of a single gene encoding a single enzyme. In Glasgow, Pontecorvo’s group selected Aspergillus nidulans (Edam) Winter as their model. Some species such as Sordaria fimicola (Roberge ex Desm.) Griffiths & Seaver were even obliging enough to provide the results of their meiotic divisions in linear ordered tetrads. The field of fungal genetics relies mainly on work performed on just a few terrestrial ascomycetes, including the yeasts Saccharomyces cerevisiae Hansen and Schizosaccharomyces pombe Lindner. Marine fungi are most precisely defined as those that can complete their entire life cycles within the sea, i.e. those that ‘‘can grow and sporulate exclusively in a marine or estuarine habitat’’ (Kohlmeyer and Volkmann-Kohlmeyer 2003). Earlier physiological work often extrapolated the behaviour of one isolate to an entire species and mutants were conspicuous by their absence. Two marine species, the filamentous
hyphomycete Dendryphiella salina (G.K.Sutherland) Nicot & Pugh and the ascomycete yeast Debaryomyces hansenii (Zopf) Lodder & Kreger – van Rij figure most prominently as physiological models (Clipson and Jennings 1992) but D. hansenii provides the bulk of references for the genetics and molecular biology of marine fungi. This article will review the main techniques available for the genetic characterisation of marine fungi and highlight the primary results of these investigations.
2.
Classical genetics
Classical genetics in fungi revolves around the isolation and characterisation of mutants. These represent defects or mutations in specific genes – characterisation of which may lead to a better understanding of particular physiological or developmental processes. For example Morales et al. (1990) described an ingenious method of isolation of mutants of D. hansenii altered in glycerol metabolism based upon changes in buoyant density allowing for their enrichment by density gradient centrifugation. Mutants that overproduced glycerol could be recognised by supporting growth of Escherichia coli glycerol requiring strains. In the major terrestrial models, mutations were used to define genes and then map these to chromosomal loci by following recombination and linkage behaviour via meiotic or
* Corresponding author. E-mail address: [email protected] 0269-915X/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycol.2006.10.002
The genetics and molecular biology of marine fungi
parasexual cycles. It took some 60 y and thousands of researchers to characterise and map perhaps less than a thousand genes to linkage maps in A. nidulans by classical techniques. No marine fungus has been characterised by classical techniques in such detail. However the advent of recombinant DNA technology in the early 1970’s offered more powerful and economical methods widely applicable to any species from which DNA or RNA could be extracted. The excellent review in The Mycologist by Bridge (2002) explains many of the molecular techniques discussed here in more detail.
3.
145
DNA PCR PRODUCTS
PCR primers to consensus sequences
Clone & DNA sequence
Molecular analysis
4.
Gene cloning techniques
Recombinant DNA techniques physically isolate genes in vitro as represented by DNA molecules rather than the more timeconsuming ‘‘classical’’ approach of studying their effects indirectly in vivo following mutation. The process of cloning allows the rapid production of identical copies in sufficient quantity for characterisation, for example by DNA sequencing reactions. From the DNA sequence a prediction may be made of the gene product and its likely function using the genetic code and on-line databases. Initially these methods concentrated on characterising individual genes, whilst more recently genomics attempts to characterise every gene. Fig. 1 outlines the strategies that have been used to clone and characterize DNA sequences from marine fungi.
DNA fragments
Clone all fragments into vector
• DNA sequence every clone – genomics • Transform host cell and select clone by complementation • DNA hybridisation
Chromosome analysis Generally the chromosomes of fungi (though not of oomycetes like Phytophthora or Pythium) were too small to be studied by conventional microscopy. However fungal chromosomes may be resolved by the technique of pulse field gel electrophoresis which relies upon the separation of chromosomes (as ‘‘bands’’) within an electric field where the orientation of the current is changed at regular intervals. Debaryomyces cultures, including 35 D. hansenii strains, were characterised this way by Corredor et al. (2003). Four to ten bands of size ranges 0.7 Mb (millions of base pairs of DNA) to 4.2 Mb could be detected within members of the genus and chromosome length polymorphisms were detected between different strains of D. hansenii. D. hansenii strains are generally haploid and following mating the transient diploid strain breaks down to give a single ascospore within an ascus (http://cbi.labri.fr/Genolevures/).
Restriction Enzymes
Fig. 1 – A summary of cloning strategies for marine fungi. Alternative approaches might focus upon the use of mRNA extracts and reverse transcription into cDNA for subtractive hybridisation or differential display, so characterising genes and transcripts that are induced under particular conditions.
suggested by the authors to be possible candidates as cloning vectors. Ricaurte and Govind (1999) constructed two plasmids (pMR95 and pMR96) by combining a naturally replicating DNA sequence from D. hansenii (an ‘‘autonomously replicating sequence’’) with a selectable marker encoding hygromycin B resistance. Additionally pMR96 carried a second selectable marker URA3 to allow the detection of the plasmid within the alternative host, S. cerevisiae. Ricaurte and Govind (1999) introduced the DNA using electroporation (a treatment of a high voltage for a few milliseconds) to demonstrate for the first time the successful uptake of these plasmids as naked DNA from outside the cell by D. hansenii. This was the first report of the ‘‘transformation’’ of a marine fungus. Baisden and Cooney (1996) screened twenty one isolates representing nine genera of marine filamentous fungi (including a strain of Dendryphiella salina) but could find evidence of plasmids in only one, a species of Lulworthia. This plasmid (pQB) was linear and appeared to be located in the mitochondria. As the plasmid replicated autonomously it too might represent a useful basis for a cloning vector. In filamentous fungi the transforming DNA is often integrated into the host genome, increasing the stability of the transformant.
Transformation
Complementation
Gene cloning relies upon the development of cloning vectors – self-replicating DNA molecules such as plasmids that can carry cloned DNA and deliver them into a host cell or mycelium. Gunge et al. (1993) characterised three linear plasmids in D. hansenii which were maintained in their hosts by osmotic stress – withdrawal of salts or polyols from the growth medium resulted in a temperature dependant loss of the plasmids by the host (Fukuda et al. 2004). As naturally occurring molecules of the fungus, these plasmids (pDHL1, 2, 3) were
Cloning a gene by complementation of an altered phenotype involves the development of a ‘‘gene library’’ of the organism, a collection of vector molecules with cloned DNA that represents every gene. The gene library is transformed into a host and then an altered phenotype is selected. Hence a direct test of gene function is possible, usually by complementing a mutation and restoring a normal or ‘‘wild type’’ phenotype. The development of a transformation system for a previously untransformed species is not simple. Suitable vectors need to
146
Fig. 2 – An example of complementation cloning. A collection of D. salina cloned DNA molecules in phage lambda were transformed into an A. nidulans strain carrying a salt sensitivity mutation (sltA1). Individual selected transformants (prefixed DS) are compared with a salt sensitive control strain (WV101) in growth upon solid medium containing 0.5 M NaCl. Four of the transformants shown here have levels of tolerance that approach wild type for the A. nidulans host (photo by courtesy D. Clement).
be constructed, a method of delivery of the DNA into the host cell established and finally a selection method is required to identify which individuals have taken up and can express the vector sequences. The development of a transformation system for a marine fungus can be avoided by expressing the gene library in a more amenable system such as A. nidulans or S. cerevisiae. Clement et al. (1999) transformed a D. salina gene library into an A. nidulans strain carrying a salt sensitivity mutation. A number of potential clones were identified that appeared to complement the salt sensitivity of their new host, restoring a measure of salt tolerance. Fig. 2 illustrates this screening procedure. In some cases the host strain carrying these clones showed an improved content of polyols as compatible solutes when grown under salt stress (Clement et al. 1999). A similar approach was used by Prista et al. (2002) when they isolated clones of D. hansenii DNA in a plasmid vector that enhanced the salt tolerance at alkali pH of a wild type strain of S. cerevisiae. Bansal and Mondal (2000) used a complementation cloning strategy to isolate the hog (regulatory gene controlling osmotic responses) homologue of D. hansenii in S. cerevisiae.
Cloning by PCR and DNA hybridization The polymerase chain reaction (PCR) has been used to clone a variety of genes from a number of species. PCR (Bridge 2002) relies on the design of short DNA sequences (primers)
P. Hooley, M. Whitehead
that match or complement sequences that are thought to flank the gene of interest. Well characterised S. cerevisiae or N. crassa homologous genes provide sufficient sequence information to design a range of likely primers. Multiple rounds of in vitro amplification using the Taq DNA polymerase enzyme then produce large quantities of the sequence of interest. This method has been used to clone genes encoding sodium pumps from D. hansenii (Almagro et al. 2001) and the superoxide detoxification enzyme superoxide dismutase from several marine Debaryomyces species (Hernandez Saavedra and Romero Geraldo 2001). Interestingly the D. hansenii sodium pump genes (DhENA1 and DhENA2) were expressed in the presence of high Naþ concentrations (0.5M) and DhENA2 also required an elevated pH (7.5) for expression. Both of these features would be desirable to maintain Naþ extrusion within the sea. Thome and Trench (1999) cloned the glycerol – 3 – phosphate dehydrogenase gene from D. hansenii. Northern Blot analysis of mRNA induced following different physiological treatments revealed that expression of the gene was inducible by NaCl indicating that salt tolerance is mediated at the level of the transcription of a gene responsible for the control of synthesis of the compatible solute glycerol. Guerrero et al. (2005) used the cloned genes from D. hansenii encoding NADP glutamate dehydrogenase and glutamine synthetase to demonstrate Naþ dependent expression which was not found in S. cerevisiae. As these genes are involved in ammonium assimilation this may represent an adaptation of nitrogen metabolism to the elevated cytosolic salt concentrations found within the marine species. Aggarwal et al. (2005) compared the homologous genes from the same two species encoding 30 (20 ), 50 -bisphosphate nucleotidase (Dhal2p), an enzyme that is particularly sensitive to salt in the terrestrial species. When S. cerevisiae was transformed with the D. hansenii gene (DHAL2), the terrestrial host became more salt tolerant. Hence cloned genes have proved useful tools in identifying changes in particular molecular processes that may result in marine adaptation. Aggarwal et al. (2005) noted that the N-terminal 54 amino acid residues of Dha12p had no homology with other yeast homologues. Yet this region can be seen to be present in the A. fumigatus (a terrestrial but salt tolerant filamentous species) homologue revealed by the recently completed genome project (Fig. 3). Other important genes recently cloned from marine fungi include the beta lactam biosynthetic genes ( pcbAB-pcbC ) from Kallichroma tethys (Kohlm. & Kohlm.) Kohlmeyer and Volkmann – Kohlmeyer (Kim et al. 2003). This group used PCR, exploiting primers whose design was based upon common (consensus) sequences from A. nidulans, Penicillium chrysogenum and Acremonium chrysogenum, to isolate a short (1,300 base pair) sequence. This was then used as a labelled probe to isolate a much larger complementary sequence by DNA hybridization. Analysis of the DNA sequences from this fragment confirmed a close relationship with orthologous genes from the cephalosporin producer Acremonium chrysogenum.
Fig. 3 – A multiple sequence alignment of three selected proteins using T-COFFEE, Notredame et al. (2000). The sequences are Dhal2p from Debaryomyces hansenii (NCBI accession no. AAR03496) and homologues from Saccharomyces cerevisiae (ARR89916) and Aspergillus fumigatus (EAL 88757). Red indicates high levels of similarity, yellow intermediate while green indicates low similarity. Asterisks indicate perfectly conserved (Cons) residues, two dots indicate very similar and full stops similar amino acid residues.
The genetics and molecular biology of marine fungi
D.hansenii S.cerevisiae A.fumigatus
147
---------MLRLMKYFTFAVYFSLLAVLLSIFISNYKIIPSWAISIL -----------------------------------------------MGPRSINTDDCCSVRRSSFSIFSCL--VLCSTLLLGVSIIFKTPPRIF
Cons
D.hansenii S.cerevisiae A.fumigatus
KIYPLKITNSSFKATMSTIPADHPYYKELEIASIAVIRTSILTKKLSD ----------------------MALERELLVATQAVRKASLLTKRIQS ASLPAASSSPNYTHT-----ANMSYQQERYIAELAVQRATLLTQKVFH
Cons
.
:*
:*
** ::::**:::
D.hansenii S.cerevisiae A.fumigatus
SIATTQKSGTHTKDDKSPVTIGDYASQAIINHAIKLNFPEDEIVGEED EVISHKDSTTITKNDNSPVTTGDYAAQTIIINAIKSNFPDDKVVGEES E----KAKGTVSKDDKSPVTIGDFGAQALIIQALRKNFPNDEIVAEEE
Cons
.
D.hansenii S.cerevisiae A.fumigatus
AEVLRKDDAEGKDLSAKVLEIISDVQSQTSQYNNRLGK--------LSSGLSD---------AFVSGILNEIKANDEVYNKNYKKDDFLFTNDQF ANSLREDKA----LSAEIWRLVKDIRLGDNESNELLGG--------LL
Cons
:. * .
D.hansenii S.cerevisiae A.fumigatus
--EKETEIYDSIDLGNSQGGSKGRFWALDPIDGTKGFLRGDQFAVCLA PLKSLEDVRQIIDFGNYEGGRKGRFWCLDPIDGTKGFLRGEQFAVCLA --PSEDAMLDIIDQGKSAGGPKGRIWALDPIDGTKGFLRGGQYAVCLG
Cons
: . * :*:*:**** **:.:*::* :*:: ***:*::*.**.
.
* :
: : ** *:
::.:::
.
*:
** ***:*.************* *:****.
D.hansenii S.cerevisiae A.fumigatus
LIEDGKVVLGVIGCPNLPATVVSNE-------EMSG--ARGGLFSAVR LIVDGVVQLGCIGCPNLVL-------SSYGAQDLKGHESFGYIFRAVR LIEDGDVKVGAIGCPNLPVDDSVAMTASIGVDQTDG-AGMGVLFSAIK
Cons
** ** * :* ******
D.hansenii S.cerevisiae A.fumigatus
GVGSFYSNLFDKQDFTPLSKQERIQMTQHTTPESLKVVEGVEKGHSSH GLGAFYSPSSDAESWT------KIHVRHLKDTKDMITLEGVEKGHSSH GQGSISRPLSNGA----LAESKPISMRPVPDIKQAVFCEGVEAAHSAQ
Cons
* *::
D.hansenii S.cerevisiae A.fumigatus
STQSQIKDKLGFNTETVSKQTINLDSQVKYCVLAKGQADIYLRLPISD DEQTAIKNKLN-----I-SKSLHLDSQAKYCLLALGLADVYLRLPIKL GDNAAVAQLLG-----ITSPSVRLDSQAKYCSIARGAGDIYLRLPVRK
Cons
. :: : : *.
D.hansenii S.cerevisiae A.fumigatus
TYREKIWDHAAGNILVYESGGLVGDI-HGNELNFGNGRHLNS-QGVVA SYQEKIWDHAAGNVIVHEAGGIHTDAMEDVPLDFGNGRTLAT-KGVIA DYQEKIWDHAAGDLIVREAGGQVTDI-YGQRLDFSKGRTLAANKGVVA
Cons
D.hansenii S.cerevisiae A.fumigatus Cons
: .*
:
* :
**** .**::
: . ::.****.*** :* * .*:*****:
*:*********:::* *:**
*
--GNKSVFKKVIEAVSDVLK---A SSGPRELHDLVVSTSCDVIQSRNA --APEAIQDQVISAVKTVLK---L . . : . *:.:
:.
. * :* *::
*::
.
*:*.:** * : :**:*
148
P. Hooley, M. Whitehead
The pcbAB gene from K. tethys was able to complement a defect in the corresponding gene encoding the ACV synthetase enzyme in A. nidulans.
5.
Phylogeny and taxonomy
Molecular methods also provide some of the most powerful tools for the identification of isolates and the examination of the taxonomic relationships of marine fungi. A comparative characterisation of at least part of the genome provides rigorous criteria, less susceptible to the transient effects suffered by morphological or biochemical markers. Michaelis et al. (1987) examined genetic variation by isoenzyme profiles that represented 14 assumed structural loci of Dendryphiella. Relatively little variation was shown with isolates from a common location usually appearing to be clonal in origin. Edwards et al. (1998) used a numerical taxonomic approach to compare a number of isolates of D. salina and D. arenaria to arrive at a quantitative description of the variation within and between a species. Again some isolates acquired from particular geographical locations were very similar if not identical. These two groups reached a common conclusion in designating their isolates into two distinct species which, in the absence of sexual reproduction, showed a somewhat limited capacity to generate genetic variation. Currently BIOLOG phenotype microarrays that examine substrate utilization and the PCR – based RAPD’s (random amplified polymorphic DNA) are amongst the techniques being used to examine variation in Dendryphiella isolates from around the world (Dela Cruz et al. 2006). PCR of particular repetitive DNA sequences (such as ‘‘microsatellites’’ or those encoding small or large ribosomal subunits and their associated ITS regions) can be used to directly assess fungal diversity in marine habitats by producing strain or species specific ‘‘DNA fingerprints’’ (Table 1). Cooke et al. (2000) described how a group of marine isolates within the genus Halophytophthora were distinct from Phytophthora and most Pythium species. The evolution of these taxonomic groups seems to be driven by host specialization. Gadanho et al. (2003) arranged 234 marine isolates into 31 groups and interestingly basidiomycetous yeasts were more common than ascomycetous yeasts. Zuccaro et al. (2003) identified marine fungi in dead and decaying plant matter. Spatafora et al. (1998) applied the analysis of rDNA sequences to suggest that the Halosphaeriales were derived from terrestrial ancestors. A number of other genes have been used for identification and taxonomic purposes, these include those encoding
Table 1 – Examples of Molecular Taxonomy in Marine Fungi Species Halosphaeriales Halosarpheia, Lignicola, Nais Halophytophthora Novel isolates Marine yeasts Various including Halosphaeriales, Hypocreales and Pleosporales
Reference Spatafora et al. 1998 Kong et al. 2000 Cooke et al. 2000 Stoeck and Epstein 2003 Gadanho et al. 2003 Zuccaro et al. 2003
polyketide synthases, beta-tubulin and laccases (e.g. Lyons et al. 2003). Developments of PCR can even amplify diagnostic DNA sequences directly from environmental samples without the prior subculture of the organism. This is presently the most common source of accession numbers for marine fungal genes on the NCBI database (http://www.ncbi.nlm.nih.gov/). Stoeck and Epstein (2003) showed how this approach may allow the detection of novel (and perhaps unculturable) isolates of marine fungi. Hence our assumptions about the relative diversity of particular groups of fungi may be tested. Molecular techniques based upon the comparative DNA sequencing of the trpC gene isolated via PCR were used to identify a fungal agent causing death of Caribbean sea fans. This analysis confirmed the pathogen as Aspergillus sydowii (Bainier & Sartory) Thom & Church, a cosmopolitan species found in both terrestrial and marine locations. Other non-marine isolates of the same species were unable to infect sea fans (Geiser et al. 1998). Hence the acquisition of at least a partial marine habit by a genetically distinct group of strains correlated with enhanced pathogenicity. Kis-Papo et al. (2003) examined the genomic diversity of strains of the cosmopolitan species Aspergillus versicolor (Vuill.) Tirab associated with the Dead Sea where a combination of high salt concentrations (up to 10 times seawater) and acidity (pH 5.9) provide an interesting contrast with the conventional marine habitat. They used a PCR based technique of AFLP’s (amplified fragment length polymorphisms) to compare the diversity of strains isolated at different salinities and from terrestrial or freshwater sites. Intriguingly, genetic exchange and diversity was positively correlated with increasing stress, only declining under the most extreme conditions of the deep waters of the Dead Sea. Molecular techniques therefore may help to refine the definition of obligate and facultative marine fungi (Kohlmeyer and Volkmann-Kohlmeyer 2003).
6.
Genomics
Genome projects aim to clone and sequence all of the DNA of an organism as individual clones and then organise such information into contiguous overlapping sections (‘‘contigs’’) that are arranged into precisely annotated chromosomes. The D. hansenii genome project has now been published (Dujon et al. 2004). Lepingle et al. (2000) compared partial clones of D. hansenii genes with the S. cerevisiae genome and discovered that the genes associated with transport functions (such as for carbon and nitrogen sources) were much more common in the marine than in the terrestrial yeast. Dujon et al. (2004) described the monumental task of comparing the entire genomes of several yeast species. Comparative genomics aims to compare all the gene sets and chromosome positions between two or more closely or disparately related organisms. The type strain D.hansenii var. hansenii CBS767 has seven chromosomes and shows a close relationship to members of the genus Candida. Full details are publically available online (Genolevures database: http://cbi.labri.fr/Genolevures/). Table 2 summarises how the increase in gene number in the marine species is not achieved by an overall increase in genome size but rather by a reduction in the proportion
The genetics and molecular biology of marine fungi
Table 2 – A simplified comparison of the genomes of Saccharomyces cerevisiae and Debaryomyces hansenii (CDS [ protein coding sequences, using data from Dujon et al. 2004)
Genome Size (Mb) Total CDS Mean Gene Density (protein coding as %) Mean CDS size (codons)
Saccharomyces cerevisiae
Debaryomyces hansenii
12.1 5807 70.3
12.2 6906 79.2
485
389
of non-coding DNA coupled to a shortening of gene length. D. hansenii was found to have many more examples of the duplication of genes, reflecting either a recent series of amplification events or a less efficient process to eliminate redundant genes. This may also reflect a more demanding environment which selects for the retention of duplicated genes with even very slight changes in encoded protein activities. Again, families encoding transporters such as multidrug resistance proteins and hexose sugar transporters were particularly expanded in relation to the other yeast species studied. A similar comparative approach for the marine bacterium Silicibacter pomeroyi Gonzalez revealed that this species had a higher proportion of genes controlling signal transduction and transport processes than did other related non-marine species (Moran et al. 2004). Survival of microorganisms in the nutrient poor marine environment demands the evolution of a wide portfolio of transport genes, with their products able to function for example at alkaline pH and under ionic stress. Another genomics approach (‘‘high-throughput shotgun sequencing’’) has provided global views of communities and the metabolic capabilities of uncultured organisms which occupy niche environments. This technique has compared fungal members of a marine environment to terrestrial environments (Tringe et al. 2005). These results again highlighted the importance of genetic elements coding for organic molecule transporters and sodium ion exporters in the marine species.
7.
Conclusions
After a slow start on the classical genetics of marine fungi we are now fortunate to have one supremely detailed genetical resource, namely the genome project for D. hansenii. The genome of D. hansenii appears markedly different to many other yeasts, suggesting a large number of mutations are needed to acquire and maintain the marine habit. However the recent appearance of apparently marine strains of an otherwise terrestrial species of Aspergillus (Kohlmeyer and Volkmann-Kohlmeyer 2003) offers the prospect of identifying a more limited number of key genetic changes required for life in the ocean. Once genome projects are completed then microarray technology (‘‘transcriptomics’’, analysing levels of mRNA specific to particular groups of induced or active genes) becomes feasible (e.g. Posas et al. 2000). However, how representative of marine fungi is one species of yeast – D. hansenii? With the much larger genomes of filamentous fungi approaching twice
149
the number of yeast genes (http://mips.gsf.de/projects/fungi/ fungi_db.html), the genome of a filamentous species would be an enticing prospect to study. A number of species may be suggested but the physiological legacy and ability to produce secondary metabolites make D. salina an attractive candidate. Given the investment required for such an undertaking it would be important that the marine fungal research community reaches a consensus. At present the genomics approach is being applied both to microbial and metazoan marine communities (Wilson et al. 2005). A number of genomes of Phytophthora species of crop plants have now been sequenced (http://www.pfgd.org/). These may be compared to selected sequences from the marine genus Halophytophthora should these become available. For example genes that influence cytoplasmic turgour and zoospore release, such as those encoding particular transcription factors or protein kinases (Judelson and Roberts 2002), would be potentially very informative to contrast between terrestrial and marine species. This is particularly intriguing when one considers that some susceptible crops are grown on salt affected soils. There is also scope to return to classical techniques to answer long – standing questions about fungal physiology, including those relating to the role of the wall as an ion sink (Clipson and Jennings 1992). It is feasible to isolate and characterise the osmotic responses of cell wall mutants in models such as D. salina. In our own laboratory we have found conventional mutagens such as MNNG, ultra violet light and nitrous acid are successful at inducing stable morphological mutants of D. salina, affecting both colony and spore characteristics. Many of the genes encoding enzymes involved in cell wall synthesis are well characterised in terrestrial models. They may now be compared directly in silico with marine counterparts on the Genolevures site. Fig. 3 illustrates how candidate domains and residues for ‘‘marine’’ adaptations in proteins can be identified. One area that deserves more attention is the induction of novel metabolites by stress responses in the sea. This area is probably receiving more attention than is reflected by the academic press due to commercial restraints. Secondary metabolite genes are stress responders controlling production at the end of lag or stationary growth phases and marine fungi are often slow growers. Many novel compounds may have been missed by conventional screens. Molecular approaches such as that of Kim et al. (2003) may give an insight into an enticing world of new antimicrobials. A search of the database at the National Centre for Biotechnology Information (http://www. ncbi.nlm.nih.gov/) reveals a number of Dendryphiella DNA sequences and their encoded haloperoxidase and protease enzymes which are part of patent applications of interest to the biotechnology industry. It has been possible to argue that the sea represents a somewhat buffered environment with relatively constant levels of salinity, temperature and pH along with predictable tidal regimes. However it is now very clear that human activities are altering this stability. For example alkaline pH values of 8.2 þ/ 0.3 in the sea already represent a 30 % increase in hydrogen ion concentration since the onset of the Industrial Revolution. As more CO2 dissolves, the pH falls and the carbonate building blocks for coral formation become less available (The Royal Society 2005). Marine fungi are ubiquitous
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colonisers of marine corals yet have been largely ignored and may damage their increasingly stressed hosts (Golubic et al. 2005). Molecular studies such as those of Geiser et al. (1998) and Kis-Papo (2003) assume a renewed importance in predicting the ability of fungi from other habitats to invade this changing marine environment. How will imperfect marine species with relatively little genetic variation such as D. salina fare once climate change takes hold? The key role of microorganisms within the global carbon cycle and in particular within the ocean is obvious and yet the contributions of marine fungi, with their increased complexity of carbon transporters are not quantified. The increased fragility of the marine ecosystem gives a renewed urgency to the study of the genetics and molecular biology of the marine fungi.
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Aggarwal M, Bansal PK, Mondal AK, 2005. Molecular cloning and biochemical characterization of a 30 (20 ), 50 -bisphosphate nucleotidase from Debaryomyces hansenii. Yeast 22: 457–470. Almagro A, Prista C, Benito B, Loureiro-Dias MC, Ramos J, 2001. Cloning and expression of two genes coding for sodium pumps in the salt tolerant yeast Debaryomyces hansenii. Journal of Bacteriology 183: 3251–3255. Baisden CM, Cooney JJ, 1996. Screening marine fungi for plasmids and characterization of a linear mitochondrial plasmid in a Lulworthia sp. Mycologia 88: 350–357. Bansal PK, Mondal AK, 2000. Isolation and sequence of the HOG1 homologue from Debaryomyces hansenii by complementation of the hog1 Delta strain of Saccharomyces cerevisiae. Yeast 16: 81–88. Bridge P, 2002. The history and application of molecular mycology. The Mycologist 16: 90–99. Clement DJ, Stanley MS, O’Neil J, Woodcock NA, Fincham DA, Clipson NJW, Hooley P, 1999. Complementation cloning of salt tolerance determinants from the marine hyphomycete Dendryphiella salina in Aspergillus nidulans. Mycological Research 103: 1252–1258. Clipson NJW, Jennings DH, 1992. Dendryphiella salina and Debaryomyces hansenii: models for ecophysical adaptation to salinity by fungi that grow in the sea. Canadian Journal of Botany 70: 2097–2105. Cooke DEL, Drenth A, Duncan JM, Wagels G, Brasier CM, 2000. A molecular phylogeny of Phytophthora and related oomycetes. Fungal Genetics and Biology 30: 17–32. Corredor M, Davila AM, Casaregola S, Gaillardin C, 2003. Chromosomal polymorphism in the yeast species Debaryomyces hansenii. Antonie Van Leeuwenhoek 84: 81–88. Dela Cruz TE, Schulz BE, Kubicek CP, Druzhinina IS, 2006. Carbon source utilization by the marine Dendryphiella species D. arenaria and D. salina. FEMS Microbiology Ecology, in press. Dujon B and 66 others, 2004. Genome evolution in yeasts. Nature 430: 35–44. Edwards J, Chamberlain D, Brosnan G, West D, Stanley MS, Clipson NJW, Hooley P, 1998. A comparative physiological and morphological study of Dendryphiella salina and D.arenaria in relation to adaptation to life in the sea. Mycological Research 102: 1198–1202. Fukuda K, Chen JS, Kawano M, Sudo K, Gunge N, 2004. Stress responses of linear plasmids from Debaryomyces hansenii. FEMS Microbiology Letters 237: 243–248. Gadanho M, Almeida JM, Sampaio JP, 2003. Assessment of yeast diversity in a marine environment in the south of Portugal by microsatellite primed PCR. Antonie Van Leeuwenhoek 84: 217–227.
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Geiser DM, Taylor JW, Ritchie KB, Smith GW, 1998. Cause of sea fan death in the West Indies. Nature 394: 137–138. Golubic S, Radtke G, Le Campion-Alsumard T, 2005. Endolithic fungi in marine ecosystems. Trends in Microbiology 13: 229–235. Gunge N, Fukuda K, Morikawa K, Takeda M, Miwa A, 1993. Osmophilic linear plasmids from the salt tolerant yeast Debaryomyces hansenii. Current Genetics 23: 443–449. Guerrero CA, Aranda C, Deluna A, Filetici P, Riego L, Anaya VH, Gonzalez A, 2005. Salt dependent expression of ammonium assimilation genes in the halotolerant yeast, Debaryomyces hansenii. Current Genetics 47: 163–171. Hernandez Saavedra NY, Romero Geraldo R, 2001. Cloning and sequencing the genomic encoding region of copper zinc superoxide dismutase enzyme from several marine strains of the genus Debaryomyces (Lodder and Kreger-van Rij). Yeast 18: 1227–1238. Judelson HS, Roberts S, 2002. Novel protein kinase induced during sporangial cleavage in the oomycete Phytophthora infestans. Eukaryotic Cell 1: 687–695. Kim CF, Lee SKY, Price J, Jack RW, Turner G, Kong RYC, 2003. Cloning and expression analysis of the pcbAB-pcbC beta lactam genes in the marine fungus Kallichroma tethys. Applied and Environmental Microbiology 69: 1308–1314. Kis-Papo T, Kirzhner V, Wasser SP, Nevo E, 2003. Evolution of genomic diversity and sex at extreme environments; fungal life under hypersaline Dead Sea stress. Proceedings of The National Academies of Science (U.S.A.) 100: 14970–14975. Kohlmeyer J, Volkmann-Kohlmeyer B, 2003. Fungi from coral reefs: a commentary. Mycological Research 107: 386–387. Kong RYC, Chan JYC, Mitchell JI, Vrijmoed LLP, Gareth Jones EB, 2000. Relationships of Halosarpheia, Lignincola and Nais inferred from partial 18S rDNA. Mycological Research 104: 35–43. Lepingle A, Casaregola S, Neuveglise C, Bon E, Nguyen HV, Artiguenave F, Wincker P, Gaillardin C, 2000. Genomic exploration of the hemiascomycetous yeasts: 14. Debaryomyces hansenii var. hansenii. FEBS Letters 487: 82–86. Lyons JI, Newell SY, Buchan A, Moran MA, 2003. Diversity of ascomycete laccase gene sequences in a southeastern U.S. salt marsh. Microbial Ecology 45: 270–281. Michaelis KC, Gessner RV, Romano MA, 1987. Population genetics and systematics of marine species of Dendryphiella. Mycologia 79: 514–518. Morales C, Andre L, Adler L, 1990. A procedure for enrichment and isolation of mutants of the salt tolerant yeast Debaryomyces hansenii having altered glycerol metabolism. FEMS Microbiology Letters 57: 73–77. Moran MA and 34 others, 2004. Genome sequence of Silicibacter pomeroyi reveals adaptations to the marine environment. Nature 432: 910–913. Notredame C, Higgins D, Heringa J, 2000. T-Coffee: A novel method for multiple sequence alignments. Journal of Molecular Biology 302: 205–217. Posas F, Chambers JR, Heyman JA, Hoeffler JP, de Nadal E, Arino J, 2000. The transcriptional response of yeast to saline stress. Journal of Biological Chemistry 275: 17249–17255. Prista C, Soeiro A, Vesely P, Almagro A, Ramos J, LoureiroDias MC, 2002. Genes from Debaryomyces hansenii increase salt tolerance in Saccharomyces cerevisiae W303. FEMS Yeast Research 2: 151–157. Ricaurte ML, Govind NS, 1999. Construction of plasmid vectors and transformation of the marine yeast Debaryomyces hansenii. Marine Biotechnology 1: 15–19. Spatafora JW, Volkmann-Kohlmeyer B, Kohlmeyer J, 1998. Independent terrestrial origins of the Halosphaeriales (Marine Ascomycota). American Journal of Botany 85: 1569–1580. Stoeck T, Epstein S, 2003. Novel Eukaryotic Lineages Inferred from Small-Subunit rRNA Analyses of Oxygen-Depleted Marine
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Environments. Applied and Environmental Microbiology 69: 2657– 2663. The Royal Society, 2005. Ocean acidification due to increasing atmospheric carbon dioxide. Policy document 12/05 (available on-line www.royalsoc.ac.uk). Thome PE, Trench RK, 1999. Osmoregulation and the genetic induction of glycerol–3–phosphate dehydrogenase by NaCl in the euryhaline yeast Debaryomyces hansenii. Marine Biotechnology 1: 230–238.
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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/mycol
The genetics and molecular biology of marine fungi Paul HOOLEY*, Michael WHITEHEAD School of Applied Sciences, University of Wolverhampton, WV1 1SB, UK
abstract Keywords: Climate change
Interest in the genetics of marine fungi has focused upon the basis of stress adaptation and
Cloning
the control of the production of secondary metabolites and enzymes. Analysis by molecu-
Debaryomyces
lar genetics has been applied to marine fungal taxonomy, phylogeny and species identifi-
Genomics
cation. The advent of the Debaryomyces hansenii genome project and the influence of
Marine fungi
climate change on this research are discussed. ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Mutants
1.
Introduction
Fungal genetics was first studied in detail in the 1940’s as fungi appeared to present ideal models to study the nature of the gene. Beadle and Tatum’s classic work on Neurospora crassa Shear and Dodge laid the foundations for the ‘‘one gene – one enzyme hypothesis’’. Nutritionally deficient mutants (auxotrophs) allowed the dissection of biochemical pathways that illustrated the stepwise conversion of precursor molecules to products, each step under the control of a single gene encoding a single enzyme. In Glasgow, Pontecorvo’s group selected Aspergillus nidulans (Edam) Winter as their model. Some species such as Sordaria fimicola (Roberge ex Desm.) Griffiths & Seaver were even obliging enough to provide the results of their meiotic divisions in linear ordered tetrads. The field of fungal genetics relies mainly on work performed on just a few terrestrial ascomycetes, including the yeasts Saccharomyces cerevisiae Hansen and Schizosaccharomyces pombe Lindner. Marine fungi are most precisely defined as those that can complete their entire life cycles within the sea, i.e. those that ‘‘can grow and sporulate exclusively in a marine or estuarine habitat’’ (Kohlmeyer and Volkmann-Kohlmeyer 2003). Earlier physiological work often extrapolated the behaviour of one isolate to an entire species and mutants were conspicuous by their absence. Two marine species, the filamentous
hyphomycete Dendryphiella salina (G.K.Sutherland) Nicot & Pugh and the ascomycete yeast Debaryomyces hansenii (Zopf) Lodder & Kreger – van Rij figure most prominently as physiological models (Clipson and Jennings 1992) but D. hansenii provides the bulk of references for the genetics and molecular biology of marine fungi. This article will review the main techniques available for the genetic characterisation of marine fungi and highlight the primary results of these investigations.
2.
Classical genetics
Classical genetics in fungi revolves around the isolation and characterisation of mutants. These represent defects or mutations in specific genes – characterisation of which may lead to a better understanding of particular physiological or developmental processes. For example Morales et al. (1990) described an ingenious method of isolation of mutants of D. hansenii altered in glycerol metabolism based upon changes in buoyant density allowing for their enrichment by density gradient centrifugation. Mutants that overproduced glycerol could be recognised by supporting growth of Escherichia coli glycerol requiring strains. In the major terrestrial models, mutations were used to define genes and then map these to chromosomal loci by following recombination and linkage behaviour via meiotic or
* Corresponding author. E-mail address: [email protected] 0269-915X/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycol.2006.10.002
The genetics and molecular biology of marine fungi
parasexual cycles. It took some 60 y and thousands of researchers to characterise and map perhaps less than a thousand genes to linkage maps in A. nidulans by classical techniques. No marine fungus has been characterised by classical techniques in such detail. However the advent of recombinant DNA technology in the early 1970’s offered more powerful and economical methods widely applicable to any species from which DNA or RNA could be extracted. The excellent review in The Mycologist by Bridge (2002) explains many of the molecular techniques discussed here in more detail.
3.
145
DNA PCR PRODUCTS
PCR primers to consensus sequences
Clone & DNA sequence
Molecular analysis
4.
Gene cloning techniques
Recombinant DNA techniques physically isolate genes in vitro as represented by DNA molecules rather than the more timeconsuming ‘‘classical’’ approach of studying their effects indirectly in vivo following mutation. The process of cloning allows the rapid production of identical copies in sufficient quantity for characterisation, for example by DNA sequencing reactions. From the DNA sequence a prediction may be made of the gene product and its likely function using the genetic code and on-line databases. Initially these methods concentrated on characterising individual genes, whilst more recently genomics attempts to characterise every gene. Fig. 1 outlines the strategies that have been used to clone and characterize DNA sequences from marine fungi.
DNA fragments
Clone all fragments into vector
• DNA sequence every clone – genomics • Transform host cell and select clone by complementation • DNA hybridisation
Chromosome analysis Generally the chromosomes of fungi (though not of oomycetes like Phytophthora or Pythium) were too small to be studied by conventional microscopy. However fungal chromosomes may be resolved by the technique of pulse field gel electrophoresis which relies upon the separation of chromosomes (as ‘‘bands’’) within an electric field where the orientation of the current is changed at regular intervals. Debaryomyces cultures, including 35 D. hansenii strains, were characterised this way by Corredor et al. (2003). Four to ten bands of size ranges 0.7 Mb (millions of base pairs of DNA) to 4.2 Mb could be detected within members of the genus and chromosome length polymorphisms were detected between different strains of D. hansenii. D. hansenii strains are generally haploid and following mating the transient diploid strain breaks down to give a single ascospore within an ascus (http://cbi.labri.fr/Genolevures/).
Restriction Enzymes
Fig. 1 – A summary of cloning strategies for marine fungi. Alternative approaches might focus upon the use of mRNA extracts and reverse transcription into cDNA for subtractive hybridisation or differential display, so characterising genes and transcripts that are induced under particular conditions.
suggested by the authors to be possible candidates as cloning vectors. Ricaurte and Govind (1999) constructed two plasmids (pMR95 and pMR96) by combining a naturally replicating DNA sequence from D. hansenii (an ‘‘autonomously replicating sequence’’) with a selectable marker encoding hygromycin B resistance. Additionally pMR96 carried a second selectable marker URA3 to allow the detection of the plasmid within the alternative host, S. cerevisiae. Ricaurte and Govind (1999) introduced the DNA using electroporation (a treatment of a high voltage for a few milliseconds) to demonstrate for the first time the successful uptake of these plasmids as naked DNA from outside the cell by D. hansenii. This was the first report of the ‘‘transformation’’ of a marine fungus. Baisden and Cooney (1996) screened twenty one isolates representing nine genera of marine filamentous fungi (including a strain of Dendryphiella salina) but could find evidence of plasmids in only one, a species of Lulworthia. This plasmid (pQB) was linear and appeared to be located in the mitochondria. As the plasmid replicated autonomously it too might represent a useful basis for a cloning vector. In filamentous fungi the transforming DNA is often integrated into the host genome, increasing the stability of the transformant.
Transformation
Complementation
Gene cloning relies upon the development of cloning vectors – self-replicating DNA molecules such as plasmids that can carry cloned DNA and deliver them into a host cell or mycelium. Gunge et al. (1993) characterised three linear plasmids in D. hansenii which were maintained in their hosts by osmotic stress – withdrawal of salts or polyols from the growth medium resulted in a temperature dependant loss of the plasmids by the host (Fukuda et al. 2004). As naturally occurring molecules of the fungus, these plasmids (pDHL1, 2, 3) were
Cloning a gene by complementation of an altered phenotype involves the development of a ‘‘gene library’’ of the organism, a collection of vector molecules with cloned DNA that represents every gene. The gene library is transformed into a host and then an altered phenotype is selected. Hence a direct test of gene function is possible, usually by complementing a mutation and restoring a normal or ‘‘wild type’’ phenotype. The development of a transformation system for a previously untransformed species is not simple. Suitable vectors need to
146
Fig. 2 – An example of complementation cloning. A collection of D. salina cloned DNA molecules in phage lambda were transformed into an A. nidulans strain carrying a salt sensitivity mutation (sltA1). Individual selected transformants (prefixed DS) are compared with a salt sensitive control strain (WV101) in growth upon solid medium containing 0.5 M NaCl. Four of the transformants shown here have levels of tolerance that approach wild type for the A. nidulans host (photo by courtesy D. Clement).
be constructed, a method of delivery of the DNA into the host cell established and finally a selection method is required to identify which individuals have taken up and can express the vector sequences. The development of a transformation system for a marine fungus can be avoided by expressing the gene library in a more amenable system such as A. nidulans or S. cerevisiae. Clement et al. (1999) transformed a D. salina gene library into an A. nidulans strain carrying a salt sensitivity mutation. A number of potential clones were identified that appeared to complement the salt sensitivity of their new host, restoring a measure of salt tolerance. Fig. 2 illustrates this screening procedure. In some cases the host strain carrying these clones showed an improved content of polyols as compatible solutes when grown under salt stress (Clement et al. 1999). A similar approach was used by Prista et al. (2002) when they isolated clones of D. hansenii DNA in a plasmid vector that enhanced the salt tolerance at alkali pH of a wild type strain of S. cerevisiae. Bansal and Mondal (2000) used a complementation cloning strategy to isolate the hog (regulatory gene controlling osmotic responses) homologue of D. hansenii in S. cerevisiae.
Cloning by PCR and DNA hybridization The polymerase chain reaction (PCR) has been used to clone a variety of genes from a number of species. PCR (Bridge 2002) relies on the design of short DNA sequences (primers)
P. Hooley, M. Whitehead
that match or complement sequences that are thought to flank the gene of interest. Well characterised S. cerevisiae or N. crassa homologous genes provide sufficient sequence information to design a range of likely primers. Multiple rounds of in vitro amplification using the Taq DNA polymerase enzyme then produce large quantities of the sequence of interest. This method has been used to clone genes encoding sodium pumps from D. hansenii (Almagro et al. 2001) and the superoxide detoxification enzyme superoxide dismutase from several marine Debaryomyces species (Hernandez Saavedra and Romero Geraldo 2001). Interestingly the D. hansenii sodium pump genes (DhENA1 and DhENA2) were expressed in the presence of high Naþ concentrations (0.5M) and DhENA2 also required an elevated pH (7.5) for expression. Both of these features would be desirable to maintain Naþ extrusion within the sea. Thome and Trench (1999) cloned the glycerol – 3 – phosphate dehydrogenase gene from D. hansenii. Northern Blot analysis of mRNA induced following different physiological treatments revealed that expression of the gene was inducible by NaCl indicating that salt tolerance is mediated at the level of the transcription of a gene responsible for the control of synthesis of the compatible solute glycerol. Guerrero et al. (2005) used the cloned genes from D. hansenii encoding NADP glutamate dehydrogenase and glutamine synthetase to demonstrate Naþ dependent expression which was not found in S. cerevisiae. As these genes are involved in ammonium assimilation this may represent an adaptation of nitrogen metabolism to the elevated cytosolic salt concentrations found within the marine species. Aggarwal et al. (2005) compared the homologous genes from the same two species encoding 30 (20 ), 50 -bisphosphate nucleotidase (Dhal2p), an enzyme that is particularly sensitive to salt in the terrestrial species. When S. cerevisiae was transformed with the D. hansenii gene (DHAL2), the terrestrial host became more salt tolerant. Hence cloned genes have proved useful tools in identifying changes in particular molecular processes that may result in marine adaptation. Aggarwal et al. (2005) noted that the N-terminal 54 amino acid residues of Dha12p had no homology with other yeast homologues. Yet this region can be seen to be present in the A. fumigatus (a terrestrial but salt tolerant filamentous species) homologue revealed by the recently completed genome project (Fig. 3). Other important genes recently cloned from marine fungi include the beta lactam biosynthetic genes ( pcbAB-pcbC ) from Kallichroma tethys (Kohlm. & Kohlm.) Kohlmeyer and Volkmann – Kohlmeyer (Kim et al. 2003). This group used PCR, exploiting primers whose design was based upon common (consensus) sequences from A. nidulans, Penicillium chrysogenum and Acremonium chrysogenum, to isolate a short (1,300 base pair) sequence. This was then used as a labelled probe to isolate a much larger complementary sequence by DNA hybridization. Analysis of the DNA sequences from this fragment confirmed a close relationship with orthologous genes from the cephalosporin producer Acremonium chrysogenum.
Fig. 3 – A multiple sequence alignment of three selected proteins using T-COFFEE, Notredame et al. (2000). The sequences are Dhal2p from Debaryomyces hansenii (NCBI accession no. AAR03496) and homologues from Saccharomyces cerevisiae (ARR89916) and Aspergillus fumigatus (EAL 88757). Red indicates high levels of similarity, yellow intermediate while green indicates low similarity. Asterisks indicate perfectly conserved (Cons) residues, two dots indicate very similar and full stops similar amino acid residues.
The genetics and molecular biology of marine fungi
D.hansenii S.cerevisiae A.fumigatus
147
---------MLRLMKYFTFAVYFSLLAVLLSIFISNYKIIPSWAISIL -----------------------------------------------MGPRSINTDDCCSVRRSSFSIFSCL--VLCSTLLLGVSIIFKTPPRIF
Cons
D.hansenii S.cerevisiae A.fumigatus
KIYPLKITNSSFKATMSTIPADHPYYKELEIASIAVIRTSILTKKLSD ----------------------MALERELLVATQAVRKASLLTKRIQS ASLPAASSSPNYTHT-----ANMSYQQERYIAELAVQRATLLTQKVFH
Cons
.
:*
:*
** ::::**:::
D.hansenii S.cerevisiae A.fumigatus
SIATTQKSGTHTKDDKSPVTIGDYASQAIINHAIKLNFPEDEIVGEED EVISHKDSTTITKNDNSPVTTGDYAAQTIIINAIKSNFPDDKVVGEES E----KAKGTVSKDDKSPVTIGDFGAQALIIQALRKNFPNDEIVAEEE
Cons
.
D.hansenii S.cerevisiae A.fumigatus
AEVLRKDDAEGKDLSAKVLEIISDVQSQTSQYNNRLGK--------LSSGLSD---------AFVSGILNEIKANDEVYNKNYKKDDFLFTNDQF ANSLREDKA----LSAEIWRLVKDIRLGDNESNELLGG--------LL
Cons
:. * .
D.hansenii S.cerevisiae A.fumigatus
--EKETEIYDSIDLGNSQGGSKGRFWALDPIDGTKGFLRGDQFAVCLA PLKSLEDVRQIIDFGNYEGGRKGRFWCLDPIDGTKGFLRGEQFAVCLA --PSEDAMLDIIDQGKSAGGPKGRIWALDPIDGTKGFLRGGQYAVCLG
Cons
: . * :*:*:**** **:.:*::* :*:: ***:*::*.**.
.
* :
: : ** *:
::.:::
.
*:
** ***:*.************* *:****.
D.hansenii S.cerevisiae A.fumigatus
LIEDGKVVLGVIGCPNLPATVVSNE-------EMSG--ARGGLFSAVR LIVDGVVQLGCIGCPNLVL-------SSYGAQDLKGHESFGYIFRAVR LIEDGDVKVGAIGCPNLPVDDSVAMTASIGVDQTDG-AGMGVLFSAIK
Cons
** ** * :* ******
D.hansenii S.cerevisiae A.fumigatus
GVGSFYSNLFDKQDFTPLSKQERIQMTQHTTPESLKVVEGVEKGHSSH GLGAFYSPSSDAESWT------KIHVRHLKDTKDMITLEGVEKGHSSH GQGSISRPLSNGA----LAESKPISMRPVPDIKQAVFCEGVEAAHSAQ
Cons
* *::
D.hansenii S.cerevisiae A.fumigatus
STQSQIKDKLGFNTETVSKQTINLDSQVKYCVLAKGQADIYLRLPISD DEQTAIKNKLN-----I-SKSLHLDSQAKYCLLALGLADVYLRLPIKL GDNAAVAQLLG-----ITSPSVRLDSQAKYCSIARGAGDIYLRLPVRK
Cons
. :: : : *.
D.hansenii S.cerevisiae A.fumigatus
TYREKIWDHAAGNILVYESGGLVGDI-HGNELNFGNGRHLNS-QGVVA SYQEKIWDHAAGNVIVHEAGGIHTDAMEDVPLDFGNGRTLAT-KGVIA DYQEKIWDHAAGDLIVREAGGQVTDI-YGQRLDFSKGRTLAANKGVVA
Cons
D.hansenii S.cerevisiae A.fumigatus Cons
: .*
:
* :
**** .**::
: . ::.****.*** :* * .*:*****:
*:*********:::* *:**
*
--GNKSVFKKVIEAVSDVLK---A SSGPRELHDLVVSTSCDVIQSRNA --APEAIQDQVISAVKTVLK---L . . : . *:.:
:.
. * :* *::
*::
.
*:*.:** * : :**:*
148
P. Hooley, M. Whitehead
The pcbAB gene from K. tethys was able to complement a defect in the corresponding gene encoding the ACV synthetase enzyme in A. nidulans.
5.
Phylogeny and taxonomy
Molecular methods also provide some of the most powerful tools for the identification of isolates and the examination of the taxonomic relationships of marine fungi. A comparative characterisation of at least part of the genome provides rigorous criteria, less susceptible to the transient effects suffered by morphological or biochemical markers. Michaelis et al. (1987) examined genetic variation by isoenzyme profiles that represented 14 assumed structural loci of Dendryphiella. Relatively little variation was shown with isolates from a common location usually appearing to be clonal in origin. Edwards et al. (1998) used a numerical taxonomic approach to compare a number of isolates of D. salina and D. arenaria to arrive at a quantitative description of the variation within and between a species. Again some isolates acquired from particular geographical locations were very similar if not identical. These two groups reached a common conclusion in designating their isolates into two distinct species which, in the absence of sexual reproduction, showed a somewhat limited capacity to generate genetic variation. Currently BIOLOG phenotype microarrays that examine substrate utilization and the PCR – based RAPD’s (random amplified polymorphic DNA) are amongst the techniques being used to examine variation in Dendryphiella isolates from around the world (Dela Cruz et al. 2006). PCR of particular repetitive DNA sequences (such as ‘‘microsatellites’’ or those encoding small or large ribosomal subunits and their associated ITS regions) can be used to directly assess fungal diversity in marine habitats by producing strain or species specific ‘‘DNA fingerprints’’ (Table 1). Cooke et al. (2000) described how a group of marine isolates within the genus Halophytophthora were distinct from Phytophthora and most Pythium species. The evolution of these taxonomic groups seems to be driven by host specialization. Gadanho et al. (2003) arranged 234 marine isolates into 31 groups and interestingly basidiomycetous yeasts were more common than ascomycetous yeasts. Zuccaro et al. (2003) identified marine fungi in dead and decaying plant matter. Spatafora et al. (1998) applied the analysis of rDNA sequences to suggest that the Halosphaeriales were derived from terrestrial ancestors. A number of other genes have been used for identification and taxonomic purposes, these include those encoding
Table 1 – Examples of Molecular Taxonomy in Marine Fungi Species Halosphaeriales Halosarpheia, Lignicola, Nais Halophytophthora Novel isolates Marine yeasts Various including Halosphaeriales, Hypocreales and Pleosporales
Reference Spatafora et al. 1998 Kong et al. 2000 Cooke et al. 2000 Stoeck and Epstein 2003 Gadanho et al. 2003 Zuccaro et al. 2003
polyketide synthases, beta-tubulin and laccases (e.g. Lyons et al. 2003). Developments of PCR can even amplify diagnostic DNA sequences directly from environmental samples without the prior subculture of the organism. This is presently the most common source of accession numbers for marine fungal genes on the NCBI database (http://www.ncbi.nlm.nih.gov/). Stoeck and Epstein (2003) showed how this approach may allow the detection of novel (and perhaps unculturable) isolates of marine fungi. Hence our assumptions about the relative diversity of particular groups of fungi may be tested. Molecular techniques based upon the comparative DNA sequencing of the trpC gene isolated via PCR were used to identify a fungal agent causing death of Caribbean sea fans. This analysis confirmed the pathogen as Aspergillus sydowii (Bainier & Sartory) Thom & Church, a cosmopolitan species found in both terrestrial and marine locations. Other non-marine isolates of the same species were unable to infect sea fans (Geiser et al. 1998). Hence the acquisition of at least a partial marine habit by a genetically distinct group of strains correlated with enhanced pathogenicity. Kis-Papo et al. (2003) examined the genomic diversity of strains of the cosmopolitan species Aspergillus versicolor (Vuill.) Tirab associated with the Dead Sea where a combination of high salt concentrations (up to 10 times seawater) and acidity (pH 5.9) provide an interesting contrast with the conventional marine habitat. They used a PCR based technique of AFLP’s (amplified fragment length polymorphisms) to compare the diversity of strains isolated at different salinities and from terrestrial or freshwater sites. Intriguingly, genetic exchange and diversity was positively correlated with increasing stress, only declining under the most extreme conditions of the deep waters of the Dead Sea. Molecular techniques therefore may help to refine the definition of obligate and facultative marine fungi (Kohlmeyer and Volkmann-Kohlmeyer 2003).
6.
Genomics
Genome projects aim to clone and sequence all of the DNA of an organism as individual clones and then organise such information into contiguous overlapping sections (‘‘contigs’’) that are arranged into precisely annotated chromosomes. The D. hansenii genome project has now been published (Dujon et al. 2004). Lepingle et al. (2000) compared partial clones of D. hansenii genes with the S. cerevisiae genome and discovered that the genes associated with transport functions (such as for carbon and nitrogen sources) were much more common in the marine than in the terrestrial yeast. Dujon et al. (2004) described the monumental task of comparing the entire genomes of several yeast species. Comparative genomics aims to compare all the gene sets and chromosome positions between two or more closely or disparately related organisms. The type strain D.hansenii var. hansenii CBS767 has seven chromosomes and shows a close relationship to members of the genus Candida. Full details are publically available online (Genolevures database: http://cbi.labri.fr/Genolevures/). Table 2 summarises how the increase in gene number in the marine species is not achieved by an overall increase in genome size but rather by a reduction in the proportion
The genetics and molecular biology of marine fungi
Table 2 – A simplified comparison of the genomes of Saccharomyces cerevisiae and Debaryomyces hansenii (CDS [ protein coding sequences, using data from Dujon et al. 2004)
Genome Size (Mb) Total CDS Mean Gene Density (protein coding as %) Mean CDS size (codons)
Saccharomyces cerevisiae
Debaryomyces hansenii
12.1 5807 70.3
12.2 6906 79.2
485
389
of non-coding DNA coupled to a shortening of gene length. D. hansenii was found to have many more examples of the duplication of genes, reflecting either a recent series of amplification events or a less efficient process to eliminate redundant genes. This may also reflect a more demanding environment which selects for the retention of duplicated genes with even very slight changes in encoded protein activities. Again, families encoding transporters such as multidrug resistance proteins and hexose sugar transporters were particularly expanded in relation to the other yeast species studied. A similar comparative approach for the marine bacterium Silicibacter pomeroyi Gonzalez revealed that this species had a higher proportion of genes controlling signal transduction and transport processes than did other related non-marine species (Moran et al. 2004). Survival of microorganisms in the nutrient poor marine environment demands the evolution of a wide portfolio of transport genes, with their products able to function for example at alkaline pH and under ionic stress. Another genomics approach (‘‘high-throughput shotgun sequencing’’) has provided global views of communities and the metabolic capabilities of uncultured organisms which occupy niche environments. This technique has compared fungal members of a marine environment to terrestrial environments (Tringe et al. 2005). These results again highlighted the importance of genetic elements coding for organic molecule transporters and sodium ion exporters in the marine species.
7.
Conclusions
After a slow start on the classical genetics of marine fungi we are now fortunate to have one supremely detailed genetical resource, namely the genome project for D. hansenii. The genome of D. hansenii appears markedly different to many other yeasts, suggesting a large number of mutations are needed to acquire and maintain the marine habit. However the recent appearance of apparently marine strains of an otherwise terrestrial species of Aspergillus (Kohlmeyer and Volkmann-Kohlmeyer 2003) offers the prospect of identifying a more limited number of key genetic changes required for life in the ocean. Once genome projects are completed then microarray technology (‘‘transcriptomics’’, analysing levels of mRNA specific to particular groups of induced or active genes) becomes feasible (e.g. Posas et al. 2000). However, how representative of marine fungi is one species of yeast – D. hansenii? With the much larger genomes of filamentous fungi approaching twice
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the number of yeast genes (http://mips.gsf.de/projects/fungi/ fungi_db.html), the genome of a filamentous species would be an enticing prospect to study. A number of species may be suggested but the physiological legacy and ability to produce secondary metabolites make D. salina an attractive candidate. Given the investment required for such an undertaking it would be important that the marine fungal research community reaches a consensus. At present the genomics approach is being applied both to microbial and metazoan marine communities (Wilson et al. 2005). A number of genomes of Phytophthora species of crop plants have now been sequenced (http://www.pfgd.org/). These may be compared to selected sequences from the marine genus Halophytophthora should these become available. For example genes that influence cytoplasmic turgour and zoospore release, such as those encoding particular transcription factors or protein kinases (Judelson and Roberts 2002), would be potentially very informative to contrast between terrestrial and marine species. This is particularly intriguing when one considers that some susceptible crops are grown on salt affected soils. There is also scope to return to classical techniques to answer long – standing questions about fungal physiology, including those relating to the role of the wall as an ion sink (Clipson and Jennings 1992). It is feasible to isolate and characterise the osmotic responses of cell wall mutants in models such as D. salina. In our own laboratory we have found conventional mutagens such as MNNG, ultra violet light and nitrous acid are successful at inducing stable morphological mutants of D. salina, affecting both colony and spore characteristics. Many of the genes encoding enzymes involved in cell wall synthesis are well characterised in terrestrial models. They may now be compared directly in silico with marine counterparts on the Genolevures site. Fig. 3 illustrates how candidate domains and residues for ‘‘marine’’ adaptations in proteins can be identified. One area that deserves more attention is the induction of novel metabolites by stress responses in the sea. This area is probably receiving more attention than is reflected by the academic press due to commercial restraints. Secondary metabolite genes are stress responders controlling production at the end of lag or stationary growth phases and marine fungi are often slow growers. Many novel compounds may have been missed by conventional screens. Molecular approaches such as that of Kim et al. (2003) may give an insight into an enticing world of new antimicrobials. A search of the database at the National Centre for Biotechnology Information (http://www. ncbi.nlm.nih.gov/) reveals a number of Dendryphiella DNA sequences and their encoded haloperoxidase and protease enzymes which are part of patent applications of interest to the biotechnology industry. It has been possible to argue that the sea represents a somewhat buffered environment with relatively constant levels of salinity, temperature and pH along with predictable tidal regimes. However it is now very clear that human activities are altering this stability. For example alkaline pH values of 8.2 þ/ 0.3 in the sea already represent a 30 % increase in hydrogen ion concentration since the onset of the Industrial Revolution. As more CO2 dissolves, the pH falls and the carbonate building blocks for coral formation become less available (The Royal Society 2005). Marine fungi are ubiquitous
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colonisers of marine corals yet have been largely ignored and may damage their increasingly stressed hosts (Golubic et al. 2005). Molecular studies such as those of Geiser et al. (1998) and Kis-Papo (2003) assume a renewed importance in predicting the ability of fungi from other habitats to invade this changing marine environment. How will imperfect marine species with relatively little genetic variation such as D. salina fare once climate change takes hold? The key role of microorganisms within the global carbon cycle and in particular within the ocean is obvious and yet the contributions of marine fungi, with their increased complexity of carbon transporters are not quantified. The increased fragility of the marine ecosystem gives a renewed urgency to the study of the genetics and molecular biology of the marine fungi.
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