Phylogenetic position and codon usage of two centrin genes from the rumen ciliate protozoan, Entodinium caudatum

FEMS Microbiology Letters 166 (1998) 147^154

Phylogenetic position and codon usage of two centrin genes from the rumen ciliate protozoan, Entodinium caudatum Sylvain C.P. Eschenlauer a , Neil R. McEwan a , Roger E. Calza b , R. John Wallace a , Ryoji Onodera c , C. James Newbold a; * b

a Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK Departments of Genetics and Cell Biology and Animal Sciences, Washington State Unversity, Pullman, WA 99164-6320, USA c Animal Science Division, Miyazaki University, Miyazaki-Shi 889-21, Japan

Received 5 July 1998; revised 17 July 1998; accepted 17 July 1998

Abstract A V phage cDNA expression library was constructed from washed suspensions of the rumen ciliate protozoan, Entodinium caudatum, which had been maintained in an isolated, monofaunated sheep. The library was screened using an anti-E. caudatum antiserum raised in rabbits against sonically disrupted protozoa. DNA sequences for two centrins or caltractins, a subfamily of the EF-hand Ca2‡ -modulated proteins which are closely related, highly conserved cytoskeletal proteins, were identified and characterised. Their phylogenetic position was established relative to other centrin gene sequences. The two proteins showed homology to Paramecium tetraurelia centrins: 50 and 52% identities and 71 and 75% similarities in the protein sequence, over 99 and 110 amino acids fragments. Codon usage and indices revealed that E. caudatum follows universal codon usage, but with a restricted number of codons, and has a low G+C content. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Rumen ; Protozoon; Centrin; Codon usage; cDNA ; Entodinium caudatum

1. Introduction The rumen plays a central role in the nutritional adaptation of ruminants to thrive on ¢brous plant materials [1]. Microbial fermentation of plant cell wall material results in the production of volatile fatty acids which act as an energy source while microbial cells leaving the rumen provide much of the

* Corresponding author. Tel.: +44 (1224) 712751; Fax: +44 (1224) 716687; E-mail: [email protected]

protein available for digestion by the host. The bacteria and fungi in the rumen have been extensively studied and many genes have been characterised from these organisms [2,3]. Ciliate protozoa comprise up to 50% of the microbial biomass, however they have been studied in much less detail, particularly at the molecular level, because it is not possible to maintain these protozoa in vitro in axenic culture [4]. Some phylogenetic relations have been established from rRNA gene sequences [5^9], but to date only two partial sequences encoding a protein, namely K-tubulin, obtained from non-de¢ned Entodinium and Epidinium spp., have been analysed [10].

0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 3 2 2 - X

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Entodinium caudatum is a prominent rumen ciliate, and also one of the simplest and smallest (ellipsoid, 50^70 Wm in length and 50^60 Wm in width [4]). E. caudatum possesses amylolytic and bacteriolytic activities, but it lacks the ¢bre-degrading ability of some other rumen ciliates [4]. Its small size means that it can be consumed by larger species, such as Entodinium bursa. When the predatory species are present, E. caudatum forms a characteristic caudal spine as a form of defence [4]. The aims of this work were to create a cDNA expression library from E. caudatum, and to characterise DNA sequences with regard to phylogenetic relations and codon usage in this organism.

USA). cDNA was synthesised and the library packaged in lambda ZAP Express1 using ZAP Express cDNA synthesis kit and ZAP Express cDNA Gigapack0 III Gold cloning kit (Stratagene, La Jolla, CA, USA). 2.2. Production of antiserum

2. Materials and methods

Protozoal antigen was prepared from washed E. caudatum cells by sonication followed by precipitation, by adding 550 g ammonium sulfate per litre and incubating at 4³C for 16 h. Precipitated protein was harvested by centrifugation (16 000Ug, 30 min, 4³C) and washing once in 50 mM potassium phosphate bu¡er, pH 6.5. Partially puri¢ed antigen was used to raise polyclonal antibody in rabbits as described by Dresser [14] and Vaitukaitis [15].

2.1. cDNA library

2.3. Antibody screening of the cDNA library

E. caudatum was originally isolated from a mixed rumen population by repeated subculture in vitro in the presence of chloramphenicol as described by Coleman [11]. Cultures were maintained in vitro for 6 months and used to inoculate previously defaunated sheep [12]. Monofaunated sheep were maintained in isolation for 2 years before the start of the experiment. One litre of rumen £uid was collected 2 h after feeding via the rumen cannula. Protozoa were prepared from rumen £uid by ¢ltration and sedimentation [4], and were resuspended in diethyl pyrocarbonate-treated Coleman's salts solution D [13]. The latter solution was prepared by mixing two separate components of Coleman's salts solution D, each with added 0.1% diethyl pyrocarbonate, after autoclaving; the ¢rst component was CaCl2 solution, and the second contained the remaining components of Coleman's salts solution D. The cells were harvested by centrifugation at 550Ug for 5 min at room temperature and resuspended in 20 ml of Trizol0 reagent (Life Technologies Inc., Grand Island, USA), then homogenised for 30 s at top speed with a top bladed homogeniser. Total RNA was extracted using Trizol reagent according to the manufacturer's instructions. The poly(A) mRNA was isolated using polyATtract0 mRNA isolation system II (Promega Corporation, Madison, WI,

Once antiserum of appropriate titre was obtained, it was treated with 10% (w/v) acetone extract [16] of Escherichia coli XL1 blue (Stratagene) for 1 h at 37³C, in order to remove possible background interference with the antiserum. The phage library was plated on 15 cm diameter petri dishes according to the manufacturer's protocol. Nitrocellulose membranes were prewashed in 10Usodium citrate bu¡er [16] and allowed to dry, then were placed on the surface of the plates for 15 min. The membranes were removed and blocked in PBS containing 0.1% (v/v) Tween 20 and 0.25% (w/v) bovine serum albumin (PBST-BSA) for 1 h. The membranes were rinsed twice in distilled water, then incubated for 2 h at 37³C with rabbit antiserum diluted 1000 times in PBST-BSA. The membranes were then washed twice in distilled water and incubated for 2 h at 37³C with goat anti-rabbit IgG (whole molecule) alkaline phosphatase conjugate diluted 15 000 times in PBST-BSA. The membranes were washed three times in distilled water and then incubated for 30 min at 37³C in 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium liquid substrate system (Sigma) diluted 5 times in distilled water. 2.4. Excision of positive clones Phagemids were excised in vitro from positive

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clones according to the manufacturer's instructions and were used to transformed E. coli XLOLR (Stratagene). The transformed bacterial cells were used to prepare plasmid DNA [16]. 2.5. DNA sequencing Selected positive clones from the antibody screening procedure were sequenced full length in both directions using ABI Prism1 BigDye terminator sequencing ready reaction kit (Perkin Elmer Corporation, Norwalk, CT, USA) with T3 and T7 primers on an ABI Prism1 377XL DNA sequencer (Perkin Elmer Corporation). 2.6. Analysis of DNA sequences DNA and protein similarity searches were performed using BLAST (http://genome.eerie.fr/bin/ blast-guess.cgi). Translations were performed by spreadsheet analysis [17]. Pair-wise alignments were performed at http://genome.eerie.fr/bin/align-guess. cgi and ClustalW multiple sequence alignment at http://www2.ebi.ac.uk/clustalw/. Phylogenetic analysis was performed using programs within PHYLIP version 3.57c [18]; bootstrapping used the Seqboot program (100 repetitions), trees were constructed using the PROTPARS (Protein Sequence Parsimony method) and a consensus tree was derived by analysing the resulting Tree¢le using the Consense program. 2.7. Codon usage and codon indices Codon usage tables were determined for protozoal genes following the method described in McEwan and Gatherer [17]. The e¡ective codon number (Nc [19]), use of G or C in the third position of a codon (GC3), use of purines in the third position of a codon (Pu3), mutational response index (MRI [20]) and overall gene G+C content were calculated. In the case of all Paramecium sequences, indices were modi¢ed to take account of the coding potential of codons TAA and TAG, i.e. the potential upper limit of Nc becomes 63, rather than 61, and in calculating the MRI glutamine is treated as being encoded by four codons, rather than two.

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3. Results and discussion 3.1. Centrin DNA sequences An antibody screen, based on antiserum raised against disrupted cells of E. caudatum, detected large numbers of recombinant phage which expressed protozoal antigens in a V cDNA expression library. It is not possible to grow E. caudatum axenically, so the original cell suspensions used to raise antibodies would have been contaminated with some bacteria which survived the washing procedure. Similarly, bacterial RNA must have been present with the original mRNA extracted from E. caudatum. The selection of poly(A) mRNA using oligo(dT) attached to magnetic beads should have eliminated the possibility of cloning bacterial messages. Two (GenBank AF065247 and AF065248) of four inserts were selected according to their size and their restriction pro¢le with EcoRI and were, following sequencing, found to have high homology to other protozoal centrin gene sequences, indicating that at least some of the cloned sequences were of protozoal origin and enabling analysis of the phylogenetic position of these centrins and of codon usage in E. caudatum. On the basis of BLAST searches, the ¢rst centrinlike sequence had up to 63% identity in the DNA sequence over a 279-nucleotide sequence (P = 1.9U10328 ) to other centrins, with up to 50% identity and 71% similarity in the protein sequence, over a 110-amino acid fragment (P = 3.3U10354 ). The second sequence had up to 61% identity in the DNA sequence over a 282-nucleotide sequence (P = 4.7U10325 ) to other centrins, with up to 56% identity and 74% similarity in the protein sequence, over a 94-amino acid fragment (P = 6.9U10354 ). The centrins from E. caudatum displayed 84.5% identity at the DNA level and 89.5% in amino acid sequence relative to each other. The ¢rst cDNA was 606 bases in length encoding 170 amino acid residues, the second 642 bases encoding 172 amino acids. Both sequences were assumed to be partial as they lacked a methionine residue at their start. Centrins (or caltractins) form a subfamily of the EF-hand Ca2‡ -modulated proteins [21]. They are acidic cytoskeletal proteins with a molecular mass of around 20 kDa, found in structures associated

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with microtubule-organising centres, including centrosomes, mitotic spindle poles and basal bodies. They are closely related and highly conserved, thus of particular interest for phylogenetic and codon usage studies. 3.2. Phylogenetic analysis of centrin sequences A phylogenetic tree was constructed for centrins using 19 sequences retrieved from protein databases and the two new translated sequences from E. caudatum. One additional protein was selected, the Sac-

charomyces cerevisiae CDC31 protein, because a BLAST search revealed that this protein had a high homology to centrins. The CDC31 protein has an involvement in the yeast cell cycle, and due to the importance of cytoskeletal proteins in the cell cycle, it is reasonable to assume that this homology implies a role in yeast similar to centrins in other eukaryotes [22]. The sequences were ¢rst aligned using ClustalW before constructing a phylogenetic tree using programs from the PHYLIP package (Fig. 1). This tree shows four distinct clusters of centrins: a cluster

Fig. 1. Bootstrap majority-rule consensus tree obtained from 100 maximum parsimony replicates (Seqboot, PROTPARS, and Consense programs) with 22 centrin sequences. Cluster I: diverging centrins ; cluster II: vertebrate centrins ; cluster III: protozoal centrins; cluster IV: plant centrins. Numbers in parentheses refer to GenBank accession numbers.

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Table 1 Codon usage and codon indices for all nine protozoal sequences known AA

Codon Entodinium caudatum

Leu

TTA TTG CTT CTC CTA CTG CGT CGC CGA CGG AGA AGG TCT TCC TCA TCG AGT AGC GTT GTC GTA GTG CCT CCC CCA CCG ACT ACC ACA ACG GCT GCC GCA GCG GGT GGC GGA GGG ATT ATC ATA TTT TTC TAT TAC CAT CAC CAA CAG TAA TAG

Arg

Ser

Val

Pro

Thr

Ala

Gly

Ile

Phe Tyr His Gln

AF065247

AF065248

6 1 4 3 0 0 0 0 0 0 4 1 1 3 0 0 0 3 3 3 0 0 0 1 0 0 5 5 0 0 5 5 0 0 1 0 5 0 8 2 2 2 7 1 1 1 0 5 1 N/A N/A

6 2 3 4 0 0 0 0 0 0 6 0 2 0 0 0 0 2 2 5 0 0 0 0 1 0 6 9 1 0 5 6 0 0 1 1 5 0 5 6 0 1 8 1 1 0 1 2 1 N/A N/A

Paramecium tetraurelia U35344 5 7 1 0 0 0 0 0 1 0 6 0 3 1 2 0 2 1 2 3 1 1 2 2 4 0 3 6 4 0 5 3 8 0 2 1 7 0 7 2 0 8 4 0 2 0 0 3 0 9 1

U76540 6 7 0 0 0 0 0 0 1 0 6 0 2 0 4 0 3 0 3 1 2 0 2 1 5 0 1 6 6 0 6 3 7 0 2 1 7 0 7 3 0 9 3 0 2 0 0 4 0 8 1

U35396

U35397

4 7 1 1 0 0 0 0 1 0 6 0 3 1 2 0 1 2 2 1 1 2 1 1 6 0 4 1 8 0 11 1 3 0 3 0 6 1 6 4 0 7 5 1 1 0 0 5 0 9 1

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4 7 1 1 0 0 0 0 1 0 6 0 2 2 2 0 2 1 2 2 1 1 1 1 6 0 4 5 3 0 9 3 3 0 1 1 8 0 7 2 1 7 5 1 1 0 0 6 0 8 1

Giardia intestinalis

Naegleria gruberi

U42428

U21725

1 1 4 3 0 3 4 7 1 1 2 2 2 2 2 2 3 1 1 0 1 3 1 2 0 0 1 1 2 2 3 6 4 2 1 3 2 3 6 1 9 7 4 1 0 1 1 1 3 N/A N/A

U59300 0 2 4 5 0 2 0 5 3 2 2 3 3 3 0 2 0 0 0 2 0 2 1 1 1 0 2 1 2 2 2 4 3 2 0 5 3 1 2 6 3 2 7 1 0 0 1 0 2 N/A N/A

3 3 1 5 0 0 2 0 1 0 4 0 3 1 3 1 2 2 1 0 1 1 0 0 1 0 2 1 4 0 4 2 6 0 7 0 4 0 10 3 0 6 5 0 1 1 0 7 1 N/A N/A

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Table 1 (Continued). Asn

AAT AAC Lys AAA AAG Asp GAT GAC Glu GAA GAG Cys TGT TGC Met ATG Trp TGG Stop TAA Stop TAG Stop TGA GC3 Pu3 MRI Nc GC

10 4 15 3 18 6 15 2 0 1 7 0 1 0 0 0.345 0.398 0.154 34.1 0.345

4 8 15 3 14 10 16 1 0 1 7 0 1 0 0 0.439 0.387 0.181 32.8 0.385

4 1 10 5 11 8 15 1 0 0 7 0 N/A N/A 1 0.308 0.538 0.185 40.1 0.368

5 0 10 5 14 5 14 2 0 0 7 0 N/A N/A 1 0.258 0.566 0.217 40.4 0.350

3 2 10 5 14 5 12 4 0 0 6 0 N/A N/A 0 0.282 0.547 0.186 43.1 0.359

1 4 11 4 17 2 13 3 0 0 6 0 N/A N/A 0 0.289 0.528 0.170 43.1 0.363

4 1 4 7 3 15 6 17 0 0 6 0 0 0 1 0.559 0.497 30.044 59.6 0.497

0 5 4 9 5 12 3 21 0 0 8 0 0 1 0 0.716 0.512 0.005 53.9 0.537

6 1 11 11 15 1 17 1 0 0 10 0 1 0 0 0.289 0.526 0.044 39.8 0.345

Accession numbers refer to GenBank database.

of diverging centrins (I), a vertebrate cluster (II), a protozoal cluster (III), and ¢nally a cluster of primarily plant and green algal sequences (IV) which also included the protozoal sequence from Giardia intestinalis (GenBank U59300). The cluster of diverging centrins (I) is similar to that found previously [22]. The position of the centrin sequences in the phylogenetic tree also con¢rmed that the original message had not been derived from a rumen fungal species, the host animal, or plant material present in the feed. 3.3. Codon usage Codon usage analysis (Table 1) concentrated on the nine protozoal centrin protein sequences. The E. caudatum and Paramecium tetraurelia sequences have a low G+C content, 34.5^38.5%, and both Giardia intestinalis sequences have a G+C content of nearer 50%. The use of a G or a C in third position (GC3) in E. caudatum is greater than in P. tetraurelia, even though the overall G+C content is not higher. This is particularly true in sequence 2 from E. caudatum. The use of a purine in position 3 is lower in both E. caudatum, around 40%, than in the other protozoa, which have values around 50%.

The mutational response index (MRI), which is a measure of the preferential bias for use of speci¢c codons, is fairly high in both E. caudatum and P. tetraurelia, 0.154^0.217, but is extremely low for both Giardia spp. and Naegleria gruberi. Where the discrepancy of G+C content from 50% of the gene is greater than 20%, an MRI value of around 0.3^0.4 is anticipated in coding sequences, with a much lower MRI in putative ORFs which are non-functional [23]. Thus, MRI values of around 0.15^0.2 might reasonably be anticipated in coding sequences with this type of G+C content. Since similar values are seen in both E. caudatum and P. tetraurelia, this is additional evidence that these sequences are likely to encode proteins. The e¡ective codon number (Nc) is an indication of the number of di¡erent codons used in a speci¢c gene. In addition to MRI, Nc has been used as an indicator of a translated ORF in sequences where the G+C content is very di¡erent from 50% of the nucleotides present [23]. Under conditions far removed from 50% G+C content, the Nc value tends towards 20 (i.e. only one codon being used for each amino acid), and deviation from this trend is indicative of a non-encoding ORF. Nc values are high for both Giardia sequences, over 50, as might be anticipated in a gene with approximately 50% G+C content,

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where synonymous use of most codons might be anticipated (total synonymity for all amino acids giving an Nc value of 61). In contrast, the genes with a greater G+C (or as in this case A+T) bias would be expected to have low Nc values. This observation is borne out, with N. gruberi and P. tetraurelia values of around 40 and E. caudatum values of less than 35. It should be noted that P. tetraurelia values are slightly higher than the other organisms with a G+C bias, but this might be explained as being a result of the Nc value being in the range 20^63, due to the additional codons used to encode glutamine. The use of codon indices adds additional weight to the argument that the original message isolated is used to encode a functional protein, due to the relatively high MRI and relatively low Nc values. From the codon usage table, although the E. caudatum sequences cluster most closely with those previously described in P. tetraurelia, they appear to use TAA as a stop codon, in keeping with universal codon usage. This would be expected to be the most frequently used stop codon in an AT-rich organism, unless there is deviation from the universal code. Certain protozoa such as P. tetraurelia utilise TAA and TAG to translate protein as opposed to stop codons making it impossible to use bacteria as expression systems for such heterologous genes.

4. Conclusions The availability of monofaunated sheep has allowed collection of su¤cient amounts of E. caudatum to perform cloning experiments. We have shown that a cDNA expression library is a useful tool for cloning genes from rumen ciliate protozoa, and can provide novel information on molecular genetics in E. caudatum. This ¢nding opens a new route for exploring the molecular biology of the hydrolytic enzymes which rumen ciliate protozoa use to carry out the nutritionally important processes of plant cell wall breakdown and bacteriolysis.

Acknowledgments We thank Freda M. McIntosh for help raising

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antibodies, and Derek Gatherer, Liverpool John Moores University, for discussions on the adaptation of the codon indices for non-universal codon usage.

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