BMC Genomics 2008, 9:200 http://www.biomedcentral.com/1471-2164/9/200 phylogenies that suggest some relationship between B. algerae and A. locustae [[25] and unpublished data]. In addition to being densely packed, E. cuniculi genes have also been shown to be shorter on average than homo- logues in the S. cerevisiae genome [10]. This has been dis- cussed in the context of genome compaction, but also suggested to be the result of a reduction in the number of proteins in the cell, which leads to smaller interaction net- works, which in turn allows proteins to reduce their number or complexity of interacting domains [13]. In yeast, it has been shown that there is a correlation between protein size and connectivity, with larger pro- teins displaying a greater number of interactions [33]. We examined the only five full-length genes identified in the B. algerae survey with homologues in yeast and found that all five were shorter than S. cerevisiae homologues, and more surprisingly most were also shorter than the E. cuniculi homologues (Figure 2). Similarly, from the E. aedis survey, only five full-length genes were found (Figure 2), and four of these were shorter than S. cerevisiae homo- logues, and comparable in size with the E. cuniculi homo- logues. The sole E. aedis protein predicted to be larger than the yeast counterpart encodes a ribosomal protein (Figure 2). Given that these genomes are not compacted, it sug- gests that proteins are either shorter due to a reduced pro- teome complexity, or that they had an ancestor with a compacted genome, or both. Gene content and coding capacity There is no experimental estimate of the E. aedis genome size, but the B. algerae genome has been estimated to be between 15–20 Mbp by pulsed-field gel electrophoresis [23]. This is much larger than the genomes of either E. cuniculi (2.9 Mbp) or A. locustae (5.4 Mbp). As we show, much of the genome size difference can be attributed to the significantly lower gene-densities of B. algerae and E. aedis. However, it is still possible that one or both of these genomes is also larger because it contains more genes than E. cuniculi. The E. cuniculi genome clearly under went a massive gene loss relative to other eukaryotes, but this gene loss may have been ancestral to microsporidia. If this were the case we would expect to find few genes in E. aedis and B. algerae that are present and conserved in other eukaryotes that are not also present in E. cuniculi. That is to say that the B. algerae and E. aedis genomes would not have more con- served genes than the pool remaining in the ancestral microsporidian after this gene loss event. Our sampling of E. aedis and B. algerae genomes shows that this scenario is quite possible. Of the protein-coding genes with identifiable homologues in some other genome that we found in B. algerae (34 cases) and E. aedis 1 0 0 Length in amino acids 4 002 030 00 5 0 0 0 Ba Cell division kinase Ec Sc Ba RRP40 EScc Ba Rho GTPase EScc Ubiquitin conjugating enzyme Ba E2-24KD EScc Ba RAD31 Ec Sp U6 snRNA-associated Ea Ec small nucleoprotein Sc 26S Proteasome Ea Ec B-type subunit Sc Ea 40S Ribosomal protein Ec Sc Ea Methyltransferase EScc Ea Transcription factor GCN5 EScc FCiogmurpear2ison of microsporidian and yeast protein lengths Comparison of microsporidian and yeast protein lengths. The number of codons for all full-length proteins found within B. algerae (Ba) and E. aedis (Ea) sequence sur- veys compared to homologues from S. cerevisiae (Sc) (or Schizosaccahromyces pombe (Sp) in cases where S. cerevisiae does not encode a homologue). (25 cases), every one is also present in E. cuniculi (Table 3). Given the sample size, it is likely that either genome could contain some genes found in other organisms but not E. cuniculi, but it is unlikely that they are abundant. This lack of excess conserved gene homologues is of inter- est because it implies that the large-scale gene loss charac- teristic of E. cuniculi took place relatively early in microsporidian evolution, in the ancestor of E. cuniculi, A. locustae, E. aedis and B. algerae. However, the B. algerae survey consisted of 20% coding sequence, so taking into account the range or estimated genome sizes for B. algerae (15–20 Mbp), this suggests between 2,786 and 3,714 genes in the Brachiola genome (assuming an average gene length of 1,077 as in E. cuniculi). The discrepancy between this predicted coding capacity of B. algerae and the observation that all the rec- ognizable genes we sampled are shared with E. cuniculi could be explained in many ways. First, our sample may be biased to gene-encoding regions, and this would lead to an overestimate of the gene-density. Second, a large number of lineage-specific ORFs could skew the estimate, Page 5 of 9 (page number not for citation purposes)