Evaluation of Three Automated Genome Annotations for Halorhabdus utahensis

Genome annotations are accumulating rapidly and depend heavily on automated annotation systems. Many genome centers offer annotation systems but no one has compared their output in a systematic way to determine accuracy and inherent errors. Errors in the annotations are routinely deposited in databases such as NCBI and used to validate subsequent annotation errors. We submitted the genome sequence of halophilic archaeon Halorhabdus utahensis to be analyzed by three genome annotation services. We have examined the output from each service in a variety of ways in order to compare the methodology and effectiveness of the annotations, as well as to explore the genes, pathways, and physiology of the previously unannotated genome. The annotation services differ considerably in gene calls, features, and ease of use. We had to manually identify the origin of replication and the species-specific consensus ribosome-binding site. Additionally, we conducted laboratory experiments to test H. utahensis growth and enzyme activity. Current annotation practices need to improve in order to more accurately reflect a genome's biological potential. We make specific recommendations that could improve the quality of microbial annotation projects

Candida albicans morphology and commensal fitness in the mammalian gut

While C. albicans has largely been studied as a pathogen, its primary role is a commensal of the gastrointestinal tract. Overall, the fungal components of the GI microbiome are less well studied than the bacterial members, despite fungi making up a significant portion of the commensal microbiota. Understanding C. albicans’ role in the GI niche will provide important insights into fungal GI commensalism.My studies investigate the role that filamentation plays in gut commensalism. C. albicans filamentation has previously been characterized as an important part of the pathogenic lifestyle of the fungus, but the role of hyphae in GI commensalism is unclear. We show that the C. albicans filamentation program is detrimental to commensal colonization, as mutants of pro-filamentation transcription factors are hyperfit in the gut. Interestingly, however, we find that hyphae are present throughout the gut. Because hyphae themselves are not detrimental to commensalism, we hypothesize that expression of hyphal specific genes mediates regulation of the commensal population. Indeed, we find that mutants of hyphal specific cell surface proteins, like Sap6, are hyperfit in the GI tract, suggesting that cell type specific markers, rather than cell shape, mediate commensal fitness. We also find that C. albicans’ interactions with other members of the microbiota, namely Lactobacilli, are important mediators of commensal fitness. Our lab previously discovered a novel phenotypic form of C. albicans, termed GUT cells, which are specialized for commensalism. Surprisingly, mice naturally colonized with high levels of Lactobacillus spp. antagonize the GUT cell commensal advantage. GUT cells, which are normally less responsive to classic filamentation cues as compared to white cells, filament robustly in the presence of Lactobacillus species in vitro. As we know that filamentation is detrimental to commensal, we hypothesized that Lactobacillus-GUT cell interactions reverse the competitive advantage of GUT cells. Indeed, we find that pre-colonizing mice with high levels of Lactobacillus, antagonizes recovery of GUT cells from GI tract. Taken together, these studies help us understand how C. albicans phenotypic transitions and interactions with other members of the gut microbiota influence commensal colonization in the mammalian GI tract

Candida albicans Morphogenesis Programs Control the Balance between Gut Commensalism and Invasive Infection

Candida albicans is a gut commensal and opportunistic pathogen. The transition between yeast and invasive hyphae is central to virulence but has unknown functions during commensal growth. In a mouse model of colonization, yeast and hyphae co-occur throughout the gastrointestinal tract. However, competitive infections of C. albicans homozygous gene disruption mutants revealed an unanticipated, inhibitory role for the yeast-to-hypha morphogenesis program on commensalism. We show that the transcription factor Ume6, a master regulator of filamentation, inhibits gut colonization, not by effects on cell shape, but by activating the expression of a hypha-specific pro-inflammatory secreted protease, Sap6, and a hyphal cell surface adhesin, Hyr1. Like a ume6 mutant, strains lacking SAP6 exhibit enhanced colonization fitness, whereas SAP6-overexpression strains are attenuated in the gut. These results reveal a tradeoff between fungal programs supporting commensalism and virulence in which selection against hypha-specific markers limits the disease-causing potential of this ubiquitous commensal-pathogen

<i>H. utahensis</i> primary contig and ORC/Cdc6 genes.

<p>Circular display of the largest contig of the <i>H. utahensis</i> genome sequence. The contig begins at the top and wraps clockwise. The red bars illustrate the location of ORC/Cdc6 orthologs. The ORC/Cdc6 gene numbered 3 lies near the origin of replication, at 2,327,225 base pairs.</p

Comparison of putative glycoside hydrolase start sites.

<p>Examination of an individual gene displays tendencies of the annotation services. RAST identifies GTG as the start codon for the gene, while IMG and JCVI select two ATG codons at different locations. Predicted start codon affects gene length.</p

Venn diagrams of gene predictions.

<p>(A) The diagram to the left shows the number of predicted protein coding genes that share stop sites with the other annotations. Overlapping regions indicate genes having same stop site between annotations. (B) The diagram to the right shows the number of predicted protein coding genes that share start site and stop site with the other annotations. Overlapping regions indicate genes having exact matches between annotations.</p

Comparison of rRNA calls.

<p>Review of predicted coding regions for ribosomal RNA for each annotation service shows that IMG and RAST have identical calls, while JCVI fails to call 23s rRNA and predicts different start and stop sites.</p

Potential comparison tool.

<p>Hypothetical comparison of start sites from multiple annotations, combined with species-specific RBS data. Start #2 would be the most likely start codon based on RBS spacing.</p