Mor N. Lurie-Weinberger

Phylogenetic- and genome-derived insight into the evolution of N-glycosylation in Archaea

N-glycosylation, the covalent attachment of oligosaccharides to target protein Asn residues, is a post-translational modification that occurs in all three domains of life. In Archaea, the N-linked glycans that decorate experimentally characterized glycoproteins reveal a diversity in composition and content unequaled by their bacterial or eukaryal counterparts. At the same time, relatively little is known of archaeal N-glycosylation pathways outside of a handful of model strains. To gain insight into the distribution and evolutionary history of the archaeal version of this universal protein-processing event, 168 archaeal genome sequences were scanned for the presence of aglB, encoding the known archaeal oligosaccharyltransferase, an enzyme key to N-glycosylation. Such analysis predicts the presence of AglB in 166 species, with some species seemingly containing multiple versions of the protein. Phylogenetic analysis reveals that the events leading to aglB duplication occurred at various points during archaeal evolution. In many cases, aglB is found as part of a cluster of putative N-glycosylation genes. The presence, arrangement and nucleotide composition of genes in aglB-based clusters in five species of the halophilic archaeon Haloferax points to lateral gene transfer as contributing to the evolution of archaeal N-glycosylation.

By their genes ye shall know them: genomic signatures of predatory bacteria

Predatory bacteria are taxonomically disparate, exhibit diverse predatory strategies and are widely distributed in varied environments. To date, their predatory phenotypes cannot be discerned in genome sequence data thereby limiting our understanding of bacterial predation, and of its impact in nature. Here, we define the ‘predatome,’ that is, sets of protein families that reflect the phenotypes of predatory bacteria. The proteomes of all sequenced 11 predatory bacteria, including two de novo sequenced genomes, and 19 non-predatory bacteria from across the phylogenetic and ecological landscapes were compared. Protein families discriminating between the two groups were identified and quantified, demonstrating that differences in the proteomes of predatory and non-predatory bacteria are large and significant. This analysis allows predictions to be made, as we show by confirming from genome data an over-looked bacterial predator. The predatome exhibits deficiencies in riboflavin and amino acids biosynthesis, suggesting that predators obtain them from their prey. In contrast, these genomes are highly enriched in adhesins, proteases and particular metabolic proteins, used for binding to, processing and consuming prey, respectively. Strikingly, predators and non-predators differ in isoprenoid biosynthesis: predators use the mevalonate pathway, whereas non-predators, like almost all bacteria, use the DOXP pathway. By defining predatory signatures in bacterial genomes, the predatory potential they encode can be uncovered, filling an essential gap for measuring bacterial predation in nature. Moreover, we suggest that full-genome proteomic comparisons are applicable to other ecological interactions between microbes, and provide a convenient and rational tool for the functional classification of bacteria.

In and out: an analysis of epibiotic vs periplasmic bacterial predators

Bdellovibrio and like organisms (BALO) are obligate predators of Gram-negative bacteria, belonging to the α- and δ-proteobacteria. BALO prey using either a periplasmic or an epibiotic predatory strategy, but the genetic background underlying these phenotypes is not known. Here we compare the epibiotic Bdellovibrio exovorus and Micavibrio aeruginosavorus to the periplasmic B. bacteriovorus and Bacteriovorax marinus. Electron microscopy showed that M. aeruginosavorus, but not B. exovorus, can attach to prey cells in a non-polar manner through its longitudinal side. Both these predators were resistant to a surprisingly high number of antibiotic compounds, possibly via 26 and 19 antibiotic-resistance genes, respectively, most of them encoding efflux pumps. Comparative genomic analysis of all the BALOs revealed that epibiotic predators have a much smaller genome (ca. 2.5 Mbp) than the periplasmic predators (ca. 3.5 Mbp). Additionally, periplasmic predators have, on average, 888 more proteins, at least 60% more peptidases, and one more rRNA operon. Fifteen and 219 protein families were specific to the epibiotic and the periplasmic predators, respectively, the latter clearly forming the core of the periplasmic ‘predatome’, which is upregulated during the growth phase. Metabolic deficiencies of epibiotic genomes include the synthesis of inosine, riboflavin, vitamin B6 and the siderophore aerobactin. The phylogeny of the epibiotic predators suggests that they evolved by convergent evolution, with M. aeruginosavorus originating from a non-predatory ancestor while B. exovorus evolved from periplasmic predators by gene loss.

Archaea in and on the Human Body: Health Implications and Future Directions.

Although they are abundant and even dominant members of animal microbiomes (microbiotas), from sponges and termites to mice and cattle, archaea in our own microbiomes have received much less attention than their bacterial counterparts. The fact that human-associated archaea have been relatively little-studied may be at least partially attributed to the lack of any established archaeal human pathogens [1,2]. Clinically oriented microbiology courses often do not mention archaea at all, and most medical school and biology students are only aware of archaea as exotic extremophiles that have strange and eukaryotic-like molecular machinery. Since archaea have been known to be associated with the human gut for several decades, one would think that human microbiome studies may unravel new facets of archaea–human interactions. However, adequate universal primers that amplify both bacterial and archaeal small 16S rRNA genes but not any host rRNA genes were only published in mid-2011 [3], and thus, many studies chose to focus on bacteria alone rather than multiply effort and expense to cover taxa that are considered secondary in importance, if not altogether rare. Here, we provide a brief overview of what is currently known about archaea in and on the human body and their potential effects on human health (for additional reviews on archaea and their potential involvement in human disease, see [4–8]).

YMAP: a pipeline for visualization of copy number variation and loss of heterozygosity in eukaryotic pathogens.

The design of effective antimicrobial therapies for serious eukaryotic pathogens requires a clear understanding of their highly variable genomes. To facilitate analysis of copy number variations, single nucleotide polymorphisms and loss of heterozygosity events in these pathogens, we developed a pipeline for analyzing diverse genome-scale datasets from microarray, deep sequencing, and restriction site associated DNA sequence experiments for clinical and laboratory strains of Candida albicans, the most prevalent human fungal pathogen. The YMAP pipeline (http://lovelace.cs.umn.edu/Ymap/) automatically illustrates genome-wide information in a single intuitive figure and is readily modified for the analysis of other pathogens with small genomes.

Contribution of lateral gene transfer to the gene repertoire of a gut-adapted methanogen

Methanobrevibacter smithii is the most abundant archaeon in the human colon. As most of its neighbors are bacterial species, it is expected that lateral gene acquisition from bacteria might have contributed to the evolution and adaptation of this archaeon. We performed a tree-based genome-wide survey of putative lateral gene transfer products in M. smithii, using a phylogenetic pipeline. Over 15% of the coding genes of M. smithii are inferred to be bacterial in origin, based on this analysis. Laterally acquired genes have had the largest contribution to surface functions, and encode glycosyl-transferases and adhesin-like proteins. In addition, several important ABC transporters, especially metal transporters are of bacterial origin. Thus, bacterial genes contributed to the host-adaptation by allowing a larger variety of surface structures and increasing the efficiency of metal ion uptake in the competitive gut niche.

Heteroresistance to Fluconazole Is a Continuously Distributed Phenotype among Candida glabrata Clinical Strains Associated with In Vivo Persistence

ABSTRACT Candida glabrata causes persistent infections in patients treated with fluconazole and often acquires resistance following exposure to the drug. Here we found that clinical strains of C. glabrata exhibit cell-to-cell variation in drug response (heteroresistance). We used population analysis profiling (PAP) to assess fluconazole heteroresistance (FLCHR) and to ask if it is a binary trait or a continuous phenotype. Thirty (57.6%) of 52 fluconazole-sensitive clinical C. glabrata isolates met accepted dichotomous criteria for FLCHR. However, quantitative grading of FLCHR by using the area under the PAP curve (AUC) revealed a continuous distribution across a wide range of values, suggesting that all isolates exhibit some degree of heteroresistance. The AUC correlated with rhodamine 6G efflux and was associated with upregulation of the CDR1 and PDH1 genes, encoding ATP-binding cassette (ABC) transmembrane transporters, implying that HetR populations exhibit higher levels of drug efflux. Highly FLCHR C. glabrata was recovered more frequently than nonheteroresistant C. glabrata from hematogenously infected immunocompetent mice following treatment with high-dose fluconazole (45.8% versus 15%, P = 0.029). Phylogenetic analysis revealed some phenotypic clustering but also variations in FLCHR within clonal groups, suggesting both genetic and epigenetic determinants of heteroresistance. Collectively, these results establish heteroresistance to fluconazole as a graded phenotype associated with ABC transporter upregulation and fluconazole efflux. Heteroresistance may explain the propensity of C. glabrata for persistent infection and the emergence of breakthrough resistance to fluconazole. IMPORTANCE Heteroresistance refers to variability in the response to a drug within a clonal cell population. This phenomenon may have crucial importance for the way we look at antimicrobial resistance, as heteroresistant strains are not detected by standard laboratory susceptibility testing and may be associated with failure of antimicrobial therapy. We describe for the first time heteroresistance to fluconazole in C. glabrata, a finding that may explain the propensity of this pathogen to acquire resistance following exposure to fluconazole and to persist despite treatment. We found that, rather than being a binary all-or-none trait, heteroresistance was a continuously distributed phenotype associated with increased expression of genes that encode energy-dependent drug efflux transporters. Moreover, we show that heteroresistance is associated with failure of fluconazole to clear infection with C. glabrata. Together, these findings provide an empirical framework for determining and quantifying heteroresistance in C. glabrata. Heteroresistance refers to variability in the response to a drug within a clonal cell population. This phenomenon may have crucial importance for the way we look at antimicrobial resistance, as heteroresistant strains are not detected by standard laboratory susceptibility testing and may be associated with failure of antimicrobial therapy. We describe for the first time heteroresistance to fluconazole in C. glabrata, a finding that may explain the propensity of this pathogen to acquire resistance following exposure to fluconazole and to persist despite treatment. We found that, rather than being a binary all-or-none trait, heteroresistance was a continuously distributed phenotype associated with increased expression of genes that encode energy-dependent drug efflux transporters. Moreover, we show that heteroresistance is associated with failure of fluconazole to clear infection with C. glabrata. Together, these findings provide an empirical framework for determining and quantifying heteroresistance in C. glabrata.

Extensive Inter-Domain Lateral Gene Transfer in the Evolution of the Human Commensal Methanosphaera stadtmanae.

Methanosphaera stadtmanae is a commensal methanogenic archaeon found in the human gut. As most of its niche-neighbors are bacteria, it is expected that lateral gene transfer (LGT) from bacteria might have contributed to the evolutionary history of this organism. We performed a phylogenomic survey of putative LGT events in M. stadtmanae, using a phylogenetic pipeline. Our analysis indicates that a substantial fraction of the proteins of M. stadtmanae are inferred to have been involved in inter-domain LGT. Laterally acquired genes have had a large contribution to surface functions, by providing novel glycosyltransferase functions. In addition, several ABC transporters seem to be of bacterial origin, including the molybdate transporter. Thus, bacterial genes contributed to the adaptation of M. stadtmanae to a host-dependent lifestyle by allowing a larger variation in surface structures and increasing transport efficiency in the gut niche which is diverse and competitive.