Dragana Gagic

Methane yield phenotypes linked to differential gene expression in the sheep rumen microbiome

Ruminant livestock represent the single largest anthropogenic source of the potent greenhouse gas methane, which is generated by methanogenic archaea residing in ruminant digestive tracts. While differences between individual animals of the same breed in the amount of methane produced have been observed, the basis for this variation remains to be elucidated. To explore the mechanistic basis of this methane production, we measured methane yields from 22 sheep, which revealed that methane yields are a reproducible, quantitative trait. Deep metagenomic and metatranscriptomic sequencing demonstrated a similar abundance of methanogens and methanogenesis pathway genes in high and low methane emitters. However, transcription of methanogenesis pathway genes was substantially increased in sheep with high methane yields. These results identify a discrete set of rumen methanogens whose methanogenesis pathway transcription profiles correlate with methane yields and provide new targets for CH4 mitigation at the levels of microbiota composition and transcriptional regulation.

Identification of the gate regions in the primary structure of the secretin pIV.

Secretins are a family of large bacterial outer membrane channels that serve as exit ports for folded proteins, filamentous phage and surface structures. Despite the large size of their substrates, secretins do not compromise the barrier function of the outer membrane, implying a gating mechanism. The region in the primary structure that forms the putative gate has not previously been determined for any secretin. To identify residues involved in gating the pIV secretin of filamentous bacteriophage f1, we used random mutagenesis of the gene followed by positive selection for mutants with compromised barrier function (‘leaky’ mutants). We identified mutations in 34 residues, 30 of which were clustered into two regions located in the centre of the conserved C‐terminal secretin family domain: GATE1 (that spanned 39 residues) and GATE2 (that spanned 14 residues). An internal deletion constructed in the GATE2 region resulted in a severely leaky phenotype. Three of the four remaining mutations are located in the region that encodes the N‐terminal, periplasmic portion of pIV and could be involved in triggering gate opening. Two missense mutations in the 24‐residue region that separates GATE1 and GATE2 were also constructed. These mutant proteins were unstable, defective in multimerization and non‐functional.

An adhesin from hydrogen-utilizing rumen methanogen Methanobrevibacter ruminantium M1 binds a broad range of hydrogen-producing microorganisms.

Symbiotic associations are ubiquitous in the microbial world and have a major role in shaping the evolution of both partners. One of the most interesting mutualistic relationships exists between protozoa and methanogenic archaea in the fermentative forestomach (rumen) of ruminant animals. Methanogens reside within and on the surface of protozoa as symbionts, and interspecies hydrogen transfer is speculated to be the main driver for physical associations observed between the two groups. In silico analyses of several rumen methanogen genomes have previously shown that up to 5% of genes encode adhesin-like proteins, which may be central to rumen interspecies attachment. We hypothesized that adhesin-like proteins on methanogen cell surfaces facilitate attachment to protozoal hosts. Using phage display technology, we have identified a protein (Mru_1499) from Methanobrevibacter ruminantium M1 as an adhesin that binds to a broad range of rumen protozoa (including the genera Epidinium and Entodinium). This unique adhesin also binds the cell surface of the bacterium Butyrivibrio proteoclasticus, suggesting a broad adhesion spectrum for this protein.

Exploring the Secretomes of Microbes and Microbial Communities Using Filamentous Phage Display.

Microbial surface and secreted proteins (the secretome) contain a large number of proteins that interact with other microbes, host and/or environment. These proteins are exported by the coordinated activities of the protein secretion machinery present in the cell. A group of bacteriophage, called filamentous phage, have the ability to hijack bacterial protein secretion machinery in order to amplify and assemble via a secretion-like process. This ability has been harnessed in the use of filamentous phage of Escherichia coli in biotechnology applications, including screening large libraries of variants for binding to “bait” of interest, from tissues in vivo to pure proteins or even inorganic substrates. In this review we discuss the roles of secretome proteins in pathogenic and non-pathogenic bacteria and corresponding secretion pathways. We describe the basics of phage display technology and its variants applied to discovery of bacterial proteins that are implicated in colonization of host tissues and pathogenesis, as well as vaccine candidates through filamentous phage display library screening. Secretome selection aided by next-generation sequence analysis was successfully applied for selective display of the secretome at a microbial community scale, the latter revealing the richness of secretome functions of interest and surprising versatility in filamentous phage display of secretome proteins from large number of Gram-negative as well as Gram-positive bacteria and archaea.

Metasecretome-selective phage display approach for mining the functional potential of a rumen microbial community

BackgroundIn silico, secretome proteins can be predicted from completely sequenced genomes using various available algorithms that identify membrane-targeting sequences. For metasecretome (collection of surface, secreted and transmembrane proteins from environmental microbial communities) this approach is impractical, considering that the metasecretome open reading frames (ORFs) comprise only 10% to 30% of total metagenome, and are poorly represented in the dataset due to overall low coverage of metagenomic gene pool, even in large-scale projects.ResultsBy combining secretome-selective phage display and next-generation sequencing, we focused the sequence analysis of complex rumen microbial community on the metasecretome component of the metagenome. This approach achieved high enrichment (29 fold) of secreted fibrolytic enzymes from the plant-adherent microbial community of the bovine rumen. In particular, we identified hundreds of heretofore rare modules belonging to cellulosomes, cell-surface complexes specialised for recognition and degradation of the plant fibre.ConclusionsAs a method, metasecretome phage display combined with next-generation sequencing has a power to sample the diversity of low-abundance surface and secreted proteins that would otherwise require exceptionally large metagenomic sequencing projects. As a resource, metasecretome display library backed by the dataset obtained by next-generation sequencing is ready for i) affinity selection by standard phage display methodology and ii) easy purification of displayed proteins as part of the virion for individual functional analysis.

Unique secreted–surface protein complex of Lactobacillus rhamnosus, identified by phage display

Proteins are the most diverse structures on bacterial surfaces; hence, they are candidates for species‐ and strain‐specific interactions of bacteria with the host, environment, and other microorganisms. Genomics has decoded thousands of bacterial surface and secreted proteins, yet the function of most cannot be predicted because of the enormous variability and a lack of experimental data that would allow deduction of function through homology. Here, we used phage display to identify a pair of interacting extracellular proteins in the probiotic bacterium Lactobacillus rhamnosus HN001. A secreted protein, SpcA, containing two bacterial immunoglobulin‐like domains type 3 (Big‐3) and a domain distantly related to plant pathogen response domain 1 (PR‐1‐like) was identified by screening of an L. rhamnosus HN001 library using HN001 cells as bait. The SpcA‐“docking” protein, SpcB, was in turn detected by another phage display library screening, using purified SpcA as bait. SpcB is a 3275‐residue cell‐surface protein that contains general features of large glycosylated Serine‐rich adhesins/fibrils from gram‐positive bacteria, including the hallmark signal sequence motif KxYKxGKxW. Both proteins are encoded by genes within a L. rhamnosus‐unique gene cluster that distinguishes this species from other lactobacilli. To our knowledge, this is the first example of a secreted‐docking protein pair identified in lactobacilli.

Improving the genetic representation of rare taxa within complex microbial communities using DNA normalization methods

Complex microbial communities typically contain a large number of low abundance species, which collectively, comprise a considerable proportion of the community. This ‘rare biosphere’ has been speculated to contain keystone species and act as a repository of genomic diversity to facilitate community adaptation. Many environmental microbes are currently resistant to cultivation, and can only be accessed via culture‐independent approaches. To enhance our understanding of the role of the rare biosphere, we aimed to improve their metagenomic representation using DNA normalization methods, and assess normalization success via shotgun DNA sequencing. A synthetic metagenome was constructed from the genomic DNA of five bacterial species, pooled in a defined ratio spanning three orders of magnitude. The synthetic metagenome was fractionated and thermally renatured, allowing the most abundant sequences to hybridize. Double‐stranded DNA was removed either by hydroxyapatite chromatography, or by a duplex‐specific nuclease (DSN). The chromatographic method failed to enrich for the genomes present in low starting abundance, whereas the DSN method resulted in all genomes reaching near equimolar abundance. The representation of the rarest member was increased by approximately 450‐fold. De novo assembly of the normalized metagenome enabled up to 18.0% of genes from the rarest organism to be assembled, in contrast to the un‐normalized sample, where genes were not able to be assembled at the same sequencing depth. This study has demonstrated that the application of normalization methods to metagenomic samples is a powerful tool to enrich for sequences from rare taxa, which will shed further light on their ecological niches.

Metasecretome Phage Display

Metasecretome is a collection of cell-surface and secreted proteins that mediate interactions between microbial communities and their environment. These include adhesins, enzymes, surface structures such as pili or flagella, vaccine targets or proteins responsible for immune evasion. Traditional approaches to exploring matasecretome of complex microbial communities via cultivation of microorganisms and screening of individual strains fail to sample extraordinary diversity in these communities, since only a limited fraction of microorganisms are represented by cultures. Advances in culture-independent sequence analysis methods, collectively referred to as metagenomics, offer an alternative approach that enables the direct analysis of collective microbial genomes (metagenome) recovered from environmental samples. This protocol describes a method, metasecretome phage display, which selectively displays the metasecretome portion of the metagenome. The metasecretome library can then be used for two purposes: (1) to sequence the entire metasecretome (using PacBio technology); (2) to identify metasecretome proteins that have a specific function of interest by affinity-screening (bio-panning) using a variety of methods described in other chapters of this volume.