Phytoplankton community structure is shaped by both bottom–up factors, such as nutrient availability, and top–down processes, such as predation. Here we show that marine viruses can blur these ...distinctions, being able to amend how host cells acquire nutrients from their environment while also predating and lysing their algal hosts. Viral genomes often encode genes derived from their host. These genes may allow the virus to manipulate host metabolism to improve viral fitness. We identify in the genome of a phytoplankton virus, which infects the small green alga Ostreococcus tauri, a host-derived ammonium transporter. This gene is transcribed during infection and when expressed in yeast mutants the viral protein is located to the plasma membrane and rescues growth when cultured with ammonium as the sole nitrogen source. We also show that viral infection alters the nature of nitrogen compound uptake of host cells, by both increasing substrate affinity and allowing the host to access diverse nitrogen sources. This is important because the availability of nitrogen often limits phytoplankton growth. Collectively, these data show that a virus can acquire genes encoding nutrient transporters from a host genome and that expression of the viral gene can alter the nutrient uptake behavior of host cells. These results have implications for understanding how viruses manipulate the physiology and ecology of phytoplankton, influence marine nutrient cycles, and act as vectors for horizontal gene transfer.
Candida species are important pathogens of humans and the fourth most commonly isolated pathogen from nosocomial blood stream infections. Although Candida albicans is the principle causative agent of ...invasive candidosis, the incidence of
infections has rapidly grown. The reason for this increase is not fully understood, but it is clear that the species has a higher innate tolerance to commonly administered azole antifungals, in addition to being highly tolerant to stresses especially oxidative stress. Taking the approach that using the model organism,
, with its intrinsic sensitivity to oxidative stress, we hypothesized that by expressing mediators of stress resistance from C. glabrata in
, it would result in induced resistance. To test this we transformed, en-masse, the
ORFeome library into
. This resulted in 1,500 stress resistant colonies and the recovered plasmids of 118 ORFs. Sequencing of these plasmids revealed a total of 16 different C. glabrata ORFs. The recovery of genes encoding known stress protectant proteins such as GPD1, GPD2 and TRX3 was predicted and validated the integrity of the screen. Through this screen we identified a C. glabrata unique ORF that confers oxidative stress resistance. We set to characterise this gene herein, examining expression in oxidative stress sensitive strains, comet assays to measure DNA damage and synthetic genetic array analysis to identify genetic interaction maps in the presence and absence of oxidative stress.
Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome ...libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, cloned into the Gateway® Entry Vector pDONR207, and sequenced, providing a total of 3022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171), in addition to genes residing at dispensable chromosomes (n= 122), or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway® Destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics, and offers unparalleled opportunities to understand the biology of Z. tritici.
Oomycete protists share phenotypic similarities with fungi, including the ability to cause plant diseases, but branch in a distant region of the tree of life. It has been suggested that multiple ...horizontal gene transfers (HGTs) from fungi-to-oomycetes contributed to the evolution of plant-pathogenic traits. These HGTs are predicted to include secreted proteins that degrade plant cell walls, a barrier to pathogen invasion and a rich source of carbohydrates. Using a combination of phylogenomics and functional assays, we investigate the diversification of a horizontally transferred xyloglucanase gene family in the model oomycete species Phytophthora sojae . Our analyses detect 11 xyloglucanase paralogs retained in P. sojae . Using heterologous expression in yeast, we show consistent evidence that eight of these paralogs have xyloglucanase function, including variants with distinct protein characteristics, such as a long-disordered C-terminal extension that can increase xyloglucanase activity. The functional variants analyzed subtend a phylogenetic node close to the fungi-to-oomycete transfer, suggesting the horizontally transferred gene was a bona fide xyloglucanase. Expression of three xyloglucanase paralogs in Nicotiana benthamiana triggers high-reactive oxygen species (ROS) generation, while others inhibit ROS responses to bacterial immunogens, demonstrating that the paralogs differentially stimulate pattern-triggered immunity. Mass spectrometry of detectable enzymatic products demonstrates that some paralogs catalyze the production of variant breakdown profiles, suggesting that secretion of variant xyloglucanases increases efficiency of xyloglucan breakdown as well as diversifying the damage-associated molecular patterns released. We suggest that this pattern of neofunctionalization and the variant host responses represent an aspect of the Red Queen host–pathogen coevolutionary dynamic.
The oomycetes are heterotrophic, filamentous protists that share morphological similarities with some fungi, but according to phylogenetic evidence, are actually relatives of diatoms and brown algae, ...within a different eukaryotic supergroup (Stramenopiles) (Förster et al., 1990; Leclerc et al., 2000; Hudspeth et al., 2000; Hudspeth et al., 2003; Thines et al., 2007; McCarthy and Fitzpatrick., 2017). Horizontal Gene Transfer (HGT) from fungi to oomycetes has been previously suggested to have played a role in the evolution of osmotrophic and phytopathogenic traits during divergence of the oomycete lineage; such transfers include secreted proteins predicted to degrade plant cell wall-specific substrates – these are the initial barriers to pathogen invasion as well as an abundant source of fixed carbon (Torto et al., 2002; Belbahri et al., 2008; Richards et al., 2011; Savory et al., 2015)The HGTs are largely expanded in hemibiotrophic oomycetes, and their selective benefit is suggested by subsequent gene duplications following acquisition (Richards et al., 2011; Savory et al., 2015) - giving rise to paralogs hypothesised to possess functional differences. Bioinformatics and computational analysis of laterally-transferred Glycoside Hydrolase 10 (GH10) and GH12 in Phytophthora sojae (oomycete hemibiotrophic parasite of soybean) discovered unique differences amongst paralogs – P. sojae_482953 (GH12 xyloglucanase) encodes a significantly disordered, 186 amino acid ‘tail’, which improves the enzymatic activity of the protein towards xyloglucan when heterologously-expressed in Saccharomyces cerevisiae. Interestingly, knockout of the gene encoding P. sojae_482953 in vivo did not affect the ability of P. sojae to utilise xyloglucan as a sole carbon source, indicating that gene duplication events are also an important mechanism for transcriptional fidelity and maintenance of function. A second GH12 paralog, P. sojae_559651 was predicted to encode a ‘second’ carbohydrate-binding site, with the prediction being conserved for orthologs encoded by P. cactorum and P. nicotiniae; interestingly, two indels (coding for alanine and serine) were identified as being important for the prediction, and their removal abolished the second binding site prediction for all three proteins. Functional diversification of variant paralogs postHGT was also suggested by differences in oligosaccharides released from xyloglucan breakdown by P. sojae_559651 and P. sojae_482953 proteins using mass spectrometry. Taken together, this work extends our understanding of the functional significance of gene duplication post-HGT in hemibiotrophic oomycetes.Of further interest is how N-terminal signal peptide sequences affect the transferability of secreted proteins by cross-phylum HGT – this work has optimised a functional agar plate screen (and demonstrated a promising microdroplet approach) in order to explore signal peptide evolution in future work.
Many microbes acquire metabolites in a “feeding” process where complex polymers are broken down in the environment to their subunits. The subsequent uptake of soluble metabolites by a cell, sometimes ...called osmotrophy, is facilitated by transporter proteins. As such, the diversification of osmotrophic microorganisms is closely tied to the diversification of transporter functions. Horizontal gene transfer (HGT) has been suggested to produce genetic variation that can lead to adaptation, allowing lineages to acquire traits and expand niche ranges. Transporter genes often encode single-gene phenotypes and tend to have low protein–protein interaction complexity and, as such, are potential candidates for HGT. Here we test the idea that HGT has underpinned the expansion of metabolic potential and substrate utilization via transfer of transporter-encoding genes. Using phylogenomics, we identify seven cases of transporter-gene HGT between fungal phyla, and investigate compatibility, localization, function, and fitness consequences when these genes are expressed in Saccharomyces cerevisiae. Using this approach, we demonstrate that the transporters identified can alter how fungi utilize a range of metabolites, including peptides, polyols, and sugars. We then show, for one model gene, that transporter gene acquisition by HGT can significantly alter the fitness landscape of S. cerevisiae. We therefore provide evidence that transporter HGT occurs between fungi, alters how fungi can acquire metabolites, and can drive gain in fitness. We propose a “transporter-gene acquisition ratchet,” where transporter repertoires are continually augmented by duplication, HGT, and differential loss, collectively acting to overwrite, fine-tune, and diversify the complement of transporters present in a genome.
Carbon Storage Regulator A (CsrA) is an RNA binding protein that acts as a global regulator of diverse genes. Using a combination of genetics and biochemistry we show that CsrA binds directly to the ...5' end of the transcript encoding AcrAB. Deletion of csrA or mutagenesis of the CsrA binding sites reduced production of both AcrA and AcrB. Nucleotide substitutions at the 5' UTR of acrA mRNA that could potentially weaken the inhibitory RNA secondary structure, allow for more efficient translation of the AcrAB proteins. Given the role of AcrAB-TolC in multi-drug efflux we suggest that CsrA is a potential drug target.
Abstract
The Gram-negative bacterium, Vibrio parahaemolyticus, is a major cause of seafood-derived food poisoning throughout the world. The pathogenicity of V. parahaemolyticus is attributed to ...several virulence factors, including two type III secretion systems (T3SS), T3SS1 and T3SS2. Herein, we compare the virulence of V. parahaemolyticusPOR strains, which harbor a mutation in the T3SS needle apparatus of either system, to V. parahaemolyticusCAB strains, which harbor mutations in positive transcriptional regulators of either system. These strains are derived from the clinical RIMD 2210633 strain. We demonstrate that each mutation affects the virulence of the bacterium in a different manner. POR and CAB strains exhibited similar levels of swarming motility and T3SS effector production and secretion, but the CAB3 and CAB4 strains, which harbor a mutation in the T3SS2 master regulator gene, formed reduced biofilm growth under T3SS2 inducing conditions. Additionally, while the cytotoxicity of the POR and CAB strains was similar, the CAB2 (T3SS1 regulatory mutant) strain was strikingly more invasive than the comparable POR2 (T3SS1 structural mutant) strain. In summary, creating structural or regulatory mutations in either T3SS1 or T3SS2 causes differential downstream effects on other virulence systems. Understanding the biological differences of strains created from a clinical isolate is critical for interpreting and understanding the pathogenic nature of V. parahaemolyticus.
We demonstrate that structural or regulatory mutations of V. parahaemolyticus type III secretion systems cause downstream effects on other virulence systems, which uncovers an interconnected relationship between these systems.
We demonstrate that structural or regulatory mutations of V. parahaemolyticus type III secretion systems cause downstream effects on other virulence systems, which uncovers an interconnected relationship between these systems.
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