The influence of microbial colonization on inflammatory versus pro-healing trajectories in combat extremity wounds.

A combination of improved body armor, medical transportation, and treatment has led to the increased survival of warfighters from combat extremity injuries predominantly caused by blasts in modern conflicts. Despite advances, a high rate of complications such as wound infections, wound failure, amputations, and a decreased quality of life exist. To study the molecular underpinnings of wound failure, wound tissue biopsies from combat extremity injuries had RNA extracted and sequenced. Wounds were classified by colonization (colonized vs. non-colonized) and outcome (healed vs. failed) status. Differences in gene expression were investigated between timepoints at a gene level, and longitudinally by multi-gene networks, inferred proportions of immune cells, and expression of healing-related functions. Differences between wound outcomes in colonized wounds were more apparent than in non-colonized wounds. Colonized/healed wounds appeared able to mount an adaptive immune response to infection and progress beyond the inflammatory stage of healing, while colonized/failed wounds did not. Although, both colonized and non-colonized failed wounds showed increasing inferred immune and inflammatory programs, non-colonized/failed wounds progressed beyond the inflammatory stage, suggesting different mechanisms of failure dependent on colonization status. Overall, these data reveal gene expression profile differences in healing wounds that may be utilized to improve clinical treatment paradigms.

Kratosvirus quantuckense: the history and novelty of an algal bloom disrupting virus and a model for giant virus research.

Since the discovery of the first "giant virus," particular attention has been paid toward isolating and culturing these large DNA viruses through Acanthamoeba spp. bait systems. While this method has allowed for the discovery of plenty novel viruses in the Nucleocytoviricota, environmental -omics-based analyses have shown that there is a wealth of diversity among this phylum, particularly in marine datasets. The prevalence of these viruses in metatranscriptomes points toward their ecological importance in nutrient turnover in our oceans and as such, in depth study into non-amoebal Nucleocytoviricota should be considered a focal point in viral ecology. In this review, we report on Kratosvirus quantuckense (née Aureococcus anophagefferens Virus), an algae-infecting virus of the Imitervirales. Current systems for study in the Nucleocytoviricota differ significantly from this virus and its relatives, and a litany of trade-offs within physiology, coding potential, and ecology compared to these other viruses reveal the importance of K. quantuckense. Herein, we review the research that has been performed on this virus as well as its potential as a model system for algal-virus interactions.

Systemic inflammation induced from remote extremity trauma is a critical driver of secondary brain injury.

Blast exposure, commonly experienced by military personnel, can cause devastating life-threatening polysystem trauma. Despite considerable research efforts, the impact of the systemic inflammatory response after major trauma on secondary brain injury-inflammation is largely unknown. The aim of this study was to identify markers underlying the susceptibility and early onset of neuroinflammation in three rat trauma models: (1) blast overpressure exposure (BOP), (2) complex extremity trauma (CET) involving femur fracture, crush injury, tourniquet-induced ischemia, and transfemoral amputation through the fracture site, and (3) BOP+CET. Six hours post-injury, intact brains were harvested and dissected to obtain biopsies from the prefrontal cortex, striatum, neocortex, hippocampus, amygdala, thalamus, hypothalamus, and cerebellum. Custom low-density microarray datasets were used to identify, interpret and visualize genes significant (p < 0.05 for differential expression [DEGs]; 86 neuroinflammation-associated) using a custom python-based computer program, principal component analysis, heatmaps and volcano plots. Gene set and pathway enrichment analyses of the DEGs was performed using R and STRING for protein-protein interaction (PPI) to identify and explore key genes and signaling networks. Transcript profiles were similar across all regions in naïve brains with similar expression levels involving neurotransmission and transcription functions and undetectable to low-levels of inflammation-related mediators. Trauma-induced neuroinflammation across all anatomical brain regions correlated with injury severity (BOP+CET > CET > BOP). The most pronounced differences in neuroinflammatory-neurodegenerative gene regulation were between blast-associated trauma (BOP, BOP+CET) and CET. Following BOP, there were few DEGs detected amongst all 8 brain regions, most were related to cytokines/chemokines and chemokine receptors, where PPI analysis revealed Il1b as a potential central hub gene. In contrast, CET led to a more excessive and diverse pro-neuroinflammatory reaction in which Il6 was identified as the central hub gene. Analysis of the of the BOP+CET dataset, revealed a more global heightened response (Cxcr2, Il1b, and Il6) as well as the expression of additional functional regulatory networks/hub genes (Ccl2, Ccl3, and Ccl4) which are known to play a critical role in the rapid recruitment and activation of immune cells via chemokine/cytokine signaling. These findings provide a foundation for discerning pathophysiological consequences of acute extremity injury and systemic inflammation following various forms of trauma in the brain.

Draft Genome Sequence of the Freshwater Diatom Fragilaria crotonensis SAG 28.96.

Here, we report the assembled and annotated genome of the freshwater diatom Fragilaria crotonensis SAG 28.96. The 61.85-Mb nuclear genome was assembled into 879 contigs, has a GC content of 47.40%, contains 26,015 predicted genes, and shows completeness of 81%.

Metagenome-Assembled Genome of a Pseudanabaena sp. from a Crimson Cyanobacterial Bloom in Lake Salubria, New York, USA.

Pseudanabaena spp. are filamentous cyanobacteria widely distributed in temperate lakes. Though infrequent, they can form harmful algal blooms. Here, we present a high-quality metagenome-assembled genome of a Pseudanabaena sp. from a toxic, crimson cyanobacterial bloom in Lake Salubria, NY.

Closed, Circular Genome Sequence of Aureococcus anophagefferens Virus, a Lytic Virus of a Brown Tide-Forming Alga.

Here, we report the genomic sequence of Aureococcus anophagefferens virus, assembled into one circular contig from both Nanopore and Illumina reads. The genome is 381,717 bp long with a GC content of 29.1%, which includes an additional 5-kb region between the previously predicted polar ends of the reference genome.

Plasmid-Mediated Stabilization of Prophages.

Mobile genetic elements (MGEs) drive bacterial evolution, alter gene availability within microbial communities, and facilitate adaptation to ecological niches. In natural systems, bacteria simultaneously possess or encounter multiple MGEs, yet their combined influences on microbial communities are poorly understood. Here, we investigate interactions among MGEs in the marine bacterium Sulfitobacter pontiacus. Two related strains, CB-D and CB-A, each harbor a single prophage. These prophages share high sequence identity with one another and an integration site within the host genome, yet these strains exhibit differences in "spontaneous" prophage induction (SPI) and consequent fitness. To better understand mechanisms underlying variation in SPI between these lysogens, we closed their genomes, which revealed that in addition to harboring different prophage genotypes, CB-A lacks two of the four large, low-copy-number plasmids possessed by CB-D. To assess the relative roles of plasmid content versus prophage genotype on host physiology, a panel of derivative strains varying in MGE content were generated. Characterization of these derivatives revealed a robust link between plasmid content and SPI, regardless of prophage genotype. Strains possessing all four plasmids had undetectable phage in cell-free lysates, while strains lacking either one plasmid (pSpoCB-1) or a combination of two plasmids (pSpoCB-2 and pSpoCB-4) produced high (>105 PFU/mL) phage titers. Homologous plasmid sequences were identified in related bacteria, and plasmid and phage genes were found to be widespread in Tara Oceans metagenomic data sets. This suggests that plasmid-dependent stabilization of prophages may be commonplace throughout the oceans. IMPORTANCE The consequences of prophage induction on the physiology of microbial populations are varied and include enhanced biofilm formation, conferral of virulence, and increased opportunity for horizontal gene transfer. These traits lead to competitive advantages for lysogenized bacteria and influence bacterial lifestyles in a variety of niches. However, biological controls of "spontaneous" prophage induction, the initiation of phage replication and phage-mediated cell lysis without an overt stressor, are not well understood. In this study, we observed a novel interaction between plasmids and prophages in the marine bacterium Sulfitobacter pontiacus. We found that loss of one or more distinct plasmids-which we show carry genes ubiquitous in the world's oceans-resulted in a marked increase in prophage induction within lysogenized strains. These results demonstrate cross talk between different mobile genetic elements and have implications for our understanding of the lysogenic-lytic switches of prophages found not only in marine environments, but throughout all ecosystems.

Aureococcus anophagefferens (Pelagophyceae) genomes improve evaluation of nutrient acquisition strategies involved in brown tide dynamics.

The pelagophyte Aureococcus anophagefferens causes harmful brown tide blooms in marine embayments on three continents. Aureococcus anophagefferens was the first harmful algal bloom species to have its genome sequenced, an advance that evidenced genes important for adaptation to environmental conditions that prevail during brown tides. To expand the genomic tools available for this species, genomes for four strains were assembled, including three newly sequenced strains and one assembled from publicly available data. These genomes ranged from 57.11 to 73.62 Mb, encoding 13,191-17,404 potential proteins. All strains shared ~90% of their encoded proteins as determined by homology searches and shared most functional orthologs as determined by KEGG, although each strain also possessed coding sequences with unique functions. Like the original reference genome, the genomes assembled in this study possessed genes hypothesized to be important in bloom proliferation, including genes involved in organic compound metabolism and growth at low light. Cross-strain informatics and culture experiments suggest that the utilization of purines is a potentially important source of organic nitrogen for brown tides. Analyses of metatranscriptomes from a brown tide event demonstrated that use of a single genome yielded a lower read mapping percentage (~30% of library reads) as compared to a database generated from all available genomes (~43%), suggesting novel information about bloom ecology can be gained from expanding genomic space. This work demonstrates the continued need to sequence ecologically relevant algae to understand the genomic potential and their ecology in the environment.

Metatranscriptome Library Preparation Influences Analyses of Viral Community Activity During a Brown Tide Bloom.

There is growing interest in the use of metatranscriptomics to study virus community dynamics. We used RNA samples collected from harmful brown tides caused by the eukaryotic alga Aureococcus anophagefferens within New York (United States) estuaries and in the process observed how preprocessing of libraries by either selection for polyadenylation or reduction in ribosomal RNA (rRNA) influenced virus community analyses. As expected, more reads mapped to the A. anophagefferens genome in polyadenylation-selected libraries compared to the rRNA-reduced libraries, with reads mapped in each sample correlating to one another regardless of preprocessing of libraries. Yet, this trend was not seen for reads mapping to the Aureococcus anophagefferens Virus (AaV), where significantly more reads (approximately two orders of magnitude) were mapped to the AaV genome in the rRNA-reduced libraries. In the rRNA-reduced libraries, there was a strong and significant correlation between reads mappings to AaV and A. anophagefferens. Overall, polyadenylation-selected libraries produced fewer viral contigs, fewer reads mapped to viral contigs, and different proportions across viral realms and families, compared to their rRNA-reduced pairs. This study provides evidence that libraries generated by rRNA reduction and not selected for polyadenylation are more appropriate for quantitative characterization of viral communities in aquatic ecosystems by metatranscriptomics.

Elevated pH Conditions Associated With Microcystis spp. Blooms Decrease Viability of the Cultured Diatom Fragilaria crotonensis and Natural Diatoms in Lake Erie.

Cyanobacterial Harmful Algal Blooms (CyanoHABs) commonly increase water column pH to alkaline levels ≥9.2, and to as high as 11. This elevated pH has been suggested to confer a competitive advantage to cyanobacteria such as Microcystis aeruginosa. Yet, there is limited information regarding the restrictive effects bloom-induced pH levels may impose on this cyanobacterium's competitors. Due to the pH-dependency of biosilicification processes, diatoms (which seasonally both precede and proceed Microcystis blooms in many fresh waters) may be unable to synthesize frustules at these pH levels. We assessed the effects of pH on the ecologically relevant diatom Fragilaria crotonensis in vitro, and on a Lake Erie diatom community in situ. In vitro assays revealed F. crotonensis monocultures exhibited lower growth rates and abundances when cultivated at a starting pH of 9.2 in comparison to pH 7.7. The suppressed growth trends in F. crotonensis were exacerbated when co-cultured with M. aeruginosa at pH conditions and cell densities that simulated a cyanobacteria bloom. Estimates demonstrated a significant decrease in silica (Si) deposition at alkaline pH in both in vitro F. crotonensis cultures and in situ Lake Erie diatom assemblages, after as little as 48 h of alkaline pH-exposure. These observations indicate elevated pH negatively affected growth rate and diatom silica deposition; in total providing a competitive disadvantage for diatoms. Our observations demonstrate pH likely plays a significant role in bloom succession, creating a potential to prolong summer Microcystis blooms and constrain diatom fall resurgence.

A comparative study of metatranscriptomic assessment methods to characterize Microcystis blooms.

Harmful algal blooms are increasing in duration and severity globally, resulting in increased research interest. The use of genetic sequencing technologies has provided a wealth of opportunity to advance knowledge, but also poses a risk to that knowledge if handled incorrectly. The vast numbers of sequence processing tools and protocols provide a method to test nearly every hypothesis, but each method has inherent strengths and weaknesses. Here, we tested six methods to classify and quantify metatranscriptomic activity from a harmful algal bloom dominated by Microcystis spp. Three online tools were evaluated (Kaiju, MG-RAST, and GhostKOALA) in addition to three local tools that included a command line BLASTx approach, recruitment of reads to individual Microcystis genomes, and recruitment to a combined Microcystis composite genome generated from sequenced isolates with complete, closed genomes. Based on the analysis of each tool presented in this study, two recommendations are made that are dependent on the hypothesis to be tested. For researchers only interested in the function and physiology of Microcystis spp., read recruitments to the composite genome, referred to as "Frankenstein's Microcystis", provided the highest total estimates of transcript expression. However, for researchers interested in the entire bloom microbiome, the online GhostKOALA annotation tool, followed by subsequent read recruitments, provided functional and taxonomic characterization, in addition to transcript expression estimates. This study highlights the critical need for careful evaluation of methods before data analysis.

Episodic Decrease in Temperature Increases mcy Gene Transcription and Cellular Microcystin in Continuous Cultures of Microcystis aeruginosa PCC 7806.

Microcystins produced during harmful cyanobacterial blooms are a public health concern. Although patterns are emerging, the environmental cues that stimulate production of microcystin remain confusing, hindering our ability to predict fluctuations in bloom toxicity. In earlier work, growth at cool temperatures relative to optimum (18°C vs. 26°C) was confirmed to increase microcystin quota in batch cultures of Microcystis aeruginosa NIES-843. Here, we tested this response in M. aeruginosa PCC 7806 using continuous cultures to examine temporal dynamics and using RNA-sequencing to investigate the physiological nature of the response. A temperature reduction from 26 to 19°C increased microcystin quota ∼2-fold, from an average of ∼464 ag µm-3 cell volume to ∼891 ag µm-3 over a 7-9 d period. Reverting the temperature to 26°C returned the cellular microcystin quota to ∼489 ag µm-3. Long periods (31-42 d) at 19°C did not increase or decrease microcystin quota beyond that observed at 7-9 d. Nitrogen concentration had little effect on the overall response. RNA sequencing indicated that the decrease in temperature to 19°C induced a classic cold-stress response in M. aeruginosa PCC 7806, but this operated on a different timescale than the increased microcystin production. Microcystin quota showed a strong 48- to 72-h time-lag correlation to mcy gene expression, but no correlation to concurrent mcy expression. This work confirms an effect of temperature on microcystin quota and extends our understanding of the physiological nature of the response.

Structural and Proteomic Studies of the Aureococcus anophagefferens Virus Demonstrate a Global Distribution of Virus-Encoded Carbohydrate Processing.

Viruses modulate the function(s) of environmentally relevant microbial populations, yet considerations of the metabolic capabilities of individual virus particles themselves are rare. We used shotgun proteomics to quantitatively identify 43 virus-encoded proteins packaged within purified Aureococcus anophagefferens Virus (AaV) particles, normalizing data to the per-virion level using a 9.5-Å-resolution molecular reconstruction of the 1900-Å (AaV) particle that we generated with cryogenic electron microscopy. This packaged proteome was used to determine similarities and differences between members of different giant virus families. We noted that proteins involved in sugar degradation and binding (e.g., carbohydrate lyases) were unique to AaV among characterized giant viruses. To determine the extent to which this virally encoded metabolic capability was ecologically relevant, we examined the TARA Oceans dataset and identified genes and transcripts of viral origin. Our analyses demonstrated that putative giant virus carbohydrate lyases represented up to 17% of the marine pool for this function. In total, our observations suggest that the AaV particle has potential prepackaged metabolic capabilities and that these may be found in other giant viruses that are widespread and abundant in global oceans.

SMRT Sequencing of Paramecium Bursaria Chlorella Virus-1 Reveals Diverse Methylation Stability in Adenines Targeted by Restriction Modification Systems.

Chloroviruses (family Phycodnaviridae) infect eukaryotic, freshwater, unicellular green algae. A unique feature of these viruses is an abundance of DNA methyltransferases, with isolates dedicating up to 4.5% of their protein coding potential to these genes. This diversity highlights just one of the long-standing values of the chlorovirus model system; where group-wide epigenomic characterization might begin to elucidate the function(s) of DNA methylation in large dsDNA viruses. We characterized DNA modifications in the prototype chlorovirus, PBCV-1, using single-molecule real time (SMRT) sequencing (aka PacBio). Results were compared to total available sites predicted in silico based on DNA sequence alone. SMRT-software detected N6-methyl-adenine (m6A) at GATC and CATG recognition sites, motifs previously shown to be targeted by PBCV-1 DNA methyltransferases M.CviAI and M. CviAII, respectively. At the same time, PacBio analyses indicated that 10.9% of the PBCV-1 genome had large interpulse duration ratio (ipdRatio) values, the primary metric for DNA modification identification. These events represent 20.6x more sites than can be accounted for by all available adenines in GATC and CATG motifs, suggesting base or backbone modifications other than methylation might be present. To define methylation stability, we cross-compared methylation status of each GATC and CATG sequence in three biological replicates and found ∼81% of sites were stably methylated, while ∼2% consistently lack methylation. The remaining 17% of sites were stochastically methylated. When methylation status was analyzed for both strands of each target, we show that palindromes existed in completely non-methylated states, fully-methylated states, or hemi-methylated states, though GATC sites more often lack methylation than CATG sequences. Given that both sequences are targeted by not just methyltransferases, but by restriction endonucleases that are together encoded by PBCV-1 as virus-originating restriction modification (RM) systems, there is strong selective pressure to modify all target sites. The finding that most instances of non-methylation are associated with hemi-methylation is congruent with observations that hemi-methylated palindromes are resistant to cleavage by restriction endonucleases. However, sites where hemi-methylation is conserved might represent a unique regulatory function for PBCV-1. This study serves as a baseline for future investigation into the epigenomics of chloroviruses and their giant virus relatives.

Internal Nitrogen Pools Shape the Infection of Aureococcus anophagefferens CCMP 1984 by a Giant Virus.

The pelagophyte Aureococcus anophagefferens blooms annually in shallow bays around the world, where it is hypothesized to outcompete other phytoplankton in part by using alternative nitrogen sources. The high proportion of natural populations that are infected during the late stages of the bloom suggest viruses cause bloom collapse. We hypothesized that the Aureococcus anophagefferens Virus (AaV) infection cycle would be negatively influenced in cultures acclimated to decreasing external nitrogen conditions, but that the real-time external nitrogen concentration would not influence the infection cycle. Cultures acclimated in NO 3 - concentrations (0.0147 mM; N:P = 0.1225) that showed reduced end point cell abundances, forward scatter (a proxy for size) and red fluorescence (a proxy for chlorophyll a), also produced fewer viruses per cell at a slower rate. Decreasing the external concentration of nitrogen post infection did not alter burst size or time to lysis. These data suggest that the nitrogen used for new viral progeny is present within host cells at the time of infection. Flow cytometric data of an infection cycle showed a reduction in red fluorescence around twelve hours post infection, consistent with degradation of nitrogen-rich chloroplasts during the infection cycle. Using cell and virus quota estimates, we determined that A. anophagefferens cells had sufficient nitrogen and carbon for the lower ranges of burst sizes determined but did not contain enough phosphorous. Consistent with this observation, expression of nitrate and sugar transporters did not increase in the publicly available transcriptome data of the infection cycle, while several phosphorus transporters were. Our data demonstrate that dynamics of viruses infecting Aureococcus over the course of a bloom is dictated by the host cell state upon infection, which is set a priori by external nutrient supplies.

The "Neglected Viruses" of Taihu: Abundant Transcripts for Viruses Infecting Eukaryotes and Their Potential Role in Phytoplankton Succession.

Drivers of algal bloom dynamics remain poorly understood, but viruses have been implicated as important players. Research addressing bloom dynamics has generally been restricted to the virus-infection of the numerically dominant (i.e. bloom forming) taxa. Yet this approach neglects a broad diversity of viral groups, limiting our knowledge of viral interactions and constraints within these systems. We examined hallmark virus marker genes in metatranscriptomic libraries from a seasonal and spatial survey of a Microcystis aeruginosa bloom in Lake Tai (Taihu) China to identify active infections by nucleocytoplasmic large DNA viruses (NCLDVs), RNA viruses, ssDNA viruses, bacteriophage, and virophage. Phylogenetic analyses revealed a diverse virus population with seasonal and spatial variability. We observed disproportionately high expression of markers associated with NCLDVs and ssRNA viruses (consistent with viruses that infect photosynthetic protists) relative to bacteriophage infecting heterotrophic bacteria or cyanobacteria during the height of the Microcystis bloom event. Under a modified kill-the-winner scheme, we hypothesize viruses infecting protists help suppress the photosynthetic eukaryotic community and allow for the proliferation of cyanobacteria such as Microcystis. Our observations provide a foundation for a little considered factor promoting algal blooms.

Influence of light on the infection of Aureococcus anophagefferens CCMP 1984 by a "giant virus".

The pelagophyte Aureococcus anophagefferens has caused recurrent brown tide blooms along the northeast coast of the United States since the mid-1980's, and more recently spread to other regions of the globe. These blooms, due to the high cell densities, are associated with severe light attenuation that destroys the sea grass beds which provide the basis for many fisheries. Data collected by transmission electron microscopy, PCR, and metatranscriptomic studies of the blooms, support the hypothesis that large dsDNA viruses play a role in bloom dynamics. While a large (~140 nm) icosahedral virus, with a 371 kbp genome, was first isolated more than a decade ago, the constraints imposed by environmental parameters on bloom infection dynamics by Aureococcus anophagefferens Virus, (AaV) remain unknown. To investigate the role light plays in infection by this virus, we acclimated A. anophagefferens to light intensities of 30 (low), 60 (medium) or 90 µmol photons m-2 s-1 (high) and infected cultures at these irradiance levels. Moreover, we completed light shift experiments where acclimated cultures were exposed to even lower light intensities (0, 5, and 15 µmol photons m-2 s-1) consistent with irradiance found during the peak of the bloom when cell concentrations are highest. The abundance of viruses produced per lytic event (burst size) was lower in the low irradiance acclimated cultures compared to the medium and high acclimated cultures. Transferring infected cultures to more-limiting light availabilities further decreased burst size and increased the length of time it took for cultures to lyse, regardless of acclimation irradiance level. A hypothetical mechanism for the reduced efficiency of the infection cycle in low light due to ribosome biogenesis was predicted from pre-existing transcriptomes. Overall, these studies provide a framework for understanding light effects on infection dynamics over the course of the summer months when A. anophagefferens blooms occur.

Pathways That Synthesize Phosphatidylethanolamine Impact Candida albicans Hyphal Length and Cell Wall Composition through Transcriptional and Posttranscriptional Mechanisms.

Candida albicans is a leading cause of systemic bloodstream infections, and synthesis of the phospholipid phosphatidylethanolamine (PE) is required for virulence. The psd1Δ/Δ psd2Δ/Δ mutant, which cannot synthesize PE by the cytidine diphosphate diacylglycerol (CDP-DAG) pathway, is avirulent in the mouse model of systemic candidiasis. Similarly, an ept1Δ/Δ mutant, which cannot produce PE by the Kennedy pathway, exhibits decreased kidney fungal burden in systemically infected mice. Conversely, overexpression of EPT1 results in a hypervirulent phenotype in this model. Thus, mutations that increase PE synthesis increase virulence, and mutations that decrease PE synthesis decrease virulence. However, the mechanism by which virulence is regulated by PE synthesis is only partially understood. RNA sequencing was performed on strains with deficient or excessive PE biosynthesis to elucidate the mechanism. Decreased PE synthesis from loss of EPT1 or PSD1 and PSD2 leads to downregulation of genes that impact mitochondrial function. Losses of PSD1 and PSD2, but not EPT1, cause significant increases in transcription of glycosylation genes, which may reflect the substantial cell wall defects in the psd1Δ/Δ psd2Δ/Δ mutant. These accumulated defects could contribute to the decreased virulence observed for mutants with deficient PE synthesis. In contrast to mutants with decreased PE synthesis, there were no transcriptional differences between the EPT1 overexpression strain and the wild type, indicating that the hypervirulent phenotype is a consequence of posttranscriptional changes. It was found that overexpression of EPT1 causes increased chitin content and increased hyphal length. These phenotypes may help to explain the previously observed hypervirulence in the EPT1 overexpressor.

Bacillus pumilus B12 Degrades Polylactic Acid and Degradation Is Affected by Changing Nutrient Conditions.

Poly-lactic acid (PLA) is increasingly used as a biodegradable alternative to traditional petroleum-based plastics. In this study, we identify a novel agricultural soil isolate of Bacillus pumilus (B12) that is capable of degrading high molecular weight PLA films. This degradation can be detected on a short timescale, with significant degradation detected within 48-h by the release of L-lactate monomers, allowing for a rapid identification ideal for experimental variation. The validity of using L-lactate as a proxy for degradation of PLA films is corroborated by loss of rigidity and appearance of fractures in PLA films, as measured by atomic force microscopy and scanning electron microscopy (SEM), respectively. Furthermore, we have observed a dose-dependent decrease in PLA degradation in response to an amino acid/nucleotide supplement mix that is driven mainly by the nucleotide base adenine. In addition, amendments of the media with specific carbon sources increase the rate of PLA degradation, while phosphate and potassium additions decrease the rate of PLA degradation by B. pumilus B12. These results suggest B. pumilus B12 is adapting its enzymatic expression based on environmental conditions and that these conditions can be used to study the regulation of this process. Together, this work lays a foundation for studying the bacterial degradation of biodegradable plastics.

Lrg1 Regulates ß (1,3)-Glucan Masking in Candida albicans through the Cek1 MAP Kinase Pathway.

Candida albicans is among the most prevalent opportunistic human fungal pathogens. The ability to mask the immunogenic polysaccharide ß (1,3)-glucan from immune detection via a layer of mannosylated proteins is a key virulence factor of C. albicans We previously reported that hyperactivation of the Cek1 mitogen-activated protein (MAP) kinase pathway promotes ß (1,3)-glucan exposure. In this communication, we report a novel upstream regulator of Cek1 activation and characterize the impact of Cek1 activity on fungal virulence. Lrg1 encodes a GTPase-activating protein (GAP) that has been suggested to inhibit the GTPase Rho1. We found that disruption of LRG1 causes Cek1 hyperactivation and ß (1,3)-glucan unmasking. However, when GTPase activation was measured for a panel of GTPases, the lrg1ΔΔ mutant exhibited increased activation of Cdc42 and Ras1 but not Rho1 or Rac1. Unmasking and Cek1 activation in the lrg1ΔΔ mutant can be blocked by inhibition of the Ste11 MAP kinase kinase kinase (MAPKKK), indicating that the lrg1ΔΔ mutant acts through the canonical Cek1 MAP kinase cascade. In order to determine how Cek1 hyperactivation specifically impacts virulence, a doxycycline-repressible hyperactive STE11ΔN467 allele was expressed in C. albicans In the absence of doxycycline, this allele overexpressed STE11ΔN467 , which induced production of proinflammatory tumor necrosis factor alpha (TNF-α) from murine macrophages. This in vitro phenotype correlates with decreased colonization and virulence in a mouse model of systemic infection. The mechanism by which Ste11ΔN467 causes unmasking was explored with RNA sequencing (RNA-Seq) analysis. Overexpression of Ste11ΔN467 caused upregulation of the Cph1 transcription factor and of a group of cell wall-modifying proteins which are predicted to impact cell wall architecture.IMPORTANCECandida albicans is an important source of systemic infections in humans. The ability to mask the immunogenic cell wall polymer ß (1,3)-glucan from host immune surveillance contributes to fungal virulence. We previously reported that the hyperactivation of the Cek1 MAP kinase cascade promotes cell wall unmasking, thus increasing strain immunogenicity. In this study, we identified a novel regulator of the Cek1 pathway called Lrg1. Lrg1 is a predicted GTPase-activating protein (GAP) that represses Cek1 activity by downregulating the GTPase Cdc42 and its downstream MAPKKK, Ste11. Upregulation of Cek1 activity diminished fungal virulence in the mouse model of infection, and this correlates with increased cytokine responses from macrophages. We also analyzed the transcriptional profile determined during ß (1,3)-glucan exposure driven by Cek1 hyperactivation. Our report provides a model where Cek1 hyperactivation causes ß (1,3)-glucan exposure by upregulation of cell wall proteins and leads to more robust immune detection in vivo, promoting more effective clearance.