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bioRxiv2018

Genetics behind the biosynthesis of nonulosonic acid containing lipooligosaccharides in Campylobacter coli

Campylobacter jejuni and Campylobacter coli are the most common cause of bacterial gastroenteritis in the world. Ganglioside mimicry by C. jejuni lipooligosaccharide (LOS) is the triggering factor of Guillain-Barre syndrome (GBS), an acute polyneuropathy. Sialyltransferases from the glycosyltransferase (GT) family 42 are essential for the expression of ganglioside mimics in C. jejuni. Recently, two novel GT-42 genes, cstIV and cstV, have been identified in C. coli. Despite being present in [~]11% of currently available C. coli genomes, the biological role of cstIV and cstV is unknown. In the present study, mutation studies in two strains expressing either cstIV or cstV were performed and mass spectrometry was used to investigate differences in the chemical composition of LOS. Attempts were made to identify donor and acceptor molecules using in vitro activity tests with recombinant GT-42 enzymes. Here, we show that CstIV and CstV are involved in C. coli LOS biosynthesis. In particular, cstV is associated with LOS sialylation, while cstIV is linked to the addition of a diacetylated nonulosonic acid residue.\n\nIMPORTANCEDespite being a major foodborne pathogen, Campylobacter coli glycobiology has been largely neglected. The genetic makeup of the C. coli lipooligosaccharide biosynthesis locus was largely unknown until recently. C. coli harbour a large set of genes associated to lipooligosaccharide biosynthesis, including several putative glycosyltransferases involved in the synthesis of sialylated lipooligosaccharide in Campylobacter jejuni. In the present study, C. coli was found to express lipooligosaccharide structures containing sialic acid and other nonulosonate acids. These findings have a strong impact in understanding C. coli ecology, host-pathogen interaction, and pathogenesis.

microbiology↗

bioRxiv2018

A role for a conserved kinase in the transcriptional control of methionine biosynthesis in Escherichia coli experiencing sustained nitrogen starvation

The initial adaptive transcriptional response to nitrogen (N) starvation in Escherichia coli involves large-scale alterations to the transcriptome mediated by the transcription activator, NtrC. One of the NtrC-activated genes is yeaG, which encodes a conserved bacterial kinase. Although it is known that YeaG is required for optimal survival under sustained N starvation, the molecular basis by which YeaG benefits N starved E. coli remains elusive. By combining transcriptomics with targeted metabolomics analyses, we demonstrate that the methionine biosynthesis pathway becomes transcriptionally dysregulated in{Delta} yeaG bacteria experiencing sustained N starvation. This results in the aberrant and energetically costly biosynthesis of methionine and associated metabolites under sustained N starvation with detrimental consequences to cell viability. It appears the activity of the master transcriptional repressor of methionine biosynthesis genes, MetJ, is compromised in{Delta} yeaG bacteria under sustained N starvation, resulting in transcriptional derepression of MetJ-regulated genes. The results suggest that YeaG is a novel regulatory factor and functions as a molecular brake in the transcriptional control of both the NtrC-regulon and methionine biosynthesis genes in E. coli experiencing sustained N starvation.

microbiology↗

bioRxiv2018

Inter- and intra-domain functional redundancy in the rumen microbiome during plant biomass degradation

BackgroundRuminant livestock is a major source of the potent greenhouse gas methane (CH4), produced by the complex rumen microbiome. Using an integrated approach, combining quantitative metatranscriptomics with gas- and volatile fatty acid (VFA) profiling, we gained fundamental insights into temporal dynamics of the cow rumen microbiome during feed degradation.\n\nResultsThe microbiome composition was highly individual and remarkably stable within each cow, despite similar gas emission and VFA profiles between cows. Gene expression profiles revealed a fast microbial growth response to feeding, reflected by drastic increases in microbial biomass, CH4 emissions and VFA concentrations. Microbiome individuality was accompanied by high inter- and intra-domain functional redundancy among pro- and eukaryotic microbiome members in the key steps of anaerobic feed degradation. Methyl-reducing but not CO2-reducing methanogens were correlated with increased CH4 emissions during plant biomass degradation.\n\nConclusionsThe major response of the rumen microbiome to feed intake was a general growth of the whole community. The high functional redundancy of the cow-individual microbiomes was possibly linked to the robust performance of the anaerobic degradation process. Furthermore, the strong response of methylotrophic methanogens is suggesting that they might play a more important role in ruminant CH4 emissions than previously assumed, making them potential targets for CH4 mitigation strategies.

microbiology↗

bioRxiv2018

The biogenesis of extracellular vesicles from Staphylococcus aureus and their application as a novel vaccine platform

Gram-positive bacteria secrete extracellular vesicles (EVs) that package diverse bacterial antigens and play key roles in bacterial pathogenesis. However, the mechanisms underlying EV production in Gram-positive bacteria are poorly understood. We purified and characterized EVs from a community-associated methicillin-resistant Staphylococcus aureus isolate (USA300) and investigated mechanisms underlying EV production. Native EVs contained 165 proteins, including cytosolic, surface, and secreted proteins, autolysins, and numerous cytolysins. Staphylococcal alpha-type phenol-soluble modulins (surfactant-like peptides) promoted EV biogenesis, presumably by acting at the cytoplasmic membrane, whereas peptidoglycan crosslinking and autolysin activity were found to increase EV production by altering the permeability of the staphylococcal cell wall. To address the immunogenicity of EVs, we created engineered EVs (eng-EVs) by expressing detoxified proteins HlaH35L and LukE in EVs generated from a nontoxic S. aureus {Delta}agr{Delta}spa mutant. Eng-EVs exhibited no cytotoxicity in vitro, and mice immunized with the eng-EVs produced toxin-neutralizing antibodies and showed reduced lethality in a mouse sepsis model. Our study reveals novel mechanisms underlying S. aureus EV production and highlights the usefulness of EVs as a novel S. aureus vaccine platform.

microbiology↗

bioRxiv2018

Harnessing a Novel P450 Fatty Acid Decarboxylase from Macrococcus caseolyticus for Microbial Biosynthesis of Odd Chain Terminal Alkenes

Alkenes are industrially important platform chemicals with broad applications. In this study, we report a microbial conversion route for direct biosynthesis of medium and long chain terminal alkenes from fermentable sugars by harnessing a novel P450 fatty acid (FA) decarboxylase from Macrococcus caseolyticus (OleTMC). We first characterized OleTMC and demonstrated its in vitro H2O2-independent activities towards linear and saturated C10:0-C18:0 FAs, with the highest activity for C16:0 and C18:0 FAs. Combining protein homology modeling, in silico residue mutation analysis, and docking simulation with direct experimental evidence, we elucidated the underlying mechanism for governing the observed substrate preference of OleTMC, which depends on the size of FA binding pocket, not the catalytic site. Next, we engineered the terminal alkene biosynthesis pathway, consisting of an engineered E. coli thioesterase (TesA*) and OleTMC, and introduced this pathway into E. coli for direct terminal alkene biosynthesis from glucose. The recombinant strain E. coli EcNN101 produced a total of 17.78 {+/-} 0.63 mg/L odd-chain terminal alkenes, comprising of 0.9% {+/-} 0.5% C11 alkene, 12.7% {+/-} 2.2% C13 alkene, 82.7% {+/-} 1.7% C15 alkene, and 3.7% {+/-} 0.8% C17 alkene, and a yield of 0.87 {+/-} 0.03 (mg/g) on glucose after 48 h in baffled shake flasks. To improve the terminal alkene production, we identified and overcame the electron transfer limitation in OleTMC, by introducing a two-component redox system, consisting of a putidaredoxin reductase CamA and a putidaredoxin CamB from Pseudomonas putida, into EcNN101, and demonstrated the terminal alkene production increased [~]2.8 fold after 48 h. Overall, this study provides a better understanding of the function of P450 FA decarboxylases and helps guide future protein and metabolic engineering for enhanced microbial production of target designer alkenes in a recombinant host.

microbiology↗

bioRxiv2018

Efficient enrichment cloning of TAL effector genes from Xanthomonas

Many plant-pathogenic xanthomonads use a type III secretion system to translocate Transcription Activator-Like (TAL) effectors into eukaryotic host cells where they act as transcription factors. Target genes are induced upon binding of a TAL effector to double-stranded DNA in a sequence-specific manner. DNA binding is governed by a highly repetitive protein domain, which consists of an array of nearly identical repeats of ca. 102 base pairs. Many species and pathovars of Xanthomonas, including pathogens of rice, cereals, cassava, citrus and cotton, encode multiple TAL effectors in their genomes. Some of the TAL effectors have been shown to act as key pathogenicity factors, which induce the expression of susceptibility genes to the benefit of the pathogen. However, due to the repetitive character and the presence of multiple gene copies, high-throughput cloning of TAL effector genes remains a challenge. In order to isolate complete TAL effector gene repertoires, we developed an enrichment cloning strategy based on O_LIgenome-informed in silico optimization of restriction digestions,\nC_LIO_LIselective restriction digestion of genomic DNA, and\nC_LIO_LIsize fractionation of DNA fragments.\nC_LI Our rapid, cheap and powerful method allows efficient cloning of TAL effector genes from xanthomonads, as demonstrated for two rice-pathogenic strains of Xanthomonas oryzae from Africa.\n\nMETHOD NAMETAL fishing

microbiology↗

bioRxiv2018

Infection By A Giant Virus Induces Widespread Physiological Reprogramming In Aureococcus anophagefferens - A Harmful Bloom Algae

While viruses with distinct phylogenetic origins and different nucleic acid types can infect and lyse eukaryotic phytoplankton, \"giant\" dsDNA viruses have been found to be associated with important ecological processes, including the collapse of algal blooms. However, the molecular aspects of giant virus - host interactions remain largely unknown. AaV, a giant virus in the Mimiviridae clade, is known to play a critical role in regulating the fate of brown tide blooms caused by the pelagophyte Aureococcus anophagefferens. To understand the physiological response of A. anophagefferens CCMP1984 upon AaV infection, we studied the transcriptomic landscape of this host-virus pair over an entire infection cycle using a RNA-sequencing approach. A massive transcriptional reprogramming of the host was evident as early as 5 min post-infection, with modulation of specific processes likely related to both host defense mechanism(s) and viral takeover of the cell. Infected Aureococcus showed a relative suppression of host-cell transcripts associated with photosynthesis, cytoskeleton formation, fatty acid and carbohydrate biosynthesis. In contrast, host cell processes related to protein synthesis, polyamine biosynthesis, cellular respiration, transcription and RNA processing were overrepresented compared to the healthy cultures at different stages of the infection cycle. A large number of redox active host-selenoproteins were overexpressed, which suggested that viral replication and assembly progresses in a highly oxidative environment. The majority (99.2%) of annotated AaV genes were expressed at some point during the infection cycle and demonstrated a clear temporal-expression pattern and an increasing relative expression for the majority of the genes through the time course. We detected a putative early promoter motif for AaV, which was highly similar to the early promoter elements of two other Mimiviridae members, indicating some degree of evolutionary conservation of gene regulation within this clade. This large-scale transcriptome study provides the insight into the Aureococcus virocell, and establishes a foundation to test hypotheses regarding metabolic and regulatory processes critical for AaV and other Mimiviridae members.

microbiology↗

bioRxiv2018

CozEa and CozEb play overlapping and essential roles in controlling cell division in Staphylococcus aureus

Staphylococcus aureus needs to control the position and timing of cell division and cell wall synthesis to maintain its spherical shape. We identified two membrane proteins, named CozEa and CozEb, which together are important for proper cell division in S. aureus. CozEa and CozEb are homologs of the cell elongation regulator CozESpn of Streptococcus pneumoniae. While cozEa and cozEb were not essential individually, the {Delta}cozEa{Delta}cozEb double mutant was lethal. To study the functions of cozEa and cozEb, we constructed a CRISPR interference (CRISPRi) system for S. aureus, allowing transcriptional knockdown of essential genes. CRISPRi knockdown of cozEa in the {Delta}cozEb strain (and vice versa) causes cell morphological defects and aberrant nucleoid staining, showing that cozEa and cozEb have overlapping functions and are important for normal cell division. We found that CozEa and CozEb interact with the cell division protein EzrA, and that EzrA-GFP mislocalizes in the absence of CozEa and CozEb. Furthermore, the CozE-EzrA interaction is conserved in S. pneumoniae, and cell division is mislocalized in cozESpn-depleted S. pneumoniae cells. Together, our results show that CozE proteins mediate control of cell division in S. aureus and S. pneumoniae, likely via interactions with key cell division proteins such as EzrA.

microbiology↗

bioRxiv2018

Exploratory re-encoding of Yellow Fever Virus genome: analysis of in vitro and in vivo replicative phenotypes

Virus attenuation by genome re-encoding is a pioneering approach for generating effective live-attenuated vaccine candidates. Its core principle is to introduce a large number of synonymous substitutions into the viral genome to produce stable attenuation of the targeted virus. Introduction of large numbers of mutations has also been shown to maintain stability of the attenuated phenotype by lowering the risk of reversion and recombination of re-encoded genomes. Identifying mutations with low fitness cost is pivotal as this increases the number that can be introduced and generates more stable and attenuated viruses. Here, we sought to identify mutations with low deleterious impact on the in vivo replication and virulence of yellow fever virus (YFV). Following comparative bioinformatic analyses of flaviviral genomes, we categorized synonymous transition mutations according to their impact on CpG/UpA composition and secondary RNA structures. We then designed 17 re-encoded viruses with 100-400 synonymous mutations in the NS2A-to-NS4B coding region of YFV Asibi and Ap7M (hamster-adapted) genomes. Each virus contained a panel of synonymous mutations designed according to the above categorisation criteria. The replication and fitness characteristics of parent and re-encoded viruses were compared in vitro using cell culture competition experiments. In vivo laboratory hamster models were also used to compare relative virulence and immunogenicity characteristics. Most of the re-encoded strains showed no decrease in replicative fitness in vitro. However, they showed reduced virulence and, in some instances, decreased replicative fitness in vivo. Importantly, the most attenuated of the re-encoded strains induced robust, protective immunity in hamsters following challenge with Ap7M, a virulent virus. Overall, the introduction of transitions with no or a marginal increase in the number of CpG/UpA dinucleotides had the mildest impact on YFV replication and virulence in vivo. Thus, this strategy can be incorporated in procedures for the finely tuned creation of substantially re-encoded viral genomes.

microbiology↗

bioRxiv2018

A proposal for a standardized bacterial taxonomy based on genome phylogeny

Taxonomy is a fundamental organizing principle of biology, which ideally should be based on evolutionary relationships. Microbial taxonomy has been greatly restricted by the inability to obtain most microorganisms in pure culture and, to a lesser degree, the historical use of phenotypic properties as the basis for classification. However, we are now at the point of obtaining genome sequences broadly representative of microbial diversity by using culture-independent techniques, which provide the opportunity to develop a comprehensive genome-based taxonomy. Here we propose a standardized bacterial taxonomy based on a concatenated protein phylogeny that conservatively removes polyphyletic groups and normalizes ranks based on relative evolutionary divergence. From 94,759 bacterial genomes, 99 phyla are described including six major normalized monophyletic units from the subdivision of the Proteobacteria, and amalgamation of the Candidate Phyla Radiation into the single phylum Patescibacteria. In total, 73% of taxa had one or more changes to their existing taxonomy.

microbiology↗

bioRxiv2018

Characterization of flagellar and toxin phase variation in Clostridium difficile ribotype 012 isolates

Clostridium difficile causes diarrheal diseases mediated in part by the secreted toxins TcdA and TcdB. C. difficile produces flagella that also contribute to motility and bacterial adherence to intestinal cells during infection. Flagellum and toxin gene expression are linked via the flagellar alternative sigma factor, SigD. Recently, we identified a \"flagellar switch\" upstream of the early flagellar biosynthesis operon that mediates phase variation of both flagellum and toxin production in C. difficile strain R20291. However, we were unable to detect flagellar switch inversion in C. difficile strain 630, a ribotype 012 strain commonly used in research labs, suggesting the strain is phase-locked. To determine whether a \"phase locked\" flagellar switch is limited to 630 or present more broadly in ribotype 012 strains, we assessed the frequency and phenotypic outcomes of flagellar switch inversion in multiple C. difficile ribotype 012 isolates. The laboratory-adapted strain JIR8094, a derivative of strain 630, and six clinical and environmental isolates were all found to be phase OFF, non-motile, and attenuated for toxin production. We isolated low frequency motile derivatives of JIR8094 with partial recovery of motility and toxin production, and found that additional changes in JIR8094 impact these processes. The clinical and environmental isolates varied considerably in the frequency by which flagellar phase ON derivatives arose, and these derivatives showed fully restored motility and toxin production. Taken together, these results demonstrate heterogeneity in flagellar and toxin phase variation among C. difficile ribotype 012 strains, and perhaps other ribotypes, which could impact disease progression and diagnosis.\n\nImportanceClostridium difficile causes diarrheal disease resulting in significant morbidity and mortality in many countries. C. difficile produces flagella that enhance bacterial motility, and secretes toxins that promote diarrheal disease symptoms. Previously, we found that production of flagella and toxins are co-regulated via a flippable DNA element termed the \"flagellar switch\", which mediates the phase variable production of these factors. C. difficile can exist as flagellated, motile, toxigenic bacteria (\"flg ON\") or aflagellate, non-motile, nontoxigenic bacteria (\"flg OFF\") due in part to the flagellar switch orientation. Here we evaluate multiple isolates of C. difficile ribotype 012 strains and find them to be primarily flg OFF. Some, but not all, of these isolates showed the ability to switch between flg ON and OFF states. These findings suggest heterogeneity in the ability of C. difficile ribotype 012 strains to phase vary flagellum and toxin production, which may broadly apply to pathogenic C. difficile.

microbiology↗

bioRxiv2018

Ferroptosis-like signaling facilitates a potent innate defense against Plasmodium infection

The facets of host control during Plasmodium liver infection remain largely unknown and conventional innate regulatory pathways are only minimally effective at eliminating parasites1-3. Ferroptosis, a recently described form of iron-dependent cell death that drives accumulation of reactive oxygen species and lipid peroxides, but has not yet been shown to function as an innate immune response4,5. Inducing ferroptosis with pharmacologicals or by genetic perturbation of its negative regulators, GPX4 and SLC7a11, dramatically reduces survival of the Plasmodium Liver Stage. In contrast, knockdown or knockout of NOX1 or knockdown of TFR1, which are required for ferroptosis, increases the number of Liver Stage parasites. Moreover, we demonstrate that blocking ferroptosis renders parasite-infected hepatocytes resistant to P53-mediated hepatocyte death. Our work establishes that ferroptotic signaling serves to control Plasmodium infection in the liver and raises the possibility that ferroptosis operates as an axis of the innate immune system to defend against intracellular pathogens.

microbiology↗

bioRxiv2018

N6-methyladenosine Binding Proteins Negatively Regulate HIV-1 Infectivity and Viral Production

BackgroundThe internal N6-methyladenosine (m6A) modification of cellular mRNA regulates post-transcriptional gene expression. The YTH domain family proteins (YTHDF1-3, or Y1-3) bind to m6A-modified cellular mRNA and modulate its metabolism and processing, thereby affecting protein translation in cells. We previously reported that HIV-1 RNA contains m6A modification and that Y1-3 proteins inhibit HIV-1 infection by decreasing HIV-1 reverse transcription. Here we extended our studies to further understand the mechanisms of Y1-3-mediated inhibition of HIV-1 infection and viral production.\n\nResultsOverexpression of Y1-3 proteins in HIV-1 target cells decreased viral genome RNA (gRNA) levels and inhibited early and late reverse transcription. Purified recombinant Y1-3 proteins preferentially bound to the m6A-modified 5 leader sequence of gRNA compared with its unmodified RNA counterpart, consistent with the strong binding of Y1-3 to HIV-1 gRNA in infected cells. HIV-1 mutants with two altered m6A modification sites in the 5 leader sequence of gRNA demonstrated significantly lower infectivity compared with wild-type HIV-1, confirming that these sites are important for viral infection. HIV-1 produced from cells with knockdown of endogenous Y1, Y3, or Y1-3 proteins together showed increased viral infectivity compared with HIV-1 produced from control cells. Interestingly, we found that Y1-3 proteins and HIV-1 Gag formed a complex with RNA in HIV-1-infected target cells.\n\nConclusionsOur results suggest the inhibitory effects of Y1-3 proteins on HIV-1 infection and provide new insight into the mechanisms of m6A modification of HIV-1 RNA in regulating viral replication, which clarify some discrepancies in the previously published studies in this area.

microbiology↗

bioRxiv2018

Regulation of (p)ppGpp hydrolysis by a conserved archetypal regulatory domain

Sensory and regulatory domains allow bacteria to adequately respond to environmental changes. The regulatory ACT domains are mainly found in metabolic-related proteins as well as in long (p)ppGpp synthetase/hydrolase (SD/HD) enzymes. Here, we investigate the functional role of the ACT domain of SpoT, the only (p)ppGpp SD/HD of Caulobacter crescentus. We show that SpoT requires the ACT domain to hydrolyse ppGpp in an efficient way. In addition, our in vivo and in vitro data show that the phosphorylated version of EIIANtr (EIIANtr~P) interacts directly with the ACT to inhibit the hydrolase activity of SpoT. Finally, we highlight the conservation of the ACT-dependent interaction between EIIANtr~P and SpoT/Rel along with the PTSNtr-dependent regulation of (p)ppGpp accumulation upon nitrogen starvation in Sinorhizobium meliloti, a plant-associated -proteobacterium. Thus, this work suggests that -proteobacteria might have inherited from a common ancestor, a PTSNtr dedicated to modulate (p)ppGpp levels.

microbiology↗

bioRxiv2018

Evidence of independent acquisition and adaption of ultra-small bacteria to human hosts across the highly diverse yet reduced genomes of the phylum Saccharibacteria

Recently, we discovered that a member of the Saccharibacteria/TM7 phylum (strain TM7x) isolated from the human oral cavity, has an ultra-small cell size (200-300nm), a highly reduced genome (705 Kbp) with limited de novo biosynthetic capabilities, and a very novel lifestyle as an obligate epibiont on the surface of another bacterium 1. There has been considerable interest in uncultivated phyla, particularly those that are now classified as the proposed candidate phyla radiation (CPR) reported to include 35 or more phyla and are estimated to make up nearly 15% of the domain Bacteria. Most members of the larger CPR group share genomic properties with Saccharibacteria including reduced genomes (<1Mbp) and lack of biosynthetic capabilities, yet to date, strain TM7x represents the only member of the CPR that has been cultivated and is one of only three CPR routinely detected in the human body. Through small subunit ribosomal RNA (SSU rRNA) gene surveys, members of the Saccharibacteria phylum are reported in many environments as well as within a diversity of host species and have been shown to increase dramatically in human oral and gut diseases. With a single copy of the 16S rRNA gene resolved on a few limited genomes, their absolute abundance is most often underestimated and their potential role in disease pathogenesis is therefore underappreciated. Despite being an obligate parasite dependent on other bacteria, six groups (G1-G6) are recognized using SSU rRNA gene phylogeny in the oral cavity alone. At present, only genomes from the G1 group, which includes related and remarkably syntenic environmental and human oral associated representatives1, have been uncovered to date. In this study we systematically captured the spectrum of known diversity in this phylum by reconstructing completely novel Class level genomes belonging to groups G3, G6 and G5 through cultivation enrichment and/or metagenomic binning from humans and mammalian rumen. Additional genomes for representatives of G1 were also obtained from modern oral plaque and ancient dental calculus. Comparative analysis revealed remarkable divergence in the host-associated members across this phylum. Within the human oral cavity alone, variation in as much as 70% of the genes from nearest oral clade (AAI 50%) as well as wide GC content variation is evident in these newly captured divergent members (G3, G5 and G6) with no environmental relatives. Comparative analyses suggest independent episodes of transmission of these TM7 groups into humans and convergent evolution of several key functions during adaptation within hosts. In addition, we provide evidence from in vivo collected samples that each of these major groups are ultra-small in size and are found attached to larger cells.

microbiology↗

bioRxiv2018

Candida albicans dispersed cells signify a developmental state distinct from biofilm and planktonic phases

Candida albicans surface-attached biofilms are sites of amplification of an infection through continuous discharge of cells capable of initiating new infectious foci. Yeast cells released from biofilms on intravenous catheters have direct access to the bloodstream. We previously reported that dispersed cells are largely lateral yeast cells that originate from the hyphal layers of the biofilm. Compared to their planktonic counterparts, these biofilm-dispersed yeast cells displayed enhanced virulence-associated gene expression and drug resistance. Little is known about the molecular properties of dispersed cells. We found that the inducer of dispersal, PES1, genetically interacts with the repressor of filamentation, NRG1, in a manner that supports a genetic definition of dispersed cells as yeast. We combined a flow biofilm model with RNA sequencing technology, to identify transcriptomic characteristics of freshly dispersed yeast cells versus biofilms or age-matched planktonic yeast cells growing in glucose-rich medium. Dispersed cells largely inherited a biofilm-like mRNA profile but with one stark difference: dispersed cells were transcriptionally reprogrammed to metabolize alternative carbon sources, while their sessile parents expressed glycolytic genes, despite exposure to the same nutritional signals. Our studies hence define dispersal cell production as an intrinsic step of biofilm development which generates propagules capable of colonizing distant host sites. This developmental step anticipates the need for virulence-associated gene expression before experiencing the associated external signals.

microbiology↗

bioRxiv2018

Divergent PAM Specificity of a Highly-Similar SpCas9 Ortholog

RNA-guided DNA endonucleases of the CRISPR-Cas system are widely used for genome engineering and thus have numerous applications in a wide variety of fields. The range of sequences that CRISPR endonucleases can recognize, however, is constrained by the need for a specific protospacer adjacent motif (PAM) flanking the target site. In this study, we demonstrate the natural PAM plasticity of a highly-similar, yet previously uncharacterized, Cas9 from Streptococcus canis (ScCas9) through rational manipulation of distinguishing motif insertions. To this end, we report a divergent affinity to 5-NNGT-3 PAM sequences and demonstrate the editing capabilities of the ortholog in both bacterial and human cells. Finally, we build an automated bioinformatics pipeline, the Search for PAMs by ALignment Of Targets (SPAMALOT), which further explores the microbial PAM diversity of otherwise-overlooked Streptococcus Cas9 orthologs. Our results establish that ScCas9 can be utilized both as an alternative genome editing tool and as a functional platform to discover novel Streptococcus PAM specificities.

microbiology↗

bioRxiv2018

Thermoelectric Heat Exchange and Growth Regulation in a Continuous Yeast Culture

We have designed a thermoelectric heat exchanger (TEHE) for microbial fermentations, able to control the temperature of a microbial continuous culture, and produce electric power. The system proved able to stably maintain both the temperature and the optical density of the culture during the exponential, highly productive phase.

microbiology↗