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bioRxiv2017

Expanding Gene Families Helps Generate The Metabolic Robustness Required For Antibiotic Biosynthesis

Introductory paragraphExpanding the genetic repertoire of an organism by gene duplication or horizontal gene transfer (HGT) can aid adaptation. Streptomyces species are prolific producers of bioactive specialised metabolites with adaptive functions in nature and some have found utility in human medicine such as antibiotics. Whilst the biosynthesis of these specialised metabolites is directed by dedicated biosynthetic gene clusters (BGCs), little attention has been focussed on how these organisms have evolved robustness into their genomes to facilitate the metabolic plasticity required to provide chemical precursors for biosynthesis. Here we show that specific expansions of gene families in central carbon metabolism have evolved and become fixed in Streptomyces bacteria to enable plasticity and robustness that maintain cell functionality whilst costly specialised metabolites are produced. These expanded gene families, in addition to being a metabolic adaptation, make excellent targets for metabolic engineering of industrial specialised metabolite producing bacteria.

microbiology↗

bioRxiv2017

Adaptation, Specialization, and Coevolution within Phytobiomes

Growth patterns of individual plants and evolutionary trajectories of plant communities are intimately linked with and are critically affected by host-associated microbiomes. Research across systems has begun to shed light on how these phytobiomes are established and under laboratory and natural conditions, and have cultivated hope that a better understanding of the governing principles for host-microbe interactions can guide attempts to engineer microbiomes to boost agricultural yields. One important, yet relatively understudied, parameter in regards to phytobiome membership is the degree to which specialization and coevolution between plant species and microbial strains structures these communities. In this article, I provide an broad overview about current knowledge concerning mechanisms enabling adaptation and specialization of phytobiome communties to host plants as well as the potential for plants themselves to recruit and cultivate interactions with beneficial microbes. I further explore the possibility of host-beneficial microbe coevolution and suggest interactions that could promote the evolution of such close-knit partnerships. It is my hope that this overview will encourage future experiments that can begin to fill in this black box of ecological and evolutionary interactions across phytobiomes.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC=\"FIGDIR/small/120493_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@16c5315org.highwire.dtl.DTLVardef@c298d7org.highwire.dtl.DTLVardef@1c073a6org.highwire.dtl.DTLVardef@1e8bd1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2017

Enterovirus-A76 of South-East Asian ancestry in a Captive Chimpanzee (Pan troglodytes) in Jos, Nigeria

We recently detected EV-A119 and EV-B111 (previously shown to co-circulate between nonhuman primates (NHPs) and humans) in Nigerian children diagnosed with acute flaccid paralysis (AFP). This study was designed to investigate and catalogue EVs present in captive NHPs in Nigeria.\n\nTwenty-seven fecal samples collected from captive NHPs in a Wild Life Park and Zoological garden at Jos, Nigeria in April 2016 were analyzed in this study. Samples were resuspended in a phosphate buffered saline (PBS)/chloroform mixture, and the clarified supernatant was subjected to RNA extraction, cDNA synthesis, a Panenterovirus 5I-UTR assay, and three different enterovirus VP1 snPCR assays. All amplicons from the snPCR assays were sequenced, and enteroviruses identified using the enterovirus genotyping tool and phylogenetic analysis.\n\nEight (29.63%) (two each from Chimpanzees, Patas Monkey, Mona Monkey and Baboon) of the 27 samples were positive for the 5I-UTR assay. One (3.70%) of the 27 samples was positive for the enterovirus VP1 snPCR assays in addition to its positivity by 5I-UTR assay. The same sample happens to be one of the two samples from Chimpanzees that tested positive for the 5I-UTR assay, and it was subsequently identified as EV-A76 of South-East Asia ancestry.\n\nThis study documents the first recorded attempt to detect and identify enteroviruses in NHPs in Nigeria. It also reports the first detection and identification of EV-A76 in Nigeria and particularly in a NHP. It is of utmost importance that the enterovirus VP1 assays be improved to enable detection of EVs that have been detected in NHPs but yet to be described in humans.

microbiology↗

bioRxiv2017

Brucella abortus Induces A Warburg Shift In Host Metabolism That Enhances Intracellular Replication Of The Pathogen

Intracellular bacterial pathogens exploit host cell resources to replicate and survive inside the host. Targeting these host systems is one promising approach to developing novel antimicrobials to treat intracellular infections. We show that human macrophage-like cells infected with Brucella abortus undergo a metabolic shift characterized by attenuated tricarboxylic acid cycle metabolism, reduced amino acid consumption, altered mitochondrial localization, and increased lactate production. This shift to an aerobic glycolytic state resembles the Warburg effect, a change in energy production that is well-described in cancer cells, and also occurs in activated inflammatory cells. B. abortus efficiently uses lactic acid as its sole carbon and energy source and requires the ability to metabolize lactate for normal survival in human macrophage-like cells. We demonstrate that chemical inhibitors of host glycolysis and lactate production do not affect in vitro growth of B. abortus in axenic culture, but decrease its survival in the intracellular niche. Our data support a model in which infection shifts host metabolism to a Warburg-like state, and B. abortus uses this change in metabolism to promote intracellular survival. Pharmacological perturbation of these features of host cell metabolism may be a useful strategy to inhibit infection by intracellular pathogens.\n\nIMPORTANCEBrucella spp. are intracellular bacterial pathogens that cause disease in a range of mammals, including livestock. Transmission from livestock to humans is common and can lead to chronic human disease. Human macrophage-like cells infected with Brucella abortus undergo a Warburg-like metabolic shift to an aerobic glycolytic state where the host cells produce lactic acid and have reduced amino acid catabolism. We provide evidence that the pathogen can exploit this change in host metabolism to support growth and survival in the intracellular niche. Drugs that inhibit this shift in host cell metabolism inhibit intracellular replication and decrease the survival of B. abortus in an in vitro infection model; these drugs may be broadly useful therapeutics for intracellular infections.

microbiology↗

bioRxiv2017

Replication Defective Viral Genomes Exploit A Cellular Pro-Survival Mechanism To Establish Viral Persistence

Replication defective viral genomes (DVGs) generated during virus replication are the primary triggers of antiviral immunity in many RNA virus infections. However, DVGs can also facilitate viral persistence. Why and how these two opposing functions of DVGs are achieved remain unknown. Here we report that during Sendai and respiratory syncytial virus infections DVGs selectively protect a subpopulation of cells from death and promote the establishment of persistent infections. We find that during Sendai virus infection this phenotype results from DVGs stimulating a MAVS-mediated TNF response that drives apoptosis of highly infected cells while extending the survival of cells enriched in DVGs. The pro-survival effect of TNF depends on the activity of the TNFR2/TRAF1 pathway that is regulated by MAVS signaling. These results identify TNF as a pivotal factor in determining cell fate during a viral infection and delineate a MAVS/TNFR2-mediated mechanism that drives the persistence of otherwise acute viruses.

microbiology↗

bioRxiv2017

Primary souring: a novel bacteria-free method for sour beer production

In the beverage fermentation industry, especially at the craft or micro level, there is a movement to incorporate as many local ingredients as possible to both capture terroir and stimulate local economies. In the case of craft beer, this has traditionally only encompassed locally sourced barley, hops, and other agricultural adjuncts. The identification and use of novel yeasts in brewing lags behind. We sought to bridge this gap by bio-prospecting for wild yeasts, with a focus on the American Midwest. We isolated 284 different strains from 54 species of yeast and have begun to determine their fermentation characteristics. During this work, we found several isolates of five species that produce lactic acid and ethanol during wort fermentation: Hanseniaspora vineae, Lachancea fermentati, Lachancea thermotolerans, Schizosaccharomyces japonicus, and Wickerhamomyces anomalus. Tested representatives of these species yielded excellent attenuation, lactic acid production, and sensory characteristics, positioning them as viable alternatives to lactic acid bacteria (LAB) for the production of sour beers. Indeed, we suggest a new LAB-free paradigm for sour beer production that we term \"primary souring\" because the lactic acid production and resultant pH decrease occurs during primary fermentation, as opposed to kettle souring or souring via mixed culture fermentation.\n\nChemical compounds studied in this article: Lactic acid (PubChem CID: 612); Ethanol (PubChem CID: 702)\n\nAbbreviations: ABV, alcohol by volume; DIC, differential interference contrast; EtOH, ethanol; FG, final gravity; gDNA, genomic DNA; IBU, international bittering unit; LAB, lactic acid bacteria; LASSO, lactic acid specific soft-agar overlay; N-J, neighbor-joining; OG, original gravity; WLN, Wallerstein Laboratories nutrient; YPD, yeast extract, peptone, and dextrose

microbiology↗

bioRxiv2017

Quantifying The Fitness Benefit And Cost Of Cefixime Resistance In Neisseria gonorrhoeae To Inform Prescription Policy

Gonorrhea is one of the most common bacterial sexually transmitted infections in England. Over 41,000 cases were recorded in 2015, more than half of which occurred in men who have sex with men (MSM). As the bacterium has developed resistance to each first-line antibiotic in turn, we need an improved understanding of fitness benefits and costs of antibiotic resistance to inform control policy and planning. Cefixime was recommended as a single dose treatment for gonorrhea from 2005 to 2010, during which time resistance increased and subsequently declined. We developed a stochastic compartmental model representing the natural history and transmission of cefixime sensitive and resistant strains of Neisseria gonorrhoeae in MSM in England, which was applied to data on diagnoses and prescriptions between 2008 and 2015. We estimated that asymptomatic carriers play a crucial role in overall transmission dynamics, with about 40% of infections remaining asymptomatic and untreated, accounting for 96% of onward transmission. The fitness cost of cefixime resistance in the absence of cefixime usage was estimated to be such that the number of secondary infections caused by resistant strains is only about half as much as for the susceptible strains, which is insufficient to maintain persistence. However, we estimated that treatment of cefixime-resistant strains with cefixime was unsuccessful in 84% of cases, representing a fitness benefit of resistance. This benefit was large enough to counterbalance the fitness cost when 31% of cases are treated with cefixime, and when more than 51% of cases were treated with cefixime the resistant strain had a net fitness advantage over the susceptible strain. Our findings have important implications for antibiotic stewardship and public health policies, and in particular suggest that cefixime could be used to treat a minority of gonorrhea cases without raising resistance levels.

microbiology↗

bioRxiv2017

Development Of Amplicon Deep Sequencing Markers And Data Analysis Pipeline For Genotyping Multi-Clonal Malaria Infections

Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. Such high resolution is needed for molecular monitoring of drug efficacy trials. Targeted sequencing of molecular marker csp and novel marker cpmp was conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol to multiplex up to 384 samples in a single sequencing run was applied. Software \"HaplotypR\" was developed for data analysis. Cpmp was highly diverse (He=0.96) in contrast to csp (He=0.57). Minority clones were robustly detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below that threshold. To reliably detect haplotypes at very low frequencies, experiments are best performed in duplicate and should aim for coverage of >10000 reads/amplicon. When compared to length polymorphic marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority clones.

microbiology↗

bioRxiv2017

Integrating Patient And Whole Genome Sequencing Data To Provide Insights Into The Epidemiology Of Seasonal Influenza A(H3N2) Viruses

Genetic surveillance of seasonal influenza is largely focused upon sequencing of the haemagglutinin gene. Consequently, our understanding of the contribution of the remaining seven gene segments to the evolution and epidemiological dynamics of seasonal influenza is relatively limited. The increased availability of next generation sequencing technologies allows rapid and economic whole genome sequencing (WGS). Here, 150 influenza A(H3N2) positive clinical specimens with linked epidemiological data, from the 2014/15 season in Scotland, were sequenced directly using both Sanger sequencing of the HA1 region and WGS using the Illumina MiSeq platform. Sequences generated by both methods were highly consistent and WGS provided on average >90% whole genome coverage. As reported in other European countries during 2014/15, all strains belonged to genetic group 3C, with subgroup 3C.2a predominating. Inter-subgroup reassortants were identified (9%), including three 3C.3 viruses descended from a single reassortment event, which had persisted in the population. Significant phylogenetic associations with cases of severe acute respiratory illness observed herein warrant further investigation. Severe cases were also more likely to be associated with reassortant viruses (odds ratio: 4.4 (1.3-15.5)) and occur later in the season. These results suggest that increased levels of WGS, linked to clinical and epidemiological data, could improve influenza surveillance.

microbiology↗

bioRxiv2017

Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis

Pathogenic bacteria have evolved highly specialized systems to extract essential nutrients from their hosts and Mycobacterium tuberculosis (Mtb) scavenges lipids (cholesterol and fatty acids) to maintain infection in mammals. While the uptake of cholesterol by Mtb is mediated by the Mce4 transporter, the route(s) of uptake of fatty acids remain unknown. Here, we demonstrate that an uncharacterized protein LucA, integrates the assimilation of both cholesterol and fatty acids in Mtb. LucA interacts with subunits of the Mce1 and Mce4 complexes to coordinate the activities of these nutrient transporters. We also demonstrate that Mce1 functions as an important fatty acid transporter in Mtb and we determine that the integration of cholesterol and fatty acid transport by LucA is required for full bacterial virulence in vivo. These data establish that fatty acid and cholesterol assimilation are inexorably linked in Mtb and reveals a key role for LucA in coordinating both transport activities.

microbiology↗

bioRxiv2017

Methicillin resistant Staphylococcus aureus emerged long before the introduction of methicillin in to clinical practice

The spread of drug-resistant bacterial pathogens pose a major threat to global health. It is widely recognised that the widespread use of antibiotics has generated selective pressures that have driven the emergence of resistant strains. Methicillin-resistant Staphylococcus aureus (MRSA) was first observed in 1960, less than one year after the introduction of this second generation {beta}-lactam antibiotic into clinical practice. Epidemiological evidence has always suggested that resistance arose around this period, when the mecA gene encoding methicillin resistance carried on an SCCmec element, was horizontally transferred to an intrinsically sensitive strain of S. aureus. Whole genome sequencing a collection of the very first MRSA isolates allowed us to reconstruct the evolutionary history of the archetypal MRSA. Bayesian phylogenetic reconstruction was applied to infer the time point at which this early MRSA lineage arose and when SCCmec was acquired. MRSA emerged in the mid 1940s, following the acquisition of an ancestral type I SCCmec element, some fourteen years prior to the first therapeutic use of methicillin. Methicillin use was not the original driving factor in the evolution of MRSA as previously thought. Rather it was the widespread use of first generation {beta}-lactams such as penicillin in the years prior to the introduction of methicillin, which selected for S. aureus strains carrying the mecA determinant. Crucially this highlights how new drugs, introduced to circumvent known resistance mechanisms, can be rendered ineffective by unrecognised adaptations in the bacterial population due to the historic selective landscape created by the widespread use of other antibiotics.

microbiology↗

bioRxiv2017

The Toxoplasma centrocone houses cell cycle regulatory factors

Our knowledge of cell cycle regulatory mechanisms in apicomplexan parasites is very limited. In this study, we describe a novel Toxoplasma gondii factor, essential for chromosome replication 1 (ECR1), that has a vital role in chromosome replication and the regulation of cytoplasmic and nuclear mitotic structures. ECR1 was discovered by complementation of a temperature sensitive (ts) mutant that suffers lethal, uncontrolled chromosome replication at 40{degrees}C similar to a ts-mutant carrying a defect in topoisomerase. ECR1 is a 52kDa protein containing divergent RING and TRAF-Sina like zinc-binding domains that is dynamically expressed in the tachyzoite cell cycle. ECR1 first appears in the centrocone compartment of the nuclear envelope in early S phase and then in the nucleus in late S phase where it reaches maximum expression. Following nuclear division, but before daughters resolve from the mother, ECR1 is down regulated and is absent in new daughter parasites. The proteomics of ECR1 identified interactions with the ubiquitin-mediated protein degradation machinery and the minichromosome maintenance complex and the loss of ECR1 led to increased stability of a key member of this complex, MCM2. ECR1 also forms a stable complex with the CDK-related kinase, TgCrk5, which shares a similar cell cycle expression and localization during tachyzoite replication. Altogether, the results of this study suggest ECR1 may be a unique E3 ligase that regulates DNA licensing and other mitotic processes. Importantly, the localization of ECR1/TgCrk5 in the centrocone indicates this Apicomplexa-specific spindle compartment houses important regulatory factors that control the parasite cell cycle.\n\nIMPORTANCEParasites of the apicomplexan family are important causes of human disease including malaria, toxoplasmosis, and cryptosporidiosis. Parasite growth is the underlying cause of pathogenesis, yet despite this importance the molecular basis for parasite replication is poorly understood. Filling this knowledge gap cannot be accomplished by mining recent whole genome sequencing because apicomplexan cell cycles differ substantially and lack many of the key regulatory factors of well-studied yeast and mammalian cell division models. We have utilized forward genetics to discover essential factors that regulate cell division in these parasites using the Toxoplasma gondii model. An example of this approach is described here with the discovery of a putative E3 ligase/protein kinase mechanism involved in regulating chromosome replication and mitotic processes of asexual stage parasites.

microbiology↗

bioRxiv2017

Horizontal Gene Transfer of Functional Type VI Killing Genes by Natural Transformation

Horizontal gene transfer can have profound effects on bacterial evolution by allowing individuals to rapidly acquire adaptive traits that shape their strategies for competition. One strategy for intermicrobial antagonism often used by Proteobacteria is the genetically-encoded contact-dependent Type VI secretion system (T6SS); a weapon used to kill heteroclonal neighbors by direct injection of toxic effectors. Here, we experimentally demonstrate that Vibrio cholerae can acquire new T6SS effector genes via horizontal transfer and utilize them to kill neighboring cells. Replacement of one or more parental alleles with novel effectors allows the recombinant strain to dramatically outcompete its parent. Through spatially-explicit simulation modeling, we show that the HGT is risky: transformation brings a cell into conflict with its former clonemates, but can be adaptive when superior T6SS alleles are acquired. More generally, we find that these costs and benefits are not symmetric, and that high rates of HGT can act as hedge against competitors with unpredictable T6SS efficacy. We conclude that antagonism and horizontal transfer drive successive rounds of weapons-optimization and selective sweeps, dynamically shaping the composition of microbial communities.

microbiology↗

bioRxiv2017

Worldwide Population Structure Of Escherichia coli Reveals Two Major Subspecies

Recombination is one of the most important mechanisms of prokaryotic species evolution but its exact roles are still in debate. Here we try to infer genome-wide recombination events within a species uti-lizing a dataset of 104 complete genomes of Escherichia coli from diverse origins, among which 45 from world-wide animal-hosts are in-house sequenced using SMRT (single-molecular real time) technology.Two major clades are identified based on evidences of ecological and physiological characteristics, as well as distinct genomic features implying scarce inter-clade genetic exchange. By comparing the synteny of identical fragments genome-widely searched for each genome pair, we achieve a fine-scale map of re-combination within the population. The recombination is rather extensive within clade, which is able to break linkages between genes but does not interrupt core genome framework and primary metabolic port-folios possibly due to natural selection for physiological compatibility and ecological fitness. Meanwhile,the recombination between clades declines drastically as the phylogenetic distance increases, generally 10-fold reduced than those of the intra-clade, which establishes genetic barrier between clades. These empirical data of recombination suggest its critical role in the early stage of speciation, where recombina-tion rate differs according to phylogentic distance. The extensive intra-clade recombination coheres sister strains into a quasi-sexual group and optimizes genes or alleles to streamline physiological activities,whereas shapely declined inter-clade recombination split the population into clades adaptive to divergent ecological niches.\n\nSignificance StatementRoles of recombination in species evolution have been debated for decades due to difficulties in inferring recombination events during the early stage of speciation, especially when recombination is always complicated by frequent gene transfer events of bacterial genomes. Based on 104 high-quality complete E. coli genomes, we infer gene-centric dynamics of recombination in the formation of two E. coli clades or subpopulations, and recombination is found to be rather intensive in a within-clade fashion, which forces them to be quasi-sexual. The recombination events can be mapped among individual genomes in the context of genes and their variations; decreased between-clade and increased intra-claderecombination engender a genetic barrier that further encourages clade-specific secondary metabolic portfolios for better environmental adaptation. Recombination is thus a major force that accelerates bacterial evolution to fit ecological diversity.

microbiology↗

bioRxiv2017

Draft Genome Sequence Of A Candidatus Brocadia Bacterium Enriched From Tropical-Climate Activated Sludge

We present the draft genome of an anaerobic ammonium-oxidizing (anammox) bacterium, cluster III Candidatus Brocadia, which was enriched in an anammox reactor. A 3.2 Mb genome sequence comprising 168 contigs was assembled, in which 2,765 gene-coding regions, 47 tRNAs, and 5S, 16S and 23S ribosomal RNAs were annotated. No evidence for the presence of a nitric oxide-forming nitrite reductase was found.

microbiology↗

bioRxiv2017

Uncovering The Repertoire Of Endogenous Flaviviral Elements In Aedes Mosquito Genomes

Endogenous viral elements derived from non-retroviral RNA viruses were described in various animal genomes. Whether they have a biological function such as host immune protection against related viruses is a field of intense study. Here, we investigated the repertoire of endogenous flaviviral elements (EFVEs) in Aedes mosquitoes, the vectors of arboviruses such as dengue and chikungunya viruses. Previous studies identified three EFVEs from Ae. albopictus and one from Ae. aegypti cell lines. However, in-depth characterization of EFVEs in wild-type mosquito populations and individuals in vivo has not been performed. We detected the full-length DNA sequence of the previously described EFVEs and their respective transcripts in several Ae. albopictus and Ae. aegypti populations from geographically distinct areas. However, EFVE-derived proteins were not detected by mass spectrometry. Using deep sequencing, we detected the production of piRNA-like small RNAs in antisense orientation, targeting the EFVEs and their flanking regions in vivo. The EFVEs were integrated in repetitive regions of the mosquito genomes, and their flanking sequences varied among mosquito populations from different geographical regions. We bioinformatically predicted several new EFVEs from a Vietnamese Ae. albopictus population and observed variation in the occurrence of those elements among mosquito populations. Phylogenetic analysis of an Ae. aegypti EFVE suggested that it integrated prior to the global expansion of the species and subsequently diverged among and within populations. Together, this study revealed substantial structural and nucleotide diversity of flaviviral integrations in Aedes genomes. Unraveling this diversity will help to elucidate the potential biological function of these EFVEs.\n\nImportanceEndogenous viral elements (EVEs) are whole or partial viral sequences integrated in host genomes. Interestingly, some EVEs have important functions for host fitness and antiviral defense. Because mosquitoes also have EVEs in their genomes, we decided to thoroughly characterized them to lay the foundation of the potential use of these EVEs to manipulate the mosquito antiviral response. Here, we focused on EVEs related to the Flavivirus genus, to which dengue and Zika viruses belong, in Aedes mosquito individuals from geographically distinct areas. We showed the existence in vivo of flaviviral EVEs previously identified in mosquito cell lines and we detected new ones. We showed that EVEs have evolved differently in each mosquito population. They produced transcripts and small RNAs, but not proteins, suggesting a function at the RNA level. Our study uncovers the diverse repertoire of flaviviral EVEs in Aedes mosquito populations and suggests a role in the host antiviral system.

microbiology↗

bioRxiv2017

A Novel Ultra High-Throughput 16S rRNA Amplicon Sequencing Library Preparation Method On The Illumina HiSeq Platform

BackgroundAdvances in sequencing technologies and bioinformatics have made the analysis of microbial communities almost routine. Nonetheless, the need remains to improve on the techniques used for gathering such data, including increasing throughput while lowering cost, and benchmarking the techniques so that potential sources of bias can be better characterized.\n\nResultsWe present a triple-index amplicon sequencing strategy that uses a two-stage PCR protocol. The strategy was extensively benchmarked through analysis of a mock community in order to assess biases introduced by sample indexing, number of PCR cycles, and template concentration. We further evaluated the method through re-sequencing of a standardized environmental sample. Finally, we evaluated our protocol on a set of fecal samples from a small cohort of healthy adults, demonstrating good performance in a realistic experimental setting. Between-sample variation was mainly related to batch effects, such as DNA extraction, while sample indexing was also a significant source of bias. PCR cycle number strongly influenced chimera formation and affected relative abundance estimates of species with high GC content. Libraries were sequenced using the Illumina HiSeq and MiSeq platforms to demonstrate that this protocol is highly scalable to sequence thousands of samples at a very low cost.\n\nConclusionsHere, we provide the most comprehensive study of performance and bias inherent to a 16S rRNA gene amplicon sequencing method to date. Triple-indexing greatly reduces the number of long custom DNA oligos required for library preparation, while the inclusion of variable length heterogeneity spacers minimizes the need for PhiX spike-in. This design results in a significant cost reduction of highly multiplexed amplicon sequencing. The biases we characterize highlight the need for highly standardized protocols. Reassuringly, we find that the biological signal is a far stronger structuring factor than the various sources of bias.

microbiology↗

bioRxiv2017

FtsEX-Mediated Regulation Of Inner Membrane Fusion And Cell Separation Reveals Morphogenetic Plasticity In Caulobacter crescentus

During its life cycle, Caulobacter crescentus undergoes a series of coordinated shape changes, including generation of a polar stalk and reshaping of the cell envelope to produce new daughter cells through the process of cytokinesis. The mechanisms by which these morphogenetic processes are coordinated in time and space remain largely unknown. Here we demonstrate that the conserved division complex FtsEX controls both the early and late stages of cytokinesis in C. crescentus, namely initiation of constriction and final cell separation. {Delta}ftsE cells display a striking phenotype: cells are chained, with skinny connections between cell bodies resulting from defects in inner membrane fusion and cell separation. Surprisingly, the thin connections in {Delta}ftsE cells share morphological and molecular features with C. crescentus stalks. Our data uncover unanticipated morphogenetic plasticity in C. crescentus, with loss of FtsE causing a stalk-like program to take over at failed division sites and yield novel cell morphology.\n\nAuthor SummaryBacterial cell shape is genetically hardwired and is critical for fitness and, in certain cases, pathogenesis. In most bacteria, a semi-rigid structure called the cell wall surrounds the inner membrane, offering protection against cell lysis while simultaneously maintaining cell shape. A highly dynamic macromolecular structure, the cell wall undergoes extensive remodeling as bacterial cells grow and divide. We demonstrate that a broadly conserved cell division complex, FtsEX, relays signals from the cytoplasm to the cell wall to regulate key developmental shape changes in the -proteobacterium Caulobacter crescentus. Consistent with studies in diverse bacteria, we observe strong synthetic interactions between ftsE and cell wall hydrolytic factors, suggesting that regulation of cell wall remodeling is a conserved function of FtsEX. Loss of FtsE causes morphological defects associated with both the early and late stages of division. Intriguingly, without FtsE, cells frequently fail to separate and instead elaborate a thin, tubular structure between cell bodies, a growth mode observed in other -proteobacteria. Overall, our results highlight the plasticity of bacterial cell shape and demonstrate how altering the activity of one morphogenetic program can produce diverse morphologies resembling those of other bacteria in nature.

microbiology↗