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bioRxiv2017

Linking soil biology and chemistry using bacterial isolate exometabolite profiles

Sequencing provides a window into microbial community structure and metabolic potential; however, linking these data to exogenous metabolites that microorganisms process and produce (the exometabolome) remains challenging. Previously, we observed strong exometabolite niche partitioning among bacterial isolates from biological soil crust (biocrust). Here we examine native biocrust to determine if these patterns are reproduced in the environment. Overall, most soil metabolites displayed the expected relationship (positive or negative correlation) with four dominant bacteria following a wetting event and across biocrust developmental stages. For metabolites that were previously found to be consumed by an isolate, 78% were negatively correlated with the abundance of in situ isolate phylotypes whereas for released metabolites, 73% were positively correlated. Our results demonstrate that metabolite profiling, sequencing and exometabolomics can be successfully integrated to functionally link metagenomes and microbial community structure with environmental chemistry.

microbiology↗

bioRxiv2017

Studies in Particle Sorting by Paramecium Cilia Arrays

Motile cilia are cell-surface organelles whose purposes, in ciliated protists and certain ciliated vertebrate epithelia, include generating fluid flow, chemosensation, mechanosensation and substance uptake. Certain properties of cilia arrays, such as beating synchronisation and manipulation of external proximate particulate matter, are considered emergent, but remain incompletely characterised despite these phenomena having being the subject of extensive modelling. This study constitutes a laboratory experimental characterisation of one of the emergent properties of motile cilia; microparticle manipulation. The work demonstrates through automated videomicrographic particle tracking that interactions between microparticles and somatic cilia arrays of the ciliated model organism Paramecium caudatum constitute a form of rudimentary sorting. Small particles are drawn into the organisms proximity by cilia-induced fluid currents at all times, whereas larger particles may be held immobile at a distance from the cell margin when the cell generates characteristic feeding currents in the surrounding media. These findings can contribute to the design and fabrication of biomimetic cilia, with potential applications to the study of ciliopathies.

microbiology↗

bioRxiv2017

Toxicity and applications of internalised magnetite nanoparticles within live Paramecium caudatum cells

The nanotechnology revolution has allowed us to speculate on the possibilities of hybridising nanoscale materials with live substrates, yet significant doubt still remains pertaining to the effects of nanomaterials on biological matter. In this investigation we cultivate the ciliated protistic pond-dwelling microorganism Paramecium caudatum in the presence of excessive quantities of magnetite nanoparticles in order to assess both potential beneficial applications for this technique as well as any deleterious effects on the organisms health. Our findings indicate that these nanoparticles are well-tolerated by paramecia, who were observed to consume in quantities exceeding 10% of their body volume: cultivation in the presence of magnetite nanoparticles does not alter P. caudatum cell volume, swim speed, growth rate or peak colony density and cultures may persist in nanoparticle-contaminated medium for many weeks. We demonstrate that P. caudatum cells ingest starch coated magnetite nanoparticles which facilitates their being magnetically immobilised whilst maintaining apparently normal ciliary dynamics, thus demonstrating that nanoparticle biohybridisation is a viable alternative to conventional forms of ciliate quieting. Ingested magnetite nanoparticle deposits appear to aggregate, suggesting that (a) the process of being internalised concentrates and therefore detoxifies nanomaterial suspensions in aquatic environments and (b) P. caudatum is a candidate organism for programmable nanomaterial manipulation and delivery.

microbiology↗

bioRxiv2017

MinE recruits, stabilizes, releases, and inhibits MinD interactions with membrane to drive oscillation

The MinD and MinE proteins of Escherichia coli self-organize into a standing-wave oscillator on the membrane to help align division at mid-cell. When unleashed from cellular confines, we find that MinD and MinE form a wide spectrum of patterns on artificial bilayers - static amoebas, traveling waves, traveling mushrooms, and bursts with standing-wave dynamics. We recently focused our cell-free studies on bursts because their dynamics closely resemble those found in vivo. The data unveiled a patterning mechanism largely governed by MinE regulation of MinD interaction with membrane. We proposed that the MinD to MinE ratio on the membrane acts as a toggle switch between MinE-stimulated recruitment or release of MinD from the membrane. Here we provide data that further refines and extends our model that explains the remarkable spectrum of patterns supported by these two simple proteins.

microbiology↗

bioRxiv2017

Are oceanic fronts ecotones? Seasonal changes along the Subtropical Front show fronts as bacterioplankton transition zones but not diversity hotspots

Ecotones are regarded as diversity hotspots in terrestrial systems, but it is unknown if this \"ecotone effect\" occurs in the marine environment. Oceanic fronts are widespread mesoscale features, present in the boundary between different water masses, and are arguably the best potential examples of ecotones in the ocean. Here we performed the first seasonal study along an oceanic front, combining 16S rRNA gene sequencing coupled with a high spatial resolution analysis of the physical properties of the water masses. Using the Subtropical Frontal Zone off New Zealand we demonstrate that fronts delimit shifts in bacterioplankton community composition between water masses, but that the strength of this effect is seasonally dependent. While creating a transition zone where physicochemical parameters and bacterioplankton communities get mixed, this ecotone does not result in increased diversity. Thus unlike terrestrial ecotones, oceanic ecotones like fronts are boundaries but not hotspot of bacterioplankton diversity in the ocean.

microbiology↗

bioRxiv2017

Bacterial outer membrane homeostasis via retrograde phospholipid transport

Biogenesis of the outer membrane (OM) in Gram-negative bacteria, which is essential for viability, requires the coordinated transport and assembly of proteins and lipids, including lipopolysaccharides (LPS) and phospholipids (PLs), into the membrane. While pathways for LPS and OM protein assembly are well-studied, how PLs are transported to and from the OM is not clear. Mechanisms that ensure OM stability and homeostasis are also unknown. The trans-envelope Tol-Pal complex, whose physiological role has remained elusive, is important for OM stability. Here, we establish that the Tol-Pal complex is required for PL transport and OM lipid homeostasis in Escherichia coli. Cells lacking the complex exhibit defects in lipid asymmetry and accumulate excess phospholipids (PLs) in the OM. This imbalance in OM lipids is due to defective retrograde PL transport in the absence of a functional Tol-Pal complex. Thus, cells ensure the assembly of a stable OM by maintaining an excess flux of PLs to the OM only to return the surplus to the inner membrane. Our findings also provide insights into the mechanism by which the Tol-Pal complex may promote OM invagination during cell division.

microbiology↗

bioRxiv2017

Local interactions lead to spatially correlated gene expression levels in bacterial groups.

Many bacteria live in spatially structured assemblies where the microenvironment of a cell is shaped by the activities of its neighbors. Bacteria regulate their gene expression based on the inferred state of the environment. This raises the question whether the phenotypes of neighboring cells can become correlated through interactions via the shared microenvironment. Here, we addressed this question by following gene expression dynamics in Escherichia coli microcolonies. We observed strong spatial correlations in the expression dynamics for pathways involved in toxin production, SOS-stress response, and metabolism. These correlations can partly be explained by a combination of shared lineage history and spatial gradients in the colony. Interestingly, we also found evidence for cell-cell interactions in SOS-stress response, methionine biosynthesis and overall metabolic activity. Together our data suggests that intercellular feedbacks can couple the phenotypes of neighboring cells, raising the question whether gene-regulatory networks have evolved to spatially organize biological functions.

microbiology↗

bioRxiv2017

The mutation rates and mutational bias of influenza A virus

Influenza virus has a high mutation rate, and this low replicative fidelity contributes to its capacity for rapid evolution. Clonal sequencing and fluctuation tests have suggested that the mutation rate of influenza A virus is 7.1 x 10-6 - 4.5 x 10-5 substitutions per nucleotide per cell infection cycle and 2.7 x 10-6 - 3.0 x 10-5 substitutions per nucleotide per strand copied (s/n/r). However, sequencing assays are biased toward mutations with minimal impacts on viral fitness and fluctuation tests typically investigate only a subset of the twelve mutational classes. We developed a fluctuation test based on reversion to fluorescence in a set of virally encoded mutant green fluorescent proteins. This method allowed us to measure the rates of selectively neutral mutations representative of all 12 mutational classes in the context of an unstructured RNA. We measured an overall mutation rate of 1.8 x 10-4 s/n/r for PR8 (H1N1) and 2.5 x 10-4 s/n/r for Hong Kong 2014 (H3N2). The replication mode was linear. The mutation rates of these divergent strains are significantly higher than previous estimates and suggest that each replicated genome will have an average of 2-3 mutations. The viral mutational spectrum is heavily biased toward A to G and U to C transitions, resulting in a transition to transversion bias of 2.7 and 3.6 for the two strains. These mutation rates were relatively constant over a range of physiological temperatures. Our high-resolution analysis of influenza virus mutation rates will enable more refined models of its molecular evolution.\n\nSignificanceThe rapid evolution of influenza virus is a major problem in public health. A key factor driving this rapid evolution is the virus very high mutation rate. We developed a new method for measuring the rates of all 12 mutational classes in influenza virus, which eliminates some of the biases of existing assays. We find that the influenza virus mutation rate is much higher than previously reported and is consistent across two distinct strains and a range of temperatures. Our data suggest that influenza viruses replicate at their maximally tolerable mutation rates, highlighting both the virus evolutionary potential and its significant constraints.

microbiology↗

bioRxiv2017

Global Proteomic Analyses of Macrophage Response to Mycobacterium tuberculosis Infection

Alveolar macrophages serve as the first line of defence against microbial infection, yet provide a unique niche for the growth of Mycobacterium tuberculosis. To better understand the evasive nature of the tubercle bacilli and its molecular manifest on the macrophage response to infection, we conducted a global quantitative proteomic profile of infected macrophages. By examining four independent controlled infection experiments, we detected 42,007 peptides resulting in the characterization of 4,868 distinct proteins. Of these, we identified 845 macrophage proteins whose expression is modulated upon infection in all replicates. We showed that the macrophages response to M. tuberculosis infection includes simultaneous and concerted upregulation of selected proteins. Using a number of statistical methods, we identified 27 proteins whose expression levels are significantly regulated outside of a 90% confidence interval about the mean. These host proteins represent the macrophage transcriptional, translational, and innate immune response to infection as well as its signaling capacity. The contribution of PtpA, an M. tuberculosis secreted virulence factor, modulated the expression levels of 11 host macrophage proteins, as categorized by RNA metabolism, translation, and cellular respiration.

microbiology↗

bioRxiv2017

Soil classification predicts differences in prokaryotic communities across a range of geographically distant soils once pH is accounted for

Agricultural land is typically managed based on visible plant life at the expense of the belowground majority. However, microorganisms mediate processes sustaining plant life and the soil environment. To understand the role of microbes we first must understand what controls soil microbial community assembly. We assessed the distribution and composition of prokaryotic communities from soils representing four geographic regions on the South Island of New Zealand. These soils are under three different uses (dairy, sheep and beef, and high country farming) and are representative of major soil classification groups (brown, pallic, gley and recent). We hypothesized that pH would account for major community patterns based on 16S profiles, but that land use and location would be secondary modifiers. Community diversity and structure was linked to pH, coinciding with land use. Soil classification correlated with microbial community structure and evenness, but not richness in high country and sheep and beef communities. The impact of land use and pH remained significant at the regional scale, but soil classification provided support for community variability not explained by either of those factors. These results suggest that several edaphic properties must be examined at multiple spatial scales to robustly examine soil prokaryotic communities.

microbiology↗

bioRxiv2017

Stable association of a Drosophila-derived microbiota with its animal partner and the nutritional environment upon transfer between populations and generations

In the past years, the fruit fly Drosophila melanogaster has been extensively used to study the relationship between animals and their associated microbes. Compared to the one of wild populations, the microbiota of laboratory-reared flies is less diverse, and comprises fewer bacterial taxa; nevertheless, the main commensal bacteria found in fly microbiota always belong to the Acetobacteraceae and Lactobacillaceae families. The bacterial communities associated with the fly are environmentally acquired, and the partners engage in a perpetual re-association process. Adult flies constantly ingest and excrete microbes from and onto their feeding substrate, which are then transmitted to the next generation developing within this shared habitat. We wanted to analyze the potential changes in the bacterial community during its reciprocal transfer between the two compartments of the niche (i.e. the fly and the diet). To address this question, we used a diverse, wild-derived microbial community and analyzed its relationship with the fly population and the nutritive substrate in a given habitat. Here we show that the community was overall well maintained upon transmission to a new niche, to a new fly population and to their progeny, illustrating the stable association of a Drosophila-derived microbiota with its fly partner and the nutritional environment. These results highlight the preponderant role of the nutritional substrate in the dynamics of Drosophila/microbiota interactions, and the need to fully integrate this variable when performing such studies.

microbiology↗

bioRxiv2017

How to name and classify your phage: an informal guide

With this informal guide, we try to assist both new and experienced phage researchers through two important stages that follow phage discovery, i.e. naming and classification. Providing an appropriate name for a bacteriophage is not as trivial as it sounds and the effects might be long-lasting in databases and in official taxon names. Phage classification is the responsibility of the Bacterial and Archaeal Viruses Subcommittee (BAVS) of the International Committee on the Taxonomy of Viruses (ICTV). While the BAVS aims at providing a holistic approach to phage taxonomy, for individual researchers who have isolated and sequenced a new phage, this can be a little overwhelming. We are now providing these researchers with an informal guide to phage naming and classification, taking a \"bottom-up\" approach from the phage isolate level.

microbiology↗

bioRxiv2017

Eelgrass leaf surface microbiomes are locally variable and highlycorrelated with epibiotic eukaryotes

Eelgrass (Zostera marina) is a marine foundation species essential for coastal ecosystem services around the northern hemisphere. Like all macroscopic organisms, it possesses a microbiome which may play critical roles in modulating the interaction of eelgrass with its environment. For example, its leaf surface microbiome could inhibit or attract eukaryotic epibionts which may overgrow the eelgrass leading to reduced primary productivity and subsequent eelgrass meadow decline. We used amplicon sequencing of the 16S and 18S rRNA genes of prokaryotes and eukaryotes to assess the leaf surface microbiome (prokaryotes) as well as eukaryotic epibionts in-and outside lagoons on the German Baltic Sea coast. Bacterial microbiomes varied substantially both between sites inside lagoons and between open coastal and lagoon sites. Water depth, leaf area and biofilm chlorophyll a concentration explained a large amount of variation in both bacterial and eukaryotic community composition. Communities of bacterial and eukaryotic epibionts were highly correlated, and network analysis revealed disproportionate co-occurrence between a limited number of eukaryotic taxa and several bacterial taxa. This suggests that eelgrass leaf surface biofilms are a mosaic of the microbiomes of several eukaryotes, in addition to that of the eelgrass itself, and underlines that eukaryotic microbial diversity should be taken into account in order to explain microbiome assembly and dynamics in aquatic environments.

microbiology↗

bioRxiv2017

A unifying mechanism for the biogenesis of prokaryotic membrane proteins co-operatively integrated by the Sec and Tat pathways

The vast majority of polytopic membrane proteins are inserted into the cytoplasmic membrane of prokaryotes by the general secretory (Sec) pathway. However, a subset of monotopic proteins that contain non-covalently-bound redox cofactors depend on the twin-arginine translocase (Tat) machinery for membrane integration. Recently actinobacterial Rieske iron-sulfur cluster-containing proteins were identified as an unusual class of membrane proteins that require both the Sec and Tat pathways for the insertion of their three transmembrane domains (TMDs). The Sec pathway inserts the first two TMDs of these proteins co-translationally, but releases the polypeptide prior to the integration of TMD3 to allow folding of the cofactor-containing domain and its translocation by Tat. Here we have investigated features of the Streptomyces coelicolor Rieske polypeptide that modulate its interaction with the Sec and Tat machineries. Mutagenesis of a highly conserved loop region between Sec-dependent TMD2 and Tat-dependent TMD3 shows that it plays no significant role in coordinating the activities of the two translocases, but that a minimum loop length of approximately eight amino acids is required for the Tat machinery to recognise TMD3. Instead we show that a combination of relatively low hydrophobicity of TMD3, coupled with the presence of C-terminal positively-charged amino acids, results in abortive insertion of TMD3 by the Sec pathway and its release at the cytoplasmic side of the membrane. Bioinformatic analysis identified two further families of polytopic membrane proteins that share features of dual Sec-Tat-targeted membrane proteins. A predicted heme-molybdenum cofactor-containing protein with five TMDs, and a polyferredoxin also with five predicted TMDs, are encoded across bacterial and archaeal genomes. We demonstrate that membrane insertion of representatives of each of these newly-identified protein families is dependent on more than one protein translocase, with the Tat machinery recognising TMD5. Importantly, the combination of low hydrophobicity of the final TMD and the presence of multiple C-terminal positive charges that serve as critical Sec-release features for the actinobacterial Rieske protein also dictate Sec release in these further protein families. Therefore we conclude that a simple unifying mechanism governs the assembly of dual targeted membrane proteins.

microbiology↗

bioRxiv2017

Altering lipid droplet homeostasis affects Coxiella burnetii intracellular growth

Coxiella burnetii is an obligate intracellular bacterial pathogen and a causative agent of culture-negative endocarditis. While C. burnetii initially infects alveolar macrophages, it has also been found in lipid droplet (LD)-containing foamy macrophages in the cardiac valves of endocarditis patients. In addition, transcriptional studies of C. burnetii-infected macrophages reported differential regulation of the LD coat protein-encoding gene perilipin 2 (plin-2). To further investigate the relationship between LDs and C. burnetii, we compared LD numbers using fluorescence microscopy in mock-infected and C. burnetii-infected alveolar macrophages. On average, C. burnetii-infected macrophages contained twice as many LDs as mock-infected macrophages. LD numbers increased as early as 24 hours post-infection, an effect reversed by blocking C. burnetii protein synthesis. The observed LD accumulation was dependent on the C. burnetii Type 4B Secretion System (T4BSS), a major virulence factor that manipulates host cellular processes by secreting bacterial effector proteins into the host cell cytoplasm. To determine the importance of LDs during C. burnetii infection, we manipulated LD homeostasis and assessed C. burnetii intracellular growth. Surprisingly, blocking LD formation with the pharmacological inhibitors triacsin C or T863, or knocking out acyl-CoA transferase-1 (acat-1) in alveolar macrophages, increased C. burnetii growth at least 2-fold. Conversely, preventing LD lipolysis by inhibiting adipose triglyceride lipase (ATGL) with atglistatin almost completely blocked bacterial growth, suggesting LD breakdown is essential for C. burnetii. Together these data suggest that maintenance of LD homeostasis, possibly via the C. burnetii T4BSS, is critical for bacterial growth.\n\nIMPORTANCEHost neutral lipid storage organelles known as lipid droplets (LDs) serve as a source of energy, nutrients, and signaling lipids. LDs are associated with infection of the intracellular bacterial pathogen Coxiella burnetii, a significant cause of culture-negative endocarditis. While C. burnetii was found in LD-rich foamy macrophages in endocarditis patients, little is known about the host LD-C. burnetii relationship. We demonstrated C. burnetii Type 4B Secretion System (T4BSS)-dependent LD accumulation in macrophages, suggesting a T4BSS-mediated regulation of host LD homeostasis. Further, manipulating LD homeostasis significantly affected bacterial growth, indicating LDs play an important role during C. burnetii infection. As C. burnetii endocarditis has a 19% mortality rate even in treated patients, exploring the LD-C. burnetii association might identify novel therapeutic targets.

microbiology↗

bioRxiv2017

Cohesion: A method for quantifying the connectivity of microbial communities

The ability to predict microbial community dynamics lags behind the quantity of data available in these systems. Most predictive models use only environmental parameters, although a long history of ecological literature suggests that community complexity should also be an informative parameter. Thus, we hypothesize that incorporating information about a communitys complexity might improve predictive power in microbial models. Here, we present a new metric, called community \"cohesion,\" that quantifies the degree of connectivity of a microbial community. We validate our approach using long-term (10+ year) phytoplankton datasets, where absolute abundance counts are available. As a case study of our metrics utility, we show that community cohesion is a strong predictor of Bray-Curtis dissimilarity (R2 = 0.47) between phytoplankton communities in Lake Mendota, WI, USA. Our cohesion metrics outperform a model built using all available environmental data collected during a long-term sampling program. The result that cohesion corresponds strongly to Bray-Curtis dissimilarity is consistent across the five lakes analyzed here. Our cohesion metrics can be used as a predictor for many community-level properties, such as phylogenetic diversity, nutrient fluxes, or ecosystem services. We explain here the calculation of our cohesion metrics and their potential uses in microbial ecology.\n\nConflict of InterestThe authors declare no conflict of interest.

microbiology↗

bioRxiv2017

Varying Effects of Common Tuberculosis Drugs on Enhancing Clofazimine Activity in vitro

Clofazimine (CFZ), originally developed as an anti-tuberculosis (TB) drug in the 1950s 1, is commonly used to treat leprosy and also nontuberculous mycobacterial (NTM) infections. 2 Although CFZ has good activity against Mycobacterium tuberculosis, it was not used in the treatment of pulmonary TB mainly because it had the side effect of skin discoloration and there were other more effective drugs like isoniazid (INH), rifampin (RIF) and pyrazinamide (PZA) already available for the treatment of TB. 2 However, the increasing emergence of multi-drug-resistant TB (MDR-TB) has revived interest in the use of CFZ to treat MDR-TB. 2,3\n\nAlthough resistance to CFZ has been shown to be mediated by mutations in Rv0678,4,5 Rv1979c, or Rv2535c (PepQ),5 the mode of action of CFZ has remained poorly understood. CFZ appears to hav ...

microbiology↗

bioRxiv2017

Activity of Sulfa Drugs and Their Combinations against Stationary Phase B. burgdorferi in vitro

Lyme disease is a most common vector borne disease in the US. Although the majority of Lyme patients can be cured with the standard 2-4 week antibiotic treatment, at least 10-20% of patients continue to suffer from prolonged post-treatment Lyme disease syndrome (PTLDS). While the cause for this is unclear, one possibility is that persisting organisms are not killed by current Lyme antibiotics. In our previous studies, we screened an FDA drug library and an NCI compound library on B. burgdorferi and found some drug hits including sulfa drugs as having good activity against B. burgdorferi stationary phase cells. In this study, we evaluated the relative activity of three commonly used sulfa drugs sulfamethoxazole (Smx), dapsone (Dps), sulfachlorpyridazine (Scp), and also trimethoprim (Tmp), and assessed their combinations with the commonly prescribed Lyme antibiotics for activities against B. burgdorferi stationary phase cells. Using the same molarity concentration, dapsone, sulfachlorpyridazine and trimethoprim showed very similar activity against stationary phase B. burgdorferi enriched in persisters, however, sulfamethoxazole was the least active drug among the three sulfa drugs tested. Interestingly, contrary to other bacterial systems, Tmp did not show synergy in drug combinations with the three sulfa drugs at their clinically relevant serum concentrations against B. burgdorferi. We found that sulfa drugs combined with other antibiotics were more active than their respective single drugs and that four-drug combinations were more active than three-drug combinations. Four drug combinations dapsone+minocycline+cefuroxime+azithromycin and dapsone+minocycline+cefuroxime+rifampin showed best activity against stationary phase B. burgdorferi in these sulfa drug combinations. However, these 4-sulfa drug containing combinations still had considerably less activity against B. burgdorferi stationary phase cells than the daptomycin+cefuroxime+doxycycline used as a positive control which completely eradicated B. burgdorferi stationary phase cells. Future studies are needed to evaluate and optimize the sulfa drug combinations in vitro and also in animal models.

microbiology↗