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bioRxiv2016

A bacterial chaperone is required for plastid function in malaria parasites

Apicomplexan parasites such as Plasmodium falciparum, the causative agent of malaria, contain a non-photosynthetic plastid known as the apicoplast that functions to produce essential metabolic compounds. It was previously reported that several members of the Clp family of chaperones and proteases localize to the apicoplast. In bacteria and in chloroplasts these proteins form complexes that degrade proteins in a proteasome-like manner to regulate key cellular processes, but their function in the apicoplast is completely unknown. In this study, we generated a conditional mutant of the P. falciparum apicoplast-targeted pfclpc gene and found that under normal conditions it localizes to the apicoplast. Knockdown of PfClpC results in growth inhibition and morphological defects, indicating that PfClpC is essential for parasite viability. Upon inhibition, PfClpC loses its apicoplast localization and appears in vesicle-like structures. Other apicoplast-targeted proteins also localize to these structures, suggesting that organelle integrity is compromised. Addition of isopentynyl pyrophosphate completely rescued the growth inhibition, indicating that the only essential function of PfClpC is related to the apicoplast. Moreover, cellular assays suggest that PfClpC inhibition interferes with the ability of the schizont-stage parasites to properly sort functional apicoplast organelles into daughter-merozoites. These data show that PfClpC is an essential gene that functions to maintain apicoplast integrity.\n\nAuthor SummaryThe deadly human malaria parasite, Plasmodium falciparum, contains a unique organelle called the apicoplast, a non-photosynthetic plastid that produces vital metabolites. Members of the prokaryotic-derived Clp family were previously reported to localize to the apicoplast. In bacteria and plant chloroplasts, Clp homologs form a proteasome-like complex that degrade proteins but their function in parasite biology is unknown. Here we took a conditional knockdown approach to study an apicoplast localized Clp proteins, PfClpC, which we found to be essential for parasite viability. Inhibition of PfClpC results in a growth arrest phenotype that correlates with a reduced replication rate. We observed that PfClpC localizes to the apicoplast, however upon inhibition it is found dispersed in vesicle-like structures suggesting a complete breakdown of organelle integrity. Our ability to rescue the phenotype by adding an essential apicoplast-derived metabolite proved that the only essential function of PfClpC is linked to apicoplast function. Furthermore, we have found evidence supporting a role for PfClpC in apicoplast sorting into daughter cells. Therefore, we propose PfClpC as a potential drug target due to its essentiality, prokaryotic origin and absence from the human host.

Microbiology↗

bioRxiv2016

Functional analysis of a biosynthetic cluster essential for production of 4-formylaminooxyvinylglycine, a germination-arrest factor from Pseudomonas fluorescens WH6

Rhizosphere-associated Pseudomonas fluorescens WH6 produces the germination-arrest factor, 4-formylaminooxyvinylglycine (FVG). FVG has previously been shown to both arrest the germination of weedy grasses and to inhibit the growth of the bacterial plant pathogen Erwinia amylovora. Very little is known about the mechanism by which FVG is produced. Although a previous study identified a region of the genome that may be involved in FVG biosynthesis, it has not yet been determined which genes within that region are sufficient and necessary for FVG production. In the current study, we explored the role of each of the putative genes encoded in that region by constructing deletion mutations. Mutant strains were assayed for their ability to produce FVG with a combination of biological assays and thin-layer chromatographic analyses. This work defined the core FVG biosynthetic gene cluster and revealed several interesting characteristics of FVG production. We determined that FVG biosynthesis requires two small open reading frames of less than 150 nucleotides and that multiple transporters have overlapping but distinct functionality. In addition, two genes in the center of the biosynthetic gene cluster are not required for FVG production, suggesting that additional products may be produced from the cluster. Transcriptional analysis indicated that at least three active promoters play a role in the expression of genes within this cluster. The results of this study enrich our knowledge regarding the diversity of mechanisms by which bacteria produce non-proteinogenic amino acids like vinylglycines.

Microbiology↗

bioRxiv2016

Structure and culture of the gut microbiome of the Mormon cricket Anabrus simplex

The gut microbiome of insects plays an important role in their ecology and evolution, participating in nutrient acquisition, immunity, and behavior. Microbial community structure within the gut is thought to be heavily influenced by differences among gut regions in morphology and physiology, which determine the niches available for microbes to colonize. We present a high-resolution analysis of the structure of the gut microbiome in the Mormon cricket Anabrus simplex, an insect known for its periodic outbreaks in the Western United States and nutrition-dependent mating system. The Mormon cricket microbiome was dominated by eleven bacterial phylotypes from the Lactobacillaceae, Enterobacteriaceae, and Streptococcaeae. While most of these were represented in all gut regions, there were marked differences in their relative abundance, with lactic-acid bacteria (Lactobacillaceae) more common in the foregut and midgut and enteric (Enterobacteriaceae) bacteria more common in the hindgut. Differences in community structure were driven by variation in the relative prevalence of three groups: a Lactobacillus phylotype in the foregut, Pediococcus lactic-acid bacteria in the midgut, and Pantoea agglomerans, an enteric bacterium, in the hindgut. These taxa have been shown to have beneficial effects on their hosts in insects and other animals by improving nutrition, increasing resistance to pathogens, and modulating social behavior. Phylogenetic analysis of 16s rRNA sequences from cultured isolates indicated low levels of divergence from sequences derived from plants and other insects, suggesting that these bacteria are likely to be exchanged between Mormon crickets and the environment. Our study provides the foundation for future work on an economically important insect and emerging model for the study of how social interaction influence host-microbe symbiosis.

microbiology↗

bioRxiv2016

Rapid and consistent evolution of colistin resistance in Pseudomonas aeruginosa during morbidostat culture

Colistin is a last resort antibiotic commonly used against multidrug-resistant strains of Pseudomonas aeruginosa. To investigate the potential for in-situ evolution of resistance against colistin and map the molecular targets of colistin resistance, we exposed two P. aeruginosa isolates to colistin using a continuous culture device known as morbidostat. Colistin resistance emerged within two weeks along with highly stereotypic yet strain specific mutation patterns. The majority of mutations hit the prmAB two component signaling system and genes involved in lipopolysaccharide synthesis, including lpxC, pmrE, and migA. In seven out of 18 cultures, we observed mutations in mutS along with a mutator phenotype that seemed to facilitate resistance evolution.

microbiology↗

bioRxiv2016

Deconstructing cell size control into physiological modules in Escherichia coli

It is generally assumed that the allocation and synthesis of total cellular resources in microorganisms are uniquely determined by the growth conditions. Adaptation to a new physiological state leads to a change in cell size via reallocation of cellular resources. However, it has not been understood how cell size is coordinated with biosynthesis and robustly adapts to physiological states. We show that cell size in Escherichia coli can be predicted for any steady-state condition by projecting all biosynthesis into three measurable variables representing replication initiation, replication-division cycle, and the global biosynthesis rate. These variables can be decoupled by selectively controlling their respective core biosynthesis using CRISPR interference and antibiotics, verifying our predictions that different physiological states can result in the same cell size. We performed extensive growth inhibition experiments, and discovered that cell size at replication initiation per origin, namely the initiation mass or \"unit cell,\" is remarkably invariant under perturbations targeting transcription, translation, ribosome content, replication kinetics, fatty acid and cell-wall synthesis, cell division, and cell shape. Based on this invariance and balanced resource allocation, we explain why the total cell size is the sum of all unit cells. These results provide an overarching framework with quantitative predictive power over cell size in bacteria.

microbiology↗

bioRxiv2016

Aer is a family of Energy-Taxis Receptors in Pseudomonas which also involves Aer-2 and CttP

Chemotaxis allows bacteria to sense gradients in their environment and respond by directing their swimming. Aer is a receptor that, instead of responding to a specific chemoattractant, allows bacteria to sense cellular energy levels and move towards favourable environments. In Pseudomonas, the number of apparent Aer homologs differs between the only two species it had been characterized in, P. aeruginosa and P. putida. Here we combined bioinformatic approaches with deletional mutagenesis in P. pseudoalcaligenes KF707 to further characterize Aer. It was determined that the number of Aer homologs varies between 0-4 throughout the Pseudomonas genus, and they were phylogenetically classified into 5 subgroups. We also used sequence analysis to show that these homologous receptors differ in their HAMP signal transduction domains. Genetic analysis also indicated that some Aer homologs have likely been subject to horizontal transfer. P. pseudoalcaligenes KF707 was unique among species for having three Aer homologs as well as the receptors CttP and McpB. Phenotypic characterization in this species showed the most prevalent homolog of Aer was key, but not essential for energy-taxis. This study demonstrates that energy-taxis in Pseudomonas varies between species and provides a new naming convention and associated phylogenetic details for Aer chemoreceptors.

microbiology↗

bioRxiv2016

Studying vertical microbiome transmission from mothers to infants by strain-level metagenomic profiling

The gut microbiome starts to be shaped in the first days of life and continues to increase its diversity during the first months. Several investigations are assessing the link between the configuration of the infant gut microbiome and infant health, but a comprehensive strain-level assessment of vertically transmitted microbes from mother to infant is still missing. We longitudinally collected fecal and breast milk samples from multiple mother-infant pairs during the first year of life, and applied shotgun metagenomic sequencing followed by strain-level profiling. We observed several specific strains including those from Bifidobacterium bifidum, Coprococcus comes, and Ruminococcus bromii, that were present in samples from the same mother-infant pair, while being clearly distinct from those carried by other pairs, which is indicative of vertical transmission. We further applied metatranscriptomics to study the in vivo expression of vertically transmitted microbes, for example Bacteroides vulgatus and Bifidobacterium spp., thus suggesting that transmitted strains are functionally active in the two rather different environments of the adult and infant guts. By combining longitudinal microbiome sampling and newly developed computational tools for strain-level microbiome analysis, we showed that it is possible to track vertical transmission of members of the microbiome from mother to infants and characterize their transcriptional activity. Our work poses the basis for surveying at larger scale the sources of microbial diversity in the infants and starts associating these transmissions with the subsequent longer-term development of a healthy or dysbiotic microbiome.\n\nImportanceEarly infant exposure is important in the acquisition and ultimate development of a healthy infant microbiome. There is increasing support that the maternal microbial reservoir is a key route of microbial transmission, yet much is inferred from the observation of shared species in mother and infant. Common species, per se, does not necessarily equate vertical transmission as species exhibit considerable strain heterogeneity and it is therefore imperative to identify shared strains. We demonstrate here the potential of shotgun metagenomics and strain-level resolution to identify vertical transmission events via the maternal source. Combined with a metatranscriptomic approach, we show the potential not only to identify and track the fate of microbes in the early infant microbiome but also identify the metabolically active members. These approaches will ultimately provide important insights into the acquisition, development and community dynamics of the infant microbiome.

microbiology↗

bioRxiv2016

Population diversity jeopardizes the efficacy of antibiotic cycling

Treatment strategies that anticipate and respond to the evolution of pathogens are promising tools for combating the global rise of antibiotic resistance1-3. Mutations conferring resistance to one drug can confer positive or negative cross-resistance to other drugs4. The sequential use of drugs exhibiting negative cross-resistance has been proposed to prevent or slow down the evolution of resistance5-8, although factors affecting its efficacy have not been investigated. Here we show that population diversity can disrupt the efficacy of negative cross-resistance-based therapies. By testing 3317 resistant Staphylococcus aureus mutants against multiple antibiotics, we show that first-step mutants exhibit diverse cross-resistance profiles: even when the majority of mutants show negative cross-resistance, rare positive cross-resistant mutants can appear. Using a drug pair showing reciprocal negative cross-resistance, we found that selection for resistance to the first drug in small populations can decrease resistance to the second drug, but identical selection conditions in large populations can increases resistance to the second drug through the appearance of rare positive cross-resistant mutants. We further find that, even with small populations and strong bottlenecks, resistance to both drugs can increase through sequential steps of negative cross-resistance cycling. Thus, low diversity is necessary but not sufficient for effective cycling therapies. While evolutionary interventions are promising tools for controlling antibiotic resistance, they can be sensitive to population diversity and the accessibility of evolutionary paths, and so must be carefully designed to avoid harmful outcomes.

microbiology↗

bioRxiv2016

First report of the East-Central South African Genotype of Chikungunya Virus in Rio de Janeiro, Brazil

BackgroundChikungunya virus (CHIKV) is an arbovirus that causes an acute febrile illness characterized by severe and debilitating arthralgia. In Brazil, the Asian and East-Central South African (ECSA) genotypes are circulating in the north and northeast of the country, respectively. In 2015, the first autochthonous cases in Rio de Janeiro, Brazil were reported but until now the circulating strains have not been characterized. Therefore, we aimed here to perform the molecular characterization and phylogenetic analysis of CHIKV strains circulating in the 2016 outbreak occurred in the municipality of Rio de Janeiro.\n\nMethodsThe cases analyzed in this study were collected at a private Hospital, from April 2016 to May 2016, during the chikungunya outbreak in Rio de Janeiro, Brazil. All cases were submitted to the Real Time RT-PCR for CHIKV genome detection and to anti-CHIKV IgM ELISA. Chikungunya infection was laboratorially confirmed by at least one diagnostic method and, randomly selected positive cases (n=10), were partially sequenced (CHIKV E1 gene) and analyzed.\n\nResultsThe results showed that all the samples grouped in ECSA genotype branch and the molecular characterization of the fragment did not reveal the A226V mutation in the Rio de Janeiro strains analyzed, but a K211T amino acid substitution was observed for the first time in all samples and a V156A substitution in two of ten samples.\n\nConclusionsPhylogenetic analysis and molecular characterization reveals the circulation of the ECSA genotype of CHIKV in the city of Rio de Janeiro, Brazil and two amino acids substitutions (K211T and V156A) exclusive to the CHIKV strains obtained during the 2016 epidemic, were reported.

microbiology↗

bioRxiv2016

Effect of common and experimental anti-tuberculosis treatments on Mycobacterium tuberculosis growing as biofilms

Much is known regarding the antibiotic susceptibility of planktonic cultures of Mycobacterium tuberculosis, the bacterium responsible for the lung disease tuberculosis (TB). As planktonically-grown M. tuberculosis are unlikely to be entirely representative of the bacterium during infection, we set out to determine how effective a range of anti-mycobacterial treatments were against M. tuberculosis growing as a biofilm, a bacterial phenotype known to be more resistant to antibiotic treatment. Light levels from bioluminescently-labelled M. tuberculosis H37Rv (strain BSG001) were used as a surrogate for bacterial viability, and were monitored before and after 1 week of treatment. After treatment, biofilms were disrupted, washed and inoculated into fresh broth and plated onto solid media to rescue any surviving bacteria. We found that in this phenotypic state M. tuberculosis was resistant to the majority of the compounds tested. Minimum inhibitory concentrations (MICs) increased by 20-fold to greater than 1000-fold, underlying the potential of this phenotype to cause significant problems during treatment.

microbiology↗

bioRxiv2016

The fitness of Pseudomonas aeruginosa quorum sensing signal cheats is influenced by the diffusivity of the environment

Experiments examining the social dynamics of bacterial quorum sensing (QS) have focused on mutants which do not respond to signals, and the role of QS-regulated exoproducts as public goods. The potential for QS signal molecules to themselves be social public goods has received much less attention. Here, we analyse how signal-deficient (lasI-) mutants of the opportunistic pathogen Pseudomonas aeruginosa interact with wild-type cells in an environment where QS is required for growth. We show that when growth requires a private intracellular metabolic mechanism activated by the presence of QS signal, lasI- mutants act as social cheats and outcompete signal-producing wild-type bacteria in mixed cultures, because they can use the signals produced by wild type cells. However, reducing the ability of signal molecules to diffuse through the growth medium, results in signal molecules becoming less accessible to mutants, leading to reduced cheating. Our results indicate that QS signal molecules can be considered as social public goods in a way that has been previously described for other exoproducts, but that spatial structuring of populations reduces exploitation by non-cooperative signal cheats.\n\nImportanceBacteria communicate via signaling molecules to regulate the expression of a whole range of genes. This process, termed quorum sensing (QS), moderates bacterial metabolism in many environmental conditions, from soil and water (where QS-regulated genes influence nutrient cycling) to animal hosts (where QS-regulated genes determine pathogen virulence). Understanding the ecology of QS could therefore yield vital clues as to how we might modify bacterial behaviour for environmental or clinical gains. Here, we demonstrate that QS signals act as shareable public goods. This means that their evolution, and therefore population-level responses to interference with QS, will be constrained by population structure. Further, we show that environmental structure (constraints on signal diffusion) alters the accessibility of QS signals and demonstrates that we need to consider population and environmental structure to help us further our understanding of QS signaling systems.

microbiology↗

bioRxiv2016

Metabolomic and proteomic analyses of a quiescent Escherichia coli cell factory reveal the mechanisms behind its production efficiency

1Quiescent (Q-Cell) Escherichia coli cultures can be created by using the signalling molecule indole to halt cell division of an hns mutant strain. This uncouples metabolism from cell growth and allows for more efficient use of carbon feedstocks. However, the reason for the increased productivity of cells in this state was previously unknown. We show here that Q-cells can maintain metabolic activity in the absence of growth for up to 24 h, leading to four times greater per-cell productivity of a model metabolite, 3-hydroxybutyrate (3HB), than a control. Metabolomic data show that by disrupting the proton-motive force, indole interrupts the tricarboxylic acid cycle, leading to the accumulation of metabolites in the glycolysis pathway that are excellent starting points for high-value chemical production. By comparing protein expression patterns between wild-type and Q-cell cultures we show that Q-cells overexpress stress response proteins, which prime them to tolerate the metabolic imbalances incurred through indole addition. Quiescent cultures produced half the cell biomass of control cultures lacking indole, but were still able to produce 39.4 g.L-1 of 3HB compared to 18.6 g.L-1 in the control. Therefore, Q-cells have high potential as a platform technology for the efficient production of a wide range of commodity and high value chemicals.

microbiology↗

bioRxiv2016

The genome-wide transcriptional response to varying RpoS levels in Escherichia coli K-12

The alternative sigma factor RpoS is a central regulator of a many stress responses in Escherichia coli. The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We show that 23% of genes in the E. coli genome are regulated by RpoS level, and we identify many RpoS-transcribed genes and promoters. We observe three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the production of only a little RpoS (\"sensitive\" genes), and genes whose expression changes very little with the production of a little RpoS (\"insensitive\"). We show that sequences outside the core promoter region determine whether a RpoS-regulated gene in sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes, and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase, and may also contribute to the diversity of stress responses directed by RpoS.\n\nImportanceThe sigma factor RpoS is a global regulator that controls the response to many stresses in Escherichia coli. Different stresses result in different levels of RpoS production, but the consequences of this variation are unknown. We describe how changing the level of RpoS does not influence all RpoS-regulated genes equally. The cause of this variation is likely the action of transcription factors that bind the promoters of the genes. We show that the sensitivity of a gene to RpoS levels explains the timing of expression as cells enter stationary phase, and that genes with different RpoS sensitivities are enriched for specific functional groups. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes in response to stresses.

microbiology↗

bioRxiv2016

The molecular mechanism of the type IVa pilus motors

Type IVa pili are protein filaments essential for virulence in many bacterial pathogens; they extend and retract from the surface of bacterial cells to pull the bacteria forward with unprecedented force. They are used for attachment, swarming and twitching motility, biofilm formation, up-regulation of other virulence factors, and natural competence. The pilus is assembled by the motor subcomplex which consists of the inner membrane protein PilC and the cytoplasmic ATPase PilB. How PilB catalyzes this process is unknown, due in part to the lack of high-resolution structural information. Phylogenetic analysis of PilB-like ATPases, including GspE, PilT, BfpD, FlaI, and archaeal GspE2 revealed highly conserved residues essential for function in this family of ATPases. Here we report the structure of the core ATPase domains of Geobacter metalloreducens PilB bound to ADP and the non-hydrolysable ATP analogue, AMPPNP, at 3.4 and 2.3[A], respectively. Importantly, these structures were determined in non-saturating nucleotide conditions, revealing important differences in nucleotide binding between chains. Analysis of these differences revealed the sequential turnover of nucleotide by the chains, and the corresponding domain movements. Our data indicate a clockwise rotation of movement in PilB, which would support the assembly of a right-handed helical pilus. Conversely, our analysis suggests a counterclockwise rotation in PilT that would enable right-handed pilus disassembly. The proposed model provides insight into how this family of ATPases can power pilus extension and retraction with extraordinary forces.

microbiology↗

bioRxiv2016

Growth-independent cross-feeding modifies boundaries for coexistence in a bacterial mutualism

Nutrient cross-feeding can stabilize microbial mutualisms, including those important for carbon cycling in nutrient-limited anaerobic environments. It remains poorly understood how nutrient limitation within natural environments impacts mutualist growth, cross-feeding levels, and ultimately mutualism dynamics. We examined the effects of nutrient limitation within a mutualism using theoretical and experimental approaches with a synthetic anaerobic coculture pairing fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris. In this coculture, E. coli and R. palustris resemble an anaerobic food web by cross-feeding essential carbon (organic acids) and nitrogen (ammonium), respectively. Organic acid cross-feeding stemming from E. coli fermentation can continue in a growth-independent manner during nutrient limitation, while ammonium cross-feeding by R. palustris is growth-dependent. When ammonium cross-feeding was limited, coculture trends changed yet coexistence persisted under both homogenous and heterogenous conditions. Theoretical modeling indicated that growth-independent fermentation was crucial to sustain cooperative growth under conditions of low nutrient exchange. We also show that growth-independent fermentation sets the upper E. coli cell density at which this mutualism is supported. Thus, growth-independent fermentation can conditionally stabilize or destabilize a mutualism, indicating the potential importance of growth-independent metabolism for nutrient-limited mutualistic communities.\n\nConflict of interestThe authors declare no conflict of interest.

microbiology↗

bioRxiv2016

Inhibition of microbial biofuel production in drought stressed switchgrass hydrolysate

BackgroundInterannual variability in precipitation, particularly drought, can affect lignocellulosic crop biomass yields and composition, and is expected to increase biofuel yield variability. However, the effect of precipitation on downstream fermentation processes has never been directly characterized. In order to investigate the impact of interannual climate variability on biofuel production, corn stover and switchgrass were collected during three years with significantly different precipitation profiles, representing a major drought year (2012) and two years with average precipitation for the entire season (2010 and 2013). All feedstocks were AFEX (ammonia fiber expansion)-pretreated, enzymatically hydrolyzed, and the hydrolysates separately fermented using xylose-utilizing strains of Saccharomyces cerevisiae and Zymomonas mobilis. A chemical genomics approach was also used to evaluate the growth of yeast mutants in the hydrolysates.\n\nResultsWhile most corn stover and switchgrass hydrolysates were readily fermented, growth of S. cerevisiae was completely inhibited in hydrolysate generated from drought stressed switchgrass. Based on chemical genomics analysis, yeast strains deficient in genes related to protein trafficking within the cell were significantly more resistant to the drought year switchgrass hydrolysate. Detailed biomass and hydrolysate characterization revealed that switchgrass accumulated greater concentrations of soluble sugars in response to the drought and these sugars were subsequently degraded to pyrazines and imidazoles during ammonia-based pretreatment. When added ex situ to normal switchgrass hydrolysate, imidazoles and pyrazines caused anaerobic growth inhibition of S. cerevisiae.\n\nConclusionsIn response to the osmotic pressures experienced during drought stress, plants accumulate soluble sugars that are susceptible to degradation during chemical pretreatments. For ammonia-based pretreatment these sugars degrade to imidazoles and pyrazines. These compounds contribute to S. cerevisiae growth inhibition in drought year switchgrass hydrolysate. This work discovered that variation in environmental conditions during the growth of bioenergy crops could have significant detrimental effects on fermentation organisms during biofuel production. These findings are relevant to regions where climate change is predicted to cause an increased incidence of drought and to marginal lands with poor water holding capacity, where fluctuations in soil moisture may trigger frequent drought stress response in lignocellulosic feedstocks.

microbiology↗

bioRxiv2016

Genome scale mutational analysis of Geobacter sulfurreducens reveals distinct molecular mechanisms for respiration of poised electrodes vs. Fe(III) oxides

Geobacter sulfurreducens generates electricity by coupling intracellular oxidation of organic acids with electron transfer to the cell exterior, while maintaining a conductive connection to electrode surfaces. This unique ability has been attributed to the bacteriums capacity to also respire extracellular terminal electron acceptors that require contact, such as insoluble metal oxides. To expand the molecular understanding of electricity generation mechanisms, we constructed Geobacter sulfurreducens transposon mutant (Tn-Seq) libraries for growth with soluble fumarate or an electrode surface as the electron acceptor. Mutant libraries with over 33,000 unique transposon insertions and an average of 9 transposon insertions per kb allowed identification of 1,214 genomic features essential for growth with fumarate, including over 270 genes with one or more functional homologs that could not be resolved by previous annotation or in silico modeling. Tn-Seq analysis of electrode-grown cells identified mutations in over 50 genes encoding cytochromes, processing systems for proline-rich proteins, sensory systems, extracellular structures, polysaccharides, metabolic enzymes and hypothetical proteins that caused at least a 50% reduction in apparent growth rate. Scarless deletion mutants of genes identified via Tn-Seq revealed a new putative c-type cytochrome conduit complex (extABCD) essential for growth with electrodes, which was not required for Fe(III)-oxide reduction. In addition, mutants lacking components of a putative methyl-accepting chemotaxis/cyclic dinucleotide sensing network (esnABCD) were defective in electrode growth, but grew normally with Fe(III)-oxides. These results suggest that G. sulfurreducens possesses distinct mechanisms for recognition, colonization, and reduction of electrodes compared to other environmental electron acceptors.

microbiology↗

bioRxiv2016

Metabolic and spatio-taxonomic response of uncultivated seafloor bacteria following the Deepwater Horizon oil spill

The release of 700 million liters of oil into the Gulf of Mexico over a few months in 2010 produced dramatic changes in the microbial ecology of the water and sediment. Previous 4 studies have examined the phylogeny and function of these changes, but until now a 5 fundamental examination of the extant hydrocarbon metabolisms that supported these 6 changes had not been performed. Here, we reconstructed the genomes of 57 widespread 7 uncultivated bacteria from post spill sediments, and recovered their gene expression 8 pattern across the seafloor. These genomes comprised a common collection of bacteria 9 that were highly enriched in heavily affected sediments around the wellhead. While rare in distal sediments, some members were still detectable at sites up to 60 km away. Many of these genomes exhibited phylogenetic clustering indicative of common trait selection by the environment, and within half we identified 264 genes associated with hydrocarbon degradation. Observed alkane degradation ability was near ubiquitous among candidate hydrocarbon degraders, while just 3 harbored elaborate gene inventories for the degradation of alkanes and (poly)aromatic hydrocarbons. Differential gene expression profiles revealed a spill-promoted microbial sulfur cycle alongside gene up-regulation associated with polyaromatic hydrocarbon degradation. Gene expression associated with alkane degradation was widespread, although active alkane degrader identities changed along the pollution gradient. The resulting analysis suggests a broad metabolic capacity to respond to oil exists across a large array of usually rare bacteria.

microbiology↗