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bioRxiv2015

Enumeration of coliform bacteria and characterization of Escherichia coli isolated from Staff Club swimming pool in Ile-Ife, Nigeria

Water recreation, though increasing globally, is strongly associated with infectious diseases. Unexpectedly, artificial water recreation systems e.g. swimming pools account for 90% of these outbreaks. It is therefore essential that pool waters be regularly monitored for deviations from microbial water quality guidelines. To assess the sanitary quality of a club swimming pool in Ile-Ife, Nigeria, I used the multiple-tube fermentation technique to determine the most probable number (MPN) of coliform bacteria in 100 ml of pool water. MPN estimates ranged from 9 to 93 with geometric mean of 38. Escherichia coli was isolated from positive presumptive tubes, indicating recent faecal contamination. The isolate elicited similar biochemical reactions as reference E. coli (ATCC-25922), except that it utilized sucrose and liquefied gelatin, which probably indicates potential pathogenicity. Also, the E. coli isolate was resistant to 13 antibiotics from 9 different classes. Finally, coliform counts and detection of E. coli clearly violates international guidelines. I recommend that pool operators increase water disinfection efficiency and educate the public on the need for improved swimmer hygiene to reduce the risk of recreational water illness transmission.\n\nConflicts of InterestThe author declares no conflict of interest.

Microbiology↗

bioRxiv2015

Metagenome-assembled genomes uncover a global brackish microbiome

Microbes are main drivers of biogeochemical cycles in oceans and lakes, yet surprisingly few bacterioplankton genomes have been sequenced, partly due to difficulties in cultivating them. Here we used automatic binning to reconstruct a large number of bacterioplankton genomes from a metagenomic time-series from the Baltic Sea. The genomes represent novel species within freshwater and marine clades, including clades not previously genome-sequenced. Their seasonal dynamics followed phylogenetic patterns, but with fine-grained lineage specific adaptations. Signs of streamlining were evident in most genomes, and estimated genome sizes correlated with abundance variation across filter size fractions. Comparing the genomes with globally distributed aquatic metagenomes suggested the existence of a global brackish metacommunity whose populations diverged from freshwater and marine relatives >100,000 years ago, hence long before the Baltic Sea was formed (8000 years). This markedly contrasts to most Baltic Sea multicellular organisms that are locally adapted populations of fresh- or marine counterparts.

Microbiology↗

bioRxiv2015

Genome analysis leads to discovery of Quorum Sensing Genes in Cedecea neteri

We have identified a strain of C. neteri SSMD04 isolated from pickled mackerel sashimi that produced N-acyl-homoserine lactone (AHL) type quorum sensing (QS) activity. Tandem mass sspectrometry revealed that C. neteri SSMD04 produced N-butyryl-homoserine lactone (C4-HSL). We identified a pair of luxIR homologues in this genome that shares the highest similarity with croIR from Citrobacter rodentium. The AHL synthase, which we named it as cneI (636 bp) and at 8bp distance from cneI is a sequence encoding a hypothetical protein, potentially the cognate receptor, a luxR homologue which we named it as cneR. We also found an orphan luxR in this isolate. To our knowledge, this is the first report on the AHL production activity in C. neteri, discovery of its luxI/R homologues and the orphan receptor.

Microbiology↗

bioRxiv2015

Tropical Soil Metagenome Library Reveals Complex Microbial Assemblage

In this work, we characterized the metagenome of a Malaysian mangrove soil sample via next generation sequencing (NGS). Shotgun NGS data analysis revealed high diversity of microbes from Bacteria and Archaea domains. The metabolic potential of the metagenome was reconstructed using the NGS data and the SEED classification in MEGAN shows abundance of virulence factor genes, implying that mangrove soil is potential reservoirs of pathogens.

Microbiology↗

bioRxiv2015

Designing and interpreting limiting dilution assays: general principles and applications to the latent reservoir for HIV-1

Limiting dilution assays are commonly used to measure the extent of infection, and in the context of HIV they represent an essential tool for studying latency and potential curative strategies. To assist investigators using dilution assays, we illustrate the major statistical method for estimating the frequency of infected cells (or other infectious units) from assay results, and we offer an online tool for computing this estimate. We then recommend a procedure for optimizing assay design to achieve any desired set of sensitivity and precision goals, subject to experimental constraints. We discuss challenges involved in interpreting experiments in which no viral growth is observed and explain how using alternative measures for viral outgrowth may make measurement of HIV latency more efficient. Finally, we discuss how biological complications - such as probabilistic growth of a small infection in culture - alter interpretations of experimental results.

Microbiology↗

bioRxiv2015

Ecological and evolutionary adaptations shape the gut microbiome of BaAka African rainforest hunter-gatherers

The gut microbiome provides access to otherwise unavailable metabolic and immune functions, likely affecting mammalian fitness and evolution. To investigate how this microbial ecosystem impacts evolutionary adaptation of humans to particular habitats, we explore the gut microbiome and metabolome of the BaAka rainforest hunter-gatherers from Central Africa. The data demonstrate that the BaAka harbor a colonic ecosystem dominated by Prevotellaceae and other taxa likely related to an increased capacity to metabolize plant structural polysaccharides, phenolics, and lipids. A comparative analysis shows that the BaAka gut microbiome shares similar patterns with that of the Hadza, another hunter-gatherer population from Tanzania. Nevertheless, the BaAka harbor significantly higher bacterial diversity and pathogen load compared to the Hadza, as well as other Western populations. We show that the traits unique to the BaAka microbiome and metabolome likely reflect adaptations to hunter-gatherer lifestyles and particular subsistence patterns. We hypothesize that the observed increase in microbial diversity and potential pathogenicity in the BaAka microbiome has been facilitated by evolutionary adaptations in immunity genes, resulting in a more tolerant immune system.\n\nSignificanceHuman ecological adaptation requires changes at the genomic level. However, the gut microbiome, the collection of microbes inhabiting the gastrointestinal tract and their functions, also responds significantly to ecological challenge. To determine how the gut microbiome responds to evolutionary adaptations in the host, we profiled gut bacterial communities of the BaAka, rainforest hunter-gatherers from Central Africa. The gut microbiome of the BaAka shows adaptations to metabolize foods rich in fiber, tannins and fats. Similarly, higher bacterial diversity and abundance of pathogenic bacteria, compared to other hunter-gatherers and western populations, suggest that the BaAka immune system evolved to coexist with increased pathogen threats. Accordingly, these results show how the gut microbiome contributes to human ecological plasticity, impacting host adaptation and evolution.

Microbiology↗

bioRxiv2015

Discovery of Pandoraea pnomenusa RB38 N-acyl homoserine Lactone Synthase (PpnI) and its Complete Genome Sequence Analysis

In this study, we sequenced the genome of P. pnomenusa RB38 and reported the finding of a pair of cognate luxI/R homologs which we firstly coined as ppnI, which is found adjacent to a luxR homolog, ppnR. An additional orphan luxR homolog, ppnR2 was also discovered. Multiple sequence alignment revealed that PpnI is a distinct cluster of AHL synthase compared to those of its nearest phylogenetic neighbor, Burkholderia spp. When expressed heterologously and analysed using high resolution tandem mass spectrometry, PpnI directs the synthesis of N-octanoylhomoserine lactone (C8-HSL). To our knowledge, this is the first documentation of the luxI/R homologs of the genus of Pandoraea.

Microbiology↗

bioRxiv2015

When does high-dose antimicrobial chemotherapy prevent the evolution of resistance?

High-dose chemotherapy has long been advocated as a means of controlling drug resistance in infectious diseases but recent empirical and theoretical studies have begun to challenge this view. We show how high-dose chemotherapy engenders opposing evolutionary processes involving the mutational input of resistant strains and their release from ecological competition. Whether such therapy provides the best approach for controlling resistance therefore depends on the relative strengths of these processes. These opposing processes lead to a unimodal relationship between drug pressure and resistance emergence. As a result, the optimal drug dose always lies at either end of the therapeutic window of clinically acceptable concentrations. We illustrate our findings with a simple model that shows how a seemingly minor change in parameter values can alter the outcome from one where high-dose chemotherapy is optimal to one where using the smallest clinically effective dose is best. A review of the available empirical evidence provides broad support for these general conclusions. Our analysis opens up treatment options not currently considered as resistance management strategies, and greatly simplifies the experiments required to determine the drug doses which best retard resistance emergence in patients.\n\nSignificance StatementThe evolution of antimicrobial resistant pathogens threatens much of modern medicine. For over one hundred years, the advice has been to hit hard, in the belief that high doses of antimicrobials best contain resistance evolution. We argue that nothing in evolutionary theory supports this as a good rule of thumb in the situations that challenge medicine. We show instead that the only generality is to either use the highest tolerable drug dose or the lowest clinically effective dose; that is, one of the two edges of the therapeutic window. This approach suggests treatment options not currently considered, and greatly simplifies the experiments required to identify the dose that best retards resistance evolution.

Microbiology↗

bioRxiv2015

Generic transcriptional response of E. coli to stress

Bacteria are often exposed to various stressors with inter-linked effects leading to cross-resistance. In order to study the common molecular response to stress in bacteria, we collated and compared transcriptional response of E. coli under a variety of biotic and abiotic stresses. Bacterial genome-wide gene expression data were retrieved from the NCBI Gene Expression Omnibus (GEO) database and interrogated to identify differentially expressed genes.\n\nOne hundred and sixty eight genes displayed distinct transcriptional response of E. coli to stress, with simultaneous down-regulation of flagellar assembly pathway and up-regulation of the global regulator rpoS.\n\nThis computational analysis summarizes stress responsive genes in E. coli and their inter-relationships.

Microbiology↗

bioRxiv2015

Probing the fabric of the rare biosphere

The relatively recent development of high-throughput sequencing (HTS) techniques has revealed a wealth of novel sequences found in very low abundance: the rare biosphere. Today, most studies of diversity of microorganisms are carried out almost exclusively with HTS techniques. However, culturing seems indispensable for diversity studies especially if the aim is exploring the rare biosphere. We have carried out a deep (1 million sequences per sample) pyrosequencing analysis of two marine bacterial samples and isolated a culture collection from one of them. We have shown that the collectors curves of the pyrosequencing data were close to reaching an asymptote. We have estimated the total richness of the samples and the sequencing effort necessary to obtain the total estimated richness experimentally. Comparing the pyrosequencing data and the isolates sequences we have found that isolation retrieves some of the rarest taxa and that the composition of rare taxa follows an annual succession. We have shown that increasing the number of tags sequenced would slowly uncover the isolated bacteria. However, even if the whole bacterial diversity was found by increasing the sampling depth, culturing would still be essential for the study of marine bacterial communities, especially if the target is the rare biosphere.

Microbiology↗

bioRxiv2015

The discovery, distribution and evolution of viruses associated with Drosophila melanogaster

AO_SCPLOWBSTRACTC_SCPLOWDrosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens, but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont--which is known to be protective against virus infections in Drosophila--we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host-virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research.\n\nDO_SCPLOWATAC_SCPLOW AO_SCPLOWVAILABILITYC_SCPLOWAll of the relevant data can be found within the paper and its Supporting Information files, with the exception of raw metagenomic sequence data which are deposited at NCBI Sequence Read Archive (SRP056120), and sequence data which are deposited at Genbank (KP714070-KP714108, KP757922-KP757936 and KP757937-KP757993)

Microbiology↗

bioRxiv2015

Regulation of yefM/yoeB toxin antitoxin system is independent of ppGpp and inorganic polyphosphate in Escherichia coli.

Bacterial persistence is a phenomenon wherein small proportion of a bacterial population attains transient antibiotic tolerance likely by virtue of metabolic minimization. Type II Toxin-Antitoxin systems (TAs), small overlapping bicistronic negative auto-regulons, were recently shown to induce the persistence state. Maisonneuve et al., 2013 reported that TAs are activated by a regulatory cascade consisting of stochastic accumulation of ppGpp leading to accumulation of inorganic polyphosphate (polyP). PolyP supposedly is essential for Lon protease-dependent-degradation of antitoxins resulting in activation of toxins and induction of persistence phenotype. In contrast, using semi-quantitative primer extension, we show that transcriptional up-regulation of yefM/yoeB loci, one of the well characterized TAs of Escherichia coli, is independent of ppGpp and polyP. Similarly, we show that chromosome-encoded YoeB-dependent target mRNA cleavage is independent of polyP. Our results and meta-analysis of literature we conclude that the regulation of yefM/yoeB TAs is independent of ppGpp and polyP.

Microbiology↗

bioRxiv2015

Species-level resolution of 16S rRNA gene amplicons sequenced through MinIONTM portable nanopore sequencer

BackgroundThe miniaturised and portable DNA sequencer MinIONTM has been released to the scientific community within the framework of an early access programme to evaluate its application for a wide variety of genetic approaches. This technology has demonstrated great potential, especially in genome-wide analyses. In this study, we tested the ability of the MinIONTM system to perform amplicon sequencing in order to design new approaches to study microbial diversity using nearly full-length 16S rDNA sequences.\n\nResultsUsing R7.3 chemistry, we generated more than 3.8 million events (nt) during a single sequencing run. These data were sufficient to reconstruct more than 90% of the 16S rRNA gene sequences for 20 different species present in a mock reference community. After read mapping and 16S rRNA gene assembly, consensus sequences and 2d reads were recovered to assign taxonomic classification down to the species level. Additionally, we were able to measure the relative abundance of all the species present in a mock community and detected a biased species distribution originating from the PCR reaction using universal primers.\n\nConclusionsAlthough nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, the MinIONTM DNA sequencer is valuable for both high taxonomic resolution and microbial diversity analysis. Improvements in nanopore chemistry, such as minimising base-calling errors and the nucleotide bias reported here for 16S amplicon sequencing, will further deliver more reliable information that is useful for the specific detection of microbial species and strains in complex ecosystems.

Microbiology↗

bioRxiv2015

Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production

Biological production of terminal alkenes has garnered a significant interest due to their industrial applications such as lubricants, detergents and fuels. Here, we engineered the yeast Saccharomyces cerevisiae to produce terminal alkenes via a one-step fatty acid decarboxylation pathway and improved the alkene production using combinatorial engineering strategies. In brief, we first characterized eight fatty acid decarboxylases to enable and enhance alkene production. We then increased the production titer 7-fold by improving the availability of the precursor fatty acids. We additionally increased the titer about 5-fold through genetic cofactor engineering and gene expression tuning in rich medium. Lastly, we further improved the titer 1.8-fold to 3.7 mg/L by optimizing the culturing conditions in bioreactors. This study represents the first report of terminal alkene biosynthesis in S. cerevisiae, and the abovementioned combinatorial engineering approaches collectively increased the titer 67.4-fold. We envision that these approaches could provide insights into devising engineering strategies to improve the production of fatty acid-derived biochemicals in S. cerevisiae.

Microbiology↗

bioRxiv2015

Unearthing the microbial ecology of soil carbon cycling with DNA-SIP

Introductory ParagraphWe explored the microbial contributions to decomposition using a sophisticated approach to DNA Stable Isotope Probing (SIP). Our experiment evaluated the dynamics and ecological characteristics of functionally defined microbial groups that metabolize labile and structural C in soils. We added to soil a complex amendment representing plant derived organic matter substituted with either 13C-xylose or 13C-cellulose to represent labile and structural C pools derived from abundant components of plant biomass. We found evidence for 13C-incorporation into DNA from 13C-xylose and 13C-cellulose in 49 and 63 operational taxonomic units (OTUs), respectively. The types of microorganisms that assimilated 13C in the 13C-xylose treatment changed over time being predominantly Firmicutes at day 1 followed by Bacteroidetes at day 3 and then Actinobacteria at day 7. These 13C-labeling dynamics suggest labile C traveled through different trophic levels. In contrast, microorganisms generally metabolized cellulose-C after 14 days and did not change to the same extent in phylogenetic composition over time. Microorganisms that metabolized cellulose-C belonged to poorly characterized but cosmopolitan soil lineages including Verrucomicrobia, Chloroflexi and Planctomycetes. We show that microbial life history traits are likely to constrain the diversity of microorganisms that participate in the soil C-cycle.

Microbiology↗

bioRxiv2015

Sulfur Isotope Effects of Dissimilatory Sulfite Reductase

The precise interpretation of environmental sulfur isotope records requires a quantitative understanding of the biochemical controls on sulfur isotope fractionation by the principle isotope-fractionating process within the S cycle, microbial sulfate reduction (MSR). Here we provide the only direct observation of the major (34S/32S) and minor (33S/32S, 36S/32S) sulfur isotope fractionations imparted by a central enzyme in the energy metabolism of sulfate reducers, dissimilatory sulfite reductase (DsrAB). Results from in vitro sulfite reduction experiments allow us to calculate the in vitro DsrAB isotope effect in 34S/32S (hereafter, 34{varepsilon}DsrAB) to be 15.3{+/-}2{per thousand}, 2{sigma}.The accompanying minor isotope effect in 33S, described as 33{lambda}DsrAB, is calculated to be 0.5150{+/-}0.0012, 2{sigma}. These observations facilitate a rigorous evaluation of the isotopic fractionation associated with the dissimilatory MSR pathway, as well as of the environmental variables that govern the overall magnitude of fractionation by natural communities of sulfate reducers. The isotope effect induced by DsrAB upon sulfite reduction is a factor of 0.3 to 0.6 times prior indirect estimates, which have ranged from 25 to 53{per thousand} in 34{varepsilon}DsrAB. The minor isotope fractionation observed from DsrAB is consistent with a kinetic or equilibrium effect. Our in vitro constraints on the magnitude of 34{varepsilon}DsrAB is similar to the median value of experimental observations compiled from all known published work, where 34{varepsilon}r-p = 16.1{per thousand} (r - p indicates reactant versus product, n = 648). This value closely matches those of MSR operating at high sulfate reduction rates in both laboratory chemostat experiments (34{varepsilon}SO4-H2S = 17.3{+/-}1.5{per thousand}) and in modern marine sediments (34{varepsilon}SO4-H2S = 17.3{+/-}3.8{per thousand}). Targeting the direct isotopic consequences of a specific enzymatic processes is a fundamental step toward a biochemical foundation for reinterpreting the biogeochemical and geobiological sulfur isotope records in modern and ancient environments.

Microbiology↗

bioRxiv2015

Genomic signatures of experimental adaptation to antimicrobial peptides in Staphylococcus aureus

ObjectivesThe evolution of resistance against antimicrobial peptides has long been considered unlikely due to their mechanism of action, yet experimental selection with AMPs results in rapid evolution of resistance in several species of bacteria. Although numerous studies have utilized mutant screens to identify loci that determine AMP susceptibility, there is a dearth of data concerning the genomic changes which accompany experimental evolution of AMP resistance.\n\nMethodsUsing genome re-sequencing we analysed the mutations which arise during experimental evolution of resistance to the cationic AMPs iseganan, melittin and pexiganan, as well as to a combination of melittin and pexiganan, or to the aminoglycoside antibiotic streptomycin.\n\nResultsAnalysis of 17 independently replicated Staphylococcus aureus selection lines, including unselected controls, showed that each AMP selected for mutations at distinct loci. We identify mutations in genes involved in the synthesis and maintenance of the cell envelope. This includes genes previously identified from mutant screens for AMP resistance, and genes involved in the response to AMPs and cell-wall-active antibiotics. Furthermore, transposon insertion mutants were used to verify that a number of the identified genes are directly involved in determining AMP susceptibility.\n\nConclusionsStrains selected for AMP resistance under controlled experimental evolution displayed consistent AMP-specific mutations in genes which determine AMP susceptibility. This suggests that different routes to evolve resistance are favored within a controlled genetic background.

Microbiology↗

bioRxiv2015

Efficient Disruption and Replacement of an Effector Gene in the Oomycete Phytophthora sojae using CRISPR/Cas9

Phytophthora sojae is a pathogenic oomycete that infects soybean seedlings as well as stems and roots of established plants, costing growers $1-2 billion per year. Due to its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. Despite the availability of several genome sequences, the lack of efficient techniques for targeted mutagenesis and gene replacement have long hampered genetic studies of pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR effector gene Avr4/6 as target, we observed that in the absence of a homologous template, the repair of Cas9-induced double-strand breaks (DSBs) in P. sojae was mediated by non-homologous end joining (NHEJ), primarily resulting in short indels. Most mutants were homozygous, presumably due to gene conversion triggered by Cas9-mediated cleavage of non-mutant alleles. When donor DNA was present, homology directed repair (HDR) was observed, which resulted in the replacement of the target gene with the donor DNA. By testing the specific virulence of several NHEJ mutants and HDR -mediated gene replacements on soybeans, we have validated the contribution of Avr4/6 to recognition by soybean R gene loci, Rps4 and Rps6, but also uncovered additional contributions to resistance by these two loci. Our results establish a powerful tool for studying functional genomics in Phytophthora, which provides new avenues for better control of this pathogen.

Microbiology↗