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bioRxiv2017

Phenotypic consequences of RNA polymerase dysregulation in Escherichia coli

Many bacterial adaptive responses to changes in growth conditions due to biotic and abiotic factors involve reprogramming of gene expression at the transcription level. The bacterial RNA polymerase (RNAP), which catalyzes transcription, can thus be considered as the major mediator of cellular adaptive strategies. But how do bacteria respond if a stress factor directly compromises the activity of the RNAP? We used a phage-derived small protein to specifically perturb bacterial RNAP activity in exponentially growing Escherichia coli. Using cytological profiling, tracking RNAP behavior at single-molecule level and transcriptome analysis, we reveal that adaptation to conditions that directly perturb bacterial RNAP performance can result in a biphasic growth behavior and thereby confer the adapted bacterial cells an enhanced ability to tolerate diverse antibacterial stresses. The results imply that while synthetic transcriptional rewiring may confer bacteria with the intended desirable properties, such approaches may also collaterally allow them to acquire undesirable traits.

microbiology↗

bioRxiv2017

Construction of soil defined media using quantitative exometabolomic analysis of soil metabolites

Exometabolomics enables analysis of metabolite utilization of low molecular weight organic substances by soil isolates. Environmentally-based defined media are needed to examine ecologically relevant patterns of substrate utilization. Here, we describe an approach for the construction of defined media using untargeted characterization of water soluble soil metabolites. To broadly characterize soil metabolites, both liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass spectrometry (GC/MS) were used. With this approach, 96 metabolites were identified, including amino acids, amino acid derivatives, sugars, sugar alcohols, mono- and di-carboxylic acids, osmolytes, nucleobases, and nucleosides. From this pool of metabolites, 25 were quantified. Water soluble organic carbon was fractionated by molecular weight and measured to determine the fraction of carbon accounted for by the quantified metabolites. This revealed that, community structures, these soil metabolites have an uneven quantitative distribution, with a single metabolite, trehalose accounting for 9.9 percent of much like soil microbial the (< 1 kDa) water extractable organic carbon. This quantitative information was used to formulate two soil defined media (SDM), one containing 23 metabolites (SDM1) and one containing 46 (SDM2). To evaluate SDM for supporting the growth of bacteria found at this field site, we examined the growth of 30 phylogenetically diverse soil isolates obtained using standard R2A medium. The simpler SDM1 supported the growth of up to 13 isolates while the more complex SDM2 supported up to 25 isolates. One isolate, Pseudomonas corrugata strain FW300-N2E2 was selected for a time-series exometabolomics analysis to investigate SDM1 substrate preferences. Interestingly, it was found that this organism preferred lower-abundance substrates such as guanine, glycine, proline and arginine and glucose and did not utilize the more abundant substrates maltose, mannitol, trehalose and uridine. These results demonstrate the viability and utility of using exometabolomics to construct a tractable environmentally relevant media. We anticipate that this approach can be expanded to other environments to enhance isolation and characterization of diverse microbial communities.\n\nHighlights O_LILC/MS and GC/MS analyses of soil extracts revealed a diversity of 96 metabolites.\nC_LIO_LISoil defined media were constructed based on water extractable soil metabolomics data.\nC_LIO_LIThe defined media supported the growth of 25 out of 30 bacterial isolates.\nC_LIO_LIExometabolomics demonstrated preferential consumption of amino acids for one isolate.\nC_LIO_LIThese media can be used to understand environmentally relevant microbial substrate preferences.\nC_LI\n\nAbbreviationsDOM dissolved organic matter; SOM soil organic matter; WEOC water extractable organic carbon; LMWOS low molecular weight organic substances; SDM(1/2) soil defined media (1 and 2); R2A Reasoners 2A agar medium; ORFRC Oak Ridge Field Research Center; LC/MS liquid chromatography mass spectrometry; GC/MS gas chromatography/ mass spectrometry; TOC total organic carbon; HILIC hydrophilic interaction liquid chromatography

microbiology↗

bioRxiv2017

EsaB is a core component of the Staphylococcus aureus Type VII secretion system

Type VII secretion systems (T7SS) are found in many bacteria and secrete proteins involved in virulence and bacterial competition. In Staphylococcus aureus the small ubiquitin-like EsaB protein has been previously implicated as having a regulatory role in the production of the EsxC substrate. Here we show that in the S. aureus RN6390 strain, EsaB does not genetically regulate production of any T7 substrates or components, but is indispensable for secretion activity. Consistent with EsaB being a core component of the T7SS, loss of either EsaB or EssC are associated with upregulation of a common set of iron acquisition genes. However, a further subset of genes were dysregulated only in the absence of EsaB. In addition, fractionation revealed that although an EsaB fusion to yellow fluorescent protein partially localised to the membrane, it was still membrane-localised when the T7SS was absent. Taken together our findings suggest that EsaB has T7SS-dependent and T7SS-independent roles in S. aureus.

microbiology↗

bioRxiv2017

Increased ParB level affects expression of stress response, adaptation and virulence operons and potentiates repression of promoters adjacent to the high affinity binding sites parS3 and parS4 in Pseudomonas aeruginosa

Similarly to its homologs in other bacteria, Pseudomonas aeruginosa partitioning protein ParB facilitates segregation of newly replicated chromosomes. Lack of ParB is not lethal but results in increased frequency of anucleate cells production, longer division time, cell elongation, altered colony morphology and defective swarming and swimming motility. Unlike in other bacteria, inactivation of parB leads to major changes of the transcriptome, suggesting that, directly or indirectly, ParB plays a role in regulation of gene expression in this organism.\n\nParB overproduction affects growth rate, cell division and motility in a similar way as ParB deficiency. To identify primary ParB targets, here we analysed the impact of a slight increase in ParB level on P. aeruginosa transcriptome. ParB excess, which does not cause changes in growth rate and chromosome segregation, significantly alters the expression of 176 loci. Most notably, the mRNA level of genes adjacent to high affinity ParB binding sites parS1-4 close to oriC is reduced. Conversely, in cells lacking either parB or functional parS sequences the orfs adjacent to parS3 and parS4 are upregulated, indicating that direct ParB-parS3/parS4 interactions repress the transcription in this region. In addition, increased ParB level brings about repression or activation of numerous genes including several transcriptional regulators involved in SOS response, virulence and adaptation. Overall, our data support the role of partitioning protein ParB as a transcriptional regulator in Pseudomonas aeruginosa.

microbiology↗

bioRxiv2017

Metagenomics reveals niche partitioning within the phototrophic zone of a microbial mat

Hypersaline photosynthetic microbial mats are stratified microbial communities known for their taxonomic and metabolic diversity and strong light-driven day-night environmental gradients. In this study of the upper photosynthetic zone of hypersaline microbial mats of Elkhorn Slough, California (USA), we show how reference-based and reference-free methods can be used to meaningfully assess microbial ecology and genetic partitioning in these complex microbial systems. Mapping of metagenome reads to the dominant Cyanobacteria observed in the system, Coleofasciculus (Microcoleus) chthonoplastes, was used to examine strain variants within these metagenomes. Highly conserved gene subsystems indicate a core genome for the species, and a number of variant genes and subsystems suggest strain level differentiation, especially for carbohydrate utilization. Metagenome sequence coverage binning was used to assess ecosystem partitioning of remaining microbes. Functional gene annotation of these bins (primarily of Proteobacteria, Bacteroidetes, and Cyanobacteria) recapitulated the known biogeochemical functions in microbial mats using a genetic basis, and also revealed evidence of novel functional diversity within the Gemmatimonadetes and Gammaproteobacteria. Combined, these two approaches show how genetic partitioning can inform biogeochemical partitioning of the metabolic diversity within microbial ecosystems.

microbiology↗

bioRxiv2017

Zika Virus in Salivary Glands of Five Different Species of Wild-Caught Mosquitoes from Mexico

Zika virus (ZIKV) is a mosquito-borne virus and Aedes agypti has been mentioned as the main vector of the disease. Other mosquito species in the Aedes and Culex genera have been suggested to have the potential for being competent vectors, based on experimental exposition of mosquitoes to an infectious blood meal containing ZIKV. Here, we report the isolation in cell culture of ZIKV from different body parts of wild-caught female mosquitoes (Ae. aegypti, Ae. vexans, Culex quinquefasciatus, Cx. coronator, and Cx. tarsalis) and whole male mosquitoes (Ae. aegypti and Cx. quinquefasciatus) in Mexico. Importantly, the virus was isolated from the salivary glands of all of these mosquitoes, strongly suggesting that these species are potential vectors for ZIKV.

microbiology↗

bioRxiv2017

A single point mutation in a TssB/VipA homolog disrupts sheath formation in the type VI secretion system of Proteus mirabilis

The type VI secretion (T6S) system is a molecular device for the delivery of proteins from one cell into another. T6S function depends on the contractile sheath comprised of TssB/VipA and TssC/VipB proteins. We previously reported on a mutant variant of TssB that disrupts T6S-dependent export of the self-identity protein, IdsD, in the bacterium Proteus mirabilis. Here we determined the mechanism underlying that initial observation. We show that T6S-dependent export of multiple self-recognition proteins is abrogated in this mutant strain. We have mapped the mutation, which is a single amino acid change, to a region predicted to be involved in the formation of the TssB-TssC sheath. We have demonstrated that this mutation does indeed inhibit sheath formation, thereby explaining the global disruption of T6S activity. We propose that this mutation could be utilized as an important tool for studying functions and behaviors associated with T6S systems.

microbiology↗

bioRxiv2017

VipA N-terminal linker and VipB-VipB interaction modulate the contraction of Type VI secretion system sheath

Secretion systems are essential for bacteria to survive and manipulate their environment. The bacterial Type VI Secretion System (T6SS) generates the force needed for protein translocation by the contraction of a long polymer called sheath, which is composed of interconnected VipA/VipB subunits forming a six-start helix. The mechanism of T6SS sheath contraction and the structure of its extended state are unknown. Here we show that elongating the N-terminal VipA linker or eliminating charge of a specific VipB residue abolished sheath contraction and delivery of effectors into target cells. The assembly of the non-contractile sheaths was dependent on the baseplate component TssE and mass-spectrometry analysis identified Hcp, VgrG and other components of the T6SS baseplate specifically associated with stable non-contractile sheaths. The ability to lock T6SS in the pre-firing state opens new possibilities for understanding its mode of action.

microbiology↗

bioRxiv2017

Viral and Bacterial Communities of Colorectal Cancer

Human viruses (those that infect human cells) have been associated with many cancers, largely due to their mutagenic and functionally manipulative abilities. Despite this, cancer microbiome studies have almost exclusively focused on bacteria instead of viruses. We began evaluating the cancer virome by focusing on colorectal cancer, a primary cause of morbidity and mortality throughout the world, and a cancer linked to altered colonic bacterial community compositions but with an unknown association with the gut virome. We used 16S rRNA gene, whole shotgun metagenomic, and purified virus metagenomic sequencing of stool to evaluate the differences in human colorectal cancer virus and bacterial community composition. Through random forest modeling we identified differences in the healthy and colorectal cancer virome. The cancer-associated virome consisted primarily of temperate bacteriophages that were also predicted to be bacteria-virus community network hubs. These results provide foundational evidence that bacteriophage communities are associated with colorectal cancer and potentially impact cancer progression by altering the bacterial host communities.

microbiology↗

bioRxiv2017

Metagenomic characterization of the effect of feed additives on the gut microbiome and antibiotic resistome of feedlot cattle

In North America, antibiotic feed additives such as monensin and tylosin are added to the finishing diets of feedlot cattle to counter the ill-effects of feeding diets with rapidly digestible carbohydrates. While these feed additives have been proven to improve feed efficiency, and reduce liver abscess incidence, how these products impact the gastrointestinal microbiota is not completely understood. Furthermore, there are concerns that antibiotic feed additives may expand the antibiotic resistome of feedlot cattle by enriching antimicrobial resistance genes in pathogenic and nonpathogenic bacteria in the gut microbiota. In this study, we analyzed the impact of providing antibiotic feed additives to feedlot cattle using metagenome sequencing of treated and untreated animals. Our results indicate that use of antibiotic feed additives does not produce discernable changes at the phylum level however treated cattle had reduced the abundance of gram-positive bacteria at the genus level. The abundance of Ruminococcus, Erysipelotrichaceae and Lachanospira in the gut of treated steers was reduced. This may impact the ability of these animals to exclude pathogens from the gut. However, our results did not show any correlation between the presence of antimicrobial resistance genes in the gut microbiota and the administration of antibiotic feed additives.

microbiology↗

bioRxiv2017

Rapid whole genome amplification and sequencing of low cell numbers in a bacteraemia model

Whilst next generation sequencing is frequently used to whole genome sequence bacteria from cultures, its rarely applied directly to clinical samples. Therefore, this study addresses the issue of applying NGS microbial diagnostics directly to blood samples. To demonstrate the potential of direct from blood sequencing a bacteria spiked blood model was developed. Horse blood was spiked with clinical samples of E. coli and S. aureus, and a process developed to isolate bacterial cells whilst removing the majority of host DNA. One sample of each isolate was then amplified using {phi}29 multiple displacement amplification (MDA) and sequenced. The total processing time, from sample to amplified DNA ready for sequencing was 3.5 hours, significantly faster than the 18-hour overnight culture step which is typically required. Both bacteria showed 100% survival through the processing. The direct from sample sequencing resulted in greater than 92% genome coverage of the pathogens whilst limiting the sequencing of host genome (less than 7% of all reads). Analysis of de novo assembled reads allowed accurate genotypic antibiotic resistance prediction. The sample processing is easily applicable to multiple sequencing platforms. Overall this model demonstrates potential to rapidly generate whole genome bacterial data directly from blood.

microbiology↗

bioRxiv2017

c-di-GMP-linked phenotypes are modulated by the interaction between a diguanylate cyclase and a polar hub protein

c-di-GMP is a major player in the decision between biofilm and motile lifestyles. Several bacteria present a large number of c-di-GMP metabolizing proteins, thus a fine-tuning of this nucleotide levels may occur. It is hypothesized that some c-di-GMP metabolizing proteins would provide the global c-di-GMP levels inside the cell whereas others would maintain a localized pool, with the resulting c-di-GMP acting at the vicinity of its production. Although attractive, this hypothesis has yet to be proven in Pseudomonas aeruginosa. We found that the diguanylate cyclase DgcP interacts with the cytosolic region of FimV, a peptidoglycan-binding protein involved in type IV pilus assembly. Moreover, DgcP is located at the cell poles in wild type cells, but scattered in the cytoplasm of cells lacking FimV. Overexpression of DgcP leads to the classical phenotypes of high c-di-GMP levels (increased biofilm and impaired motilities) in the wild-type strain, but not in a AfimV background. Therefore, our findings strongly suggest that DgcP is regulated by FimV and may provide the local c-di-GMP pool that can be sensed by other proteins at the cell pole, bringing to light a specialized function for a specific diguanylate cyclase.

microbiology↗

bioRxiv2017

Bacteria facilitate viral co-infection of mammalian cells and promote genetic recombination

Intestinal bacteria promote infection of several mammalian enteric viruses, but the mechanisms and consequences are unclear. We screened a panel of 41 bacterial strains as a platform to determine how different bacteria impact enteric viruses. We found that most bacterial strains bound poliovirus, a model enteric virus. Given that each bacterium bound multiple virions, we hypothesized that bacteria may deliver multiple viral genomes to a mammalian cell even when very few virions are present, such as during the first replication cycle after inter-host transmission. We found that exposure to certain bacterial strains increased viral co-infection even when the ratio of virus to host cells was low. Bacteria-mediated viral co-infection correlated with bacterial adherence to cells. Importantly, bacterial strains that induced viral co-infection facilitated viral fitness restoration through genetic recombination. Thus, bacteria-virus interactions may increase viral fitness through viral recombination at initial sites of infection, potentially limiting abortive infections.

microbiology↗

bioRxiv2017

Bottled aqua incognita: Microbiota assembly and dissolved organic matter diversity in natural mineral waters

BackgroundNon-carbonated natural mineral waters contain microorganisms that regularly grow after bottling despite low concentrations of dissolved organic matter (DOM). Yet, the compositions of bottled water microbiota and organic substrates that fuel microbial activity, and how both change after bottling, are still largely unknown.\n\nResultsWe performed a multifaceted analysis of microbiota and DOM diversity in twelve natural mineral waters from six European countries. 16S rRNA gene-based analyses showed that less than ten species-level operational taxonomic units (OTUs) dominated the bacterial communities in the water phase and associated with the bottle wall after a short phase of post-bottling growth. Members of the betaproteobacterial genera Curvibacter, Aquabacterium, and Polaromonas (Comamonadaceae) grew in most waters and represent ubiquitous, mesophilic, heterotrophic aerobes in bottled waters. Ultrahigh-resolution mass spectrometry of DOM in bottled waters and their corresponding source waters identified thousands of molecular formulae characteristic of mostly refractory, soil-derived DOM.\n\nConclusionsThe bottle environment, including source water physicochemistry, selected for growth of a similar low-diversity microbiota across various bottled waters. Relative abundance changes of hundreds of multi-carbon molecules were related to growth of less than ten abundant OTUs. We thus speculate that individual bacteria cope with oligotrophic conditions by simultaneously consuming diverse DOM molecules.

microbiology↗

bioRxiv2017

Community dynamics in parallelized, aerobic, single carbon-source enrichments

Here we seek to test the extent to which laboratory enrichments mimic natural community processes and the degree to which the initial structure of a community determines its response to a press disturbance via the addition of environmentally-relevant carbon compounds. By utilizing aerobic substrate arrays to examine the effect of carbon amendment on microbial communities taken from six distinct environments (soil from a temperate prairie and forest, tropical forest soil, subalpine forest soil, and surface water and soil from a palustrine emergent wetland), we examined how carbon amendment and inoculum source shape the composition of the community in each enrichment. Dilute subsamples from each environment were used to inoculate 96-well microtiter plates containing triplicate wells amended with one of 31 carbon sources from 6 different classes of organic compound (phenols, polymers, carbohydrates, carboxylic acids, amines, amino acids). After incubating each well aerobically in the dark for 72 hours, we analyzed the composition of the microbial communities on the substrate arrays as well as the initial inocula by sequencing 16S rRNA gene amplicons using the Illumina MiSeq platform. Comparisons of alpha and beta diversity in these systems showed that, while the composition of the communities that grow to inhabit the wells in each substrate array diverges sharply from that of the original community in the inoculum, these enrichment communities are still is strongly affected by the inoculum source. We found most enrichments were dominated by one or several OTUs most closely related to aerobes or facultative anaerobes from the Proteobacteria (e.g. Pseudomonas, Burkholderia, and Ralstonia) or Bacteroidetes (e.g. Chryseobacterium). Comparisons within each substrate array based on the class of carbon source further show that the communities inhabiting wells amended with a carbohydrate differ significantly from those enriched with a phenolic compound. Niche selection therefore seems to play a strong role in shaping the communities in the substrate arrays, although some stochasticity is seen whereby several replicate wells within a single substrate array display strongly divergent community compositions. Overall, the use of highly parallel substrate arrays offers a promising path forward to study the response of microbial communities to a changing environment.

microbiology↗

bioRxiv2017

Whole genome sequencing-based detection of antimicrobial resistance and virulence in non-typhoidal Salmonella enterica isolated from wildlife

The aim of this study was to generate a reference set of Salmonella enterica genomes isolated from wildlife from the United States and to determine the antimicrobial resistance and virulence gene profile of the isolates from the genome sequence data. We sequenced the whole genomes of 103 Salmonella isolates sampled between 1988 and 2003 from wildlife and exotic pet cases that were submitted to the Oklahoma Animal Disease Diagnostic Laboratory, Stillwater, Oklahoma. Among 103 isolates, 50.48% were from wild birds, 0.9% was from fish, 24.27% each were from reptiles and mammals. 50.48% isolates showed resistance to at least one antibiotic. Resistance against the aminoglycoside streptomycin was most common while 9 isolates were found to be multi-drug resistant having resistance against more than three antibiotics. Determination of virulence gene profile revealed that the genes belonging to csg operons, the fim genes that encode for type 1 fimbriae and the genes belonging to type III secretion system were predominant among the isolates. The universal presence of fimbrial genes and the genes encoded by pathogenicity islands 1-2 among the isolates we report here indicates that these isolates could potentially cause disease in humans. Therefore, the genomes we report here could be a valuable reference point for future traceback investigations when wildlife is considered to be the potential source of human Salmonellosis.

microbiology↗

bioRxiv2017

Systematic identification of novel regulatory interactions controlling biofilm formation in the bacterium Escherichia coli

Here, we investigated novel interactions of three global regulators of the network that controls biofilm formation in the model bacterium Escherichia coli using computational network analysis, an in vivo reporter assay and physiological validation experiments. We were able to map critical nodes that govern planktonic to biofilm transition and identify 8 new regulatory interactions for CRP, IHF or Fis responsible for the control of the promoters of rpoS, rpoE, flhD, fliA, csgD and yeaJ. Additionally, an in vivo promoter reporter assay and motility analysis revealed a key role for IHF as a repressor of cell motility through the control of FliA sigma factor expression. This investigation of first stage and mature biofilm formation indicates that biofilm structure is strongly affected by IHF and Fis, while CRP seems to provide a fine-tuning mechanism. Taken together, the analysis presented here shows the utility of combining computational and experimental approaches to generate a deeper understanding of the biofilm formation process in bacteria.

microbiology↗

bioRxiv2017

Suppressor screen connects HAD protein function to metabolic control in Plasmodium falciparum

In the malaria parasite Plasmodium falciparum, synthesis of isoprenoids from glycolytic intermediates is essential for survival. The antimalarial fosmidomycin (FSM) inhibits isoprenoid synthesis. In P. falciparum, we identify a loss-of-function mutation in HAD2 (PF3D7_1226300) as necessary for FSM resistance. Enzymatic characterization reveals that HAD2, a member of the haloacid dehalogenase-like hydrolase (HAD) superfamily, is a phosphatase. Harnessing a growth defect in resistant parasites, we select for suppression of HAD2-mediated FSM resistance and uncover hypomorphic suppressor mutations in the locus encoding the glycolytic enzyme phosphofructokinase (PFK9). Metabolic profiling demonstrates that FSM resistance is achieved via increased steady-state levels of MEP pathway and glycolytic intermediates and confirms reduced PFK9 function in the suppressed strains. We identify HAD2 as a novel regulator of malaria parasite metabolism and drug sensitivity and uncover PFK9 as a novel site of genetic metabolic plasticity in the parasite. Our study informs the biological functions of an evolutionarily conserved family of metabolic regulators and reveals a previously undescribed strategy by which malaria parasites adapt to cellular metabolic dysregulation.\n\nIMPORTANCEUnique and essential aspects of parasite metabolism are excellent targets for development of new antimalarials. An improved understanding of parasite metabolism and drug resistance mechanisms are urgently needed. The antibiotic fosmidomycin targets the synthesis of essential isoprenoid compounds from glucose and is a candidate for antimalarial development. Our study identifies a novel mechanism of drug resistance and further describes a family of metabolic regulators in the parasite. Using a novel forward genetic approach, we also uncover mutations that suppress drug resistance in the glycolytic enzyme PFK9. Thus, we identify an unexpected genetic mechanism of adaptation to metabolic insult that influences parasite fitness and tolerance to antimalarials.

microbiology↗