SEARCH · bioRxiv Science

Results for “Microbiology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 991 records · Page 55Linked to original sources

bioRxiv2017

Wine yeast phenomics: a standardized fermentation method for assessing quantitative traits of Saccharomyces cerevisiae strains in enological conditions.

This work describes the set up of a small scale fermentation methodology for measuring quantitative traits of hundreds of samples in an enological context. By using standardized screw cap vessels, the alcoholic fermentation kinetics of Saccharomyces cerevisiae strains were measured by following the weight loss over time. Preliminary results showed that the kinetic parameters measured are in agreement with those observed in larger scale vats. The small volume used did not impair any analysis of the strain performance. Indeed, this fermentation system was coupled with robotized enzymatic assays and 8 end-point metabolites of enological interest were measured accurately. Moreover, the vessel used offers the possibility to assay 32 volatiles compounds using a headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry approach. Data presented demonstrates that the shaking conditions significantly impacted the mean and the variance of kinetic parameters, primary metabolites, and the production of volatile compounds. This effect was likely due to an enhanced transfer of dissolved oxygen during the first hours of the alcoholic fermentation. To test the efficiency of this experimental design, the phenotypic response of 35 wine industrial starters was measured in five grape juices from the Bordeaux area. A multivariate analysis illustrated that strains were better discriminated by some grape must, than others. The technological performances as well as the phenotypic robustness of these strains was measured and discussed. The optimized methodology developed allows investigating multiple fermentation traits for numerous yeast strains and conditions and greatly contributes in achieving quantitative genetics studies as well as yeast selection programs.

microbiology↗

bioRxiv2017

Proteomic identification of Axc, a novel beta-lactamase with carbapenemase activity in a meropenem-resistant clinical isolate of Achromobacterxylosoxidans

The development of antibiotic resistance during treatment is a threat to patients and their environment. Insight in the mechanisms of resistance development is important for appropriate therapy and infection control. Here, we describe how through the application of mass spectrometry-based proteomics, a novel beta-lactamase Axc was identified as an indicator of acquired carbapenem resistance in a clinical isolate of Achromobacter xylosoxidans.\n\nComparative proteomic analysis of consecutively collected susceptible and a resistant isolates from the same patient revealed that high Axc protein levels were only observed in the resistant isolate. Heterologous expression of Axc in Escherichia coli significantly increased the resistance towards carbapenems. Importantly, direct Axc mediated hydrolysis of imipenem was demonstrated using pH shift assays and 1H-NMR, confirming Axc as a legitimate carbapenemase. Whole genome sequencing revealed that the susceptible and resistant isolates were remarkably similar.\n\nTogether these findings provide a molecular context for the fast development of meropenem resistance in A. xylosoxidans during treatment and demonstrate the use of mass spectrometric techniques in identifying novel resistance determinants.

microbiology↗

bioRxiv2017

Genetic diversity and mother-child overlap of the gut associated microbiota determined by reduced genome sequencing

The genetic diversity and sharing of the mother-child associated microbiota remain largely unexplored. This severely limits our functional understanding of gut microbiota transmission patterns. The aim of our work was therefore to use a novel reduced metagenome sequencing in combination with shotgun and 16S rRNA gene sequencing to determine both the metagenome genetic diversity and the mother-to-child sharing of the microbiota. For a cohort of 17 mother-child pairs we found an increase of the collective metagenome size from about 100 Mbp for 4-day-old children to about 500 Mbp for mothers. The 4-day-old children shared 7% of the metagenome sequences with the mothers, while the metagenome sequence sharing was more than 30% among the mothers. We found 15 genomes shared across more than 50% of the mothers, of which 10 belonged to Clostridia. Only Bacteroides showed a direct mother-child association, with B. vulgatus being abundant in both 4-day-old children and mothers. In conclusion, our results support a common pool of gut bacteria that are transmitted from adults to infants, with most of the bacteria being transmitted at a stage after delivery.

microbiology↗

bioRxiv2017

Prasinovirus attack of Ostreococcus is furtive by day but savage by night

Prasinoviruses are large DNA viruses that infect diverse genera of green microalgae worldwide in aquatic ecosystems, but molecular knowledge of their life-cycles is lacking. Several complete genomes of both these viruses and their marine algal hosts are now available and have been used to show the pervasive presence of these species in microbial metagenomes. We have analysed the life-cycle of OtV5, a lytic virus, using RNA-Seq from 12 time points of healthy or infected Ostreococcus tauri cells over a day/night cycle in culture. In the day, viral gene transcription remained low while host nitrogen metabolism gene transcription was initially strongly repressed for two successive time points before being induced for 8 hours, but in the night viral transcription increased steeply while host nitrogen metabolism genes were repressed and many host functions that are normally reduced in the night appeared to be compensated either by genes expressed from the virus or by increased expression of a subset of 4.4 % of the hosts genes. Some host cells lysed progressively during the night, but a larger proportion lysed the following morning. Our data suggest that the life-cycles of algal viruses mirror the diurnal rhythms of their hosts.

microbiology↗

bioRxiv2017

Comprehensive analysis of central carbon metabolism reveals multiple connections between nutrients, biosynthetic capacity, and cell morphology in Escherichia coli

Bacterial morphology is a complex trait that is highly sensitive to changes in the environment. For heterotrophic organisms, such as Escherichia coli, increases in nutrient levels are frequently accompanied by several-fold increases in both size and growth rate. Despite the dramatic nature of these changes, how alterations in nutrient availability translate into changes in growth and morphology remains a largely open question. To understand the signaling networks coupling nutrient availability with size and shape, we examined the impact of deletions in the entirety of non-essential central carbon metabolic genes on E. coli growth rate and cell size. Our data reveal the presence of multiple metabolic nodes that play important yet distinctive roles in shaping the cell. Consistent with recent work from our lab and others, although both are sensitive to nutrient availability, size and growth rate vary independently. Cell width and length also appear to be independent phenomena, influenced by different aspects of central carbon metabolism. These findings highlight the diversity of factors that can impact cell morphology and provide a foundation for further studies.\n\nAuthor summaryOften taken for granted, the shape of bacterial cells is a complex trait that is highly sensitive to environmental perturbations. Nutrients in particular, strongly impact bacterial morphology together with growth rate. The ubiquitous, rod-shaped bacteria Escherichia coli increases both length and width several fold upon a shift from nutrient poor to nutrient rich medium, a change accompanied by an equally dramatic increase in growth rate. Central carbon metabolism is an obvious site for the integration of nutrient dependent signals that dictate cell size and shape. To develop a clearer picture of the molecular mechanisms coupling nutrient assimilation with cell growth and morphology, we screened the entirety of nonessential carbon metabolic genes for their contribution to growth rate and cell shape. Our data reveal the presence of multiple regulatory circuits coordinating different metabolic pathways with specific aspects of cell growth and morphology. Together, these data firmly establish a role for central carbon metabolism as an environmentally sensitive sculptor of bacterial cells.

microbiology↗

bioRxiv2017

Identification of environmental and genetic factors important for Cryptococcus neoformans titan cell formation using new in vitro inducing conditions

The pathogenic fungus Cryptococcus neoformans exhibits morphological changes in cell size during lung infection, producing both typical size 5 to 7 {micro}m cells and large titan cells (> 10 {micro}m and up to 100 {micro}m). We found and optimized in vitro conditions that produce titan cells in order to identify the ancestry of titan cells, the environmental determinants, and the key gene regulators of titan cell formation. Titan cells generated in vitro harbor the main characteristics of titan cells produced in vivo including their large cell size (>10 {micro}m), polyploidy with a single nucleus, large vacuole, dense capsule, and thick cell wall. Here we show titan cells derived from the enlargement of progenitor cells in the population independent of yeast growth rate. Change in the incubation medium, hypoxia, nutrient starvation and low pH were the main factors that trigger titan cell formation, while quorum sensing factors like the initial inoculum concentration, pantothenic acid, and the quorum sensing peptide Qsp1p also impacted titan cell formation. Inhibition of ergosterol, protein and nucleic acid biosynthesis altered titan cell formation, as did serum, phospholipids and anti-capsular antibodies in our settings. We explored genetic factors important for titan cell formation using three approaches. Using H99-derivative strains with natural genetic differences, we showed that titan cell formation was dependent on LMP1 and SGF29 genes. By screening a gene deletion collection, we also confirmed that GPR4/5-RIM101, and CAC1 genes were required to generate titan cells and that the PKR1, TSP2, USV101 genes negatively regulated titan cell formation. Furthermore, analysis of spontaneous Pkr1 loss-of-function clinical isolates confirmed the important role of the Pkr1 protein as a negative regulator of titan cell formation. Through development of a standardized and robust in vitro assay, our results provide new insights into titan cell biogenesis with the identification of multiple important factors/pathways.\n\nAuthor SummaryCryptococcus neoformans is a yeast that is capable of morphological change upon interaction with the host. Particularly, in the lungs of infected mice, a subpopulation of yeast enlarges, producing cells up to 100 {micro}m in cell body diameter - referred to as titan cells. Along with their large size, the titan cells have other unique characteristics such as thickened cell wall, dense capsule, polyploidization, large vacuole with peripheral nucleus and cellular organelles. The generation of a large number of such cells outside the lungs of mice has been described but was not reproducible nor standardized. Here we report standardized, reproducible, robust conditions for generation of titan cells and explored the environmental and genetic factors underlying the genesis of these cells. We showed that titan cells were generated upon stresses such as change in the incubation medium, nutrient deprivation, hypoxia and low pH. Using collections of well characterized reference strains and clinical isolates, we validated with our model that the cAMP/PKA/Rim101 pathway is a major genetic determinant of titan cell formation. This study opens the way for a more comprehensive picture of the ontology of morphological changes in Cryptococcus neoformans and its impact on pathobiology of this deadly pathogen.

microbiology↗

bioRxiv2017

Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity

Type IV pili are expressed by a wide range of prokaryotes, including the opportunistic pathogen Pseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The pilus is composed mainly of major pilin subunits while the low abundance minor pilins FimU-PilVWXE and the putative adhesin PilY1 prime pilus assembly and are proposed to form the pilus tip. The minor pilins and PilY1 are encoded in an operon that is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 is proposed to be a mechanosensory component that - in conjunction with minor pilins - triggers up-regulation of acute virulence phenotypes upon surface attachment. Here, we investigated the link between the minor pilins and virulence. pilW, pilX, and pilY1 mutants had reduced virulence towards Caenorhabditis elegans relative to wild type or a major pilin mutant, implying a role in pathogenicity that is independent of pilus assembly. We hypothesized that loss of specific minor pilins relieves feedback inhibition on FimS-AlgR, increasing transcription of the minor pilin operon and other members of the AlgR regulon. Reporter assays confirmed that FimS-AlgR were required for the increased expression from the minor pilin operon promoter upon loss of select minor pilins. Overexpression of AlgR or its hyperactivation via point mutation reduced virulence, and the virulence defects of pilW, pilX, and pilY1 mutants were dependent on FimS-AlgR expression and activation. We propose that PilY1 and the minor pilins inhibit their own expression, and that loss of these proteins leads to FimS-mediated activation of AlgR and reduced expression of acute-phase virulence factors. This mechanism could contribute to adaptation of P. aeruginosa in chronic lung infections, as mutations in the minor pilin operon result in the loss of piliation and increased expression of AlgR-dependent virulence factors - such as alginate - that are characteristic of such infections.\n\nAuthor summaryPseudomonas aeruginosa causes dangerous infections, including chronic lung infections in cystic fibrosis patients. It uses many strategies to infect its hosts, including deployment of grappling hook-like fibres called type IV pili. Among the components involved in assembly and function of the pilus are five proteins called minor pilins that - along with a larger protein called PilY1 - may help the pilus attach to surfaces. In a roundworm infection model, loss of PilY1 and specific minor pilins delayed killing, while loss of other pilus proteins did not. We traced this effect to increased activation of the FimS-AlgR regulatory system that inhibits expression of virulence factors used to initiate infections, while positively regulating chronic infection traits such as alginate production, a phenotype called mucoidy. A disruption in the appropriate timing of FimS-AlgR-dependent virulence factor expression when select minor pilins or PilY1 are missing may explain why those pilus-deficient mutants have reduced virulence compared with others whose products are not under FimS-AlgR control. Increased FimS-AlgR activity upon loss of PilY1 and specific minor pilins could help to explain the frequent co-occurrence of the non-piliated and mucoid phenotypes that are hallmarks of chronic P. aeruginosa lung infections.

microbiology↗

bioRxiv2017

USA300 MRSA lineages persist on multiple body sites following infection

Abstract\n\nBACKGROUNDUSA300 methicillin-resistant Staphylococcus aureus (MRSA) is a community- and hospital- acquired pathogen that frequently causes infections but also can survive on the human body asymptomatically as a part of the normal flora. We devised a comparative genomic strategy to track colonizing USA300 at different body sites after S. aureus infection.\n\nMETHODSWe sampled ST8 S. aureus from subjects at the site of a first known MRSA infection. Within 60 days of this infection and again 12 months later, each subject was tested for asymptomatic colonization in the nose, throat and perirectal region. 93 S. aureus strains underwent whole genome shotgun sequencing.\n\nRESULTSGenome sequencing revealed that 23 patients carried USA300 intra-subject lineages (ISLs), defined as having an index infection isolate (III) and closely related strains. Pairwise distance between strains in different ISLs was 48 to 162 single nucleotide polymorphisms (SNPs), whereas within the same ISL it was 0 to 26 SNPs. At the initial sampling time among 23 subjects, we isolated S. aureus from the nose, throat and perirectal sites from 15, 11 and 15 of them, respectively. Twelve months later we isolated S. aureus within the same ISL from 9 subjects, with 6, 3 and 3 strains from the nose, throat and perirectal area, respectively. The median time from initial acquisition of the S. aureus USA300 strains to culture of the index infection was estimated at 18 weeks. Strains in ISLs from the same subject differed in plasmid and prophage content, and contained deletions that removed the mecA-containing SCCmec and ACME regions. Five strains contained frameshift mutations in agr toxin-regulating genes. Persistence of an ISL was not associated with clinical or demographic subject characteristics.\n\nCONCLUSIONClonal lineages of USA300 may continue to colonize people at one or more anatomic sites up to a year after an initial infection and experience loss of the SCCmec, loss and gain of other mobile genetic elements, and mutations in the agr operon.

microbiology↗

bioRxiv2017

Clofazimine targets essential nucleoid associated protein, mycobacterial integration host factor (mIHF), in Mycobacterium tuberculosis

Clofazimine (CFZ) is a phenazine derivative used for treatment of leprosy, MDR-TB and XDR-TB. There is recent interest in understanding how CFZ works following the demonstration of its unique ability to shorten the treatment of MDR-TB. However, the target of CFZ in mycobacteria has remained elusive. Here, we show that CFZ binds to mycobacterial integration host factor (mIHF), which is an essential nucleoid associated protein in mycobacteria involved in DNA protection, chromosome organization and global gene regulation. We demonstrate that CFZ inhibits mIHF binding to DNA and interferes with mycobacterial gene expression. This mode of action is unique among all antibiotics including antimycibacterial agents and may help to explain its unusual action against Mycobacterium tuberculosis. Our study provides new insight about the mechanism of action of this intriguing drug and has implications for developing more effective treatment of TB.

microbiology↗

bioRxiv2017

A near complete haplotype-phased genome of the dikaryotic wheat stripe rust fungus Puccinia striiformis f. sp. tritici reveals high inter-haplome diversity

A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here we present a diploidaware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long-reads using the FALCON-Unzip assembler. RNA-seq datasets were used to infer high quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N50 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high inter-haplotype diversity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When investigating genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors, and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales. In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant pathogenic fungus previously hidden in collapsed and fragmented genome assemblies.\n\nImportanceCurrent representations of eukaryotic microbial genomes are haploid, hiding the genomic diversity intrinsic to diploid and polyploid life forms. This hidden diversity contributes to the organisms evolutionary potential and ability to adapt to stress conditions. Yet it is challenging to provide haplotype-specific information at a whole-genome level. Here, we take advantage of long-read DNA sequencing technology and a tailored-assembly algorithm to disentangle the two haploid genomes of a dikaryotic pathogenic wheat rust fungus. The two genomes display high levels of nucleotide and structural variations, which leads to allelic variation and the presence of genes lacking allelic counterparts. Non-allelic candidate effector genes, which likely encode important pathogenicity factors, display distinct genome localization patterns and are less likely to be evolutionary conserved than those which are present as allelic pairs. This genomic diversity may promote rapid host adaptation and/or be related to the age of the sequenced isolate since last meiosis.

microbiology↗

bioRxiv2017

Contribution of the β-glucosidase BglC to the Onset of the Pathogenic Lifestyle of Streptomyces scabies

Common scab disease on root and tuber plants is caused by Streptomyces scabies and related species which use the cellulose synthase inhibitor thaxtomin A as main phytotoxin. Thaxtomin production is primarily triggered by the import of cello-oligosaccharides. Once inside the cell, the fate of the cello-oligosaccharides is dichotomized into i) fueling glycolysis with glucose for the saprophytic lifestyle through the action of {beta}-glucosidase(s) (BG), and ii) eliciting the pathogenic lifestyle by inhibiting the CebR-mediated transcriptional repression of thaxtomin biosynthetic genes. Here we investigated the role of scab57721 encoding a putative BG (BglC) in the onset of the pathogenicity of S. scabies. Enzymatic assays showed that BglC was able to release glucose from cellobiose, cellotriose and all other cello-oligosaccharides tested. Its inactivation resulted in a phenotype opposite to what was expected as we monitored reduced production of thaxtomin when the mutant was cultivated on media containing cello-oligosaccharides as unique carbon source. This unexpected phenotype could be attributed to the highly increased activity of alternative intracellular BGs, probably as a compensation of bglC inactivation, which then prevented cellobiose and cellotriose accumulation to reduce the activity of CebR. In contrast, when the bglC null mutant was cultivated on media devoid of cello-oligosaccharides it instead constitutively produced thaxtomin. This observed hypervirulent phenotype does not fit with the proposed model of the cello-oligosaccharide-mediated induction of thaxtomin production and suggests that the role of BglC in the route to the pathogenic lifestyle of S. scabies is more complex than currently presented.

microbiology↗

bioRxiv2017

Social behavior in bees influences the abundance of Sodalis (Enterobacteriaceae) symbionts

Social interactions can facilitate transmission of microbes between individuals, reducing variation in gut communities within social groups. Thus, the evolution of social behaviors and symbiont community composition have the potential to be tightly linked. We explored this connection by characterizing the diversity of bacteria associated with both social and solitary bee species within the behaviorally variable family Halictidae using 16S amplicon sequencing. Contrary to expectations, we found few differences in bacterial abundance or variation between social forms, and most halictid species appear to share similar gut bacterial communities. However, several strains of Sodalis, a genus described as a symbiont in a variety of insects but yet to be characterized in bees, differ in abundance between social and solitary bees. Phylogenetic reconstructions based on whole-genome alignments indicate that Sodalis has independently colonized halictids at least three times. These strains appear to be mutually exclusive within individual bees, although they are not host-species-specific and no signatures of vertical transmission were observed, suggesting that Sodalis strains compete for access to hosts. De novo genome assemblies indicate that these three lineages are subject to widespread relaxed selection and that Sodalis is undergoing genome degeneration during the colonization of these hosts.

microbiology↗

bioRxiv2017

Two rsaM homologues encode central regulatory elements modulating quorum sensing expression in Burkholderia thailandensis

The bacterium Burkholderia thailandensis possesses three conserved N-acyl-L-homoserine lactone (AHL) quorum sensing (QS) systems designated BtaI1/BtaR1 (QS-1), BtaI2/BtaR2 (QS-2), and BtaI3/BtaR3 (QS-3). These QS-systems are associated with the biosynthesis of N-octanoyl-homoserine lactone (C8-HSL), N-3-hydroxy-decanoyl-homoserine lactone (3OHC10-HSL), as well as N-3-hydroxy-octanoyl-homoserine lactone (3OHC8-HSL), which are produced by the LuxI-type synthase BtaI1, BtaI2, and BtaI3, and modulated by the LuxR-type transcriptional regulators BtaR1, BtaR2, and BtaR3. Both btaR1/btaI1 and btaR2/btaI2 gene clusters contain an additional gene that is conserved in the Burkholderia genus, homologous to a gene coding for the negative AHL biosynthesis modulatory protein RsaM originally identified in the phytopathogen Pseudomonas fuscovaginae, and hence designated rsaM1 and rsaM2. We have characterized the function of these two rsaM homologues and demonstrated their involvement in the regulation of AHLs biosynthesis in B. thailandensis strain E264. We measured the production of C8-HSL, 3OHC10-HSL, and 3OHC8-HSL by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in the wild-type strain and in the rsaM1- and rsaM2- mutants, and monitored the transcription of btaI1, btaI2, and btaI3 using chromosomal mini-CTX-lux transcriptional reporters. The expression of btaR1, btaR2, and btaR3 was also measured by quantitative everse-transcription PCR (qRT-PCR). We demonstrate that the QS-1 system is repressed by RsaM1, whereas RsaM2 principally represses the QS-2 system. We also found that both rsaM1 and rsaM2 are QS-controlled, as well as negatively auto-regulated. We conclude that RsaM1 and RsaM2 are an integral part of the QS modulatory circuitry of B. thailandensis, and play a major role in the hierarchical and homeostatic organization of the QS-1, QS-2, and QS-3 systems.\n\nImportanceQuorum sensing (QS) is a global regulatory mechanism of genes expression depending on bacterial density. QS is commonly involved in the coordination of genes expression associated with the establishment of host-pathogen interactions and acclimatization to the environment. We present the functional characterization of the two rsaM homologues designated rsaM1 and rsaM2 in the regulation of the multiple QS systems coexisting in the non-pathogenic bacterium Burkholderia thailandensis, widely used as a model system for the study of the pathogen Burkholderia pseudomallei. We found that inactivation of these rsaM homologues, which are clustered with the other QS genes, profoundly affects the QS regulatory circuity of B. thailandensis. It is proposed that these genes code for QS repressors and we conclude that they constitute essential regulatory components of the QS modulatory network of B. thailandensis, and provide additional layers of regulation to modulate the expression of QS-controlled genes, including those encoding virulence/survival factors and linked to environmental adaptation in B. pseudomallei.

microbiology↗

bioRxiv2017

Filling Gaps in Bacterial Amino Acid Biosynthesis Pathways with High-throughput Genetics

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.

microbiology↗

bioRxiv2017

Cryptococcus neoformans can form titan-like cells in vitro in response to multiple signals that require the activation of several transduction pathways

Cryptococcus neoformans is an encapsulated pathogenic yeast that can change the size of the cells during infection. In particular, this process can occur by enlarging the size of the capsule without modifying the size of the cell body, or by increasing the diameter of the cell body, which is normally accompanied by an increase of the capsule too. This last process leads to the formation of cells of an abnormal enlarged size denominated titan cells. Previous works characterized titan cell formation during pulmonary infection but research on this topic has been hampered due to the difficulty to obtain them in vitro. In this work, we describe in vitro conditions (low nutrient, serum supplemented medium at neutral pH) that promote the transition from regular to titan-like cells. Moreover, addition of azide and static incubation of the cultures in a CO2 enriched atmosphere favored cellular enlargement. This transition occurred at low cell densities, suggesting that the process was regulated by quorum sensing molecules and was independent of the cryptococcal serotype/species. Transition to titan-like cell formation was impaired by pharmacological inhibition of PKC and TOR signaling pathways. Mutants affected in capsule synthesis did not form titan-like cells. Analysis of the gene expression profile in titan-like cells indicated that they overexpressed membrane proteins and transporters, being the gene encoding the Cig1 mannoprotein involved in iron uptake from heme groups the gene most differentially expressed compared to cells of regular size. We also investigated the gene expression profile of titan-like cells isolated from mice, and observed that during infection these cells mainly overexpressed genes related to metabolism and respiration. In summary, our work provides a new alternative method to investigate titan cell formation devoid the bioethical problems that involve animal experimentation.\n\nAUTHOR SUMMARYCryptococcus neoformans is a fungal pathogen that has a significant incidence in HIV+ patients in particular, in Subsaharian Africa, Asia and South America. This yeast poses an excellent model to investigate fungal virulence because it develops many strategies to adapt to the host and evade the immune response. One of the adaptation mechanisms involves the formation of Titan Cells, which are yeast of an abnormal large size. However, research on these cells has been limited to in vivo studies (mainly in mice) because they were not reproducibly found in vitro. In this work, we describe several conditions that induce the appearance of cells that mimic titan cells, and that we denominated as titan-like cells. The main factor that induced titan-like cells was the addition of serum to nutrient limited media. This has allowed to easily performing new approaches to characterize several signaling pathways involved in their development. We found that the formation of these cells is regulated by quorum sensing molecules, and that pathways such as PKC and TOR kinases regulate the process of cellular enlargement. We have also to perform transcriptomic analysis, which led to the identification of new genes that could be involved in the process. This work will open different research lines that will contribute to the elucidation of the role of these cells during infection and on the development of cryptococcal disease.

microbiology↗

bioRxiv2017

Direct PCR offers a fast and reliable alternative to conventional DNA isolation methods for animal gut microbiomes

The gut microbiome of animals is emerging as an important factor influencing ecological and evolutionary processes. A major bottleneck in obtaining microbiome data from large numbers of samples is the time-consuming laboratory procedures, specifically the isolation of DNA and generation of amplicon libraries. Recently, direct PCR kits have been developed that circumvent conventional DNA extraction steps, thereby streamlining the laboratory process by reducing preparation time and costs. However, the reliability and efficacy of the direct PCR method for measuring host microbiomes has not yet been investigated other than in humans with 454-sequencing. Here, we conduct a comprehensive evaluation of the microbial communities obtained with direct PCR and the widely used MoBio PowerSoil DNA extraction kit in five distinct gut sample types (ileum - caecum - colon - faeces - cloaca) from 20 juvenile ostriches, using 16S rRNA Illumina MiSeq sequencing. We found that direct PCR was highly comparable over a range of measures to the DNA extraction method in caecal, colon, and faecal samples. However, the two methods recovered significantly different microbiomes in cloacal, and especially ileal samples. We also sequenced 100 replicate sample pairs to evaluate repeatability during both extraction and PCR stages, and found that both methods were highly consistent for caecal, colon, and faecal samples (rs > 0.7), but had low repeatability for cloacal (rs = 0.39) and ileal (rs = -0.24) samples. This study indicates that direct PCR provides a fast, cheap, and reliable alternative to conventional DNA extraction methods for retrieving 16S data, which will aid future gut microbiome studies of animals.

microbiology↗

bioRxiv2017

Isolation of phosphate solubilizing Pseudomonas strains from apple rhizosphere in the Trans Himalayan region of Himachal Pradesh, India

Total fifteen phosphate solubilizing bacteria were isolated from rhizosphere soil of apple tree on Kings B medium belonging to genus Pseudomonas spp. They were characterized on the basis of morphological and biochemical characteristics. Preliminary selection as phosphate solubilizing bacteria were done on the basis of formation of transparent zone around the colony on Pikovskayas agar medium containing 0.5% triclcium phosphate (TCP). Maximum in vitro phosphate solubilization on Pikovskayas agar plates after 72h incubation at 28{degrees}C was shown by An-15-Mg (46 mm), whereas in case of broth, again this strain showed maximum tricalcium phosphate (TCP) solubilization (76 g/ml). The pH of each inoculated broth was recorded daily and dropped significantly (pH 7.0-3.99). All the Pseudomonas isolates were further evaluated for overall plant growth promoting traits. Greatest siderophore activity was exhibited by An-14-Mg (71.23 %SU) followed by An-15-Mg (70.12 %SU) which were statiscally at par with each other. Whereas, maximum IAA production was observed again in An-15-Mg (95 g/ml). This isolate also showed the maximum production of HCN and ammonia. 16S rDNA and phylogenetic analysis showed that strain An-15-Mg exhibits 99% level of similarity with Pseudomonas aeruginosa. Therefore, it was designated as Pseudomonas aeruginosa strain An-15-Mg. HPLC analysis showed that the Pseudomonas aeruginosa strain An-15-Mg produced maximum concentration of succinic acid, malonic, citric and malic acid with small amounts of schimic, quinic, tartaric, fumaric and lactic acids. The strain An-15-Mg possessed phosphate solubilization as major PGP trait along with different PGP traits. The potential phosphate solubilizing strain of Pseudomonas aeruginosa was reported first time as PGPR which lives in close association with apple tree without harming the plant, therefore could be used as a promising phosphate solubilizer and biofertilizer in apple crop grown in high hills of Himachal Pradesh.\n\nAuthors contributionAll authors made significant efforts towards the completion of this research work and preparation of manuscript timely. Author Ranjna Sharma has done collection of soil samples of apple tree from two districts, isolation and characterization of phosphate solubilizing Pseudomonas isolates and their screening for different PGPTs, phenotypic characteriazation, 16S rRNA analysis and statistical analysis. Author S P Singh has done HPLC analysis at their institute. Author Ankita has done sequence analysis and phylogenetic tree preparation. Author Mohinder Kaur is my mentor and generated idea regarding this work. She guided me in completing this work successfully as well as manuscript writing.

microbiology↗

bioRxiv2017

Histone deacetylase inhibition reduces deleterious cytokine release induced by ingenol stimulation

IntroductionLatency reversal agents (LRAs), such as protein kinase C (PKC) agonists, constitute a promising strategy for exposing and eliminating the HIV-1 latent reservoir. PKC agonists activate NF-{kappa}B and, in turn, induce deleterious pro-inflammatory cytokine production. Adjuvant pharmacological agents, such as ruxolitinib, a JAK inhibitor, and rapamycin, an mTOR inhibitor, have previously been combined with LRAs to reduce deleterious pro-inflammatory cytokine secretion without inhibiting HIV-1 viral reactivation in vitro. Histone deacetylase inhibitors (HDACi) are known to dampen pro-inflammatory cytokine secretion in the context of other diseases and can synergize with other LRAs to bring dormant proviruses out of latency. In this study we investigated whether a broad panel of epigenetic modifiers, including HDACi, could effectively dampen PKC-induced pro-inflammatory cytokine secretion during latency reversal. MethodsWe screened an epigenetic modifier library to identify compounds that reduced intracellular IL-6 production induced by the PKC agonist Ingenol-3,20-dibenzoate. We further tested the most promising epigenetic inhibitor class, HDACi, for their ability to reduce a broad panel of pro-inflammatory cytokines and reactivate latent HIV-1 ex vivo. ResultsWe identified nine epigenetic modulators that reduced PKC-induced intracellular IL-6. In cells from aviremic individuals living with HIV-1, the HDAC1-3 inhibitor, suberohydroxamic acid (SBHA), reduced secretion of pro-inflammatory cytokines TNF-, IL-5, IL-2r, and IL-17 but did not significantly reactivate latent HIV-1 when used in combination with Ingenol-3,20-dibenzoate. ConclusionThe addition of SBHA to Ingenol-3,20-dibenzoate reduces deleterious cytokine production during latency reversal but does not induce significant viral reactivation in aviremic donor PBMCs. The ability of SBHA to reduce PKC-induced pro-inflammatory cytokines when used in combination with Ingenol-3,20-dibenzoate suggests that SBHA can be used to reduced PKC induced pro-inflammatory cytokines but not to achieve latency reversal in the context of HIV-1.

microbiology↗