ResearchPad - bacillus-subtilis https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[iterb-PPse: Identification of transcriptional terminators in bacterial by incorporating nucleotide properties into PseKNC]]> https://www.researchpad.co/article/elastic_article_14750 Terminator is a DNA sequence that gives the RNA polymerase the transcriptional termination signal. Identifying terminators correctly can optimize the genome annotation, more importantly, it has considerable application value in disease diagnosis and therapies. However, accurate prediction methods are deficient and in urgent need. Therefore, we proposed a prediction method “iterb-PPse” for terminators by incorporating 47 nucleotide properties into PseKNC-Ⅰ and PseKNC-Ⅱ and utilizing Extreme Gradient Boosting to predict terminators based on Escherichia coli and Bacillus subtilis. Combing with the preceding methods, we employed three new feature extraction methods K-pwm, Base-content, Nucleotidepro to formulate raw samples. The two-step method was applied to select features. When identifying terminators based on optimized features, we compared five single models as well as 16 ensemble models. As a result, the accuracy of our method on benchmark dataset achieved 99.88%, higher than the existing state-of-the-art predictor iTerm-PseKNC in 100 times five-fold cross-validation test. Its prediction accuracy for two independent datasets reached 94.24% and 99.45% respectively. For the convenience of users, we developed a software on the basis of “iterb-PPse” with the same name. The open software and source code of “iterb-PPse” are available at https://github.com/Sarahyouzi/iterb-PPse.

]]>
<![CDATA[Identification of soil bacteria capable of utilizing a corn ethanol fermentation byproduct]]> https://www.researchpad.co/article/5c8c1951d5eed0c484b4d3e6

A commercial corn ethanol production byproduct (syrup) was used as a bacterial growth medium with the long-term aim to repurpose the resulting microbial biomass as a protein supplement in aquaculture feeds. Anaerobic batch reactors were used to enrich for soil bacteria metabolizing the syrup as the sole nutrient source over an eight-day period with the goal of obtaining pure cultures of facultative organisms from the reactors. Amplification of the V4 variable region of the 16S rRNA gene was performed using barcoded primers to track the succession of microbes enriched for during growth on the syrup. The resulting PCR products were sequenced using Illumina MiSeq protocols, analyzed via the program QIIME, and the alpha-diversity was calculated. Seven bacterial families were the most prevalent in the bioreactor community after eight days of enrichment: Clostridiaceae, Alicyclobacillaceae, Ruminococcaceae, Burkholderiaceae, Bacillaceae, Veillonellaceae, and Enterobacteriaceae. Pure culture isolates obtained from the reactors, and additional laboratory stock strains, capable of facultative growth, were grown aerobically in microtiter plates with the syrup substrate to monitor growth yield. Reactor isolates of interest were identified at a species level using the full 16S rRNA gene and other biomarkers. Bacillus species, commonly used as probiotics in aquaculture, showed the highest biomass yield of the monocultures examined. Binary combinations of monocultures yielded no apparent synergism between organisms, suggesting competition for nutrients instead of cooperative metabolite conversion.

]]>
<![CDATA[Toxicity and oviposition deterrence of essential oils of Clinopodium nubigenum and Lavandula angustifolia against the myiasis-inducing blowfly Lucilia sericata]]> https://www.researchpad.co/article/5c76fe5ad5eed0c484e5b930

Cutaneous myiasis is a severe worldwide medical and veterinary issue. In this trial the essential oil (EO) of the Andean medicinal plant species Clinopodium nubigenum (Kunth) Kuntze was evaluated for its bioactivity against the myiasis-inducing blowfly Lucilia sericata (Meigen) (Diptera Calliphoridae) and compared with that of the well-known medicinal plant species Lavandula angustifolia Mill. The EOs were analysed and tested in laboratory for their oviposition deterrence and toxicity against L. sericata adults. The physiology of EO toxicity was evaluated by enzymatic inhibition tests. The antibacterial and antifungal properties of the EOs were tested as well. At 0.8 μL cm-2, both EOs completely deterred L. sericata oviposition up to 3 hours. After 24 h, the oviposition deterrence was still 82.7% for L. angustifolia and the 89.5% for C. nubigenum. The two EOs were also toxic to eggs and adults of L. sericata. By contact/fumigation, the EOs, the LC50 values against the eggs were 0.07 and 0.48 μL cm-2 while, by topical application on the adults, LD50 values were 0.278 and 0.393 μL per individual for C. nubigenum and L. angustifolia EOs, respectively. Inhibition of acetylcholine esterase of L. sericata by EOs (IC50 = 67.450 and 79.495 mg L-1 for C. nubigenum and L. angustifolia, respectively) suggested that the neural sites are targets of the EO toxicity. Finally, the observed antibacterial and antifungal properties of C. nubigenum and L. angustifolia EOs suggest that they could also help prevent secondary infections.

]]>
<![CDATA[Protein fortification with mealworm (Tenebrio molitor L.) powder: Effect on textural, microbiological, nutritional and sensory features of bread]]> https://www.researchpad.co/article/5c5df30cd5eed0c484580bee

In the present study, inclusion of mealworm (Tenebrio molitor L.) powder into bread doughs at 5 and 10% substitution level of soft wheat (Triticum aestivum L.) flour was tested to produce protein fortified breads. The addition of mealworm powder (MP) did not negatively affect the technological features of either doughs or breads. All the tested doughs showed the same leavening ability, whereas breads containing 5% MP showed the highest specific volume and the lowest firmness. An enrichment in protein content was observed in experimental breads where the highest values for this parameter were recorded in breads containing 10% MP. Breads fortified with 10% MP also exhibited a significant increase in the content of free amino acids, and especially in the following essential amino acids: tyrosine, methionine, isoleucine, and leucine. By contrast, no differences in nutritional quality of lipids were seen between fortified and control breads. Results of sensory analyses revealed that protein fortification of bread with MP significantly affected bread texture and overall liking, as well as crust colour, depending on the substitution level. Overall, proof of concept was provided for the inclusion of MP into bread doughs started with different leavening agents (sourdough and/or baker’s yeast), at 5 or 10% substitution level of soft wheat flour. Based on the Technology Readiness Level (TRL) scale, the proposed bread making technology can be situated at level 4 (validation in laboratory environment), thus suggesting that the production of breads with MP might easily be scaled up at industrial level. However, potential spoilage and safety issues that need to be further considered were highlighted.

]]>
<![CDATA[Multi-study inference of regulatory networks for more accurate models of gene regulation]]> https://www.researchpad.co/article/5c536a85d5eed0c484a47592

Gene regulatory networks are composed of sub-networks that are often shared across biological processes, cell-types, and organisms. Leveraging multiple sources of information, such as publicly available gene expression datasets, could therefore be helpful when learning a network of interest. Integrating data across different studies, however, raises numerous technical concerns. Hence, a common approach in network inference, and broadly in genomics research, is to separately learn models from each dataset and combine the results. Individual models, however, often suffer from under-sampling, poor generalization and limited network recovery. In this study, we explore previous integration strategies, such as batch-correction and model ensembles, and introduce a new multitask learning approach for joint network inference across several datasets. Our method initially estimates the activities of transcription factors, and subsequently, infers the relevant network topology. As regulatory interactions are context-dependent, we estimate model coefficients as a combination of both dataset-specific and conserved components. In addition, adaptive penalties may be used to favor models that include interactions derived from multiple sources of prior knowledge including orthogonal genomics experiments. We evaluate generalization and network recovery using examples from Bacillus subtilis and Saccharomyces cerevisiae, and show that sharing information across models improves network reconstruction. Finally, we demonstrate robustness to both false positives in the prior information and heterogeneity among datasets.

]]>
<![CDATA[A rapid method for post-antibiotic bacterial susceptibility testing]]> https://www.researchpad.co/article/5c40f7bdd5eed0c4843867df

Antibiotic susceptibility testing is often performed to determine the most effective antibiotic treatment for a bacterial infection, or perhaps to determine if a particular strain of bacteria is becoming drug resistant. Such tests, and others used to determine efficacy of candidate antibiotics during the drug discovery process, have resulted in a demand for more rapid susceptibility testing methods. Here, we have developed a susceptibility test that utilizes chemiluminescent determination of ATP release from bacteria and the overall optical density (OD600) of the bacterial solution. Bacteria release ATP during a growth phase or when they are lysed in the presence of an effective antibiotic. Because optical density increases during growth phase, but does not change during bacterial lysing, an increase in the ATP:optical density ratio after the bacteria have reached the log phase of growth (which is steady) would indicate antibiotic efficacy. Specifically, after allowing a kanamycin-resistant strain of Escherichia coli (E.coli) to pass through the growth phase and reach steady state, the addition of levofloxacin, an antibiotic to which E. coli is susceptible, resulted in a significant increase in the ATP:OD600 ratio in comparison to the use of kanamycin alone (1.80 +/- 0.50 vs. 1.12 +/- 0.28). This difference could be measured 20 minutes after the addition of the antibiotic, to which the bacteria are susceptible, to the bacterial sample. Furthermore, this method also proved useful with gram positive bacteria, as the addition of kanamycin to a chloramphenicol-resistant strain of Bacillus subtilis (B. subtilis) resulted in an ATP:OD600 ratio of 2.14 +/- 0.26 in comparison to 0.62 +/- 0.05 for bacteria not subjected to the antibiotic to which the bacteria are susceptible. Collectively, these results suggest that measurement of the ATP:OD600 ratio may provide a susceptibility test for antibiotic efficacy that is more rapid and quantitative than currently accepted techniques.

]]>
<![CDATA[CRISPR-Cas9 In Situ engineering of subtilisin E in Bacillus subtilis]]> https://www.researchpad.co/article/5c3d0133d5eed0c48403922d

CRISPR-Cas systems have become widely used across all fields of biology as a genome engineering tool. With its recent demonstration in the Gram positive industrial workhorse Bacillus subtilis, this tool has become an attractive option for rapid, markerless strain engineering of industrial production hosts. Previously described strategies for CRISPR-Cas9 genome editing in B. subtilis have involved chromosomal integrations of Cas9 and single guide RNA expression cassettes, or construction of large plasmids for simultaneous transformation of both single guide RNA and donor DNA. Here we use a flexible, co-transformation approach where the single guide RNA is inserted in a plasmid for Cas9 co-expression, and the donor DNA is supplied as a linear PCR product observing an editing efficiency of 76%. This allowed multiple, rapid rounds of in situ editing of the subtilisin E gene to incorporate a salt bridge triad present in the Bacillus clausii thermotolerant homolog, M-protease. A novel subtilisin E variant was obtained with increased thermotolerance and activity.

]]>
<![CDATA[Structural characterization of a pathogenicity-related superoxide dismutase codified by a probably essential gene in Xanthomonas citri subsp. citri]]> https://www.researchpad.co/article/5c3d015bd5eed0c48403a8e5

Citrus canker is a plant disease caused by the bacteria Xanthomonas citri subsp. citri that affects all domestic varieties of citrus. Some annotated genes from the X. citri subsp. citri genome are assigned to an interesting class named "pathogenicity, virulence and adaptation". Amongst these is sodM, which encodes for the gene product XcSOD, one of four superoxide dismutase homologs predicted from the genome. SODs are widespread enzymes that play roles in the oxidative stress response, catalyzing the degradation of the deleterious superoxide radical. In Xanthomonas, SOD has been associated with pathogenesis as a counter measure against the plant defense response. In this work we initially present the 1.8 Å crystal structure of XcSOD, a manganese containing superoxide dismutase from Xanthomonas citri subsp. citri. The structure bears all the hallmarks of a dimeric member of the MnSOD family, including the conserved hydrogen-bonding network residues. Despite the apparent gene redundancy, several attempts to obtain a sodM deletion mutant were unsuccessful, suggesting the encoded protein to be essential for bacterial survival. This intriguing observation led us to extend our structural studies to the remaining three SOD homologs, for which comparative models were built. The models imply that X. citri subsp. citri produces an iron-containing SOD which is unlikely to be catalytically active along with two conventional Cu,ZnSODs. Although the latter are expected to possess catalytic activity, we propose they may not be able to replace XcSOD for reasons such as distinct subcellular compartmentalization or differential gene expression in pathogenicity-inducing conditions.

]]>
<![CDATA[Experimental studies addressing the longevity of Bacillus subtilis spores – The first data from a 500-year experiment]]> https://www.researchpad.co/article/5c1028ebd5eed0c4842487a5

The ability to form endospores allows certain Gram-positive bacteria (e.g. Bacillus subtilis) to challenge the limits of microbial resistance and survival. Thus, B. subtilis is able to tolerate many environmental extremes by transitioning into a dormant state as spores, allowing survival under otherwise unfavorable conditions. Despite thorough study of spore resistance to external stresses, precisely how long B. subtilis spores can lie dormant while remaining viable, a period that potentially far exceeds the human lifespan; is not known although convincing examples of long term spore survival have been recorded. In this study, we report the first data from a 500-year microbial experiment, which started in 2014 and will finish in 2514. A set of vials containing a defined concentration of desiccated B. subtilis spores is opened and tested for viability every two years for the first 24 years and then every 25 years until experiment completion. Desiccated baseline spore samples were also exposed to environmental stresses, including X-rays, 254 nm UV-C, 10% H2O2, dry heat (120°C) and wet heat (100°C) to investigate how desiccated spores respond to harsh environmental conditions after long periods of storage. Data from the first 2 years of storage show no significant decrease in spore viability. Additionally, spores of B. subtilis were subjected to various short-term storage experiments, revealing that space-like vacuum and high NaCl concentration negatively affected spore viability.

]]>
<![CDATA[Characterization of biodegradation in a 17th century easel painting and potential for a biological approach]]> https://www.researchpad.co/article/5c117b5ed5eed0c484698e58

It is important to characterize the microorganisms involved in biodeterioration processes to understand their effects on cultural assets and to define an efficient strategy for protecting artworks, monuments, and buildings from microbiological recolonization. In this study, we analyzed the microbial communities dwelling on the verso (front) and recto (back) sides of a 17th century easel painting attributed to Carlo Bononi, an Italian artist of the first Baroque period. Cultivable bacteria and fungi colonizing the painting were isolated and identified in order to characterize the microbial community possibly involved in deteriorating the pictorial layer of the painting. The isolated bacterial strains belonged to the Staphylococcus and Bacillus genera. Furthermore, culture-dependent techniques and SEM/EDS analyses revealed the presence of filamentous fungi of the genera Aspergillus, Penicillium, Cladosporium, and Alternaria. The chemical compositions of pigments were consistent with typical 17th century paintings, and some of the identified pigments, namely red lac and red and yellow earths, could be exploited as nutrient sources by painting-associated microorganisms. The study also evaluated, in vitro, the potential decontaminating activity of a biocompound, containing spores of Bacillus subtilis, Bacillus pumilus, and Bacillus megaterium. The results indicated the ability of this biocompound to counteract the growth of contaminating microorganisms that are potentially dangerous to the painting, suggesting the potential use of these microorganisms to prevent biodeterioration of artworks.

]]>
<![CDATA[The positioning of the asymmetric septum during sporulation in Bacillus subtilis]]> https://www.researchpad.co/article/5c0c04e4d5eed0c48481ced8

Probably one of the most controversial questions about the cell division of Bacillus subtilis, a rod-shaped bacterium, concerns the mechanism that ensures correct division septum placement–at mid-cell during vegetative growth but closer to one end during sporulation. In general, bacteria multiply by binary fission, in which the division septum forms almost exactly at the cell centre. How the division machinery achieves such accuracy is a question of continuing interest. We understand in some detail how this is achieved during vegetative growth in Escherichia coli and B. subtilis, where two main negative regulators, nucleoid occlusion and the Min system, help to determine the division site, but we still do not know exactly how the asymmetric septation site is determined during sporulation in B. subtilis. Clearly, the inhibitory effects of the nucleoid occlusion and Min system on polar division have to be overcome. We evaluated the positioning of the asymmetric septum and its accuracy by statistical analysis of the site of septation. We also clarified the role of SpoIIE, RefZ and MinCD on the accuracy of this process. We determined that the sporulation septum forms approximately 1/6 of a cell length from one of the cell poles with high precision and that SpoIIE, RefZ and MinCD have a crucial role in precisely localizing the sporulation septum. Our results strongly support the idea that asymmetric septum formation is a very precise and highly controlled process regulated by a still unknown mechanism.

]]>
<![CDATA[Discovery of a dual protease mechanism that promotes DNA damage checkpoint recovery]]> https://www.researchpad.co/article/5b4a2865463d7e4513b897e6

The DNA damage response is a signaling pathway found throughout biology. In many bacteria the DNA damage checkpoint is enforced by inducing expression of a small, membrane bound inhibitor that delays cell division providing time to repair damaged chromosomes. How cells promote checkpoint recovery after sensing successful repair is unknown. By using a high-throughput, forward genetic screen, we identified two unrelated proteases, YlbL and CtpA, that promote DNA damage checkpoint recovery in Bacillus subtilis. Deletion of both proteases leads to accumulation of the checkpoint protein YneA. We show that DNA damage sensitivity and increased cell elongation in protease mutants depends on yneA. Further, expression of YneA in protease mutants was sufficient to inhibit cell proliferation. Finally, we show that both proteases interact with YneA and that one of the two proteases, CtpA, directly cleaves YneA in vitro. With these results, we report the mechanism for DNA damage checkpoint recovery in bacteria that use membrane bound cell division inhibitors.

]]>
<![CDATA[Conserved Units of Co-Expression in Bacterial Genomes: An Evolutionary Insight into Transcriptional Regulation]]> https://www.researchpad.co/article/5989da96ab0ee8fa60ba2148

Genome-wide measurements of transcriptional activity in bacteria indicate that the transcription of successive genes is strongly correlated beyond the scale of operons. Here, we analyze hundreds of bacterial genomes to identify supra-operonic segments of genes that are proximal in a large number of genomes. We show that these synteny segments correspond to genomic units of strong transcriptional co-expression. Structurally, the segments contain operons with specific relative orientations (co-directional or divergent) and nucleoid-associated proteins are found to bind at their boundaries. Functionally, operons inside a same segment are highly co-expressed even in the apparent absence of regulatory factors at their promoter regions. Remote operons along DNA can also be co-expressed if their corresponding segments share a transcriptional or sigma factor, without requiring these factors to bind directly to the promoters of the operons. As evidence that these results apply across the bacterial kingdom, we demonstrate them both in the Gram-negative bacterium Escherichia coli and in the Gram-positive bacterium Bacillus subtilis. The underlying process that we propose involves only RNA-polymerases and DNA: it implies that the transcription of an operon mechanically enhances the transcription of adjacent operons. In support of a primary role of this regulation by facilitated co-transcription, we show that the transcription en bloc of successive operons as a result of transcriptional read-through is strongly and specifically enhanced in synteny segments. Finally, our analysis indicates that facilitated co-transcription may be evolutionary primitive and may apply beyond bacteria.

]]>
<![CDATA[Artificial Polyploidy Improves Bacterial Single Cell Genome Recovery]]> https://www.researchpad.co/article/5989da01ab0ee8fa60b740a1

Background

Single cell genomics (SCG) is a combination of methods whose goal is to decipher the complete genomic sequence from a single cell and has been applied mostly to organisms with smaller genomes, such as bacteria and archaea. Prior single cell studies showed that a significant portion of a genome could be obtained. However, breakages of genomic DNA and amplification bias have made it very challenging to acquire a complete genome with single cells. We investigated an artificial method to induce polyploidy in Bacillus subtilis ATCC 6633 by blocking cell division and have shown that we can significantly improve the performance of genomic sequencing from a single cell.

Methodology/Principal Findings

We inhibited the bacterial cytoskeleton protein FtsZ in B. subtilis with an FtsZ-inhibiting compound, PC190723, resulting in larger undivided single cells with multiple copies of its genome. qPCR assays of these larger, sorted cells showed higher DNA content, have less amplification bias, and greater genomic recovery than untreated cells.

Significance

The method presented here shows the potential to obtain a nearly complete genome sequence from a single bacterial cell. With millions of uncultured bacterial species in nature, this method holds tremendous promise to provide insight into the genomic novelty of yet-to-be discovered species, and given the temporary effects of artificial polyploidy coupled with the ability to sort and distinguish differences in cell size and genomic DNA content, may allow recovery of specific organisms in addition to their genomes.

]]>
<![CDATA[Fused Regression for Multi-source Gene Regulatory Network Inference]]> https://www.researchpad.co/article/5989d9e5ab0ee8fa60b6b09f

Understanding gene regulatory networks is critical to understanding cellular differentiation and response to external stimuli. Methods for global network inference have been developed and applied to a variety of species. Most approaches consider the problem of network inference independently in each species, despite evidence that gene regulation can be conserved even in distantly related species. Further, network inference is often confined to single data-types (single platforms) and single cell types. We introduce a method for multi-source network inference that allows simultaneous estimation of gene regulatory networks in multiple species or biological processes through the introduction of priors based on known gene relationships such as orthology incorporated using fused regression. This approach improves network inference performance even when orthology mapping and conservation are incomplete. We refine this method by presenting an algorithm that extracts the true conserved subnetwork from a larger set of potentially conserved interactions and demonstrate the utility of our method in cross species network inference. Last, we demonstrate our method’s utility in learning from data collected on different experimental platforms.

]]>
<![CDATA[Extracellular DNA Release by Undomesticated Bacillus subtilis Is Regulated by Early Competence]]> https://www.researchpad.co/article/5989daeeab0ee8fa60bc0670

Extracellular DNA (eDNA) release is a widespread capacity described in many microorganisms. We identified and characterized lysis-independent eDNA production in an undomesticated strain of Bacillus subtilis. DNA fragments are released during a short time in late-exponential phase. The released eDNA corresponds to whole genome DNA, and does not harbour mutations suggesting that is not the result of error prone DNA synthesis. The absence of eDNA was linked to a spread colony morphology, which allowed a visual screening of a transposon library to search for genes involved in its production. Transposon insertions in genes related to quorum sensing and competence (oppA, oppF and comXP) and to DNA metabolism (mfd and topA) were impaired in eDNA release. Mutants in early competence genes such as comA and srfAA were also defective in eDNA while in contrast mutations in late competence genes as those for the DNA uptake machinery had no effect. A subpopulation of cells containing more DNA is present in the eDNA producing strains but absent from the eDNA defective strain. Finally, competent B. subtilis cells can be transformed by eDNA suggesting it could be used in horizontal gene transfer and providing a rationale for the molecular link between eDNA release and early-competence in B. subtilis that we report.

]]>
<![CDATA[How Quorum Sensing Connects Sporulation to Necrotrophism in Bacillus thuringiensis]]> https://www.researchpad.co/article/5989dad1ab0ee8fa60bb6380

Bacteria use quorum sensing to coordinate adaptation properties, cell fate or commitment to sporulation. The infectious cycle of Bacillus thuringiensis in the insect host is a powerful model to investigate the role of quorum sensing in natural conditions. It is tuned by communication systems regulators belonging to the RNPP family and directly regulated by re-internalized signaling peptides. One such RNPP regulator, NprR, acts in the presence of its cognate signaling peptide NprX as a transcription factor, regulating a set of genes involved in the survival of these bacteria in the insect cadaver. Here, we demonstrate that, in the absence of NprX and independently of its transcriptional activator function, NprR negatively controls sporulation. NprR inhibits expression of Spo0A-regulated genes by preventing the KinA-dependent phosphorylation of the phosphotransferase Spo0F, thus delaying initiation of the sporulation process. This NprR function displays striking similarities with the Rap proteins, which also belong to the RNPP family, but are devoid of DNA-binding domain and indirectly control gene expression via protein-protein interactions in Bacilli. Conservation of the Rap residues directly interacting with Spo0F further suggests a common inhibition of the sporulation phosphorelay. The crystal structure of apo NprR confirms that NprR displays a highly flexible Rap-like structure. We propose a molecular regulatory mechanism in which key residues of the bifunctional regulator NprR are directly and alternatively involved in its two functions. NprX binding switches NprR from a dimeric inhibitor of sporulation to a tetrameric transcriptional activator involved in the necrotrophic lifestyle of B. thuringiensis. NprR thus tightly coordinates sporulation and necrotrophism, ensuring survival and dissemination of the bacteria during host infection.

]]>
<![CDATA[An integrated overview of the midgut bacterial flora composition of Phlebotomus perniciosus, a vector of zoonotic visceral leishmaniasis in the Western Mediterranean Basin]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcde0

Background

The Leishmania developmental life cycle within its sand fly vector occurs exclusively in the lumen of the insect’s digestive tract in the presence of symbiotic bacteria. The composition of the gut microbiota and the factors that influence its composition are currently poorly understood. A set of factors, including the host and its environment, may influence this composition. It has been demonstrated that the insect gut microbiota influences the development of several human pathogens, such as Plasmodium falciparum. For sand flies and Leishmania, understanding the interactions between the parasite and the microbial environment of the vector midgut can provide new tools to control Leishmania transmission.

Methodology/Principal findings

The midguts of female Phlebotomus perniciosus from laboratory colonies or from the field were collected during the months of July, September and October 2011 and dissected. The midguts were analyzed by culture-dependent and culture-independent methods. A total of 441 and 115 cultivable isolates were assigned to 30 and 11 phylotypes from field-collected and colonized P. perniciosus, respectively. Analysis of monthly variations in microbiota composition shows a species diversity decline in October, which is to the end of the Leishmania infantum transmission period. In parallel, a compilation and a meta-analysis of all available data concerning the microbiota of two Psychodidae genera, namely Phlebotomus and Lutzomyia, was performed and compared to P. perniciosus, data obtained herein. This integrated analysis did not reveal any substantial divergences between Old and New world sand flies with regards to the midgut bacterial phyla and genera diversity. But clearly, most bacterial species (>76%) are sparsely distributed between Phlebotominae species.

Conclusion/Significance

Our results pinpoint the need for a more exhaustive understanding of the bacterial richness and abundance at the species level in Phlebotominae sand flies in order to capture the role of midgut bacteria during Leishmania development and transmission. The occurrence of Bacillus subtilis in P. perniciosus and at least two other sand fly species studied so far suggests that this bacterial species is a potential candidate for paratransgenic or biolological approaches for the control of sand fly populations in order to prevent Leishmania transmission.

]]>
<![CDATA[Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions]]> https://www.researchpad.co/article/5989da8dab0ee8fa60b9ed54

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria.

]]>
<![CDATA[The Spatial Architecture of Bacillus subtilis Biofilms Deciphered Using a Surface-Associated Model and In Situ Imaging]]> https://www.researchpad.co/article/5989da93ab0ee8fa60ba0c6c

The formation of multicellular communities known as biofilms is the part of bacterial life cycle in which bacteria display cooperative behaviour and differentiated phenotypes leading to specific functions. Bacillus subtilis is a Gram-positive bacterium that has served for a decade as a model to study the molecular pathways that control biofilm formation. Most of the data on B. subtilis biofilms have come from studies on the formation of pellicles at the air-liquid interface, or on the complex macrocolonies that develop on semi-solid nutritive agar. Here, using confocal laser scanning microcopy, we show that B. subtilis strains of different origins are capable of forming biofilms on immersed surfaces with dramatically protruding “beanstalk-like” structures with certain strains. Indeed, these structures can reach a height of more than 300 µm with one undomesticated strain from a medical environment. Using 14 GFP-labeled mutants previously described as affecting pellicle or complex colony formation, we have identified four genes whose inactivation significantly impeded immersed biofilm development, and one mutation triggering hyperbiofilm formation. We also identified mutations causing the three-dimensional architecture of the biofilm to be altered. Taken together, our results reveal that B. subtilis is able to form specific biofilm features on immersed surfaces, and that the development of these multicellular surface-associated communities involves regulation pathways that are common to those governing the formation of pellicle and/or complex colonies, and also some specific mechanisms. Finally, we propose the submerged surface-associated biofilm as another relevant model for the study of B. subtilis multicellular communities.

]]>