High-resolution time series of Pseudomonas aeruginosa gene expression and rhamnolipid secretion through growth curve synchronization

<p>Abstract</p> <p>Background</p> <p>Online spectrophotometric measurements allow monitoring dynamic biological processes with high-time resolution. Contrastingly, numerous other methods require laborious treatment of samples and can only be carried out offline. Integrating both types of measurement would allow analyzing biological processes more comprehensively. A typical example of this problem is acquiring quantitative data on rhamnolipid secretion by the opportunistic pathogen <it>Pseudomonas aeruginosa</it>. <it>P. aeruginosa </it>cell growth can be measured by optical density (OD₆₀₀) and gene expression can be measured using reporter fusions with a fluorescent protein, allowing high time resolution monitoring. However, measuring the secreted rhamnolipid biosurfactants requires laborious sample processing, which makes this an offline measurement.</p> <p>Results</p> <p>Here, we propose a method to integrate growth curve data with endpoint measurements of secreted metabolites that is inspired by a model of exponential cell growth. If serial diluting an inoculum gives reproducible time series shifted in time, then time series of endpoint measurements can be reconstructed using calculated time shifts between dilutions. We illustrate the method using measured rhamnolipid secretion by <it>P. aeruginosa </it>as endpoint measurements and we integrate these measurements with high-resolution growth curves measured by OD₆₀₀and expression of rhamnolipid synthesis genes monitored using a reporter fusion. Two-fold serial dilution allowed integrating rhamnolipid measurements at a ~0.4 h^-1frequency with high-time resolved data measured at a 6 h^-1frequency. We show how this simple method can be used in combination with mutants lacking specific genes in the rhamnolipid synthesis or quorum sensing regulation to acquire rich dynamic data on <it>P. aeruginosa </it>virulence regulation. Additionally, the linear relation between the ratio of inocula and the time-shift between curves produces high-precision measurements of maximum specific growth rates, which were determined with a precision of ~5.4%.</p> <p>Conclusions</p> <p>Growth curve synchronization allows integration of rich time-resolved data with endpoint measurements to produce time-resolved quantitative measurements. Such data can be valuable to unveil the dynamic regulation of virulence in <it>P. aeruginosa</it>. More generally, growth curve synchronization can be applied to many biological systems thus helping to overcome a key obstacle in dynamic regulation: the scarceness of quantitative time-resolved data.</p

Analysis of a marine phototrophic biofilm by confocal laser scanning microscopy using the new image quantification software PHLIP

BACKGROUND: Confocal laser scanning microscopy (CLSM) is the method of choice to study interfacial biofilms and acquires time-resolved three-dimensional data of the biofilm structure. CLSM can be used in a multi-channel modus where the different channels map individual biofilm components. This communication presents a novel image quantification tool, PHLIP, for the quantitative analysis of large amounts of multichannel CLSM data in an automated way. PHLIP can be freely downloaded from RESULTS: PHLIP is an open source public license Matlab toolbox that includes functions for CLSM imaging data handling and ten image analysis operations describing various aspects of biofilm morphology. The use of PHLIP is here demonstrated by a study of the development of a natural marine phototrophic biofilm. It is shown how the examination of the individual biofilm components using the multi-channel capability of PHLIP allowed the description of the dynamic spatial and temporal separation of diatoms, bacteria and organic and inorganic matter during the shift from a bacteria-dominated to a diatom-dominated phototrophic biofilm. Reflection images and weight measurements complementing the PHLIP analyses suggest that a large part of the biofilm mass consisted of inorganic mineral material. CONCLUSION: The presented case study reveals new insight into the temporal development of a phototrophic biofilm where multi-channel imaging allowed to parallel monitor the dynamics of the individual biofilm components over time. This application of PHLIP presents the power of biofilm image analysis by multi-channel CLSM software and demonstrates the importance of PHLIP for the scientific community as a flexible and extendable image analysis platform for automated image processing

Effect of temperature on growth, photosynthesis and biochemical composition of Nannochloropsis oceanica, grown outdoors in tubular photobioreactors

Since temperature is an important factor affecting microalgal growth, photosynthetic rate and biomass composition, this study has accordingly focused on its effects on biomass yield and nighttime biomass loss, as well as photochemical changes, using Nannochloropsis oceanica as model species, grown in two outdoor 50-L tubular photobioreactors (PBR). In two independent trials, cultures were subjected to a diurnal light:dark cycle, under a constant temperature of 28 degrees C and, on the second trial, at 18 degrees C. Changes in culture performance were assessed by measuring growth, lipid and fatty acid composition of the biomass in both morning and evening. Our results revealed that N. oceanica shows a wide temperature tolerance with relevant nighttime biomass loss, that decreased at lower temperatures, at the expenses of its daily productivity. Fluorescence measurements revealed reversible damage to photosystem II in cells growing in the PBR under optimal thermal conditions, whereas microalgae grown at suboptimal ones exhibited an overall lower photosynthetic activity. Lipids were partially consumed overnight to support cell division and provide maintenance energy. Eicosapentaenoic acid (EPA) catabolism reached a maximum after the dark period, as opposed to their saturated counterparts; whereas lower temperatures led to higher EPA content which reached the maximum in the morning. These findings are relevant for the production of Nannochloropsis at industrial scale.European Cooperation in Science and Technology (COST) Action: European network for algal-bio-products (EUALGAE) [ES1408]Portuguese national funds from the Foundation for Science and Technology (FCT) [SFRH/BD/129952/2017]Laboratory for Process Engineering, Environment, Biotechnology and Energy -LEPABE -by the FCT/MCTES (PIDDAC) [UIDB/00511/2020]project: "LEPABE-2-ECO-INNOVATION" - North Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) [NORTE-01-0145-FEDER-000005]project: "DINOSSAUR" - ERDF through Programa Operacional Competitividade e Internacionalizacao (COMPETE2020) [PTDC/BBB-EBB/1374/2014-POCI-01-0145-FEDER-016640]project: "SABANA"- European Union [727874][UID/Multi/04326/2019]info:eu-repo/semantics/publishedVersio

A Mutational Hotspot and Strong Selection Contribute to the Order of Mutations Selected for during Escherichia coli Adaptation to the Gut

The relative role of drift versus selection underlying the evolution of bacterial species within the gut microbiota remains poorly understood. The large sizes of bacterial populations in this environment suggest that even adaptive mutations with weak effects, thought to be the most frequently occurring, could substantially contribute to a rapid pace of evolutionary change in the gut. We followed the emergence of intra-species diversity in a commensal Escherichia coli strain that previously acquired an adaptive mutation with strong effect during one week of colonization of the mouse gut. Following this first step, which consisted of inactivating a metabolic operon, one third of the subsequent adaptive mutations were found to have a selective effect as high as the first. Nevertheless, the order of the adaptive steps was strongly affected by a mutational hotspot with an exceptionally high mutation rate of 10-5. The pattern of polymorphism emerging in the populations evolving within different hosts was characterized by periodic selection, which reduced diversity, but also frequency-dependent selection, actively maintaining genetic diversity. Furthermore, the continuous emergence of similar phenotypes due to distinct mutations, known as clonal interference, was pervasive. Evolutionary change within the gut is therefore highly repeatable within and across hosts, with adaptive mutations of selection coefficients as strong as 12% accumulating without strong constraints on genetic background. In vivo competitive assays showed that one of the second steps (focA) exhibited positive epistasis with the first, while another (dcuB) exhibited negative epistasis. The data shows that strong effect adaptive mutations continuously recur in gut commensal bacterial species.Deutsche Forschungs gemeinschaft grant: (SFB 680); Fundação para a Ciência e a Tecnologia grant: (SFRH/BPD/1117 25/2015)

Universality in Bacterial Colonies

The emergent spatial patterns generated by growing bacterial colonies have been the focus of intense study in physics during the last twenty years. Both experimental and theoretical investigations have made possible a clear qualitative picture of the different structures that such colonies can exhibit, depending on the medium on which they are growing. However, there are relatively few quantitative descriptions of these patterns. In this paper, we use a mechanistically detailed simulation framework to measure the scaling exponents associated with the advancing fronts of bacterial colonies on hard agar substrata, aiming to discern the universality class to which the system belongs. We show that the universal behavior exhibited by the colonies can be much richer than previously reported, and we propose the possibility of up to four different sub-phases within the medium-to-high nutrient concentration regime. We hypothesize that the quenched disorder that characterizes one of these sub-phases is an emergent property of the growth and division of bacteria competing for limited space and nutrients.Comment: 12 pages, 5 figure

The first steps of adaptation of Escherichia coli to the gut are dominated by soft sweeps

The accumulation of adaptive mutations is essential for survival in novel environments. However, in clonal populations with a high mutational supply, the power of natural selection is expected to be limited. This is due to clonal interference - the competition of clones carrying different beneficial mutations - which leads to the loss of many small effect mutations and fixation of large effect ones. If interference is abundant, then mechanisms for horizontal transfer of genes, which allow the immediate combination of beneficial alleles in a single background, are expected to evolve. However, the relevance of interference in natural complex environments, such as the gut, is poorly known. To address this issue, we studied the invasion of beneficial mutations responsible for Escherichia coli's adaptation to the mouse gut and demonstrate the pervasiveness of clonal interference. The observed dynamics of change in frequency of beneficial mutations are consistent with soft sweeps, where a similar adaptive mutation arises repeatedly on different haplotypes without reaching fixation. The genetic basis of the adaptive mutations revealed a striking parallelism in independently evolving populations. This was mainly characterized by the insertion of transposable elements in both coding and regulatory regions of a few genes. Interestingly in most populations, we observed a complete phenotypic sweep without loss of genetic variation. The intense clonal interference during adaptation to the gut environment, here demonstrated, may be important for our understanding of the levels of strain diversity of E. coli inhabiting the human gut microbiota and of its recombination rate.Howard Hughes Medical Institute (HHMI-55007436), LAO/ITQB, FCT Grants: FRH/BD/80257/2011 and SFRH/BPD/14299/2003

Plant aqueous extracts: antioxidant capacity via haemolysis and bacteriophage P22 protection

The bacteriophage P22/Salmonella Typhimurium system, as well as human erythrocytes have been used to assay for protection, against forced oxidation caused by hydrogen peroxide, brought about by several aqueous extracts of selected adventitious plants grown in Portugal. This study proved, for the first time, that the aforementioned bacteriophage-based system is a suitable method to assess the antioxidant activity of plant extracts; among the 12 plants tested, raspberry (Rubus idaeus), sage (Salvia sp.), savory (Satureja montana) and yarrow (Achillea millefolium) were found to effectively protect against oxidative damage caused by H2O2. Haemolysis was inhibited via pre-treatment with every plant extract tested, except heath at 0.1% (w/v). The two analytical methods produced different results – and for some plants, there was a dependence (either direct or inverse) of the quantitative protection effect on extract concentration, whereas for others no significant dependence was found at all. Savory yielded the most promising results, using either method. Therefore, the P22/Salmonella system can be used as a suitable in vivo assay, and human erythrocytes as a suitable in vitro assay to confirm (or not) the antioxidant capacity of plant extracts in biological matrices.info:eu-repo/semantics/acceptedVersio

Aryl hydrocarbon receptor and cysteine redox dynamics underlie (Mal)adaptive mechanisms to chronic intermittent hypoxia in kidney cortex

Funding Information: Funding: This work was supported by Fundação para Ciência e Tecnologia [PTDC/MED-TOX/30418/2017] and iNOVA4Health [UID/Multi/04462/2013]. M.J.C., F.L.-C., N.R.C., C.G.-D. and J.M. are supported by FCT grants [SFRH/BD/131331/2017, PD/BD/128337/2017, PD/BD/114257/2016, and PD/BD/105892/2014, PTDC/MED-TOX/30418/2017 respectively]. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.We hypothesized that an interplay between aryl hydrocarbon receptor (AhR) and cysteine-related thiolome at the kidney cortex underlies the mechanisms of (mal)adaptation to chronic intermittent hypoxia (CIH), promoting arterial hypertension (HTN). Using a rat model of CIH-HTN, we investigated the impact of short-term (1 and 7 days), mid-term (14 and 21 days, pre-HTN), and long-term intermittent hypoxia (IH) (up to 60 days, established HTN) on Cyp1a1 protein level (a sensitive hallmark of AhR activation) and cysteine-related thiol pools. We found that acute and chronic IH had opposite effects on Cyp1a1 and the thiolome. While short-term IH decreased Cyp1a1 and increased protein-S-thiolation, long-term IH increased Cyp1a1 and free oxidized cysteine. In addition, an in vitro administration of cystine, but not cysteine, to human endothelial cells increased Cyp1a1 expression, supporting cystine as a putative AhR activator. This study supports Cyp1a1 as a biomarker of obstructive sleep apnea (OSA) severity and oxidized pools of cysteine as risk indicator of OSA-HTN. This work contributes to a better understanding of the mechanisms underlying the phenotype of OSA-HTN, mimicked by this model, which is in line with precision medicine challenges in OSA.publishersversionpublishe

Physical rehabilitation programs for bedridden patients with prolonged immobility: a scoping review

Bedridden patients usually stay in bed for long periods, presenting several problems caused by immobility, leading to a long recovery process. Thus, identifying physical rehabilitation programs for bedridden patients with prolonged immobility requires urgent research. Therefore, this scoping review aimed to map existing physical rehabilitation programs for bedridden patients with prolonged immobility, the rehabilitation domains, the devices used, the parameters accessed, and the context in which these programs were performed. This scoping review, guided by the Joanna Briggs Institute’s (JBI) methodology and conducted in different databases (including grey literature), identified 475 articles, of which 27 were included in this review. The observed contexts included research institutes, hospitals, rehabilitation units, nursing homes, long-term units, and palliative care units. Most of the programs were directed to the musculoskeletal domain, predominantly toward the lower limbs. The devices used included lower limb mobilization, electrical stimulation, inclined planes, and cycle ergometers. Most of the evaluated parameters were musculoskeletal, cardiorespiratory, or vital signs. The variability of the programs, domains, devices and parameters found in this scoping review revealed no uniformity, a consequence of the personalization and individualization of care, which makes the development of a standard intervention program challenging.This research was co-financed by the European Regional Development Fund (ERDF) through the partnership agreement Portugal 2020—Operational Programme for Competitiveness and Internationalization (COMPETE2020) under the project POCI-01-0247-FEDER-047087 ABLEFIT: Desenvolvimento de um Sistema avançado para Reabilitação