Response of predominant soil bacteria to grassland succession as monitored by ribosomal RNA analyses

The research described in this thesis was aimed to provide insight into the effects of grassland succession on the composition of the soil bacteria community in the Drentse A agricultural research area. The Drentse A meadows represent grassland succession at different stages. Since 30 years particular plots have been taken out of intense agricultural production and were not fertilized anymore. However, the grass-vegetation was continuously removed once per year. This caused a progressive depletion of nutrients in the soil. In order to reveal the effect of grassland succession on soil microbes, the main bacteria in Drentse A grassland soils were identified by a molecular strategy based on detection and quantification of 16S rRNA. Instead of only using genomic 16S rDNA to reveal present sequences, we focused on rRNA to quantify the activity of the predominant bacteria. The ribosome is considered to be a useful marker for the overall metabolic activity of bacteria. In bacterial cultures the amount of ribosomes per cell has been found to be roughly proportional to growth activity. In our approach the activity is defined as total activity of one ribotype in relation to the bacterial community and not as activity per cell. Hence, bacteria of low activity per cell but extraordinary high cell number might be assessed as very active. Following direct ribosome isolation from soil, several different methods like RTPCR, separation of amplicons by temperature gradient gel electrophoresis (TGGE), different hybridization methods, cloning and sequencing were applied simultaneously to reveal the predominant 16S rRNA sequences for taxonomic identification. Quantitative dot blot hybridization with taxonspecific oligonucleotide probes revealed dominance of low G+C Gram-positives while other important groups appeared to be aProteobacteria and high G+C Gram-positives (Chapter 5). However, this approach did not demonstrate clear tendencies of community structure shifts by quantifying the rRNA of the major taxa. Therefore, a more sensitive method has been chosen, based on RT-PCR amplicons of bacterial 16S rRNA. The sequence-specific separation of these amplicons by TGGE reproducibly yielded characteristic band patterns from hundreds of soil samples (Chapter 3). Although the TGGE signals were very complex due to the high bacterial diversity in soil, different 16S rRNA fingerprints from a single plot were highly similar, while reproducible differences between plots of different history were observed. A parallel approach with PCRamplified genomic 16S rDNA led to similar results. The presence and activity of prominent bacteria in test fields of several hundreds m 2 were found to be quite homogeneous. Only one gram of soil was found to be representative for the predominant bacteria in large homogeneous grassland areas. After the high reproducibility of presence and activity was demonstrated for the main soil bacteria, representative TGGE fingerprints were compared to TGGE signals from a clone library of genes coding for 16S rRNA (Chapter 5). Cloned 16S rDNA amplicons matching the intense bands in the fingerprint were sequenced. The relationship of these sequences to those of cultured organisms of known phylogeny were determined. Approximately one half of the amplicons represented sequences closely related to those of cultured Bacillus -species, indicating that most of the active bacteria apparently belonged to the genus Bacillus . Other sequences similar to Gram-positive bacteria with high G+C-content were only rernotely related to those of cultured bacteria, as is illustrated by clone DA079 that could be affiliated to uncultured Actinobacteria from peat (Chapter 6). Another important group of sequenees was related to Proteobacteria, mainly the α-subclass. Several sequences could not be related to cultured organisms hut to the Holophaga/Acidobacterium - or the Verrucomicrobiales -cluster (Chapters 7 and 8).The parallel application of RT-PCR/TGGE and 16S rDNA-cloning to reveal the most abundant 16S rRNA sequences was found to be a powerful combination. The clone screening on TGGE was convenient and efficient, offering access to the almost complete 16S rRNA sequence. The subsequently performed V6-hybridization was a relatively simple approach to prove the identity of bands even in complex fingerprints (Chapter 6). The most predominant Bacillus -like ribotype DA001 in Drentse A grassland soils could also be detected by fluorescent whole-cell in situ hybridization (Chapter 10). Prominent rod-shaped cells of approximately 2 μm length could be identified in bacterial suspensions from soil with a multiple 16S rRNA probing approach. The specific DA001-signals represented about 5% of all microbial particles, which were visualized by the universal DNA-dye DAPI. Indeed, the sequences detected by the PCRbased methods represented abundant bacteria in soil. The most predominant Bacillus -like 16S rRNA sequence DA001 apparently originated from active, vegetative cells and not from endospores.The possibility to draw quantitative information about the microbial community from the complex TGGE fingerprints has been explored. A novel approach has been developed to quantify rRNA sequences in complex bacterial communities by multiple competitive RT-PCR and subsequent TGGE analysis (Chapter 4). The used primer pair (U968-GC and L1401) was carefully tested and found to amplify with the same efficiency 16S rRNAs from bacterial cultures of different taxa as well as the cloned 16S rDNA amplicons from soil samples. The sequence-specific efficiency of amplification was followed by monitoring the amplification kinetics via kinetic PCR. The primer-specific amplification efficiency was assessed by competitive PCR and RT-PCR, and identical input amounts of different 16S rRNAs were found to result in equal amplicon yields. We applied this method as multiple competitive RTPCR to TGGE fingerprints from soil bacteria to estimate the ratios of their 16S rRNAs (Chapter 9). This was done for different stages of grassland succession in the Dutch Drentse A area. The 16S rRNA amounts g -1 soil of 20 predominant ribotypes were monitored via multiple competitive RT-PCR in six plots of different succession stage. The 20 monitored 16S rRNA levels represented approximately half of all bacterial soil rRNA. The different Drentse A meadows, representing progressing stages of grassland succession, showed highly reproducible shifts of ribotype composition. In general, the rRNA levels were found to be doubled after the first years without fertilization. During the further progression of grassland succession the rRNA amounts were found to decline again. The 20 ribotypes showed remarkably different succession histories, causing the differences in TGGE fingerprints from different plots. While organic carbon and available nitrogen were declining during grassland succession, some bacteria were apparently suffering much more than the average. However, other bacteria showed an increased contribution to the bacterial rRNA pool, indicating that some bacteria could improve their position when less nutrients were available. The general increase in bacterial ribosomes in the first years after fertilization-stop was correlating to the increase of other parameters related to bacterial activity, i. e. carbon mineralization and microbial biomass. This suggested a true correlation between the total activity of bacterial communities in soil and the amount of ribosomes. This study provides extended information about uncultured bacteria in soil and describes the application and evaluation of several novel approaches in molecular microbial ecology. The following six conclusions are highlighting the major achievements and findings:1. Representative rRNA and rDNA fingerprints can be generated for homogeneous landscapes of large scale. This demonstrates that the often tiny sample size in molecular studies has not to be a limitation for microbial ecology. Nucleic acid extraction from small soil samples can be applied to characterize a several magnitudes larger environmental matrix. The composition of bacterial communities might be quite homogeneous for kilometers of grassland with heterogeneous vegetation and cultivation history. This conclusion is of general importance for molecular rnicrobial ecology and landscape ecology.2. The rRNA cycle (see Introduction, Fig. 2) for the predominant soil Bacillus recognized as ribotype DA001 has been completed. Its rRNA not only has been identified as predominant in the isolated fraction of soil ribosomes, but was also detected in an abundant type of bacterial eells by whole-cell hybridization to a fluorescently labeled, 16S rRNA-targeted oligonucleotide probe.3. The genus Bacillus appears to be dominant in the microbial community of Drentse A grassland soils. Uncultured members of the B. benzoevorans -line of descent are predominant among these bacilli. This group of Bacillus -ribotypes accounted for approximately 20% of all bacterial ribosomes in Drentse A soil. Such a predominant cluster of very closely related bacteria has never been observed before in soils. The reasons and circumstances of this special community composition remained unexplored.4. Prominent clusters of hitherto uncultured environmental bacteria were also detected in Drentse A soils. The Holophaga/Acidobacterium -cluster, the Verrucomicrobiales and a peat-related Actinobacteria-cluster had already been known from different locations all over the world. lt is the first time that these hitherto uncultured bacteria were identified as prominent contributors to the ribosome fraction in soil. Since these organisms apparently contain considerable amounts of intact ribosomes they are likely to be metabolically active. Some of these novel ribotypes belong to the most intense bands in the TGGE fingerprints, which may suggest a major role in environmental nutrient fluxes. This finding also contributes to the discussion about the unculturability of environmental bacteria, since based on the presented results it appears unlikely that the reason for their unculturability is a lack of viability. lt is more probable that suitable culture conditions are still not found yet. Since these bacteria are abundantly detected all over the world, future research should be aimed to attempt to culture them.5. A novel approach has been developed to quantify the predominant rRNA molecules of environmental bacteria communities. The multiple competitive RT-PCR allowed to quantify in a highly-specific way many different rRNA molecules within one assay. The RT-PCR-related possibilities of bias were investigated and excluded for the applied primer pair. Therefore, amplification by RT-PCR could be excluded as a major source of bias in this study. Other uncertainties are the selectivity of the applied primers and probes and the cell lysis efficiency. The selection of all the oligonucleotide probes and primers is based on only a few thousand 16S rRNA sequences of different length and quality. Although the available 16S rRNA sequence data are limited, the presenee of hitherto unknown bacteria with novel 16S rRNA sequences not matching the used primers is not indicated. The combined results of cloning, TGGE and dot blot hybridization, all achieved with different probes or primers, do not reveal possibly neglected groups of organisms. A serious bias caused by incomplete cell lysis may not be excluded. However, the majority of ribosomes originated from Gram-positives, indicating the lysis of bacteria with resistant cell walls. The possibility that highly resistant resting stages like endospores might have been missed is not relevant, since this study aimed to detect the most active bacteria.6. Multiple competitive RT-PCR revealed activity shifts for the predominant soil bacteria during Drentse A grassland succession. Some species responded to the nutrient depletion during grassland succession. Though the depleting nutritious matter and the changing vegetation, the overall impact of grassland succession did not cause a correspondingly drastic impact on the microbial community composition. Reproducible shifts of ribosome levels could be demonstrated, but the composition of the bacterial community remained remarkably stable. Evidence for major competition or replacement of species could not be found

Les points de vue des étudiants en médecine autochtones sur une voie d’admission au programme postdoctoral réservée aux candidats autochtones

Objective: The objective of this study is to assess the perceptions of Indigenous medical students on postgraduate admissions through an Indigenous admissions pathway (IAP), and to determine what factors may influence Indigenous medical students’ choice of residency training program.       Methods: We distributed a survey to self-identified Indigenous students at settler Canadian medical schools. The survey questioned the students’ acceptability of an IAP, and what factors would influence application through an IAP. Analysis included descriptive statistics and thematic analysis of open-ended questions. Results: Thirty-six participants responded to the survey. Location and proximity to family or support system were the most important factors in choosing a residency program. Participants identified mentorship from Indigenous physicians and community involvement as being important features of a residency program that has an IAP. Eighty-one percent of participants felt the availability of an IAP would influence their choice of residency program. Fear of judgement or stigma, concern about entrance requirements, and program logistics were identified as barriers to applying to residency through an IAP. All participants believed that an IAP would have a positive influence on the healthcare system more broadly. Conclusions: An IAP appears to be an acceptable residency application format to Indigenous students but cannot exist in isolation. It is important for programs to consider the needs and safety of Indigenous trainees within residency programs. Objectif : L’objectif de cette étude est d’interroger les perceptions des étudiants en médecine autochtones d’une voie d’admission aux études postdoctorales réservée aux candidats autochtones et de relever les facteurs qui influenceraient leur choix de programme de résidence. Méthodes : Nous avons fait parvenir un questionnaire aux étudiants inscrits dans les facultés de médecine canadiennes qui se définissent comme Autochtones. L’enquête portait sur l’acceptabilité par les étudiants d’un programme d’admission pour candidats autochtones (PACA) et sur les facteurs qui influenceraient leur choix de poser leur candidature dans le cadre d’un PACA. L’examen portait sur des statistiques descriptives et sur une analyse thématique de questions ouvertes. Résultats : Trente-six participants ont répondu à l’enquête. Le lieu et la proximité de la famille ou du réseau de soutien ressortent comme les facteurs les plus importants dans le choix d’un programme de résidence. Ils attachent également de l’importance à la présence de mentorat de la part de médecins autochtones et à la participation de la communauté dans le cadre d’un programme de résidence et par conséquent ils voient un intérêt à ce qu’un tel programme soit accessible par le biais d’un PACA. Quatre-vingt-un pour cent des participants ont estimé que la présence d’un PACA influencerait leur choix de programme de résidence. La crainte d’être jugé ou stigmatisé, l’inquiétude par rapport aux conditions d’admission et la logistique du programme ont été identifiées comme des obstacles à la présentation d’une demande de résidence dans le cadre d’un PACA. Ils sont unanimes quant à l’influence positive d’un tel programme sur le système de soins de santé en général. Conclusions : Les étudiants autochtones trouvent la formule d’une voie d’admission à la résidence réservée aux candidats autochtones acceptable, à condition qu’elle ne soit pas un élément isolé et qu’on tienne compte des besoins et de la sécurité des étudiants dans le cadre des programmes de résidence eux-mêmes

A brief mindfulness-based intervention reduces eating disorder symptoms and improves eating self-efficacy and emotion regulation among adults seeking bariatric surgery

Background Up to 64% of bariatric (weight-loss) surgery-seeking adults report eating disorder (ED) symptoms (i.e., binge eating, emotional eating, addictive-like eating, and grazing) that can interfere with surgery outcomes. Well-designed pre-surgical interventions targeting eating behaviours may reduce ED symptoms and protect against suboptimal surgery outcomes. Objectives Provide proof-of-concept data to inform the design and optimization of a pre-surgical mindfulness-based intervention (MBI) for ED symptoms. Evaluate whether the MBI produces meaningful improvements in ED symptoms and clarify the mechanisms-of-action by which the MBI impacts ED symptoms. Methods Twenty-one pre-surgical patients with obesity and ED symptoms referred to a MBI completed self-report measures of addictive-like eating, binge eating, emotional eating, grazing, mindful eating, eating self-efficacy, and emotion regulation pre-(T1) and post-(T2) MBI. Results Repeated-measures ANOVAs revealed improvements in binge eating symptoms (F (1,20) = 30.38, ηp2 = .60, p < .001) and grazing (F (1,20) = 7.57, ηp2 = .28, p = .012), pre- to post-MBI. Adjusting for multiple comparisons, no significant improvements were found for addictive-like eating or emotional eating. Eating self-efficacy (F (1,20) = 29.70, ηp2 = .60, p < .001) and emotion regulation (F (1,20) = 7.18, ηp2 = .26, p = .014) improved, while mindful eating decreased (F (1,20) = 16.25, ηp2 = .45, p = .001), following the MBI. Bivariate correlations found associations between improvements in the mechanism of eating self-efficacy and improvements in the ED symptom of grazing pre- to post-MBI (r = 0.46, p < .05).  As well, improvements in emotion regulation were associated with positive changes in binge and emotional eating and grazing (r = 0.55, p < .001, r = 0.66, p < .001, r = 0.61, p < .05, respectively). Conclusions After participating in the MBI binge eating, grazing, eating self-efficacy, and emotion regulation abilities improved. Further work is needed to understand and mitigate deterioration in mindful eating. Moreover, acceptability and feasibility of the MBI should be assessed prior to testing the MBI in a large-scale efficacy trial. Future research should assess the the impact of this intervention on post-surgery weight-loss, weight-loss maintenance, and maintenance of improvements in ED symptoms

Partnering to Enhance Education and Public Engagement Programs

Collaborating with partners is a fundamental aspect of the Lunar and Planetary Institute's (LPI) educational and public engagement efforts. Such partnerships enable scientists and educators to include members of the audience in program planning and execution. Ultimately, partnerships strengthen programs by providing diverse resources, expertise, and expanding the potential audience

Response of a soil bacterial community to grassland succession as monitored by 16S rRNA levels of the predominant ribotypes

The composition of predominant soil bacteria during grassland succession was investigated in the Dutch Drentse A area. Five meadows, taken out of agricultural production at different time points, and one currently fertilized plot represented different stages of grassland succession. Since fertilization and agricultural production were stopped, the six plots showed a constant decline in the levels of nutrients and vegetation changes. The activity of the predominant bacteria was monitored by direct ribosome isolation from soil and temperature gradient gel electrophoresis of reverse transcription (RT)-PCR products generated from bacterial 16S rRNA. The amounts of 16S rRNA of 20 predominant ribosome types per gram of soil were monitored via multiple competitive RT-PCR in six plots at different succession stages. These ribosome types mainly represented Bacillus and members of the Acidobacterium cluster and the subclass of the class Proteobacteria. The 20 16S rRNA molecules monitored represented approximately half of all bacterial soil rRNA which was estimated by dot blot hybridizations of soil rRNA with the Bacteria probe EUB338. The grasslands showed highly reproducible and specific shifts of bacterial ribosome type composition. The total bacterial ribosome level increased during the first years after agricultural production and fertilization stopped. This correlated with the collapse of the dominant Lolium perenne population and an increased rate of mineralization of organic matter. The results indicate that there is a true correlation between the total activity of the bacterial community in soil and the amount of bacterial ribosomes

Development and application of a selective pcr-denaturing gradient gel electrophoresis approach to detect a recently cultivated Bacillus group predominant in soil

The worldwide presence of a hitherto-nondescribed group of predominant soil microorganisms related to Bacillus benzoevorans was analyzed after development of two sets of selective primers targeting 16S rRNA genes in combination with denaturing gradient gel electrophoresis (DGGE). The high abundance and cultivability of at least some of these microorganisms makes them an appropriate subject for studies on their biogeographical dissemination and diversity. Since cultivability can vary significantly with the physiological state and even between closely related strains, we developed a culture-independent 16S rRNA gene-targeted DGGE fingerprinting protocol for the detection of these bacteria from soil samples. The composition of the B. benzoevorans relatives in the soil samples from The Netherlands, Bulgaria, Russia, Pakistan, and Portugal showed remarkable differences between the different countries. Differences in the DGGE profiles of these communities in archived soil samples from the Dutch Wieringermeer polder were observed over time during which a shift from anaerobic to aerobic and from saline to freshwater conditions occurred. To complement the molecular methods, we additionally cultivated B. benzoevorans-related strains from all of the soil samples. The highest number of B. benzoevorans relatives was found in the soils from the northern part of The Netherlands. The present study contributes to our knowledge of the diversity and abundance of this interesting group of microbes in soils throughout the world

Functional abilities of cultivable plant growth promoting bacteria associated with wheat (Triticum aestivum L.) crops

Abstract In the pursuit of sustainable agriculture, bioinoculants usage as providers of a crop's needs is a method to limit environmental damage. In this study, a collection of cultivable putative plant growth promoting (PGP) bacteria associated with wheat crops was obtained and this bacterial sample was characterized in relation to the functional diversity of certain PGP features. The isolates were obtained through classical cultivation methods, identified by partial 16S rRNA gene sequencing and characterized for PGP traits of interest. Functional diversity characterization was performed using Categorical Principal Component Analysis (CatPCA) and Multiple Correspondence Analysis (MCA). The most abundant genera found among the 346 isolates were Pseudomonas, Burkholderia, and Enterobacter. Occurrence of PGP traits was affected by genus, niche, and sampling site. A large number of genera grouped together with the ability to produce indolic compounds; phosphate solubilization and siderophores production formed a second group related to fewer genera, in which the genus Burkholderia has a great importance. The results obtained may help future studies aiming prospection of putative plant growth promoting bacteria regarding the desired organism and PGP trait

Amplification by PCR Artificially Reduces the Proportion of the Rare Biosphere in Microbial Communities

The microbial world has been shown to hold an unimaginable diversity. The use of rRNA genes and PCR amplification to assess microbial community structure and diversity present biases that need to be analyzed in order to understand the risks involved in those estimates. Herein, we show that PCR amplification of specific sequence targets within a community depends on the fractions that those sequences represent to the total DNA template. Using quantitative, real-time, multiplex PCR and specific Taqman probes, the amplification of 16S rRNA genes from four bacterial species within a laboratory community were monitored. Results indicate that the relative amplification efficiency for each bacterial species is a nonlinear function of the fraction that each of those taxa represent within a community or multispecies DNA template. Consequently, the low-proportion taxa in a community are under-represented during PCR-based surveys and a large number of sequences might need to be processed to detect some of the bacterial taxa within the ‘rare biosphere’. The structure of microbial communities from PCR-based surveys is clearly biased against low abundant taxa which are required to decipher the complete extent of microbial diversity in nature

Metabarcoding analysis on European coastal samples reveals new molecular metazoan diversity

Although animals are among the best studied organisms, we still lack a full description of their diversity, especially for microscopic taxa. This is partly due to the time-consuming and costly nature of surveying animal diversity through morphological and molecular studies of individual taxa. A powerful alternative is the use of high-throughput environmental sequencing, providing molecular data from all organisms sampled. We here address the unknown diversity of animal phyla in marine environments using an extensive dataset designed to assess eukaryotic ribosomal diversity among European coastal locations. A multi-phylum assessment of marine animal diversity that includes water column and sediments, oxic and anoxic environments, and both DNA and RNA templates, revealed a high percentage of novel 18S rRNA sequences in most phyla, suggesting that marine environments have not yet been fully sampled at a molecular level. This novelty is especially high among Platyhelminthes, Acoelomorpha, and Nematoda, which are well studied from a morphological perspective and abundant in benthic environments. We also identified, based on molecular data, a potentially novel group of widespread tunicates. Moreover, we recovered a high number of reads for Ctenophora and Cnidaria in the smaller fractions suggesting their gametes might play a greater ecological role than previously suspected

Isolation and characterization of two plant growth-promoting bacteria from the rhizoplane of a legume (Lupinus albescens) in sandy soil

Duas linhagens bacterianas que apresentaram amplificação de parte do gene nifH, RP1p e RP2p, pertencentes aos gêneros Enterobacter e Serratia, foram isoladas do rizoplano de Lupinus albescens. Essas bactérias são Gram-negativas, com formato de bastonete, móveis, anaeróbias facultativas e apresentam multiplicação rápida, com colônias alcançando diâmetros de 3–4 mm em 24 h de incubação a 28 ºC. RP1p e RP2p também foram capazes de multiplicação em temperaturas elevadas, como 40 ºC, na presença de alta concentração de NaCl (2–3 % v/v) e em valores de pH que variaram de 4 a 10. A linhagem RP1p foi capaz de utilizar 10 das 14 fontes de carbono avaliadas, enquanto a linhagem RP2p utilizou nove. Os isolados produziram sideróforos e compostos indólicos, mas foram incapazes de solubilizar fosfatos. A inoculação de L. albescens com as linhagens RP1p e RP2p resultou em aumento significativo do peso das plantas secas, o que demonstra que essas bactérias apresentam propriedades que favorecem o crescimento vegetal.Two bacterial strains that amplified part of the nifH gene, RP1p and RP2p, belonging to the genus Enterobacter and Serratia, were isolated from the rhizoplane of Lupinus albescens. These bacteria are Gram-negative, rod-shaped, motile, facultative anaerobic, and fast-growing; the colonies reach diameters of 3–4 mm within 24 h of incubation at 28 °C. The bacteria were also able to grow at temperatures as high as 40 °C, in the presence of high (2–3 % w/v) NaCl concentrations and pH 4 -10. Strain RP1p was able to utilize 10 of 14 C sources, while RP2p utilized nine. The isolates produced siderophores and indolic compounds, but none of them was able to solubilize phosphate. Inoculation of L. albescens with RP1p and RP2p strains resulted in a significant increase in plant dry matter, indicating the plant-growth-promoting abilities of these bacteria