Bayesian prediction of bacterial growth temperature range based on genome sequences

<p>Abstract</p> <p>Background</p> <p>The preferred habitat of a given bacterium can provide a hint of which types of enzymes of potential industrial interest it might produce. These might include enzymes that are stable and active at very high or very low temperatures. Being able to accurately predict this based on a genomic sequence, would thus allow for an efficient and targeted search for production organisms, reducing the need for culturing experiments.</p> <p>Results</p> <p>This study found a total of 40 protein families useful for distinction between three thermophilicity classes (thermophiles, mesophiles and psychrophiles). The predictive performance of these protein families were compared to those of 87 basic sequence features (relative use of amino acids and codons, genomic and 16S rDNA AT content and genome size). When using naïve Bayesian inference, it was possible to correctly predict the optimal temperature range with a Matthews correlation coefficient of up to 0.68. The best predictive performance was always achieved by including protein families as well as structural features, compared to either of these alone. A dedicated computer program was created to perform these predictions.</p> <p>Conclusions</p> <p>This study shows that protein families associated with specific thermophilicity classes can provide effective input data for thermophilicity prediction, and that the naïve Bayesian approach is effective for such a task. The program created for this study is able to efficiently distinguish between thermophilic, mesophilic and psychrophilic adapted bacterial genomes.</p

On the molecular mechanism of GC content variation among eubacterial genomes

Background: As a key parameter of genome sequence variation, the GC content of bacterial genomes has been investigated for over half a century, and many hypotheses have been put forward to explain this GC content variation and its relationship to other fundamental processes. Previously, we classified eubacteria into dnaE-based groups (the dimeric combination of DNA polymerase III alpha subunits), according to a hypothesis where GC content variation is essentially governed by genome replication and DNA repair mechanisms. Further investigation led to the discovery that two major mutator genes, polC and dnaE2, may be responsible for genomic GC content variation. Consequently, an in-depth analysis was conducted to evaluate various potential intrinsic and extrinsic factors in association with GC content variation among eubacterial genomes. Results: Mutator genes, especially those with dominant effects on the mutation spectra, are biased towards either GC or AT richness, and they alter genomic GC content in the two opposite directions. Increased bacterial genome size (or gene number) appears to rely on increased genomic GC content; however, it is unclear whether the changes are directly related to certain environmental pressures. Certain environmental and bacteriological features are related to GC content variation, but their trends are more obvious when analyzed under the dnaE-based grouping scheme. Most terrestrial, plant-associated, and nitrogen-fixing bacteria are members of the dnaE1|dnaE

유산균의 진화적 및 기능적 특성에 대한 다중 유전체학적 접근

학위논문(박사) -- 서울대학교대학원 : 농업생명과학대학 농생명공학부, 2022. 8. 김희발.In this study, genome comparison analysis and microbial experiments were performed to obtain a comprehensive understanding of the bacterial evolution and their functionality at the molecular level. Lactic acid bacteria (LAB) are a species-rich in useful functions for humans and are widely used as health functional foods. Since LAB have many useful functions for humans, it is valuable to study. In addition, it is suitable for genome research as its short genome make it easier to understand the entire genome compared to other individuals. Therefore, research on the genome of LAB will not only increase the utilization of resources useful to humans, but also contribute to the genetic understanding of higher organisms with complex genomes. Therefore, this study was conducted to provide a multifaceted understanding of the evolution and functionality of LAB useful in humans through various genome analyses. In Chapter 2, the full-length genome sequence of Lactiplantibacillus plantarum GB-LP3 (Lactobacillus plantarum), a functional lactic acid bacterium, was sequenced and its evolutionary characteristics were detected by comparing the published L. plantarum complete genomes. It was confirmed that it has the closest evolutionary distance to the genome of L. plantarum ZJ316 identified in infant fecal samples, and possesses several functional genes and an evolutionarily accelerated ATP transporter. Based on comparative analysis, it was possible to infer the adaptation of L. plantarum in the special environment such as Korean fermented food. In Chapter 3, it was confirmed that Lactobacillus delbrueckii subsp. bulgaricus and Limosilactobacillus fermentum, which are widely used as health functional foods, have higher GC content compared to their genome size compared to other LAB species. It was confirmed that the high GC content was due to the difference in the 3rd nucleotide of the triplet code encoding the amino acid, and the difference in energy caused by this was compared. Through this, it was inferred that L. bulgaricus and Lm. fermentum evolved toward having a high GC content to adapt to the environment. In Chapter 4, in order to confirm the phenotypic and genotype changes for the evolutionary pressure, a Lactobacillus acidophilus strain with increased heat resistance was developed by artificially exposing it to a high temperature environment. The heat adapted strain showed a significant increase in survival rate at a high temperature of 65 degrees or more compared to the wild-type. Two SNPs were found in the vicinity of genes related to the cell wall through whole-genome comparison. Based on these results, it was suggested that the L. acidophilus strain evolved in the direction to harden the cell wall to adapt to heat stimulation. In Chapter 5, the cognitive ability of mice fed Lactobacillus acidophilus, Lacticaseibacillus paracasei, and Lacticaseibacillus rhamnosus for 8 weeks was evaluated and changes in intestinal microbial composition were compared to confirm the effect of LAB on the experimental animal in vivo. Among the experimental groups, the group fed L. acidophilus showed the highest cognitive ability improvement, and 16 bacterial species showed a significant difference in the intestinal microbial flora comparison comparing control group. Many of the bacteria with the changed ratio are involved in the production of substances for the synthesis of neuronal substances acting on the animal's brain. Based on these results, evidences that orally ingested LAB can affect cognitive ability were indicated. From Chapters 2 to 5 of this dissertation, the evolution and functional characteristics of LAB were presented through 3rd generation sequencing and genomic analysis. In detail, de novo whole-genome sequencing, phylogenetic tree construction, genome comparison analysis, and metagenome analysis were performed and these were applied to understanding for LAB. These studies will provide a deeper understanding of the evolution and characteristics of microorganisms through sequencing. Through these studies, it was possible not only to present the functional and evolutionary characteristics of LAB through genome analysis but also to identify the expected functionalities through experiments and to infer genetic factors. Through this research, a comprehensive understanding of the characteristics of microorganisms and genome analysis was provided.본 연구는 분자 수준에서 미생물의 진화와 그 기능성에 대한 포괄적 이해를 얻기 위해 실험적 증명 및 유전체 비교 분석을 수행되었다. 특히, 유산균은 인간에게 유용한 기능이 풍부한 종으로써 건강기능식품 등으로 많이 사용되고 있다. 유산균은 인간에게 유용한 기능성이 풍부하기 때문에, 학술적 및 상업적 연구 가치가 충분하다. 또한 유전체 크기가 작기 때문에 다른 개체에 비해 유전체 전체를 이해하기 용이하여, 유전체 연구에도 적합하다. 따라서 유산균의 유전체 연구는 인간에게 유용한 자원의 활용도를 높일 뿐만 아니라 복잡한 유전체를 가진 고등생물의 유전적 이해를 하는 데에도 기여를 할 것이다. 따라서 본 연구는 다양한 유전체 분석을 통해 인간에게 유용한 유산균의 진화와 기능에 대한 다면적 이해를 제공하고자 수행되었다. 2장에서는 기능성 유산균인 Lactiplantibacillus plantarum GB-LP3 의 전장 유전체 서열을 해독하고, 기존에 해독된 L. plantarum 전장 유전체들과 비교하여 진화적 특성을 판별하였다. Infant fecal samples에서 동정된 ZJ316의 유전체와 가장 가까운 진화적 거리를 가지고 있으며, 다수의 기능성 유전자 및 진화적으로 가속화된 ATP transporter를 보유함을 확인하였다. 이를 토대로 발효식품이라는 특수한 환경 속에서 L. plantarum의 적응 방식을 추론할 수 있었다. 3장에서는 건강기능식품으로 널리 사용 중인 Lactobacillus bulgaricus와 Limosilactobacillus fermentum이 다른 유산균 종들에 비해 Genome size 대비 높은 GC content를 보유함을 확인하였다. 높은 GC content는 아미노산을 암호화하는 triplet code의 세 번째 nucleotide의 차이 때문임을 확인하고 이로 인해 발생하는 에너지의 차이를 비교하였다. 이를 통해 L. bulgaricus와 L. fermentum가 환경에 적응하기 위해 높은 GC content를 갖는 쪽으로 진화하였음을 추론하였다. 4장에서는 진화압에 대한 표현형과 유전자형의 변화를 확인하기 위해, 인위적으로 고온에 노출시켜 내열성을 높인 Lactobacillus acidophilus 균주를 개발하였다. 열적응 균주는 야생형에 비해 65도 이상의 고온에서 생존율이 유의하게 증가하였다. 전장 유전체 비교를 통해 세포벽에 관련된 유전자 부근에서 2개의 SNP를 확인하였다. 이를 토대로 L. acidophilus 균주가 열자극에 적응하기 위하여 세포벽을 단단하게 하는 방향으로 진화하였음을 제시하였다. 5장에서는 Lactobacillus acidophilus, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus를 8주간 투여한 쥐의 인지능력을 평가하고 장내미생물 조성의 변화를 비교하여 LAB가 in vivo 실험동물에 미치는 영향을 확인하였다. 실험군 중 L. acidophilus를 먹인 균주에서 가장 높은 인지능력의 향상을 보였으며 이와 함께 두 그룹간 장내미생물 균총 비교에서 16개의 박테리아 종이 유의미하게 차이를 나타냈다. 비율이 변한 박테리아의 상당수가 동물의 뇌에 작용하는 신경물질 합성에 필요한 물질 생산에 관여하는 것으로, 장내에 늘어난 L. acidophilus 균총 증가의 영향으로 신경물질의 합성량이 증가했고 그로 인해 인지능력이 향상되었음을 추론 및 제시하였다. 본 논문의 2장부터 5장까지는 3세대 염기서열분석과 유전체 분석을 통해 유산균의 진화와 기능적 특성을 제시하였다. 구체적으로 전장 유전체 해독, 계통수 작성, 유전체 비교 분석, metagenome 분석을 수행하여 유산균에 대한 이해에 적용하였다. 이러한 연구를 통해 유전자 분석을 통해 유산균의 기능적, 진화적 특성을 제시할 수 있을 뿐만 아니라 실험을 통해 기대되는 기능을 규명하고 유전적 요인을 유추할 수 있었다. 이 연구를 통해 미생물의 특성과 유전체 분석에 대한 포괄적인 이해가 깊어지기를 바란다.Chapter 1. Literature Review １ 1.1. Bioinformatics ２ 1.1.1. Genome sequencing ２ 1.1.2. Genomic data analysis ７ 1.2. Lactic acid bacteria １２ 1.2.1. Lactic acid bacteria as probiotics １２ 1.2.2. Functionality and role of LAB １３ 1.2.3. NGS for LAB application １４ Chapter 2. Comparative genome analysis of Lactobacillus plantarum GB-LP3 provides candidates of survival-related genetic factors １７ 2.1. Abstract １８ 2.2. Introduction ２０ 2.3. Materials and Methods ２２ 2.3.1. Sample isolation and whole genome sequencing ２２ 2.3.2. Annotation and identification of GB-LP3 genome ２３ 2.3.3. Comparative genome analysis ２３ 2.3.4. dN/dS Analysis ２５ 2.4. Results ２６ 2.4.1. General features of L. plantarum GB-LP3 ２６ 2.4.2. Phylogenetic trees among L. plantarum strains ３０ 2.4.3. Comparative genome analysis with L. plantarum ZJ316 ３２ 2.4.4. Investigation of GB-LP3 specific genes ３７ 2.5. Discussion ４２ 2.5.1. Genomic islands in GB-LP3 genome ４２ 2.5.2. Genetic factors related to survival fitness ４３ Chapter 3. Comparative genomic analysis of Lactobacillus delbrueckii subsp. bulgaricus and Limosilactobacillus fermentum with elevated GC contents among lactic acid bacteria ７１ 3.1. Abstract ７２ 3.2. Introduction ７４ 3.3. Materials and Methods ７７ 3.3.1. Data collection and construction of a phylogenetic tree ７７ 3.3.2. Genome comparison of Lactobacillaceae family ７９ 3.3.3. Analysis of codon usage and amino acid pattern ７９ 3.3.4. Calculation of relative synonymous codon usage and effective number of codon ８０ 3.4.5. Identification of specific genes found in L. bulgaricus and Lm. fermentum ８２ 3.4.6. Statistical analysis ８３ 3.4. Results ８４ 3.4.1. Species identification with high GC contents ８４ 3.4.2. Comparison of genetic factors ８９ 3.4.3. Comparison of codon and amino acid patterns ９２ 3.4.4. Analysis of codon usage bias ９５ 3.4.5. Detection of a candidate gene related to elevated GC content and classification of isolation source ９９ 3.5. Discussion １００ Chapter 4. Complete Genome Sequence of the Newly Developed Lactobacillus acidophilus Strain With Improved Thermal Adaptability １１３ 4.1. Abstract １１４ 4.2. Introduction １１６ 4.3. Materials and Methods １２０ 4.3.1. Strain identification and bacterial culture １２０ 4.3.2. Adaptive Laboratory Evolution and screening a thermal adapted strain １２３ 4.3.3. Assessment of phenotypical changes １２４ 4.3.4. Bacterial kinetics １２５ 4.3.5. Statistical analysis １２５ 4.3.6. Whole genome sequencing １２６ 4.3.7. Annotation of genomic information １２７ 4.3.8. Comparative genomic analysis with Lactobacilaceae family １２８ 4.4. Results １３０ 4.4.1. Development of heat-resistant L. acidophilus strain based on ALE method １３０ 4.4.2. Overcoming the limit of thermal resistance of L. acidophilus EG004 strain １３２ 4.4.3. Complete genomic analysis for L. acidophilus EG004 and EG008 strains １３４ 4.4.4. Potential genetic factor related of heat resistance improvement through comparative genome analysis １３９ 4.5. Discussion １４２ Chapter 5. Positive Effect of Lactobacillus acidophilus EG004 on Cognitive Ability of Healthy Mice by Fecal Microbiome Analysis Using Full-Length 16S-23S rRNA Metagenome Sequencing １５５ 5.1. Abstract １５６ 5.2. Introduction １５８ 5.3. Materials and Methods １６２ 5.3.1. Animals １６２ 5.3.2. Bacterial treatment １６２ 5.3.3. Animal treatment １６４ 5.3.4. Y maze (Spontaneous alternation; SA) １６６ 5.3.5. Novel object recognition test (NOR) １６６ 5.3.6. Passive avoidance task (PAT) １６７ 5.3.7. Y maze (Forced alternation; FA) １６９ 5.3.8. Feces collection and cognitive ability evolution １６９ 5.3.9. Statistics １７０ 5.3.10. Full 16S-23S rRNA sequencing １７０ 5.3.11. Metagenome analysis １７２ 5.3.12. SCFA identification in bacterial culture １７３ 5.3.13. Whole-genome sequencing of EG005 and EG006 and Whole-genome sequence of EG004 １７３ 5.3.14. Comparative analysis of bacterial genome sequences １７５ 5.3.15. Data availability １７５ 5.4. Results １７６ 5.4.1. Bacterial and animal treatments １７６ 5.4.2. Cognitive behavioral tests １７８ 5.4.3. Full 16S-23S rRNA sequencing and biological diversity １８３ 5.4.4. Microbial composition １８６ 5.4.5. Functional profiling and correlation analysis １８７ 5.4.6. Comparative analysis of genetic contents in bacterial whole-genome sequences １９２ 5.5. Discussion １９５ General discussion ２１２ References ２１６ 국문초록 ２３３박

Crystallographic studies on two hyperthermophilic enzymes

Dissertation presented to obtain the Ph.D degree in BiochemistryWhile Aristotle cautioned “everything in moderation”, the Romans, known for their eccentricities, coined the word “extremus”, the superlative of exter, “being on the outside”. By the fifteenth century “extreme” had arrived to English, via Middle French. At the beginning of the 21st century, we know that Earth contains environmental extremes unimaginable to our ancestors of the 19th century. Even more unimaginable to them would be the fact that there are organisms that live, and grow, in these environmental extremes. R. D. MacElroy named these organisms lovers (from the Greek “philos”), “extremophiles” as in “lovers of extreme environments”. The discovery of extremophiles has put vitality in the biotechnology industry as this discipline has exploded in the past 20 years. Several reviews have been published on extremophiles and an increasing number of meetings and conferences are organised around the theme. Genomes of extremophiles have been sequenced, patents have been filed and several funding programmes have been launched namely the US National Science Foundation and NASA’s programmes in “Life in Extreme Environments, Exobiology and Astrobiology”, and the European Union’s “Biotechnology of Extremophiles” and “Extremophiles as Cell Factories”(...)Fundação para a Ciência e a Tecnologia (FCT), e Fundo Social Europeu (FSE), o apoio financeiro no âmbito do Quadro Comunitário de apoio (Bolsa de Doutoramento SFRH/BD/30512/2006

Modelling evolution of genome size in prokaryotes in response to changes in their abiotic environment

The size of the genomes of known free-living prokaryotes varies from � 1:3 Mbp to � 13 Mbp. This thesis proposes a possible explanation of this variation due to variability of the physical conditions of the environment. In a stable environment, competition for the resource becomes the main force of selection and smaller (thus cheaper) genomes are favoured. In more variable conditions larger genomes will be preferred, as they have a wider range of response to a less predictable environment. An agent-based model (ABM) of genome evolution in an free-living prokaryotic population has been proposed. Using the classic Hutchinson niche space model, a gene was defined as a Gaussian function over a corresponding niche dimension. The cell can have more than one gene along a given dimension, and the envelope of all the corresponding responses is considered a full description of a cell’s phenotype over that dimension. Gene deletion, gene duplication, and modifying mutations are permitted during reproduction, so the number of genes and their phenotypic effect (height and position of the Gaussian envelope) are free to evolve. The surface under the curve is fixed to prevent ‘supergenes’ from occurring. Change of the environmental conditions is simulated as a bounded random walk with a varying length of the step (a parameter representing variability of the environment). Using this approach, the model is able to reproduce the phenomenon of genome streamlining in more stable environments (analogical to e.g. oligotrophic gyre regions of the ocean) and genome complexification in variable environments. Horizontal gene transfer (HGT) was also introduced, but was found to act in a similar manner as gene duplication and shown no important contribution to the speed of evolution and the adaptive potential of the population

Comparative genomics reveals intraspecific divergence of Acidithiobacillus ferrooxidans: insights from evolutionary adaptation

Acidithiobacillus ferrooxidans serves as a model chemolithoautotrophic organism in extremely acidic environments, which has attracted much attention due to its unique metabolism and strong adaptability. However, little was known about the divergences along the evolutionary process based on whole genomes. Herein, we isolated six strains of A. ferrooxidans from mining areas in China and Zambia, and used comparative genomics to investigate the intra-species divergences. The results indicated that A. ferrooxidans diverged into three groups from a common ancestor, and the pan-genome is ‘open’. The ancestral reconstruction of A. ferrooxidans indicated that genome sizes experienced a trend of increase in the very earliest days before a decreasing tendency during the evolutionary process, suggesting that both gene gain and gene loss played crucial roles in A. ferrooxidans genome flexibility. Meanwhile, 23 single-copy orthologous groups (OGs) were under positive selection. The differences of rusticyanin (Rus) sequences (the key protein in the iron oxidation pathway) and type IV secretion system (T4SS) composition in the A. ferrooxidans were both related to their group divergences, which contributed to their intraspecific diversity. This study improved our understanding of the divergent evolution and environmental adaptation of A. ferrooxidans at the genome level in extreme conditions, which provided theoretical support for the survival mechanism of living creatures at the extreme

SuspensionFeeding Benthic Species’ Physiological and Microbiome Response to Salmon Farming and Associated Environmental Changes

Caged salmon farming is increasingly undertaken in water bodies with strong hydrodynamics where hard and mixed substrate habitats are more prevalent. Yet, these structurally complex and heterogeneous habitats support diverse benthic communities including several cnidarians and sponges that remain poorly characterized. This study used a combination of respirometry measurements, gas chromatography and 16S rRNA metabarcoding to define the respiration rate, stable carbon (δ13C) and nitrogen isotopes (δ15N), fatty acid (FA) and microbial profiles, and assess the impact of salmon farming on four important epibenthic suspension-feeders along the western Norwegian coast: the sponges Craniella and Weberella, the soft coral Duva florida and the anemone Hormathia digitata. Our results showed striking differences in fatty acid profiles and host microbiome communities in terms of identity, functional capabilities and genetic properties across the suspension-feeders. We found evidence of increased mortality rate in specimens located near fish farm activities and of a species-specific effect on respiration rate, with D. florida showing increased activity under the farm. Effects of fish farming on the suspension feeders were also species-specific and particularly evidenced by functional microbial turnover and by alteration of overall FA profiles in the soft coral and sea anemone. In particular, D. florida showed reduced level of FAs close to the farm (0-350 m), with significant difference in composition along a distance gradient. Only H. digitata showed evidence of incorporation of organic material from the fish farm waste via fatty acids trophic markers (FATM) and stable isotope analysis. Overall, our study demonstrates that suspension feeders have taxon-specific sensitivity towards the effect of salmon farming, and identified several potential molecular indicators that could be used as surrogate of impact gradient upon further research and validation. It also provides a wealth of ecological and physiological information on some of the most common sessile epibenthic organisms within Arctic and sub-Arctic regions, enabling us to better understand their response and evaluate their resilience to environmental changes.publishedVersio

Phenotypic Switching of Bacterial Cells in Extreme Environments

A large number of terrestrial microbial lives thrive in extremes of environmental conditions, including extremes of pressure, temperature, salinity, pH, and a combination of them. For example, all the marine biomass thrive at high hydrostatic pressure depending on depth. The temperature in the ocean can be very high near the hydrothermal vents and salinity and pH depends on the composition of salt in the surrounding areas. On the surface, hot springs, lakes and geysers provide high temperature conditions, while many places are permafrost regions with subzero temperatures. There is an emerging body of work on the viability, genomics, and metagenomics of these organisms, also called extremophiles due to their love for extremes of physicochemical conditions. However, these studies only provide a small window into their adaptation. A better insight into their adaptation to these conditions can be obtained by investigating the effect of these environments on cellular processes of mesophiles, organisms that are not adapted to extremes. Usually, extremes of environmental conditions lead to stresses on mesophilic cells, and therefore, application of these environments may reveal the bottleneck cellular processes that are prone to fail. The effect of pressure, temperature, and salinity on various cellular processes including metabolisms, growth, cell division, and gene expression of a mesophilic bacterium, Escherichia coli was studied. This work provides a quantitative picture of these cellular processes of phenotypes obtained under different extremes, and sets a foundation for long-term laboratory evolution of a mesophilic bacterium to the extremes of environmental conditions. Furthermore, the results presented here are also useful in assessing the kind of microbial lifeforms that may exist elsewhere in our solar system, such as Mars, Europa, Ceres, and Enceladus, where the presence of liquid water is known