The spatial evolution of the chemotaxis proteins of the Bacillus subtilis group

The aim of this work was to study spatial evolution of the chemotaxis proteins of a group of plant-associated soil-dwelling bacteria vernacularly referred to as the B. subtilis group. This was achieved by creating homology models for the chemotaxis proteins if a suitable template was available, and by analysing the selective forces (positive, purifying or neutral) acting upon the chemotaxis proteins. Chemotaxis is the phenomenon in which bacteria direct their movement towards more favourable conditions, and is critical for processes such as obtaining nutrients, escaping toxic compounds, host colonization and bio-film formation. Members of the B. subtilis group exhibit different preferences for certain host plants, and it is therefore feasible that their chemotactic machinery are fine-tuned to respond optimally to the conditions of the various niches that the strains inhabit. Homology models were inferred for the plant growth promoting B. amyloliquefaciens FZB42 proteins CheB, CheC, CheD, CheR, CheW and CheY. The interactions between: CheC-CheD, the P1 and P2 domains of CheA with CheY and CheB, and the P4 and P5 domains of CheA with CheW were also modelled. The hydrophobic interactions contributing to intra- and inter-protein contacts were analysed. The models of the interactions between CheB and the various domains of CheA are of particular interest, because to date no structures have been solved that show an interaction between a histidine kinase (such as CheA) and a multidomain response regulator (such as CheB). Furthermore, evidence that phospho-CheB may inhibit the formation of phospho-CheY by competitively binding to the P2 domain of CheA is also presented. Proteins were analysed to determine if individual amino acid sites are under positive, neutral or purifying selection. The Methyl Accepting Chemotaxis Proteins (MCPs), CheA and CheV were also analyzed, but due to a lack of suitable templates, no homology models were constructed. Site-specific positive and purifying selection were estimated by comparing the ratios of non-synonymous to synonymous substitutions at each site in the sequences for the chemotaxis proteins as well as for the receptors McpA, McpB, and McpC. Homology models were coloured according to intensity of selective forces. It was found that the chemotaxis proteins of member of the B. subtilis group are under strong evolutionary constraints, hence it is unlikely that positive selection in these proteins are responsible for the differences in habitat preference that these organism exhibit

ORCAE-AOCC : a centralized portal for the annotation of African orphan crop genomes

ORCAE (Online Resource for Community Annotation of Eukaryotes) is a public genome annotation curation resource. ORCAE-AOCC is a branch that is dedicated to the genomes published as part of the African Orphan Crops Consortium (AOCC). The motivation behind the development of the ORCAE platform was to create a knowledge-based website where the research-community can make contributions to improve genome annotations. All changes to any given gene-model or gene description are stored, and the entire annotation history can be retrieved. Genomes can either be set to “public” or “restricted” mode; anonymous users can browse public genomes but cannot make any changes. Aside from providing a user- friendly interface to view genome annotations, the platform also includes tools and information (such as gene expression evidence) that enables authorized users to edit and validate genome annotations. The ORCAE-AOCC platform will enable various stakeholders from around the world to coordinate their efforts to annotate and study underutilized crops

Draft genomes of two Artocarpus plants, jackfruit (A. heterophyllus) and breadfruit (A. altilis)

Two of the most economically important plants in the Artocarpus genus are jackfruit (A. heterophyllus Lam.) and breadfruit (A. altilis (Parkinson) Fosberg). Both species are long-lived trees that have been cultivated for thousands of years in their native regions. Today they are grown throughout tropical to subtropical areas as an important source of starch and other valuable nutrients. There are hundreds of breadfruit varieties that are native to Oceania, of which the most commonly distributed types are seedless triploids. Jackfruit is likely native to the Western Ghats of India and produces one of the largest tree-borne fruit structures (reaching up to 45 kg). To-date, there is limited genomic information for these two economically important species. Here, we generated 273 Gb and 227 Gb of raw data from jackfruit and breadfruit, respectively. The high-quality reads from jackfruit were assembled into 162,440 scaffolds totaling 982 Mb with 35,858 genes. Similarly, the breadfruit reads were assembled into 180,971 scaffolds totaling 833 Mb with 34,010 genes. A total of 2822 and 2034 expanded gene families were found in jackfruit and breadfruit, respectively, enriched in pathways including starch and sucrose metabolism, photosynthesis, and others. The copy number of several starch synthesis-related genes were found to be increased in jackfruit and breadfruit compared to closely-related species, and the tissue-specific expression might imply their sugar-rich and starch-rich characteristics. Overall, the publication of high-quality genomes for jackfruit and breadfruit provides information about their specific composition and the underlying genes involved in sugar and starch metabolism

First draft genome assembly of the desert locust, Schistocerca gregaria

Background: At the time of publication, the most devastating desert locust crisis in decades is affecting East Africa, the Arabian Peninsula and South-West Asia. The situation is extremely alarming in East Africa, where Kenya, Ethiopia and Somalia face an unprecedented threat to food security and livelihoods. Most of the time, however, locusts do not occur in swarms, but live as relatively harmless solitary insects. The phenotypically distinct solitarious and gregarious locust phases differ markedly in many aspects of behaviour, physiology and morphology, making them an excellent model to study how environmental factors shape behaviour and development. A better understanding of the extreme phenotypic plasticity in desert locusts will offer new, more environmentally sustainable ways of fighting devastating swarms. Methods: High molecular weight DNA derived from two adult males was used for Mate Pair and Paired End Illumina sequencing and PacBio sequencing. A reliable reference genome of Schistocerca gregaria was assembled using the ABySS pipeline, scaffolding was improved using LINKS. Results: In total, 1,316 Gb Illumina reads and 112 Gb PacBio reads were produced and assembled. The resulting draft genome consists of 8,817,834,205 bp organised in 955,015 scaffolds with an N50 of 157,705 bp, making the desert locust genome the largest insect genome sequenced and assembled to date. In total, 18,815 protein-encoding genes are predicted in the desert locust genome, of which 13,646 (72.53%) obtained at least one functional assignment based on similarity to known proteins. Conclusions: The desert locust genome data will contribute greatly to studies of phenotypic plasticity, physiology, neurobiology, molecular ecology, evolutionary genetics and comparative genomics, and will promote the desert locust’s use as a model system. The data will also facilitate the development of novel, more sustainable strategies for preventing or combating swarms of these infamous insects

First draft genome assembly of the desert locust, Schistocerca Gregaria

BACKGROUND: At the time of publication, the most devastating desert locust crisis in decades is affecting East Africa, the Arabian Peninsula and South-West Asia. The situation is extremely alarming in East Africa, where Kenya, Ethiopia and Somalia face an unprecedented threat to food security and livelihoods. Most of the time, however, locusts do not occur in swarms, but live as relatively harmless solitary insects. The phenotypically distinct solitarious and gregarious locust phases differ markedly in many aspects of behaviour, physiology and morphology, making them an excellent model to study how environmental factors shape behaviour and development. A better understanding of the extreme phenotypic plasticity in desert locusts will offer new, more environmentally sustainable ways of fighting devastating swarms. METHODS: High molecular weight DNA derived from two adult males was used for Mate Pair and Paired End Illumina sequencing and PacBio sequencing. A reliable reference genome of Schistocerca gregaria was assembled using the ABySS pipeline, scaffolding was improved using LINKS. RESULTS: In total, 1,316 Gb Illumina reads and 112 Gb PacBio reads were produced and assembled. The resulting draft genome consists of 8,817,834,205 bp organised in 955,015 scaffolds with an N50 of 157,705 bp, making the desert locust genome the largest insect genome sequenced and assembled to date. In total, 18,815 proteinencoding genes are predicted in the desert locust genome, of which 13,646 (72.53%) obtained at least one functional assignment based on similarity to known proteins. CONCLUSIONS: The desert locust genome data will contribute greatly to studies of phenotypic plasticity, physiology, neurobiology, molecular ecology, evolutionary genetics and comparative genomics, and will promote the desert locust’s use as a model system. The data will also facilitate the development of novel, more sustainable strategies for preventing or combating swarms of these infamous insects.The Special Research Fund of KU Leuven, the Research Foundation of Flanders, the Special Research Fund of Ghent University, the Department of Research and Innovation of the University of Pretoria, the U.S. National Science Foundation, the U.S. Department of Agriculture and the Biotechnology and Biological Sciences Research Council UK.http://f1000research.compm2021BiochemistryGeneticsMicrobiology and Plant Patholog

The draft genomes of five agriculturally important African orphan crops

Background: Continuous growth of the world population is expected to double the worldwide demand for food by 2050. Eighty-eight percent of countries current face a serious burden of malnutrition, especially in Africa and South and South-East Asia. About 95% of the food energy needs of humans are fulfilled by just 30 species, of which wheat, maize and rice provide the majority of calories. Therefore, to diversify and stabilize global food supply, enhance agricultural productivity and tackle malnutrition, greater use of neglected or underutilized local plants (so-called 'orphan crops‘, but also including a few plants of special significance to agriculture, agroforestry and nutrition) could be a partial solution.Results: Here, we present draft genome information from five agriculturally, biologically, medicinally and economically important underutilized plants native to Africa; Vigna subterranea, Lablab purpureus, Faidherbia albida, Sclerocarya birrea, and Moringa oleifera. Assembled genomes range in size from 217 to 654 Mb. In V. subterranea, L. purpureus, F. albida, S. birrea and M. oleifera we have predicted 31707, 20946, 28979, 18937, 18451 protein-coding genes, respectively. By further analysing the expansion and contraction of selected gene families, we have characterized root nodule symbiosis genes, transcription factors and starch biosynthesis-related genes in these genomes.Conclusions: These genome data will be useful to identify and characterize agronomically important genes and understand their modes of action, enabling genomics-based, evolutionary studies, and breeding strategies to design faster, more focused and predictable crop improvement programs

Neutralization profiles of HIV-1 viruses from the VRC01 Antibody Mediated Prevention (AMP) trials

The VRC01 Antibody Mediated Prevention (AMP) efficacy trials conducted between 2016 and 2020 showed for the first time that passively administered broadly neutralizing antibodies (bnAbs) could prevent HIV-1 acquisition against bnAb-sensitive viruses. HIV-1 viruses isolated from AMP participants who acquired infection during the study in the sub-Saharan African (HVTN 703/HPTN 081) and the Americas/European (HVTN 704/HPTN 085) trials represent a panel of currently circulating strains of HIV-1 and offer a unique opportunity to investigate the sensitivity of the virus to broadly neutralizing antibodies (bnAbs) being considered for clinical development. Pseudoviruses were constructed using envelope sequences from 218 individuals. The majority of viruses identified were clade B and C; with clades A, D, F and G and recombinants AC and BF detected at lower frequencies. We tested eight bnAbs in clinical development (VRC01, VRC07-523LS, 3BNC117, CAP256.25, PGDM1400, PGT121, 10–1074 and 10E8v4) for neutralization against all AMP placebo viruses (n = 76). Compared to older clade C viruses (1998–2010), the HVTN703/HPTN081 clade C viruses showed increased resistance to VRC07-523LS and CAP256.25. At a concentration of 1μg/ml (IC80), predictive modeling identified the triple combination of V3/V2-glycan/CD4bs-targeting bnAbs (10-1074/PGDM1400/VRC07-523LS) as the best against clade C viruses and a combination of MPER/V3/CD4bs-targeting bnAbs (10E8v4/10-1074/VRC07-523LS) as the best against clade B viruses, due to low coverage of V2-glycan directed bnAbs against clade B viruses. Overall, the AMP placebo viruses represent a valuable resource for defining the sensitivity of contemporaneous circulating viral strains to bnAbs and highlight the need to update reference panels regularly. Our data also suggests that combining bnAbs in passive immunization trials would improve coverage of global viruses

Comparative structural bioinformatics analysis of Bacillus amyloliquefaciens chemotaxis proteins within Bacillus subtilis group

Chemotaxis is a process in which bacteria sense their chemical environment and move towards more favorable conditions. Since plant colonization by bacteria is a multifaceted process which requires a response to the complex chemical environment, a finely tuned and sensitive chemotaxis system is needed. Members of the Bacillus subtilis group including Bacillus amyloliquefaciens are industrially important, for example, as bio-pesticides. The group exhibits plant growth-promoting characteristics, with different specificity towards certain host plants. Therefore, we hypothesize that while the principal molecular mechanisms of bacterial chemotaxis may be conserved, the bacterial chemotaxis system may need an evolutionary tweaking to adapt it to specific requirements, particularly in the process of evolution of free-living soil organisms, towards plant colonization behaviour. To date, almost nothing is known about what parts of the chemotaxis proteins are subjected to positive amino acid substitutions, involved in adjusting the chemotaxis system of bacteria during speciation. In this novel study, positively selected and purified sites of chemotaxis proteins were calculated, and these residues were mapped onto homology models that were built for the chemotaxis proteins, in an attempt to understand the spatial evolution of the chemotaxis proteins. Various positively selected amino acids were identified in semi-conserved regions of the proteins away from the known active sites

Perturbations in nucleotide biosynthesis inhibits Salmonella biofilm formation

The overall goal of this project was to investigate the molecular link between nucleotide metabolism and biofilm formation, with a particular focus on pyrimidine starvation. The potential of using nucleoside based drugs as antibacterial compounds was also explored. In order to determine which intermediates in the de novo pyrimidine biosynthesis pathways are required for biofilm formation, a set of single gene knockout mutants was evaluated for its ability to form biofilms. The molecular links between nucleotide biosynthesis and biofilm formation were probed by assessing the effect of disruption of nucleotide biosynthesis pathways on the expression of a diverse set of genes. Gene expression levels were measured with RNA sequencing, GFP-promoter fusions and RT-qPCR. Transcriptome profiles were analysed using a network based approach that integrates a variety of “omics” data. Furthermore metabolite analysis and mutant phenotyping were also used. Our results indicate that pyrimidine starvation is a limiting factor for biofilm formation and that biofilm inhibition is associated with a repression of the transcription of csgD. Interestingly csgD down-regulation occurs despite an increase in c-di-GMP, which is known to stimulate biofilm formation. We explored the possible causes behind the increased c-di-GMP level and concluded that it is propelled by an increase of the precursor GTP. In fact, not only GTP but also other purine pathway intermediates showed an increased intracellular concentration. We propose that this increase is driven by an unknown factor that prevents repression of purine and PRPP biosynthesis genes by PurR. We also explored possible mechanisms behind the down-regulation of csgD despite high c-di-GMP levels. Based on transcriptome data analyzed with a network-based approach, we hypothesize that the global regulator Fis is involved. Fis negatively regulates rpoS expression and RpoS is known to activate csgD transcription. Transcription of fis is sensitive to nucleotide availability, providing a link between pyrimdine starvation, csgD down regulation and biofilm inhibition. Our results indicate that interfering with nucleotide biosynthesis and signalling is a feasible way to combat biofilms, as demonstrated with 5-FU.Acknowledgementsi Summary iii Samenvatting v List of abbreviations vii Chapter 11 1 A general introduction to Salmonella and biofilm formation and outline of the thesis 1 1.1 Abstract 1 1.2 Aim and scope of thesis 1 1.3 Introduction 2 1.3.1 Salmonella taxonomy 2 1.3.2 Clinical and economic relevance of NTS Salmonella infections 4 1.4 Biofilm formation 6 1.4.1 Biofilm associated problems 6 1.4.2 The switch to a biofilm mode of life 7 1.4.1 Relevance of Salmonella biofilms 8 1.5 Models for studying biofilm formation 10 1.5.1 Congo red Agar 10 1.5.2 Calcofluor agar 11 1.5.3 Crystal violet staining 12 1.5.4 Fluorescent probes that bind specific EPS structures 13 1.6 Regulation of Salmonella biofilm formation 13 1.6.1 CsgD, the master regulator 15 1.6.2 Coordinating motility, virulence and biofilm formation 16 1.7 Concluding remarks 17 Chapter 2 19 2 The importance of nucleotides in biofilm formation .19 2.1 Abstract 19 2.2 Background 19 2.2.1 Introduction to the purine and pyrimidine biosynthesis 19 2.2.2 Purine synthesis and regulation 21 2.2.3 Pyrimidine synthesis and regulation 26 2.3 The role of nucleotide biosynthesis in biofilm formation 34 2.3.1 Nucleotide derived second messengers 36 2.3.2 The role of c-di-GMP signaling in Salmonella 46 2.3.3 Regulation of biofilm formation by c-di-GMP in Salmonella 49 Chapter 3 55 3 Repurposing of nucleoside- and nucleobase-derivative drugs as antibiotics and biofilm inhibitors 55 3.1 Abstract 55 3.2 Introduction 55 3.2.1 Nucleobase and nucleoside analogs have been in clinical use for several decades 56 3.2.2 Repurposing NNADs could overcome hurdles in drug discovery 57 3.3 Examples of approved NNADs with activity against bacteria 61 3.3.1 5-Fluorouracil 61 3.3.2 5-Fluorocytosine 64 3.3.3 5-Azacytidine 65 3.3.4 Azathioprine and 6-Mercaptopurine .68 3.3.5 Caffeine 69 3.3.6 Azidothymidine 68 3.3.7 Gemcitabine 70 3.4 Resistance development against NNADs 71 3.4.1 NNADs can have multiple targets within the bacterial cell .72 3.4.2 Combining treatment with antibiotics can prevent the establishment of resistance against NNADs 72 3.4.3 Mutations providing resistance to NNADs are potentially disadvantageous 73 3.4.4 NNADs can inhibit biofilm formation without impacting growth 73 3.5 Conclusion 73 Chapter 4 81 4 Pyrimidine starvation inhibits biofilm formation despite high c-di-GMP levels81 4.1 Abstract 81 4.2 Introduction 81 4.3 Results .84 4.3.1 Pyrimidine starvation is a limiting factor for biofilm formation 84 4.3.2 UDP glucose levels are unaffected by pyrimdine starvation 90 4.3.3 A lack of pyrimidines is associated with an increase in c-di-GMP 91 4.4 Conclusion .92 4.5 Materials and Methods 93 4.5.1 Bacterial strains and growth conditions 93 4.5.2 Construction of deletion and complementation mutants 92 4.5.3 Phenotypic switching between planktonic and biofilm mode of growt.96 4.5.4 Assaying biofilm formation 97 4.5.5 Microscopy 98 4.5.6 Metabolite extraction and mass spectrometry analysis .98 Chapter 5 .101 5 The impact of pyrimidine starvation on the transcriptome 101 5.1 Abstract 101 5.2 Introduction .101 5.3 Results .103 5.3.1 Pyrimidine limitation affects global regulators 105 5.3.2 Pyrimidine starvation leads to an increase in c-di-GMP 112 5.3.3 Pyrimidine starvation impacts the transcription of csgD 121 5.3.4 Other processes that are affected by pyrimdine starvation 130 5.4 Conclusion 134 5.5 Materials and Methods 136 5.5.1 Bacterial strains and growth conditions 136 5.5.2 Construction of deletion and complementation mutants 137 5.5.3 Temporal quantification of gene transcription .137 5.5.4 Metabolite extraction .138 5.5.5 Experimental setup for RNA extraction 139 5.5.6 RNA extraction .139 5.5.7 RNA sequencing 140 5.5.8 Transcriptome assembly and analysis 140 5.5.9 Network based- analysis 140 5.5.10 Primer extension analysis 141 Chapter 6 143 6 Prevention of Salmonella biofilm formation by 5-FU 143 6.1 Abstract 143 6.2 Introduction 143 6.3 Results144 6.3.1 5-FU inhibits Salmonella biofilm formation.144 6.3.2 Differential gene expression in response to 5-FU treatment 145 6.4 Conclusion 146 6.5 Materials and methods .147 6.5.1 Assaying biofilm formation .148 6.5.2 High throughput biofilm assay 149 6.5.3 Microscopy 149 6.5.4 Temporal quantification of gene transcription 149 6.6 Acknowledgements 150 Chapter 7 151 7 Concluding remarks 151 7.1 General conclusion 151 7.1.1 Nucleotides and biofilm formation .151 7.1.2 Possible polar effects on carB due to carA deletion 154 7.2 Future perspectives 154 7.2.1 Linking fis expression and nucleotide levels to biofilm formation 154 7.2.2 Regulatory interplay between the purine and pyrimidine pathways 155 7.2.3 Partitioning of nucleotide resources during biofilm formation 156 7.2.4 Screening NNADs 156 7.2.5 Effect on virulence 156 7.3 Final remarks 157 Appendix 159 List of references 191 List of publications 225status: publishe

Agaric acid reduces Salmonella biofilm formation by inhibiting flagellar motility

status: Published onlin