Catalogue of antimicrobial resistance genes in species of Bacillus used to produce food enzymes and feed additives

A key step in the characterisation of bacterial strains used in the food and feed chain is the identification of antimicrobial resistance (AMR) genes in their genomes. The presence of acquired AMR genes influences important aspects of the risk assessment, such as the applicability of the qualified presumption of safety (QPS) approach, which can have a direct impact on the data requirements. Aiming to implement the EFSA approach to discriminate between ‘intrinsic’ and ‘acquired’ AMR genes, a bioinformatics pipeline was developed and applied to the species of the genus Bacillus that are most frequently subjects of applications for regulated products submitted to EFSA. The results are presented as a catalogue of genes indicating their abundance and distribution among complete and confirmed genomes publicly available for each species. The results of this work are aimed to support the evaluation of AMR genes in a consistent and harmonised way

On the track of β-lactam resistance: Studies on the regulation of methicillinresistance in Staphylococcus aureus

Dissertação para obtenção do Grau de Doutor em BiologiaMethicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen, causing a wide range of infections. MRSA has not only developed an intrinsic resistance to all β-lactams, but has also acquired resistance to virtually all classes of antimicrobial agents. The characteristic MRSA phenotype is conferred by the presence of mecA gene which is regulated by a sensor-inducer (MecR1) and a repressor (MecI). However, mecA induction by its cognate sensor/inducer is very inefficient and, therefore, it is believed that optimal expression of β-lactam resistance in MRSA requires a non-functional MecR1-MecI system. Surprisingly, no correlation was found between the presence of functional MecR1- MecI and the level of β-lactam resistance in a representative collection of epidemic MRSA strains, suggesting the existence of other mecA regulators. In these studies, we show that the mecA regulatory locus is not a two-component system but, actually, it is a three-component system containing besides mecR1-mecI, the previously unidentified antirepressor mecR2. The crystal structure of MecR2 reveals a three-domain architecture, with an N-terminal DNA-binding-like domain, an intermediate scaffold domain, and a C-terminal dimerization domain, important to the functional dimeric oligomerization state. MecR2 disturbs the binding of the repressor MecI to the mecA promoter, which leads to its proteolytic inactivation independently from MecR1, presumably by non-specific cytoplasmatic proteases. Our data also demonstrates that in the presence of functional mecR1-mecI genes, mecR2 is essential for a robust induction of mecA transcription and, consequently, for the optimal expression of resistance phenotype in MRSA. These observations point to a revision of the current model for the transcriptional control of the mecA gene.Fundação para a Ciência e a Tecnologia - Doctoral Project (SFRH/BD/38316/2007

Synthetic bacterial communities for plant growth promotion

PhD ThesisIncreasing food demands have driven the adoption of new global strategies to intensify productivity without relying on heavy chemical treatments. In the last decades, plant-growth promoting rhizobacteria (PGPR) have emerged as potential biofertilisers and biopesticides in agriculture. The overall aim of this study was to research and develop approaches to genetically engineer PGPR to improve their beneficial activities toward the plant partner. A simplified PGPR community, a Bacillus consortium of three strains, was adopted to study the complexity of the interactions occurring within the consortium and the plant microbiome. Firstly, the comparative genomic analysis of the consortium highlighted the unique and shared features responsible for plant promotion, microbial interaction and cooperation among the strains (niche partitioning, organisation in biofilms with cooperative mechanisms of quorum sensing, cell density control and antibiotic detoxification). Flux balance analysis identified cross-feeding interactions among the strains and the metabolic capability of the consortium to provide nitrogen to the plant, transforming it into forms available for plant utilisation. The consortium PGP potential was then investigated in vitro (LEAP mesocosm assay) and in vivo (pot experiment) on the vegetable crop Brassica rapa. These tests show increased plant growth when the strains were inoculated together rather than individually and when the consortium was used as a supplement of the natural bulk soil microbiome. The in silico study and the plant experiments highlighted areas for genetic improvement of the consortium genomes. Lastly, this work describes the development of a conjugation system that could be used to efficiently engineer non-domesticated bacteria and bacterial communities, such as rhizobacteria and plant microbiomes. The system, based on the plasmid pLS20, was developed in Bacillus subtilis 168 and successfully tested on twenty-three wild type Bacillus strains and three rhizobacillus communities. The research presented here provides tools and approaches for the genetic manipulation of rhizobacterial communities, with the ultimate aim of generating sustainable agricultural bioformulations and sheds light on the complex interactions that can occur in a model microbial PGPR consortia

SubtilNet2, a functional interaction model for the validation of potential therapeutic targets

Systems Biology- the study of interactions between components of biological systems, and how these can produce new functions and behaviours, is beginning to produce a more comprehensive understanding of biology. Its development is enabling many new opportunities, including the discovery and development of more effective and targeted therapeutics for a range of different conditions. It was in this context that this investigation began, with focus placed upon identifying therapeutic targets in Bacillus subtilis that could be used to limit the development and spread of infection, so called anti-infective targets. Using an in silico data driven Systems Biology approach, our industrial collaborators, e- Therapeutics predicted pairs of genes from B. subtilis that could act as anti-infective targets when targeted together. This investigation was tasked with the development and testing of experimental models and approaches that could be used to validate these potential targets. In a separate collaboration with the Integrative Bioinformatics Group at Newcastle University, a functional interaction network model for B. subtilis- SubtilNet2, was generated and tested. Compiled from a range of experimental, bioinformatical and literature based sources, it represented all known functional interactions known to occur within B. subtilis. This network was applied to investigate the selection of the predicted targets, and determine any biological basis for the experimental results seen. A single predicted target acting by itself was confirmed to be successful. As a second component to this investigation, Systems Biology was used to complement traditional hypothesis driven research, specifically the possibility of directed targeting and channelling of substrates between two biosynthetic pathways. This was explored by studying the synthesis of carbamoyl phosphate (CP), an intermediate in both the arginine and uracil biosynthetic pathways. Typically, prokaryotes encode a single heterodimeric carbamoyl phosphate synthetase (CPS) that is used by both the arginine and pyrimidine biosynthetic pathways. B. subtilis and its close relatives are unique in encoding arginine- and uracil-specific copies of this enzyme. Moreover, the genes encoding the respective arginine (carA and carB) and uracil (pyrAA and pyrAB) specific CPSs are clustered with the other genes in their respective pathways (e.g. argC,J,B,D-carA,B-argF and pyrB,C,AA,AB,K,D,F,E) This degree of clustering is not found in bacteria with single CPSs. Experimental and SubtilNet2 analysis approaches were developed to express and individually test for the presence of any interaction between the subunits of each systems CPS’s, as well as to other components within associated gene clusters. The presence or absence of interaction would be used to determine if CP produced by one system could be shared with the opposite system. If it couldn’t, could the unusual cluster of genes seen to surround each CPS be used to encode a macromolecular complex structure with a single point of entry and exit to channel CP and other substrates within a biosynthetic system? A failure despite repeated attempts and strategies to produce soluble CPS subunits and other biosynthesis proteins, when expressed independently of one another, suggested a need for the presence of other members of each pathway. SubtilNet2 testing of these components and their functional associations didn’t identify any distinct groups or systems being supplied with system specific CP, however this is more likely to result from limitations of the associated approaches, rather than genuine a biological property.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Integrating distributed post-genomic data to infer the molecular basis of bacterial phenotypes

The aim of the project described in this thesis is to understand and predict the characteristics and behaviour of a family of bacteria through an analysis of genome wide data from a variety of sources. The focus of the research is a family of bacteria, Bacillus, whose members show a diverse range of phenotypes, from the non-pathogenic B. subtilis to B. anthrncis, the causative agent of anthrax. Specifically, the focus was on the genomic scale identification and characterisation of secreted proteins from Bacillus species. Firstly, the application of Grid-based computational approaches to problems in genomic analysis and annotation was investigated, applying mllGrid technology to a biological problem not previously addressed using this approach. e-Science workflows and a service-oriented approach were developed and applied to predict and characterise secreted proteins, and the results automatically integrated into a custom relational database. An associated Web portal was also developed to facilitate expert curation, results browsing and querying over the database. Workflow technology was also used to classify the putative secreted proteins into families and to study the relationships between and within these families. The design of the workflows, the architecture and the reasoning behind the approach used to build this system, called BaSPP, are discussed. Analysis of the putative Bacillus secretomes revealed clear distinctions between proteins present in the pathogens and those in the non-pathogens. The properties of the protein families present in all Bacillus secretomes, as well as those specific either to the pathogens or to the non-pathogens were investigated. Many of the protein families contained members of unknown function. In the iv second part of the project, these families were investigated in more depth, using additional data integration strategies not previously applied to these organisms. The secretomes were modelled at the system level, in the broader context of interactomes. A system called SubtilNet was therefore developed, using B. subtilis as the reference organism. As part of SubtilNet, a toolkit and architecture were developed and implemented for building and analysing probabilistic functional integrated networks (PFINs). The PFINs built for each Bacillus species using this system were subsequently used to delve further into the interactions specific to the secreted proteins by extracting and exploring the cross-species PFINs of these proteins. The cross-species PFINs for the protein families specific to the pathogens and non-pathogens were explored, with particular emphasis on the core PrsA-like protein family, which acted as a use case to show how the PFIN s can be used to shed light on protein function. The addition of orthologous links between species was demonstrated to facilitate network clustering and analysis, enabling putative annotations to be applied to proteins previously of unknown function.EThOS - Electronic Theses Online ServiceNorth East Regional e-Science Centre : European Commission (LSHC-CT-2004-503468) : EPSRC : Non-Linear DynamicsGBUnited Kingdo

Studies towards the development of a host:vector system for the genus Clostridium

Clostridia are widely recognised as organisms of biotechnological importance. This potential, however, cannot be fully exploited until reliable methods have been developed for the transfer of genetic information into and between members of the genus. As with other Gram-positive bacteria, attempts to develop host:vector systems have focused on protoplast transformation procedures. This approach, however, has been hampered by the lack of suitable plasmid vectors. Studies have been initiated to construct such a vector. As a potential source of a clostridial replicon, the cryptic plasmid of C. butvricum NCIB 7423 (pCBlOl, 6.05 kb) has been examined. The complete nucleotide sequence of pCBlOl was determined and it's minimal replicon characterised together with the elucidation of some of the mechanisms involved in it's replication. The erythromycin resistance determinant (Emr) of the Grampositive R-factor pAM/3l was chosen as a selectable marker and the entire nucleotide sequence of this gene was determined. The Ferredoxin (Fd) gene of Clostridium pasteurianum was cloned and employed in the construction of an expression cartridge. The effectiveness of this cartridge in promoting the expression of heterologous genes was examined using a promoter-less xvlE gene. The conjugative Streptococcus faecalis plasmid pAM/Jl was used to mobilise vectors carrying the xvlE gene (under the transcriptional control of the Fd promoter) into Clostridium acetobutvlicum where the gene was expressed. This represents one of the first reports of directed gene expression in ç, acetqbutyllgmn

Characterisation of the enzymes involved in methanol dissimilation in Bacillus methanolicus: Assessment of enzyme-based biohydrogen production for a cell-free biofuel cell

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The role of SecA2 in targeting substrates to the Sec-dependent protein translocase

PhD ThesisThe Sec pathway is the major route for secretion of proteins by bacteria. Its major components are: (i) the translocation channel comprising the SecYEG proteins; (ii) SecA: a chaperone/targeting/molecular motor that drives the movement of proteins across the membrane with participation of SecB or SRP (signal recognition particle) chaperones. Following translocation, proteins are folded to their mature conformation by folding factors such as PrsA. When compared with B. subtilis, the secretory translocase of B. anthracis contains homologues of several Sec pathway components: (i) two homologues of SecA (SecA1 and SecA2), (ii) two homologues of SecY (SecY1 and SecY2), (iii) three homologues of the PrsA foldase (PrsAA, PrsAB and PrsAC). In previous studies, SecA2 was shown to be specific for secretion of S-layer proteins: Sap and EA1, while SecY2 has not been shown to have any substrate specificity. Instead, it seems to ensure high levels of protein secretion in later phases of the growth cycle. A combination of approaches was used to continue the analysis of functioning of the SecA2 secretion pathway. They involved analysis of deletion mutants of B. anthracis, complementation studies, gene expression analysis, protein interaction investigation. We found that a novel protein BA0881 facilitates secretion of Sap and EA1, but is not essential for the processes, and was renamed SecH. Moreover, protein interaction and complementation studies revealed putative interactions between (i) SecA2 and EA1, (ii) SecA2 and SecH, (iii) EA1 and SecH, (iv) SecA1 and SecA2, raising the possibility that SecA2 and SecA1 form a dimer, which might be a functional entity for the secretion of Sap and EA1, with SecH having a role in enhancing interaction between SecA1/SecA2 dimer and its substrates. The role of PrsA-like foldases on secretion was also investigated. PrsAB was found to show substrate specificity for Sap and EA1, while PrsAA showed substrate specificity for penicillin binding proteins. Analysis of cell morphology suggests that PrsAB and SecA2 may also have substrates other than those of Sap and EA1 as their null-mutants show changes in cell length and shape. Lastly, analysis of gene expression showed that the deletion of genes encoding elements of the translocation sytem: SecA2, SecH, PrsAA, PrsAB, PrsAC, SecY2 leads to changes in the level of expression of sap and eag.BBSRC-CASE studentship,University of Newcastle, and Novozymes, Denmark