Microbial Genetics, Genomics and Biotechnologies

Groups leaders: Alessandra Albertini, Cinzia Calvio, Davide Sassera, Claudio Seppi

Collaborators: Giulia Barbieri (Postdoctoral fellow), Paolo Gabrieli (Postdoctoral fellow), Elisabetta Andreoli (technician), Giuliano Gasperi (adjunct professor), Luca Longanesi (assegnista), Francesco Comandatore (Postdoctoral fellow), Stefano Gaiarsa (PhD student), Leone De Marco (PhD student), Emanuela Clementi (technician), Luciano Sacchi (adjunct professor)

RESEARCH LINES

 

REGULATION OF GENE EXPRESSION IN THE GRAM + MODEL ORGANISM Bacillus subtilis

A. Albertini; in collaboration with A.L. Sonenshein and B.R. Belitsky (Department of Molecular Biology and Microbiology Tufts University of Boston MA, USA)

One of our main research interests is the study of regulation of gene expression in Bacillus subtilis. Bacteria can use different strategies to cope with difficult environmental conditions and with scarcity of nutrients. These include the ability to move, the synthesis of degradative enzymes and transporters able to import the products of degradation, and the production of antibiotics. At the onset of stationary phase, Bacillus subtilis is also able to differentiate into competent cells capable of taking up DNA from the environment, or into metabolically dormant spores, highly resistant to external stresses. Our group is interested in the study of the regulatory pathways that control these differentiation processes at the beginning of stationary phase. In collaboration with professors A.L. Sonenshein and B. Belitsky, we recently demonstrated that the global transcriptional regulator CodY, one of the main regulators of the nutritional stress response, is involved in the regulation of four extracellular proteases in B. subtilis.

Related papers

  1. Barbieri G, Albertini AM, Ferrari E, Sonenshein AL, Belitsky BR. Interplay of CodY and ScoC in the Regulation of Major Extracellular Protease Genes of Bacillus subtilis. J Bacteriol. Jan 4;198(6):907-20. doi: 10.1128/JB.00894-15, 2016
  2. Belitsky BR, Barbieri G, Albertini AM, Ferrari E, Strauch MA, Sonenshein AL. Interactive regulation by the Bacillus subtilis global regulators CodY and ScoC. Mol Microbiol., Aug;97(4):698-716. doi: 10.1111/mmi.13056 4.419, 2015
  3. Barbieri G, Voigt B, Albrecht D, Hecker M, Albertini AM, Sonenshein AL, Ferrari E, Belitsky BR. CodY regulates expression of the Bacillus subtilis extracellular proteases Vpr and Mpr. J Bacteriol. Apr;197(8):1423-32. doi: 10.1128/JB.02588-14. 2.808, 2015

IDENTIFICATION OF NEW BIOLOGICAL INSECTICIDES BY BACTERIA FOR THE CONTROL OF Aedes albopictus (ASIAN TIGER MOSQUITO)

A. Albertini, G. Gasperi

The project, funded by the Bussolera-Branca Foundation, interests two laboratories with complementary skills of the Department of Biology and Biotechnology, and aims to the identification of new bacterial strains able to produce molecules characterized by specific insecticidal activity for the control of Aedes albopictus. For this purpose, bacteria with larvicidal activity are sought in nature (soil, standing water, etc.) tested and  validated on standard tiger mosquito strain larvae of Italian origin, available in the Insects Genomics and Biotechnology Laboratory. Subsequently will be assayed the bacterial strains and the compounds endowed with bio-insecticide activities isolated from soil, for toxicity against other natural populations of Aedes albopictus, as well as for the safety for vertebrates, insects and other animal and plants species of agronomic interest, in view of the validation of a possible field use.

Genomic and metagenomic approaches are employed to identify genes involved in the production of these bacterial secondary metabolites; to improve the level of expression in a bacterium suitable for the production on a commercial scale, such as B. subtilis, will be set up cloning and “genome shuffling” methods.

 

BACTERIAL CELL FACTORIES FOR CATALYSTS PRODUCTION AND MUTAGENESIS. 

 A. Albertini and C. Calvio; in collaboration with D. Ubiali and T. Bavaro (Department of Drug Science, University of Pavia) and C. Morelli and G. Speranza (Department of Chemistry, Statale University of Milano)

E. coli is well suited for the expression of foreign genes, due to the vast knowledge of its metabolism and the availability of a large collection of different vectors and strains.

The group of Microbial Genetics and Biotechnology collaborated with the Biocatalysis Laboratory of the Department of Drug Sciences for the production of new biocatalysts,  such as PGA (Penicillin G-acylase, catalyst for the production of β lactam nuclei, to be used for the synthesis of semi-synthetic antibiotics), or purine and pyrimidine phosphorylases, catalysts for the transglycosilation reactions between a nucleoside (natural or modified in the sugar moiety) and a natural or modified purine or pyrimidine base.

Aims of these projects are the production of new immobilized catalysts trough the search of new genes for purine and pyrimidine phosphorylases from different bacterial sources, and new strategies coupling in vitro site directed mutagenesis and immobilization for the rational design of more efficient biocatalysts.

Recently, in collaboration with C. Morelli (Department of Chemistry, Statale University of Milano), a CARIPLO research grant is funding studies on the trans-peptidation reactions catalyzed by g-glutamyl transpeptidases (GGTs) of microbial origin to improve through mutagenesis the catalytic properties of the enzymes to produce new g-glutamyl derivatives of interest in the food and pharma industry. The mutagenesis and selection of recombinant GGTs will allow the immobilization for the development of large scales processes.

Related papers

  1. Biagiotti M, Borghese G, Francescato P, Morelli C F, Albertini AM, Bavaro T, Ubiali D, Mendichi R and Speranza G. Esterification of poly(γ-glutamic acid) (γ-PGA) mediated by its tetrabutylammonium salt. RSC Adv., 6, 43954-43958. doi: 10.1039/C6RA08567A, 2016
  2. Ubiali D., Morelli C.F., Rabuffetti M., Cattaneo G., Serra I., Bavaro T., Albertini A.M. and Speranza G. Substrate Specificity of a Purine Nucleoside Phosphorylase from Aeromonas hydrophila Toward 6-Substituted Purines and its Use as a Biocatalyst in the Synthesis of the Corresponding Ribonucleosides, Curr. Org. Chem. 19 (22): 2220 – 2225,. 2.157, 2015
  3. Serra I., Ubiali D., Cecchini D.A., Calleri E., Albertini A.M., Terreni M., Temporini C. Assessment of immobilized PGA orientation via the LC-MS analysis of tryptic digests of the wild type and its 3K-PGA mutant assists in the rational design of a high-performance biocatalyst. Anal Bioanal Chem. Jan; 405(2-3):745-53. doi: 10.1007/s00216-012-6143-z , 2013
  4. Serra I., Bavaro T., Cecchini D. A., Daly S., Albertini A.M., Terreni M., Ubiali D., A Comparison between Immobilized Pyrimidine Nucleoside Phosphorylase from Bacillus subtilis and Thymidine Phosphorylase from Escherichia coli in the Synthesis of 5-Substituted Pyrimidine 2′-Deoxyribonucleosides. Journal of Molecular Catalysis B: Enzymatic, 95, 16-22, DOI information: 10.1016/j.molcatb.2013.05.007, 2013
  5. Serra I., Ubiali D., Piskur J., Christoffersen S., Lewkowicz E., Iribarren A. M.,. Albertini A. M, Terreni M., Developing a Collection of Immobilized Nucleoside Phosphorylases for the preparation of Nucleoside Analogues: Enzymatic Synthesis of Arabinosyladenine and 2′,3′-Dideoxyinosine, Chem Plus Chem, 78 (2), 157-165. DOI: 10.1002/cplu.201200278, 2013
  6. Serra I., Ubiali D., Cecchini D.A., Calleri E., Albertini A.M., Terreni M., Temporini C. Assessment of immobilized PGA orientation via the LC-MS analysis of tryptic digests of the wild type and its 3K-PGA mutant assists in the rational design of a high-performance biocatalyst. Anal Bioanal Chem., 405(2-3):745-53, DOI: 10.1007/s00216-012-6143-z, 2012
  7. Ubiali D., Serra C.D., Serra I., Morelli C.F., Terreni M., Albertini A.M., Manitto P. and Speranza G. Production, characterization and synthetic applicatio of a purine nucleoside phosphorylase from Aereomonas hydrophyla. Adv Synth. Catal. 354, 96-1041, 2012
  8. Serra I, Cecchini DA, Ubiali D, Manazza EM, Albertini AM, Terreni M. Coupling of site-directed mutagenesis and immobilization for the rational design of more efficient biocatalysts. The case of immobilized 3G3K PGA from coli. Eur. J. Org. Chem., 9:1384 -1389, 2009
  9. Cecchini DA, Serra I, Ubiali D, Terreni M and Albertini AM. New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase. BMC Biotechnol.7: 54, 2007.

 

Bacillus subtilis STRAIN IMPROVEMENT FOR γ-PGA PRODUCTION

C. Calvio, C. Seppi; in collaboration with P. Mustarelli, Chemistry Dept. and P. Magni, Electrical Computer and Biomedical Engineering (University of Pavia), and G. Mazzini (IGM-CNR, Pavia)

The need of safer raw material, derived from renewable sources is the motor driving the growing interest towards natural biopolymers. γ-PGA is an anionic polymer produced by Bacilli, formed by thousands glutamic acid units. Due to its non-toxicity, the water solubility and biodegradability it finds application in several biotechnological fields as: flocculant for heavy metal removal, cryoprotectant, humectant, thickening additive in cosmetics and food industries, as biological glue, as drug or vaccine carrier or scaffold for biomedical engineering. However, for its full industrial exploitation it is mandatory to reduce production costs, both increasing bacterial productivity and reducing fermentation costs. Our Lab has obtained a producer strain derived from the B. subtilis lab strain, 168, fully characterized. The availability of a well defined strain, which is genetically amendable, offers the opportunity to apply genetic engineering to improve productivity and rationalize metabolic pathways for lowering fermentation costs. By introducing specific mutations we already obtained strains that show high product yield. Now, the aim is to obtain a producer able to ferment organic components contained in some agro-industrial by-products. We plan to take advantage of both rice straw, an abundant biomass currently under-exploited, and raw glycerol, a co-product in the biodiesel industry as bacterial feedstock. The meeting of the above objectives will not only lead to cheaper γ-PGA, but will also contribute to the valorization of the rice and biodiesel production chains and reinforce the development of new bio-economy sectors. This research line is currently funded by two CARIPLO FOUNDATION grants.

 

γ-PGA-HYDROLASES AS ANTIBACTERIAL TOOLS

  1. C. Calvio, C. Seppi; in collaboration with C. Morelli form the Dept. of Chemistry (Statale University of Milan) and A. Pastore, Dept. of Molecular Medicine (University of Pavia).

We identified and characterized four new genes of B. subtilis that code for γ-PGA hydrolases. We found that those gene are phage-derived and spread across bacteria through horizontal gene transfer. We also identified γ-PGA coding capacity in several microbial species, among which several pathogens. We are now tryng to analyze the role of γ-PGA  in the virulence behavior in some of those species and the possible use of the γ-PGA  hydrolase and antibacterial agents.

 

THE ROLE OF SwrA, A RECENTLY DICOVERED Bacillus subtilis REGULATORY FACTOR

C. Calvio

In Bacillus subtilis the two-component system DegS-DegU controls the expression of one hundred of genes involved in the exponential-to stationary phase of growth transition, coordinates single cells differentiation in multicellular communities and in pathogenic species, as Listeria monocytogenes or Bacillus anthracis, is involved in virulence. It has been shown that DegU regulates B. subtilis motility in a complex way. Also SwrA a protein which has no similarity to previously characterized proteins, is involved in such complex regulation. We have shown that there is a functional and molecular interaction between the two proteins, DegU and SwrA, in motility and now would like to extend our analyses to other genetic pathways DegU-regulated.

 

GENOMIC EPIDEMIOLOGY AND COMPARATIVE GENOMICS OF  NOSOCOMIAL PATHOGENS

D. Sassera; in partnership with P. Marone (IRCCS Policlinico S. Matteo in Pavia), S. Brisse (Institut Pasteur, Paris), E. Feil (University of Bath)

This research line is focused on whole-genome sequencing of large numbers of strains of nosocomial pathogens. The genomes thus generated are analyzed using bioinformatics tools, some developed ad-hoc, for the comparative genomic and genomic epidemiology analysis. The genomics of nosocomial pathogens allows to evaluate the genomic variability, the presence and mobility of virulence factors and antibiotic resistance, describe recombination events and overall architecture of genomic variations. This approach leads to applicative outcomes, such as the ability to reconstruct epidemic events and to identify transmission patterns, to characterize strains of interest based on the resistance and virulence profiles, and to approach the objective of using genomics in diagnostic microbiology.

 

ROLE OF SYMBIOSIS IN PARASITES

D. Sassera; in collaboration with C. Bandi (Statale University of Milan), B. Makepeace (University of Liverpool), O. Plantard (INRA, Nantes), G. Favia (University of Camerino)

Symbiotic microorganisms are widespread in many organisms, where they assume different roles, from parasitism to obligate mutualism. In the context of this research line, we use an interdisciplinary approach to identify new symbionts and to understand their role in the biology of the hosts. Integrating methods of light and electron microscopy, molecular biology, genomics and transcriptomics we are currently working on different microorganisms:

Midichloria mitochondrii: bacterium order Rickettsiales, symbiont of the tick Ixodes ricinus, has the unique ability to localize within the mitochondria of host cells. We are currently working to understand the role of bacteria in the physiology of the tick, through an integrated approach of electron microscopy, transcriptomics and proteomics. In parallel we are looking for bacteria phylogenetically related to M. mitochondrii other host with genomic techniques.

Symbiotic yeasts: yeast strains may have a role of symbionts in different species of arthropods, including vectors of important diseases. With a multidisciplinary project coupling electron microscopy and comparative genomics, we are investigating the presence and role of the yeast species Wickeramomyces anomalus and Meyerozyma guilliermondii in mosquitoes and sandflies.

 

TRANSCRIPTOMICS OF ARTHROPOD VECTORS

D. Sassera; in collaboration with S. Epis (University of Milan), S. Urbanelli and D. Porretta (University of Rome La Sapienza) , Favia G. and I. Ricci (University of Camerino)

The use of the RNA-seq methodology allows the evaluation of changes in gene expression at the whole transcriptome level. In this line of research we investigate these changes in arthropods that are vectors of diseases of great medical interest, mosquitoes and ticks, during the course of the life cycle or in response to specific stimuli, such as the presence of insecticide molecules.  The goal is to understand fundamental aspects of the biology of these vectors, as well as specific responses to treatments, in order to design innovative control strategies.