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Understanding and engineering bacterial genome architecture and gene regulation

Medicaco sative root nodules pictures
Photo: B. Frage, D. Kapp
Medicago sativa root nodules (left), root nodule section (middle), and electron microscopy of Sinorhizobium meliloti bacteroids (left).

Bacterial gene regulation

Bacteria virtually colonize all habitats on earth, from hospitable environments to those that are very hostile. Among their habitats are all complex life forms on Earth, which either benefit from or are harmed by the bacterial invaders and colonizers. Bacteria either act as highly specialized individual cells, as communities, or form complex multi-cellular structures. In natural habitats, major challenges for bacteria are physical changes in the ecosystem, fluctuations in the availability of nutrients, and transitions between lifestyles, such as switching from planktonic to surface associated or from free-living to host-associated states. Bacteria have evolved an array of specialized molecular mechanisms to sense and interpret environmental changes, and mount adaptive responses to master imposed challenges and exploit opportunities.

We explore and engineer bacterial regulatory network architectures and investigate the underlying molecular mechanisms.

Nucleotide second messengers

Lab picture, nucleotids
Photo: S. Schäper

Nucleotide second messengers are key components of the signaling networks that link sensory input with the regulatory output. We study second messenger signaling in alphaproteobacterial root-nodule symbionts, which possess an exceptional high number of enzymes making and breaking nucleotide second messengers. Our studies center on the role and regulatory mechanisms of cyclic-di-GMP signaling in switching between motile and sessile states, and of cAMP and cGMP signaling in the symbiotic interaction with the host plant.

We are member of the DFG-funded Priority Programme SPP 1879 "Nucleotide Second Messenger Signaling in Bacteria".

Diffusible Signals

Table Overview Diffusible Signals project
Photo: Anke Becker

Klebsiella pneumoniae is an important opportunistic pathogen frequently causing nosocomial infections of urinary and respiratory tract. In the framework of the LOEWE Research Cluster “Diffusible Signals”, we investigate factors influencing biosynthesis and properties of capsular polysaccharide (CPS) in the K. pneumoniae – host interaction. Second messenger signaling plays an important role in regulation of CPS biosynthesis. More than 100 capsule types have been identified in K. pneumoniae. CPS is a major determinant of virulence and a potential therapeutic target component.

Selected publications:

Schäper S, Wendt H, Bamberger J, Sieber V, Schmid J, Becker A (2019) A bifunctional UDP-sugar 4-epimerase supports biosynthesis of multiple cell surface polysaccharides in Sinorhizobium meliloti. J Bacteriol 201: e00801-18

Schäper S, Steinchen W, Krol E, Altegoer F, Skotnicka D, Søgaard-Andersen L, Bange G, Becker A (2017) AraC-like transcriptional activator CuxR binds c‑di‑GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production. Proc Natl Acad Sci USA 114: E4822-E4831

Krol E, Klaner C, Gnau P, Kaever V, Essen LO, Becker A (2016) Cyclic mononucleotide- and Clr-dependent gene regulation in Sinorhizobium meliloti. Microbiology SGM 162: 1840-1856

Schäper S, Krol E, Skotnicka D, Kaever V, Hilker R, Søgaard-Andersen L, Becker A (2016) Cyclic di-GMP regulates multiple cellular functions in the symbiotic α‑proteobacterium Sinorhizobium meliloti. J Bacteriol 198: 521-535

Quorum sensing

Person working in the Lab, SPP1617 Logo
Photo: V. Bettenworth, Rolf K. Wegst

Intercellular communication by means of small signal molecules synchronizes gene expression and coordinates functions among bacteria. This population density-dependent regulation is known as quorum sensing. Quorum sensing is frequently mediated by acyl homoserine lactone (AHL) autoinducers. Alphaproteobacterial rhizobia possess AHL-based quorum sensing systems controlling multiple functions including exopolysaccharide biosynthesis and motility. We investigate the molecular mechanisms, biological role, and regulation of quorum sensing. A special focus is on the role of quorum sensing in phenotypic heterogeneity, which is a phenomenon where genetically identical cells form subpopulations of distinct phenotypes.

Selected publications

Bettenworth V, McIntosh M, Becker A, Eckhardt B (2018) Front-propagation in bacterial inter-colony communication. Chaos 28: 106316

Charoenpanich P, Soto MJ, Becker A, McIntosh M (2015) Quorum sensing restrains growth and is rapidly inactivated during domestication of Sinorhizobium meliloti. Environ Microbiol Rep 7: 373-382

Schlüter J-P, Czuppon P, Schauer O, Pfaffelhuber P, McIntosh M, Becker A (2015) Classification of phenotypic subpopulations in isogenic bacterial cultures by triple promoter probing at single cell level. J Biotechnol 198: 3-14

Charoenpanich P, Meyer S, Becker A, McIntosh M (2013) Temporal expression program of quorum sensing-based transcription regulation in Sinorhizobium meliloti. J Bacteriol 195: 3224-3236

Alternative sigma factors

Picture alternative sigma factors and Logo ERASynBio
Rights: ERA SynBio

Reprogramming gene transcription by directing the RNA polymerase to specific promoters through alternative extracytoplasmic function (ECF) σ-factors is a wide-spread bacterial strategy of adaptation to stress factors. We study ECF σ-factor-mediated gene regulation in Alphaproteobacteria and explore these alternative σ-factors as orthogonal parts for implementing synthetic regulatory switches and gene circuits. This work was previously ERASynBio-funded in the framework of the ECFexpress consortium (Thorsten Mascher - Technische Universität Dresden; Anke Becker - Universität Marburg; Mark Buttner - John Innes Centre, Norwich; Georg Fritz - previously Universität Marburg, now The University of Western Australia; Alexander Goesmann - Universität Giessen; Carol Gross - University of California at San Francisco).

Selected publications

Meier D, Casas-Pastor D, Fritz G, Becker A (2020) Gene regulation by extracytoplasmic function (ECF) σ factors in alpha-rhizobia. Advances in Botanical Research 94: 289-321.

RNA-based regulation

Picture RNA
Photo: J. Reinkensmeier

Massive parallel cDNA sequencing (RNA-seq) has revolutionized global transcriptomic analysis and revealed an unexpected complexity of the prokaryotic transcriptome landscape.  Apart from mRNAs, ribosomal RNAs, and tRNAs, prokaryotes express a heterogeneous group of non-coding RNA species. Many of these have roles in post-transcriptional gene regulation, thereby contributing to the adjustment of bacterial physiology to changing environments. We investigate the transcriptome landscape of Alphaproteobacteria and functionally analyze non-coding regulatory RNAs.

Selected publications

Robledo M, Schlüter JP, Loehr LO, Linne U, Albaum S, Jiménez-Zurdo JI, Becker A (2018) An sRNA and cold shock protein homolog-based feedforward loop post-transcriptionally controls cell cycle master regulator CtrA. Front Microbiol 9:763

Robledo M, Peregrina A, Millán V, García-Tomsig NI, Torres-Quesada O, Mateos PF, Becker A, Jiménez-Zurdo JI (2017) A conserved α-proteobacterial small RNA contributes to osmoadaptation and symbiotic efficiency of rhizobia on legume roots. Environ Microbiol 19: 2661-2680

Saramago M, Peregrina A, Robledo M, Matos RG, Hilker R, Serrania J, Becker A, Arraiano CM, Jiménez-Zurdo JI (2017) Sinorhizobium meliloti YbeY is an endoribonuclease with unprecedented catalytic features, acting as silencing enzyme in riboregulation. Nucl Acids Res 45: 1371-1391

Robledo M, Frage B, Wright PR, Becker A (2015) A stress-induced small RNA modulates alpha-rhizobial cell cycle progression. PLoS Genetics 11: e1005153

Schlüter JP, Reinkensmeier J, Barnett MJ, Lang C, Krol E, Giegerich R, Long SR, Becker A (2013) Global mapping of transcription start sites and promoter motifs in the symbiotic -proteobacterium Sinorhizobium meliloti 1021. BMC Genomics 14: 156

Root nodule symbiosis

Image Roots
Photo: E. Krol

Legumes establish beneficial nitrogen-fixing symbioses with rhizobia that supply nitrogen for the plant within root nodules. Early stages of this interaction require crucial molecular exchanges in the rhizosphere before the host plant reprograms for bacterial root entry. Most rhizobia infect the roots of their host via a newly formed root hair intracellular apoplastic compartment called infection thread. We explore molecular mechanisms of root colonization and early infection stages. We are member of the ANR/DFG-funded Life-Switch consortium (Anke Becker - Universität Marburg; Macarena Marin - LMU München; Joëlle Fournier, Fernanda de Carvalho-Niebel - CNRS, LIPM, Toulouse).

Selected publications

Salas MA, Lozano MJ, Lopez JL, Draghi W, Serrania J, Torres Tejerizo GA, Albicoro FJ, Nilsson JF, Pistorio M, del Papa MF, Parisi G, Becker A, Lagares A (2017) Specificity traits consistent with legume-rhizobia coevolution displayed by Sinorhizobium meliloti rhizosphere colonization. Environ Microbiol 19: 3423-3438

Bacterial genome architecture

Chromids and megaplasmids: fundamental studies and applications

Picture of chromids
Photo: M. Wagner

About 10% of the sequenced bacterial species maintain multipartite genomic DNA. Among these bacteria are plant symbionts (e.g. many rhizobia), plant pathogens (e.g. Agrobacterium), and animal and human pathogens (e.g. Brucella, Burkholderia, Vibrio). In most cases, acquisition of the secondary replicons is attributed to ancestral plasmid domestication. They are called mega­plas­mids if larger than 350 Mb and chromids if containing core essential genes. The largest megaplasmids and chromids identified in bacteria to date even exceed 3 Mb. The large size of these secondary replicons raises the questions how the multipartite genomic DNA is spatially organized in the bacterial cell and how the secondary replicons are integrated in the cell cycle, i.e. how replication and segregation of the main chromosome and secondary replicon(s) is coordinated. We address these fundamental questions in various model bacteria of the α‑proteo­bac­terial Rhizobiales, and employ this knowledge to engineer synthetic secondary neo-replicons and integrate these into the bacterial cell cycle.

Selected publications

Döhlemann J, Wagner M, Happel C, Carrillo M, Sobetzko P, Erb TJ, Thanbichler M, Becker A (2017) A family of single copy repABC-type shuttle vectors stably maintained in the alpha-proteobacterium Sinorhizobium meliloti. ACS Synth Biol 6: 968-984

Frage B, Döhlemann J, Robledo M, Lucena D, Sobetzko P, Graumann PL, Becker A (2016) Spatiotemporal choreography of chromosome and megaplasmids in the Sinorhizobium meliloti cell cycle. Mol Microbiol 100:808-823

Alpha-rhizobial cell growth

Picture Alpharhizobium
Photo: E. Krol

The life cycle of bacteria includes genome replication and approximate duplication of cell size followed by cell division. Increasing the cell volume relies on cell wall growth, which requires elongation of the peptido­glycan sacculus. Most of the rod-shaped bacteria elongate by incorporating new PG in a dispersed manner along the sidewall. In contrast, the alphaproteobacterial Rhizobiales include species that elongate unipolarly at the new pole of the rod-shaped cells, including Sinorhizobium meliloti, Agrobacterium tumefaciens and Brucella abortus. We investigate the molecular processes determining the cell wall growth zone and how these processes are coordinated with cell division in space and time.

Selected publications

Krol E, Yau HCL, Lechner M, Schäper S, Bange G, Vollmer W, Becker A (2020) Tol-Pal system and Rgs proteins interact to promote unipolar growth and cell division in Sinorhizobium meliloti. mBio 11: e00306-20

Schäper S, Yau H, Krol E, Skotnicka D, Heimerl T, Gray J, Kaever V, Søgaard-Andersen L, Vollmer W, Becker A (2018) Seven-transmembrane receptor protein RgsP and cell wall-binding protein RgsM promote unipolar growth in Rhizobiales. PLOS Genetics 14: e1007594

We are member of the DFG-funded Transregio Collaborative Research Center TRR 174.

Alphaproteobacterial chassis for biotechnology

Team members working at the robotics unit
Photo: A. Becker, A. Kautz

We develop alphaproteobacterial chassis, such as Sinorhizobia and Methylobacteria, and drive development of laboratory automation processes for synthetic microbiology and biotechnology. Sinorhizobium is of special interest as plant-growth promoting bacterium, exopolysaccharide and vitamin producer, and platform for Mb-scale cloning, whereas the methylotroph Methylorubrum is of interest for its ability to reduce one-carbon compounds, such as methanol. We develop tools and methods for efficient engineering of alphaproteobacterial production strains to foster synthetic biology-driven knowledge-based bioeconomy.

We are member of the BMBF-funded SynBioTech consortium in the BioBall Innovationsraum and member of the European Commission H2020 Research & Innovation Programme-funded BioRoboost initiative on standards in Synthetic Biology. 

Selected publications

Carillo M, Wagner M, Petit F, Dransfeld A, Becker A, Erb T (2019) Design and control of extrachromosomal elements in Methylorubrum extorquens AM1. ACS Synth Biol 8: 2451-2456

Döhlemann J, Wagner M, Happel C, Carrillo M, Sobetzko P, Erb TJ, Thanbichler M, Becker A (2017) A family of single copy repABC-type shuttle vectors stably maintained in the alpha-proteobacterium Sinorhizobium meliloti. ACS Synth Biol 6: 968-984

Döhlemann J, Brennecke M, Becker A (2016) Cloning-free genome engineering in Sinorhizobium meliloti advances applications of Cre/loxP site-specific recombination. J Biotechnol 233: 160-170