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Research Report Laboratory Prof. Dr. B. Rak

Universität Freiburg
Institut für Biologie III
Schänzlestrasse 1
D-79104 Freiburg i. Br.
Germany
rak at uni-freiburg.de
phone +49 761 203 2729

Molecular Genetics of Bacteria

Team:  Thomas Bahr (bahr at uni-freiburg.de), Elge Koalick (elge.koalick at biologie.uni-freiburg.de, Henrik Mülders (henrik.muelders at biologie.uni-freiburg.de), Bodo Rak
 

Former members: Stefan Armbruster (until 11/2005),  Daniel Breustedt (until 9/00), Agnes Eber (until 11/00), Boris Görke (until 12/01), Ruth Jaehne (until 4/00); Michael Lorenz (until 4/99), Markus Haas (until 9/2005),  Walter Maerz (until 10/2009), Margarida Neto-Coelho (until 7/98), Jana Reinhardt (until 7/02); Esther Reuss (until 2/97), Claudia Schempp  (until 2/2003), Petra Steinke (until 10/2008), Susanne Zimmermann (until 9/00). 
 

Projects
 


Publications
 


The general aim of our research is to contribute to our knowledge of the functioning and evolution of genetic information. Our model organism is the bacterium Escherichia coli K-12. Bacteria can be propagated fast and easily and possess a rather small genome. The non-pathogenic bacterium E.coli K-12 has the additional advantage of having been investigated in much detail for several decades. Many of the basic genetic discoveries have been and are still being worked out with E.coli. In order to expand our principle understanding of the processes taking place at and around the genetic information we are focussing on two different projects.
 
 

Mobile DNA-Elements

In nature specialized DNA segments exist which are able to change their location within a genome. Such "vagabounding genes" have surprisingly been found in such different systems as bacteria, fungi, plants, flies and archae. Most likely they are components of the genomes of all kinds of living cells.Their presence is revealed by the diverse mutagenic effects which they exert when changing their location. In the simplest case an insertion mutation is found at the new location to which an element has moved. Since such sequences can also hop to viral or plasmidic sequences and are able to mobilize any piece of adjacent genetic information, they facilitate the horizontal exchange of genetic information. Mobile DNA elements thus play an important role in evolution.

We are investigating molecular details of their functioning, the different types and effects of mutations which they cause, and the mechanisms which control their mutagenic potential. The data suggest that the mobility of such elements is subject to complex control mechanisms, which sense the physiological condition of their hosts. Very surprising is the compact genetic organization which we have detected in the course of a detailed analysis of such elements of E.coli (Rak and von Reutern, EMBO J.3:807-811, 1984;Vögele et al, 1991; Fig. 1).

While studying mobile elements we came across the beta-glucoside operon of E.coli, which became an additional central research project (see below).

Click for Figure 1

Fig. 1: Genes on IS5 and IS150. (A) IS5 contains three genes which are encapsulated as shown. Moreover, expression of gene ins5A leads to two products: the 37 kD transposase Ins5A is being proteolytically cleaved, resulting in a stable N-terminal protein which acts as an inhibitor of transposition. Proteolysis is controlled by the host's growth state. (B) IS150 also contains three open reading frames, which are transcribed and translated. In addition to Ins150A and Ins150B a fusion protein is jointly encoded by ins150A and ins150B. This is accomplished by translational frameshifting into the -1 phase at a specific position, 11 codons 5' to the ins150A stop codon. The mechanism responsible for frameshifting is remarkably efficient - 50% of the ribosomes perform this shift. Analogous strategies for the synthesis of fusion proteins are used by retroviruses. Moreover, retroviral integrases show homologies to the Ins150B domain. Ins150A acts inhibitory on transpositional activity, while Ins150AB carries the essential transposition function. The mayor role of the InsA protein, however, seems to restrict excision of IS150 to a productive transposition event, since an element defective for Ins150A produces with high rates excised linear as well as circularized copies of IS150. Ins150B is also essential, but only in the presence of Ins150A. In addition, synthesis of Ins150B is controlled by the physiology of the host and functions in the decision between simple transposition and other reactions which resemble transpositional cointegrates. Interestingly, IS5 and IS150 reach the same goal, i.e. the synthesis of a set of specifically interrelated proteins, by fundamentally different mechanisms. Top
 

The beta-glucoside (bgl) Operon

Wild type strains of E.coli are unable to utilize beta-glucosidic sugars. Spontaneous mutants arise, however, which are able to grow on certain beta-glucosides, revealing the presence of a cryptic operon called bgl. This operon encodes all functions necessary for the regulated uptake and degradation of aryl-beta-glucosides. We have shown that inactivity is due to repression (silencing) of the bgl promoter by its sequence context. Spontaneous activation of the operon is mainly caused by insertion of mobile DNA elements into the promoter region, and we demonstrated that this mutative activation can in some cases be described as an enhancer-analogous amplification of the activity of the bgl promoter (Schnetz and Rak, 1992). Our detailed analysis of the operon included sequencing and functional assignment of its genes (Schnetz et al., J.Bacteriol.169:2579-2590, 1987; Andersen et al., 1999), analysis of the beta-glucoside transport system encoded by the operon (Schnetz et al, 1990, Sutrina et al., 1990), and substrate-dependent regulation of the operon (Schnetz and Rak 1988 EMBO J.7:3271-3277, 1988; Schnetz and Rak, 1990, Goerke and Rak, 1999; Fig. 2). Recently, we have characterized a system from the gram-positive bacterium Bacillus subtilis, which is closely related in its regulatory functions to those of the bgl operon. (Schnetz et al, 1996).

Click for Figure 2

Fig. 2: Regulation of the bgl operon as an example of a signal transduction network. The operon encodes six genes (lower panel), three of which are necessary and sufficient for the regulated uptake and utilization of aryl-beta-glucosides such as salicin and arbutin: bglB codes for a phospho-beta-glucosidase, the degradating enzyme, bglF codes for the transport protein, Enzyme IIBgl  (EIIBgl), which is part of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). Within the PTS,  EI first autophosphorylates with phosphoenolpyruve (PEP) in a slow reaction. The phosphoryl group is subsequently transferred to enzyme HPr, from where it is relayed to the sugar-specific EIIs. Note, that the high-energy phosphoryl groups can reversibly flow within the PTS between its protein members. In the case of EIIBgl, HPr phosphorylates a histidine within the A-domain. The phosphoryl group is then transferred to a cysteine within the B-domain and from there to the incoming sugar, which passes through the C-domain (see upper panel). Gene bglG encodes a transcriptional antiterminator which prevents termination at two terminators (t1 and t2) flanking bglG. (A) In the absence of any PTS sugar, proteins of the PTS are all maintained in a phosphorylated state. Since phosphate cannot be transferred to beta-glucosidic substrate, EIIBgl phosphorylates BglG, which leads to its monomer formation, the inactive form. In addition HPr may phosphorylate BglG at a second site. (B) In the presence of beta-glucosides (but absence of other PTS substrates), EIIBgl dephosphorylates BglG, which is necessary but not sufficient for its activation. In addition, HPr directly transfers phosphoryl groups to a distinct site within BglG, which allows it to dimerize to the active form and alleviate transcription termination within the bgl operon. The operon is induced. This model also includes a subtle mode of bgl operon autoregulation, since both, phosphate flow to the sugar during transport and activation of BglG by HPr-catalyzed reversible phosphorylation have to share the limited pool of phosphoryl groups provided by the slowly autophosphorylating EI. (C) Appearance of any other PTS-sugar leads to additional competition for phosphoryl groups. Since the KM of EIIBgl is higher than that reported for any other EII, it is conceivable, that phosphoryl groups are massively drained away from EIIBgl in favor of the other sugar transport. This does not only lead to severe reduction of beta-glucoside transport but in addition inhibits the capacity of EIIBgl to phosphorylate BglG. As a consequence, EIIBgl is not able to repress BglG activity any more. It is therefore necessary to additionally couple control of activity of BglG to the general state of the PTS via the phosphorylation state of HPr. When phosphoryl groups become limited due to transport of other PTS sugars, HPr is underphosphorylated and therefore unable to activate BglG by phosphorylation. BglG is inactive. Top
 

Acknowledgements

This work was financed by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, the Land Baden-Württemberg and the EC (HCM).
 

Staatsexamina
 

  • Assem, Bayya (1990) Funktionelle Analyse einer neuen repetitiven DNA-Sequenz aus Escherichia coli

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  • Neto Coelho, Margarida (1998) Vorarbeiten zur Entwicklung eines neuartigen genetischen Systems zur Detektion von Protein-Protein-Interaktionen.

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Diploma Theses
 

  • Schnetz, Karin (1985) Zur Aktivierung des kryptischen bgl Operons von Escherichia coli durch Insertionsmutationen. 
  • Brombacher, Frank (1986) Das bakterielle mobile DNA-Element IS5: Zur Funktion seiner Genprodukte. 
  • Fuchs, Klaus (1986) Das bakterielle Insertionselement IS5: Studien zur Expression des Gens ins5A
  • Ronecker, Hans-Joerg (1986) Selektion und physikalische Analyse einer repräsentativen Anzahl spontaner Bgl+ Mutationen.
  • Welz, Caroline (1988) Das bakterielle mobile DNA-Element IS150: Studien zur Genepression. 
  • Meinhof, Carl-Georg (1989) Konstruktion eines konditional replizierenden Lambda-Klonierungsvektors zur Entwicklung eines neuen Transpositions-Testsystems.
  • Krieg, Marion (1990) Selektion und Charakterisierung regulatorischer Mutanten im bgl-Operon von Escherichia coli
  • Fischer, Christian (1991) Entwicklung und molekularbiologischer Test einesComputer Programmes zur physikalischen Kartierung von Loci auf dem Eschichia coli-Chromosom.
  • Hölzle, Johannes (1991) Aufbau eines Systems zur Analyse der transpositionellen Spezifität und Induktion von IS150.
  • Görke, Boris (1995) Das Escherichia coli Antiterminationsprotein BglG: In vitro-Studien und Untersuchungen zur zellulären Lokalisation.
  • Haas, Markus (1996) Das bakterielle Insertionselement IS150: Überexpression seiner Genprodukte und Analyse von in vivo generierten Excisionsprodukten.
  • Huchler, Manfred (1996) Zur Steuerung der Aktivität des Antiterminationsproteins BglG aus Escherichia coli.
  • Reuss, Esther (1997) Funktionsanalyse von Deletionsderivaten des Antiterminatorproteins BglG aus Escherichia coli.
  • Lorenz,  Michael (1999) Studien zur Interaktion zwischen dem Antiterminatorprotein BglG und EIIBgl aus Escherichia coli.
  • Breustedt,  Daniel (2000) Substitution der PTS-Phosphotransferasen EI und HPr von E. coli durch die homologen Transferasen aus einem Gram-positiven Bakterium.
  • Reinhardt, Jana (2002) Repression des lac-Operons von der cytoplasmatischen Membran aus.
  •  Maerz, Walter (2002) Etablierung eines Erythromycin-Resistenztransposons und Charakterisierung von Transposon-generierten Escherichia-coli-Mutanten mit Einfluss auf die Aktivitaetssteuerung des transkriptionellen Antiterminatorproteins LicT.
  • Schempp, Claudia (2003) Transposition und Excision des Insertionselementes IS150 aus dem bgl-Operon von Escherichia coli.
  • Muelders, Henrik (2005) Targetpraeferenzen des Insertionselementes IS150 und die Abhaengigkeit seiner Transpositionsaktivitaet vom Integrationsort. 
  • Armbruster, Stefan (2005) In vivo-Studien zur Integrationsaktivität künstlicher Transpositionsintermediate des Insertionselementes IS150 aus Escherichia coli.
  • Bahr, Thomas (2005) Etablierung und Validierung von Methoden zur Analyse der regulation des bgl-Operons von Escherichia coli
     
     
     
     
     
     
     
     
     
     
     
     

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    Ph.D. Theses
     

    • Schnetz, Karin (1988) Das beta-Glukosid-Operon von Escherichia coli K-12. This thesis was awarded the Gödecke prize as the best thesis out of the faculty within two years. 
    • Schwartz, Edward (1988) Gal+ revertanten der polaren Insertionsmutation galOP-306::IS1. Molekulare Analyse der Mutationsereignisse und Charakterisierung eines neuen, mobilen Promotorelementes. 
    • Ronecker, Hans-Jörg (1991) Transpositionelle Destabilisierung eines genomischen Bereiches als mögliche Folge eines DNA-Transfers von Escherichia coli B nach K-12.
    • Krieg, Marion (1993) Zu Mechanismus und Steuerung der transkriptionellen Antitermination im bgl-Operon von Escherichia coli
    • Vögele, Karl (1993) Molekulare Analyse der Genexpression des bakteriellen Insertionselementes IS150
    • Welz, Caroline (1993) Funktionelle Analyse des bakteriellen Insertionelementes IS150
    • Meinhof, Carl-Georg (1994) Studien zur IS5-Transposase und Entwicklung eines Informations-Managementsystems für rekombinante DNA. 
    • Görke, Boris (2000) Signaltransduktion am bgl-Operon von Escherichia coli. This thesis was awarded the Goedecke Prize as the best thesis out of the faculty within two years. 
    • Haas, Markus (2001) Zwei-Stufen-Transposition des Insertionselementes IS150 ueber zirkulaere und lineare Intermediate
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      Publications
       

      • Schnetz, K., Toloczyki, C. and Rak,B. (1987) Beta-glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes. J. Bacteriol. 169: 2579-2590. <Medline> 
      • Ronecker, H.J., and Rak,B. (1987) Genetic organization of insertion element IS2 based on a revised nucleotide sequence. Gene 59:291-296. <Medline> 
      • Schwartz, E., Herberger,C., and Rak,B. (1988) Second-element turn-on of gene expression in an IS1 insertion mutant. Mol. Gen. Genet. 211: 282-289. <Medline> 
      • Schnetz,K. and Rak.B. (1988) Cleavage by EcoO109 and DraII is inhibited by overlapping dcm methylation. Nucleic Acids Res. 16: 1623. <Medline> 
      • Saier,M.H.Jr, Yamada,M.; Suda,K.; Erni,B.; Rak,B.; Lengeler,J.; Stewart,G.C.; Waygood,E.B.; Rapoport,G. (1988) Bacterial proteins with N-terminal leader sequences resembling mitochondrial targeting sequences of eukaryotes. Biochimie 70: 1743-1748. <Medline> 
      • Schwartz,E., Kroger,M., and Rak,B. (1988) IS150: distribution, nucleotide sequence and phylogenetic relationships of a new E. coli insertion element. Nucleic Acids Res. 16: 6789-6802. <Medline> 
      • Schnetz,K. and Rak,B. (1988) Regulation of the bgl operon of Escherichia coli by transcriptional antitermination. EMBO J. 7: 3271-3277. <Medline> 
      • Junne, T., Schnetz, K., and Rak, B. (1990). Location of the bglA gene on the physical map of Escherichia coli. J. Bacteriol. 172,6615-6616. <Medline>
      • Schnetz, K., Sutrina, S.L., Saier, M.H.,Jr., and Rak, B. (1990). Identification of catalytic residues in the beta-glucoside permease of Escherichiacoli by site-specific mutagenesis and demonstration of interdomain crossreactivity between the beta-glucoside and glucose systems. J. Biol. Chem.265, 13464-13471. <Medline>
      • Schnetz, K. and Rak, B. (1990). beta-glucoside permease represses the bgl operon of E.coli by phosphorylation of the antiterminatorprotein and also interacts with enzymeIIIglc, the key element in catabolite control. Proc. Natl. Acad. Sci. USA 87, 5074-5078. <Medline>
      • Sutrina, S.L., Schnetz, K., Rak, B., and Saier, M.H.,Jr. (1990). Mechanism of sugar transport and phosphorylation via permeases of the bacterial phosphotransferase system: catalytic residues in the beta-glucoside-specific permease as defined by site-specific mutagenesis. Research in Microbiology141, 368-374. <Medline> 
      • Vogele, K., Schwartz, E., Welz, C., Schiltz, E., and Rak, B.(1991).High-level ribosomal frameshifting directs the synthesis of IS150 gene products. Nucleic Acids Res. 19, 4377-4385. <Medline>
      •  Schnetz, K. and Rak, B. (1992). IS5: a mobile enhancer of transcription in Escherichia coli. Proc. Natl. Acad. Sci. USA 89, 1244-1248. <Medline> 
      • Rak, B., Schnetz, K., Ronecker, H.-J., and Welz, C. (1993). Insertion sequences may dictate chromosomal domains. J. Cell. Biochem. 17E, 282. 
      • Schnetz, K., Rak, B., and Wang, J.C. (1993). What keeps the promoter of the bgl operon cryptic? J. Cell. Biochem. 17E, 304. 
      • Koch, S., Sutrina, S.L., Wu, L.-F., Reizer, J., Schnetz, K., Rak, B., andSaier, M.H., Jr. (1996). Identification of a site in the phosphocarrierprotein, HPr, which influences its interactions with sugar permeases of the bacterial phosphotransferase system: kinetic analyses employing site-specificmutants. J. Bacteriol. 178, 1126-1133. <Medline> 
      • Schnetz, K., Stülke, J., Gertz, S., Krüger, S., Krieg, M., Hecker,M., and Rak, B. (1996). LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG Family. J. Bacteriol. 178,1971-1979. <Medline> 
      • Andersen, C. Rak, B. and Benz, R. (1999) The gene bglH present in the bgl-operon of Escherichia coli, responsible for uptake and fermentation of b-glucosides encodes for a carbohydrate-specific outer membrane porin. Molec. Microbiol. 31,499-510.<Medline> 
      • Goerke, B., and Rak, B. (1999) Catabolite control of Escherichia coli regulatory protein BglG activity by antagonistically acting phosphorylations. EMBO J. 18, 3370-3379. <Medline><Supplementary Materials>
      • Goerke, B., and Rak, B. (2001) Efficient Transcriptional Antitermination from the Escherichia coli cytoplasmic membrane. J. Mol. Biol. 308, 131-145. <Medline>
      • Tanja Egener, José Granado, Marie-Christine Guitton, Annette Hohe, Hauke Holtorf, Jan M Lucht, Stefan A Rensing, Katja Schlink, Julia Schulte, Gabriele Schween, Susanne Zimmermann, Elke Duwenig, Bodo Rak and Ralf Reski. (2002) High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library. BMC Plant Biology 2:6 <open access link >
      • Haas, M. and B. Rak (2002) Escherichia coli Insertion Sequence IS150: Transposition via Circular and Linear Intermediates. J. Bacteriol. 184:5833-5841. <Medline><Supplemetary Materials>
      • Goerke, B  (2003) .Regulation of the Escherichia coli Antiterminator Protein BglG by Phosphorylation at Multiple Sites and Evidence for Transfer of Phosphoryl Groups between Monomers. J. Biol. Chem. 278: 46219-46229. <medline> 
      • Goerke, B., Reinhardt, J., and Rak, B. (2005). Activity of Lac repressor anchored to the cytoplasmic membrane. Nucleic Acids Res. 33: 2504-2511. <open access link> 
      • Birte Reichenbach, B.,  Breustedt, D.A., Stuelke, J, Rak, B., and Goerke, B. (2007). Genetic Dissection of Specificity Determinants in the Interaction of HPr with Enzymes II of the bacterial Phosphoenolpyruvate:Sugar Phosphotransferase System in Escherichia coli J Bacteriol. 189: 4603-13.  [Abstract] 
      • Kalamorz, F., Reichenbach, B., Maerz, W., Rak, B., and Goerke, B. (2007) Feedback control of glucosamine-6-phosphate synthase GlmS expression depends on the small RNA GlmZ and involves the novel protein YhbJ in Escherichia coli. Mol. Microbiol. 65:1518-1533. PubMed http://www.ncbi.nlm.nih.gov/pubmed/17824929?dopt=Citation Suggested by Faculty of Thousand (must read): http://f1000biology.com/article/id/1091823/evaluation
      •  Bahr,T., Luettmann,D., Maerz,W., Rak,B. and Goerke,B. (2011). Insight into bacterial phosphotransferase system  mediated signaling by inter-species transplantation of a transcriptional regulator. J. Bacteriol. in press.
      Patent
      • Rak, B., Reski, R., Zimmermann, S., Guitton, M-C., Duwenig, E. and Freund, A. (2001) Method for the mutagenesis of nucleotide sequences from plants, algae or fungi.  Patent WO 01/38509 A1. 
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