Specific binding of the regulatory protein ExpG to promoter regions of the galactoglucan biosynthesis gene cluster of Sinorhizobium meliloti: a combined molecular biology and force spectroscopy investigation

Bartels FW, Baumgarth B, Anselmetti D, Ros R, Becker A. Specific binding of the regulatory protein ExpG to promoter regions of the galactoglucan biosynthesis gene cluster of Sinorhizobium meliloti: a combined molecular biology and force spectroscopy investigation. Journal of structural biology. 2003;143(2):145-152.Specific protein-DNA interaction is fundamental for all aspects of gene transcription. We focus on a regulatory DNA-binding protein in the Gram-negative soil bacterium Sinorhizobium meliloti 2011, which is capable of fixing molecular nitrogen in a symbiotic interaction with alfalfa plants. The ExpG protein plays a central role in regulation of the biosynthesis of the exopolysaccharide galactoglucan, which promotes the establishment of symbiosis. ExpG is a transcriptional activator of exp gene expression. We investigated the molecular mechanism of binding of ExpG to three associated target sequences in the exp gene cluster with standard biochemical methods and single molecule force spectroscopy based on the atomic force microscope (AFM). Binding of ExpG to expA1, expG-expD1, and expE1 promoter fragments in a sequence specific manner was demonstrated, and a 28 bp conserved region was found. AFM force spectroscopy experiments confirmed the specific binding of ExpG to the promoter regions, with unbinding forces ranging from 50 to 165pN in a logarithmic dependence from the loading rates of 70-79000 pN/s. Two different regimes of loading rate-dependent behaviour were identified. Thermal off-rates in the range of k off = (1.2 ± 1.0) × 10 -3 s -1 were derived from the lower loading rate regime for all promoter regions. In the upper loading rate regime, however, these fragments exhibited distinct differences which are attributed to the molecular binding mechanism

Detailed studies of the binding mechanism of the Sinorhizobium meliloti transcriptional activator ExpG to DNA

Baumgarth B, Bartels FW, Anselmetti D, Becker A, Ros R. Detailed studies of the binding mechanism of the Sinorhizobium meliloti transcriptional activator ExpG to DNA. Microbiology. 2005;151(1):259-268.The exopolysaccharide galactoglucan promotes the establishment of symbiosis between the nitrogen-fixing Gram-negative soil bacterium Sinorhizobium meliloti 2011 and its host plant alfalfa. The transcriptional regulator ExpG activates expression of galactoglucan biosynthesis genes by direct binding to the expA1, expG/expD1 and expE1 promoter regions. ExpG is a member of the MarR family of regulatory proteins. Analysis of target sequences of an ExpG(His)6 fusion protein in the exp promoter regions resulted in the identification of a binding site composed of a conserved palindromic region and two associated sequence motifs. Association and dissociation kinetics of the specific binding of ExpG(His)6 to this binding site were characterized by standard biochemical methods and by single-molecule spectroscopy based on the atomic force microscope (AFM). Dynamic force spectroscopy indicated a distinct difference in the kinetics between the wild-type binding sequence and two mutated binding sites, leading to a closer understanding of the ExpG-DNA interaction

Molekularbiologische Charakterisierung des ExpG-Proteins aus Sinorhizobium meliloti und Untersuchungen zur komplexen Regulation der exp-Genexpression durch MucR, PhoB und ExpG

Baumgarth B. Molekularbiologische Charakterisierung des ExpG-Proteins aus Sinorhizobium meliloti und Untersuchungen zur komplexen Regulation der exp-Genexpression durch MucR, PhoB und ExpG. Bielefeld (Germany): Bielefeld University; 2004.Die Grundlage der vorliegenden Arbeit war die molekularbiologische Charakterisierung der Funktion des an der Galactoglucan-Biosynthese (EPS II) beteiligten transkriptionellen Aktivators ExpG aus Sinorhizobium meliloti. Das ExpG-Protein zeigt Ähnlichkeiten zu Transkriptionsregulatoren der MarR-Familie, die an die DNA mittels eines helix-turn-helix (HTH)-Motivs binden. Das expG-Genprodukt weist ein C-terminales HTH-MarR-Motiv auf. Im Rahmen der Untersuchungen wurde ExpG als N-terminales und C-terminales His6-Fusionsprotein in Escherichia coli heterolog exprimiert. Durch Sequenzvergleich konnte eine AT-reiche, 21 bp umfassende palindromische Kernregion in den Promotorregionen vor dem expA-, expE- und expD-Operon sowie vor dem expG-Gen identifiziert werden. Mit Box1 und Box2 wurden zwei weitere Bereiche, die hohe Übereinstimmungen zeigen, stromaufwärts bzw. stromabwärts dieser Kernregion lokalisiert. Nach affinitätschromatographischer Aufreinigung des jeweiligen Fusionsproteins konnte eine direkte Bindung an die exp-Promotorregionen nachgewiesen werden. Die Aktivierung der Transkription der EPS II-Biosynthesegene erfolgt durch die spezifische Bindung von ExpG an die expA1- und expE1-Promotorregionen sowie an die intergenische Region zwischen expG und expD. Die palindromische Kernregion ist essentiell für diese Protein-DNA-Interaktion, die aber erst vollständig durch die vermutlich stabilisierende Wirkung der beiden Motive Box1 und Box2 ermöglicht wird. Aus den in dieser Arbeit ermittelten Assoziations- und Dissoziationsraten ließen sich Dissoziationskonstanten für die ExpG-DNA-Komplexe errechnen, die vergleichbar zu transkriptionellen Regulatoren aus anderen Protein-Familien sind. Im zweiten Teil dieser Arbeit konnte durch Promotoranalysen die komplexe Regulation der exp-Genexpression durch MucR, PhoB und ExpG weiter aufgeklärt werden. Die Überlappung der ExpG-Bindestellen mit den potentiellen PHO Boxen in den exp-Promotorregionen lässt auf ein Zusammenspiel der beiden Regulatoren ExpG und PhoB schließen. Der regulatorische Einfluss von MucR erfolgt vermutlich indirekt über die zusätzlich identifizierte palindromische Sequenz in den expA1- und expE1-Promotorregionen, die möglicherweise als Bindestelle für ein weiteres regulatorisches Protein fungiert. Basierend auf den Ergebnissen der untersuchten Promotorregionen im Wildtyp-, phoB-, mucR-, [Delta]G- und [Delta]G/mucR-Hintergrund ließ sich ein Modell zur komplexen Regulation der Transkription der Galactoglucan-Biosynthesegene sowohl unter Phosphat-suffizienten als auch unter Phosphat-limitierenden Bedingungen entwickeln. Der transkriptionelle Regulator ExpG kann bei Phosphatmangel und bei ausreichenden Phosphatkonzentrationen zum einen als Aktivator und zum anderen als Repressor auf die potentiellen Promotoren in den exp-Promotorregionen wirken. Der reprimierende Einfluss des zentralen Regulators MucR wird sehr wahrscheinlich durch den kooperativen Effekt von PhoB und ExpG unter Phosphat-limitierenden Bedingungen teilweise wieder aufgehoben und es erfolgt die Aktivierung der Galactoglucan-Biosynthese

Fine-tuning of galactoglucan biosynthesis in Sinorhizobium meliloti by differential wggR (expG)-, PhoB-, and mucR-dependent regulation of two promoters

Bahlawane C, Baumgarth B, Serrania J, Rueberg S, Becker A. Fine-tuning of galactoglucan biosynthesis in Sinorhizobium meliloti by differential wggR (expG)-, PhoB-, and mucR-dependent regulation of two promoters. JOURNAL OF BACTERIOLOGY. 2008;190(10):3456-3466

Low molecular weight plant extract induces metabolic changes and the secretion of extracellular enzymes, but has a negative effect on the expression of the type-III secretion system in Xanthomonas campestris pv. campestris

Watt TF, Vucur M, Baumgarth B, Watt SA, Niehaus K. Low molecular weight plant extract induces metabolic changes and the secretion of extracellular enzymes, but has a negative effect on the expression of the type-III secretion system in Xanthomonas campestris pv. campestris. Journal of Biotechnology. 2009;140(1-2):59-67.Xanthomonas campestris pathovar campestris (Xcc) is a plant pathogenic bacterium and as such has to adapt to a variety of environments. During the course of disease, Xcc colonizes the Surface of its host, infects the xylem in the early stages, and develops a fully saprophytic life-style, aided by secreted degradative enzymes, in the late stages. To get some insight into this complex regulation, Xcc was cultivated in the presence of low molecular weight host plant extract (<10 kDa). From this experiments it could be observed, that malate and Sucrose are taken LIP preferably in such an environment. Furthermore, it was demonstrated, that the plant extract has a negative effect on the gene expression of the hrp-gene cluster, although the activator hrpG was induced. Also, the secretion of degradative enzymes was shown to be upregulated. These observations indicate, that a low molecular weight plant extract (<10 kDa) is a sufficient signal to regulate metabolic pathways and the secretion of enzymes relevant for the development of virulence in Xanthomonas, but has a negative effect oil the expression of genes involved in type-III secretion, (C) 2009 Elsevier B.V. All rights reserved

Expression of small RNAs in Rhizobiales and protection of a small RNA and its degradation products by Hfq in Sinorhizobium meliloti

Voss B, Holscher M, Baumgarth B, et al. Expression of small RNAs in Rhizobiales and protection of a small RNA and its degradation products by Hfq in Sinorhizobium meliloti. Biochemical and Biophysical Research Communications. 2009;390(2):331-336.Regulatory RNA plays a pivotal role in the regulation of bacterial gene expression. Here, five small RNAs were studied in Sinorhizobium meliloti - SmrC15, SmrC16, Sra33, 6S and the signal recognition particle (SRP) RNA, which are conserved among at least seven different Rhizobium and Sinorhizobium species. The amount of SmrC16 decreased in stationary phase, while the other RNAs were up-regulated. The smallest changes, maximally 2-fold, were observed for 6S RNA. In the distantly related Bradyrhizobium japonicum, the amount of 6S RNA was not increased in stationary phase, suggesting some functional divergence in the roles of this molecule in Rhizobiales in comparison to Escherichia coli. Different decay rates were observed for SmrC15 and SmrC16 of S. meliloti upon rifampicin treatment, revealing posttranscriptional regulation during growth. The use of a Deltahfq mutant showed that Hfq protects full-length SmrC16 from degradation and stabilises its specific degradation products

Single molecule force spectroscopy on ligand-DNA complexes: from molecular binding mechanisms to biosensor applications

Ros R, Eckel R, Bartels FW, et al. Single molecule force spectroscopy on ligand-DNA complexes: from molecular binding mechanisms to biosensor applications. Journal of Biotechnology. 2004;112(1-2):5-12.Recent developments in single molecule force spectroscopy (SMFS) allow direct observation and measurements of forces that hold protein-DNA complexes together. Furthermore, the mechanics of double-stranded (ds) DNA molecules in the presence of small binding ligands can be detected. The results elucidate molecular binding mechanisms and open the way for ultra sensitive and powerful biosensor applications

Relationship of Bacillus amyloliquefaciens clades associated with strains DSM 7T and FZB42T: a proposal for Bacillus amyloliquefaciens subsp. amyloliquefaciens subsp. nov. and Bacillus amyloliquefaciens subsp. plantarum subsp. nov. based on complete genome sequence comparisons

Borriss R, Chen X-H, Rückert C, et al. Relationship of Bacillus amyloliquefaciens clades associated with strains DSM 7T and FZB42T: a proposal for Bacillus amyloliquefaciens subsp. amyloliquefaciens subsp. nov. and Bacillus amyloliquefaciens subsp. plantarum subsp. nov. based on complete genome sequence comparisons. Int J Syst Evol Microbiol. 2011;61(8):1786-1801.The whole-genome-sequenced rhizobacterium Bacillus amyloliquefaciens FZB42(T) (Chen et al., 2007) and other plant-associated strains of the genus Bacillus described as belonging to the species Bacillus amyloliquefaciens or Bacillus subtilis are used commercially to promote the growth and improve the health of crop plants. Previous investigations revealed that a group of strains represented a distinct ecotype related to B. amyloliquefaciens; however, the exact taxonomic position of this group remains elusive (Reva et al., 2004). In the present study, we demonstrated the ability of a group of Bacillus strains closely related to strain FZB42(T) to colonize Arabidopsis roots. On the basis of their phenotypic traits, the strains were similar to Bacillus amyloliquefaciens DSM 7(T) but differed considerably from this type strain in the DNA sequences of genes encoding 16S rRNA, gyrase subunit A (gyrA) and histidine kinase (cheA). Phylogenetic analysis performed with partial 16S rRNA, gyrA and cheA gene sequences revealed that the plant-associated strains of the genus Bacillus, including strain FZB42(T), formed a lineage, which could be distinguished from the cluster of strains closely related to B. amyloliquefaciens DSM 7(T). DNA-DNA hybridizations (DDH) performed with genomic DNA from strains DSM 7(T) and FZB42(T) yielded relatedness values of 63.7-71.2 %. Several methods of genomic analysis, such as direct whole-genome comparison, digital DDH and microarray-based comparative genomichybridization (M-CGH) were used as complementary tests. The group of plant-associated strains could be distinguished from strain DSM 7(T) and the type strain of B. subtilis by differences in the potential to synthesize non-ribosomal lipopeptides and polyketides. Based on the differences found in the marker gene sequences and the whole genomes of these strains, we propose two novel subspecies, designated B. amyloliquefaciens subsp. plantarum subsp. nov., with the type strain FZB42(T) ( = DSM 23117(T) = BGSC 10A6(T)), and B. amyloliquefaciens subsp. amyloliquefaciens subsp. nov., with the type strain DSM 7(T)( = ATCC 23350(T) = Fukumoto Strain F(T)), for plant-associated and non-plant-associated representatives, respecitvely. This is in agreement with results of DDH and M-CGH tests and the MALDI-TOF MS of cellular components, all of which suggested that the ecovars represent two different subspecies