TY  - JOUR
A1  - Fröhlich, Kathrin S.
A1  - Papenfort, Kai
A1  - Berger, Allison A.
A1  - Vogel, Jörg
T1  - A conserved RpoS-dependent small RNA controls the synthesis of major porin OmpD
JF  - Nucleic Acids Research
N2  - A remarkable feature of many small non-coding RNAs (sRNAs) of Escherichia coli and Salmonella is their accumulation in the stationary phase of bacterial growth. Several stress response regulators and sigma factors have been reported to direct the transcription of stationary phase-specific sRNAs, but a widely conserved sRNA gene that is controlled by the major stationary phase and stress sigma factor, Sigma(S) (RpoS), has remained elusive. We have studied in Salmonella the conserved SdsR sRNA, previously known as RyeB, one of the most abundant stationary phase-specific sRNAs in E. coli. Alignments of the sdsR promoter region and genetic analysis strongly suggest that this sRNA gene is selectively transcribed by Sigma(S). We show that SdsR down-regulates the synthesis of the major Salmonella porin OmpD by Hfq-dependent base pairing; SdsR thus represents the fourth sRNA to regulate this major outer membrane porin. Similar to the InvR, MicC and RybB sRNAs, SdsR recognizes the ompD mRNA in the coding sequence, suggesting that this mRNA may be primarily targeted downstream of the start codon. The SdsR-binding site in ompD was localized by 3'-RACE, an experimental approach that promises to be of use in predicting other sRNA-target interactions in bacteria.
KW  - shock sigma factor
KW  - general stress response
KW  - down regulation
KW  - stationary phase
KW  - salmonella enterica
KW  - messenger RNA
KW  - escherichia coli
KW  - enterica serovar typhimurium
KW  - outer-membrane proteins
KW  - small noncoding RNAs
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134230
VL  - 40
IS  - 8
ER  - 
TY  - JOUR
A1  - Jäger, Dominik
A1  - Pernitzsch, Sandy R.
A1  - Richter, Andreas S.
A1  - Backofen, Rolf
A1  - Sharma, Cynthia M.
A1  - Schmitz, Ruth A.
T1  - An archaeal sRNA targeting cis- and trans-encoded mRNAs via two distinct domains
JF  - Nucleic Acids Research
N2  - We report on the characterization and target analysis of the small (s) RNA\(_{162}\) in the methanoarchaeon Methanosarcina mazei. Using a combination of genetic approaches, transcriptome analysis and computational predictions, the bicistronic MM2441-MM2440 mRNA encoding the transcription factor MM2441 and a protein of unknown function was identified as a potential target of this sRNA, which due to processing accumulates as three stabile 5' fragments in late exponential growth. Mobility shift assays using various mutants verified that the non-structured single-stranded linker region of sRNA\(_{162}\) (SLR) base-pairs with the MM2440-MM2441 mRNA internally, thereby masking the predicted ribosome binding site of MM2441. This most likely leads to translational repression of the second cistron resulting in dis-coordinated operon expression. Analysis of mutant RNAs in vivo confirmed that the SLR of sRNA\(_{162}\) is crucial for target interactions. Furthermore, our results indicate that sRNA\(_{162}\)-controlled MM2441 is involved in regulating the metabolic switch between the carbon sources methanol and methylamine. Moreover, biochemical studies demonstrated that the 50 end of sRNA\(_{162}\) targets the 5'-untranslated region of the cis-encoded MM2442 mRNA. Overall, this first study of archaeal sRNA/mRNA-target interactions unraveled that sRNA\(_{162}\) acts as an antisense (as) RNA on cis- and trans-encoded mRNAs via two distinct domains, indicating that cis-encoded asRNAs can have larger target regulons than previously anticipated.
KW  - strain
KW  - escherichia coli
KW  - methanosarcina mazei GO1
KW  - methanol methyltransferase isozymes
KW  - small nucleolar RNAs
KW  - acetivorans C2A
KW  - antisense RNAs
KW  - GO1
KW  - transcriptional regulator
KW  - translational initiation
KW  - pyrococcus furiosus
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134972
VL  - 40
IS  - 21
ER  - 
TY  - JOUR
A1  - Becker, Svetlana
A1  - Oelschlaeger, Tobias A.
A1  - Wullaert, Andy
A1  - Pasparakis, Manolis
A1  - Wehkamp, Jan
A1  - Stange, Eduard F.
A1  - Gersemann, Michael
T1  - Bacteria Regulate Intestinal Epithelial Cell Differentiation Factors Both In Vitro and In Vivo
JF  - PLoS ONE
N2  - Background: The human colon harbours a plethora of bacteria known to broadly impact on mucosal metabolism and function and thought to be involved in inflammatory bowel disease pathogenesis and colon cancer development. In this report, we investigated the effect of colonic bacteria on epithelial cell differentiation factors in vitro and in vivo. As key transcription factors we focused on Hes1, known to direct towards an absorptive cell fate, Hath1 and KLF4, which govern goblet cell. 
Methods: Expression of the transcription factors Hes1, Hath1 and KLF4, the mucins Muc1 and Muc2 and the defensin HBD2 were measured by real-time PCR in LS174T cells following incubation with several heat-inactivated E. coli strains, including the probiotic E. coli Nissle 1917+/- flagellin, Lactobacilli and Bifidobacteria. For protein detection Western blot experiments and chamber-slide immunostaining were performed. Finally, mRNA and protein expression of these factors was evaluated in the colon of germfree vs. specific pathogen free vs. conventionalized mice and colonic goblet cells were counted. 
Results: Expression of Hes1 and Hath1, and to a minor degree also of KLF4, was reduced by E. coli K-12 and E. coli Nissle 1917. In contrast, Muc1 and HBD2 expression were significantly enhanced, independent of the Notch signalling pathway. Probiotic E. coli Nissle 1917 regulated Hes1, Hath1, Muc1 and HBD2 through flagellin. In vivo experiments confirmed the observed in vitro effects of bacteria by a diminished colonic expression of Hath1 and KLF4 in specific pathogen free and conventionalized mice as compared to germ free mice whereas the number of goblet cells was unchanged in these mice. 
Conclusions: Intestinal bacteria influence the intestinal epithelial differentiation factors Hes1, Hath1 and KLF4, as well as Muc1 and HBD2, in vitro and in vivo. The induction of Muc1 and HBD2 seems to be triggered directly by bacteria and not by Notch.
KW  - stem cells
KW  - inflammatory-bowel-disease
KW  - Ileal Crohns-disease
KW  - coli nissel 1917
KW  - ulcreative colitis
KW  - escherichia coli
KW  - porphyromonas gingivalis
KW  - antimicrobial peptides
KW  - colorectal cancer
KW  - alpha defensins
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131168
VL  - 8
IS  - 2
ER  - 
TY  - JOUR
A1  - Szambowska, Anna
A1  - Tessmer, Ingrid
A1  - Kursula, Petri
A1  - Usskilat, Christian
A1  - Prus, Potr
A1  - Pospiech, Helmut
A1  - Grosse, Frank
T1  - DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding
JF  - Nucleic Acids Research
N2  - The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3' protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3'-5' polarity and, thereby acts as a molecular 'wedge' to initiate DNA strand displacement.
KW  - protein secondary structure
KW  - circular dichroism spectra
KW  - small-angle scattering
KW  - single-stranded-DNA
KW  - cyclin-dependent kinases
KW  - ray solution scattering
KW  - saccharmyces cerevisiae
KW  - escherichia coli
KW  - recj exonuclease
KW  - s-phase
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117538
SN  - 1362-4962
VL  - 42
IS  - 4
ER  - 
TY  - JOUR
A1  - Fan, Ben
A1  - Li, Lei
A1  - Chao, Yanjie
A1  - Förstner, Konrad
A1  - Vogel, Jörg
A1  - Borriss, Rainer
A1  - Wu, Xiao-Qin
T1  - dRNA-Seq Reveals Genomewide TSSs and Noncoding RNAs of Plant Beneficial Rhizobacterium Bacillus amyloliquefaciens FZB42
JF  - PLoS One
N2  - Bacillus amyloliquefaciens subsp. plantarum FZB42 is a representative of Gram-positive plant-growth-promoting rhizobacteria (PGPR) that inhabit plant root environments. In order to better understand the molecular mechanisms of bacteria-plant symbiosis, we have systematically analyzed the primary transcriptome of strain FZB42 grown under rhizospheremimicking conditions using differential RNA sequencing (dRNA-seq). Our analysis revealed 4,877 transcription start sites for protein-coding genes, identified genes differentially expressed under different growth conditions, and corrected many previously mis-annotated genes. We also identified a large number of riboswitches and cis-encoded antisense RNAs, as well as trans-encoded small noncoding RNAs that may play important roles in the gene regulation of Bacillus. Overall, our analyses provided a landscape of Bacillus primary transcriptome and improved the knowledge of rhizobacteria-host interactions.
KW  - gene expression
KW  - subtilis genome
KW  - enterica serovar thphimurium
KW  - small regulatory RNAs
KW  - binding protein HFQ
KW  - escherichia coli
KW  - messenger RNA
KW  - transcriptional landscape
KW  - mycobacterium tuberculosis
KW  - listeria monocytogenes
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-138369
VL  - 10
IS  - 11
ER  - 
TY  - JOUR
A1  - Schmidtke, Cornelius
A1  - Findeiß, Sven
A1  - Sharma, Cynthia M.
A1  - Kuhfuss, Juliane
A1  - Hoffmann, Steve
A1  - Vogel, Jörg
A1  - Stadler, Peter F.
A1  - Bonas, Ulla
T1  - Genome-wide transcriptome analysis of the plant pathogen Xanthomonas identifies sRNAs with putative virulence functions
JF  - Nucleic Acids Research
N2  - The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is an important model to elucidate the mechanisms involved in the interaction with the host. To gain insight into the transcriptome of the Xcv strain 85-10, we took a differential RNA sequencing (dRNA-seq) approach. Using a novel method to automatically generate comprehensive transcription start site (TSS) maps we report 1421 putative TSSs in the Xcv genome. Genes in Xcv exhibit a poorly conserved -10 promoter element and no consensus Shine-Dalgarno sequence. Moreover, 14% of all mRNAs are leaderless and 13% of them have unusually long 5'-UTRs. Northern blot analyses confirmed 16 intergenic small RNAs and seven cis-encoded antisense RNAs in Xcv. Expression of eight intergenic transcripts was controlled by HrpG and HrpX, key regulators of the Xcv type III secretion system. More detailed characterization identified sX12 as a small RNA that controls virulence of Xcv by affecting the interaction of the pathogen and its host plants. The transcriptional landscape of Xcv is unexpectedly complex, featuring abundant antisense transcripts, alternative TSSs and clade-specific small RNAs.
KW  - SUBSP carotovora
KW  - regulatory RNA
KW  - gene-cluster
KW  - campestris PV vesicatoria
KW  - escherichia coli
KW  - determines pathgenicity
KW  - hypersensitive response
KW  - ralstonia solanacearum
KW  - extracellular enzymes
KW  - secretion systems
KW  - transcription initiation site
KW  - RNA sequence analyses
KW  - messanger RNA
KW  - plants
KW  - libraries
KW  - genome
KW  - genes
KW  - gene expression profiling
KW  - genetic transcription
KW  - northern blotting
KW  - untranslated regions
KW  - xanthomonas
KW  - xanthomonas campestris
KW  - bacteria
KW  - virulence
KW  - pathogenetic organism
KW  - RNA
KW  - small RNA
KW  - pathogenicity
KW  - type III secretion system pathways
KW  - maps
KW  - consesus
KW  - host (organism)
KW  - type III protein secretion system complex
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131781
VL  - 40
IS  - 5
SP  - 2020
EP  - 2031
ER  - 
TY  - JOUR
A1  - Aistleitner, Karin
A1  - Heinz, Christian
A1  - Hoermann, Alexandra
A1  - Heinz, Eva
A1  - Montanaro, Jacqueline
A1  - Schulz, Frederik
A1  - Maier, Elke
A1  - Pichler, Peter
A1  - Benz, Roland
A1  - Horn, Matthias
T1  - Identification and Characterization of a Novel Porin Family Highlights a Major Difference in the Outer Membrane of Chlamydial Symbionts and Pathogens
JF  - PLoS ONE
N2  - The Chlamydiae constitute an evolutionary well separated group of intracellular bacteria comprising important pathogens of humans as well as symbionts of protozoa. The amoeba symbiont Protochlamydia amoebophila lacks a homologue of the most abundant outer membrane protein of the Chlamydiaceae, the major outer membrane protein MOMP, highlighting a major difference between environmental chlamydiae and their pathogenic counterparts. We recently identified a novel family of putative porins encoded in the genome of P. amoebophila by in silico analysis. Two of these Protochlamydia outer membrane proteins, PomS (pc1489) and PomT (pc1077), are highly abundant in outer membrane preparations of this organism. Here we show that all four members of this putative porin family are toxic when expressed in the heterologous host Escherichia coli. Immunofluorescence analysis using antibodies against heterologously expressed PomT and PomS purified directly from elementary bodies, respectively, demonstrated the location of both proteins in the outer membrane of P. amoebophila. The location of the most abundant protein PomS was further confirmed by immuno-transmission electron microscopy. We could show that pomS is transcribed, and the corresponding protein is present in the outer membrane throughout the complete developmental cycle, suggesting an essential role for P. amoebophila. Lipid bilayer measurements demonstrated that PomS functions as a porin with anion-selectivity and a pore size similar to the Chlamydiaceae MOMP. Taken together, our results suggest that PomS, possibly in concert with PomT and other members of this porin family, is the functional equivalent of MOMP in P. amoebophila. This work contributes to our understanding of the adaptations of symbiotic and pathogenic chlamydiae to their different eukaryotic hosts.
KW  - cell wall
KW  - protochlamydia amoebophila
KW  - escherichia coli
KW  - matrix protein porin
KW  - gram negative bacteria
KW  - single channel analysis
KW  - developmental cycle
KW  - mycobacterium smegmatis
KW  - monoclonal antibodies
KW  - signal peptides
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131176
VL  - 8
IS  - 1
ER  - 
TY  - JOUR
A1  - Khan, Irfan
A1  - Suhasini, Avvaru N.
A1  - Banerjee, Taraswi
A1  - Sommers, Joshua A.
A1  - Kaplan, Daniel L.
A1  - Kuper, Jochen
A1  - Kisker, Caroline
A1  - Brosh, Jr., Robert M.
T1  - Impact of Age-Associated Cyclopurine Lesions on DNA Repair Helicases
JF  - PLOS ONE
N2  - 8,5' cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ helicases (RECQ1, BLM, WRN, RecQ) were inhibited by cPu in the helicase translocating strand, whereas helicases from SF1 (UvrD) and SF4 (DnaB) tolerated cPu in either strand. SF2 Fe-S helicases (FANCJ, DDX11 (ChlR1), DinG, XPD) displayed marked differences in their ability to unwind the cPu DNA substrates. Archaeal Thermoplasma acidophilum XPD (taXPD), homologue to the human XPD helicase involved in NER DNA damage verification, was impeded by cPu in the non-translocating strand, while FANCJ was uniquely inhibited by the cPu in the translocating strand. Sequestration experiments demonstrated that FANCJ became trapped by the translocating strand cPu whereas RECQ1 was not, suggesting the two SF2 helicases interact with the cPu lesion by distinct mechanisms despite strand-specific inhibition for both. Using a protein trap to simulate single-turnover conditions, the rate of FANCJ or RECQ1 helicase activity was reduced 10-fold and 4.5-fold, respectively, by cPu in the translocating strand. In contrast, single-turnover rates of DNA unwinding by DDX11 and UvrD helicases were only modestly affected by the cPu lesion in the translocating strand. The marked difference in effect of the translocating strand cPu on rate of DNA unwinding between DDX11 and FANCJ helicase suggests the two Fe-S cluster helicases unwind damaged DNA by distinct mechanisms. The apparent complexity of helicase encounters with an unusual form of oxidative damage is likely to have important consequences in the cellular response to DNA damage and DNA repair.
KW  - nucleotide excision-repair
KW  - replication fork
KW  - substrate specificity
KW  - translesion synthesis
KW  - genomic stability
KW  - Warsaw breakage syndrome
KW  - escherichia coli
KW  - xeroderma-pigmentosum
KW  - human cells
KW  - biochemical characterization
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114635
VL  - 9
IS  - 11
ER  - 
TY  - JOUR
A1  - Benz, Roland
A1  - Jones, Michael D.
A1  - Younas, Farhan
A1  - Maier, Elke
A1  - Modi, Niraj
A1  - Mentele, Reinhard
A1  - Lottspeich, Friedrich
A1  - Kleinekathöfer, Ulrich
A1  - Smit, John
T1  - OmpW of Caulobacter crescentus functions as an outer membrane channel for cations
JF  - PLoS ONE
N2  - Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two known structures with the model of OmpW of C. crescentus suggested that it has a more hydrophilic interior and possibly a larger diameter.
KW  - matrix protein porin
KW  - amino acid sequence
KW  - escherichia coli
KW  - selective channel
KW  - molecular basis
KW  - lipid bilayer membranes
KW  - S-layer protein
KW  - pseudomonas aeruginosa
KW  - ionic selectivity
KW  - cell wall
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-145114
VL  - 10
IS  - 11
ER  - 
TY  - JOUR
A1  - Abda, Ebrahim M.
A1  - Krysciak, Dagmar
A1  - Krohn-Molt, Ines
A1  - Mamat, Uwe
A1  - Schmeisser, Christel
A1  - Förstner, Konrad U.
A1  - Schaible, Ulrich E.
A1  - Kohi, Thomas A.
A1  - Nieman, Stefan
A1  - Streit, Wolfgang R.
T1  - Phenotypic Heterogeneity Affects Stenotrophomonas maltophilia K279a Colony Morphotypes and \(\beta\)-Lactamase Expression
JF  - Frontiers in Microbiology
N2  - Phenotypic heterogeneity at the cellular level in response to various stresses, e.g., antibiotic treatment has been reported for a number of bacteria. In a clonal population, cell-to-cell variation may result in phenotypic heterogeneity that is a mechanism to survive changing environments including antibiotic therapy. Stenotrophomonas rnaltophilia has been frequently isolated from cystic fibrosis patients, can cause numerous infections in other organs and tissues, and is difficult to treat due to antibiotic resistances. S. maltophilia K279a produces the Li and L2 beta-lactamases in response to beta-lactam treatment. Here we report that the patient isolate S. rnaltophilia K279a diverges into cellular subpopulations with distinct but reversible morphotypes of small and big colonies when challenged with ampicillin. This observation is consistent with the formation of elongated chains of bacteria during exponential growth phase and the occurrence of mainly rod-shaped cells in liquid media. RNA-seq analysis of small versus big colonies revealed differential regulation of at least seven genes among the colony morphotypes. Among those, bleu and bla(L2) were transcriptionally the most strongly upregulated genes. Promoter fusions of b/a(L1) and b/a(L2) genes indicated that expression of both genes is also subject to high levels of phenotypic heterogeneous expression on a single cell level. Additionally, the comE homolog was found to be differentially expressed in homogenously versus heterogeneously bla(L2) expressing cells as identified by RNA(seq) analysis. Overexpression of cornE in S. maltophilia K279a reduced the level of cells that were in a bla(L2)-ON mode to 1% or lower. Taken together, our data provide strong evidence that S. maltophilia K279a populations develop phenotypic heterogeneity in an ampicillin challenged model. This cellular variability is triggered by regulation networks including b/a(L1), b/a(L2), and comE.
KW  - xanthomonas maltophilia
KW  - gram-negative bacteria
KW  - RNA-seq
KW  - pseudomas aeruginosa
KW  - antibiotic resistance
KW  - colony morphotypes
KW  - beta-lactamases
KW  - K279a
KW  - Stenotrophomonas maltophilia
KW  - phenotypic heterogeneity
KW  - persister cells
KW  - streptococcus pneumoniae
KW  - nosocomial pathogen
KW  - membrane vesicles
KW  - sinorhizobium fredii NGR234
KW  - red fluorescent protein
KW  - escherichia coli
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136446
VL  - 6
IS  - 1373
ER  -