Publications by authors named "Franz Narberhaus"

123 Publications

Promiscuous phospholipid biosynthesis enzymes in the plant pathogen Pseudomonas syringae.

Biochim Biophys Acta Mol Cell Biol Lipids 2021 Mar 22;1866(7):158926. Epub 2021 Mar 22.

Microbial Biology, Ruhr University Bochum, Bochum, Germany. Electronic address:

Bacterial membranes are primarily composed of phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). In the canonical PE biosynthesis pathway, phosphatidylserine (PS) is decarboxylated by the Psd enzyme. CL formation typically depends on CL synthases (Cls) using two PG molecules as substrates. Only few bacteria produce phosphatidylcholine (PC), the hallmark of eukaryotic membranes. Most of these bacteria use phospholipid N-methyltransferases to successively methylate PE to PC and/or a PC synthase (Pcs) to catalyze the condensation of choline and CDP-diacylglycerol (CDP-DAG) to PC. In this study, we show that membranes of Pseudomonas species able to interact with eukaryotes contain PE, PG, CL and PC. More specifically, we report on PC formation and a poorly characterized CL biosynthetic pathway in the plant pathogen P. syringae pv. tomato. It encodes a Pcs enzyme responsible for choline-dependent PC biosynthesis. CL formation is catalyzed by a promiscuous phospholipase D (PLD)-type enzyme (PSPTO_0095) that we characterized in vivo and in vitro. Like typical bacterial CL biosynthesis enzymes, it uses PE and PG for CL production. This enzyme is also able to convert PE and glycerol to PG, which is then combined with another PE molecule to synthesize CL. In addition, the enzyme is capable of converting ethanolamine or methylated derivatives into the corresponding phospholipids such as PE both in P. syringae and in E. coli. It can also hydrolyze CDP-DAG to yield phosphatidic acid (PA). Our study adds an example of a promiscuous Cls enzyme able to synthesize a suite of products according to the available substrates.
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http://dx.doi.org/10.1016/j.bbalip.2021.158926DOI Listing
March 2021

A Salmonella Typhi RNA thermosensor regulates virulence factors and innate immune evasion in response to host temperature.

PLoS Pathog 2021 Mar 2;17(3):e1009345. Epub 2021 Mar 2.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

Sensing and responding to environmental signals is critical for bacterial pathogens to successfully infect and persist within hosts. Many bacterial pathogens sense temperature as an indication they have entered a new host and must alter their virulence factor expression to evade immune detection. Using secondary structure prediction, we identified an RNA thermosensor (RNAT) in the 5' untranslated region (UTR) of tviA encoded by the typhoid fever-causing bacterium Salmonella enterica serovar Typhi (S. Typhi). Importantly, tviA is a transcriptional regulator of the critical virulence factors Vi capsule, flagellin, and type III secretion system-1 expression. By introducing point mutations to alter the mRNA secondary structure, we demonstrate that the 5' UTR of tviA contains a functional RNAT using in vitro expression, structure probing, and ribosome binding methods. Mutational inhibition of the RNAT in S. Typhi causes aberrant virulence factor expression, leading to enhanced innate immune responses during infection. In conclusion, we show that S. Typhi regulates virulence factor expression through an RNAT in the 5' UTR of tviA. Our findings demonstrate that limiting inflammation through RNAT-dependent regulation in response to host body temperature is important for S. Typhi's "stealthy" pathogenesis.
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http://dx.doi.org/10.1371/journal.ppat.1009345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954313PMC
March 2021

A Novel, Universally Active C-terminal Protein Degradation Signal Generated by Alternative Splicing.

J Mol Biol 2021 Apr 23;433(8):166890. Epub 2021 Feb 23.

Department of Biology, Technical University of Darmstadt, Darmstadt 64287, Germany. Electronic address:

Proteome integrity is crucial for cellular homeostasis and adaptation to stress conditions such as hypoxia. One mechanism for rapid adaptation of the proteome in response to changing environmental signals is alternative splicing. In addition to generating different protein isoforms, alternative splicing is also capable of controlling total protein levels by the regulated synthesis of non-productive mRNA isoforms. The hypoxia-induced isoform E of the tumor suppressor MAX is produced by retention and translation of the last intron. This leads to an alternative C-terminus that harbors a potent C-degron, the isoE degron. Strikingly, the isoE degron represents a universal protein degradation signal that is not only functional in mammalian cells, but also in yeast and even in bacteria. Essential for efficient protein decay is a conserved (F/W)xxW motif. Degradation of isoE tagged proteins is mediated by the proteasome in eukaryotes and Lon protease in bacteria. Thus, the isoE degron is a broadly applicable and highly efficient tool in protein analyses.
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http://dx.doi.org/10.1016/j.jmb.2021.166890DOI Listing
April 2021

A LysR-type transcriptional regulator controls the expression of numerous small RNAs in Agrobacterium tumefaciens.

Mol Microbiol 2021 Feb 9. Epub 2021 Feb 9.

Microbial Biology, Ruhr University Bochum, Bochum, Germany.

Small RNAs (sRNAs) are universal posttranscriptional regulators of gene expression and hundreds of sRNAs are frequently found in each and every bacterium. In order to coordinate cellular processes in response to ambient conditions, many sRNAs are differentially expressed. Here, we asked how these small regulators are regulated using Agrobacterium tumefaciens as a model system. Among the best-studied sRNAs in this plant pathogen are AbcR1 regulating numerous ABC transporters and PmaR, a regulator of peptidoglycan biosynthesis, motility, and ampicillin resistance. We report that the LysR-type regulator VtlR (also known as LsrB) controls expression of AbcR1 and PmaR. A vtlR/lsrB deletion strain showed growth defects, was sensitive to antibiotics and severely compromised in plant tumor formation. Transcriptome profiling by RNA-sequencing revealed more than 1,200 genes with altered expression in the mutant. Consistent with the function of VtlR/LsrB as regulator of AbcR1, many ABC transporter genes were affected. Interestingly, the transcription factor did not only control the expression of AbcR1 and PmaR. In the mutant, 102 sRNA genes were significantly up- or downregulated. Thus, our study uncovered VtlR/LsrB as the master regulator of numerous sRNAs. Thereby, the transcriptional regulator harnesses the regulatory power of sRNAs to orchestrate the expression of distinct sub-regulons.
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http://dx.doi.org/10.1111/mmi.14695DOI Listing
February 2021

Arginine-Rich Small Proteins with a Domain of Unknown Function, DUF1127, Play a Role in Phosphate and Carbon Metabolism of Agrobacterium tumefaciens.

J Bacteriol 2020 10 22;202(22). Epub 2020 Oct 22.

Microbial Biology, Ruhr University Bochum, Bochum, Germany

In any given organism, approximately one-third of all proteins have a yet-unknown function. A widely distributed domain of unknown function is DUF1127. Approximately 17,000 proteins with such an arginine-rich domain are found in 4,000 bacteria. Most of them are single-domain proteins, and a large fraction qualifies as small proteins with fewer than 50 amino acids. We systematically identified and characterized the seven DUF1127 members of the plant pathogen They all give rise to authentic proteins and are differentially expressed as shown at the RNA and protein levels. The seven proteins fall into two subclasses on the basis of their length, sequence, and reciprocal regulation by the LysR-type transcription factor LsrB. The absence of all three short DUF1127 proteins caused a striking phenotype in later growth phases and increased cell aggregation and biofilm formation. Protein profiling and transcriptome sequencing (RNA-seq) analysis of the wild type and triple mutant revealed a large number of differentially regulated genes in late exponential and stationary growth. The most affected genes are involved in phosphate uptake, glycine/serine homeostasis, and nitrate respiration. The results suggest a redundant function of the small DUF1127 paralogs in nutrient acquisition and central carbon metabolism of They may be required for diauxic switching between carbon sources when sugar from the medium is depleted. We end by discussing how DUF1127 might confer such a global impact on cell physiology and gene expression. Despite being prevalent in numerous ecologically and clinically relevant bacterial species, the biological role of proteins with a domain of unknown function, DUF1127, is unclear. Experimental models are needed to approach their elusive function. We used the phytopathogen , a natural genetic engineer that causes crown gall disease, and focused on its three small DUF1127 proteins. They have redundant and pervasive roles in nutrient acquisition, cellular metabolism, and biofilm formation. The study shows that small proteins have important previously missed biological functions. How small basic proteins can have such a broad impact is a fascinating prospect of future research.
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http://dx.doi.org/10.1128/JB.00309-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7585064PMC
October 2020

Lead-seq: transcriptome-wide structure probing in vivo using lead(II) ions.

Nucleic Acids Res 2020 07;48(12):e71

Microbial Biology, Ruhr University Bochum, 44780 Bochum, Germany.

The dynamic conformation of RNA molecules within living cells is key to their function. Recent advances in probing the RNA structurome in vivo, including the use of SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) or kethoxal reagents or DMS (dimethyl sulfate), provided unprecedented insights into the architecture of RNA molecules in the living cell. Here, we report the establishment of lead probing in a global RNA structuromics approach. In order to elucidate the transcriptome-wide RNA landscape in the enteric pathogen Yersinia pseudotuberculosis, we combined lead(II) acetate-mediated cleavage of single-stranded RNA regions with high-throughput sequencing. This new approach, termed 'Lead-seq', provides structural information independent of base identity. We show that the method recapitulates secondary structures of tRNAs, RNase P RNA, tmRNA, 16S rRNA and the rpsT 5'-untranslated region, and that it reveals global structural features of mRNAs. The application of Lead-seq to Y. pseudotuberculosis cells grown at two different temperatures unveiled the first temperature-responsive in vivo RNA structurome of a bacterial pathogen. The translation of candidate genes derived from this approach was confirmed to be temperature regulated. Overall, this study establishes Lead-seq as complementary approach to interrogate intracellular RNA structures on a global scale.
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http://dx.doi.org/10.1093/nar/gkaa404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7337928PMC
July 2020

Lon Protease Removes Excess Signal Recognition Particle Protein in Escherichia coli.

J Bacteriol 2020 06 25;202(14). Epub 2020 Jun 25.

Microbial Biology, Ruhr University Bochum, Bochum, Germany

Correct targeting of membrane proteins is essential for membrane integrity, cell physiology, and viability. Cotranslational targeting depends on the universally conserved signal recognition particle (SRP), which is a ribonucleoprotein complex comprised of the protein component Ffh and the 4.5S RNA in About 25 years ago it was reported that Ffh is an unstable protein, but the underlying mechanism has never been explored. Here, we show that Lon is the primary protease responsible for adjusting the cellular Ffh level. When overproduced, Ffh is particularly prone to degradation during transition from exponential to stationary growth and the cellular Ffh amount is lowest in stationary phase. The Ffh protein consists of two domains, the NG domain, responsible for GTP hydrolysis and docking to the membrane receptor FtsY, and the RNA-binding M domain. We find that the NG domain alone is stable, whereas the isolated M domain is degraded. Consistent with the importance of Lon in this process, the M domain confers synthetic lethality to the mutant. The Ffh homolog from the model plant , which forms a protein-protein complex rather than a protein-RNA complex, is stable, suggesting that the RNA-binding ability residing in the M domain of Ffh is important for proteolysis. Our results support a model in which excess Ffh not bound to 4.5S RNA is subjected to proteolysis until an appropriate Ffh concentration is reached. The differential proteolysis adjusts Ffh levels to the cellular demand and maintains cotranslational protein transport and membrane integrity. Since one-third of all bacterial proteins reside outside the cytoplasm, protein targeting to the appropriate address is an essential process. Cotranslational targeting to the membrane relies on the signal recognition particle (SRP), which is a protein-RNA complex in bacteria. We report that the protein component Ffh is a substrate of the Lon protease. Regulated proteolysis of Ffh provides a simple mechanism to adjust the concentration of the essential protein to the cellular demand. This is important because elevated or depleted SRP levels negatively impact protein targeting and bacterial fitness.
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http://dx.doi.org/10.1128/JB.00161-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317043PMC
June 2020

An RNA thermometer dictates production of a secreted bacterial toxin.

PLoS Pathog 2020 01 17;16(1):e1008184. Epub 2020 Jan 17.

Microbial Biology, Ruhr University Bochum, Bochum, Germany.

Frequent transitions of bacterial pathogens between their warm-blooded host and external reservoirs are accompanied by abrupt temperature shifts. A temperature of 37°C serves as reliable signal for ingestion by a mammalian host, which induces a major reprogramming of bacterial gene expression and metabolism. Enteric Yersiniae are Gram-negative pathogens accountable for self-limiting gastrointestinal infections. Among the temperature-regulated virulence genes of Yersinia pseudotuberculosis is cnfY coding for the cytotoxic necrotizing factor (CNFY), a multifunctional secreted toxin that modulates the host's innate immune system and contributes to the decision between acute infection and persistence. We report that the major determinant of temperature-regulated cnfY expression is a thermo-labile RNA structure in the 5'-untranslated region (5'-UTR). Various translational gene fusions demonstrated that this region faithfully regulates translation initiation regardless of the transcription start site, promoter or reporter strain. RNA structure probing revealed a labile stem-loop structure, in which the ribosome binding site is partially occluded at 25°C but liberated at 37°C. Consistent with translational control in bacteria, toeprinting (primer extension inhibition) experiments in vitro showed increased ribosome binding at elevated temperature. Point mutations locking the 5'-UTR in its 25°C structure impaired opening of the stem loop, ribosome access and translation initiation at 37°C. To assess the in vivo relevance of temperature control, we used a mouse infection model. Y. pseudotuberculosis strains carrying stabilized RNA thermometer variants upstream of cnfY were avirulent and attenuated in their ability to disseminate into mesenteric lymph nodes and spleen. We conclude with a model, in which the RNA thermometer acts as translational roadblock in a two-layered regulatory cascade that tightly controls provision of the CNFY toxin during acute infection. Similar RNA structures upstream of various cnfY homologs suggest that RNA thermosensors dictate the production of secreted toxins in a wide range of pathogens.
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http://dx.doi.org/10.1371/journal.ppat.1008184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992388PMC
January 2020

Regulation of OmpA Translation and Shigella dysenteriae Virulence by an RNA Thermometer.

Infect Immun 2020 02 20;88(3). Epub 2020 Feb 20.

Department of Biomedical Sciences, Ohio University Heritage College of Osteopathic Medicine, Athens, Ohio, USA.

RNA thermometers are -acting riboregulators that mediate the posttranscriptional regulation of gene expression in response to environmental temperature. Such regulation is conferred by temperature-responsive structural changes within the RNA thermometer that directly result in differential ribosomal binding to the regulated transcript. The significance of RNA thermometers in controlling bacterial physiology and pathogenesis is becoming increasingly clear. This study combines , molecular genetics, and biochemical analyses to characterize both the structure and function of a newly identified RNA thermometer within the transcript of First identified by structural predictions, genetic analyses have demonstrated that the RNA thermometer is a functional riboregulator sufficient to confer posttranscriptional temperature-dependent regulation, with optimal expression observed at the host-associated temperature of 37°C. Structural studies and ribosomal binding analyses have revealed both increased exposure of the ribosomal binding site and increased ribosomal binding to the transcript at permissive temperatures. The introduction of site-specific mutations predicted to alter the temperature responsiveness of the RNA thermometer has predictable consequences for both the structure and function of the regulatory element. Finally, tissue culture-based analyses implicate the RNA thermometer as a bona fide virulence factor in this bacterial pathogen. Given that is highly conserved among Gram-negative pathogens, these studies not only provide insight into the significance of riboregulation in controlling virulence, but they also have the potential to facilitate further understanding of the physiology and/or pathogenesis of a wide range of bacterial species.
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http://dx.doi.org/10.1128/IAI.00871-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035939PMC
February 2020

An unconventional RNA-based thermosensor within the 5' UTR of Staphylococcus aureus cidA.

PLoS One 2019 1;14(4):e0214521. Epub 2019 Apr 1.

Infection and Topical Disease Institute, Ohio University, Athens, OH, United States of America.

Staphylococcus aureus is a Gram-positive bacterial pathogen of global concern and a leading cause of bacterial infections worldwide. Asymptomatic carriage of S. aureus on the skin and in the anterior nares is common and recognized as a predisposing factor to invasive infection. Transition of S. aureus from the carriage state to that of invasive infection is often accompanied by a temperature upshift from approximately 33°C to 37°C. Such a temperature shift is known in other pathogens to influence gene expression, often resulting in increased production of factors that promote survival or virulence within the host. One mechanism by which bacteria modulate gene expression in response to temperature is by the regulatory activity of RNA-based thermosensors, cis-acting riboregulators that control translation efficiency. This study was designed to identify and characterize RNA-based thermosensors in S. aureus. Initially predicted by in silico analyses of the S. aureus USA300 genome, reporter-based gene expression analyses and site-specific mutagenesis were performed to demonstrate the presence of a functional thermosensor within the 5' UTR of cidA, a gene implicated in biofilm formation and survival of the pathogen. The nucleic sequence composing the identified thermosensor are sufficient to confer temperature-dependent post-transcriptional regulation, and activity is predictably altered by the introduction of site-specific mutations designed to stabilize or destabilize the structure within the identified thermosensor. The identified regulator is functional in both the native bacterial host S. aureus and in the distally related species Escherichia coli, suggesting that its regulatory activity is independent of host-specific factors. Interestingly, unlike the majority of bacterial RNA-based thermosensors characterized to date, the cidA thermosensor facilitates increased target gene expression at lower temperatures. In addition to the characterization of the first RNA-based thermosensor in the significant pathogen S. aureus, it highlights the diversity of function within this important class of ribo-regulators.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214521PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443170PMC
December 2019

The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer .

J Bacteriol 2019 06 8;201(11). Epub 2019 May 8.

Microbial Biology, Ruhr University Bochum, Bochum, Germany

Riboregulation involving regulatory RNAs, RNA chaperones, and ribonucleases is fundamental for the rapid adaptation of gene expression to changing environmental conditions. The gene coding for the RNase YbeY belongs to the minimal prokaryotic genome set and has a profound impact on physiology in a wide range of bacteria. Here, we show that the gene is not essential. Deletion of the gene in the plant pathogen reduced growth, motility, and stress tolerance. Most interestingly, YbeY is crucial for -mediated T-DNA transfer and tumor formation. Comparative proteomics by using isobaric tags for relative and absolute quantitation (iTRAQ) revealed dysregulation of 59 proteins, many of which have previously been found to be dependent on the RNA chaperone Hfq. YbeY and Hfq have opposing effects on production of these proteins. Accumulation of a 16S rRNA precursor in the mutant suggests that YbeY is involved in rRNA processing. RNA coimmunoprecipitation-sequencing (RIP-Seq) showed binding of YbeY to the region immediately upstream of the 16S rRNA. Purified YbeY is an oligomer with RNase activity. It does not physically interact with Hfq and thus plays a partially overlapping but distinct role in the riboregulatory network of the plant pathogen. Although gene belongs to the universal bacterial core genome, its biological function is incompletely understood. Here, we show that YbeY is critical for fitness and host-microbe interaction in the plant pathogen Consistent with the reported endoribonuclease activity of YbeY, YbeY acts as a RNase involved in maturation of 16S rRNA. This report adds a worldwide plant pathogen and natural genetic engineer of plants to the growing list of bacteria that require the conserved YbeY protein for host-microbe interaction.
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http://dx.doi.org/10.1128/JB.00730-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509659PMC
June 2019

Intricate Crosstalk Between Lipopolysaccharide, Phospholipid and Fatty Acid Metabolism in Modulates Proteolysis of LpxC.

Front Microbiol 2018 14;9:3285. Epub 2019 Jan 14.

Microbial Biology, Ruhr University Bochum, Bochum, Germany.

Lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria provide the first line of defense against antibiotics and other harmful compounds. LPS biosynthesis critically depends on LpxC catalyzing the first committed enzyme in this process. In , the cellular concentration of LpxC is adjusted in a growth rate-dependent manner by the FtsH protease making sure that LPS biosynthesis is coordinated with the cellular demand. As a result, LpxC is stable in fast-growing cells and prone to degradation in slow-growing cells. One of the factors involved in this process is the alarmone guanosine tetraphosphate (ppGpp) but previous studies suggested the involvement of yet unknown factors in LpxC degradation. We established a quantitative proteomics approach aiming at the identification of proteins that are associated with LpxC and/or FtsH at high or low growth rates. The identification of known LpxC and FtsH interactors validated our approach. A number of proteins involved in fatty acid biosynthesis and degradation, including the central regulator FadR, were found in the LpxC and/or FtsH interactomes. Another protein associated with LpxC and FtsH was WaaH, a LPS-modifying enzyme. When overproduced, several members of the LpxC/FtsH interactomes were able to modulate LpxC proteolysis. Our results go beyond the previously established link between LPS and phospholipid biosynthesis and uncover a far-reaching network that controls LPS production by involving multiple enzymes in fatty acid metabolism, phospholipid biosynthesis and LPS modification.
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http://dx.doi.org/10.3389/fmicb.2018.03285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6339880PMC
January 2019

A Small Regulatory RNA Controls Cell Wall Biosynthesis and Antibiotic Resistance.

mBio 2018 11 13;9(6). Epub 2018 Nov 13.

Department of Microbial Biology, Ruhr University Bochum, Bochum, Germany

Small regulatory RNAs play an important role in the adaptation to changing conditions. Here, we describe a differentially expressed small regulatory RNA (sRNA) that affects various cellular processes in the plant pathogen Using a combination of bioinformatic predictions and comparative proteomics, we identified nine targets, most of which are positively regulated by the sRNA. According to these targets, we named the sRNA PmaR for peptidoglycan biosynthesis, motility, and ampicillin resistance regulator. spp. are long known to be naturally resistant to high ampicillin concentrations, and we can now explain this phenotype by the positive PmaR-mediated regulation of the beta-lactamase gene Structure probing revealed a spoon-like structure of the sRNA, with a single-stranded loop that is engaged in target interaction and Several riboregulators have been implicated in antibiotic resistance mechanisms, such as uptake and efflux transporters, but PmaR represents the first example of an sRNA that directly controls the expression of an antibiotic resistance gene. The alphaproteobacterium is able to infect various eudicots causing crown gall tumor formation. Based on its unique ability of interkingdom gene transfer, serves as a crucial biotechnological tool for genetic manipulation of plant cells. The presence of hundreds of putative sRNAs in this organism suggests a considerable impact of riboregulation on physiology. Here, we characterized the biological function of the sRNA PmaR that controls various processes crucial for growth, motility, and virulence. Among the genes directly targeted by PmaR is coding for a beta-lactamase that confers ampicillin resistance, suggesting that the sRNA is crucial for fitness in the competitive microbial composition of the rhizosphere.
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http://dx.doi.org/10.1128/mBio.02100-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234868PMC
November 2018

Virulence of Agrobacterium tumefaciens requires lipid homeostasis mediated by the lysyl-phosphatidylglycerol hydrolase AcvB.

Mol Microbiol 2019 01 14;111(1):269-286. Epub 2018 Nov 14.

Institute for Microbiology, Technische Universität Braunschweig, 38106, Braunschweig, Germany.

Agrobacterium tumefaciens transfers oncogenic T-DNA via the type IV secretion system (T4SS) into plants causing tumor formation. The acvB gene encodes a virulence factor of unknown function required for plant transformation. Here we specify AcvB as a periplasmic lysyl-phosphatidylglycerol (L-PG) hydrolase, which modulates L-PG homeostasis. Through functional characterization of recombinant AcvB variants, we showed that the C-terminal domain of AcvB (residues 232-456) is sufficient for full enzymatic activity and defined key residues for catalysis. Absence of the hydrolase resulted in ~10-fold increase in L-PG in Agrobacterium membranes and abolished T-DNA transfer and tumor formation. Overproduction of the L-PG synthase gene (lpiA) in wild-type A. tumefaciens resulted in a similar increase in the L-PG content (~7-fold) and a virulence defect even in the presence of intact AcvB. These results suggest that elevated L-PG amounts (either by overproduction of the synthase or absence of the hydrolase) are responsible for the virulence phenotype. Gradually increasing the L-PG content by complementation with different acvB variants revealed that cellular L-PG levels above 3% of total phospholipids interfere with T-DNA transfer. Cumulatively, this study identified AcvB as a novel virulence factor required for membrane lipid homeostasis and T-DNA transfer.
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http://dx.doi.org/10.1111/mmi.14154DOI Listing
January 2019

Coordinated regulation of nitrogen fixation and molybdate transport by molybdenum.

Mol Microbiol 2019 01 14;111(1):17-30. Epub 2018 Nov 14.

Microbial Biology, Ruhr University Bochum, Bochum, Germany.

Biological nitrogen fixation, the reduction of chemically inert dinitrogen to bioavailable ammonia, is a central process in the global nitrogen cycle highly relevant for life on earth. N reduction to NH is catalyzed by nitrogenases exclusively synthesized by diazotrophic prokaryotes. All diazotrophs have a molybdenum nitrogenase containing the unique iron-molybdenum cofactor FeMoco. In addition, some diazotrophs encode one or two alternative Mo-free nitrogenases that are less efficient at reducing N than Mo-nitrogenase. To permit biogenesis of Mo-nitrogenase and other molybdoenzymes when Mo is scarce, bacteria synthesize the high-affinity molybdate transporter ModABC. Generally, Mo supports expression of Mo-nitrogenase genes, while it represses production of Mo-free nitrogenases and ModABC. Since all three nitrogenases and ModABC can reach very high levels at suitable Mo concentrations, tight Mo-mediated control saves considerable resources and energy. This review outlines the similarities and differences in Mo-responsive regulation of nitrogen fixation and molybdate transport in diverse diazotrophs.
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http://dx.doi.org/10.1111/mmi.14152DOI Listing
January 2019

Next-Generation Trapping of Protease Substrates by Label-Free Proteomics.

Methods Mol Biol 2018 ;1841:189-206

Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität Bochum, Bochum, Germany.

AAA proteases (ATPases associated with various cellular activities) shape the cellular protein pool in response to environmental conditions. A prerequisite for understanding the underlying recognition and degradation principles is the identification of as many protease substrates as possible. Most previous studies made use of inactive protease variants to trap substrates, which were identified by 2D-gel based proteomics. Since this method is known for limitations in the identification of low-abundant proteins or proteins with many transmembrane domains, we established a trapping approach that overcomes these limitations. We used a proteolytically inactive FtsH variant (FtsH) of Escherichia coli (E. coli) that is still able to bind and translocate substrates into the proteolytic chamber but no longer able to degrade proteins. Proteins associated with FtsH or FtsH (proteolytically active FtsH) were purified, concentrated by an 1D-short gel, and identified by LC-coupled mass spectrometry (LC-MS) followed by label-free quantification. The identification of four known FtsH substrates validated this approach and suggests that it is generally applicable to AAA proteases.
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http://dx.doi.org/10.1007/978-1-4939-8695-8_14DOI Listing
April 2019

A phosphatidic acid-binding protein is important for lipid homeostasis and adaptation to anaerobic biofilm conditions in .

Biochem J 2018 06 6;475(11):1885-1907. Epub 2018 Jun 6.

Institute of Microbiology, Technische Universität Braunschweig, Spielmannstraße 7, D-38106 Braunschweig, Germany

A quantitative proteomics approach revealed increased abundance of the so-far uncharacterized protein PA3911 in anaerobic biofilms grown under conditions of the cystic fibrosis lung. Physiological relevance of ORF PA3911 was demonstrated, , using phenotype microarray experiments. The mutant strain showed increased susceptibility in the presence of antimicrobials (minocycline, nafcillin, oxacillin, chloramphenicol and thiamphenicol), enhanced twitching motility and significantly impaired biofilm formation. PA3911 is a soluble, cytoplasmic protein in In protein-lipid overlay experiments, purified PA3911 bound specifically to phosphatidic acid (PA), the central hub of phospholipid metabolism. Structure-guided site-directed mutagenesis was used to explore the proposed ligand-binding cavity of PA3911. Protein variants of Leu, Leu, Val and Leu were shown to impair PA interaction. A comparative shotgun lipidomics approach demonstrated a multifaceted response of to anaerobic conditions at the lipid head group and fatty acid level. Lipid homeostasis in the PA3911 mutant strain was imbalanced with respect to lysophosphatidylcholine, phosphatidylcholine and diacylglycerol under anaerobic and/or aerobic conditions. The impact of the newly identified PA-binding protein on lipid homeostasis and the related macroscopic phenotypes of are discussed.
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http://dx.doi.org/10.1042/BCJ20180257DOI Listing
June 2018

An Integrated Proteomic Approach Uncovers Novel Substrates and Functions of the Lon Protease in Escherichia coli.

Proteomics 2018 07 6;18(13):e1800080. Epub 2018 Jun 6.

Department of Microbial Biology, Ruhr University Bochum, Universitätsstraße 150, D-44801, Bochum, Germany.

Controlling the cellular abundance and proper function of proteins by proteolysis is a universal process in all living organisms. In Escherichia coli, the ATP-dependent Lon protease is crucial for protein quality control and regulatory processes. To understand how diverse substrates are selected and degraded, unbiased global approaches are needed. We employed a quantitative Super-SILAC (stable isotope labeling with amino acids in cell culture) mass spectrometry approach and compared the proteomes of a lon mutant and a strain producing the protease to discover Lon-dependent physiological functions. To identify Lon substrates, we took advantage of a Lon trapping variant, which is able to translocate substrates but unable to degrade them. Lon-associated proteins were identified by label-free LC-MS/MS. The combination of both approaches revealed a total of 14 novel Lon substrates. Besides the identification of known pathways affected by Lon, for example, the superoxide stress response, our cumulative data suggests previously unrecognized fundamental functions of Lon in sulfur assimilation, nucleotide biosynthesis, amino acid and central energy metabolism.
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http://dx.doi.org/10.1002/pmic.201800080DOI Listing
July 2018

RNA Thermometers in Bacterial Pathogens.

Microbiol Spectr 2018 04;6(2)

Microbial Biology, Ruhr University, Bochum, Germany.

Temperature variation is one of the multiple parameters a microbial pathogen encounters when it invades a warm-blooded host. To survive and thrive at host body temperature, human pathogens have developed various strategies to sense and respond to their ambient temperature. An instantaneous response is mounted by RNA thermometers (RNATs), which are integral sensory structures in mRNAs that modulate translation efficiency. At low temperatures outside the host, the folded RNA blocks access of the ribosome to the translation initiation region. The temperature shift upon entering the host destabilizes the RNA structure and thus permits ribosome binding. This reversible zipper-like mechanism of RNATs is ideally suited to fine-tune virulence gene expression when the pathogen enters or exits the body of its host. This review summarizes our present knowledge on virulence-related RNATs and discusses recent developments in the field.
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http://dx.doi.org/10.1128/microbiolspec.RWR-0012-2017DOI Listing
April 2018

Design of a Temperature-Responsive Transcription Terminator.

ACS Synth Biol 2018 02 12;7(2):613-621. Epub 2017 Dec 12.

Microbial Biology, Ruhr University Bochum , 44780 Bochum, Germany.

RNA structures regulate various steps in gene expression. Transcription in bacteria is typically terminated by stable hairpin structures. Translation initiation can be modulated by metabolite- or temperature-sensitive RNA structures, called riboswitches or RNA thermometers (RNATs), respectively. RNATs control translation initiation by occlusion of the ribosome binding site at low temperatures. Increasing temperatures destabilize the RNA structure and facilitate ribosome access. In this study, we exploited temperature-responsive RNAT structures to design regulatory elements that control transcription termination instead of translation initiation in Escherichia coli. In order to mimic the structure of factor-independent intrinsic terminators, naturally occurring RNAT hairpins were genetically engineered to be followed by a U-stretch. Functional temperature-responsive terminators (thermoterms) prevented mRNA synthesis at low temperatures but resumed transcription after a temperature upshift. The successful design of temperature-controlled terminators highlights the potential of RNA structures as versatile gene expression control elements.
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http://dx.doi.org/10.1021/acssynbio.7b00356DOI Listing
February 2018

One gene, two proteins: coordinated production of a copper chaperone by differential transcript formation and translational frameshifting in Escherichia coli.

Mol Microbiol 2017 Nov 6;106(4):635-645. Epub 2017 Oct 6.

Department of Biophysics, Ruhr University Bochum, Universitätsstr. 150, Bochum, D-44801, Germany.

Programmed ribosomal frameshifting (PRF) is a translational anomaly causing the ribosome to shift into an alternative reading frame. PRFs are common in viral genomes, using a single nucleotide sequence to code for two proteins in overlapping frames. In bacteria and eukaryota, PRFs are less frequent. We report on a PRF in the copper detoxification system of Escherichia coli where a metallochaperone is generated out of the first 69 amino acids and a C-terminal out-of-frame glycine of the gene copA. copA besides codes for the P -ATPase CopA, a membrane-integral protein and principal interaction target of the chaperone. To enhance the production of the frameshift-generated cytosolic copper binding protein a truncated transcript is produced from the monocistronic copA gene. This shorter transcript is essential for producing sufficient amounts of the chaperone to support the membrane pump. The findings close the gap in our understanding of the molecular physiology of cytoplasmic copper transport in E. coli, revealing that a chaperone-like entity is required for full functionality of the P -ATPase copper pump. We, moreover, demonstrate that the primary transcriptional response to copper results in formation of the small transcript and concurrently, the metallochaperone plays a key role in resistance against copper shock.
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http://dx.doi.org/10.1111/mmi.13841DOI Listing
November 2017

Dissection of membrane-binding and -remodeling regions in two classes of bacterial phospholipid N-methyltransferases.

Biochim Biophys Acta Biomembr 2017 Dec 11;1859(12):2279-2288. Epub 2017 Sep 11.

Microbial Biology, Faculty of Biology, Ruhr University Bochum, Bochum, Germany. Electronic address:

Bacterial phospholipid N-methyltransferases (Pmts) catalyze the formation of phosphatidylcholine (PC) via successive N-methylation of phosphatidylethanolamine (PE). They are classified into Sinorhizobium-type and Rhodobacter-type enzymes. The Sinorhizobium-type PmtA protein from the plant pathogen Agrobacterium tumefaciens is recruited to anionic lipids in the cytoplasmic membrane via two amphipathic helices called αA and αF. Besides its enzymatic activity, PmtA is able to remodel membranes mediated by the αA domain. According to the Heliquest program, αA- and αF-like amphipathic helices are also present in other Sinorhizobium- and Rhodobacter-type Pmt enzymes suggesting a conserved architecture of α-helical membrane-binding regions in these methyltransferases. As representatives of the two Pmt families, we investigated the membrane binding and remodeling capacity of Bradyrhizobium japonicum PmtA (Sinorhizobium-type) and PmtX1 (Rhodobacter-type), which act cooperatively to produce PC in consecutive methylation steps. We found that the αA regions in both enzymes bind anionic lipids similar to αA of A. tumefaciens PmtA. Membrane binding of PmtX1 αA is enhanced by its substrate monomethyl-PE indicating a substrate-controlled membrane association. The αA regions of all investigated enzymes remodel spherical liposomes into tubular filaments suggesting a conserved membrane-remodeling capacity of bacterial Pmts. Based on these results we propose that the molecular details of membrane-binding and remodeling are conserved among bacterial Pmts.
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http://dx.doi.org/10.1016/j.bbamem.2017.09.013DOI Listing
December 2017

The Copper Efflux Regulator CueR Is Subject to ATP-Dependent Proteolysis in .

Front Mol Biosci 2017 28;4. Epub 2017 Feb 28.

Microbial Biology, Ruhr University Bochum Bochum, Germany.

The trace element copper serves as cofactor for many enzymes but is toxic at elevated concentrations. In bacteria, the intracellular copper level is maintained by copper efflux systems including the Cue system controlled by the transcription factor CueR. CueR, a member of the MerR family, forms homodimers, and binds monovalent copper ions with high affinity. It activates transcription of the copper tolerance genes and via a conserved DNA-distortion mechanism. The mechanism how CueR-induced transcription is turned off is not fully understood. Here, we report that CueR is prone to proteolysis by the AAA proteases Lon, ClpXP, and ClpAP. Using a set of CueR variants, we show that CueR degradation is not altered by mutations affecting copper binding, dimerization or DNA binding of CueR, but requires an accessible C terminus. Except for a twofold stabilization shortly after a copper pulse, proteolysis of CueR is largely copper-independent. Our results suggest that ATP-dependent proteolysis contributes to copper homeostasis in by turnover of CueR, probably to allow steady monitoring of changes of the intracellular copper level and shut-off of CueR-dependent transcription.
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http://dx.doi.org/10.3389/fmolb.2017.00009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5329002PMC
February 2017

Membrane Remodeling by a Bacterial Phospholipid-Methylating Enzyme.

mBio 2017 02 14;8(1). Epub 2017 Feb 14.

Microbial Biology, Faculty of Biology, Ruhr University Bochum, Bochum, Germany

Membrane deformation by proteins is a universal phenomenon that has been studied extensively in eukaryotes but much less in prokaryotes. In this study, we discovered a membrane-deforming activity of the phospholipid -methyltransferase PmtA from the plant-pathogenic bacterium PmtA catalyzes the successive three-step -methylation of phosphatidylethanolamine to phosphatidylcholine. Here, we defined the lipid and protein requirements for the membrane-remodeling activity of PmtA by a combination of transmission electron microscopy and liposome interaction studies. Dependent on the lipid composition, PmtA changes the shape of spherical liposomes either into filaments or small vesicles. Upon overproduction of PmtA in , vesicle-like structures occur in the cytoplasm, dependent on the presence of the anionic lipid cardiolipin. The N-terminal lipid-binding α-helix (αA) is involved in membrane deformation by PmtA. Two functionally distinct and spatially separated regions in αA can be distinguished. Anionic interactions by positively charged amino acids on one face of the helix are responsible for membrane recruitment of the enzyme. The opposite hydrophobic face of the helix is required for membrane remodeling, presumably by shallow insertion into the lipid bilayer. The ability to alter the morphology of biological membranes is known for a small number of some bacterial proteins. Our study adds the phospholipid -methyltransferase PmtA as a new member to the category of bacterial membrane-remodeling proteins. A combination of and methods reveals the molecular requirements for membrane deformation at the protein and phospholipid level. The dual functionality of PmtA suggests a contribution of membrane biosynthesis enzymes to the complex morphology of bacterial membranes.
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http://dx.doi.org/10.1128/mBio.02082-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5312082PMC
February 2017

Systematic probing of the bacterial RNA structurome to reveal new functions.

Curr Opin Microbiol 2017 Apr 1;36:14-19. Epub 2017 Feb 1.

Department of Microbial Biology, Ruhr University Bochum, Bochum, Germany. Electronic address:

RNA folds into intricate structures. Recent discoveries using next-generation sequencing (NGS) approaches have revealed unprecedented structural complexity with a pivotal role in regulating RNA function and stability. Here, we present new discoveries from the transcriptome-wide determination of RNA structuromes in bacteria and discuss emerging concepts in the role of mRNA structures in regulating transcription, translation and degradation. We also provide critical viewpoints on the use of NGS approaches for elucidating of RNA structuromes at the systems level.
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http://dx.doi.org/10.1016/j.mib.2017.01.003DOI Listing
April 2017

When, how and why? Regulated proteolysis by the essential FtsH protease in Escherichia coli.

Biol Chem 2017 05;398(5-6):625-635

Microbial Biology, Ruhr University Bochum, Universitätsstr. 150, NDEF 06/783, D-44801 Bochum.

Cellular proteomes are dynamic and adjusted to permanently changing conditions by ATP-fueled proteolytic machineries. Among the five AAA+ proteases in Escherichia coli FtsH is the only essential and membrane-anchored metalloprotease. FtsH is a homohexamer that uses its ATPase domain to unfold and translocate substrates that are subsequently degraded without the need of ATP in the proteolytic chamber of the protease domain. FtsH eliminates misfolded proteins in the context of general quality control and properly folded proteins for regulatory reasons. Recent trapping approaches have revealed a number of novel FtsH substrates. This review summarizes the substrate diversity of FtsH and presents details on the surprisingly diverse recognition principles of three well-characterized substrates: LpxC, the key enzyme of lipopolysaccharide biosynthesis; RpoH, the alternative heat-shock sigma factor and YfgM, a bifunctional membrane protein implicated in periplasmic chaperone functions and cytoplasmic stress adaptation.
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http://dx.doi.org/10.1515/hsz-2016-0302DOI Listing
May 2017

Modular arrangement of regulatory RNA elements.

RNA Biol 2017 03 23;14(3):287-292. Epub 2016 Dec 23.

a Microbial Biology, Ruhr University Bochum , Bochum , Germany.

Due to their simple architecture and control mechanism, regulatory RNA modules are attractive building blocks in synthetic biology. This is especially true for riboswitches, which are natural ligand-binding regulators of gene expression. The discovery of various tandem riboswitches inspired the design of combined RNA modules with activities not yet found in nature. Riboswitches were placed in tandem or in combination with a ribozyme or temperature-responsive RNA thermometer resulting in new functionalities. Here, we compare natural examples of tandem riboswitches with recently designed artificial RNA regulators suggesting substantial modularity of regulatory RNA elements. Challenges associated with modular RNA design are discussed.
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http://dx.doi.org/10.1080/15476286.2016.1274853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5367253PMC
March 2017

In vivo trapping of FtsH substrates by label-free quantitative proteomics.

Proteomics 2016 12;16(24):3161-3172

Ruhr-Universität Bochum, Lehrstuhl Biologie der Mikroorganismen, Bochum, Germany.

FtsH is the only membrane-bound and essential protease in Escherichia coli. It is responsible for the degradation of regulatory proteins and enzymes such as the heat-shock sigma factor RpoH or LpxC, the key enzyme of lipopolysaccharide biosynthesis. To find new FtsH targets, we trapped substrates in E. coli cells from exponential and stationary growth phase by using a proteolytically inactive FtsH variant. Subsequent analysis of the isolated FtsH-substrate complexes by label-free nanoLC-MS/MS revealed more than 50 putative FtsH substrates, among them five already known substrates. Four out of thirty-seven tested candidates were found to be novel FtsH substrates as shown by in vivo degradation experiments. Six other candidates were degraded by one or more other protease(s). The FtsH substrates SecD and ExbD are involved in transport processes across the membrane, whereas the physiological roles of YlaC and YhbT are yet unknown. The presence of the previously identified YfgM degron in two of the novel substrates suggests general rules for substrate recognition of this unique protease.
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http://dx.doi.org/10.1002/pmic.201600316DOI Listing
December 2016

Temperature-responsive in vitro RNA structurome of Yersinia pseudotuberculosis.

Proc Natl Acad Sci U S A 2016 06 13;113(26):7237-42. Epub 2016 Jun 13.

Department of Microbial Biology, Ruhr University Bochum, 44801 Bochum, Germany;

RNA structures are fundamentally important for RNA function. Dynamic, condition-dependent structural changes are able to modulate gene expression as shown for riboswitches and RNA thermometers. By parallel analysis of RNA structures, we mapped the RNA structurome of Yersinia pseudotuberculosis at three different temperatures. This human pathogen is exquisitely responsive to host body temperature (37 °C), which induces a major metabolic transition. Our analysis profiles the structure of more than 1,750 RNAs at 25 °C, 37 °C, and 42 °C. Average mRNAs tend to be unstructured around the ribosome binding site. We searched for 5'-UTRs that are folded at low temperature and identified novel thermoresponsive RNA structures from diverse gene categories. The regulatory potential of 16 candidates was validated. In summary, we present a dynamic bacterial RNA structurome and find that the expression of virulence-relevant functions in Y. pseudotuberculosis and reprogramming of its metabolism in response to temperature is associated with a restructuring of numerous mRNAs.
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http://dx.doi.org/10.1073/pnas.1523004113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4932938PMC
June 2016

Molybdate uptake by Agrobacterium tumefaciens correlates with the cellular molybdenum cofactor status.

Mol Microbiol 2016 09 10;101(5):809-22. Epub 2016 Jun 10.

Microbial Biology, Ruhr University Bochum, Bochum, Germany.

Many enzymes require the molybdenum cofactor, Moco. Under Mo-limiting conditions, the high-affinity ABC transporter ModABC permits molybdate uptake and Moco biosynthesis in bacteria. Under Mo-replete conditions, Escherichia coli represses modABC transcription by the one-component regulator, ModE, consisting of a DNA-binding and a molybdate-sensing domain. Instead of a full-length ModE protein, many bacteria have a shorter ModE protein, ModE(S) , consisting of a DNA-binding domain only. Here, we asked how such proteins sense the intracellular molybdenum status. We show that the Agrobacterium tumefaciens ModE(S) protein Atu2564 is essential for modABC repression. ModE(S) binds two Mo-boxes in the modA promoter as shown by electrophoretic mobility shift assays. Northern analysis revealed cotranscription of modE(S) with the upstream gene, atu2565, which was dispensable for ModE(S) activity. To identify genes controlling ModE(S) function, we performed transposon mutagenesis. Tn5 insertions resulting in derepressed modA transcription mapped to the atu2565-modE(S) operon and several Moco biosynthesis genes. We conclude that A. tumefaciens ModE(S) activity responds to Moco availability rather than to molybdate concentration directly, as is the case for E. coli ModE. Similar results in Sinorhizobium meliloti suggest that Moco dependence is a common feature of ModE(S) regulators.
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http://dx.doi.org/10.1111/mmi.13421DOI Listing
September 2016