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Berlin Brandenburg

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  • 1
    In: mBio, 2018, Vol.9(6)
    Description: The alphaproteobacterium Agrobacterium tumefaciens is able to infect various eudicots causing crown gall tumor formation. Based on its unique ability of interkingdom gene transfer, Agrobacterium 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 A. tumefaciens 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 ampC 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. ABSTRACT 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 Agrobacterium tumefaciens . 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. Agrobacterium 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 ampC . Structure probing revealed a spoon-like structure of the sRNA, with a single-stranded loop that is engaged in target interaction in vivo and in vitro . 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.
    Keywords: Research Article ; Molecular Biology And Physiology ; Antibiotic Resistance ; Gene Regulation ; Plant-Microbe Interaction ; Posttranscriptional Control ; Regulatory Rna
    ISSN: 21612129
    E-ISSN: 2150-7511
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  • 2
    In: mBio, 2018, Vol.9(6)
    Description: P. aeruginosa is a soil dwelling bacterium and a plant pathogen, and it also causes life-threatening infections in humans. Thus, P. aeruginosa thrives in diverse environments and over a broad range of temperatures. Some P. aeruginosa strains rely on the CRISPR-Cas adaptive immune system as a phage defense mechanism. Our discovery that low temperatures increase CRISPR adaptation suggests that the rarely occurring but crucial naive adaptation events may take place predominantly under conditions of slow growth, e.g., during the bacterium’s soil dwelling existence and during slow growth in biofilms. ABSTRACT Clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) systems are adaptive defense systems that protect bacteria and archaea from invading genetic elements. In Pseudomonas aeruginosa , quorum sensing (QS) induces the CRISPR-Cas defense system at high cell density when the risk of bacteriophage infection is high. Here, we show that another cue, temperature, modulates P. aeruginosa CRISPR-Cas. Increased CRISPR adaptation occurs at environmental (i.e., low) temperatures compared to that at body (i.e., high) temperature. This increase is a consequence of the accumulation of CRISPR-Cas complexes, coupled with reduced P. aeruginosa growth rate at the lower temperature, the latter of which provides additional time prior to cell division for CRISPR-Cas to patrol the cell and successfully eliminate and/or acquire immunity to foreign DNA. Analyses of a QS mutant and synthetic QS compounds show that the QS and temperature cues act synergistically. The diversity and level of phage encountered by P. aeruginosa in the environment exceed that in the human body, presumably warranting increased reliance on CRISPR-Cas at environmental temperatures.
    Keywords: Research Article ; Molecular Biology And Physiology ; Crispr ; Phage ; Pseudomonas ; Quorum Sensing ; Growth Rate
    ISSN: 21612129
    E-ISSN: 2150-7511
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  • 3
    In: mBio, 2017, Vol.8(4)
    Description: ABSTRACT L7Ae is a universal archaeal protein that recognizes and stabilizes kink-turn (k-turn) motifs in RNA substrates. These structural motifs are widespread in nature and are found in many functional RNA species, including ribosomal RNAs. Synthetic biology approaches utilize L7Ae/k-turn interactions to control gene expression in eukaryotes. Here, we present results of comprehensive RNA immunoprecipitation sequencing (RIP-Seq) analysis of genomically tagged L7Ae from the hyperthermophilic archaeon Sulfolobus acidocaldarius . A large set of interacting noncoding RNAs was identified. In addition, several mRNAs, including the l7ae transcript, were found to contain k-turn motifs that facilitate L7Ae binding. In vivo studies showed that L7Ae autoregulates the translation of its mRNA by binding to a k-turn motif present in the 5′ untranslated region (UTR). A green fluorescent protein (GFP) reporter system was established in Escherichia coli and verified conservation of L7Ae-mediated feedback regulation in Archaea . Mobility shift assays confirmed binding to a k-turn in the transcript of nop5-fibrillarin , suggesting that the expression of all C/D box sRNP core proteins is regulated by L7Ae. These studies revealed that L7Ae-mediated gene regulation evolved in archaeal organisms, generating new tools for the modulation of synthetic gene circuits in bacteria. IMPORTANCE L7Ae is an essential archaeal protein that is known to structure ribosomal RNAs and small RNAs (sRNAs) by binding to their kink-turn motifs. Here, we utilized RIP-Seq methodology to achieve a first global analysis of RNA substrates for L7Ae. Several novel interactions with noncoding RNA molecules (e.g., with the universal signal recognition particle RNA) were discovered. In addition, L7Ae was found to bind to mRNAs, including its own transcript’s 5′ untranslated region. This feedback-loop control is conserved in most archaea and was incorporated into a reporter system that was utilized to control gene expression in bacteria. These results demonstrate that L7Ae-mediated gene regulation evolved originally in archaeal organisms. The feedback-controlled reporter gene system can easily be adapted for synthetic biology approaches that require strict gene expression control.
    Keywords: Research Article ; Archaea ; Rna Binding Proteins ; Rna Structure ; Gene Regulation
    E-ISSN: 2150-7511
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  • 4
    Language: English
    In: mBio, 01 December 2017, Vol.8(6), p.e01964-17
    Description: Bacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drives Escherichia coli persister formation through protease Lon and activation of toxin-antitoxin (TA) modules. This model found considerable support among researchers studying persisters but also generated controversy as part of recent debates in the field. In this study, we therefore used our previous work as a model to critically examine common experimental procedures to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variations in culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection of mutant strains with bacteriophage ϕ80, a notorious laboratory contaminant, apparently caused several of the phenotypes that we reported in our previous studies. We therefore reconstructed all infected mutants and probed the validity of our model of persister formation in a refined assay setup that uses robust culture conditions and unravels the dynamics of persister cells through all bacterial growth stages. Our results confirm the importance of (p)ppGpp and Lon but no longer support a role of TA modules in E. coli persister formation under unstressed conditions. We anticipate that the results and approaches reported in our study will lay the ground for future work in the field.
    Keywords: Biology
    E-ISSN: 2150-7511
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  • 5
    Language: English
    In: mBio, 08/29/2014, Vol.5(4)
    Description: Transcription start sites (TSSs) lying inside annotated genes, on the same or opposite strand, have been observed in diverse bacteria, but the function of these unexpected transcripts is unclear. Here, we use the metal-reducing bacterium Shewanella oneidensis MR-1 and its relatives to study the evolutionary conservation of unexpected TSSs. Using high-resolution tiling microarrays and 5'-end RNA sequencing, we identified 2,531 TSSs in S. oneidensis MR-1, of which 18% were located inside coding sequences (CDSs). Comparative transcriptome analysis with seven additional Shewanella species revealed that the majority (76%) of the TSSs within the upstream regions of annotated genes (gTSSs) were conserved. Thirty percent of the TSSs that were inside genes and on the sense strand (iTSSs) were also conserved. Sequence analysis around these iTSSs showed conserved promoter motifs, suggesting that many iTSS are under purifying selection. Furthermore, conserved iTSSs are enriched for regulatory motifs, suggesting that they are regulated, and they tend to eliminate polar effects, which confirms that they are functional. In contrast, the transcription of antisense TSSs located inside CDSs (aTSSs) was significantly less likely to be conserved (22%). However, aTSSs whose transcription was conserved often have conserved promoter motifs and drive the expression of nearby genes. Overall, our findings demonstrate that some internal TSSs are conserved and drive protein expression despite their unusual locations, but the majority are not conserved and may reflect noisy initiation of transcription rather than a biological function. Importance: The first step of gene expression is the initiation of transcription from promoters, which have been traditionally thought to be located upstream of genes. Recently, studies showed that in diverse bacteria, promoters are often located inside genes. It has not been clear if these unexpected promoters are important to the organism or if they result from transcriptional noise. Here, we identify and examine promoters in eight related bacterial species. Promoters that lie within genes on the sense strand are often conserved as locations and in their sequences. Furthermore, these promoters often affect the bacterium's growth. Thus, many of these unexpected promoters are likely functional. Fewer promoters that lie within genes on the antisense strand are conserved, but the conserved ones seem to drive the expression of nearby genes.
    Keywords: Biology;
    ISSN: mBio
    E-ISSN: 2150-7511
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  • 6
    Language: English
    In: mBio, 04/30/2019, Vol.10(2)
    Description: Proteins comprised of 50 or fewer amino acids have been shown to interact with and modulate the functions of larger proteins in a range of organisms. Despite the possible importance of small proteins, the true prevalence and capabilities of these regulators remain unknown as the small size of the proteins places serious limitations on their identification, purification, and characterization. Here, we present a ribosome profiling approach with stalled initiation complexes that led to the identification of 38 new small proteins.Small proteins consisting of 50 or fewer amino acids have been identified as regulators of larger proteins in bacteria and eukaryotes. Despite the importance of these molecules, the total number of small proteins remains unknown because conventional annotation pipelines usually exclude small open reading frames (smORFs). We previously identified several dozen small proteins in the model organism Escherichia coli using theoretical bioinformatic approaches based on sequence conservation and matches to canonical ribosome binding sites. Here, we present an empirical approach for discovering new proteins, taking advantage of recent advances in ribosome profiling in which antibiotics are used to trap newly initiated 70S ribosomes at start codons. This approach led to the identification of many novel initiation sites in intergenic regions in E. coli. We tagged 41 smORFs on the chromosome and detected protein synthesis for all but three. Not only are the corresponding genes intergenic but they are also found antisense to other genes, in operons, and overlapping other open reading frames (ORFs), some impacting the translation of larger downstream genes. These results demonstrate the utility of this method for identifying new genes, regardless of their genomic context.
    Keywords: Ribo-Seq ; Small Protein ; Alternate Orfs ; Antisense ; Genome Annotation ; Leader Peptide ; Biology;
    ISSN: mBio
    ISSN: 21612129
    E-ISSN: 2150-7511
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  • 7
    Language: English
    In: mBio, 04/30/2019, Vol.10(2)
    Description: The ability of bacteria to sense and respond to environmental signals is critical for survival. Bacteria use cyclic dinucleotides as second messengers to regulate a number of physiological processes, such as the fundamental life style transition between motility and sessility (biofilm formation). cGAMP, which is synthesized by a dinucleotide cyclase called DncV, is a newly discovered second messenger involved in virulence and chemotaxis in the Vibrio cholerae biovar El Tor causing the current 7th cholera pandemic. However, to what extent cGAMP exists and participates in physiological processes in other bacteria is still unknown. In this study, we found an elevated cGAMP level to possibly regulate biofilm formation and motility in the animal commensal E. coli strain ECOR31. Thus, we detected a novel role for cGAMP signaling in regulation of physiological processes other than those previously reported in proteobacterial species.Cyclic dinucleotides (cDNs) act as intracellular second messengers, modulating bacterial physiology to regulate the fundamental life style transition between motility and sessility commonly known as biofilm formation. Cyclic GMP-AMP (cGAMP), synthesized by the dinucleotide cyclase DncV, is a newly discovered cDN second messenger involved in virulence and chemotaxis in Vibrio cholerae O1 biovar El Tor. Here we report a novel role for horizontally transferred DncV in cGAMP production and regulation of biofilm formation and motility in the animal commensal strain Escherichia coli ECOR31. ECOR31 expresses a semiconstitutive temperature-independent rdar (red, dry, and rough) morphotype on Congo red agar plates characterized by the extracellular matrix components cellulose and curli fimbriae which requires activation by the major biofilm regulator CsgD and cyclic di-GMP signaling. In contrast, C-terminal His-tagged DncV negatively regulates the rdar biofilm morphotype and cell aggregation via downregulation of csgD mRNA steady-state level. Furthermore, DncV sequentially promotes and inhibits adhesion to the abiotic surface after 24 h and 48 h of growth, respectively. DncV also suppresses swimming and swarming motility posttranscriptional of the class 1 flagellum regulon gene flhD. Purified DncV produced different cDNs, cyclic di-GMP, cyclic di-AMP, an unknown product(s), and the dominant species 3′3′-cGAMP. In vivo, only the 3′3′-cGAMP concentration was elevated upon short-term overexpression of dncV, making this work a first report on cGAMP production in E. coli. Regulation of rdar biofilm formation and motility upon overexpression of untagged DncV in combination with three adjacent cotransferred gene products suggests a novel temperature-dependent cGAMP signaling module in E. coli ECOR31.
    Keywords: Csgd ; Dncv ; Biofilm Formation ; Cgamp ; Cyclic Di-Gmp ; Motility ; Rdar ; Biology;
    ISSN: mBio
    ISSN: 21612129
    E-ISSN: 2150-7511
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  • 8
    In: mBio, 2019, Vol.10(3)
    Description: Dual RNA sequencing (RNA-seq) offers the promise of determining an interactome at a transcriptional level between a bacterium and the host but has yet to be done on any bacterial infection in human tissue. We performed dual RNA-seq and metabolomics analyses on wounded and infected sites following experimental infection of the arm with H. ducreyi . Our results suggest that H. ducreyi survives in an abscess by utilizing l -ascorbate as an alternative carbon source, possibly taking advantage of host ascorbic acid recycling, and that H. ducreyi also adapts by upregulating genes involved in anaerobic metabolism and inorganic ion and nutrient transport. To our knowledge, this is the first description of an interaction network between a bacterium and the human host at a site of infection. ABSTRACT A major gap in understanding infectious diseases is the lack of information about molecular interaction networks between pathogens and the human host. Haemophilus ducreyi causes the genital ulcer disease chancroid in adults and is a leading cause of cutaneous ulcers in children in the tropics. We developed a model in which human volunteers are infected on the upper arm with H. ducreyi until they develop pustules. To define the H. ducreyi and human interactome, we determined bacterial and host transcriptomic and host metabolomic changes in pustules. We found that in vivo H. ducreyi transcripts were distinct from those in the inocula, as were host transcripts in pustule and wounded control sites. Many of the upregulated H. ducreyi genes were found to be involved in ascorbic acid and anaerobic metabolism and inorganic ion/nutrient transport. The top 20 significantly expressed human pathways showed that all were involved in immune responses. We generated a bipartite network for interactions between host and bacterial gene transcription; multiple positively correlated networks contained H. ducreyi genes involved in anaerobic metabolism and host genes involved with the immune response. Metabolomic studies showed that pustule and wounded samples had different metabolite compositions; the top ion pathway involved ascorbate and aldarate metabolism, which correlated with the H. ducreyi transcriptional response and upregulation of host genes involved in ascorbic acid recycling. These data show that an interactome exists between H. ducreyi and the human host and suggest that H. ducreyi exploits the metabolic niche created by the host immune response.
    Keywords: Research Article ; Host-Microbe Biology ; Dual Rna-Seq ; Haemophilus Ducreyi ; Human Infection Model ; Interactome ; Metabolome
    ISSN: 21612129
    E-ISSN: 2150-7511
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