A Natural Plasmid Uniquely Encodes Two Biosynthetic Pathways Creating a Potent Anti-MRSA Antibiotic

Background Understanding how complex antibiotics are synthesised by their producer bacteria is essential for creation of new families of bioactive compounds. Thiomarinols, produced by marine bacteria belonging to the genus Pseudoalteromonas, are hybrids of two independently active species: the pseudomonic acid mixture, mupirocin, which is used clinically against MRSA, and the pyrrothine core of holomycin. Methodology/Principal Findings High throughput DNA sequencing of the complete genome of the producer bacterium revealed a novel 97 kb plasmid, pTML1, consisting almost entirely of two distinct gene clusters. Targeted gene knockouts confirmed the role of these clusters in biosynthesis of the two separate components, pseudomonic acid and the pyrrothine, and identified a putative amide synthetase that joins them together. Feeding mupirocin to a mutant unable to make the endogenous pseudomonic acid created a novel hybrid with the pyrrothine via “mutasynthesis” that allows inhibition of mupirocin-resistant isoleucyl-tRNA synthetase, the mupirocin target. A mutant defective in pyrrothine biosynthesis was also able to incorporate alternative amine substrates. Conclusions/Significance Plasmid pTML1 provides a paradigm for combining independent antibiotic biosynthetic pathways or using mutasynthesis to develop a new family of hybrid derivatives that may extend the effective use of mupirocin against MRSA

Transcriptional control of the H-NS antagonists LeuO and RcsB-BglJ in Escherichia coli

The bacterial nucleoid-associated protein (NAP) H-NS is involved in the organization and compaction of the bacterial chromatin and acts as a global respressor, mainly of genes that have been acquired by horizontal gene transfer and that are related to stress responses and pathogenicity. Binding of H-NS to the DNA and formation of a nucleoprotein complex at promoter regions leads to repression. This repressor effect of H-NS can be antagonized by gene-specific transcription factors (H-NS antagonists) that activate transcription of H NS-repressed genes by competing with H-NS for binding or by disturbing formation of the nucleoprotein complex. Two examples of such H NS antagonists are the LysR-type transcription factor LeuO and the FixJ/NarL-type transcription factor heterodimer RcsB-BglJ. LeuO is a pleiotropic regulator of stress responses and virulence determinants. RcsB-BglJ activates transcription of the H NS-repressed bgl (aryl-β,D-glucoside) operon. In this work, novel targets of RcsB-BglJ were identified in Escherichia coli by microarray analyses. The results suggest that heterodimerization of RcsB and BglJ is essential for regulation. Further, in addition to genes related to unknown or predicted function in the membrane the leuO gene was identified as a target gene. Detailed analysis of transcriptional regulation of leuO demonstrated that RcsB-BglJ strongly activates transcription of leuO by binding proximal to a newly mapped leuO promoter. Thus RcsB-BglJ antagonizes repression of leuO by H-NS and the H-NS-like protein StpA. Additional data presented here show that LeuO negatively autoregulates its own expression and inhibits activation of leuO by RcsB-BglJ. Regulation of leuO by RcsB-BglJ and autoregulation by LeuO, as shown here, as well as activation of bglJ by LeuO, as published previously, indicates a feedback control mechanism of two global transcriptional regulators and H-NS antagonists.This feedback regulation may ensure turn on of their expression in response to specific environmental signals. Screens to search for novel regulators or upstream signals were performed by transposon mutagenesis and by using a genomic expression library. These screens indicate that additional factors may be involved in the regulation of this leuO-bglJ feedback loop

PHLPP1 counter-regulates STAT1-mediated inflammatory signaling.

Inflammation is an essential aspect of innate immunity but also contributes to diverse human diseases. Although much is known about the kinases that control inflammatory signaling, less is known about the opposing phosphatases. Here we report that deletion of the gene encoding PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) protects mice from lethal lipopolysaccharide (LPS) challenge and live Escherichia coli infection. Investigation of PHLPP1 function in macrophages reveals that it controls the magnitude and duration of inflammatory signaling by dephosphorylating the transcription factor STAT1 on Ser727 to inhibit its activity, reduce its promoter residency, and reduce the expression of target genes involved in innate immunity and cytokine signaling. This previously undescribed function of PHLPP1 depends on a bipartite nuclear localization signal in its unique N-terminal extension. Our data support a model in which nuclear PHLPP1 dephosphorylates STAT1 to control the magnitude and duration of inflammatory signaling in macrophages

Molecular mechanisms of transcription initiation—structure, function, and evolution of TFE/TFIIE-like factors and open complex formation

Transcription initiation requires that the promoter DNA is melted and the template strand is loaded into the active site of the RNA polymerase (RNAP), forming the open complex (OC). The archaeal initiation factor TFE and its eukaryotic counterpart TFIIE facilitate this process. Recent structural and biophysical studies have revealed the position of TFE/TFIIE within the pre-initiation complex (PIC) and illuminated its role in OC formation. TFE operates via allosteric and direct mechanisms. Firstly, it interacts with the RNAP and induces the opening of the flexible RNAP clamp domain, concomitant with DNA melting and template loading. Secondly, TFE binds physically to single-stranded DNA in the transcription bubble of the OC and increases its stability. The identification of the β-subunit of archaeal TFE enabled us to reconstruct the evolutionary history of TFE/TFIIE-like factors, which is characterised by winged helix (WH) domain expansion in eukaryotes and loss of metal centres including iron-sulfur clusters and Zinc ribbons. OC formation is an important target for the regulation of transcription in all domains of life. We propose that TFE and the bacterial general transcription factor CarD, although structurally and evolutionary unrelated, show interesting parallels in their mechanism to enhance OC formation. We argue that OC formation is used as a way to regulate transcription in all domains of life, and these regulatory mechanisms coevolved with the basal transcription machinery

Genetic and functional characterization of the gene cluster directing the biosynthesis of putisolvin I and II in Pseudomonas putida strain PCL1445

Pseudomonas putida PCL1445 secretes two cyclic lipopeptides, putisolvin I and putisolvin II, which possess a surface-tension-reducing ability, and are able to inhibit biofilm formation and to break down biofilms of Pseudomonas species including Pseudomonas aeruginosa. The putisolvin synthetase gene cluster (pso) and its surrounding region were isolated, sequenced and characterized. Three genes, termed psoA, psoB and psoC, were identified and shown to be involved in putisolvin biosynthesis. The gene products encode the 12 modules responsible for the binding of the 12 amino acids of the putisolvin peptide moiety. Sequence data indicate that the adenylation domain of the 11th module prioritizes the recognition of Val instead of Leu or Ile and consequently favours putisolvin I production over putisolvin II. Detailed analysis of the thiolation domains suggests that the first nine modules recognize the D form of the amino acid residues while the two following modules recognize the L form and the last module the L or D form, indifferently. The psoR gene, which is located upstream of psoA, shows high similarity to luxR-type regulatory genes and is required for the expression of the pso cluster. In addition, two genes, macA and macB, located downstream of psoC were identified and shown to be involved in putisolvin production or export

Structural and functional studies on the transcriptional regulation of flagellar motility and biofilm formation

Part 1: Numerical regulation in the monotrichous bacterium Shewanella putrefaciens Microorganisms have the ability to adapt to changing environmental conditinos. This has enabled them to colonize virtually nearly every niche on the planet Earth. Key to this ability is bacterial motility, which allows bacteria to move away from unfavourable conditions and to move towards favourable conditions. In connection with a sensory system, which detects chemical cues and other stimuli, bacteria can move towards nutrients. Bacterial motility is largely enabled by flagella. The biogenesis of a flagellum is a very costly process, which is for this reason highly regulated. In the monotrichous bacterium Shewanella putrefaciens, FlhF and FlhG are responsible for maintaining number and location of the single polar flagellum. In the course of this work, it could be shown that FlhG limits the number of flagella to one by directly interacting with the master transcriptional regulator of the flagellum, FlrA. Furthermore, FlhG is implicated in assembly of the cytosolic face of the flagellum, the C-Ring. The transcriptional control via FlrA as well as the C-Ring assembly via FliM occur through the same binding site on FlhG. This highlights the central role of FlhG and shows that FlhG integrates the two processes to regulate flagellar number. Taken together, these observations represent an important step towards a complete conceptual description of flagellar biogenesis. Thereby, these results also form the basis for further research. Part 2: Transcriptional regulation of biofilms is mediated by RemA, which interacts with DNA in a histone-like manner Instead of a motile lifestyle, bacteria can also establish a multicellular, sessile lifestyle in the form of biofilms. In biofilms, bacterial cells establish a division of labour and establish an increased resistance against antibiotics and environmental hazardous conditions. This is mediated by the secretion of extracellular proteins and other biological molecules. The protein RemA is central to this process, as it activates the secretion of these extracellular components. Furthermore, RemA is implicated in processes which enable a cellular protection against osmotic pressure, which occurs during biofilm formation. In the context of this work, the structure of RemA from Geobacillus thermodenitrificans could be elucidated. RemA interacts with DNA in a novel and unique way, which is reminiscent of DNA-looping by histone-complexes. By means of biochemical methods, crucial residues of RemA responsible for DNA interaction could be functionally investigated. Furthermore, the structural fate of amino acid mutations, which impair the functionality of RemA, could be investigated. Taken together, this work represents an important step towards the understanding of the transcriptional processes that govern biofilm-formation and osmoprotection in Bacillus subtilis. This work also provides the basis to further investigate the function of RemA in the cellular context. In the future, the structural investigation of RemA-DNA-interaction is facilitated by the insights obtained in the context of this work. Part 3: Membrane protein biogenesis is regulated by a structurally unique, co-translational state of FtsY. Membrane proteins are translated by ribosomes and predominantly inserted into the membrane by the SecYEG-translocon. A factor critical for this process is the SRP-receptor FtsY, which enables co-translational targeting to the translocon in coopration with the SRP-particle FFH and SRP-RNA. In the context of this work it could be shown that a co-translational state of FtsY, the helical domain N2-4, critically mediates membrane targeting of the receptor. By means of crystallographic analyses and studies in solution, it could be shown that the subdomain of N2-4 possesses a different fold when isolated than in the context of the G-domain of FtsY. This observation represents a unique paradigm, which indicates that nascent N2-4 executes a different function during its own translation than when N2-4 is part of the mature FtsY-receptor. These results are an important step towards the conceptual understanding of membrane protein biogenesis and –targeting. Further work could elucidate, whether this concept also applies to homologs of FtsY such as FlhF

A common variant associated with dyslexia reduces expression of the KIAA0319 gene

This work was supported by the Wellcome Trust (MYD, SP, TSS, JCK, RWM, PC, SB, and APM), the Intramural Research Programs of the National Human Genome Research Institute (MYD and EDG) and National Cancer Institute (MPO), and the NIH/Ox-Cam Graduate Partnership Program (MYD).Numerous genetic association studies have implicated the KIAA0319 gene on human chromosome 6p22 in dyslexia susceptibility. The causative variant(s) remains unknown but may modulate gene expression, given that (1) a dyslexia-associated haplotype has been implicated in the reduced expression of KIAA0319, and (2) the strongest association has been found for the region spanning exon 1 of KIAA0319. Here, we test the hypothesis that variant(s) responsible for reduced KIAA0319 expression resides on the risk haplotype close to the gene's transcription start site. We identified seven single-nucleotide polymorphisms on the risk haplotype immediately upstream of KIAA0319 and determined that three of these are strongly associated with multiple reading-related traits. Using luciferase-expressing constructs containing the KIAA0319 upstream region, we characterized the minimal promoter and additional putative transcriptional regulator regions. This revealed that the minor allele of rs9461045, which shows the strongest association with dyslexia in our sample (max p-value = 0.0001), confers reduced luciferase expression in both neuronal and non-neuronal cell lines. Additionally, we found that the presence of this rs9461045 dyslexia-associated allele creates a nuclear protein-binding site, likely for the transcriptional silencer OCT-1. Knocking down OCT-1 expression in the neuronal cell line SHSY5Y using an siRNA restores KIAA0319 expression from the risk haplotype to nearly that seen from the non-risk haplotype. Our study thus pinpoints a common variant as altering the function of a dyslexia candidate gene and provides an illustrative example of the strategic approach needed to dissect the molecular basis of complex genetic traits.PostprintPeer reviewe

LOCAL AND GLOBAL GENE REGULATION ANALYSIS OF THE AUTOINDUCER-2 MEDIATED QUORUM SENSING MECHANISM IN ESCHERICHIA COLI

The term `quorum sensing' (QS) is used to define a population density based communication mechanism which uses chemical signal molecules called autoinducers to trigger unique and varied changes in gene expression. Although several communication methods have been identified in bacteria that are unique to a particular species, one type of signal molecule, autoinducer-2 (AI-2) is linked to interspecies communication, indicating its potential as a universal signal for cueing a QS response among multiple bacterial types. In E. coli, AI-2 acts as an effector by binding to the QS repressor LsrR. As a result, LsrR unbinds and relieves repression of the lsr regulon, stimulating a subsequent QS gene expression cascade. In this dissertation, LsrR structure and in vitro binding activity are examined. Genomic binding and DNA microarray analyses are conducted and three novel sites putatively regulated by LsrR, yegE-udk, mppA and yihF, are revealed. Two cAMP receptor protein (CRP) binding locations in intergenic region of the lsr regulon are also confirmed. The role of each CRP site in divergent expression is qualified, indicating the lsr intergenic region to be a class III CRP-dependent promoter. Also, four specific DNA binding sites for LsrR in the lsr intergenic region are proposed, and reliance upon simultaneous binding to these various sites and the resulting effects on LsrR repression is presented. Finally, a complex model for regulation of the lsr regulon is depicted incorporating LsrR, CRP, DNA looping, and a predicted secondary layer of repression by an integration host factor (IHF)-like protein. Further understanding of this QS genetic mechanism may potentially be used for inhibiting bacterial proliferation and infection, modifying the natural genetic system to elicit alternate desired responses, or extracted and applied to a highly customizable and sensitive in vitro biosensor

Genetic engineering and characterization of LysR-type transcriptional regulators

Thesis (Ph.D.) University of Alaska Fairbanks, 2000This thesis describes research aimed at understanding the structure and function of LysR-type transcriptional regulators. I studied two LysR-type proteins. One from the archaeon Methanococcus jannaschii, MJ-LysR. The other is from Burkholderia cepacia, DgdR. The MJ-LysR is the first putative LysR-type transcriptional regulator found in archaea. It is surprising that a prokaryotic transcriptional regulator is present in archaea, whose basal transcription machinery and RNA polymerase are more closely related to those of eukaryotes. To elucidate the structure and function of M-LysR protein, the gene was subcloned and expressed in E. coli. The gene product was isolated and purified by heat treatment and size exclusion chromatography. An in vitro binding assay showed that the purified protein bound to the intergenic region between the lysR gene and its upstream gene specifically and selectively. The results also showed that the protein maintained its binding activity even at 94C̊. The DNA footprinting data demonstrated a 30 bp protected region. Thus, this protein probably regulates expression of its own structural gene and perhaps the adjacent upstream gene. DgdR protein from Burkholderia cepacia had been previously characterized. The previous study showed that 2-methylalanine, the inducer for the DgdR regulated dgdA gene expression, but not D or L-alanine induced the conformational changes on DNA-protein complex. To further confirm this result, eleven amino acids with structures similar to 2-methylalainine were tested for their ability on affecting the binding of the DgdR protein to its operator site. Among these amino acids tested, only 2-methylalanine, 1-aminocyclopentane-1-carboxylic acid, S-2-aminobutanoic acid, RS-isovaline, and 2-trifluoromethyl-2-aminobutanoic acid generated the measurable band shifting. D- or L-norvaline, 2,2-diethyl glycine, and 2-trifluoromethylalanine did not cause any measurable change. It was concluded that both alkyl side chain size and hydrophobicity are important for the inducer recognition and binding in this protein. To solve the problem in DgdR protein purification caused by low solubility of this protein, a dgdR fusion gene to malE gene was constructed. This fusion gene provides a useful tool to further study and crystallize the DgdR protein