of different types of functional RNAs, the definition of the polymerase; sRNA, small regulatory RNA; TEC, transcription elongation complex. .. tein H-NS and transcription factor NusG were shown to .. polymerase: a love-hate relationship ?. Bacterial RNA polymerase (RNAP) consists of four core subunits (2α, β, β′) . and a nucleotide rna1 transcript from the origin of plasmid replication. of H ‐NS and (ii) the absence of alternating DNase I‐sensitive sites at David C Grainger, H-NS and RNA polymerase: a love–hate relationship?. We found that the presence of the DNA binding protein H-NS, which preferentially .. Further studies are warranted to precisely define the molecular factors that govern . H-NS and RNA polymerase: a love-hate relationship?.
Observation Transposon insertion sequencing TIS is a powerful tool purported to enable the unbiased and comprehensive identification of genetic loci required for bacterial fitness 12. TIS employs deep sequencing of transposon insertion sites within a complex population of insertion mutants in order to determine the frequency with which genetic loci are disrupted.
P1 Ref Endonuclease: A Molecular Mechanism for Phage-Enhanced Antibiotic Lethality - Dimensions
Subsequent genome-wide statistical analysis of relative insertion frequency enables identification of loci underrepresented for transposon insertion 3. Transposon insertion typically confers a loss-of-function phenotype; consequently, loci with a low frequency of transposon insertion are often presumed to contribute to bacterial fitness under the conditions assayed.
Reliable gene classification has been bolstered by extensive optimization of TIS analysis methodologies, which have all but eliminated the technical artifacts that bias the detection of insertion frequency 3. However, biological factors that may alter the frequency of insertion, irrespective of the contributions of the loci to bacterial fitness, remain largely unexplored. Many TIS studies have employed Mariner-based transposons, which insert exclusively at TA dinucleotides without additional sequence site restrictions 34.
For example, several such screens were performed to identify loci that promote in vitro growth of Vibrio cholerae, the cause of the diarrheal disease cholera, as well as to identify loci required by the pathogen for intestinal colonization of infant rabbits, a model host 5— 8.
- Structural insights into the regulation of foreign genes in Salmonella by the Hha/H-NS complex.
- The Nucleoid Binding Protein H-NS Biases Genome-Wide Transposon Insertion Landscapes
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However, these genes included several well-characterized virulence loci that are dispensable for in vitro fitness, including the cholera toxin-encoding genes ctxAB and the tcp operon that enables production of a colonization-linked pilus.
These findings suggest that the frequency of insertion at a locus may reflect factors in addition to the fitness of corresponding insertion mutants; however, the nature of such factors has not been defined.
Notably, the underrepresented virulence-associated loci were acquired via horizontal gene transfer, and, like many horizontally acquired sequences, they have GC content markedly lower than that of the ancestral chromosomes into which they have integrated 10 In many bacterial species, horizontally acquired sequences are bound by the histone-like nucleoid structuring H-NS protein 12which preferentially associates with AT-rich sequences through recognition of structural features of the minor groove of the DNA helix H-NS can oligomerize along, or form cross-bridges between, AT-rich regions, thereby producing filamentous nucleoprotein complexes and stabilizing DNA hairpins H-NS binding is associated with transcriptional silencing, due both to reduced access of transcription machinery to H-NS-bound promoters and to Rho-dependent transcriptional termination resulting from increased transcriptional pausing in bound regions H-NS-mediated repression is overcome by the activity of transcription factors and other DNA binding proteins that compete with H-NS for target sites For example, in V.
H-NS contributes to the regulation of diverse processes, and mutants lacking H-NS typically display attenuated growth, which in some cases has been specifically linked to overexpression of horizontally acquired sequences The known association between H-NS and many horizontally acquired sequences prompted us to consider the possibility that H-NS binding might account for the dearth of insertion mutants recovered for these loci.
Previous studies have linked H-NS to transposition; for example, H-NS promotes the activity of Tn10 through interactions with transpososome proteins Additionally, H-NS is thought to modify IS target site selection, although this conclusion is based on an analysis of relatively few insertion sites conducted prior to the availability of high-throughput sequencing Regulation of transcription of the leuO gene is complex.
Mutual activation of leuO and bglJ resembles a double-positive feedback network, which theoretically can result in bi-stability and heterogeneity, or be maintained in a stable OFF or ON states by an additional signal.
H-NS and RNA polymerase: a love-hate relationship?
Here we performed quantitative and single-cell expression analyses to address the antagonistic regulation and feedback control of leuO transcription by BglJ-RcsB and LeuO using a leuO promoter mVenus reporter fusion and finely tunable bglJ and leuO expression plasmids.
The data revealed uniform regulation of leuO expression in the population that correlates with the relative cellular concentration of BglJ and LeuO. The data are in agreement with a straightforward model of antagonistic regulation of leuO expression by the two regulators, LeuO and BglJ-RcsB, by independent mechanisms. Further, the data suggest that at standard laboratory growth conditions feedback regulation of leuO is of minor relevance and that silencing of leuO and bglJ by H-NS and StpA keeps these loci in the OFF state.
Introduction LeuO is a conserved and pleiotropic LysR-type transcription factor that has been best characterized in Escherichia coli and Salmonella enterica. LeuO functions both as activator and as repressor, and is presumably a tetramer, similar to other LysR-type regulators Maddocks and Oyston, ; Guadarrama et al. The biological role of LeuO is pleiotropic. LeuO is relevant for pathogenicity in S.
In accordance with the pleiotropic role of LeuO, transcription of leuO is tightly controlled. Moderate upregulation of leuO expression was observed in stationary phase and under amino acid starvation Fang and Wu, ; Fang et al.
In addition, positive autoregulation by LeuO and transcriptional coupling of leuO expression to expression of neighboring genes by DNA supercoiling has been reported Fang and Wu, ; Chen et al.
Thus, LeuO is also a negative autoregulator Stratmann et al. BglJ-RcsB is a heterodimer that activates transcription of various loci in E. BglJ-RcsB consists of RcsB, the response regulator of the Rcs two-component phosphorelay system Majdalani and Gottesman,and BglJ, which has initially been found as an activator of the bgl operon Giel et al. A Regulation of leuO by interlocked double-positive and negative feedback loops.
Mutual positive regulation represents a double-positive feedback loop. To monitor leuO transcription a PleuO mVenus fusion was constructed by replacement of the native leuO gene with mVenus.
Expression of bglJ and leuO, respectively, was induced with gradually increasing concentrations of the inducers arabinose and IPTG, respectively. This double-positive feedback loop is interlocked with a negative feedback loop which is based on negative autoregulation by LeuO Figure 1. Such a network motif can function like a switch that is stable both in the OFF as well as in the ON state.
Often an external signal locks such feedback loops in one state. Further, bi-stability resulting in population heterogeneity and oscillation can be based on interlocked positive and negative feedback loops Angeli et al. In this study we addressed the antagonistic regulation of leuO transcription by BglJ-RcsB and LeuO, which is presumably a crucial element in the complex control of leuO expression. For quantitative and single-cell expression analysis, we established a reporter fusion of the leuO promoter region PleuO to mVenus and expressed bglJ and leuO in trans using tightly controlled and gradually inducible plasmidic expression systems.
Expression analyses of the PleuO mVenus reporter at steady state growth conditions revealed uniform expression. The data are in agreement with a straightforward model of antagonistic regulation by the two regulators that act independently of each other. To address regulation of leuO transcription that is directed by at least two promoters PleuO in dependence of the concentrations of BglJ and LeuO, a suitable experimental system was established.
Second, BglJ and LeuO were ectopically expressed from two different sets of plasmids. The genes encoding the AraC and the LacI regulators, respectively, are also carried on these plasmids. Likewise, the arabinose regulon was modified to ensure a gradual induction of the PBAD promoter with arabinose, as described before Khlebnikov et al.
Briefly, the PBAD promoter is known to have a stochastic behavior when induced with arabinose. This stochastic behavior is caused by the araE and araFGH genes encoding the arabinose transporters, because induction of the transporter genes by arabinose leads to a higher arabinose uptake and thus positive feedback Siegele and Hu, ; Megerle et al.
In addition, a negative feedback caused by fermentation of intracellular arabinose through the AraBAD enzymes leads to a non-gradual induction Siegele and Hu, Further, the low affinity arabinose transporter araE was put under the control of constitutive promoter Pcp8, as described Khlebnikov et al.
Using this strain the expression level of PleuO mVenus was measured by flow-cytometry to quantify the cellular fluorescence in the population. Further, to ensure steady state conditions, cultures were grown in nutrient-poor tryptone medium. In this medium cultures that were inoculated from fresh overnight cultures to OD of 0.
Expression of bglJ was either not induced or induced by gradually increasing inducer concentrations. Activation of leuO transcription by BglJ. Expression was analyzed after 5 h of growth in tryptone medium without and with indicated inducer concentrations at an optical density OD of approximately 0. Yellow fluorescence X-axis is given in arbitrary units and the Y-axis gives the number of cells that were counted.
The median of the fluorescence intensity is given in the upper right corner of the graph. B Plot of the median fluorescence values that are shown in C solid line with filled dots and D solid line with filled squares PleuO mVenus and dashed line with open squares PleuO leuO:: The arabinose concentration used for induction of bglJ expression is given underneath the panels. Shown are representative data. Flow cytometry revealed a slight increase in PleuO mVenus expression at low levels of induction of plasmidic leuO Figure 3.