Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity

<p>Abstract</p> <p>Background</p> <p>Lysogenic mode of life cycle of a temperate bacteriophage is generally maintained by a protein called 'repressor'. Repressor proteins of temperate lambdoid phages bind to a few symmetric operator DNAs in order to regulate their gene expression. In contrast, repressor molecules of temperate mycobacteriophages and some other phages bind to multiple asymmetric operator DNAs. Very little is known at present about the structure-function relationship of any mycobacteriophage repressor.</p> <p>Results</p> <p>Using highly purified repressor (CI) of temperate mycobacteriophage L1, we have demonstrated here that L1 CI harbors an N-terminal domain (NTD) and a C-terminal domain (CTD) which are separated by a small hinge region. Interestingly, CTD is more compact than NTD at 25°C. Both CTD and CI contain significant amount of α-helix at 30°C but unfold partly at 42°C. At nearly 200 nM concentration, both proteins form appreciable amount of dimers in solution. Additional studies reveal that CI binds to <it>O</it>₆₄and <it>O</it>_{<it>L </it>}types of asymmetric operators of L1 with variable affinity at 25°C. Interestingly, repressor – operator interaction is affected drastically at 42°C. The conformational change of CI is most possibly responsible for its reduced operator binding affinity at 42°C.</p> <p>Conclusion</p> <p>Repressors encoded by mycobacteriophages differ significantly from the repressor proteins of λ and related phages at functional level but at structural level they are nearly similar.</p

Activation of the SMU.1882 Transcription by CovR in Streptococcus mutans

In Streptococcus mutans, the global response regulator CovR plays an important role in biofilm formation, stress-tolerance response, and caries production. We have previously shown that CovR acts as a transcriptional repressor by binding to the upstream promoter regions of its target genes. Here, we report that in vivo, CovR activates the transcription of SMU.1882, which encodes a small peptide containing a double-glycine motif. We also show that SMU.1882 is transcriptionally linked to comA that encodes a putative ABC transporter protein. Several genes from man gene clusters that encode mannose phosphotranferase system flank SMU.1882 -comA genes. Genomic comparison with other streptococci indicates that SMU.1882 is uniquely present in S. mutans, while the man operon is conserved among all streptococci, suggesting that a genetic rearrangement might have taken place at this locus. With the use of a transcriptional reporter system and semi-quantitative RT-PCR, we demonstrated the transcriptional regulation of SMU.1882 by CovR. In vitro gel shift and DNase I foot-printing analyses with purified CovR suggest that CovR binds to a large region surrounding the -10 region of the P1882. Using this information and comparing with other CovR regulated promoters, we have developed a putative consensus binding sequence for CovR. Although CovR binds to P1882, in vitro experiments using purified S. mutans RpoD, E. coli RNA polymerase, and CovR did not activate transcription from this promoter. Thus, we speculate that in vivo, CovR may interfere with the binding of a repressor or requires a cofactor

TLR2 and Nod2 Mediate Resistance or Susceptibility to Fatal Intracellular Ehrlichia Infection in Murine Models of Ehrlichiosis

Our murine models of human monocytic ehrlichiosis (HME) have shown that severe and fatal ehrlichiosis is due to generation of pathogenic T cell responses causing immunopathology and multi-organ failure. However, the early events in the liver, the main site of infection, are not well understood. In this study, we examined the liver transcriptome during the course of lethal and nonlethal infections caused by Ixodes ovatus Ehrlichia and Ehrlichia muris, respectively. On day 3 post-infection (p.i.), although most host genes were down regulated in the two groups of infected mice compared to naïve counterparts, lethal infection induced significantly higher expression of caspase 1, caspase 4, nucleotide binding oligomerization domain-containing proteins (Nod1), tumor necrosis factor-alpha, interleukin 10, and CCL7 compared to nonlethal infection. On day 7 p.i., lethal infection induced highly significant upregulation of type-1 interferon, several inflammatory cytokines and chemokines, which was associated with increased expression levels of Toll-like receptor-2 (TLR2), Nod2, MyD88, nuclear factor-kappa B (NF-kB), Caspase 4, NLRP1, NLRP12, Pycard, and IL-1β, suggesting enhanced TLR signals and inflammasomes activation. We next evaluated the participation of TLR2 and Nod2 in the host response during lethal Ehrlichia infection. Although lack of TLR2 impaired bacterial elimination and increased tissue necrosis, Nod2 deficiency attenuated pathology and enhanced bacterial clearance, which correlated with increased interferon-γ and interleukin-10 levels and a decreased frequency of pathogenic CD8+ T cells in response to lethal infection. Thus, these data indicate that Nod2, but not TLR2, contributes to susceptibility to severe Ehrlichia-induced shock. Together, our studies provide, for the first time, insight into the diversity of host factors and novel molecular pathogenic mechanisms that may contribute to severe HME. © 2013 Chattoraj et al

Transcriptional analysis of SMU.1882 locus.

<p>(A) Schematic diagram of SMU.1882 locus. Open reading frames (orf) are designated by block arrows, and their orientations indicate the direction of transcription. The sequence upstream of SMU.1882 orf is shown below the diagram. (B) Stability of SMU.1882 transcript. The stability of SMU.1882 transcript was measured using cultures at mid-exponential growth phase (70 KU). RNA was extracted from <i>S. mutans</i> wild type strain UA159 at the time (in minutes) indicated above the lanes following addition of rifampin to block <i>de novo</i> transcription. The mRNA decay curve was plotted as a function of time (in minutes). (C) Northern blotting was carried out using the SMU.1882 orf as a probe. A major band of 1.5 kb and two minor bands (3.0 kb and 6.0 kb) were obtained. (D) Transcriptional linkage analysis. Transcription linkage between SMU.1882/<i>comA</i>, and SMU.1884/SMU.1882 was analyzed using the primer pair 1+2 and 3+4, respectively. K-DNA denotes genomic DNA. (E) Amino acid sequence encoded by SMU.1882. The conserved GG-motif, the putative site for the signal peptide cleavage, is underlined.</p

CovR is required for SMU.1882 expression.

<p>(A). Semi-quantitative RT-PCR analysis of SMU.1882, SMU.1883, and <i>gyrA</i> for the strains UA159, Δ<i>covR</i> (IBS603) and Δ<i>covR</i> complemented with pIB30 (IBS603/pIB30). The <i>gyrA</i> gene was included as an internal control to ensure that equal amounts of RNA were loaded per lane. Experiments were repeated at least twice with two independent RNA isolations. (B) Quantitative real-time PCR analysis of SMU.1882 transcript in wild type, Δ<i>covR</i> (IBS603) and Δ<i>covR</i> complemented with pIB30 (IBS603/pIB30). The expression of SMU.1882 was normalized to the expression level of <i>gyrA</i> as an internal reference. Relative fold changes in gene expression are represented as histograms with error bars. Each histogram is the mean of four biological replicates. (C) Reporter fusion assay. The putative promoter of SMU.1882 was fused to a promoter-less <i>gusA</i> reporter gene and inserted into the <i>S. mutans</i> UA159 chromosome. Expression driven from the promoters was evaluated by measuring Gus activity. Gus values were measured from at least three independent cultures; mean values with standard deviations are shown.</p

Presence of SMU.1882 locus in different strains of <i>S. mutans</i>.

<p>(A) Southern hybridization blot of <i>Xmn</i>I digested chromosomal DNA from different <i>S. mutans</i> strains belonging to three serotypes (c, e and f). A 267-bp internal SMU.1882 sequence was used as a probe against the digested DNA. Semi-quantitative RT-PCR analysis (B) and quantitative real-time PCR analysis (C) of <i>S. mutans</i> strain GS-5, its isogenic Δ<i>covR</i> strain (IBS07) and the complementing strain (IBS07/pIB30). (D) Quantitative real-time PCR analysis of SMU.1882 transcript in NG-8, its isogenic Δ<i>covR</i> strain (IBS06) and Δ<i>covR</i> complemented with pIB30 (IBS06/pIB30). The expression of SMU.1882 was normalized to the expression level of <i>gyrA</i> as an internal reference for quantitative real-time PCR analyses.</p

Primers used in the analysis.

<p>Primers used in the analysis.</p

Effect of CovR on transcription from P<i>₁₈₈₂</i>.

<p>In vitro transcription was carried out using P<i>₁₈₈₂</i> and P<i>_ami</i> template DNA according to the protocol as described in the text. Lanes 1–3: no CovR; lanes 4–6: increasing concentration of CovR (1, 4, and 10 pmole, respectively). The experiments were repeated at least twice and a representative gel is shown.</p

Potential consensus sequence for CovR binding.

<p>(A) WebLogo representation of the position weight matrices derived from five CovR regulated promoters using GLAM2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015528#pone.0015528-Frith1" target="_blank">[36]</a>. (B) The location of the consensus sequence in the promoter region of the corresponding genes.</p

CovR binds to SMU.1882 promoter region.

<p>(A) In vitro binding of CovR to the promoter of SMU.1882. EMSA was performed with His-tagged CovR as described in the text. Lanes: 1, no CovR; 2, 14.97 pmol CovR; 3, 29.94 pmol CovR; 4, 59.88 pmol CovR; 5, 89.82 pmol CovR; 6, 119.76 pmol CovR; 7 and 8, 29.94 pmol CovR. 0.5 pmol labelled P₁₈₈₂ was used in all the reactions. 25-fold molar excess of cold P₁₈₈₂ and P_nlmA were used in lanes 7 and 8 respectively. B: bound DNA; F: free DNA. (B) DNase I protection assay of the putative SMU.1882 promoter. 0.5 pmol labelled P<i>₁₈₈₂</i> was used in all the reactions. Lane: 1, no CovR; 2, 59.88 pmol CovR; 3, 89.82 pmol CovR; 4, 119.76 pmol CovR; 5, 187.125 pmol CovR. Footprints were run in an 8% sequencing gel next to sequencing ladders (G, A, T, and C). CovR binding regions are indicated by vertical lines at the side of the sequencing gels, while horizontal lines mark -10 and -35 regions of the promoter. All experiments were carried out at least thrice, and a representative gel is shown.</p