Surface (S)-layer proteins of Deinococcus radiodurans and their utility as vehicles for surface localization of functional proteins

AbstractThe radiation resistant bacterium, Deinococcus radiodurans contains two major surface (S)-layer proteins, Hpi and SlpA. The Hpi protein was shown to (a) undergo specific in vivo cleavage, and (b) closely associate with the SlpA protein. Using a non-specific acid phosphatase from Salmonella enterica serovar Typhi, PhoN as a reporter, the Surface Layer Homology (SLH) domain of SlpA was shown to bind deinococcal peptidoglycan-containing cell wall sacculi. The association of SlpA with Hpi on one side and peptidoglycan on the other, localizes this protein in the ‘interstitial’ layer of the deinoccocal cell wall. Gene chimeras of hpi-phoN and slh-phoN were constructed to test efficacy of S-layer proteins, as vehicles for cell surface localization in D. radiodurans. The Hpi-PhoN protein localized exclusively in the membrane fraction, and displayed cell-based phosphatase activity in vivo. The SLH-PhoN, which localized to both cytosolic and membrane fractions, displayed in vitro activity but no cell-based in vivo activity. Hpi, therefore, emerged as an efficient surface localizing protein and can be exploited for suitable applications of this superbug

A novel serralysin metalloprotease from Deinococcus radiodurans

A hypothetical protein (DR2310) from the radiation resistant organism Deinococcus radiodurans harbors highly conserved Zn<SUP>+2</SUP>-binding (HEXXH) domain and Met-turn (SVMSY), characteristic of the serralysin family of secreted metalloproteases from Gram negative bacteria. Deletion mutagenesis of DR2310 confirmed that the ORF is expressed in Deinococcus radiodurans as a secreted protease of 85 kDa. Biochemical analysis revealed DR2310 to be a Ca<SUP>+2</SUP> and Zn<SUP>+2</SUP>-requiring metalloprotease. Unique features such as a long N-terminus, replacement of the highly conserved C-terminal glycine rich Ca<SUP>+2</SUP>-binding repeats with a single N-terminal aspartate rich eukaryotic thrombospondin type-3 Ca<SUP>+2</SUP>-binding repeat and absence of C-terminal secretion signals make it a novel member of serralysin family. This is the first report of a functional serralysin family metalloprotease from a Gram positive organism

Modulation of Glucose Transport Causes Preferential Utilization of Aromatic Compounds in Pseudomonas putida CSV86▿

Pseudomonas putida CSV86 utilizes aromatic compounds in preference to glucose and coutilizes aromatics and organic acids. Protein analysis of cells grown on different carbon sources, either alone or in combination, revealed that a 43-kDa periplasmic-space protein was induced by glucose and repressed by aromatics and succinate. Two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis identified this protein as closely resembling the sugar ABC transporter of Pseudomonas putida KT2440. A partially purified 43-kDa protein showed glucose binding activity and was specific for glucose. The results demonstrate that the aromatic- and organic acid-mediated repression of a periplasmic-space glucose binding protein and consequent inhibition of glucose transport are responsible for this strain's ability to utilize aromatics and organic acids in preference to glucose

Construction, analysis and validation of co-expression network to understand stress adaptation in Deinococcus radiodurans R1.

Systems biology based approaches have been effectively utilized to mine high throughput data. In the current study, we have performed system-level analysis for Deinococcus radiodurans R1 by constructing a gene co-expression network based on several microarray datasets available in the public domain. This condition-independent network was constructed by Weighted Gene Co-expression Network Analysis (WGCNA) with 61 microarray samples from 9 different experimental conditions. We identified 13 co-expressed modules, of which, 11 showed functional enrichments of one or more pathway/s or biological process. Comparative analysis of differentially expressed genes and proteins from radiation and desiccation stress studies with our co-expressed modules revealed the association of cyan with radiation response. Interestingly, two modules viz darkgreen and tan was associated with radiation as well as desiccation stress responses. The functional analysis of these modules showed enrichment of pathways important for adaptation of radiation or desiccation stress. To decipher the regulatory roles of these stress responsive modules, we identified transcription factors (TFs) and then calculated a Biweight mid correlation between modules hub gene and the identified TFs. We obtained 7 TFs for radiation and desiccation responsive modules. The expressions of 3 TFs were validated in response to gamma radiation using qRT-PCR. Along with the TFs, selected close neighbor genes of two important TFs, viz., DR_0997 (CRP) and DR_2287 (AsnC family transcriptional regulator) in the darkgreen module were also validated. In our network, among 13 hub genes associated with 13 modules, the functionality of 5 hub genes which are annotated as hypothetical proteins (hypothetical hub genes) in D. radiodurans genome has been revealed. Overall the study provided a better insight of pathways and regulators associated with relevant DNA damaging stress response in D. radiodurans

[T_n]-rich region present in the <i>kdp</i> promoter region in various bacteria.

<p>(A) [T_n]-rich region is shown in pale blue box. The number after the [T_n]-rich region indicate the number of bases between the [T_n]-rich region and the start codon. [T_n]-rich and [A_n]-rich <i>Kdp</i>E binding site of <i>E</i>. <i>coli</i> and <i>M</i>. <i>smegmatis</i>, respectively, are shown in blue boxes. [T_n]-rich sequences in the upstream regions of <i>kdpB</i> gene in other bacteria are shown in red bold letters. (B) Binding of RR protein to the P<i>kdpB</i>-200 of <i>D</i>. <i>radiodurans</i>. The indicted concentrations of RR protein were incubated with P<i>kdpB</i>-200 promoter (45 ng of DIG-labeled 200 bp dsDNA) at 37°C for 1 h and the DNA–protein complexes were resolved by 10% native PAGE. The amount of DNA-protein complexes were estimated using GelQuant software. Substrate DNA and DNA-RR complex are shown by “—” and “←”, respectively, while wells of the gels are marked by asterisk. (C) The representative graph for DNA protein complexes. The data-points were fitted into Hill’s equation (dotted line) to determine K_D value. (D) Titration of RR-promoter complexes with unlabeled promoter DNA. (E) Interaction of RR or non-specific protein BSA with P<i>kdpB</i>-200 (specific target) or non-specific DNA sequence. For (D) and (E), the DNA-protein complexes were resolved as described in legend to Fig 4B.</p

KdpB and RR expression under different growth conditions.

<p>Expression of KdpB (A) or RR (B) proteins in <i>D</i>. <i>radiodurans</i> cells incubated in TGY, K20 or K0 media. Time course of induction of KdpB (C) or RR (D) proteins in <i>D</i>. <i>radiodurans</i> cells following shift from TGY to K0 medium. Expression of KdpB (E) or RR (F) proteins in <i>D</i>. <i>radiodurans</i> cells incubated in TGY, K0, K1 or K2 media. Localization of KdpB (G) or RR (H) proteins in <i>D</i>. <i>radiodurans</i> cells incubated in K20 or K0 media. Expression of KdpB (I) or RR (J) proteins in <i>D</i>. <i>radiodurans</i> cells grown either in K20 or K0 media, or exposed to ionic (-I, 0.1M NaCl) or osmotic (-O, 0.2M sucrose) stresses in K1 medium. The cellular proteins (100 μg) were resolved by 12% SDS-PAGE, electroblotted onto nitrocellulose membrane and immuno-stained using anti-KdpB or anti-RR antibodies as detailed in materials and methods. The top most protein band (~ 125 kDa) in the corresponding Coomassie stained gel is shown below Fig 2A, 2B, 2C, 2D, 2E, 2F, 2I and 2J, as loading control. For membrane or cytosolic protein extracts, 63 kDa protein band or 44 kDa protein band, respectively, are shown as loading controls (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188998#pone.0188998.s002" target="_blank">S2 Fig</a> for details on loading controls). Red bold numbers below the KdpB or RR immuno-stained bands indicate fold increase in their levels over the lanes in which these bands were not observed (denoted by 1).</p

Construction and confirmation of ΔRR mutant.

<p>(A) Schematic representation of the RR gene (DR_B0081) in wild type <i>D</i>. <i>radiodurans</i> (a) and its replacement with kanamycin resistance cassette (<i>aph</i>) in ΔRR <i>D</i>. <i>radiodurans</i> (b). The primers used for the PCR confirmation of the mutant are shown. (B) Confirmation of complete deletion of RR gene in ΔRR <i>D</i>. <i>radiodurans</i> as compared to wild type <i>D</i>. <i>radiodurans</i>, using primer pair shown in Fig 3A. (C) Expression of KdpB or RR proteins in wild type or ΔRR <i>D</i>. <i>radiodurans</i> cells incubated in K0 or K20 media. Details of immuno-staining, loading controls and fold change levels were same as described in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188998#pone.0188998.g002" target="_blank">Fig 2</a>.</p

Regulation of potassium dependent ATPase (<i>kdp</i>) operon of <i>Deinococcus radiodurans</i>

<div><p>The genome of <i>D</i>. <i>radiodurans</i> harbors genes for structural and regulatory proteins of Kdp ATPase, in an operon pattern, on Mega plasmid 1. Organization of its two-component regulatory genes is unique. Here we demonstrate that both, the structural as well as regulatory components of the <i>kdp</i> operon of <i>D</i>. <i>radiodurans</i> are expressed quickly as the cells experience potassium limitation but are not expressed upon increase in osmolarity. The cognate DNA binding response regulator (RR) effects the expression of <i>kdp</i> operon during potassium deficiency through specific interaction with the <i>kdp</i> promoter. Deletion of the gene encoding RR protein renders the mutant <i>D</i>. <i>radiodurans</i> (ΔRR) unable to express <i>kdp</i> operon under potassium limitation. The ΔRR <i>D</i>. <i>radiodurans</i> displays no growth defect when grown on rich media or when exposed to oxidative or heat stress but shows reduced growth following gamma irradiation. The study elucidates the functional and regulatory aspects of the novel <i>kdp</i> operon of this extremophile, for the first time.</p></div

Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth

<div><p>Infection of the genitourinary tract with Group B <i>Streptococcus</i> (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs <i>ex vivo</i> resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.</p></div