The PhoBR two-component system regulates antibiotic biosynthesis in Serratia in response to phosphate

<p>Abstract</p> <p>Background</p> <p>Secondary metabolism in <it>Serratia </it>sp. ATCC 39006 (<it>Serratia </it>39006) is controlled via a complex network of regulators, including a LuxIR-type (SmaIR) quorum sensing (QS) system. Here we investigate the molecular mechanism by which phosphate limitation controls biosynthesis of two antibiotic secondary metabolites, prodigiosin and carbapenem, in <it>Serratia </it>39006.</p> <p>Results</p> <p>We demonstrate that a mutation in the high affinity phosphate transporter <it>pstSCAB-phoU</it>, believed to mimic low phosphate conditions, causes upregulation of secondary metabolism and QS in <it>Serratia </it>39006, via the PhoBR two-component system. Phosphate limitation also activated secondary metabolism and QS in <it>Serratia </it>39006. In addition, a <it>pstS </it>mutation resulted in upregulation of <it>rap</it>. Rap, a putative SlyA/MarR-family transcriptional regulator, shares similarity with the global regulator RovA (regulator of virulence) from <it>Yersina </it>spp. and is an activator of secondary metabolism in <it>Serratia </it>39006. We demonstrate that expression of <it>rap</it>, <it>pigA-O </it>(encoding the prodigiosin biosynthetic operon) and <it>smaI </it>are controlled via PhoBR in <it>Serratia </it>39006.</p> <p>Conclusion</p> <p>Phosphate limitation regulates secondary metabolism in <it>Serratia </it>39006 via multiple inter-linked pathways, incorporating transcriptional control mediated by three important global regulators, PhoB, SmaR and Rap.</p

The sub-cellular localization of Sulfolobus DNA replication

Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated

In vivo protein interactions and complex formation in the Pectobacterium atrosepticum subtype I-F CRISPR/Cas System.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated proteins (Cas; CRISPR associated) are a bacterial defense mechanism against extra-chromosomal elements. CRISPR/Cas systems are distinct from other known defense mechanisms insofar as they provide acquired and heritable immunity. Resistance is accomplished in multiple stages in which the Cas proteins provide the enzymatic machinery. Importantly, subtype-specific proteins have been shown to form complexes in combination with small RNAs, which enable sequence-specific targeting of foreign nucleic acids. We used Pectobacterium atrosepticum, a plant pathogen that causes soft-rot and blackleg disease in potato, to investigate protein-protein interactions and complex formation in the subtype I-F CRISPR/Cas system. The P. atrosepticum CRISPR/Cas system encodes six proteins: Cas1, Cas3, and the four subtype specific proteins Csy1, Csy2, Csy3 and Cas6f (Csy4). Using co-purification followed by mass spectrometry as well as directed co-immunoprecipitation we have demonstrated complex formation by the Csy1-3 and Cas6f proteins, and determined details about the architecture of that complex. Cas3 was also shown to co-purify all four subtype-specific proteins, consistent with its role in targeting. Furthermore, our results show that the subtype I-F Cas1 and Cas3 (a Cas2-Cas3 hybrid) proteins interact, suggesting a protein complex for adaptation and a role for subtype I-F Cas3 proteins in both the adaptation and interference steps of the CRISPR/Cas mechanism

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

The Csy1-3 and Cas6f proteins of <i>P. atrosepticum</i> form a complex <i>in vivo</i>.

<p>(A) Scale schematic representation of the CRISPR/Cas system in <i>P. atrosepticum</i> strain SCRI1043. The 3 CRISPR loci are denoted CRISPR1-3 in order of decreasing length and the direction of transcription indicated by the directionality of the arrows. The universal and type-specific genes, <i>cas1</i> and <i>cas2-cas3</i> are shown in blue and the subtype I-F-specific genes are depicted in light blue (<i>csy1-3</i>) and orange (<i>cas6f</i>). Between CRISPR2 and CRISPR3 is a putative toxin-antitoxin system (<i>eca3686-7</i>). (B) Co-purification of Csy1-3 and Cas6f proteins using Ni-NTA agarose. Coomassie stained SDS-PAGE gel of elution fractions from the <i>P. atrosepticum</i> Δ<i>cas</i> mutants expressing untagged Csy1-3 and Cas6f (pJSC11) and either one of the four different His-tagged bait Csy and Cas6f proteins (plasmids pJSC3-6 encode His-tagged Csy1-3 and Cas6f, respectively) or an His-tagged SdhE control (pMAT4). Proteins were identified by MS as indicated and results also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049549#pone-0049549-t003" target="_blank">Table 3</a>.</p

Archaeal orthologs of Cdc45 and GINS form a stable complex that stimulates the helicase activity of MCM

The regulated recruitment of Cdc45 and GINS is key to activating the eukaryotic MCM(2-7) replicative helicase. We demonstrate that the homohexameric archaeal MCM helicase associates with orthologs of GINS and Cdc45 in vivo and in vitro. Association of these factors with MCM robustly stimulates the MCM helicase activity. In contrast to the situation in eukaryotes, archaeal Cdc45 and GINS form an extremely stable complex before binding MCM. Further, the archaeal GINS•Cdc45 complex contains two copies of Cdc45. Our analyses give insight into the function and evolution of the conserved core of the archaeal/eukaryotic replisome

Cas1 and Cas3 interact.

<p>(A) Predicted Cas2, HD nuclease and helicase domains present in <i>P. atrosepticum</i> Cas3 based on structural homology using Phyre2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049549#pone.0049549-Kelley1" target="_blank">[50]</a>. (B) Secondary structure of <i>Desulfovibrio vulgaris</i> (DvuCas2) and multiple sequence alignment of the N-terminal 110 aa of Cas3 with Cas2 homologues for which there is structural data. Blue arrows indicate β–sheets and orange barrels α–helices. Residues identified to be involved in protein function are marked with asterisks. Conserved residues are depicted in red, functionally similar residues in yellow. (C) Co-purification of His-Cas1 and Cas3 following expression in the Δ<i>cas</i> mutant (PCF80, pJSC10). Proteins in the soluble fraction (lane 1) were loaded onto Ni-NTA-agarose and washed with 40 mM imidazole. Proteins bound specifically were eluted with an imidazole gradient: 62.5 mM (lane 2), 125 mM (lane 3), 187.5 mM (lane 4) and 250 mM (lanes 5 and 6). (D) Co-purification of His-Cas1 and Cas3 in the presence of Csy1-3 and Cas6f following expression in the Δ<i>cas</i> mutant with pJSC10 (Cas1,3) and pJSC11 (Csy1-3, Cas6f). (E) Gel filtration fraction of His-Cas1 and Cas3 following an initial Ni-NTA purification. All samples were separated by SDS-PAGE and proteins visualized by Coomassie staining.</p

Summary of Cas and Csy protein Co-IP results.

a<p>− no detection of His-prey with FLAG-bait.</p>b<p>+ detection of His-prey with FLAG-bait.</p>c<p>C-term FLAG-tagged Csy2 protein was detected as truncated.</p

Csy1-3 and Cas6f protein-protein interactions in WT and Δ<i>cas</i>

<p><b>strains.</b> N- or C-terminally FLAG-tagged Csy proteins were expressed in the presence of N-terminally His-tagged Csy and Cas6f proteins. Proteins were expressed, cells were lysed, proteins purified on anti-FLAG agarose, washed and eluted. Fractions were separated by SDS-PAGE and proteins were detected by Western blotting. Lanes indicate protein expression (Total), the final wash (Wash) and the elution fraction (Elution). (A) Csy1 and Csy2 interact in the absence of other Cas or Csy proteins. (B) Csy3 and Csy1 interact in the WT but not in the Δ<i>cas</i> mutant background. (C) Cas6f and Csy3 interact in the WT but not in the Δ<i>cas</i> mutant background. (D) Csy3 self-assembles.</p