Tolerance engineering in Deinococcus geothermalis by heterologous efflux pumps.

Producing industrially significant compounds with more environmentally friendly represents a challenging task. The large-scale production of an exogenous molecule in a host microfactory can quickly cause toxic effects, forcing the cell to inhibit production to survive. The key point to counter these toxic effects is to promote a gain of tolerance in the host, for instance, by inducing a constant flux of the neo-synthetized compound out of the producing cells. Efflux pumps are membrane proteins that constitute the most powerful mechanism to release molecules out of cells. We propose here a new biological model, Deinococcus geothermalis, organism known for its ability to survive hostile environment; with the aim of coupling the promising industrial potential of this species with that of heterologous efflux pumps to promote engineering tolerance. In this study, clones of D. geothermalis containing various genes encoding chromosomal heterologous efflux pumps were generated. Resistant recombinants were selected using antibiotic susceptibility tests to screen promising candidates. We then developed a method to determine the efflux efficiency of the best candidate, which contains the gene encoding the MdfA of Salmonella enterica serovar Choleraesuis. We observe 1.6 times more compound in the external medium of the hit recombinant than that of the WT at early incubation time. The data presented here will contribute to better understanding of the parameters required for efficient production in D. geothermalis.

Boutiques: a flexible framework to integrate command-line applications in computing platforms.

We present Boutiques, a system to automatically publish, integrate, and execute command-line applications across computational platforms. Boutiques applications are installed through software containers described in a rich and flexible JSON language. A set of core tools facilitates the construction, validation, import, execution, and publishing of applications. Boutiques is currently supported by several distinct virtual research platforms, and it has been used to describe dozens of applications in the neuroinformatics domain. We expect Boutiques to improve the quality of application integration in computational platforms, to reduce redundancy of effort, to contribute to computational reproducibility, and to foster Open Science.

RefZ facilitates the switch from medial to polar division during spore formation in Bacillus subtilis.

During sporulation, Bacillus subtilis redeploys the division protein FtsZ from midcell to the cell poles, ultimately generating an asymmetric septum. Here, we describe a sporulation-induced protein, RefZ, that facilitates the switch from a medial to a polar FtsZ ring placement. The artificial expression of RefZ during vegetative growth converts FtsZ rings into FtsZ spirals, arcs, and foci, leading to filamentation and lysis. Mutations in FtsZ specifically suppress RefZ-dependent division inhibition, suggesting that RefZ may target FtsZ. During sporulation, cells lacking RefZ are delayed in polar FtsZ ring formation, spending more time in the medial and transition stages of FtsZ ring assembly. A RefZ-green fluorescent protein (GFP) fusion localizes in weak polar foci at the onset of sporulation and as a brighter midcell focus at the time of polar division. RefZ has a TetR DNA binding motif, and point mutations in the putative recognition helix disrupt focus formation and abrogate cell division inhibition. Finally, chromatin immunoprecipitation assays identified sites of RefZ enrichment in the origin region and near the terminus. Collectively, these data support a model in which RefZ helps promote the switch from medial to polar division and is guided by the organization of the chromosome. Models in which RefZ acts as an activator of FtsZ ring assembly near the cell poles or as an inhibitor of the transient medial ring at midcell are discussed.

Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis.

Rod-shaped bacteria elongate by the action of cell wall synthesis complexes linked to underlying dynamic MreB filaments. To understand how the movements of these filaments relate to cell wall synthesis, we characterized the dynamics of MreB and the cell wall elongation machinery using high-precision particle tracking in Bacillus subtilis. We found that MreB and the elongation machinery moved circumferentially around the cell, perpendicular to its length, with nearby synthesis complexes and MreB filaments moving independently in both directions. Inhibition of cell wall synthesis by various methods blocked the movement of MreB. Thus, bacteria elongate by the uncoordinated, circumferential movements of synthetic complexes that insert radial hoops of new peptidoglycan during their transit, possibly driving the motion of the underlying MreB filaments.

Nucleoid occlusion prevents cell division during replication fork arrest in Bacillus subtilis.

How bacteria respond to chromosome replication stress has been traditionally studied using temperature-sensitive mutants and chemical inhibitors. These methods inevitably arrest all replication and lead to induction of transcriptional responses and inhibition of cell division. Here, we used repressor proteins bound to operator arrays to generate a single stalled replication fork. These replication roadblocks impeded replisome progression on one arm, leaving replication of the other arm and re-initiation unaffected. Remarkably, despite robust generation of RecA-GFP filaments and a strong block to cell division during the roadblock, patterns of gene expression were not significantly altered. Consistent with these findings, division inhibition was not mediated by the SOS-induced regulator YneA nor by RecA-independent repression of ftsL. In support of the idea that nucleoid occlusion prevents inappropriate cell division during fork arrest, immature FtsZ-rings formed adjacent to the DNA mass but rarely on top of it. Furthermore, mild alterations in chromosome compaction resulted in cell division that guillotined the DNA. Strikingly, the nucleoid occlusion protein Noc had no discernable role in division inhibition. Our data indicate that Noc-independent nucleoid occlusion prevents inappropriate cell division during replication fork arrest. They further suggest that Bacillus subtilis normally manages replication stress rather than inducing a stress response.

Resistance to bacitracin in Bacillus subtilis: unexpected requirement of the BceAB ABC transporter in the control of expression of its own structural genes.

The Bacillus subtilis BceAB ABC transporter involved in a defense mechanism against bacitracin is composed of a membrane-spanning domain and a nucleotide-binding domain. Induction of the structural bceAB genes requires the BceR response regulator and the BceS histidine kinase of a signal transduction system. However, despite the presence of such a transduction system and of bacitracin, no transcription from an unaltered bceA promoter is observed in cells lacking the BceAB transporter. Expression in trans of the BceAB transporter in these bceAB cells restores the transcription from the bceA promoter. Cells possessing a mutated nucleotide-binding domain of the transporter are also no longer able to trigger transcription from the bceA promoter in the presence of bacitracin, although the mutated ABC transporter is still bound to the membrane. In these cells, expression of the bceA promoter can no longer be detected, indicating that the ABC transporter not only must be present in the cell membrane, but also must be expressed in a native form for the induction of the bceAB genes. Several hypotheses are discussed to explain the simultaneous need for bacitracin, a native signal transduction system, and an active BceAB ABC transporter to trigger transcription from the bceA promoter.

BcrC from Bacillus subtilis acts as an undecaprenyl pyrophosphate phosphatase in bacitracin resistance.

Overexpression of the BcrC(Bs) protein, formerly called YwoA, in Escherichia coli or in Bacillus subtilis allows these bacteria to stand higher concentrations of bacitracin. It was suggested that BcrC(Bs) was a membrane-spanning domain of an ATP binding cassette (ABC) transporter involved in bacitracin resistance. However, we hypothesized that this protein has an undecaprenyl pyrophosphate (UPP) phosphatase activity able to compete with bacitracin for UPP. We found that overexpression of a recombinant His6-BcrC(Bs) protein in E. coli (i) increased the resistance of the cells to bacitracin and (ii) increased UPP phosphatase activity in membrane preparations by 600-fold. We solubilized and prepared an electrophoretically pure protein exhibiting a strong UPP phosphatase activity. BcrC(Bs), which belongs to the type 2 phosphatidic acid phosphatase (PAP2) phosphatase superfamily (PF01569), differs totally from the already known BacA UPP phosphatase from E. coli, a member of the PF02673 family of the Protein family (Pfam) database. Thus, BcrC(Bs) and its orthologs form a new class of proteins within the PAP2 phosphatase superfamily, and likely all of them share a UPP phosphatase activity.

YtsCD and YwoA, two independent systems that confer bacitracin resistance to Bacillus subtilis.

The Bacillus subtilis yts, yxd and yvc gene clusters encode a putative ABC transporter and a functionally coupled two-component system. When tested for their sensitivity towards a series of antibiotics, null yts mutants were found to be sensitive to bacitracin. Real-time polymerase chain reaction (PCR) experiments demonstrated that the presence of bacitracin in the growth medium strongly stimulates the expression of the ytsCD genes encoding the ABC transporter and that this stimulation strictly depends on the YtsA response regulator. The ywoA gene encodes a protein known to confer some resistance to bacitracin on the bacterium. When it was mutated in a null yts background, the ywoA yts double mutant was found to be five times more sensitive than the yts one. We propose that (i) the YtsCD ABC transporter exports the bacitracin; (ii) YwoA, the protein that contains an acidPPc (PAP2 or PgpB) domain, is not part of an ABC transporter but competes with bacitracin for the dephosphorylation of the C55-isoprenyl pyrophosphate (IPP); (iii) the two resistance mechanisms are independent and complementary.