Bio-engineering of bacterial microcompartments: a mini review.

Bacterial microcompartments (BMCs) are protein-bound prokaryotic organelles, discovered in cyanobacteria more than 60 years ago. Functionally similar to eukaryotic cellular organelles, BMCs compartment metabolic activities in the cytoplasm, foremost to increase local enzyme concentration and prevent toxic intermediates from damaging the cytosolic content. Advanced knowledge of the functional and structural properties of multiple types of BMCs, particularly over the last 10 years, have highlighted design principles of microcompartments. This has prompted new research into their potential to function as programmable synthetic nano-bioreactors and novel bio-materials with biotechnological and medical applications. Moreover, due to the involvement of microcompartments in bacterial pathogenesis and human health, BMCs have begun to gain attention as potential novel drug targets. This mini-review gives an overview of important synthetic biology developments in the bioengineering of BMCs and a perspective on future directions in the field.

Atomic Structure of Type VI Contractile Sheath from Pseudomonas aeruginosa.

Pseudomonas aeruginosa has three type VI secretion systems (T6SSs), H1-, H2-, and H3-T6SS, each belonging to a distinct group. The two T6SS components, TssB/VipA and TssC/VipB, assemble to form tubules that conserve structural/functional homology with tail sheaths of contractile bacteriophages and pyocins. Here, we used cryoelectron microscopy to solve the structure of the H1-T6SS P. aeruginosa TssB1C1 sheath at 3.3 Å resolution. Our structure allowed us to resolve some features of the T6SS sheath that were not resolved in the Vibrio cholerae VipAB and Francisella tularensis IglAB structures. Comparison with sheath structures from other contractile machines, including T4 phage and R-type pyocins, provides a better understanding of how these systems have conserved similar functions/mechanisms despite evolution. We used the P. aeruginosa R2 pyocin as a structural template to build an atomic model of the TssB1C1 sheath in its extended conformation, allowing us to propose a coiled-spring-like mechanism for T6SS sheath contraction.

TssA forms a gp6-like ring attached to the type VI secretion sheath.

The type VI secretion system (T6SS) is a supra-molecular bacterial complex that resembles phage tails. It is a killing machine which fires toxins into target cells upon contraction of its TssBC sheath. Here, we show that TssA1 is a T6SS component forming dodecameric ring structures whose dimensions match those of the TssBC sheath and which can accommodate the inner Hcp tube. The TssA1 ring complex binds the T6SS sheath and impacts its behaviour in vivo In the phage, the first disc of the gp18 sheath sits on a baseplate wherein gp6 is a dodecameric ring. We found remarkable sequence and structural similarities between TssA1 and gp6 C-termini, and propose that TssA1 could be a baseplate component of the T6SS Furthermore, we identified similarities between TssK1 and gp8, the former interacting with TssA1 while the latter is found in the outer radius of the gp6 ring. These observations, combined with similarities between TssF and gp6N-terminus or TssG and gp53, lead us to propose a comparative model between the phage baseplate and the T6SS.

A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K(D) of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens.

Coevolution of the ATPase ClpV, the sheath proteins TssB and TssC, and the accessory protein TagJ/HsiE1 distinguishes type VI secretion classes.

The type VI secretion system (T6SS) is a bacterial nanomachine for the transport of effector molecules into prokaryotic and eukaryotic cells. It involves the assembly of a tubular structure composed of TssB and TssC that is similar to the tail sheath of bacteriophages. The sheath contracts to provide the energy needed for effector delivery. The AAA(+) ATPase ClpV disassembles the contracted sheath, which resets the systems for reassembly of an extended sheath that is ready to fire again. This mechanism is crucial for T6SS function. In Vibrio cholerae, ClpV binds the N terminus of TssC within a hydrophobic groove. In this study, we resolved the crystal structure of the N-terminal domain of Pseudomonas aeruginosa ClpV1 and observed structural alterations in the hydrophobic groove. The modification in the ClpV1 groove is matched by a change in the N terminus of TssC, suggesting the existence of distinct T6SS classes. An accessory T6SS component, TagJ/HsiE, exists predominantly in one of the classes. Using bacterial two-hybrid approaches, we showed that the P. aeruginosa homolog HsiE1 interacts strongly with ClpV1. We then resolved the crystal structure of HsiE1 in complex with the N terminus of HsiB1, a TssB homolog and component of the contractile sheath. Phylogenetic analysis confirmed that these differences distinguish T6SS classes that resulted from a functional co-evolution between TssB, TssC, TagJ/HsiE, and ClpV. The interaction of TagJ/HsiE with the sheath as well as with ClpV suggests an alternative mode of disassembly in which HsiE recruits the ATPase to the sheath.

Concerted transfer of the virulence Ti plasmid and companion At plasmid in the Agrobacterium tumefaciens-induced plant tumour.

The plant pathogen Agrobacterium tumefaciens C58 harbours three independent type IV secretion (T4SS) machineries. T4SST-DNA promotes the transfer of the T-DNA to host plant cells, provoking tumour development and accumulation of opines such as nopaline and agrocinopines. T4SSpTi and T4SSpAt control the bacterial conjugation of the Ti and At plasmids respectively. Expression of T4SSpTi is controlled by the agrocinopine-responsive transcriptional repressor AccR. In this work, we compared the genome-wide transcriptional profile of the wild-type A. tumefaciens strain C58 with that of its accR KO-mutant to delineate the AccR regulon. In addition to the genes that encode agrocinopine catabolism and T4SSpTi , we found that AccR also regulated genes coding for nopaline catabolism and T4SSpAt . Further opine detection and conjugation assays confirmed the enhancement of nopaline consumption and At plasmid conjugation frequency in accR. Moreover, co-regulation of the T4SSpTi and T4SSpAt correlated with the co-transfer of the At and Ti plasmids both in vitro and in plant tumours. Finally, unlike T4SSpTi , T4SSpAt activation does not require quorum-sensing. Overall this study highlights the regulatory interplays between opines, At and Ti plasmids that contribute to a concerted dissemination of the two replicons in bacterial populations colonizing the plant tumour.

Deep sequencing revealed genome-wide single-nucleotide polymorphism and plasmid content of Erwinia amylovora strains isolated in Middle Atlas, Morocco.

Erwinia amylovora causes economic losses that affect pear and apple production in Morocco. Here, we report comparative genomics of four Moroccan E. amylovora strains with the European strain CFBP1430 and North-American strain ATCC49946. Analysis of single nucleotide polymorphisms (SNPs) revealed genetic homogeneity of Moroccan's strains and their proximity to the European strain CFBP1430. Moreover, the collected sequences allowed the assembly of a 65 kpb plasmid, which is highly similar to the plasmid pEI70 harbored by several European E. amylovora isolates. This plasmid was found in 33% of the 40 E. amylovora strains collected from several host plants in 2009 and 2010 in Morocco.

In planta fitness-cost of the Atu4232-regulon encoding for a selective GABA-binding sensor in Agrobacterium.

GABA (gamma-aminobutyric acid) mediates cell-to-cell communication in eukaryotes and interspecies communication in host-microbe interactions. Agrobacterium tumefaciens induces the development of plant tumor in which GABA accumulates. Two periplasmic binding proteins Atu2422 and Atu4243 and their appropriate ABC-transporter are involved in the binding and importation of GABA. The structure of the selective GABA-binding Atu4243 reveals a GABA conformation similar to a proposed model of GABA bound to the mammalian GABAC receptor. The A. tumefaciens atu4243 mutant is affected for GABA uptake, aggressiveness on plant host and GABA-induced degradation of the quorum-sensing signal, hence for horizontal transfer of the tumor-inducing plasmid. Here, we report that a de-repression of atu4243 and its co-regulated neighbor genes affect the fitness of A. tumefaciens during tumor colonization. Atu4243-orthologs are present in several species of the Agrobacterium genus. This addendum highlights the recent data on the GABA transport in the A. tumefaciens plant-pathogen.

Structural basis for selective GABA binding in bacterial pathogens.

GABA acts as an intercellular signal in eukaryotes and as an interspecies signal in host-microbe interactions. Structural characteristics of selective eukaryotic GABA receptors and bacterial GABA sensors are unknown. Here, we identified the selective GABA-binding protein, called Atu4243, in the plant pathogen Agrobacterium tumefaciens. A constructed atu4243 mutant was affected in GABA transport and in expression of the GABA-regulated functions, including aggressiveness on two plant hosts and degradation of the quorum-sensing signal. The GABA-bound Atu4243 structure at 1.28 Å reveals that GABA adopts a conformation never observed so far and interacts with two key residues, Arg(203) and Asp(226) of which the role in GABA binding and GABA signalling in Agrobacterium has been validated using appropriate mutants. The conformational GABA-analogue trans-4-aminocrotonic acid (TACA) antagonizes GABA activity, suggesting structural similarities between the binding sites of the bacterial sensor Atu4243 and mammalian GABA(C) receptors. Exploration of genomic databases reveals Atu4243 orthologues in several pathogenic and symbiotic proteobacteria, such as Rhizobium, Azospirillum, Burkholderia and Pseudomonas. Thus, this study establishes a structural basis for selective GABA sensors and offers opportunities for deciphering the role of the GABA-mediated communication in several host-pathogen interactions.

Systematic deletion of homeobox genes in Podospora anserina uncovers their roles in shaping the fruiting body.

Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures.

A conserved mechanism of GABA binding and antagonism is revealed by structure-function analysis of the periplasmic binding protein Atu2422 in Agrobacterium tumefaciens.

Bacterial periplasmic binding proteins (PBPs) and eukaryotic PBP-like domains (also called as Venus flytrap modules) of G-protein-coupled receptors are involved in extracellular GABA perception. We investigated the structural and functional basis of ligand specificity of the PBP Atu2422, which is implicated in virulence and transport of GABA in the plant pathogen Agrobacterium tumefaciens. Five high-resolution x-ray structures of Atu2422 liganded to GABA, Pro, Ala, and Val and of point mutant Atu2422-F77A liganded to Leu were determined. Structural analysis of the ligand-binding site revealed two essential residues, Phe(77) and Tyr(275), the implication of which in GABA signaling and virulence was confirmed using A. tumefaciens cells expressing corresponding Atu2422 mutants. Phe(77) restricts ligand specificity to α-amino acids with a short lateral chain, which act as antagonists of GABA signaling in A. tumefaciens. Tyr(275) specifically interacts with the GABA γ-amino group. Conservation of these two key residues in proteins phylogenetically related to Atu2422 brought to light a subfamily of PBPs in which all members could bind GABA and short α-amino acids. This work led to the identification of a fingerprint sequence and structural features for defining PBPs that bind GABA and its competitors and revealed their occurrence among host-interacting proteobacteria.

Glutathione-dependent redox status of frataxin-deficient cells in a yeast model of Friedreich's ataxia.

Friedreich's ataxia is a neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulphur cluster defects and high sensitivity to oxidative stress. Glutathione is a major protective agent against oxidative damage and glutathione-related systems participate in maintaining the cellular thiol/disulfide status and the reduced environment of the cell. Here, we present the first detailed biochemical study of the glutathione-dependent redox status of wild-type and frataxin-deficient cells in a yeast model of the disease. There were five times less total glutathione (GSH+GSSG) in frataxin-deficient cells, imbalanced GSH/GSSG pools and higher glutathione peroxidase activity. The pentose phosphate pathway was stimulated in frataxin-deficient cells, glucose-6-phosphate dehydrogenase activity was three times higher than in wild-type cells and this was coupled to a defect in the NADPH/NADP(+) pool. Moreover, analysis of gene expression confirms the adaptative response of mutant cells to stress conditions and we bring evidence for a strong relation between the glutathione-dependent redox status of the cells and iron homeostasis. Dynamic studies show that intracellular glutathione levels reflect an adaptation of cells to iron stress conditions, and allow to distinguish constitutive stress observed in frataxin-deficient cells from the acute response of wild-type cells. In conclusion, our findings provide evidence for an impairment of glutathione homeostasis in a yeast model of Friedreich's ataxia and identify glutathione as a valuable indicator of the redox status of frataxin-deficient cells.