Gouzel Karimova

Interaction Network among Escherichia coli Membrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis

Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Several of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. Although these proteins appear to be recruited to the division site in a hierarchical order, the molecular interactions underlying the assembly of the cell division machinery remain mostly unspecified. In the present study, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to unravel the molecular basis of septum assembly by analyzing the protein interaction network among E. coli cell division proteins. Our results indicate that the Fts proteins are connected to one another through multiple interactions. A deletion mapping analysis carried out with two of these proteins, FtsQ and FtsI, revealed that different regions of the polypeptides are involved in their associations with their partners. Furthermore, we showed that the association between two Fts hybrid proteins could be modulated by the coexpression of a third Fts partner. Altogether, these data suggest that the cell division machinery assembly is driven by the cooperative association among the different Fts proteins to form a dynamic multiprotein structure at the septum site. In addition, our study shows that the cAMP-based two-hybrid system is particularly appropriate for analyzing molecular interactions between membrane proteins.

[5] A bacterial two-hybrid system that exploits a cAMP signaling cascade in Escherichia coli

Publisher Summary Most biological processes involve specific protein–protein interactions. The yeast two-hybrid system represents a powerful in vivo approach to analyze interactions among macromolecules and screen for polypeptides that bind to a given bait protein. Bacterial equivalents to the yeast two-hybrid system have not been developed yet. This chapter describes a novel bacterial two-hybrid system that allows an easy in vivo screening and selection of functional interactions between two proteins. This system, because of its sensitivity and simplicity, could have broad application in the studies of structure–function relationships in biological macromolecules, in the functional analysis of genomes, and in high-throughput screening of interacting ligands or new therapeutic agents.

Chlamydia co-opts the rod shape-determining proteins MreB and Pbp2 for cell division.

Chlamydiae are obligate intracellular bacterial pathogens that have extensively reduced their genome in adapting to the intracellular environment. The chlamydial genome contains only three annotated cell division genes and lacks ftsZ. How this obligate intracellular pathogen divides is uncharacterized. Chlamydiae contain two high‐molecular‐weight (HMW) penicillin binding proteins (Pbp) implicated in peptidoglycan synthesis, Pbp2 and Pbp3/FtsI. We show here, using HMW Pbp‐specific penicillin derivatives, that both Pbp2 and Pbp3 are essential for chlamydial cell division. Ultrastructural analyses of antibiotic‐treated cultures revealed distinct phenotypes: Pbp2 inhibition induced internal cell bodies within a single outer membrane whereas Pbp3 inhibition induced elongated phenotypes with little internal division. Each HMW Pbp interacts with the Chlamydia cell division protein FtsK. Chlamydiae are coccoid yet contain MreB, a rod shape‐determining protein linked to Pbp2 in bacilli. Using MreB‐specific antibiotics, we show that MreB is essential for chlamydial growth and division. Importantly, co‐treatment with MreB‐specific and Pbp‐specific antibiotics resulted in the MreB‐inhibited phenotype, placing MreB upstream of Pbp function in chlamydial cell division. Finally, we showed that MreB also interacts with FtsK. We propose that, in Chlamydia, MreB acts as a central co‐ordinator at the division site to substitute for the lack of FtsZ in this bacterium.

Phosphorylation‐dependent binding of BvgA to the upstream region of the cyaA gene of Bordetella pertussis

In Bordetella pertussis,transcription of virulence‐associated genes is regulated by the BvgS and BvgA proteins, members of the bacterial two‐component signal‐transduction family. BvgS is the transmembrane sensor and BvgA, in its phosphorylated form, is believed to be the key transcriptional activator in B. pertussis. However, the BvgA recognition sites in most virulence promoters have not yet been identified. To investigate the interaction of BvgA with the upstream region of cyaA, the gene encoding adenylate cyclase haemolysin, we have produced large amounts of BvgA in Escherichia coli. The protein was purified from inclusion bodies and then phosphorylated by acetyl phosphate. Using electrophoretic mobility‐shift and footprinting assays, we provide evidence that BvgA cannot bind to the cyaA promoter unless it is phosphorylated. The phosphorylated form of BvgA (BvgA‐P) is able to bind specifically to the upstream region of cyaA. Analysis of this region revealed that an unexpectedly large sequence, from −137 to −51, appears to be the target for BvgA‐P binding, and probably contains multiple binding sites.

Characterization of YmgF, a 72-Residue Inner Membrane Protein That Associates with the Escherichia coli Cell Division Machinery

Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Many of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. In the present study, we attempted to identify a novel putative component(s) of the E. coli cell division machinery by searching for proteins that could interact with known Fts proteins. To do that, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to perform a library screening in order to find putative partners of E. coli cell division protein FtsL. Here we report the characterization of YmgF, a 72-residue integral membrane protein of unknown function that was found to associate with many E. coli cell division proteins and to localize to the E. coli division septum in an FtsZ-, FtsA-, FtsQ-, and FtsN-dependent manner. Although YmgF was previously shown to be not essential for cell viability, we found that when overexpressed, YmgF was able to overcome the thermosensitive phenotype of the ftsQ1(Ts) mutation and restore its viability under low-osmolarity conditions. Our results suggest that YmgF might be a novel component of the E. coli cell division machinery.

Large‐scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly

The functionally versatile type IV pili (Tfp) are one of the most widespread virulence factors in bacteria. However, despite generating much research interest for decades, the molecular mechanisms underpinning the various aspects of Tfp biology remain poorly understood, mainly because of the complexity of the system. In the human pathogen Neisseria meningitidis for example, 23 proteins are dedicated to Tfp biology, 15 of which are essential for pilus biogenesis. One of the important gaps in our knowledge concerns the topology of this multiprotein machinery. Here we have used a bacterial two‐hybrid system to identify and quantify the interactions between 11 Pil proteins from N. meningitidis. We identified 20 different binary interactions, many of which are novel. This represents the most complex interaction network between Pil proteins reported to date and indicates, among other things, that PilE, PilM, PilN and PilO, which are involved in pilus assembly, indeed interact. We focused our efforts on this subset of proteins and used a battery of assays to determine the membrane topology of PilN and PilO, map the interaction domains between PilE, PilM, PilN and PilO, and show that a widely conserved N‐terminal motif in PilN is essential for both PilM–PilN interactions and pilus assembly. Finally, we show that PilP (another protein involved in pilus assembly) forms a complex with PilM, PilN and PilO. Taken together, these findings have numerous implications for understanding Tfp biology and provide a useful blueprint for future studies.

Sensitive Genetic Screen for Protease Activity Based on a Cyclic AMP Signaling Cascade in Escherichia coli

We describe a genetic system that allows in vivo screening or selection of site-specific proteases and of their cognate-specific inhibitors in Escherichia coli. This genetic test is based on the specific proteolysis of a signaling enzyme, the adenylate cyclase (AC) of Bordetella pertussis. As a model system we used the human immunodeficiency virus (HIV) protease. When an HIV protease processing site, p5, was inserted in frame into the AC polypeptide, the resulting ACp5 protein retained enzymatic activity and, when expressed in an E. coli cya strain, restored the Cya(+) phenotype. The HIV protease coexpressed in the same cells resulted in cleavage and inactivation of ACp5; the cells became Cya(-). When the entire HIV protease, including its adjacent processing sites, was inserted into the AC polypeptide, the resulting AC-HIV-Pr fusion protein, expressed in E. coli cya, was autoproteolysed and inactivated: the cells displayed Cya(-) phenotype. In the presence of the protease inhibitor indinavir or saquinavir, AC-HIV-Pr autoproteolysis was inhibited and the AC activity of the fusion protein was preserved; the cells were Cya(+). Protease variants resistant to particular inhibitors could be easily distinguished from the wild type, as the cells displayed a Cya(-) phenotype in the presence of these inhibitors. This genetic test could represent a powerful approach to screen for new proteolytic activities and for novel protease inhibitors. It could also be used to detect in patients undergoing highly active antiretroviral therapy the emergence of HIV variants harboring antiprotease-resistant proteases.

The Escherichia coli Regulator of Sigma 70 Protein, Rsd, Can Up-Regulate Some Stress-Dependent Promoters by Sequestering Sigma 70

The Escherichia coli Rsd protein forms complexes with the RNA polymerase sigma(70) factor, but its biological role is not understood. Transcriptome analysis shows that overexpression of Rsd causes increased expression from some promoters whose expression depends on the alternative sigma(38) factor, and this was confirmed by experiments with lac fusions at selected promoters. The LP18 substitution in Rsd increases the Rsd-dependent stimulation of these promoter-lac fusions. Analysis with a bacterial two-hybrid system shows that the LP18 substitution in Rsd increases its interaction with sigma(70). Our experiments support a model in which the role of Rsd is primarily to sequester sigma(70), thereby increasing the levels of RNA polymerase containing the alternative sigma(38) factor.

Characterization of recombinant Bordetella pertussis adenylate cyclase toxins carrying passenger proteins

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400 amino acid residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver CD8+ T-cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted cytotoxic T-cell (CTL) responses in vivo. To explore the tolerance of CyaA to insertion of polypeptides of larger size, we constructed and characterized different recombinant CyaA toxins with protein inserts of 87 to 206 amino acids in length. Several of these recombinant CyaA toxins were found to be invasive. Furthermore, we showed that the unfolding of the passenger protein is a prerequisite for the translocation of the recombinant toxins into eukaryotic cells. Our results highlight the remarkable tolerance of the CyaA toxin and suggest that CyaA might be used to deliver proteins into eukaryotic cells.

Genetic systems for analyzing protein-protein interactions in bacteria.

Analysis of protein-protein interactions has been revolutionized by the yeast two-hybrid system introduced by Fields and coworkers. In recent years, similar genetic assays have been developed in bacteria. We describe here several of these systems and highlight some potential applications of these technologies.