Biology subjects

Tammam, S.

Publications and source records attributed to Tammam, S..

5 recordsLinked to original sources

bioRxiv2023

Pseudomonas aeruginosa AlgF is a protein-protein interaction mediator required for acetylation of the alginate exopolysaccharide

Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well-known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro, and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.

microbiology↗

bioRxiv2020

TsaP binds PilQ and is involved in cyclic di-GMP signaling

The type IV pilus machinery is a multi-protein complex that polymerizes and depolymerizes a pilus fibre used for attachment, twitching motility, phage adsorption, natural competence, protein secretion, and surface-sensing. An outer membrane secretin pore is required for passage of the pilus fibre out of the cell. Herein, the structure of the tetradecameric secretin, PilQ, from the Pseudomonas aeruginosa type IVa pilus system was determined to 4.3 [A] and 4.4 [A] resolution in the presence and absence of C7 symmetric spokes, respectively. The heptameric spokes were found to be the two tandem C-terminal domains of TsaP. TsaP forms a belt around PilQ and while the protein is not essential for twitching motility, over-expression of TsaP triggers a signal cascade upstream of PilY1 leading to cyclic di-GMP up-regulation. These results resolve the identity of the spokes identified with Proteobacterial PilQ homologs and may reveal a new component of the surface-sensing cyclic di-GMP signal cascade. IMPACT STATEMENTThe type IV pilus is critical for bacterial virulence. The co-structure of the pilus secretin PilQ and TsaP is determined. Characterization of TsaP implicates it in surface-sensing signal transduction.

microbiology↗

bioRxiv2019

Multiple conformations facilitate PilT function in the type IV pilus

Type IV pilus-like systems are protein complexes that polymerize a fibre of pilins. They are critical for virulence in many pathogens. Pilin polymerization and depolymerization are powered by motor PilT-like ATPases thought to possess C2 symmetry. However, most PilT-like ATPases crystallize with either C3 or C6 symmetry and the relevance of these conformations is unclear. Here we determined the X-ray structures of PilT in four unique conformations and used these structures to classify the conformation of available PilT-like ATPase structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT revealed condition-dependent preferences for C2, C3, and C6 conformations. The physiologic importance of these conformations was validated by co-evolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C2 conformation. With these data we propose a comprehensive model of PilT function with broad implications for PilT-like ATPases.

microbiology↗

bioRxiv2016

The type IVa pilus machinery is pre-installed during cell division

Type IV pili (T4aP) are ubiquitous microbial appendages used for adherence, twitching motility, DNA uptake, and electron transfer. Many of these functions depend on dynamic assembly and disassembly of the pilus by a megadalton-sized, cell envelope-spanning protein complex located at the poles of rod-shaped bacteria. How the T4aP assembly complex becomes integrated into the cell envelope in the absence of dedicated peptidoglycan (PG) hydrolases is unknown. After ruling out potential involvement of housekeeping PG hydrolases in installation of the T4aP machinery in P. aeruginosa, we discovered that key components of inner (PilMNOP) and outer (PilQ) membrane subcomplexes are recruited to future sites of cell division. Mid-cell recruitment of a fluorescently tagged alignment subcomplex component, mCherry-PilO, depended on PilQ secretin monomers - specifically, their N-terminal PG-binding AMIN domains. PilP, which connects PilO to PilQ, was required for recruitment, while PilM, which is structurally similar to divisome component FtsA, was not. Recruitment preceded secretin oligomerization in the outer membrane, as loss of the PilQ pilotin, PilF, had no effect on localization. These results were confirmed in cells chemically blocked for cell division prior to outer membrane invagination. The hub protein FimV and a component of the Polar Organelle Coordinator complex - PocA - were independently required for mid-cell recruitment of PilO and PilQ. Together, these data reveal an integrated, energy-efficient strategy for the targeting and pre-installation - rather than retrofit - of the T4aP system into nascent poles, without the need for dedicated PG-remodelling enzymes.

microbiology↗

bioRxiv2016

The molecular mechanism of the type IVa pilus motors

Type IVa pili are protein filaments essential for virulence in many bacterial pathogens; they extend and retract from the surface of bacterial cells to pull the bacteria forward with unprecedented force. They are used for attachment, swarming and twitching motility, biofilm formation, up-regulation of other virulence factors, and natural competence. The pilus is assembled by the motor subcomplex which consists of the inner membrane protein PilC and the cytoplasmic ATPase PilB. How PilB catalyzes this process is unknown, due in part to the lack of high-resolution structural information. Phylogenetic analysis of PilB-like ATPases, including GspE, PilT, BfpD, FlaI, and archaeal GspE2 revealed highly conserved residues essential for function in this family of ATPases. Here we report the structure of the core ATPase domains of Geobacter metalloreducens PilB bound to ADP and the non-hydrolysable ATP analogue, AMPPNP, at 3.4 and 2.3[A], respectively. Importantly, these structures were determined in non-saturating nucleotide conditions, revealing important differences in nucleotide binding between chains. Analysis of these differences revealed the sequential turnover of nucleotide by the chains, and the corresponding domain movements. Our data indicate a clockwise rotation of movement in PilB, which would support the assembly of a right-handed helical pilus. Conversely, our analysis suggests a counterclockwise rotation in PilT that would enable right-handed pilus disassembly. The proposed model provides insight into how this family of ATPases can power pilus extension and retraction with extraordinary forces.

microbiology↗