Inner membrane YfgM-PpiD heterodimer acts as a functional unit that associates with the SecY/E/G translocon and promotes protein translocation.

PpiD and YfgM are inner membrane proteins that are both composed of an N-terminal transmembrane segment and a C-terminal periplasmic domain. Escherichia coli YfgM and PpiD form a stable complex that interacts with the SecY/E/G (Sec) translocon, a channel that allows protein translocation across the cytoplasmic membrane. Although PpiD is known to function in protein translocation, the functional significance of PpiD-YfgM complex formation as well as the molecular mechanisms of PpiD-YfgM and PpiD/YfgM-Sec translocon interactions remain unclear. Here, we conducted genetic and biochemical studies using yfgM and ppiD mutants and demonstrated that a lack of YfgM caused partial PpiD degradation at its C-terminal region and hindered the membrane translocation of Vibrio protein export monitoring polypeptide (VemP), a Vibrio secretory protein, in both E. coli and Vibrio alginolyticus. While ppiD disruption also impaired VemP translocation, we found that the yfgM and ppiD double deletion exhibited no additive or synergistic effects. Together, these results strongly suggest that both PpiD and YfgM are required for efficient VemP translocation. Furthermore, our site-directed in vivo photocrosslinking analysis revealed that the tetratricopeptide repeat domain of YfgM and a conserved structural domain (NC domain) in PpiD interact with each other and that YfgM, like PpiD, directly interacts with the SecG translocon subunit. Crosslinking analysis also suggested that PpiD-YfgM complex formation is required for these proteins to interact with SecG. In summary, we propose that PpiD and YfgM form a functional unit that stimulates protein translocation by facilitating their proper interactions with the Sec translocon.

First Penicillin-Binding Protein Occupancy Patterns for 15 ß-Lactams and ß-Lactamase Inhibitors in Mycobacterium abscessus.

Mycobacterium abscessus causes serious infections that often require over 18 months of antibiotic combination therapy. There is no standard regimen for the treatment of M. abscessus infections, and the multitude of combinations that have been used clinically have had low success rates and high rates of toxicities. With ß-lactam antibiotics being safe, double ß-lactam and ß-lactam/ß-lactamase inhibitor combinations are of interest for improving the treatment of M. abscessus infections and minimizing toxicity. However, a mechanistic approach for building these combinations is lacking since little is known about which penicillin-binding protein (PBP) target receptors are inactivated by different ß-lactams in M. abscessus We determined the preferred PBP targets of 13 ß-lactams and 2 ß-lactamase inhibitors in two M. abscessus strains and identified PBP sequences by proteomics. The Bocillin FL binding assay was used to determine the ß-lactam concentrations that half-maximally inhibited Bocillin binding (50% inhibitory concentrations [IC50s]). Principal component analysis identified four clusters of PBP occupancy patterns. Carbapenems inactivated all PBPs at low concentrations (0.016 to 0.5 mg/liter) (cluster 1). Cephalosporins (cluster 2) inactivated PonA2, PonA1, and PbpA at low (0.031 to 1 mg/liter) (ceftriaxone and cefotaxime) or intermediate (0.35 to 16 mg/liter) (ceftazidime and cefoxitin) concentrations. Sulbactam, aztreonam, carumonam, mecillinam, and avibactam (cluster 3) inactivated the same PBPs as cephalosporins but required higher concentrations. Other penicillins (cluster 4) specifically targeted PbpA at 2 to 16 mg/liter. Carbapenems, ceftriaxone, and cefotaxime were the most promising ß-lactams since they inactivated most or all PBPs at clinically relevant concentrations. These first PBP occupancy patterns in M. abscessus provide a mechanistic foundation for selecting and optimizing safe and effective combination therapies with ß-lactams.

The transpeptidase PbpA and noncanonical transglycosylase RodA of Mycobacterium tuberculosis play important roles in regulating bacterial cell lengths.

The cell wall of Mycobacterium tuberculosis (Mtb) is a complex structure that protects the pathogen in hostile environments. Peptidoglycan (PG), which helps determine the morphology of the cell envelope, undergoes substantial remodeling under stress. This meshwork of linear chains of sugars, cross-linked through attached peptides, is generated through the sequential action of enzymes termed transglycosylases and transpeptidases. The Mtb genome encodes two classical transglycosylases and four transpeptidases, the functions of which are not fully elucidated. Here, we present work on the yet uncharacterized transpeptidase PbpA and a nonclassical transglycosylase RodA. We elucidate their roles in regulating in vitro growth and in vivo survival of pathogenic mycobacteria. We find that RodA and PbpA are required for regulating cell length, but do not affect mycobacterial growth. Biochemical analyses show PbpA to be a classical transpeptidase, whereas RodA is identified to be a member of an emerging class of noncanonical transglycosylases. Phosphorylation of RodA at Thr-463 modulates its biological function. In a guinea pig infection model, RodA and PbpA are found to be required for both bacterial survival and formation of granuloma structures, thus underscoring the importance of these proteins in mediating mycobacterial virulence in the host. Our results emphasize the fact that whereas redundant enzymes probably compensate for the absence of RodA or PbpA during in vitro growth, the two proteins play critical roles for the survival of the pathogen inside its host.

A photo-cross-linking approach to monitor folding and assembly of newly synthesized proteins in a living cell.

Many proteins form multimeric complexes that play crucial roles in various cellular processes. Studying how proteins are correctly folded and assembled into such complexes in a living cell is important for understanding the physiological roles and the qualitative and quantitative regulation of the complex. However, few methods are suitable for analyzing these rapidly occurring processes. Site-directed in vivo photo-cross-linking is an elegant technique that enables analysis of protein-protein interactions in living cells with high spatial resolution. However, the conventional site-directed in vivo photo-cross-linking method is unsuitable for analyzing dynamic processes. Here, by combining an improved site-directed in vivo photo-cross-linking technique with a pulse-chase approach, we developed a new method that can analyze the folding and assembly of a newly synthesized protein with high spatiotemporal resolution. We demonstrate that this method, named the pulse-chase and in vivo photo-cross-linking experiment (PiXie), enables the kinetic analysis of the formation of an Escherichia coli periplasmic (soluble) protein complex (PhoA). We also used our new technique to investigate assembly/folding processes of two membrane complexes (SecD-SecF in the inner membrane and LptD-LptE in the outer membrane), which provided new insights into the biogenesis of these complexes. Our PiXie method permits analysis of the dynamic behavior of various proteins and enables examination of protein-protein interactions at the level of individual amino acid residues. We anticipate that our new technique will have valuable utility for studies of protein dynamics in many organisms.

Site-Specific Photocrosslinking to Investigate Toxin Delivery Mediated by the Bacterial ß-Barrel Assembly Machine.

Contact-dependent inhibition (CDI) is a mechanism of interbacterial competition in Gram-negative organisms that relies on a specific interaction between a CdiA protein on the surface of one cell and a ß-barrel protein on the surface of a neighboring cell. This interaction triggers the transport of a protein toxin into the neighboring cell where it exerts its lethal activity. Several classes of CdiA proteins that bind to different ß-barrel receptors have been identified, but the molecular mechanism by which they deliver their toxins across the outer membranes of their target cells is poorly understood. Here we describe the use of site-specific photocrosslinking to characterize the interaction between a CdiA protein and its receptor. We describe the method for an E. coli CdiA that utilizes BamA as its receptor. BamA's central role in assembling ß-barrel proteins in the outer membrane makes its role in CDI particularly intriguing; it suggests that these two different protein transport processes might share mechanistic features. Our in vitro photocrosslinking method is useful in elucidating early steps in the CDI mechanism, but it could be adapted to study later steps or to study other CdiA-receptor pairs.

A Photo-Crosslinking Approach to Monitoring the Assembly of an LptD Intermediate with LptE in a Living Cell.

Elucidating the dynamic behavior of proteins in living cells is extremely important for understanding the physiological roles of biological processes. The site-specific in vivo photo-crosslinking approach using a photoreactive unnatural amino acid enables the analysis of protein interactions with high spatial resolution in vivo. Recently, by improving the photo-crosslinking technique, we developed the "PiXie" method for the analysis of dynamic interactions of newly synthesized proteins. Here, we describe the detailed protocols of the "PiXie" method and its application to the analysis of the assembly processes of the lipopolysaccharide translocon components, a ß-barrel outer membrane protein, LptD, and a lipoprotein, LptE.

Edge-strand of BepA interacts with immature LptD on the ß-barrel assembly machine to direct it to on- and off-pathways.

The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier. Escherichia coli periplasmic Zn-metallopeptidase BepA contributes to the maintenance of OM integrity through its involvement in the biogenesis and degradation of LptD, a ß-barrel protein component of the lipopolysaccharide translocon. BepA either promotes the maturation of LptD when it is on the normal assembly pathway (on-pathway) or degrades it when its assembly is compromised (off-pathway). BepA performs these functions probably on the ß-barrel assembly machinery (BAM) complex. However, how BepA recognizes and directs an immature LptD to different pathways remains unclear. Here, we explored the interactions among BepA, LptD, and the BAM complex. We found that the interaction of the BepA edge-strand located adjacent to the active site with LptD was crucial not only for proteolysis but also, unexpectedly, for assembly promotion of LptD. Site-directed crosslinking analyses indicated that the unstructured N-terminal half of the ß-barrel-forming domain of an immature LptD contacts with the BepA edge-strand. Furthermore, the C-terminal region of the ß-barrel-forming domain of the BepA-bound LptD intermediate interacted with a 'seam' strand of BamA, suggesting that BepA recognized LptD assembling on the BAM complex. Our findings provide important insights into the functional mechanism of BepA.

A photo-cross-linking approach to monitor protein dynamics in living cells.

BACKGROUND: Proteins, which comprise one of the major classes of biomolecules that constitute a cell, interact with other cellular factors during both their biogenesis and functional states. Studying not only static but also transient interactions of proteins is important to understand their physiological roles and regulation mechanisms. However, only a limited number of methods are available to analyze the dynamic behaviors of proteins at the molecular level in a living cell. The site-directed in vivo photo-cross-linking approach is an elegant technique to capture protein interactions with high spatial resolution in a living cell. SCOPE OF REVIEW: Here, we review the in vivo photo-cross-linking approach including its recent applications and the potential problems to be considered. We also introduce a new in vivo photo-cross-linking-based technique (PiXie) to study protein dynamics with high spatiotemporal resolution. MAJOR CONCLUSIONS: In vivo photo-cross-linking enables us to capture weak/transient protein interactions with high spatial resolution, and allows for identification of interacting factors. Moreover, the PiXie approach can be used to monitor rapid folding/assembly processes of proteins in living cells. GENERAL SIGNIFICANCE: In vivo photo-cross-linking is a simple method that has been used to analyze the dynamic interactions of many cellular proteins. Originally developed in Escherichia coli, this system has been extended to studies in various organisms, making it a fundamental technique for investigating dynamic protein interactions in many cellular processes. This article is part of a Special issue entitled "Novel major techniques for visualizing 'live' protein molecules" edited by Dr. Daisuke Kohda.

Interrogating the Dimerization Interface of the Prion Protein Via Site-Specific Mutations to p-Benzoyl-L-Phenylalanine.

Transmissible spongiform encephalopathies are centered on the conformational transition of the prion protein from a mainly helical, monomeric structure to a ß-sheet rich ordered aggregate. Experiments indicate that the main infectious and toxic species in this process are however shorter oligomers, formation of which from the monomers is yet enigmatic. Here, we created 25 variants of the mouse prion protein site-specifically containing one genetically-incorporated para-benzoyl-phenylalanine (pBpa), a cross-linkable non-natural amino acid, in order to interrogate the interface of a prion protein-dimer, which might lie on the pathway of oligomerization. Our results reveal that the N-terminal part of the prion protein, especially regions around position 127 and 107, is integral part of the dimer interface. These together with additional pBpa-containing variants of mPrP might also facilitate to gain more structural insights into oligomeric and fibrillar prion protein species including the pathological variants.

Insights into the function of FhaA, a cell division-associated protein in mycobacteria.

FhaA is a forkhead-associated domain-containing protein, the depletion of which leads to accumulation of peptidoglycan (PG) precursors at the septum and poles in Mycobacterium smegmatis (M. smegmatis), by a mechanism undefined thus far. To elucidate its function, we constructed an fhaA (MSMEG_0035) knockout (ΔfhaA) strain in M. smegmatis and demonstrated that this gene is dispensable for in vitro growth. The mutant showed a short cell length phenotype due to a probable defect in cell elongation/cell wall synthesis, which was reversed by complementation with both M. smegmatis and Mycobacterium tuberculosis (M. tb) fhaA (Rv0020c), confirming their association with the observed phenotype. The identification of penicillin binding protein A (PbpA), a PG biosynthesis enzyme as an interacting partner for mycobacterial FhaA, provided a hint into the functioning of FhaA. A drastic reduction in the levels of ectopically expressed PbpA in the ΔfhaA mutant vs wild-type M. smegmatis suggested that FhaA interacts with and stabilises PbpA. In addition, the fhaA deletion mutant was sensitive to multiple classes of antibiotics pointing to a general permeability defect. Our findings uncover a role for FhaA in PG biosynthesis and suggest its involvement in the maintenance of mycobacterial cell envelope integrity.

Interactions of Flagellar Structural Subunits with the Membrane Export Machinery.

During assembly of the bacterial flagellum, structural subunits synthesized inside the cell must be exported across the cytoplasmic membrane before they can crystallize into the nascent flagellar structure. This export process is facilitated by a specialized Flagellar Type III Secretion System (fT3SS) located at the base of each flagellum. Here, we describe three methods-isothermal titration calorimetry, photo-crosslinking using unnatural amino acids, and a subunit capture assay-used to investigate the interactions of flagellar structural subunits with the membrane export machinery component FlhB.

Polishing the craft of genetic diversity creation in directed evolution.

Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating "smart" libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field.