Uncovering the membrane-integrated SecAN protein that plays a key role in translocating nascent outer membrane proteins.

A large number of nascent polypeptides have to get across a membrane in targeting to the proper subcellular locations. The SecYEG protein complex, a homolog of the Sec61 complex in eukaryotic cells, has been viewed as the common translocon at the inner membrane for targeting proteins to three extracytoplasmic locations in Gram-negative bacteria, despite the lack of direct verification in living cells. Here, via unnatural amino acid-mediated protein-protein interaction analyses in living cells, in combination with genetic studies, we unveiled a hitherto unreported SecAN protein that seems to be directly involved in translocationg nascent outer membrane proteins across the plasma membrane; it consists of the N-terminal 375 residues of the SecA protein and exists as a membrane-integrated homooligomer. Our new findings place multiple previous observations related to bacterial protein targeting in proper biochemical and evolutionary contexts.

The Caenorhabditis elegans 12-kDa small heat shock proteins with little in vitro chaperone activity play crucial roles for its dauer formation, longevity, and reproduction.

Small heat shock proteins (sHSPs) are known to exhibit in vitro chaperone activity by suppressing the aggregation of misfolded proteins. The 12-kDa sHSPs (Hsp12s) subfamily members from Caenorhabditis elegans, including Hsp12.2, Hsp12.3, and Hsp12.6, however, are devoid of such chaperone activity, and their in vivo functions are poorly understood. Here we verified that Hsp12.1, similar to its homologs Hsp12.2, Hsp12.3, and Hsp12.6, hardly exhibited any chaperone activity. Strikingly, we demonstrated that these Hsp12s seem to play crucial physiological roles in C. elegans, for suppressing dauer formation and promoting both longevity and reproduction. A unique sHSP gene from Filarial nematode worm Brugia malayi was identified such that it encodes two products, one as a full-length Hsp12.6 protein and the other one having an N-terminal arm of normal length but lacks the C-terminal extension. This gene may represent an intermediate form in evolution from a common sHSP to a Hsp12. Together, our study offers insights on what biological functions the chaperone-defective sHSPs may exhibit and also implicates an evolutionary scenario for the unique Hsp12s subfamily.

A high-throughput genetically directed protein crosslinking analysis reveals the physiological relevance of the ATP synthase 'inserted' state.

ATP synthase, a highly conserved protein complex that has a subunit composition of α3 ß3 γδεab2 c8-15 for the bacterial enzyme, is a key player in supplying energy to living organisms. This protein complex consists of a peripheral F1 sector (α3 ß3 γδε) and a membrane-integrated Fo sector (ab2 c8-15 ). Structural analyses of the isolated protein components revealed that, remarkably, the C-terminal domain of its ε-subunit seems to adopt two dramatically different structures, but the physiological relevance of this conformational change remains largely unknown. In an attempt to decipher this, we developed a high-throughput in vivo protein photo-cross-linking analysis pipeline based on the introduction of the unnatural amino acid into the target protein via the scarless genome-targeted site-directed mutagenesis technique, and probing the cross-linked products via the high-throughput polyacrylamide gel electrophoresis technique. Employing this pipeline, we examined the interactions involving the C-terminal helix of the ε-subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the bacterial ATP synthase exists as an equilibrium between the 'inserted' and 'noninserted' state in cells, maintaining a moderate but significant level of net ATP synthesis when shifting to the former upon exposing to unfavorable energetically stressful conditions. Such a mechanism allows the bacterial ATP synthases to proportionally and instantly switch between two reversible functional states in responding to changing environmental conditions. Importantly, this high-throughput approach could allow us to decipher the physiological relevance of protein-protein interactions identified under in vitro conditions or to unveil novel physiological context-dependent protein-protein interactions that are unknown before.

Rapid Freezing Enables Aminoglycosides To Eradicate Bacterial Persisters via Enhancing Mechanosensitive Channel MscL-Mediated Antibiotic Uptake.

Bacterial persisters exhibit noninherited antibiotic tolerance and are linked to the recalcitrance of bacterial infections. It is very urgent but also challenging to develop antipersister strategies. Here, we report that 10-s freezing with liquid nitrogen dramatically enhances the bactericidal action of aminoglycoside antibiotics by 2 to 6 orders of magnitude against many Gram-negative pathogens, with weaker potentiation effects on Gram-positive bacteria. In particular, antibiotic-tolerant Escherichia coli and Pseudomonas aeruginosa persisters-which were prepared by treating exponential-phase cells with ampicillin, ofloxacin, the protonophore cyanide m-chlorophenyl hydrazone (CCCP), or bacteriostatic antibiotics-can be effectively killed. We demonstrated, as a proof of concept, that freezing potentiated the aminoglycosides' killing of P. aeruginosa persisters in a mouse acute skin wound model. Mechanistically, freezing dramatically increased the bacterial uptake of aminoglycosides regardless of the presence of CCCP, indicating that the effects are independent of the proton motive force (PMF). In line with these results, we found that the effects were linked to freezing-induced cell membrane damage and were attributable, at least partly, to the mechanosensitive ion channel MscL, which was able to directly mediate such freezing-enhanced aminoglycoside uptake. In view of these results, we propose that the freezing-induced aminoglycoside potentiation is achieved by freezing-induced cell membrane destabilization, which, in turn, activates the MscL channel, which is able to effectively take up aminoglycosides in a PMF-independent manner. Our work may pave the way for the development of antipersister strategies that utilize the same mechanism as freezing but do so without causing any injury to animal cells.IMPORTANCE Antibiotics have long been used to successfully kill bacterial pathogens, but antibiotic resistance/tolerance usually has led to the failure of antibiotic therapy, and it has become a severe threat to human health. How to improve the efficacy of existing antibiotics is of importance for combating antibiotic-resistant/tolerant pathogens. Here, we report that 10-s rapid freezing with liquid nitrogen dramatically enhanced the bactericidal action of aminoglycoside antibiotics by 2 to 6 orders of magnitude against many bacterial pathogens in vitro and also in a mouse skin wound model. In particular, such combined treatment was able to effectively kill persister cells of Escherichia coli and Pseudomonas aeruginosa, which are per se tolerant of conventional treatment with bactericidal antibiotics for several hours. We also demonstrated that freezing-induced aminoglycoside potentiation was apparently linked to freezing-induced cell membrane damage that may have activated the mechanosensitive ion channel MscL, which, in turn, was able to effectively uptake aminoglycoside antibiotics in a proton motive force-independent manner. Our report sheds light on the development of a new strategy against bacterial pathogens by combining existing antibiotics with a conventional physical treatment or with MscL agonists.

Degp degrades a wide range of substrate proteins in Escherichia coli under stress conditions.

DegP, a periplasmic dual-functional protease and chaperone in Gram-negative bacteria, is critical for bacterial stress resistance, but the precise underlying mechanisms are not fully understood. Here, we show that the protease function of DegP is critical for Escherichia coli cells to maintain membrane integrity, particularly under heat shock conditions (42°C). Site-directed photo-cross-linking, mass spectrometry and immunoblotting analyses reveal that both periplasmic proteins (e.g. OppA and MalE) and ß-barrel outer membrane proteins (OMPs) are DegP-interacting proteins and that OppA is degraded by DegP in vitro and in vivo at 42°C. In addition, OmpA and BamA, chimeric ß-barrel OMPs containing a soluble periplasmic domain, are bound to DegP in both unfolded and folded forms, whereas only the unfolded forms are degradable by DegP. The presence of folded OmpA as a substrate of DegP is attributed to its periplasmic domain, which is resistant to DegP degradation and even generally protects pure ß-barrel OMPs from degradation in an intra-molecular way. Furthermore, a pair of residues (R262 and V328) in the PDZ domain-1 of DegP play important roles for binding unfolded and folded ß-barrel OMPs, with R262 being critical. Our study, together with earlier reports, indicates that DegP plays a critical role in protein quality control in the bacterial periplasm by degrading both periplasmic proteins and ß-barrel OMPs under stress conditions and likely also by participating in the folding of chimeric ß-barrel OMPs. A working model is proposed to illustrate the finely tuned functions of DegP with respect to different substrate proteins.

Biogenesis, quality control, and structural dynamics of proteins as explored in living cells via site-directed photocrosslinking.

Protein biogenesis and quality control are essential to maintaining a functional pool of proteins and involve numerous protein factors that dynamically and transiently interact with each other and with the substrate proteins in living cells. Conventional methods are hardly effective for studying dynamic, transient, and weak protein-protein interactions that occur in cells. Herein, we review how the site-directed photocrosslinking approach, which relies on the genetic incorporation of a photoreactive unnatural amino acid into a protein of interest at selected individual amino acid residue positions and the covalent trapping of the interacting proteins upon ultraviolent irradiation, has become a highly efficient way to explore the aspects of protein contacts in living cells. For example, in the past decade, this approach has allowed the profiling of the in vivo substrate proteins of chaperones or proteases under both physiologically optimal and stressful (e.g., acidic) conditions, mapping residues located at protein interfaces, identifying new protein factors involved in the biogenesis of membrane proteins, trapping transiently formed protein complexes, and snapshotting different structural states of a protein. We anticipate that the site-directed photocrosslinking approach will play a fundamental role in dissecting the detailed mechanisms of protein biogenesis, quality control, and dynamics in the future.

Subunit interactions as mediated by "non-interface" residues in living cells for multiple homo-oligomeric proteins.

Protein-protein interaction, including protein homo-oligomerization, is commonly believed to occur through a specific interface made of a limited number of amino acid residues. Here our systematic in vivo photo-crosslinking analysis via genetically incorporated unnatural amino acids unexpectedly shows that the dimerization of HdeA, an acid stress chaperone, is mediated by the residues along its whole polypeptide. These include those "forbidden" residues that are far away from the dimerization interface as judged according to the reported 3-D structure. We demonstrate that such dimerization, though intriguing, is neither a result of protein over-expression nor of any structural disturbance caused by the residue replacement. Similar unexpected dimerization also occurs for two other oligomeric proteins, IbpB (a molecular chaperone existing as polydispersed oligomers in vitro) and DegP (a protease existing as hexamers in vitro). In contrast to these three proteins, dimerization of a few other oligomeric proteins (e.g., OmpF, LamB, SurA, FtsZ and FkpA) that we similarly examined in living cells seems to be mediated only by specific residues. Together, our unexpected observations suggest that, for some oligomeric proteins such as HdeA, IbpB and DegP, their subunit interactions in living cells can also be mediated by residues other than those located at the interfaces as revealed by in vitro structure determination. Our observations might be partially explained by the formation of "encounter complex" or by protein conformational dynamics. Our findings provide new insights on understanding protein-protein interactions and encounter complex formation in living cells.

Regrowth-delay body as a bacterial subcellular structure marking multidrug-tolerant persisters.

Bacteria have long been recognized to be capable of entering a phenotypically non-growing persister state, in which the cells exhibit an extended regrowth lag and a multidrug tolerance, thus posing a great challenge in treating infectious diseases. Owing to their non-inheritability, low abundance of existence, lack of metabolic activities, and high heterogeneity, properties of persisters remain poorly understood. Here, we report our accidental discovery of a subcellular structure that we term the regrowth-delay body, which is formed only in non-growing bacterial cells and sequesters multiple key proteins. This structure, that dissolves when the cell resumes growth, is able to be viewed as a marker of persisters. Our studies also indicate that persisters exhibit different depth of persistence, as determined by the status of their regrowth-delay bodies. Our findings imply that suppressing the formation and/or promoting the dissolution of regrowth-delay bodies could be viable strategies for eradicating persisters.

DegP functions as a critical protease for bacterial acid resistance.

To counteract the lethal acid stress, bacteria explore such strategies as cytoplasmic decarboxylase-catalyzed proton consumption and periplasmic chaperone-assisted protein refolding. Here, we report a periplasmic protease-mediated acid resistance mechanism in Escherichia coli. Deletion of the protease gene degP dramatically decreases the viability of late log or early stationary phase cells against the extreme acid stress (pH 2.3), which can only be minimally rescued by complementary expression of the protease-deficient DegP(S210A) mutant protein. Similarly, DegQ, a homolog of DegP, also contributes to the bacterial acid resistance, but SurA as an important periplasmic chaperone hardly exhibits protection effect. In vitro studies reveal that DegP completely loses its protease activity under acidic condition but is able to partially reactivate upon neutralization. Importantly, we demonstrate the interaction of DegP with typical cellular substrate proteins in cells during acid stress and/or recovery stages by using unnatural amino acid-mediated in vivo photo-crosslinking, as well as the degradation of periplasmic proteins by DegP during recovery after acidic denaturation. These data illustrate the role of DegP in bacterial acid resistance conceivably via degrading those acid-induced misfolded proteins. Our findings, together with earlier reports, suggest a comprehensive acid resistance strategy adopted by bacteria such that in the ATP-deficient extra-cytoplasm, the inevitable misfolded proteins induced by acid stress are refolded by a chaperone (e.g., HdeA/HdeB) and/or cleaved by a protease (e.g., DegP/DegQ) while in the cytoplasm excessive protons are directly consumed or exported.

Lateral interactions between protofilaments of the bacterial tubulin homolog FtsZ are essential for cell division.

The prokaryotic tubulin homolog FtsZ polymerizes into protofilaments, which further assemble into higher-order structures at future division sites to form the Z-ring, a dynamic structure essential for bacterial cell division. The precise nature of interactions between FtsZ protofilaments that organize the Z-ring and their physiological significance remain enigmatic. In this study, we solved two crystallographic structures of a pair of FtsZ protofilaments, and demonstrated that they assemble in an antiparallel manner through the formation of two different inter-protofilament lateral interfaces. Our in vivo photocrosslinking studies confirmed that such lateral interactions occur in living cells, and disruption of the lateral interactions rendered cells unable to divide. The inherently weak lateral interactions enable FtsZ protofilaments to self-organize into a dynamic Z-ring. These results have fundamental implications for our understanding of bacterial cell division and for developing antibiotics that target this key process.

The function of the DegP (HtrA) protein: Protease versus chaperone.

The DegP (or HtrA) is a highly conserved family of proteins functioning in all living organisms. It was initially identified as a protease functioning in the periplasmic space of the Gram-negative bacterial cells. It was later reported to also exhibit chaperone activity and thus has been designated as a bifunctional protein. However, recent studies demonstrated that in living cells it more likely functions only as a protease with hardly detectable chaperone activities. In this review, I will summarize the evidences clarifying that DegP more likely only functions as a protease rather than as a chaperone in cells. © 2016 IUBMB Life, 68(11):904-907, 2016.

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of ß-Barrel Outer Membrane Proteins in Bacteria.

ß-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent ß-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent ß-barrel OMPs largely via its N-domain but with ß-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent ß-barrel OMPs interact with SurA via their N- and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving ß-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for ß-barrel OMP biogenesis.

A reciprocating motion-driven rotation mechanism for the ATP synthase.

The ATP synthase (having a typical subunit composition of α3ß3γδεab2c8-15) employs an intriguing rotary mechanism for the generation of ATP from ADP and Pi, using energy stored in a transmembrane proton gradient. The conventional rotary model, although being generally accepted, remains difficult to explain certain experimental observations. Here we propose an alternative rotary model for the ATP synthase such that what rotates is the catalytic α3ß3 cylinder rather than the central stalk and the membrane-embedded c-ring. Specifically, the membrane translocation of protons would induce a cycled conformational change in the c-ring, leading to a reciprocating motion of the attached central stalk, which in turn drives the unidirectional rotation of the α3ß3 cylinder. Such a reciprocating motion-driven rotation mechanism is somehow analogous to the working mechanism of a retractable click ballpoint pen. Our new model not only explains the experimental observations that have been difficult to reconcile with the conventional model but also avoids its theoretical illogicality.