Correlative montage parallel array cryo-tomography for in situ structural cell biology.

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here we present robust tools for montage parallel array cryo-tomography (MPACT) tailored for vitrified specimens. The combination of correlative cryo-fluorescence microscopy, focused-ion-beam milling, substrate micropatterning, and MPACT supports studies that contextually define the three-dimensional architecture of cells. To further extend the flexibility of MPACT, tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

Atomic-level architecture of Caulobacter crescentus flagellar filaments provide evidence for multi-flagellin filament stabilization.

Flagella are dynamic, ion-powered machines with assembly pathways that are optimized for efficient flagella production. In bacteria, dozens of genes are coordinated at specific times in the cell lifecycle to generate each component of the flagellum. This is the case for Caulobacter crescentus, but little is known about why this species encodes six different flagellin genes. Furthermore, little is known about the benefits multi-flagellin species possess over single flagellin species, if any, or what molecular properties allow for multi-flagellin filaments to assemble. Here we present an in-depth analysis of several single flagellin filaments from C. crescentus, including an extremely well-resolved structure of a bacterial flagellar filament. We highlight key molecular interactions that differ between each bacterial strain and speculate how these interactions may alleviate or impose helical strain on the overall architecture of the filament. We detail conserved residues within the flagellin subunit that allow for the synthesis of multi-flagellin filaments. We further comment on how these molecular differences impact bacterial motility and highlight how no single flagellin filament achieves wild-type levels of motility, suggesting C. crescentus has evolved to produce a filament optimized for motility comprised of six flagellins. Finally, we highlight an ordered arrangement of glycosylation sites on the surface of the filaments and speculate how these sites may protect the ß-hairpin located on the surface exposed domain of the flagellin subunit.

The role of CenKR in the coordination of Rhodobacter sphaeroides cell elongation and division.

Cell elongation and division are essential aspects of the bacterial life cycle that must be coordinated for viability and replication. The impact of misregulation of these processes is not well understood as these systems are often not amenable to traditional genetic manipulation. Recently, we reported on the CenKR two-component system (TCS) in the Gram-negative bacterium Rhodobacter sphaeroides that is genetically tractable, widely conserved in α-proteobacteria, and directly regulates the expression of components crucial for cell elongation and division, including genes encoding subunit of the Tol-Pal complex. In this work, we show that overexpression of cenK results in cell filamentation and chaining. Using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), we generated high-resolution two-dimensional (2D) images and three-dimensional (3D) volumes of the cell envelope and division septum of wild-type cells and a cenK overexpression strain finding that these morphological changes stem from defects in outer membrane (OM) and peptidoglycan (PG) constriction. By monitoring the localization of Pal, PG biosynthesis, and the bacterial cytoskeletal proteins MreB and FtsZ, we developed a model for how increased CenKR activity leads to changes in cell elongation and division. This model predicts that increased CenKR activity decreases the mobility of Pal, delaying OM constriction, and ultimately disrupting the midcell positioning of MreB and FtsZ and interfering with the spatial regulation of PG synthesis and remodeling. IMPORTANCE By coordinating cell elongation and division, bacteria maintain their shape, support critical envelope functions, and orchestrate division. Regulatory and assembly systems have been implicated in these processes in some well-studied Gram-negative bacteria. However, we lack information on these processes and their conservation across the bacterial phylogeny. In R. sphaeroides and other α-proteobacteria, CenKR is an essential two-component system (TCS) that regulates the expression of genes known or predicted to function in cell envelope biosynthesis, elongation, and/or division. Here, we leverage unique features of CenKR to understand how increasing its activity impacts cell elongation/division and use antibiotics to identify how modulating the activity of this TCS leads to changes in cell morphology. Our results provide new insight into how CenKR activity controls the structure and function of the bacterial envelope, the localization of cell elongation and division machinery, and cellular processes in organisms with importance in health, host-microbe interactions, and biotechnology.

Conserved Proline Residues of Bacillus subtilis Intramembrane Metalloprotease SpoIVFB Are Important for Substrate Interaction and Cleavage.

Intramembrane metalloproteases (IMMPs) regulate diverse biological processes by cleaving membrane-associated substrates within the membrane or near its surface. SpoIVFB is an intramembrane metalloprotease of Bacillus subtilis that cleaves Pro-σK during endosporulation. Intramembrane metalloproteases have a broadly conserved NPDG motif, which in the structure of an archaeal enzyme is located in a short loop that interrupts a transmembrane segment facing the active site. The aspartate residue of the NPDG motif acts as a ligand of the zinc ion involved in catalysis. The functions of other residues in the short loop are less well understood. We found that the predicted short loop of SpoIVFB contains two highly conserved proline residues, P132 of the NPDG motif and P135. Mutational analysis revealed that both proline residues are important for Pro-σK cleavage in Escherichia coli engineered to synthesize the proteins. Substitutions for either residue also impaired the Pro-σK interaction with SpoIVFB in copurification assays. Disulfide cross-linking experiments showed that the predicted short loop of SpoIVFB is in proximity to the N-terminal pro-sequence region (Proregion) of Pro-σK. Alanine substitutions for N129 and P132 of the SpoIVFB NPDG motif reduced cross-linking between its predicted short loop and the Proregion more than a P135A substitution. Conversely, the SpoIVFB P135A substitution reduced Pro-σK cleavage more than the N129A and P132A substitutions during sporulation of B. subtilis. We conclude that all three conserved residues of SpoIVFB are important for substrate interaction and cleavage, and we propose that P135 is necessary to position D137 to act as a zinc ligand. IMPORTANCE Intramembrane metalloproteases (IMMPs) function in numerous signaling pathways. Bacterial IMMPs govern stress responses, including the sporulation of some species, thus enhancing the virulence and persistence of pathogens. Knowledge of IMMP-substrate interactions could aid therapeutic design, but structures of IMMP·substrate complexes are unknown. We examined the interaction of the IMMP SpoIVFB with its substrate Pro-σK, whose cleavage is required for Bacillus subtilis endosporulation. We found that conserved proline residues in a short loop predicted to interrupt a SpoIVFB transmembrane segment are important for Pro-σK binding and cleavage. The corresponding residues of the Escherichia coli IMMP RseP have also been shown to be important for substrate interaction and cleavage, suggesting that this is a broadly conserved feature of IMMPs, potentially suitable as a therapeutic target.

Flagellar Structures from the Bacterium Caulobacter crescentus and Implications for Phage ϕ CbK Predation of Multiflagellin Bacteria.

Caulobacter crescentus is a Gram-negative alphaproteobacterium that commonly lives in oligotrophic fresh- and saltwater environments. C. crescentus is a host to many bacteriophages, including ϕCbK and ϕCbK-like bacteriophages, which require interaction with the bacterial flagellum and pilus complexes during adsorption. It is commonly thought that the six paralogs of the flagellin gene present in C. crescentus are important for bacteriophage evasion. Here, we show that deletion of specific flagellins in C. crescentus can indeed attenuate ϕCbK adsorption efficiency, although no single deletion completely ablates ϕCbK adsorption. Thus, the bacteriophage ϕCbK likely recognizes a common motif among the six known flagellins in C. crescentus with various degrees of efficiency. Interestingly, we observe that most deletion strains still generate flagellar filaments, with the exception of a strain that contains only the most divergent flagellin, FljJ, or a strain that contains only FljN and FljO. To visualize the surface residues that are likely recognized by ϕCbK, we determined two high-resolution structures of the FljK filament, with and without an amino acid substitution that induces straightening of the filament. We observe posttranslational modifications on conserved surface threonine residues of FljK that are likely O-linked glycans. The possibility of interplay between these modifications and ϕCbK adsorption is discussed. We also determined the structure of a filament composed of a heterogeneous mixture of FljK and FljL, the final resolution of which was limited to approximately 4.6 Å. Altogether, this work builds a platform for future investigations of how phage ϕCbK infects C. crescentus at the molecular level.IMPORTANCE Bacterial flagellar filaments serve as an initial attachment point for many bacteriophages to bacteria. Some bacteria harbor numerous flagellin genes and are therefore able to generate flagellar filaments with complex compositions, which is thought to be important for evasion from bacteriophages. This study characterizes the importance of the six flagellin genes in C. crescentus for infection by bacteriophage ϕCbK. We find that filaments containing the FljK flagellin are the preferred substrate for bacteriophage ϕCbK. We also present a high-resolution structure of a flagellar filament containing only the FljK flagellin, which provides a platform for future studies on determining how bacteriophage ϕCbK attaches to flagellar filaments at the molecular level.

Channels modestly impact compartment-specific ATP levels during Bacillus subtilis sporulation and a rise in the mother cell ATP level is not necessary for Pro-σK cleavage.

Starvation of Bacillus subtilis initiates endosporulation involving formation of mother cell (MC) and forespore (FS) compartments. During engulfment, the MC membrane migrates around the FS and protein channels connect the two compartments. The channels are necessary for postengulfment FS gene expression, which relieves inhibition of SpoIVFB, an intramembrane protease that cleaves Pro-σK , releasing σK into the MC. SpoIVFB has an ATP-binding domain exposed to the MC cytoplasm, but the role of ATP in regulating Pro-σK cleavage has been unclear, as has the impact of the channels on MC and FS ATP levels. Using luciferase produced separately in each compartment to measure relative ATP concentrations during sporulation, we found that the MC ATP concentration rises about twofold coincident with increasing cleavage of Pro-σK , and the FS ATP concentration does not decline. Mutants lacking a channel protein or defective in channel protein turnover exhibited modest and varied effects on ATP levels, which suggested that low ATP concentration does not explain the lack of postengulfment FS gene expression in channel mutants. Furthermore, a rise in the MC ATP level was not necessary for Pro-σK cleavage by SpoIVFB, based on analysis of mutants that bypass the need for relief of SpoIVFB inhibition.

Interaction of intramembrane metalloprotease SpoIVFB with substrate Pro-σK.

Intramembrane proteases (IPs) cleave membrane-associated substrates in nearly all organisms and regulate diverse processes. A better understanding of how these enzymes interact with their substrates is necessary for rational design of IP modulators. We show that interaction of Bacillus subtilis IP SpoIVFB with its substrate Pro-σK depends on particular residues in the interdomain linker of SpoIVFB. The linker plus either the N-terminal membrane domain or the C-terminal cystathione-ß-synthase (CBS) domain of SpoIVFB was sufficient for the interaction but not for cleavage of Pro-σK Chemical cross-linking and mass spectrometry of purified, inactive SpoIVFB-Pro-σK complex indicated residues of the two proteins in proximity. A structural model of the complex was built via partial homology and by using constraints based on cross-linking data. In the model, the Proregion of Pro-σK loops into the membrane domain of SpoIVFB, and the rest of Pro-σK interacts extensively with the linker and the CBS domain of SpoIVFB. The extensive interaction is proposed to allow coordination between ATP binding by the CBS domain and Pro-σK cleavage by the membrane domain.

Bacillus subtilis Intramembrane Protease RasP Activity in Escherichia coli and In Vitro.

RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli, we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli, including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His6-MBP-RsiW(73-118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His6-MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL.IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are typically inferred from genetic studies in the native bacterium. Evidence for direct effects has come sometimes from coexpression of the enzyme and potential substrate in a heterologous host and rarely from biochemical reconstitution of cleavage in vitro We applied these two approaches to the B. subtilis enzyme RasP and its proposed substrates RsiW and FtsL. We discovered potential pitfalls and solutions in heterologous coexpression experiments in E. coli, providing evidence that both substrates are cleaved by RasP in vivo but, surprisingly, that only RsiW was cleaved in vitro, suggesting that FtsL has an additional requirement.

Complex Formed between Intramembrane Metalloprotease SpoIVFB and Its Substrate, Pro-σK.

Intramembrane metalloproteases (IMMPs) are conserved from bacteria to humans and control many important signaling pathways, but little is known about how IMMPs interact with their substrates. SpoIVFB is an IMMP that cleaves Pro-σ(K) during Bacillus subtilis endospore formation. When catalytically inactive SpoIVFB was coexpressed with C-terminally truncated Pro-σ(K)(1-126) (which can be cleaved by active SpoIVFB) in Escherichia coli, the substrate dramatically improved solubilization of the enzyme from membranes with mild detergents. Both the Pro(1-20) and σ(K)(21-126) parts contributed to improving SpoIVFB solubilization from membranes, but only the σ(K) part was needed to form a stable complex with SpoIVFB in a pulldown assay. The last 10 residues of SpoIVFB were required for improved solubilization from membranes by Pro-σ(K)(1-126) and for normal interaction with the substrate. The inactive SpoIVFB·Pro-σ(K)(1-126)-His6 complex was stable during affinity purification and gel filtration chromatography. Disulfide cross-linking of the purified complex indicated that it resembled the complex formed in vivo Ion mobility-mass spectrometry analysis resulted in an observed mass consistent with a 4:2 SpoIVFB·Pro-σ(K)(1-126)-His6 complex. Stepwise photobleaching of SpoIVFB fused to a fluorescent protein supported the notion that the enzyme is tetrameric during B. subtilis sporulation. The results provide the first evidence that an IMMP acts as a tetramer, give new insights into how SpoIVFB interacts with its substrate, and lay the foundation for further biochemical analysis of the enzyme·substrate complex and future structural studies.

Evolution of the Stx2-encoding prophage in persistent bovine Escherichia coli O157:H7 strains.

Escherichia coli O157:H7 is a human pathogen that resides asymptomatically in its bovine host. The level of Shiga toxin (Stx) produced is variable in bovine-derived strains in contrast to human isolates that mostly produce high levels of Stx. To understand the genetic basis for varied Stx production, chronological collections of bovine isolates from Wisconsin dairy farms, R and X, were analyzed for multilocus prophage polymorphisms, stx(2) subtypes, and the levels of stx(2) transcript and toxin. The E. coli O157:H7 that persisted on both farms were phylogenetically distinct and yet produced little to no Stx2 due to gene deletions in Stx2c-encoding prophage (farm R) or insertional inactivation of stx(2a) by IS1203v (farm X). Loss of key regulatory and lysis genes in Stx2c-encoding prophage abolished stx(2c) transcription and induction of the prophage and stx(2a)::IS1203v in Stx2a-encoding prophage generated a truncated stx(2a) mRNA without affecting phage production. Stx2-producing strains were transiently present (farm R) and became Stx2 negative on farm X (i.e., stx(2a)::IS1203v). To our knowledge, this is the first study that details the evolution of E. coli O157:H7 and its Stx2-encoding prophage in a chronological collection of natural isolates. The data suggest the bovine and farm environments can be niches where Stx2-negative E. coli O157:H7 emerge and persist, which explains the Stx variability in bovine isolates and may be part of an evolutionary step toward becoming bovine specialists.