Novel LC8 Mutations Have Disparate Effects on the Assembly and Stability of Flagellar Complexes

LC8 functions as a dimer crucial for a variety of molecular motors and non-motor complexes. Emerging models, founded on structural studies, suggest that the LC8 dimer promotes the stability and refolding of dimeric target proteins in molecular complexes, and its interactions with selective target proteins, including dynein subunits, is regulated by LC8 phosphorylation, which is proposed to prevent LC8 dimerization. To test these hypotheses in vivo, we determine the impacts of two new LC8 mutations on the assembly and stability of defined LC8-containing complexes in Chlamydomonas flagella. The three types of dyneins and the radial spoke are disparately affected by dimeric LC8 with a C-terminal extension. The defects include the absence of specific subunits, complex instability, and reduced incorporation into the axonemal super complex. Surprisingly, a phosphomimetic LC8 mutation, which is largely monomeric in vitro, is still dimeric in vivo and does not significantly change flagellar generation and motility. The differential defects in these flagellar complexes support the structural model and indicate that modulation of target proteins by LC8 leads to the proper assembly of complexes and ultimately higher level complexes. Furthermore, the ability of flagellar complexes to incorporate the phosphomimetic LC8 protein and the modest defects observed in the phosphomimetic LC8 mutant suggest that LC8 phosphorylation is not an effective mechanism for regulating molecular complexes.     

Flagellar assembly in Salmonella typhimurium

The bacterial flagellum is a motility apparatus in which a long helical filament - the propeller - is driven by a rotary motor embedded in the cell surface. Out of more than 40 genes required for construction of a fully functional flagellum in Salmonella typhimurium, only 18 gene products have been identified in the mature structure. Some other flagellar proteins play logistical roles during construction, which involves the selective export of flagellar components through a central hole in the flagellum. The whole structure is constructed from base to tip by linear assembly; that is, by adding new components on the growing end, resulting in the distal growth of each substructure. Components of the substructures do not necessarily self-assemble, but often demand the help of other proteins. Recent progress in the understanding of flagellar assembly, which has been most extensively studied in S. typhimurium, is reviewed.     

Assembly of Hybrid Flagellar Filaments

The distribution of flagellin subunits in flagellar filaments synthesized by merodiploid strains carrying two distinguishable hag loci has been examined. The filament was found to be a homogeneous co-polymer of the two subunits. This suggests that the subunits may be able to mix freely before being assembled.     

Chaperone Recycling in Late-Stage Flagellar Assembly

The flagellum is a sophisticated nanomachine responsible for motility in Gram-negative bacteria. Flagellar assembly is a strictly choreographed process, in which the motor and export gate are formed first, followed by the extracellular propeller structure. Extracellular flagellar components are escorted to the export gate by dedicated molecular chaperones for secretion and self-assembly at the apex of the emerging structure. The detailed mechanisms of chaperone-substrate trafficking at the export gate remain poorly understood. Here, we structurally characterized the interaction of Salmonella enterica late-stage flagellar chaperones FliT and FlgN with the export controller protein FliJ. Previous studies showed that FliJ is absolutely required for flagellar assembly since its interaction with chaperone-client complexes controls substrate delivery to the export gate. Our biophysical and cell-based data show that FliT and FlgN bind FliJ cooperatively, with high affinity and on specific sites. Chaperone binding completely disrupts the FliJ coiled-coil structure and alters its interactions with the export gate. We propose that FliJ aids the release of substrates from the chaperone and forms the basis of chaperone recycling during late-stage flagellar assembly.     

Effect of Hook Subunit Concentration on Assembly and Control of Length of the Flagellar Hook of Salmonella

The flagellar hook of Salmonella is a filamentous polymer made up of subunits of the protein FlgE. Hook assembly is terminated when the length reaches about 55 nm. After our recent study of the effect of cellular levels of the hook length control protein FliK, we have now analyzed the effect of cellular levels of FlgE itself. When FlgE was overproduced in a wild-type strain, a fliC (flagellin) mutant, or a fliD (hook-associated protein 2 [HAP2], filament capping protein) mutant, the hooks remained at the wild-type length. In a fliK (hook length control protein) mutant, which produces long hooks (polyhooks), the overproduction of FlgE resulted in extraordinarily long hooks (superpolyhooks). In a flgK (HAP1, first hook-filament junction protein) mutant or a flgL (HAP3, second hook-filament junction protein) mutant, the overproduction of FlgE also resulted in longer than normal hooks. Thus, at elevated hook protein levels not only FliK but also FlgK and FlgL are necessary for the proper termination of hook elongation. When FlgE was severely underproduced, basal bodies without hooks were often observed. However, those hooks that were seen were of wild-type length, demonstrating that FlgE underproduction decreases the probability of the initiation of hook assembly but not the extent of hook elongation.     

Effect of Flagellar Mutations on Yersinia enterocolitica Biofilm Formation

Yersinia enterocolitica biovar 1B is one of a number of strains pathogenic to humans in the genus Yersinia. It has three different type III secretion systems, Ysc, Ysa, and the flagella. In this study, the effect of flagella on biofilm formation was evaluated. In a panel of 31 mutant Y. enterocolitica strains, we observed that mutations that abolish the structure or rotation of the flagella greatly reduce biofilm formation when the bacteria are grown under static conditions. These results were further evaluated by assessing biofilm formation under continuous culture using a flow cell chamber. The results confirmed the important contribution of flagella to the initiation of biofilm production but indicated that there are differences in the progression of biofilm development between static growth and flow conditions. Our results suggest that flagella play a critical role in biofilm formation in Y. enterocolitica.     

Synthetic Effects of secG and secY2 Mutations on Exoproteome Biogenesis in Staphylococcus aureus

The Gram-positive pathogen Staphylococcus aureus secretes various proteins into its extracellular milieu. Bioinformatics analyses have indicated that most of these proteins are directed to the canonical Sec pathway, which consists of the translocation motor SecA and a membrane-embedded channel composed of the SecY, SecE, and SecG proteins. In addition, S. aureus contains an accessory Sec2 pathway involving the SecA2 and SecY2 proteins. Here, we have addressed the roles of the nonessential channel components SecG and SecY2 in the biogenesis of the extracellular proteome of S. aureus. The results show that SecG is of major importance for protein secretion by S. aureus. Specifically, the extracellular accumulation of nine abundant exoproteins and seven cell wall-bound proteins was significantly affected in an secG mutant. No secretion defects were detected for strains with a secY2 single mutation. However, deletion of secY2 exacerbated the secretion defects of secG mutants, affecting the extracellular accumulation of one additional exoprotein and one cell wall protein. Furthermore, an secG secY2 double mutant displayed a synthetic growth defect. This might relate to a slightly elevated expression of sraP, encoding the only known substrate for the Sec2 pathway, in cells lacking SecG. Additionally, the results suggest that SecY2 can interact with the Sec1 channel, which would be consistent with the presence of a single set of secE and secG genes in S. aureus.Staphylococcus aureus is a well-represented component of the human microbiota as nasal carriage of this Gram-positive bacterium has been shown for 30 to 40% of the population (32). This organism can, however, turn into a dangerous pathogen that is able to infect almost every tissue in the human body. S. aureus has become particularly notorious for its high potential to develop resistance against commonly used antibiotics (20, 49). Accordingly, the S. aureus genome encodes an arsenal of virulence factors that can be expressed when needed at different stages of growth. These include surface proteins and invasins that are necessary for colonization of host tissues, surface-exposed factors for evasion of the immune system, exotoxins for the subversion of protective host barriers, and resistance proteins for protection against antimicrobial agents (37, 57).Most proteinaceous virulence factors of S. aureus are synthesized as precursors with an N-terminal signal peptide to direct their transport from the cytoplasm across the membrane to an extracytoplasmic location, such as the cell wall or the extracellular milieu (38, 45). As shown for various Gram-positive bacteria, the signal peptides of S. aureus are generally longer and more hydrophobic than those of Gram-negative bacteria (38, 54). On the basis of signal peptide predictions using a variety of algorithms, it is believed that most exoproteins of S. aureus are exported to extracytoplasmic locations via the general secretory (Sec) pathway (38). This seems to involve precursor targeting to the Sec machinery via the signal recognition particle instead of the well-characterized proteobacterial chaperone SecB, which is absent from Gram-positive bacteria (16, 19, 53). The preproteins are then bound by the translocation motor protein SecA (38, 45). Through repeated cycles of ATP binding and hydrolysis, SecA pushes the protein in an unfolded state through the membrane-embedded SecYEG translocation channel (12, 30, 33, 52). Upon initiation of the translocation process, the proton motive force is thought to accelerate preprotein translocation through the Sec channel (26). Recently, the structure of the SecA/SecYEG complex from the Gram-negative bacterium Thermotoga maritima was solved at 4.5 Å resolution (58). In this structure, one SecA molecule is bound to one set of SecYEG channel proteins. The core of the Sec translocon consists of the SecA, SecY, and SecE proteins, which are essential for growth and viability of bacteria, such as Escherichia coli and Bacillus subtilis (6, 9, 22). In contrast, the channel component SecG is dispensable for growth, cell viability, and protein translocation (26, 48). Nevertheless, SecG does enhance the efficiency of preprotein translocation through the SecYE channel (26, 48). This is of particular relevance at low temperatures and in the absence of a proton motive force (17). Several studies suggest that E. coli SecG undergoes topology inversion during preprotein translocation (25, 27, 43). Even so, van der Sluis et al. reported that SecG cross-linked to SecY is fully functional despite its fixed topology (46). During or shortly after membrane translocation of a preprotein through the Sec channel, the signal peptide is removed by signal peptidase. This is a prerequisite for the release of the translocated protein from the membrane (1, 47).Several pathogens, including Streptococcus gordonii, Streptococcus pneumoniae, Bacillus anthracis, Bacillus cereus, and S. aureus, contain a second set of chromosomal secA and secY genes named secA2 and secY2, respectively (39). Comparison of the amino acid sequences of the SecY1 and SecY2 proteins shows that their similarity is low (about 20% identity) and that the conserved regions are mainly restricted to the membrane-spanning domains. It has been shown for S. gordonii that the transport of at least one protein is dependent on the presence of SecA2 and SecY2. This protein, GspB, is a large cell surface glycoprotein that is involved in platelet binding (4). The protein contains an unusually long N-terminal signal peptide of 90 amino acids, large serine-rich repeats, and a C-terminal LPXTG motif for covalent cell wall binding. The gspB gene is located in a gene cluster with the secA2 and secY2 genes. Two other genes in this cluster encode the glycosylation proteins GftA and GftB, which seem to be necessary for stabilization of pre-GspB. Furthermore, the asp4 and asp5 genes in the secA2 secY2 gene cluster show similarity to secE and secG, and they are important for GspB export by S. gordonii (44). Despite this similarity, SecE and SecG cannot complement for the absence of Asp4 and Asp5, respectively. The secA2-secY2 gene cluster is also present in S. aureus, but homologues of the asp4 and asp5 genes are lacking. This seems to suggest that SecA2 and SecY2 of S. aureus share the SecE and SecG proteins with SecA1 and SecY1. The sraP gene in the secA2-secY2 gene cluster of S. aureus encodes a protein with features similar to those described for GspB. Siboo and colleagues (41) have shown that SraP is glycosylated and capable of binding to platelets. Importantly, the disruption of sraP resulted in a decreased ability to initiate infective endocarditis in a rabbit model. Consistent with the findings in S. gordonii, SraP export was shown to depend on SecA2/SecY2 (40). However, it has remained unclear whether other S. aureus proteins are also translocated across the membrane in an SecA2/SecY2-dependent manner.The present studies were aimed at defining the roles of two Sec channel components, SecG and SecY2, in the biogenesis of the S. aureus exoproteome. The results show that secG and secY2 are not essential for growth and viability of S. aureus. While the absence of SecY2 by itself had no detectable effect, the absence of SecG had a profound impact on the composition of the exoproteome of S. aureus. Various extracellular proteins were present in decreased amounts in the growth medium of secG mutant strains, which is consistent with impaired Sec channel function. However, a few proteins were present in increased amounts. Furthermore, the absence of secG caused a serious decrease in the amounts of the cell wall-bound Sbi protein. Most notable, a secG secY2 double mutant strain displayed synthetic growth and secretion defects.     

A Trimeric Lipoprotein Assists in Trimeric Autotransporter Biogenesis in Enterobacteria

Trimeric autotransporter adhesins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. TAAs form fibrous, adhesive structures on the bacterial cell surface. Their N-terminal extracellular domains are exported through a C-terminal membrane pore; the insertion of the pore domain into the bacterial outer membrane follows the rules of β-barrel transmembrane protein biogenesis and is dependent on the essential Bam complex. We have recently described the full fiber structure of SadA, a TAA of unknown function in Salmonella and other enterobacteria. In this work, we describe the structure and function of SadB, a small inner membrane lipoprotein. The sadB gene is located in an operon with sadA; orthologous operons are only found in enterobacteria, whereas other TAAs are not typically associated with lipoproteins. Strikingly, SadB is also a trimer, and its co-expression with SadA has a direct influence on SadA structural integrity. This is the first report of a specific export factor of a TAA, suggesting that at least in some cases TAA autotransport is assisted by additional periplasmic proteins.     

Immunofluorescent Staining of Salmonella Species with Flagellar Sera

Salmonellae stained with flagellar sera by either the direct or indirect fluorescent-antibody technique fluoresced at the cell surface. Investigations showed that fimbriae, flagellar material, and capsule antigens were not the participating antigens in this reaction. Cell surface staining was inhibited by mannose but was unaffected by glucose, galactose, lactose, maltose, and sucrose. Specific absorption of the flagellar sera by intact cells and purified somatic antigen showed that O antigens were the site of the surface staining. The advantages of using flagellar sera in fluorescent-antibody screening of materials for the presence of salmonellae are discussed.     

Regulation of Flagellar Biogenesis by a Calcium Dependent Protein Kinase in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, a bi-flagellated green alga, is a model organism for studies of flagella or cilia related activities including cilia-based signaling, flagellar motility and flagellar biogenesis. Calcium has been shown to be a key regulator of these cellular processes whereas the signaling pathways linking calcium to these cellular functions are less understood. Calcium-dependent protein kinases (CDPKs), which are present in plants but not in animals, are also present in ciliated microorganisms which led us to examine their possible functions and mechanisms in flagellar related activities. By in silico analysis of Chlamydomonas genome we have identified 14 CDPKs and studied one of the flagellar localized CDPKs – CrCDPK3. CrCDPK3 was a protein of 485 amino acids and predicted to have a protein kinase domain at the N-terminus and four EF-hand motifs at the C-terminus. In flagella, CrCDPK3 was exclusively localized in the membrane matrix fraction and formed an unknown 20 S protein complex. Knockdown of CrCDPK3 expression by using artificial microRNA did not affect flagellar motility as well as flagellar adhesion and mating. Though flagellar shortening induced by treatment with sucrose or sodium pyrophosphate was not affected in RNAi strains, CrCDPK3 increased in the flagella, and pre-formed protein complex was disrupted. During flagellar regeneration, CrCDPK3 also increased in the flagella. When extracellular calcium was lowered to certain range by the addition of EGTA after deflagellation, flagellar regeneration was severely affected in RNAi cells compared with wild type cells. In addition, during flagellar elongation induced by LiCl, RNAi cells exhibited early onset of bulbed flagella. This work expands new functions of CDPKs in flagellar activities by showing involvement of CrCDPK3 in flagellar biogenesis in Chlamydomonas.     

The Role of Preassembled Cytoplasmic Complexes in Assembly of Flagellar Dynein Subunits

Previous work has revealed a cytoplasmic pool of flagellar precursor proteins capable of contributing to the assembly of new flagella, but how and where these components assemble is unknown. We tested Chlamydomonas outer-dynein arm subunit stability and assembly in the cytoplasm of wild-type cells and 11 outer dynein arm assembly mutant strains (oda1-oda11) by Western blotting of cytoplasmic extracts, or immunoprecipitates from these extracts, with five outer-row dynein subunit-specific antibodies. Western blots reveal that at least three oda mutants (oda6, oda7, and oda9) alter the level of a subunit that is not the mutant gene product. Immunoprecipitation shows that large preassembled flagellar complexes containing all five tested subunits (three heavy chains and two intermediate chains) exist within wild-type cytoplasm. When the preassembly of these subunits was examined in oda strains, we observed three patterns: complete coassembly (oda 1, 3, 5, 8, and 10), partial coassembly (oda7 and oda11), and no coassembly (oda2, 6, and 9) of the four tested subunits with HCβ. Our data, together with previous studies, suggest that flagellar outer-dynein arms preassemble into a complete M_r 2 × 10⁶ dynein arm that resides in a cytoplasmic precursor pool before transport into the flagellar compartment.