A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

In Vivo Display of a Multisubunit Enzyme Complex on Biogenic Magnetic Nanoparticles

Magnetosomes are unique bacterial organelles comprising membrane-enveloped magnetic crystals produced by magnetotactic bacteria. Because of several desirable chemical and physical properties, magnetosomes would be ideal scaffolds on which to display highly complicated biological complexes artificially. As a model experiment for the functional expression of a multisubunit complex on magnetosomes, we examined the display of a chimeric bacterial RNase P enzyme composed of the protein subunit (C5) of Escherichia coli RNase P and the endogenous RNA subunit by expressing a translational fusion of C5 with MamC, a known magnetosome protein, in the magnetotactic bacterium Magnetospirillum gryphiswaldense. As intended, the purified C5 fusion magnetosomes, but not wild-type magnetosomes, showed apparent RNase P activity and the association of a typical bacterial RNase P RNA. Our results demonstrate for the first time that magnetosomes can be employed as scaffolds for the display of multisubunit complexes.Magnetosomes are unique organelles comprising membrane-enveloped magnetic crystals of iron minerals (Fe₃O₄ or Fe₃S₄) produced by magnetotactic bacteria (1, 11). The bacteria employ magnetosomes to sense the environmental magnetic field, probably in order to recognize their favorite environments. Compared with chemically or physically synthesized magnetic nanoparticles, magnetosomes have a variety of desirable features, including their genetically controlled uniform size and morphology, characteristic crystal habits, and their coverage by a biological membrane that can be addressed by functionalization (1, 4, 11). Based on these features, magnetosomes would be ideal scaffolds on which to display biological molecules artificially.Until now, several heterologous target proteins have been examined for artificial display on magnetosomes (1, 11). For example, reporter proteins such as luciferase and green fluorescent protein were employed to analyze the targeting, expression, and stability of chimeric proteins displayed on magnetosomes (14, 18, 23, 30, 41). For more-practical applications, general antibody-binding proteins (protein A and protein G) were displayed to capture desired antibodies (16, 17, 25, 33, 34, 37, 41). Such antibody-captured magnetosomes are applicable for the magnetic separation of target molecules and cells. Displays of G protein-coupled receptors (the D1 dopamine receptor and the ligand binding domain of the estrogen receptor) were also examined for screening of drugs targeting these receptors (38, 39, 40).There are two major strategies for the construction of functionalized magnetosomes: subsequent chemical modifications of purified magnetosomes (3) and in vivo expression of modified magnetosome proteins (1, 19). The latter approach is confined to biological molecules that can be expressed as a genetic fusion with a magnetosome protein inside a magnetotactic bacterium. By this approach, the target-displaying magnetosomes can be constructed inside cells or under physiological conditions in the presence of a variety of chaperons, are recoverable under mild conditions employing a magnetic field, and provide control by genetic means. Thus, the approach is highly promising for the display of a naïve target such as a multisubunit complex. To date, however, experimental evidence that magnetosomes can be employed as scaffolds for the display of such targets is still lacking. In order to demonstrate this potential of magnetosomes, here, we examined the display of a holoenzyme of bacterial RNase P, one of the simplest complexes composed of a single RNA and a single protein subunit (10, 12), by expressing a fusion of a protein component of the RNase P and a magnetosome membrane protein.     

Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization

Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.The ability of a cell to rapidly respond to extracellular cues and direct cytoskeletal rearrangements is dependent on an array of signaling complexes that control actin assembly (58). The protrusive structures at the leading edges of motile cells are broadly defined as lamellipodia or filopodia (14). Lamellae are sheet-like protrusions composed of dendritic actin arrays that drive membrane expansion, with the “lamellipodium” representing a narrow region at the edge of the cell (in culture) characterized by rapid actin polymerization. This F-actin assembly is suggested to require Arp2/3 activity that nucleates new actin filaments from the sides of existing ones (58, 71) and capping proteins that limit the length of these new filaments and stabilize them (7). Arp2/3 activity in turn is regulated by the WASP/WAVE family of proteins, such as N-WASP and WAVE2 (68), whose regulation is a subject of intense interest (12, 29, 36, 41, 56, 76).Filopodia contain parallel bundles of actin filaments containing fascin (22). These are dynamic structures that emanate from the periphery of the cell and are retracted, with occasional attachment (to the dish in culture). Thus, they have been thought to have a sensory or exploratory role during cell migration (28). This is the case for neuronal growth cones, where filopodia sense attractant or repulsive cues and dictate direction in axonal path finding (9, 17, 25, 35). Filopodia have been shown to be important in the context of dendritic-spine development (64, 77), epithelial-sheet closure (26, 60, 79), and cell invasion/metastasis (80, 83).Lamellipodia have been well characterized since the pioneering work of Abercrombie et al. in the early 1970s (2, 3, 4). Filopodia require symmetry breaking at the leading edge (initiation), followed by elongation driven by a filopodial-tip protein complex (14, 28). A few proteins have been identified in this complex; Mena/Vasp serve to prevent capping at the barbed ends of bundled actin filaments (7, 53), and Dia2 promotes F-actin elongation (57, 85). Termination of filopodial elongation is not understood but nonetheless is likely to be tightly regulated. In the absence of F-actin elongation, retraction of the filopodium takes place by a rearward flow of F-actin and filament depolymerization (22).IRSp53 is in a position to play a pivotal role in generating filopodia; this brain-enriched protein was discovered as a substrate of the insulin receptor (87). Subsequently, IRSp53 was identified as an effector for Rac1 (50) and Cdc42 (27, 38), where it participates in filopodium and lamellipodium production (38, 51, 54, 86), neurite extension (27), dendritic-spine morphogenesis (1, 15, 66, 67), cell motility and invasiveness (24). The N terminus of IRSp53 contains a conserved helical domain that is found in five different gene products and is referred to as the IRSp53/MIM homology domain (IMD) (51, 70). This domain has been postulated to bind to Rac1 (50, 70) in a nucleotide-independent manner (52), but no convincing effector-like region has been identified. A Cdc42-specific CRIB-like sequence that does not bind Rac1 (27, 38) allows coupling of this and perhaps related Rho GTPases. The structure of the IMD reveals a zeppelin-shaped dimer that could bind “bent” membranes; thus, its potential as an F-actin-bundling domain (51, 82) could be an in vitro artifact often attributed to proteins with basic patches (46). Although there are reports of F-actin binding at physiological ionic strength (ca. 100 mM KCl) (82, 19), this region when expressed in isolation does not decorate F-actin in vivo.Two reports showed the IMD to be an “inverse-BAR” domain. BAR (Bin-amphipysin-Rvs) domains are found in proteins involved in endocytic trafficking, such as amphipysin and endophilin, and stabilize positively bent membranes, such as those on endocytic vesicles (31, 47). The IMD domains of both IRSp53 (70) and MIM-B (46) associate with lipids and can induce tubulations of PI(3,4,5)P₃ or PI(4,5)P₂-rich membranes, respectively. These tubulations are equivalent to membrane protrusions and are also referred to as negatively bent membranes. Ectopic expression of the IMD from IRSp53 (51, 70, 82, 86) or two other family members, MIM-B (11, 46) and IRTKS (52), can give rise to cells with many peripheral extensions. MIM-B is said to stimulate lamellipodia (11), while IRTKS generates “short actin clusters” at the cell periphery (52).In IRSp53 is a CRIB-like motif that mediates binding to Cdc42 (27, 38), but the function of this interaction in unclear. Cdc42 could relieve IRSp53 autoinhibition as described for N-Wasp (38), but there is little evidence for this. It has been suggested that Cdc42 controls IRSp53 localization and actin remodeling (27, 38), but another study indicated that these events are Cdc42 independent (19). IRSp53 contains a central SH3 domain that may bind proline-rich proteins, such as Dia1 (23), Mena (38), WAVE2 (49, 50, 69), and Eps8 (19, 24). However, it seems unlikely that all of these represent bona fide partners, and side-by-side comparison is provided in this study. Mena is involved in filopodium production (37), Dia1 in stress fiber formation (81), and WAVE2 in lamellipodium extension (72). Thus, Mena is a better candidate as a partner for IRSp53-mediated filopodia than Dia1 or WAVE2.There is good evidence for IRSp53 as a cellular partner for Eps8 (19). Eps8 is an adaptor protein containing an N-terminal PTB domain that can associate with receptor tyrosine kinases (65), and perhaps β integrins (13), and a C-terminal SH3 domain that can associate with Abi1 (30). Binding of the general adaptor Abi1 appears to positively regulate the actin-capping domain at the C terminus of Eps8 (18). It has been suggested that IRSp53 and Eps8 as a complex regulate cell motility, and perhaps Rac1 activation, via SOS (24); more recently, their roles in filopodium formation have been addressed (19). The involvement of IRSp53, but not MIM-B or IRTKS, in filopodium formation might be related to its role as a Cdc42 effector. We show here that, surprisingly, the CRIB motif is not essential for this activity, but rather, the ability of IRSp53 to associate via its SH3 domain is required, and that this domain is controlled by 14-3-3 binding.We have focused on the regulation of Cdc42 effectors that bind 14-3-3, including IRSp53 and PAK4, which are found as 14-3-3 targets in various proteomic projects (32, 44). In this study, we characterize the binding of 14-3-3 to IRSp53 and uncover how this activity regulates IRSp53 function. The phosphorylation-dependent 14-3-3 binding is GSK3β dependent, and 14-3-3 blocks the accessibility of both the CRIB and SH3 domains of IRSp53, thus indicating its primary function in controlling IRSp53 partners. This regulation of the SH3 domain by 14-3-3 is critical in the proper localization and termination of IRSp53 function to promote filopodium dynamics.     

Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the β-clamp, consistent with its canonical C-terminal β-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N²-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.The emergence and global spread of multi- and extensively drug-resistant strains of Mycobacterium tuberculosis have further complicated the already daunting challenge of controlling tuberculosis (TB) (15). The mechanisms that underlie the evolution of drug resistance in M. tuberculosis by chromosomal mutagenesis and their association with the conditions that tubercle bacilli encounter during the course of infection are poorly understood (6). It has been postulated that hypoxia, low pH, nutrient deprivation, and nitrosative and oxidative stress impose environmental and host immune-mediated DNA-damaging insults on infecting bacilli (64). In addition, the observed importance of excision repair pathways for the growth and survival of M. tuberculosis in murine models of infection (13, 55) and the upregulation of M. tuberculosis genes involved in DNA repair and modification in pulmonary TB in humans provide compelling evidence that the in vivo environment is DNA damaging (51).Damage tolerance constitutes an integral component of an organism''s response to genotoxic stress, preventing collapse of the replication fork at persisting, replication-blocking lesions through the engagement of specialized DNA polymerases that are able to catalyze translesion synthesis (TLS) across the sites of damage (19, 21, 60). Most TLS polymerases belong to the Y family, which comprises a wide range of structurally related proteins present in bacteria, archaea, and eukaryotes (44). Of these, the DinB subfamily of Y family polymerases, whose founder member is Escherichia coli Pol IV (63), is conserved among all domains of life (44). The association of Y family polymerases with inducible mutagenesis has implicated these enzymes in the adaptation of bacteria to environmental stress (17, 20, 39, 54, 58, 59, 66). Their key properties are exemplified in E. coli Pol IV: the polymerase catalyzes efficient and accurate TLS across certain N²-dG adducts (27, 28, 34, 40, 45, 67) and has been implicated in the tolerance of alkylation damage (4); furthermore, overexpression of Pol IV significantly increases mutation rates in E. coli (reviewed in references 21 and 26), and dinB is the only SOS-regulated gene required at induced levels for stress-induced mutagenesis in this organism (20). Furthermore, overproduction of E. coli Pol IV inhibits replication fork progression through replacement of the replicative polymerase to form an alternate replisome in which Pol IV modulates the rate of unwinding of the DnaB helicase (25) and also reduces colony-forming ability (61).The M. tuberculosis genome encodes two Y family polymerase homologs belonging to the DinB subfamily, designated herein as DinB1 (DinX, encoded by Rv1537) and DinB2 (DinP, encoded by Rv3056), as well as a third, distantly related homolog encoded by Rv3394c (see Fig. S1 in the supplemental material) (9). On the basis of sequence similarity with their counterparts from E. coli (63) and Pseudomonas aeruginosa (54), including the complete conservation of key acidic residues essential for catalysis, DinB1 and DinB2 may be functional DNA polymerases (see Fig. S1). In contrast, Rv3394c lacks these residues and as such is unlikely to have polymerase activity (see Fig. S1). Unlike most Y family polymerase-encoding genes investigated with other bacteria (17, 26, 54, 58), dinB1 and dinB2 expression in M. tuberculosis is not dependent on RecA, the SOS response, or the presence of DNA damage (5, 7, 52). That these genes are regulated by other mechanisms and so may serve distinct roles in DNA metabolism in M. tuberculosis is suggested by the observation that dinB1 is differentially expressed in pulmonary TB (51) and is a member of the SigH regulon (30), whereas expression of dinB2 is induced following exposure to novobiocin (5).In this study, we adopted a genetic approach to investigate the function of dinB1 and dinB2 in M. tuberculosis. Mutants with altered complements or expression levels of dinB1 and/or dinB2 were analyzed in vitro and in vivo under conditions predicted to be phenotypically revealing based on DinB function established with other model organisms. The lack of discernible phenotypes in any of the assays employed suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.     

Investigation of the Three-Dimensional Architecture of the Collagen Adhesin EmaA of Aggregatibacter actinomycetemcomitans by Electron Tomography

The periodontal pathogen Aggregatibacter actinomycetemcomitans displays on the bacterial surface a nonfimbrial adhesin, EmaA, which is required for collagen binding. In this study, electron tomography was used to characterize the three-dimensional (3D) architecture of this adhesin. The antenna-like surface appendages, corresponding to EmaA, were found to be composed of an ellipsoidal domain capping a rod-like domain that adopts either a straight or a bent conformation at various positions along the length. The most common flexible point along the length of the EmaA appendage was localized 29.4 nm away from the distal end. One-fifth of the appendages were straight and the remaining showed angles distributed between 140° and 170° at this location. Deletion analysis mapped this bend to amino acids 611 to 640 of the protein sequence. The 3D structure of the collagen binding domain of EmaA was generated by alignment and averaging of 9 subvolumes of the adhesin extracted from tomograms. The structure contains three subdomains: a globular structure with a diameter of ∼5 nm and a cylindrical domain (∼4.4 nm by 5.8 nm) separated by a linker region with a diameter of ∼3 nm, followed by a cylindrical domain (∼4.6 nm by 6.6 nm). This is the first 3D structure of a trimeric autotransporter protein of A. actinomycetemcomitans.Bacterial adhesion to host receptors, a crucial step for colonization and infection, is mediated by fimbrial and nonfimbrial adhesins. These adhesins are proteinaceous appendages displayed on the surface of bacteria and contain the receptor binding domains. Aggregatibacter actinomycetemcomitans, a gram-negative, nonmotile bacterium is found associated with periodontal diseases and other extraoral infections (12, 23, 32, 40). When isolated from the oral cavity, the bacterium exists as a fimbriated form and switches to an afimbriated form upon planktonic subculturing (5, 14). A. actinomycetemcomitans fimbriae mediate the nonspecific adherence of the bacterium to abiotic and organic surfaces and decorate the bacterial surface with long fibrils of 5 to 7 nm in diameter (14, 15). In addition to fimbriae, nonfimbrial adhesins, which mediate the specific binding to host cells and tissues, have been identified in this bacterium (1, 6, 19, 27, 29). Among these nonfimbrial adhesins, only the extracellular matrix protein adhesin A, EmaA, has been visualized forming structures on the bacterial surface by transmission electron microscopy (29).EmaA is an outer membrane collagen adhesin unique to A. actinomycetemcomitans; however, orthologous proteins exist in other bacterial genera (13, 18, 21, 26, 33, 38). The protein is encoded by a 6-kb gene present in all A. actinomycetemcomitans strains investigated (36). Genetic heterogeneity within the gene exists between different strains, which are based on the serotype of the organism. Based on this heterogeneity, two molecular forms of the protein have been identified: a full-length and an intermediate form. The prototypic or full-length protein exists as a 202-kDa protein and shares 75% amino acid homology with the intermediate form. The intermediate protein form (173 kDa) contains an in-frame 279-amino-acid deletion but maintains collagen binding activity and surface appendages similar to the prototypic form (36).EmaA is associated with the binding of A. actinomycetemcomitans to both isolated acid-soluble collagen and collagen found in tissues (19, 29, 35, 39). The specificity of EmaA for collagen was demonstrated using a rabbit cardiac valve tissue model (35). Valves with an intact endothelium bound equal amounts of the wild type or emaA isogenic mutants. Removal of the endothelium by trypsin treatment, thereby exposing the underlying collagen, did not affect the level of binding of the mutant. However, the number of wild-type bacteria bound to the exposed collagen was five times the number of mutant bacteria. This represents a 10-fold increase with respect to the number of bacteria bound to the endothelium. The role of EmaA as a virulence determinant in A. actinomycetemcomitans infection was demonstrated in a rabbit endocarditis infection model, in which the wild-type bacterium outcompeted the binding of the mutant 10-fold (35).Sequence analysis indicates that EmaA belongs to the Oca (oligomeric coiled-coil adhesin) family of autotransporter adhesins (19). Multimers of EmaA oligomerize to form appendages on the bacterial surface and are visible as long rods or antenna-like structures capped by an ellipsoidal domain (29). A strong correlation exists between the translocated region of the protein (head and stalk domains) and the structural features. The head domain, consisting of amino acids 70 to 386, forms the ellipsoidal ending of the appendage, which is essential for collagen binding, while amino acids 387 to 1900 form the stalk domain (39).Contained within the translocation domain of EmaA are three “neck” sequences, which are conserved in the Oca family protein members (21, 29, 33). These sequences are considered to stabilize the oligomer and transition between β-rolls and coiled-coil regions of the molecule (21, 26). In the EmaA sequence, two “neck” sequences are found within the first 628 amino acids of the protein sequence (19, 29). The third sequence is located in the stalk domain adjacent to the carboxy-terminal membrane anchor domain, which comprises amino acids 1901 to 1965 (19, 29). The membrane anchor domains of three or four monomers are proposed to form β-barrels that are required for pore formation and protein translocation (18, 29, 37).The translocated domain of EmaA has been subjected to a two-dimensional (2D) study by transmission electron microscopy, and the overall dimensions of the EmaA appendages have been determined by the analysis of a large number of micrographs (29). The ellipsoidal ending shows diameters of 2.8 by 4.6 nm, and the stalk domain, which is at least 150 nm long, has a diameter of 4.1 nm. Several conformations of the stalk domain were present in the micrographs: either straight or containing a bend at 29.2 nm from the distal end. This bend position was correlated with amino acids localized between the first two neck sequences (29).In this study, electron tomography was used to characterize the 3D structure of the EmaA appendages of A. actinomycetemcomitans in situ. The functional domain of EmaA was found to be composed of three distinct subdomains followed by a long stalk domain. Distinct regions of the molecule were identified that provide flexibility for the molecule and allow for the deformation or bending of the adhesin. A correlation between these flexible regions and specific amino acids in the sequence was ascertained.