Classification of a Haemophilus influenzae ABC Transporter HI1470/71 through Its Cognate Molybdate Periplasmic Binding Protein, MoIA

molA (HI1472) from H. influenzae encodes a periplasmic binding protein (PBP) that delivers substrate to the ABC transporter MolB_2C_2 (formerly HI1470/71). The structures of MolA with molybdate and tungstate in the binding pocket were solved to 1.6 and 1.7 Å resolution, respectively. The MolA-binding protein binds molybdate and tungstate, but not other oxyanions such as sulfate and phosphate, making it the first class III molybdate-binding protein structurally solved. The ~100 μM binding affinity for tungstate and molybdate is significantly lower than observed for the class II ModA molybdate-binding proteins that have nanomolar to low micromolar affinity for molybdate. The presence of two molybdate loci in H. influenzae suggests multiple transport systems for one substrate, with molABC constituting a low-affinity molybdate locus

Structural determination of the phage 186 repressor protein

Bacteriophage 186 is closely related to the non-inducible P2 family of temperate phages based on genome homology. Unlike other P2 family members, phage 186 is SOS-inducible (in response to DNA damaging signals) and requires a cII gene for efficient lysogeny, similar to the lambda (λ) family. 186 and λ have common features that serve the same role in both phages: ensuring stable lysogeny and allowing for efficient switching to lytic growth upon induction. The lysogenic state of 186, like λ, is maintained by a repressor protein, the product of the gene cI. However, the λ and 186 operons differ in promoter arrangement, DNA binding sites, and the SOS response. Since bacteriophage λ has been extensively studied, the differences between λ and 186 provide insight into gene regulation and an additional model for the “genetic switch”. In an effort to gain insight into the structural basis of cooperativity and to help elucidate the details of this genetic switch, we pursued the structural determination of the 186 repressor, cI. Biochemical studies indicate that 186 cI has two domains: an N-terminal DNA binding domain and a C-terminal oligomerization domain. The X-ray structure of the C-terminal domain was solved by multiwavelength anomalous diffraction (MAD) to 2.Å resolution. By introducing a cooperativity mutant identified by genetic screens to the full length construct of 186 cI, we were able to obtain crystals of the full length dimer (E146K). The X-ray structure of the full length dimer was solved by molecular replacement (MR) to 1.95 Å resolution. A comparison of the crystallographic structures indicate the overall fold of the C-terminal domain and helix-turn-helix (HTH) motif from 186 cI is remarkably similar to λ repressor. Although similar in their overall fold, the difference in sequence provides insight into how 186 cI works to maintain lysogeny. The structures not only deepen our understanding of the genetic studies, but also provide insight into the framework for understanding the mechanism by which repression occurs

Structural determination of the phage 186 repressor protein

Bacteriophage 186 is closely related to the non-inducible P2 family of temperate phages based on genome homology. Unlike other P2 family members, phage 186 is SOS-inducible (in response to DNA damaging signals) and requires a cII gene for efficient lysogeny, similar to the lambda (λ) family. 186 and λ have common features that serve the same role in both phages: ensuring stable lysogeny and allowing for efficient switching to lytic growth upon induction. The lysogenic state of 186, like λ, is maintained by a repressor protein, the product of the gene cI. However, the λ and 186 operons differ in promoter arrangement, DNA binding sites, and the SOS response. Since bacteriophage λ has been extensively studied, the differences between λ and 186 provide insight into gene regulation and an additional model for the “genetic switch”. In an effort to gain insight into the structural basis of cooperativity and to help elucidate the details of this genetic switch, we pursued the structural determination of the 186 repressor, cI. Biochemical studies indicate that 186 cI has two domains: an N-terminal DNA binding domain and a C-terminal oligomerization domain. The X-ray structure of the C-terminal domain was solved by multiwavelength anomalous diffraction (MAD) to 2.Å resolution. By introducing a cooperativity mutant identified by genetic screens to the full length construct of 186 cI, we were able to obtain crystals of the full length dimer (E146K). The X-ray structure of the full length dimer was solved by molecular replacement (MR) to 1.95 Å resolution. A comparison of the crystallographic structures indicate the overall fold of the C-terminal domain and helix-turn-helix (HTH) motif from 186 cI is remarkably similar to λ repressor. Although similar in their overall fold, the difference in sequence provides insight into how 186 cI works to maintain lysogeny. The structures not only deepen our understanding of the genetic studies, but also provide insight into the framework for understanding the mechanism by which repression occurs

Microevolution in response to transient heme-iron restriction enhances intracellular bacterial community development and persistence.

Bacterial pathogens must sense, respond and adapt to a myriad of dynamic microenvironmental stressors to survive. Adaptation is key for colonization and long-term ability to endure fluctuations in nutrient availability and inflammatory processes. We hypothesize that strains adapted to survive nutrient deprivation are more adept for colonization and establishment of chronic infection. In this study, we detected microevolution in response to transient nutrient limitation through mutation of icc. The mutation results in decreased 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in nontypeable Haemophilus influenzae (NTHI). In a preclinical model of NTHI-induced otitis media (OM), we observed a significant decrease in the recovery of effusion from ears infected with the icc mutant strain. Clinically, resolution of OM coincides with the clearance of middle ear fluid. In contrast to this clinical paradigm, we observed that the icc mutant strain formed significantly more intracellular bacterial communities (IBCs) than the parental strain early during experimental OM. Although the number of IBCs formed by the parental strain was low at early stages of OM, we observed a significant increase at later stages that coincided with absence of recoverable effusion, suggesting the presence of a mucosal reservoir following resolution of clinical disease. These data provide the first insight into NTHI microevolution during nutritional limitation and provide the first demonstration of IBCs in a preclinical model of chronic OM

Antimicrobial Peptide Recognition Motif of the Substrate Binding Protein SapA from Nontypeable <i>Haemophilus influenzae</i>

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen associated with respiratory diseases, including otitis media and exacerbations of chronic obstructive pulmonary disease. NTHi exhibits resistance to killing by host antimicrobial peptides (AMPs) mediated by SapA, the substrate binding protein of the sensitivity to antimicrobial peptides (Sap) transporter. However, the specific mechanisms by which SapA selectively binds various AMPs such as defensins and cathelicidin are unknown. In this study, we report mutational analyses of both defensin AMPs and the SapA binding pocket to define the specificity of AMP recognition. Bactericidal assays revealed that NTHi lacking SapA are more susceptible to human beta defensins and LL-37, while remaining highly resistant to a human alpha defensin. In contrast to homologues, our research underscores the distinct specificity of NTHi SapA, which selectively recognizes and binds to peptides containing the charged-hydrophobic motif PKE and RRY. These findings provide valuable insight into the divergence of SapA among bacterial species and NTHi SapA’s ability to selectively interact with specific AMPs to mediate resistance

The structural basis of cooperative regulation at an alternate genetic switch

Bacteriophage λ is a paradigm for understanding the role of cooperativity in gene regulation. Comparison of the regulatory regions of λ and the unrelated temperate bacteriophage 186 provides insight into alternate ways to assemble functional genetic switches. The structure of the C-terminal domain of the 186 repressor, determined at 2.7 Å resolution, reveals an unusual heptamer of dimers, consistent with presented genetic studies. In addition, the structure of a cooperativity mutant of the full-length 186 repressor, identified by genetic screens, was solved to 1.95 Å resolution. These structures provide a molecular basis for understanding lysogenic regulation in 186. Whereas the overall fold of the 186 and λ repressor monomers is remarkably similar, the way the two repressors cooperatively assemble is quite different and explains in part the differences in their regulatory activity.Heather W. Pinkett, Keith E. Shearwin, Steven Stayrook, Ian B. Dodd, Tom Burr, Ann Hochschild, J. Barry Egan and Mitchell Lewishttp://www.molecule.org

Structural and functional diversity calls for a new classification of ABC transporters.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs