Repurposing a chemosensory macromolecular machine

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identify ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we trace the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost

Estimation of Influenza Vaccine Effectiveness from Routine Surveillance Data

BACKGROUND: Influenza vaccines are reviewed each year, and often changed, in an effort to maintain their effectiveness against drifted influenza viruses. There is however no regular review of influenza vaccine effectiveness during, or at the end of, Australian influenza seasons. It is possible to use a case control method to estimate vaccine effectiveness from surveillance data when all patients in a surveillance system are tested for influenza and their vaccination status is known. METHODOLOGY/PRINCIPAL FINDINGS: Influenza-like illness (ILI) surveillance is conducted during the influenza season in sentinel general practices scattered throughout Victoria, Australia. Over five seasons 2003-7, data on age, sex and vaccination status were collected and nose and throat swabs were offered to patients presenting within three days of the onset of their symptoms. Swabs were tested using a reverse transcriptase polymerase chain reaction (RT-PCR) test. Those positive for influenza were sent to the World Health Organization (WHO) Collaborating Centre for Reference and Research on Influenza where influenza virus culture and strain identification was attempted. We used a retrospective case control design in five consecutive influenza seasons, and estimated influenza vaccine effectiveness (VE) for patients of all ages to be 53% (95% CI 38-64), but 41% (95% CI 19-57) adjusted for age group and year. The adjusted VE for all adults aged at least 20 years, the age groups for whom a benefit of vaccination could be shown, was 51% (95% CI 34-63). Comparison of VE estimates with vaccine and circulating strain matches across the years did not reveal any significant differences. CONCLUSIONS/SIGNIFICANCE: These estimates support other field studies of influenza vaccine effectiveness, given that theoretical considerations suggest that these values may underestimate true effectiveness, depending on test specificity and the ratio of the influenza ILI attack rate to the non-influenza ILI attack rate. Incomplete recording of vaccination status and under-representation of children in patients from whom a swab was collected limit the data. Improvements have been implemented for prospective studies

Repurposing a chemosensory macromolecular machine

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identify ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we trace the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost

Structure-Function Relationships in the HAMP and Proximal Signaling Domains of the Aerotaxis Receptor Aer▿

Aer, the Escherichia coli aerotaxis receptor, faces the cytoplasm, where the PAS (Per-ARNT-Sim)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximal signaling domains was probed to elucidate structure-function relationships important for signaling. Aer residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two α-helices separated by a structured loop. The proximal signaling domain consisted of two α-helices separated by a short undetermined structure. The Af1503 HAMP domain from Archaeoglobus fulgidus was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for the Aer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helix HAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the β-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed

HAMP Domain Conformers That Propagate Opposite Signals in Bacterial Chemoreceptors

<div><p>HAMP domains are signal relay modules in >26,000 receptors of bacteria, eukaryotes, and archaea that mediate processes involved in chemotaxis, pathogenesis, and biofilm formation. We identify two HAMP conformations distinguished by a four- to two-helix packing transition at the C-termini that send opposing signals in bacterial chemoreceptors. Crystal structures of signal-locked mutants establish the observed structure-to-function relationships. Pulsed dipolar electron spin resonance spectroscopy of spin-labeled soluble receptors active in cells verify that the crystallographically defined HAMP conformers are maintained in the receptors and influence the structure and activity of downstream domains accordingly. Mutation of HR2, a key residue for setting the HAMP conformation and generating an inhibitory signal, shifts HAMP structure and receptor output to an activating state. Another HR2 variant displays an inverted response with respect to ligand and demonstrates the fine energetic balance between “on” and “off” conformers. A DExG motif found in membrane proximal HAMP domains is shown to be critical for responses to extracellular ligand. Our findings directly correlate in vivo signaling with HAMP structure, stability, and dynamics to establish a comprehensive model for HAMP-mediated signal relay that consolidates existing views on how conformational signals propagate in receptors. Moreover, we have developed a rational means to manipulate HAMP structure and function that may prove useful in the engineering of bacterial taxis responses.</p> </div

Inter-spin distance measurements by PDS.

<p>Shown are experimentally determined distances of spin-labeled proteins and Cα–Cα distances from the Aer2 1–172 crystal structure. The values shown in parentheses refer to the width (Å) at half the maximum peak height, and qualify peak broadening and conformational heterogeneity. Small values represent narrow peaks and a homogeneous conformation. Large values represent broad peaks consistent with more heterogeneous populations.</p>a<p>Attachment of the MTSSL spin labels can add up to 13 Å to the Cα–Cα separation, or equivalently 6.5 Å each.</p

H1D and H1 V33G receptors both respond to attractant, but with normal and inverse responses, respectively.

<p>(A) Swim assays of ATC receptors on tryptone agar plates. Colonies with functional chemoreceptors generate a characteristic ring near the leading edge of an expanding colony as cells consume Asp and swim towards higher Asp concentrations. H1 V33G generates an inverted ring, in comparison to Tar, which suggests an inverted CCW-to-CW response to Asp. (B) Temporal assays of transmembrane receptors showing response and adaptation kinetics. CheRB+ cells expressing various receptors were allowed to reach adaptation equilibrium before Asp was added. Tumbling frequencies alter if receptors are capable of receiving and relaying signal input from TM2 to the output KCM. Tar responds in the normal direction, switching from 12.5% to 1% CW bias. After ∼300 s, the adaptation system restores Tar CW bias to 12.5%. H1D has a normal Asp response, switching from 17.5% to 2.5% CW bias. H1 V33G displays an inverted response, switching from 16% to 100% CW bias upon Asp addition. A lower concentration of Asp is representative of increased receptor sensitivity.</p

Model for HAMP domain signal relay in bacterial chemoreceptors.

<p>The HAMP domains of MCPs exchange between HAMP1 and HAMP2 states to regulate bacterial chemotaxis. The conformation of HAMP2 imparts a two-helix coiled coil across the AS2/KCM junction, which results in CheA kinase inhibition and CCW flagella rotation. A dynamic HAMP1 forms a continuous four-helix coiled coil across the junction to generate kinase activation and CW flagella rotation.</p

Repurposing a chemosensory macromolecular machine

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identify ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we trace the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost.ISSN:2041-172