A Docking Model Based on Mass Spectrometric and Biochemical Data Describes Phage Packaging Motor Incorporation

The molecular mechanism of scaffolding protein-mediated incorporation of one and only one DNA packaging motor/connector dodecamer at a unique vertex during lambdoid phage assembly has remained elusive because of the lack of structural information on how the connector and scaffolding proteins interact. We assembled and characterized a φ29 connector-scaffolding complex, which can be incorporated into procapsids during in vitro assembly. Native mass spectrometry revealed that the connector binds at most 12 scaffolding molecules, likely organized as six dimers. A data-driven docking model, using input from chemical cross-linking and mutagenesis data, suggested an interaction between the scaffolding protein and the exterior of the wide domain of the connector dodecamer. The connector binding region of the scaffolding protein lies upstream of the capsid binding region located at the C terminus. This arrangement allows the C terminus of scaffolding protein within the complex to both recruit capsid subunits and mediate the incorporation of the single connector vertex.The DNA packaging motor of double-stranded DNA bacteriophages translocates genomic DNA into a preformed procapsid to near crystalline density and is the strongest motor characterized to date. The packaging motor of the Bacillus subtilis phage φ29 can work against 57 piconewtons of internal force and translocate 2 bp of DNA per ATP hydrolyzed at a maximum velocity of 103 bp/s (1, 2). The motor complex is assembled on a dodecamer of the connector protein, which replaces a pentameric vertex in the procapsid and serves both as a portal for DNA passage and the docking site for the other packaging components (3).To successfully package a full-length genome, incorporation of one and only one connector vertex is essential (4). In vivo, nearly every assembled procapsid has one and only one connector vertex and is able to package DNA and mature into an infectious phage (5). This narrow distribution in which 95% of particles have a single connector vertex cannot be explained by random statistical incorporation. The control mechanism is coupled to the procapsid assembly process. Procapsid assembly requires the copolymerization of hundreds of copies each of the capsid and scaffolding proteins as well as a dodecamer of the portal or connector protein. The scaffolding protein acts to both activate the coat protein for assembly and ensure proper form determination. In the absence of scaffolding protein, uncontrolled polymerization results in the assembly of aberrant structures. In a properly assembled procapsid, the portal protein is located at one vertex, whereas scaffolding protein occupies the bulk of the interior space and is subsequently removed during DNA packaging by either proteolysis or simple release. Mutational studies have indicated that scaffolding protein is involved either directly or indirectly in the incorporation of the connector vertex during procapsid assembly in a variety of phages (6–8).In φ29, the connector vertex is specifically incorporated at one of the two 5-fold vertices lying on the long axis of a prolate procapsid composed of 235 copies of capsid protein and containing ∼180 copies of scaffolding protein (9, 10). The structure of the 33-kDa connector protein subunit consists of three long central α-helices bridging wide and narrow domains that are rich in β-sheets and extended polypeptides (Fig. 1A) (10–12). The 12 subunits are arranged to form a 75-Å-long tapered grommet-shaped structure with an external diameter of 69 Å at the wide end and 33 Å at the narrow end. By fitting the crystal structure of the connector dodecamer into the cryo-EM¹ density of the procapsid, the orientation of connector at the unique vertex of the procapsid was revealed. The wide domain of connector protein lies inside the procapsid, and the narrow domain is exposed to the exterior and makes contacts with the other parts of the motor complex (11). The 11-kDa scaffolding protein subunits form nanomolar affinity homodimers resembling arrows in solution. Each subunit contributes one side of the arrowhead and one-half of the long coiled coil shaft (Fig. 1B) (13). The subunit structure consists of three helical segments. A three-turn N-terminal helix (α1) followed by a five-residue loop, and an antiparallel five-turn helix (α2) makes up the arrowhead and part of the proximal part of the shaft. A three-residue loop and a seven-turn helix (α3) complete the shaft. The C-terminal 15 residues, which interact with capsid protein as determined in the in vitro assembly assay, are disordered in the crystal structure (14).Open in a separate windowFig. 1.The x-ray crystal structures of connector protein (Protein Data Bank code 1FOU, chains A and B) (A) and scaffolding protein (Protein Data Bank code 1NO4, chains A and B) (B).We have recently reported the development of an in vitro assembly system for phage φ29 in which purified connector protein complex can be successfully incorporated (15). The addition of connector protein dodecamers to coat and scaffolding subunits accelerated the rate of assembly and lowered the critical concentration, suggesting involvement in nucleation of assembly (15). Here we used native mass spectrometry, chemical cross-linking, and mutational analysis to characterize the interactions between the connector and the scaffolding proteins and develop a model of the scaffolding-connector complex, which provides a molecular model of how scaffolding protein might mediate stringent incorporation of one and only one connector dodecamer.     

Identification of subunit-subunit interactions in bacteriophage P22 procapsids by chemical cross-linking and mass spectrometry

Viral capsids are dynamic structures which self-assemble and undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Chemical cross-linking analyzed by mass spectrometry was used to identify residue specific inter- and intra-subunit interactions in the P22 procapsids. All the intersubunit cross-links occurred between residues clustered in a loop region (residues 157-207) which was previously identified by mass spectrometry based on hydrogen/deuterium exchange and biochemical experiments. DSP and BS3 which have similar distance constraints (12 angstroms and 11.4 angstroms, respectively) cross-linked the same residues between two subunits in the procapsids (K183-K183), whereas DST, a shorter cross-linker, cross-linked lysine 175 in one subunit to lysine 183 in another subunit. The replacement of threonine with a cysteine at residue 182 immediately adjacent to the K183 cross-linking site resulted in slow spontaneous disulfide bond formation in the procapsids without perturbing capsid integrity, thus suggesting flexibility within the loop region and close proximity between neighboring loop regions. To build a detailed structure model, we have predicted the secondary structure elements of the P22 coat protein, and attempted to thread the prediction onto identified helical elements of cryoEM 3D reconstruction. In this model, the loop regions where chemical cross-linkings occurred correspond to the extra density (ED) regions which protrude upward from the outside of the capsids and face one another around the symmetry axes.     

Deconstructing Ixodes ricinus: a partial matrix model allowing mapping of tick development,mortality and activity rates

A stage‐structured Leslie matrix model of a partial, discrete population of Ixodes ricinus (Linnaeus) (Ixodida: Ixodidae) ticks was developed to elucidate the impact of climate trends on the distribution and phenology of this species in the western Palaearctic. The model calculates development and mortality rates for each instar and evaluates recruitment rates based on the development of the tick population. The model captures the changes in development and mortality rates, providing a coherent index of performance correlated with the tick's geographic range. Maximum development rates are recorded for latitudes south of 36 °N and are spatially correlated with sites of maximum temperature, highest saturation deficit and highest mortality. The maximum available developmental time (the total annual time during which temperature allows development) for I. ricinus in the western Palaearctic is < 45% of the total year. North of 60 °N, available developmental time decreases sharply to only 15% of the year. The latitudinal boundary at which survival rates sharply drop is 43–46 °N, clearly delimiting the classically recognized extent of the main tick populations. The pattern of activity for larval–nymphal synchrony shows a clear west–east pattern. The model demonstrates the impact of climate according to tick stage and geographic location, and provides a practical framework for testing how the tick's lifecycle is affected by climate change.     

Long‐proboscid brachyceran flies in Cretaceous amber (Diptera: Stratiomyomorpha: Zhangsolvidae)

The monophyletic family Zhangsolvidae comprises stout‐bodied brachyceran flies with a long proboscis and occurring only in the Cretaceous, originally known in shale from the Early Cretaceous Laiyang Formation (Fm.) in China (Zhangsolva Nagatomi & Yang), subsequently from limestones of the Early Cretaceous Crato Fm. of Brazil. Cratomyoides Wilkommen is synonymized with Cratomyia Mazzarolo & Amorim, both from the Crato Fm.; Cratomyiidae is synonymized with Zhangsolvidae. Two genera and three species of Zhangsolvidae are described: Buccinatormyia magnifica Arillo, Peñalver & Pérez‐de la Fuente, gen. et sp.n. and B. soplaensis Arillo, Peñalver & Pérez‐de la Fuente, sp.n. , in Albian amber from Las Peñosas Fm. in Spain; and Linguatormyia teletacta Grimaldi, gen. et sp.n. , in Upper Albian–Lower Cenomanian amber from Hukawng Valley in Myanmar. Buccinatormyia soplaensis and Linguatormyia teletacta are unique among all Brachycera, extant or extinct, by their remarkably long, flagellate antennae, about 1.6× the body length in the latter species. A phylogenetic analysis of 52 morphological characters for 35 taxa is presented, 11 taxa being Cretaceous species, which supports placement of the family within Stratiomyomorpha, although not to any particular family within the infraorder. This published work has been registered in Zoobank, http://zoobank.org/urn:lsid:zoobank.org:pub:F32CF887‐7C37‐45D5‐BD6B‐135FE9B729A7 .     

Distribution of streptothricin acetyltransferase encodi determinants among environmental bacteria

In this paper we report about a screening for streptothricin- (St)-resistant phenotypes and genotypes among environmental bacteria from a St virgin area. St-resistant bacteria were isolated from river water, sewage, manure and soil by selective plating. The resistance quotient was typical of an area without selective pressure. The occurrence of streptothricin acetyltransferase-encoding determinants and their localization on a Tn7-like transposon was tested by the application of a set of gene probes. Sat genes could be detected in 22.5% of the tested St-resistant bacteria but in 100% of the checked Enterobacteriaceae. However, we could not detect sat genes in St-resistant bacteria from soil samples. Surprisingly the sat genes were found to be located on conjugative or mobilizable plasmids for a rather high number of strains. The determined plasmid species and their restriction patterns showed a high degree of similarities to those observed from an area of strong selective pressure.     

Resolving UV Bioactinometric Results with Intensity and Time Distributions

A UV reactor with an annular design, a total liquid volume of 460[emsp4 ]ml, and outfitted with a single lamp with 1690[emsp4 ]mW of germicidal power was tested. Coliphage MS2 was used as a bioactinometer to measure the UV dose at a flow rate of 56.7[emsp4 ]ml/sec in water with a very low absorbance. The Beers Law coefficient was A₁₀0.003. The measured dose (MS2 bioactinometry) was 35.2±1.1[emsp4 ]mW-sec/cm².A retention time distribution was generated with a dye tracer study. The reactor was modeled as if flow was confined to ten equal volume paths existing as concentric rings around the lamp. The UV intensity along each path (ith intensity) was calculated to generate a simulated distribution of UV intensity in the reactor. The retention time distribution was subdivided to estimate the retention time associated with each decile jth time) of the total flow.Seven methods of associating the ith intensity with the jth retention time were used to produce simulated dose distributions for the reactor. The average UV dose for each distribution was calculated as the average of the products of I and t (AP protocol) and by the apparent survival (AS protocol), in which the predicted survival along each path was averaged to back-calculate dose from the reference batch inactivation curve. The average dose predicted assuming that time and intensity were independent was 51.5[emsp4 ]mW-sec/cm² based on the arithmetic average (AP protocol). Using the apparent survival method, the predicted dose for the independent distribution (I independent of t) was 36.4[emsp4 ]mW-sec/cm². Three methods of developing dependent structure between time and intensity were tested. In the best possible case for stratified flow (I negatively correlated with t) the calculated (AS) intensity was 46.3[emsp4 ]mW-sec/cm^2. In the worst case for stratified flow (I positively correlated with t) the AS intensity was 32.0[emsp4 ]mW-sec/cm². In a rational case where flows were assumed to be distributed parabolically (low flow at the wall and at the lamp) produced an AS intensity of 37.7[emsp4 ]mW-sec/cm². When either time or intensity was averaged, while the other variable was allowed to keep its distribution, the (AS) dose (time averaged 43.3[emsp4 ]mW-sec/cm², intensity averaged 41.0[emsp4 ]mW-sec/cm²), yielded a poor prediction compared to the measured value.The errors associated with averaging time, intensity, or both, far outweigh the errors associated with choosing a rational distribution or an independent distribution of time and intensity in the prediction. This observation is generally true whenever an organism is exposed to UV light in a flow through reactor such that the range of doses is within the portion of the inactivation curve exhibiting strong exponential decay.