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The Filamentous Phage pIV Multimer Visualized by Scanning Transmission Electron Microscopy

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Science  28 Nov 1997:
Vol. 278, Issue 5343, pp. 1635-1638
DOI: 10.1126/science.278.5343.1635

Abstract

A family of homomultimeric outer-membrane proteins termed secretins mediates the secretion of large macromolecules such as enzymes and filamentous bacteriophages across bacterial outer membranes to the extracellular milieu. The secretin encoded by filamentous phage f1 was purified. Mass determination of individual molecules by scanning transmission electron microscopy revealed two forms, a unit multimer composed of about 14 subunits and a multimer dimer. The secretin is roughly cylindrical and has an internal diameter of about 80 angstroms, which is large enough to accommodate filamentous phage (diameter of 65 angstroms).

Filamentous phage–encoded pIV is an outer-membrane protein required for phage assembly that is not a part of the virus particle. It has sequence similarity to a family of bacterial proteins that are essential components of the type II and type III protein secretion systems that have been identified in many pathogenic Gram-negative species (1, 2). The members of this protein family (called secretins) are believed to play similar roles in mediating translocation of substrates across the outer membrane. Gene IV could be derived from a bacterial gene because some phages contain the gene in different parts of their otherwise colinear genomes and CTX, the lysogenic filamentous phage ofVibrio cholerae that encodes cholera toxin (3), lacks it entirely; CTX may use a V. cholerae secretin for phage assembly. In addition to a secretin, the type II, type III, and phage assembly systems include a protein containing an essential nucleotide-binding motif. The remaining components (∼13 for type II secretion, ∼20 for type III secretion, and 9 encoded by filamentous phage) are related within, but not between, systems.

Bacteria productively infected by filamentous phage remain viable and can continue to grow and divide indefinitely while producing and releasing phage particles. During assembly, the cytoplasmic single-stranded DNA phage genome is extruded through the cytoplasmic membrane where it becomes coated with the phage-encoded capsid proteins that reside in the cytoplasmic membrane before their incorporation into phage (4). Secretion across the outer membrane appears to be concomitant with assembly because periplasmic phage has not been detected. Thus, phage assembly and the type II and III systems all require transport of macromolecules across two bacterial membranes.

The pIV protein exists as a homomultimer that has been previously estimated to consist of 10 to 12 monomers (5). Several of the bacterial homologs form mixed multimers with pIV in vivo, implying structural relatedness and suggesting that they themselves also function as multimers. For several homologs, direct evidence of multimer formation is now available (6-8), and one (9) was shown to be a multimer a decade before it was identified as a member of this protein family. The pIV multimer is unusually resistant to dissociation, as are several other secretins (7, 10, 11). Domain swap experiments between pIVs from related phages implicate the NH2-terminus as a specificity determinant for the choice of filamentous phage to be assembled (12), whereas the NH2-terminus of a type II homolog is necessary for an interaction with substrates, a finding that can account for the species specificity of secretion (8). The fact that secretins exist as large multimers and that in each assembly-secretion system they appear to be the sole integral outer-membrane component suggests that they may form channels in the outer membrane through which secreted enzymes or assembling structures pass. Of necessity, secretin channels would have to be much larger than previously characterized outer-membrane channels, which allow passage of small molecules (∼600 kD) and have internal diameters of ∼11 to 20 Å (13). Filamentous phage is 65 Å in diameter (type IV pili are 55 Å), and several of the folded proteins secreted by type II systems are quite large. Structure-based calculations suggest that 10 to 12 pIV monomers could be arranged to accommodate a sufficiently large pore (14).

Functional His-tagged pIV (monomer molecular weight of 44,619) derived from filamentous phage f1 was solubilized from crude Escherichia coli membranes in nonionic detergent and purified by nickel-chelate and size exclusion chromatography (15,16). More than 90% of the pIV eluted at or above ∼600 kD, which is consistent with previous results indicating that all pIV extracted from cells is multimeric. The purity of pIV, estimated by comparison of immunoblots and stained samples from acrylamide and agarose gels, was ≥95%.

pIV was imaged in the scanning transmission electron microscope (STEM). The STEM is unique in its ability to visualize individual biological molecules directly without staining, fixing, or shadowing. The microscope operates in a dark-field mode, and high-efficiency annular detectors collect nearly all the scattered electrons. The number of electrons scattered is directly proportional to the mass thickness at that point, so that molecular weights of individual objects (or portions of them) can be calculated. The images are recorded digitally, in focus, and as such are directly interpretable. A typical image is shown in Fig. 1. Aside from the tobacco mosaic virus (TMV), which was added as a standard, several species are distinguishable: Rectangular objects and pairs of rectangles predominate, and some rings are also present. The double rectangles and rare brighter rings have similar masses, ∼1240 kD (Table 1), suggesting that they represent two views of the same object. The single rectangles and less intense rings have masses of ∼615 kD, which suggests that there are about 14 pIV monomers per multimer. This finding is consistent with previous estimates of the pIV multimer on the basis of its S value and elution from sizing columns (5, 15). The 1240-kD structures are presumably dimers of multimers, which may reflect formation of head-to-head or tail-to-tail complexes; indirect evidence suggests that dimer formation occurs during purification (15).

Figure 1

Dark-field STEM images of unstained pIV. A 3-μl sample of pIV in 1% CHAPS, 25 mM Hepes (pH 8.0), 0.5 M NaCl, 0.5 mM EDTA, and 0.5 mM benzamidine was injected into a drop of buffer on a thin carbon film (2 to 3 nm) supported by a holey thick carbon film on a titanium grid to which TMV had been previously applied as an internal mass calibration standard (21). The grid was washed five times with 5 μl of 100 mM ammonium acetate and 10 times with 5 μl of 20 mM ammonium acetate, quick frozen in a liquid nitrogen slush, and dried under vacuum. The STEM images were made with the Brookhaven Biotechnology Resource instrument (21). The full scale of the portion of the micrograph shown is 325 nm by 390 nm and the bar indicates 200 Å. The double-headed arrow indicates double rectangles, arrows indicate single rectangles, and arrowheads indicate rings.

Table 1

Dimensions and mass of pIV. Digital images were recorded from the bright-field detector (for stained samples) or the annular dark-field detector (for unstained samples). The dimensions of both TMV and specimen particles in methylamine vanadate–stained samples (Fig. 2) were measured with the program NIH Image and converted to angstroms with the diameter of TMV (180 Å) as a standard. For mass measurements, unstained samples were used. Areas with clean background and adequate numbers of both TMV and specimen particles (Fig. 1) were selected. The background was computed for clear areas and subtracted from the intensity summed over each particle. The STEM calibration factor was checked in each image against that of TMV; the summed intensities (minus the background) multiplied by this calibration factor give mass values for the specimens. ND, not determined.

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Specimens stained with methylamine vanadate and examined in the STEM provide more detail (Fig. 2). The most abundant structure is roughly rectangular and has dimensions similar to those of the rectangle pairs seen in the unstained preparations. It consists of two long curved rods connected by two straight crosspieces and probably connected at the ends as well. Single units of this “ladder-like” structure are presumably the 615-kD species. The single rectangles (Fig. 3B) look remarkably like cartoons of the pIV multimer drawn to interpret its physical properties (2, 8, 17). The ringlike species is also present; examples in Fig. 3C illustrate the large central cavity, which was not apparent in the lower resolution unstained images.

Figure 2

Methylamine vanadate–stained STEM images of pIV. A sample of pIV was applied to a grid to which TMV had already been added and washed as described in Fig. 1, except that the final wash was with 2% methylamine vanadate and the grid was air dried. Full scale of the micrograph is 512 nm in (A) and 128 nm in (B); the bars indicate 200 Å. The arrows are as in Fig. 1.

Figure 3

Montage of pIV species from bright-field STEM images of methylamine vanadate–stained specimens as described in Fig.2. (A) 1240-kD species; (B) 615-kD species; and (C) top or ring views.

An extract from cells that lacked pIV was subjected to the same two-column purification procedure used to isolate pIV, and the corresponding sample was examined in the STEM. The control preparation had one-tenth the amount of protein, and even concentrated samples lacked structures with the mass and appearance of those seen in the authentic preparation (18). A sample of pIV was incubated with polyclonal rabbit antibody to pIV, reisolated on a size exclusion column to remove unbound antibody, and examined in the STEM. The images were complex because of cross-linking by the antibody, and only isolated rectangles were used for mass measurements. Their average mass was substantially larger than the value determined for rectangles in the absence of antibody (1580 ± 190 versus 1250 ± 40 for dimers and 790 ± 40 versus 610 ± 20 for monomers); the greater standard deviation of measurements for pIV plus antiserum is presumably due to variable numbers of antibody molecules bound per pIV multimer. Taken together, these controls provide strong evidence that the structures detected in the STEM are pIV multimers.

The dimensions of the methylamine vanadate–stained structures were quite regular (Table 1). With the added TMV (diameter of 180 Å) as a reference standard, the ring form has an external diameter of ∼140 Å and an internal diameter of ∼80 Å. The external diameter of the ladder along the crosspieces is ∼130 Å (internal diameter of ∼80 Å). These values also suggest that the two forms are different views of the same object. They indicate that pIV is cylindrical and has an internal hole sufficient to accommodate filamentous phage. As measured in the STEM with TMV as a standard, filamentous phage was 66 to 68 Å in diameter (18).

The contour length of the double rectangle is ∼210 Å, whereas that of the single is ∼70 Å. The single rectangles have curved ends and a single crosspiece with the same dimensions as the larger structure, but the portion of the larger structure that lies between the two crosspieces is rarely visible in the single rectangle (compare Fig. 3, A and B); hence, the average length of single rectangles is less than half that of the double. Perhaps the hidden ends of this segment are hydrophobic and either bury themselves within a single multimer or self-associate to form dimers of multimers. This phenomenon may be related to the oriented rosettes that form when detergent is removed from purified influenza hemagglutinin and neuraminidase, leaving the hydrophobic domains at the core (19). This, in turn, would suggest that the “sticky” end is the COOH-terminal portion of the protein, which is normally embedded in the outer membrane, and thus that the curved arms constitute the periplasmic domain that confers specificity to the secretion-assembly process and potentially gates the channel. A single ∼70 Å–long multimer could span the outer membrane, which is ∼25 Å thick (20), and still have a substantial periplasmic domain. The crosspiece itself, which encircles the central channel, could serve as a reinforcing ring that provides the resistance to dissociation that appears to be characteristic of most secretins.

  • * To whom correspondence should be addressed. E-mail: russelm{at}rockvax.rockefeller.edu

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