Evidence for bacteriophage T7 tail extension during DNA injection
© Serwer et al; licensee BioMed Central Ltd. 2008
Received: 21 March 2008
Accepted: 26 June 2008
Published: 26 June 2008
Electron micrographs of bacteriophage T7 reveal a tail shorter than needed to reach host cytoplasm during infection-initiating injection of a T7 DNA molecule through the tail and cell envelope. However, recent data indicate that internal T7 proteins are injected before the DNA molecule is injected. Thus, bacteriophage/host adsorption potentially causes internal proteins to become external and lengthen the tail for DNA injection. But, the proposed adsorption-induced tail lengthening has never been visualized.
In the present study, electron microscopy of particles in T7 lysates reveals a needle-like capsid extension that attaches partially emptied bacteriophage T7 capsids to non-capsid vesicles and sometimes enters an attached vesicle. This extension is 40–55 nm long, 1.7–2.4× longer than the T7 tail and likely to be the proposed lengthened tail. The extension is 8–11 nm in diameter, thinner than most of the tail, with an axial hole 3–4 nm in diameter. Though the bound vesicles are not identified by microscopy, these vesicles resemble the major vesicles in T7 lysates, found to be E. coli outer membrane vesicles by non-denaturing agarose gel electrophoresis, followed by mass spectrometry.
The observed lengthened tail is long enough to reach host cytoplasm during DNA injection. Its channel is wide enough to be a conduit for DNA injection and narrow enough to clamp DNA during a previously observed stalling/re-starting of injection. However, its outer diameter is too large to explain formation by passing of an intact assembly through any known capsid hole unless the hole is widened.
A double-stranded DNA bacteriophage starts an infection by injecting its DNA genome into a host cell; progeny later assemble by packaging a genome. The protein movements that cause DNA injection are usually assumed to be restricted primarily to an external tail; the protein movements of DNA packaging are usually assumed to be restricted primarily to a multimeric DNA packaging ATPase [reviewed in references [1–3]]. However, as previously discussed , the protruding portion of the tail of bacteriophage T7 (23 nm long ) appears not long enough to reach from the outer shell of the bacteriophage, through the outer host membrane, the periplasmic space/cell wall and then the inner membrane, total distance about 24 nm [6, 7]. Thus, DNA injection appears to require lengthening of the T7 tail.
The injected internal proteins potentially lengthen the T7 tail for DNA injection and also possibly DNA protection after injection [8, 15]. However, evidence for such tail lengthening was not reported in the studies of references 4, 8, 9, 13 and 14. Intriguingly, a capsid has been observed with needle-like extension long enough to be a lengthened T7 tail . However, the extension was not bound to outer membrane, as it would be if injecting DNA. In the present study, a similar extension is observed by electron microscopy to attach and sometimes penetrate vesicles likely to be from the outer host membrane.
Electron microscopy revealed that the region between CII and dimers in Figure 2b had vesicle-like particles, possibly host outer membrane vesicles [16, 17]. Thus, gradient fractions were screened for bacteriophage capsids bound to vesicle-like particles in a complex that had the following characteristics expected of a DNA injection complex (IC): (a) either a bacteriophage tail or a lengthened tail connected bacteriophage to vesicle-like particle (to be called a vesicle) and (b) DNA was at least partially emptied from the capsid. Apparent IC's were found concentrated in the region of the gradient indicated with the bracket labeled IC and coincident with a band of light scattering in Figure 2b (1.359 g/ml).
The bacteriophage particles indicated by arrows 2 and 3 in Figure 3 have tails (note the tapering) and are attached via their DNA-containing outer shells (not the tails) to an object previously characterized [5, 18] as a product (called polycapsid) of mis-assembly of the major protein (gp10) of the capsid's outer shell. The polycapsids have vesicles within them, a point previously noted [5, 18]. The polycapsid-adsorbed bacteriophages have not injected DNA and this adsorption is assumed not to have significance for biological injection.
For the two bracket-marked capsids in Figure 3, the needle-like extension appears less tapered than the tail, an observation also made for all 27 other vesicle attached extensions observed. The total extension length cannot be determined for the bracket-marked capsids because of partial obscuring of part of the extension, especially by material inside of the vesicle. Although partially obscured by this material, the extension of the left bracket-marked capsid in Figure 3 does appear to extend in a straight line through the proximal surface of the vesicle, completely to the distal vesicle surface, producing a total length of 40–50 nm.
When analyzed by non-denaturing agarose gel electrophoresis, the vesicle-containing fractions of the gradient in Figure 2b all produced a single, broad "vesicle" band that was further from the origin than capsids (not shown). To characterize the vesicles, vesicle proteins were analyzed after vesicle purification by both the centrifugation of Figure 2a, b and then preparative non-denaturing two-dimensional agarose gel electrophoresis (2d-AGE). The sample for 2d-AGE was a solution of Alexa 488-stained particles with a density of 1.265 g/ml (density of capsid II). The procedure of 2d-AGE uses a dilute (0.3%) gel for the first dimension and a more concentrated (1.8%) gel for the second; details are described and reviewed in reference 19.
The fluorescence profile of Alexa 488-stained particles after 2d-AGE had primarily two features. (a) A band of a capsid not further investigated had a small percentage of the fluorescence (arrow inside Figure 5a). (b) A streak of vesicles had most fluorescence (bracket with capital letters in Figure 5a). The RE standard is capsid I (boxed band in Figure 5a). The capsid I had been run in parallel in a gel embedded in the same agarose frame (different quadrant) in which the gel of Figure 5a was embedded ; the image of the capsid I band was then overlaid on the image of Figure 5a. A streak of the type observed above for vesicles is expected for particles that are uniform in σ and variable in RE . The range of RE values for streak-producing particles was determined by quantification of θ values at the two ends of the streak and was found to be 20–50 nm, within the range of values previously found for E. coli outer membrane vesicles (10–100 nm ).
For further analysis, the gel at the position of the streak of Figure 5a was cut into five slices, indicated by letters in Figure 5a. The slices were boiled and the proteins were analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDSPAGE). After staining with SYPRO Ruby protein gel stain (Figure 5b), the proteins of the indicated bands were excised, digested in situ with trypsin and identified by HPLC-electrospray-tandem mass spectrometry. The two major proteins identified for slices A-E were ompA  and ompF , as indicated in Figure 5b; these proteins were not detected in several non-staining areas of the agarose gel of Figure 5a that were analyzed (not shown).
The next most abundant protein was the 60 Kd chaperonin (indicated by chaperonin in Figure 5b). However, this protein was found in several locations in the agarose gel, even where no band or streak was detected. The 60 Kd chaperonin is, therefore, not significantly associated with the vesicles. Additional, vesicle-associated proteins were ferrichrome iron receptor protein, product of the FhuA gene (FhuA in Figure 5b)  and ferrienterobactin outer-membrane receptor precursor, product of the FepA gene (FepA in Figure 5b) . OmpA, ompF, FhuA and FepA proteins are all E. coli outer membrane proteins. Thus, we conclude that the vesicles are formed from the E. coli outer membrane.
The length of the observed needle-like extension is great enough to more than traverse the host's outer membrane, periplasmic space/cell wall and inner membrane. Thus, we hypothesize that this extension lengthens the tail for DNA injection and that the extension contains at least some of the proteins that others [8, 9] have found to be injected. The small size of its channel supports the hypothesis  that a constriction causes the pre-transcription delay of injection. The extension is too short to directly explain protection of the initially injected 850–1,000 base pairs.
If gp6.7, gp14, gp15 and gp16 are part of the lengthened tail, the question remains: Did these proteins cross the capsid's outer shell while assembled as in Figure 1? If so, were the assembled proteins unfolded, as previously proposed ? Or, did these proteins cross as single protein molecules and subsequently assemble to form the needle? If the proteins cross while both assembled and folded, the outer shell/tail connector (a ring of gp8; Figure 1) would have to widen its central hole that varies from 3.5–4.5 nm in diameter . In either case, formation of the tail extension would require protein motion not previously observed.
For support, we thank the National Institutes of Health (GM24365 to PS), the Welch Foundation (AQ-764 to PS) and The Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation (PS). Protein identifications were conducted in the UTHSCSA Institutional Mass Spectrometry Laboratory.
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