RRout
4.2 Various Mechanisms for Genome Injection
function of time using estimates of the spatially-varying ejection force from recent theories of phage packaging energetics. We find that the translocation times are 2 to 3 orders of magnitude faster than the diffusional time. We also treat the case of ejection into a volume comparable to the capsid size (mimicking, say, studies in which phage are made to eject into small vesicles that have been reconstituted with receptor protein [15, 102]) and find the ejection time dependence on the relative sizes of the phage capsid and the vesicle. In Section 4.4 we treat the further speed-up in translocation due to ideal ratcheting and reversible particle binding. We find that the simple ratcheting effect is small compared to that arising from the entropic force of reversible particle binding. The effect of reversible particle binding decreases the translocation time by another order of magnitude beyond that due to capsid pressure effects. We conclude in Section 4.5 with a discussion of related work by others, of additional contributions to ejection dynamics that will be studied in future theoretical work (in particular, the effect of RNA polymerase acting on the ejected DNA), and of experiments planned to test the various predictions made in the present work.
the vesicle. The vesicle is filled with a dye called Ethidium Bromide which fluoresces upon binding to the DNA. The saturation in the observed fluorescence was obtained in about a minute, which was concluded to be the ejection time. In its fully packed configuration, at this salt concentration, phage λhas an internal force of the order 10 pN on its genome [61, 28]. Since there is nothing else to bolster the injection, it is reasonable to assume that the DNA is passively ejected from the capsid into the vesicle as a result of the internal force, which biases the diffusive motion of the DNA through the phage tail. Though this experiment is extremely elegant, it must be pointed out that there are some problems in interpreting the results. It was pointed out by P. Grayson (personal communication) that the amount of Ethidium Bromide in the vesicles was insufficient to fully bind to the total DNA length of the phage, as a result of which, it is not possible to interpret the data as an unambiguous measure of ejection time. On the other hand, elegant single phage experiments conducted by P.
Grayson in our lab have shown that under similar salt conditions,λ ejects its complete genome is around 10−15 seconds.
The interaction of T5 virions with their receptor, FhuA, causes rapid ejection of the phage genomein vitro[15]. If FhuA is incorporated into lipsomes, the amount of DNA translocated from the phage head into the liposome interior is dependent on its volume. This is consistent with the idea that the forces in the phage virion drive DNA ejection until the resistive forces from the DNA already inserted into the liposome are balanced. Also, individual T5 phage have been observed by Mangenot et al. [104] to eject DNA at an extremely high rate of around 75kb/s. A fraction of the genomes ejected paused at distinct regions, which correlate well with sites of the major single-strand nicks on the T5 genome. The nicks supposedly provide an energetic barrier to the in vitro DNA ejection process.
4.2.2 In vivo Ejection Studies
The DNA ejection in phageλoccurs in a single step at a rate of around 0.5kb/s. The DNA ejection is supposedly effected by the internal pressure in the phage capsid. On the other hand, it was seen in the previous chapter that only 60% of the phage genome is ejected at around 3atm, the approximate osmotic pressure in the bacterial cell. This would mean that some other mechanism should aid the phage to eject the DNA into the cell. Unfortunately, the presence of such a mechanism has not been experimentally demonstrated.
In the case of T4, the phage adsorbs onto the bacterial membrane and binds to its receptor lipopolysaccharide, which triggers a contraction of the tail. The tail contraction helps puncture the outer membrane and brings its tip close to the cytoplasmic membrane [39, 105]. The 172 kbp DNA then crosses the membrane in about 30 seconds at 37◦C through a phage protein gp5, which forms a voltage gated channel across the membrane [40]. This represents an extremely high rate if around 6 kb/s, observed for DNA transport, and is significantly faster than if effected by enzymes [40].
Also, since the normal transcription times for the RNAP are of the order of minutes [27], 30 seconds seems to leave insufficient time for the enzymes to mediate infection. Further, it has been found experimentally that the phage does not internalize its DNA in the absence of a potential difference across the membrane [40]. This observation led to the speculation that the DNA injection is caused by the membrane potential. However, it was subsequently shown that the voltage serves to open the voltage-gated channel formed by the phage protein. Hence, it appears that DNA ejection in the T4 phage is governed by the tight internal packing of the DNA inside the capsid, resulting in a driving force tied to the free energy release when the DNA is liberated from the capsid.
Injection in the case of phage T7 is more complicated. T7 has a genome of about 40 kbp, and its capsid is icosahedral with a diameter of around 60 nm. It has an inner cylindrical core of about 28 nm×10 nm formed of three proteins. Experimental data on T7 suggests that this phage first binds to the bacterial outer membrane. A signal is then passed through the phage tail and it releases some proteins from the capsid. This in turn triggers ejection of the cylindrical core, which penetrates the bacterial membrane and forms a channel for injection of the DNA. The internalization of the phage DNA is based upon a tripartite mechanism. First 850 bp of DNA, which has promoters for the E. coli RNA polymerase (RNAP), gets ejected by a proton motive force. The transcription due to the bacterial RNAP pulls out another 7 kbp of the phage DNA and leads to the manufacturing of T7 RNAP. The exposed DNA has promoters for T7 RNAP. The T7 RNAP then binds onto these promoters and internalizes the remaining DNA into the bacterial cell. The total time of injection for wild-type T7 is around 10 minutes at 30◦C [16, 106].
Phage T5 presents yet another example of the richness of the infection mechanisms adopted by bacteriophage. In this case, the genome length is roughly 86 kbp. As noted above, the phage binds to a cell surface receptor FhuA, which triggers the DNA ejection. The ejection process in T5 occurs in two steps. The first step transfer (FST), which involves 8% of the total DNA, is thought to be effected by the internal pressure. After the first step there is a pause for about 4 minutes (at 37 ◦C) during which time the proteins encoded by this part of the DNA are synthesized. Two of the proteins (A1 and A2) then transfer the remaining 92% DNA during a process called second step transfer (SST). The pause of 4 minutes is believed due to the fact that the FST DNA forms stem-and-loop structures that can jam the DNA and thus protect the viral DNA from the bacterial restriction system [40].
The ejection in φ29 phage is argued to be accomplished by the following two step process [95].
In the first step, about 65% of the DNA is injected into the cell, most likely by the high pressure inside the φ29 capsid [95, 11]. The genes associated with the first part of the ejected DNA are used to manufacture proteins and at least one protein,P17, participates in the molecular machinery that pulls the remaining DNA inside the bacterial cell [95]. The total time for the entire process is observed to be around half an hour.
Phage Hypothesized Genome Ejection Ejection Mechanism Length (kbp) Time (sec) Rate (kbp/sec)
λ Pressure 48.5 60 0.8
T4 Pressure 169 30 5.6
T7 Enzyme 40 6001 0.06
T5 Pressure+Protein 121 3602 0.3
φ29 Pressure+Enzyme 19 1800 0.05
Table 4.1: Tabulation of different types of ejection behavior in different phages and their average rates of ejection. It can be seen that the phages show different types ejection mechanism, and a wide variation in the average ejection rates.
We thus have seen that the ejection behavior in bacteriophage follow a rich behavior pattern. A compilation of the rates and hypothesized mechanisms for different phages is made in Table. 4.1.