FINALIDADES DE LA NUEVA REGULACIÓN

From an experimental perspective, it has been argued that there are a variety of mechanisms by

which phage either passively push their genomes into the host cell or, alternatively, have it actively

pulled into the cell [40, 16, 95, 103]. While the ejection in many phages like T4 and λoccurs in a

single step, the ejection in other phages like T5 andφ29 occurs in more than one step with different

ejection mechanisms operative in each step. The aim of this section is to provide an overview of the

in vitroandin vivostudies on the ejection mechanisms in different phages.

4.2.1

In vitro

Studies of Ejection Kinetics

The DNA ejection in λ and T5 phages is triggered by an outer membrane receptor protein that

can be isolated. The receptor protein maintains its viability even if suspended in free solution

or incorporated into liposomes, i.e., it triggers the DNA ejection from the corresponding phage even under such conditions [14]. This makes it possible to study the in vitro ejection behavior

of these phages. Most other phages, on the other hand, have their ejection triggered by certain

polysaccharides. These polysaccharides change their structure upon isolation, thus rendering them

incapable of initiating the DNA ejection. This is the main reason whyin vitro studies of the DNA

ejection process from virions have been restricted toλandT5 [103].

First, we consider the case of bacteriophage λ. In the experiment performed by Novick et

al. [14], phage λwas suspended in a solution containing 10 mM MgCl2 in the presence of vesicles

reconstituted with LamB, the outer membrane receptor protein that triggersλphage ejection. Theλ

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

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

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.