Escala de Evaluación de Funcionamiento Familiar

As well as regulators of community structures, bacteriophages have long been accepted as drivers of prokaryotic evolution. Some of the mechanisms by which they achieve this are outlined below.

1.5.1 Phages as mediators of lateral gene transfer

The phenomenon of phage-mediated lateral gene transfer (LGT) or transduction has been known about for almost 60 years (Zinder and Lederberg, 1952) and is described in many marine phage-host systems. The genetic material transferred can have repercussions on the individual host e.g. conferring toxicity and in turn altering microbial genetic diversity. A recent example can be found in a study of vibriophages in southern California coastal waters. Here it was shown that environmental phage isolates were able to infect toxigenic V. cholerae strains and transfer a genetic marker, CTXΦ, to another environmental non-toxic strain (Choi et al., 2010).

Another study carried out by Jiang and Paul (1998) quantified the transduction frequency of a plasmid in two concentrated samples of mixed bacteria as being between 1.58 x 10-8 to 3.7 x 10-8transductants PFU-1. Using known bacterial and viral concentrations, as well as the water volume of the Tampa Bay Estuary, the authors calculated the number of transduction events per year to be up to 1.3 x 1014. Such high rates would make transduction an important mechanism for gene evolution in the marine environment and highlights the role of phages in exchanging DNA between bacterial populations.

1.5.2 Phage resistance mechanisms

When a phage encounters a bacterium, successful infection is not always guaranteed due to a number of host defence mechanisms. Figure 1.7 shows the basic infection cycle of a lytic phage and each step of this cycle can fall foul of an antiphage system. Brief explanations and examples of some of these strategies will outlined below, for a more comprehensive review of phage resistance mechanisms see Labrieet al.(2010).

Figure 1.6 Lytic phage replication cycle.

Each step of the cycle can be targeted by antiphage mechanisms. Hosts can contain multiple mechanisms, though the effect of such combinations has rarely been assessed. Taken from Labrieet al.

(2010).

1.5.2.1 Blocking phage adsorption

By altering or completely removing the cell surface structures to which phage attach, bacteria can halt infection at the first step. A mutant strain of Roseobacter denitrificansOCh114, M1 that is resistant to infection by phage RDJLΦ1 was isolated by Huang et al., (2010). Comparative proteomics of wild type and the M1 strain revealed that five membrane proteins were down-regulated in the resistant strain which suggests that one of more of these proteins were the phage receptors.

Alternatively, other molecules may be produced by the potential host to mask the receptor and reduce (but not completely prevent) phage binding e.g. the immunoglobulin G-binding protein A produced by Staphylococcus aureus

(Nordström and Forsgren, 1974) which is covalently bound to mucopeptide, thus masking the O-acetyl groups of the mucopeptide that are required for phage adsorption. Other bacteria restrict access to potential receptors by the production of structured extracellular polymers. For example, phage infection of the soil-dwelling

Azotobacter chroococcum was reduced when immobilised in sodium alginate, an exopolysaccharide produced by severalAzotobacter spp., compared to that of liquid cultures (Hammad, 1998)

Finally, molecules naturally present in the environment, such as microcins (small bacteriocins, comprised of a few peptides), can be competitive inhibitors of phages. Microcin J25 binds to the E. coli iron transporter, FhuA which is also the receptor for phages T1, T5 and Φ80. This was demonstrated in vitro, by the pre- incubation of purified FhuA with J25 at varying concentrations, and YO-PRO-1 (a fluorescent DNA dye) followed by the addition of phage T5. Fluorescence, which is proportional to the release of phage DNA through binding, was found to decrease concomitantly with increasing concentration of J25 to FhuA. The authors calculated phage adsorption decreased from 100 to 45% when concentrations of J25 increased from 0.1 to 3.2μM (Destoumieus-Garzónet al., 2005)

1.5.2.2 Preventing DNA entry

Blocking the entry of phage DNA into a host cell, prevents it from sequestering the host cellular machinery; such systems are known as superinfection exclusion (Sie) systems and involve membrane-anchored or membrane-associated proteins. Interestingly, the genes responsible for such proteins are often found in prophages (Labrie, et al., 2010). One such example can be found in T4 phage- resistantE. coli already infected with T4, which has two Sie systems encoded by the

immandspgenes. Imm acts in conjunction with another membrane protein, to change the conformation of the phage DNA injection site (Lu et al., 1993), whereas Sp inhibits the T4 lysozyme preventing the creation of new holes in the host cell wall. These two systems not only prevents superinfection by T4, but all T-even-like phages (Lu and Henning, 1994).

Figure 1.7 Blocking the entry phage of DNA using E. coli proteins Imm and Sp. Taken from Labrieet al., 2010. The Imm protein binds to the phage DNA injection protein and blocks DNA entry whilst the Sp protein inhibits the phage lysozyme so the peptidoglycan layer cannot be breached.

1.5.2.3 Targeting phage nucleic acids

Two systems are known to target phage nucleic acids; the restriction modification system (of which there are at least four types) recognises foreign DNA and degrades it and the CRISPR-Cas system (Clustered Regularly Interspaces Short Palindromic Repeats), whose mechanisms are not yet fully elucidated, for a recent review on CRISPRs see Vale and Little (2010). Though the latter system was first described in 1987, its role in phage-resistance was not fully appreciated until recently. In the last three years it has been shown that bacterial cells are able to incorporate new spacers that are 100% identical to phage genetic material after infection (Barrangouet al., 2007). Acquisition of the new spacer promotes host resistance to further phage infection, whilst removal of the spacer renders it susceptible again. It was also shown that mutation of the phage genome is sufficient to counter the newly acquired resistance (Deveau et al., 2008). The antiphage response is mediated by CRISPR- associated (Cas) proteins; a helicase, Cas3, and the mature CRISPR RNAs work in tandem to interfere with phage replication (Brouns et al., 2008). Regardless of the system used, R/M or CRISPR-Cas, both allow bacterial cells to destroy foreign genetic material thus halting any phage infection.

1.5.2.4 Abortive infection systems

Unlike the examples described above, abortive infection systems (Abis) result in the “altruistic” death of the infected cell and so work on the community, not the individual level. Most Abis have been found inLactococcus lactis (23 to date, Labrie

et al., 2010), but all appear to target stages in phage multiplication. Perhaps the best characterised system is the two component Rex system found in λ-lysogenic E. coli

strains. During phage infection, the RexA protein is activated which in turn switches on an ion channel RexB. Loss of membrane potential due to RexB activation, results in a drop in ATP level which causes abortion of phage infection followed by cell death (Molineux, 1991). In contrast, the Abis in Lactococcus spp. as well as the E. coliLit and Prr systems appear to work on the transcriptional level. In these systems, during phage infection, otherwise dormant enzymes are activated and cleave highly conserved, essential components of the translational machinery. Consequently, protein synthesis is halted, phage infection is aborted and the infected cell dies (Chopinet al., 2005)

Despite the variety of the antiphage mechanisms found in prokaryotes, of which only a small number have been described above, phages have found strategies by which they can overcome all of these barriers. Consequently, in turn, bacteria must alter their strategies in order to survive resulting in a cyclic arms-race or the “Red queen effect” where there is a continuous cycle of co-evolution maintaining the genetic diversity of both bacteria and phages (Van Valen, 1973).

1.5.3 Effect of prophages on host fitness

For over 30 years, the ability of prophages to enhance host fitness has been observed as many contain genes that are not essential for the phage lifecycle. These lysogen conversion factors include virulence proteins, metabolic enzymes and transcriptional repressors which can down-regulate essential genes. Though this last point may seem at first counter-intuitive a recent study by Chen et al. (2005) found that during phage λ infection, expression of the host pckA gene (critical in gluconeogenesis) was suppressed by the phage cI repressor. They postulated silencing of this gene resulted in slower growth in the challenging glucose-free environment, thus ensuring the survival of the lysogen over its uninfected clone. Other mechanisms by which prophages can enhance host fitness are discussed in reviews by Brussowet al., (2004) and Paul (2008) and in Chapter 8.

Since high numbers of cultivable marine bacteria have been found to contain prophage-like elements (as discussed in Section 1.4.6) it has been proposed that prophages are not dangerous molecular time bombs, as traditionally viewed, but are instead advantageous to their hosts. They allow their hosts to survive in resource- replete conditions by repressing metabolic genes and provide mechanisms by which

the host (and prophage) can sense changes in the environment. As well as this, as discussed in Section 1.6.1, prophages can mediate transduction, improving host fitness thus helping to drive bacterial evolution in the sea.