Phage abortive infection in lactococci: variations on a theme

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Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance.

Introduction

Bacteria have developed many different types of mechanisms to avoid being killed by bacteriophages, which are ubiquitous and frequently outnumber bacteria in the environment. One of these mechanisms, usually denoted as exclusion or abortive infection (Abi), is characterised by a normal start of infection (i.e. phage adsorbs and injects its DNA into the host cell) followed by an interruption of phage development leading to the release of few or no progeny particles and to the death of the infected cell. As a consequence, further propagation of phage is prevented and the bacterial population survives. These mechanisms have been found in many bacterial species including Escherichia coli [1], Lactococcus lactis [2], Bacillus subtilis [3], Bacillus licheniformis [4], Shigella dysenteriae [5], Streptococcus pyogenes [6] and Vibrio cholerae [7], which indicates that they are most probably widespread in bacteria.

Most known Abis have been found in L. lactis. Bacteria from this species constitute the spontaneous dominant flora of milk left at ambient temperature and transform lactose to lactic acid. This feature has been used by man for millenniums to prepare various types of cheese [8]. Nowadays, most cheese manufacture is performed within large dairy plants, some of them processing up to 106 litres milk per day, leading to the generation of 1018 lactococcal cells. Milk fermentation by lactococci therefore represents the largest volume of bacterial culture controlled by man. Not surprisingly, this process, which is conducted under non-aseptic conditions, is highly susceptible to infection by bacteriophages (first recognised in 1935, [9]). It is likely that the huge quantity of lactococcal cells produced and the long-term challenge from phage have selected many varied and efficient mechanisms of phage resistance in this species. Because of the serious economic consequences of this problem for the dairy industry, many efforts have been devoted to the study of lactococcal phages and to the analysis of lactococcal phage defence mechanisms. This most probably explains why as many as twenty Abi systems, designated AbiA to AbiU, have been isolated in L. lactis and the corresponding genes sequenced (Table 1) [2].

In this review, we summarise the present knowledge regarding the mode of action of lactococcal Abi systems. We show that these apparently diverse mechanisms share common features, also present in the well-studied LitA and PrrC E. coli exclusion systems.

Section snippets

Lactococci contain many different Abis

Almost all known lactococcal Abis are plasmid encoded. Dairy lactococci contain many plasmids, representing a significant part of their genetic information. In particular, plasmids carry most of the genes required for optimal growth in milk (e.g. coding for enzymes required for lactose and milk proteins utilisation) and often can be readily transferred by conjugation between strains [10]. They are therefore regarded as major contributors to the adaptation of lactococci to the dairy environment.

Effects of lactococcal Abis on the phage cycle

All lactococcal phages described to date are double-stranded DNA phages. Most fall into three well-established groups of DNA homology [15]. Two of these groups, designated 936 and c2, are composed of virulent phages, and the third group, designated P335, is composed of mostly temperate phages. Phages of these three groups share very limited homology, both at the DNA and the protein level. The genetic structure of the populations is also different, depending on the phage lifestyle. Temperate and

AbiA and AbiK

AbiA and AbiK are active against lactococcal phages from all three main groups. Proteins from AbiA and AbiK share 23% identity. P335 phages resistant to these mechanisms have mutations in functionally related genes suggesting that AbiA and AbiK could have a similar mode of action. abiA and abiK are both constitutively transcribed [12•, 25] and their efficiency increases with their copy number [19, 26]. However, when abiK is cloned on a high-copy number plasmid under the control of a strong

AbiB

AbiB is only active against the 936 group of phages. Transcription of abiB, initiated at a promoter localised in an upstream ISS1 element [31], is constitutive and does not increase following phage infection [32]. Protein production, followed in vivo using specific antibodies, was also shown to be constitutive and not to increase following phage infection. Overexpression of AbiB is toxic for both L. lactis and E. coli [32].

In AbiB+ cells, phage DNA replication and transcription proceeds

AbiD1

AbiD1, active on 936 and c2 groups of phages, has been mainly studied using phage bIL66 (936 group). Expression of the abiD1 gene is toxic to the cell and is tightly regulated at both transcriptional [34] and translational (E Bidnenko et al., unpublished) levels (Figure 1b). abiD1 expression is induced following phage bIL66 infection [23], and is achieved at the translational level by the first gene (orf1) expressed from the middle operon of the bIL66 phage (E Bidnenko et al., unpublished) (

Similarity behind diversity

Behind their apparent diversity, lactococcal Abis possess some general conserved features. These features are highlighted by comparing AbiD1 with the best-studied E. coli exclusion systems Lit and Prr. In both systems, dormant enzymes are activated following phage infection, which results in the cleavage of essential and highly conserved components of the cellular translational apparatus. Protein synthesis is arrested and phage infection aborts. Activated LitA protease and PrrC anticodon

Conclusions

Abi and exclusion mechanisms, discovered for their activity against phages, have been considered as ‘altruistic death modules’ that favour cell population survival following phage infection [44]. However, the latent PrrC nuclease has recently been shown to be also induced by normal cell constituents, which suggests that this enzyme could play additional roles other than warding off phage T4 infection [45]. Interestingly, toxin–antitoxin (TA) modules, discovered for their ability to stabilise

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We thank Sylvain Moineau for the kind communication of a manuscript prior to publication.

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