R EV I EW AR T I C L E

The Lactococcus lactis plasmidome: much learnt, yet still lots todiscover

Stuart Ainsworth1, Stephen Stockdale1, Francesca Bottacini2, Jennifer Mahony1 & Douwe vanSinderen1,2

1Department of Microbiology, University College Cork, Cork, Ireland; and 2Alimentary Pharmabiotic Centre, Biosciences Institute, University

College Cork, Cork, Ireland

Correspondence: Douwe van Sinderen,

Room 4.05, Biosciences Institute, University

College Cork, Western Road, Cork, Ireland.

Tel.: +353 21 4901365;

fax: +353 21 4903101;

e-mail: d.vansinderen@ucc.ie

Received 13 February 2014; revised 17 April

2014; accepted 7 May 2014.

DOI: 10.1111/1574-6976.12074

Editor: Grzegorz Wegrzyn

Keywords

plasmid; bacteriophage; dairy fermentation;

abortive infection; conjugation; transduction.

Abstract

Lactococcus lactis is used extensively worldwide for the production of a variety

of fermented dairy products. The ability of L. lactis to successfully grow and

acidify milk has long been known to be reliant on a number of plasmid-

encoded traits. The recent availability of low-cost, high-quality genome

sequencing, and the quest for novel, technologically desirable characteristics,

such as novel flavour development and increased stress tolerance, has led to a

steady increase in the number of available lactococcal plasmid sequences. We

will review both well-known and very recent discoveries regarding plasmid-

encoded traits of biotechnological significance. The acquired lactococcal plas-

mid sequence information has in recent years progressed our understanding of

the origin of lactococcal dairy starter cultures. Salient points on the acquisition

and evolution of lactococcal plasmids will be discussed in this review, as well

as prospects of finding novel plasmid-encoded functions.

Introduction

Lactococcus lactis is a member of a diverse bacterial group

known as the lactic acid bacteria (LAB), which is a func-

tional group of Gram-positive, micro-aerophilic coccoid

and rod-shaped bacteria that produce lactic acid as the

main end product of hexose fermentation (Makarova

et al., 2006). Many LAB species are highly valuable due to

the biotechnological properties they impart on fermented

food products. These properties include organoleptic and

rheological qualities, in addition to shelf-life extension.

The latter is enabled by the ability of LAB to decrease the

pH by lactic acid production and, in certain cases, the

production of other antimicrobial substances such as bac-

teriocins (Cotter et al., 2005; de Vos, 2011).

Amongst the LAB, L. lactis is one of the most widely

applied starter cultures in the dairy industry and for this

reason has enjoyed extensive scientific scrutiny. The sheer

quantity of fermented foodstuffs produced annually

makes L. lactis one of the most economically important

bacterial species, with recent estimates published in 2011

putting the collective economic value of cheese products

alone (largely involving L. lactis strains) in the region of

€55 billion (de Vos, 2011). Furthermore, the recent

appraisal for the potential application of L. lactis strains

in oral vaccine delivery may expand the importance of

lactococcal investigations into the medical/pharmaceutical

arena (Bermudez-Humaran et al., 2013).

Lactococcus lactis is found in many environments,

although the original niche for L. lactis is now widely

accepted to be plant based (Price et al., 2011; Siezen

et al., 2011). Lactococcus lactis strains are readily isolated

from fermenting plant material and minimally processed

fresh fruit and vegetables, such as mung bean sprouts or

corn, and in some cases have been presumed to represent

the dominant element of the microbiota associated with

these products (Kelly et al., 1998).

Lactococcus lactis is currently divided into several sub-

species (Price et al., 2011). The classification of the two

dominant subspecies, that is, subsp. lactis and cremoris,

was originally based on industrially relevant phenotypic

traits (Kelly et al., 2010). However, genetic analysis has

highlighted that amongst dairy strains, two dominant

genetic lineages (genotypes) of L. lactis strains exist,

which are also appropriately termed lactis and cremoris

(Samarzija et al., 2002; Rademaker et al., 2007; Passerini

FEMS Microbiol Rev && (2014) 1–23 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

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et al., 2010). In general, the genotypic distinction is

matched by the phenotypic designations; however, atypi-

cal strains, whose phenotype and genotypes do not

match, also exist (Wegmann et al., 2007).

Lactococci have unwittingly been exploited by humans

for millennia for the production of an extensive range of

dairy-based, fermented foodstuffs, such as cheese, and as

a result enjoy a so-called generally recognised as safe

(GRAS) status (Wegmann et al., 2007). Lactococcal

strains that are used in the dairy industry appear to have

undergone extensive adaptation to the nutrient-rich dairy

environment through a process of reductive evolution

(Bachmann et al., 2012), which, when compared to lacto-

coccal strains isolated from plant material, appears to

have resulted in a smaller genome size, a higher number

of pseudogenes and acquisition of a much more extensive

plasmid complement (Bolotin et al., 2004; Makarova

et al., 2006; Kelly et al., 2010; Ainsworth et al., 2013).

Plasmids are semi-autonomously replicating extrachromo-

somal DNA entities, which are normally dispensable for

bacterial growth (Pinto et al., 2012). Plasmids typically

confer traits that impart important niche-specific pheno-

types (Siezen et al., 2011) and can often form the basis

for individuality amongst strains (Kelly et al., 2010). The

associated phenotypic traits may include industrially

important survival strategies, metabolic capabilities, viru-

lence factors and antibiotic resistances (Grohmann et al.,

2003; Mills et al., 2006). Furthermore, genetic engineering

of lactococcal plasmids has led to many highly efficient

and successful biological tools (for reviews see Mierau &

Kleerebezem, 2005; Mills et al., 2006; Morello et al.,

2007), which has enabled many lactococcal functions and

processes to be defined.

The following review aims to cover, amongst others,

the most recently reported progress and discoveries

related to lactococcal plasmids. In particular, the possible

origins and evolution of lactococcal plasmids will be dis-

cussed, in addition to prospects for further discoveries of

plasmid-associated traits.

Plasmid frequency

As mentioned above, the vast majority of dairy-associated

lactococcal isolates carry extensive plasmid complements

(estimated to be up to 14 per individual isolate; Kelly

et al., 2010), commonly constituting more than 150 kb of

extrachromosomal DNA and representing up to 9% of

the total (i.e. chromosome plus plasmids) genetic material

(Price et al., 2011; Ainsworth et al., 2013). These plas-

mids have sizes ranging from 2 kb to over 100 kb and

have in several cases been shown to be mobilisable and

transmissible by conjugation (Grohmann et al., 2003).

The availability of complete plasmid complement

sequences from several dairy-derived lactococcal strains

has reaffirmed that key industrial traits, such as lactose

metabolism, casein utilisation and many bacteriophage

resistance systems, are plasmid-encoded, thereby demon-

strating the extensive adaptation of such dairy strains to

the milk environment (Siezen et al., 2005).

In contrast, plasmid profiling surveys of plant-derived

lactococcal strains have highlighted that plasmids occur at

a lower frequency (typically one or two, or no plasmids;

Kelly et al., 2010) in plant isolates of L. lactis compared

with their dairy counterparts (Kelly et al., 2010). This

seems particularly true for plasmids of < 10 kb, which

are common in dairy isolates (Siezen et al., 2005; Gorecki

et al., 2011; Fallico et al., 2012; Ainsworth et al., 2013).

Indeed, L. lactis KW2, a fermented corn isolate whose

complete genome was recently sequenced, harbours no

plasmids (Kelly et al., 2013). Nevertheless, plasmids of

several plant and dairy isolates have been sequenced, and

plasmids of lactococcal strains isolated from dairy sam-

ples, particularly strains isolated from raw milk products,

hint at a plant-associated origin (Fallico et al., 2011) as

will be discussed later.

There are currently more than 80 individual, com-

pletely sequenced lactococcal plasmids present in public

databases, including the entire plasmid complement of

five dairy isolates and a single human isolate (Table 1).

Additionally, partial sequences of many lactococcal plas-

mids are also available. Due to the fact that, as mentioned

above, lactococcal plant isolates harbour relatively few, if

any, plasmids (Kelly et al., 2010), and because only a rela-

tively small number of plant isolates have been

sequenced, such plasmid sequences are clearly under-

represented. Currently, DNA sequences of just two plant-

associated lactococcal plasmids are publicly available

(Tanous et al., 2007; Siezen et al., 2010). The complete

plasmid complements of two human lactococcal isolates,

L. lactis CV56 (three plasmids; Gao et al., 2011) and Lac-

tococcus garviae 21881 (five plasmids; Aguado-Urda et al.,

2012), have recently been made available, revealing many

plasmid-encoded features that are common amongst the

plasmids found in such human isolates and those present

in their dairy-derived relatives.

Replication of lactococcal plasmids

Lactococcal plasmids, like the majority of plasmids, repli-

cate via two alternative modes of replication: rolling circle

replication (RCR; Leenhouts et al., 1991) or theta-type

replication (Kiewiet et al., 1993). Both replication mecha-

nisms require certain functions from the DNA replication

machinery of the host, and a brief outline of these two

replication methods is given below (for a detailed review

on lactococcal plasmid replication, see Mills et al., 2006).

FEMS Microbiol Rev && (2014) 1–23ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

2 S. Ainsworth et al.

Table 1. List of all sequenced Lactococcus lactis plasmids to date. Data obtained from the GenBank L. lactis annotation report (http://www.ncbi.

nlm.nih.gov/genome/genomes/156?subset=plasmids&details=on&)

Name Accession number Size (Kbp) GC% Genes Niche Replication mode*

pKF147A NC_013657.1 37.51 32.4 29 Plant Theta

pSK111 NC_008503.1 14.041 34.4 10 Dairy Theta

pSK112 NC_008504.1 9.554 30.4 6 Dairy Theta

pSK113 NC_008505.1 74.75 35.4 61 Dairy Theta

pSK114 NC_008506.1 47.208 34.8 35 Dairy Theta

pSK115 NC_008507.1 14.206 33.5 8 Dairy Theta

pCV56A NC_017483.1 44.098 32.1 39 Human Theta

pCV56B NC_017487.1 35.934 34.5 28 Human Theta

pCV56C NC_017484.1 31.442 32.5 29 Human Theta

pCV56D NC_017485.1 5.543 32.2 8 Human Theta

pCV56E NC_017488.1 2.262 33.8 3 Human Theta

pQA504 NC_017497.1 3.978 37.8 3 Dairy ?

pQA518 NC_017495.1 17.661 37.4 12 Dairy Theta

pQA549 NC_017493.1 49.219 35.1 42 Dairy Theta

pQA554 NC_017496.1 53.63 34.9 69 Dairy Theta

pCIS8 NC_019430.1 80.592 34 67 Dairy Theta

pCIS7 NC_019431.1 53.051 32.4 41 Dairy Theta

pCIS1 NC_019438.1 4.263 32 2 Dairy Theta

pCIS5 NC_019432.1 11.676 34.1 11 Dairy Theta

pCIS3 NC_019433.1 6.159 35.9 4 Dairy Theta

pCIS2 NC_019434.1 5.461 30.1 5 Dairy Theta

pCIS6 NC_019436.1 38.673 37.1 27 Dairy Theta

pCIS4 NC_019437.1 7.045 38.4 8 Dairy Theta

pMRC01 NC_001949.1 60.232 30.1 63 Dairy Theta

pVF18 NC_015900.1 18.977 33.9 21 Dairy Theta

pVF22 NC_015901.1 22.166 35.1 19 Dairy Theta

pVF50 NC_015902.1 53.876 34.5 41 Dairy Theta

pVF21 NC_015912.1 21.728 33.6 14 Dairy Theta

pAW153 NC_017494.1 7.122 31.4 8 ? Theta

pIL6 NC_019308.1 28.434 33.6 25 Dairy Theta

pAF04 NC_019347.1 3.801 32 4 Dairy Theta

pAF07 NC_019348.1 7.435 36.4 6 Dairy Theta

pAF12 NC_019349.1 12.067 33.3 11 Dairy Theta

pAF14 NC_019350.1 14.419 34.1 11 Dairy Theta

pAF22 NC_019351.1 22.388 34.9 23 Dairy Theta

pLP712 NC_019377.1 55.395 37.4 44 Dairy Theta

pIL105 NC_000906.2 8.506 29.8 7 Dairy Theta

pNZ4000 NC_002137.1 42.81 33.3 45 Dairy Theta

pAG6 NC_007191.1 8.663 33.7 8 ? Theta

pS7a NC_004652.1 7.302 33.4 5 Dairy Theta

pWV02 NC_002193.1 3.826 31.3 1 Dairy Theta

pSRQ800 NC_004960.1 7.858 31.3 7 Dairy Theta

pHP003 NC_004847.1 13.433 40.1 6 Dairy Theta

pGdh442 NC_009435.1 68.319 35.1 63 Plant Theta

pMN5 NC_004922.1 5.67 30.3 4 Dairy RCR

pCI305 NC_002502.1 8.694 32.4 8 Dairy Theta

pBL1 NC_004955.1 10.899 32.6 8 Dairy Theta

pCL2.1 NC_004981.2 2.047 34 2 ? RCR

pDR1-1 NC_004164.2 7.412 33.7 6 Dairy Theta

pAH82 NC_004966.1 20.331 34.4 17 Dairy Theta

pSRQ900 NC_004959.1 10.836 31.1 11 Dairy Theta

pKL001 NC_011610.1 6.068 32.9 4 ? Theta

pWV01 NC_002192.1 2.178 33.4 4 Dairy RCR

pNP40 NC_010901.1 64.98 32.3 62 Dairy Theta

FEMS Microbiol Rev && (2014) 1–23 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

The lactococcal plasmidome 3

The replicon of a typical rolling circle plasmid is com-

posed of a replication protein (encoded by rep) and a

double-stranded origin (dso) of replication, which con-

tains a so-called nic site, composed of (an) inverted

repeat(s), and a Rep-binding site, which is comprised of a

set of two to three direct repeats or an inverted repeat

(del Solar et al., 1993; Mills et al., 2006). The Rep initi-

ates replication by introducing a single-stranded break

into the nic site of the dso, generating a free 30 hydroxylgroup, which is subsequently used in leading strand syn-

thesis by the host replication machinery. Replication dis-

places the parental ‘plus’ strand and continues until the

newly reconstituted dso is reached. Lagging-strand replica-

tion on the displaced parental strand occurs from a non-

coding region, which can generate a stem loop structure,

termed the single-stranded origin (sso; del Solar et al.,

1998). Rolling-circle-type plasmids always replicate in a

unidirectional manner and represent the minority of

sequenced (seven of 82 analysed lactococcal plasmids are

known or predicted to replicate via the RCR mechanism)

lactococcal plasmids (Siezen et al., 2005; Gorecki et al.,

2011). Most lactococcal rolling-circle-type plasmids

belong to the pWV01 family of replicons and exhibit a

broad host range, being able to replicate in a range of

Gram-positive and Gram-negative bacteria. However, they

have a limited replicon size (< 10 kb) and are incompati-

ble with other RCR plasmids (Leenhouts et al., 1991),

limiting each strain to a maximum of just a single RCR

replicon. These limitations, coupled to their intrinsic

structural and segregational instability, are amongst the

likely reasons for the under-representation of rolling

circle replicons in currently sequenced L. lactis plasmids.

The majority (75 of 82) of lactococcal plasmids appear

to replicate via the theta-type mechanism of replication

(Seegers et al., 1994; Table 1). Large (80 kb) theta repli-

con plasmids have been sequenced (Ainsworth et al.,

2013) reflecting their superior structural stability relative

to RCR-type plasmids. They appear to be highly related

and are described as pWV02-type replicons, being named

after the plasmid that serves as the prototype of this

group of replicons (Kiewiet et al., 1993). Unlike RCR

plasmids, these replicons have a limited host range (Kiew-

iet et al., 1993). The typical replicon of a lactococcal

theta-type-replicating plasmid comprises of a replication

initiator protein, encoded by a gene that is often termed

repB, and an origin of replication (ori) which is com-

prised of an AT-rich region and three and a half iterons

of 22 bp in length (Fig. 2b). Additionally, two small

Table 1. Continued

Name Accession number Size (Kbp) GC% Genes Niche Replication mode*

pAR141 NC_013783.1 1.594 36.1 2 Dairy RCR

pS7b NC_004653.1 7.264 33.6 5 Dairy Theta

pIL1 NC_015860.1 6.382 32.3 7 Dairy Theta

pIL3 NC_015861.1 19.244 35.1 20 Dairy Theta

pIL5 NC_015863.1 23.395 34.5 22 Dairy Theta

pIL7 NC_015864.1 28.546 34.1 26 Dairy Theta

pIL4 NC_015862.1 48.978 35.1 47 Dairy Theta

pDBORO NC_009137.1 16.404 35.2 15 ? Theta

pDR1-1B NC_004163.1 7.344 33.7 6 ? Theta

pK214 NC_009751.1 29.871 32.4 29 ? Theta

pIL2 NC_017489.1 8.277 34.8 10 Dairy Theta

pSK11L NC_017478.1 47.165 34.8 40 Dairy Theta

pCRL291.1 NC_002799.1 4.64 33.5 3 ? Theta

pCRL1127 NC_003101.1 8.278 34.8 7 ? Theta

pBM02 NC_004930.1 3.854 35.7 6 Dairy RCR

pND324 NC_008436.1 3.602 33.4 3 ? Theta

pSRQ700 NC_002798.1 7.784 34.2 9 Dairy Theta

pAH33 NC_002150.1 6.159 35.9 7 Dairy Theta

pKP1 NC_016042.1 16.181 35.9 7 Dairy Theta

pCIS3 NC_002138.1 6.159 35.9 3 Dairy Theta

pSK11A NC_017498.1 10.372 30.9 13 Dairy Theta

pSK11P NC_017500.1 75.814 35.4 61 Dairy Theta

pSK11B NC_013551.1 13.332 34.3 14 Dairy Theta

pL2 NC_008594.1 5.299 32.5 5 Dairy Theta

pWC1 NC_004980.1 2.846 29.5 1 Dairy RCR

pCD4 NC_002748.1 6.094 33.4 5 Dairy Theta

p1 NC_022587.1 4.094 30.0 6 Dairy RCR

*Presumed replication mode. ? = unknown.

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4 S. Ainsworth et al.

inverted repeats overlap the -35 site of the repB promoter

and the ribosome binding site (Kiewiet et al., 1993; Mills

et al., 2006). DNA synthesis of theta plasmids can be uni-

or bidirectional and may initiate from multiple origins;

synthesis of the leading strand is continuous, while lag-

ging-strand synthesis is discontinuous.

In contrast to the rolling circle plasmids, multiple theta

replicons may coexist in the same lactococcal strain or

even on the same plasmid (Seegers et al., 1994). The

sequencing of complete plasmid complements has high-

lighted the extent to which highly related theta replicons

can coexist within the same host strain or plasmid. For

example, the plasmid complement of L. lactis SK11 con-

tains six theta-type replicons across four plasmids, with

two of such replicons being present on pSK11L and

pSK11B (Siezen et al., 2005), while the L. lactis IL594

seven plasmid complement harbours nine theta-type repl-

icons (Gorecki et al., 2011). These observations are in

apparent contrast to the generally accepted notion that

the more related two replicons are, the greater their

degree of incompatibility. This phenomenon is well

described (Seegers et al., 1994; Gravesen et al., 1997;

Emond et al., 2001; Gorecki et al., 2011), and Gravesen

et al. (1997) performed a screen for incompatible theta

replicons, identifying two pairs of incompatible plasmids

in their study. Analysis of these incompatible replicons

suggested that the incompatibility determinant was con-

tained within the 22-bp direct repeats and/or in the

inverted repeat IR1 of the ori, which has been proposed

to interact with a specific 13 amino acid region of RepB

(Gravesen et al., 1997). Accordingly, analysis of this 13

amino acid region revealed nine variable residues

amongst compatible plasmids, whereas incompatible plas-

mids were shown to harbour identical amino acid

sequences within this region. Analysis of the nucleotide

sequences of direct and inverted repeat regions of the

nine IL594 replicons revealed that they all varied slightly

in nucleotide sequence (Gorecki et al., 2011). The authors

suggest that each ori region interacts uniquely and specifi-

cally with its corresponding RepB, allowing coexistence of

several related replicons within one cell.

The key traits of dairy lactococci: lactoseand casein utilisation

The ability to rapidly ferment lactose, a typical feature of

LAB, is a plasmid-encoded and well-defined characteristic

amongst dairy-associated lactococci (Cords et al., 1974).

The genes encoding lactose acquisition and utilisation are

located in a single operon, lacABCDFEGX, which is regu-

lated by the repressor LacRii encoded by the divergently

oriented lacRii gene. Lactose is acquired through the phos-

phoenolpyruvate-phosphotransferase system (PEP-PTS),

which is encoded by lacEF, and which catalyses the syn-

thesis of lactose-phosphate as part of lactose transport

across the cell membrane. Once inside the cell, it is uti-

lised by the tagatose-6-phosphate enzymes (encoded by

lacABCD) through the action of phospho-b-galactosidase(lacG), which cleaves lactose-phosphate to galactose-

6-phosphate and glucose (Mills et al., 2006).

Recently, the lactose PEP-PTS system has been impli-

cated in galactose uptake and metabolism (Neves et al.,

2010). A mutation in galP, encoding the high-affinity

galactose permease, in L. lactis NZ9000 was shown not to

abolish galactose uptake in the presence of the lactose

PEP-PTS. Further investigation determined that the lac-

tose PEP-PTS system possesses a low affinity for galac-

tose. Once transported inside the cell, galactose can be

metabolised via the tagatose pathway. However, this sys-

tem alone cannot sustain growth on galactose, and it has

been hypothesised that such growth is due to low fructose

1,6-bisphosphatase activity, which impedes gluconeogenic

use of galactose (Benthin et al., 1994; Neves et al., 2010).

Loss of the lactose-metabolising phenotype is a well-

documented occurrence (McKay et al., 1972). Sequencing

of entire lactococcal plasmid complements from dairy

isolates has shown that lactose utilisation is predomi-

nantly associated with one of the larger plasmids

harboured by a given strain, where the same plasmid in

most investigated cases also encodes proteins crucial for

casein degradation (Siezen et al., 2005; Wegmann et al.,

2012; Ainsworth et al., 2013). The absence of the lactose

utilisation operon in the plasmid complements of human

isolates L. lactis CV56 (Gao et al., 2011) and L. garviae

21881 (Aguado-Urda et al., 2012), and the plant isolate

L. lactis KF147 (Siezen et al., 2010) is indicative of the

specific, nondairy niche that such strains occupy.

The primary source of amino acids in the dairy envi-

ronment is in the form of milk proteins, the majority of

which is casein (Swaisgood, 1982). Casein hydrolysis by

lactococcal proteases and peptidases is vital for desirable

flavour development due to the resulting formation of

aroma compounds from peptide/amino acid catabolism

of casein breakdown products (Steele et al., 2013). The

major L. lactis extracellular, cell wall-anchored protease,

PrtP (for a recent review see Steele et al., 2013), is

responsible for casein hydrolysis and is almost always

plasmid-encoded (Savijoki et al., 2006). PrtP exhibits

broad substrate specificity and can cleave caseins into

over 100 different oligopeptides (Juillard et al., 1995;

Savijoki et al., 2006). The extracellular hydrolysis prod-

ucts are then taken into the cell by the oligopeptide

uptake and transport system, encoded by the opp operon

(Yu et al., 1996), after which the internalised peptides are

further digested by intracellular peptidases with varying

substrate specificities (Savijoki et al., 2006; Steele et al.,

FEMS Microbiol Rev && (2014) 1–23 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

The lactococcal plasmidome 5

2013). The opp operon is generally plasmid-encoded, and

the opp operon, the prtP-specified proteinase and its asso-

ciated plasmid-encoded maturase PrtM are required for

the proteolysis phenotype (Yu et al., 1996). The recent

availability of complete plasmid complements of individ-

ual strains has revealed that the opp operon and prtP are

often located on separate plasmids, and such strains are

unable to grow on casein as a sole source of nitrogen/

amino acids if one of these plasmids is lost from a given

strain (Siezen et al., 2005; Ainsworth et al., 2013). The

dispersal of this essential (in a dairy environment) pheno-

type across two plasmids may thus act as a crude plasmid

stability system amongst dairy lactococci.

In addition to PrtP and Opp, as described above, the

proteolytic system of L. lactis also constitutes various

peptidases which further act upon the internalised casein

hydrolysis products (Steele et al., 2013). A genomic com-

parison of the proteolytic systems of 39 L. lactis strains

using CGH analysis detected variability in the presence or

absence of genes encoding the peptidases Pcp, PepO2,

PepF2 and PepX2, which the authors hypothesised was

directly due to the presence or absence of plasmids har-

bouring these genes (Liu et al., 2010).

In addition to characterised peptidase-encoding genes,

plasmid sequencing has revealed several hypothetical pro-

teins which possess peptidase-like domains (Siezen et al.,

2005; Ainsworth et al., 2013). These uncharacterised pro-

teases are often strain-/plasmid-specific and may be asso-

ciated with specific organoleptic profiles of particular

fermented dairy products (Steele et al., 2013). An exam-

ple of a plasmid-associated gene specifying a peptidase-

like domain is that harboured by L. lactis UC509.9

plasmid pCIS8. Bioinformatic analysis of the plasmid-

borne open reading frame (ORF) uc509_p8059 shows that

its protein product is a likely extracellular protein, con-

taining a C-terminal hydrophobic region with a conserved

tryptophan (ChW) repeat adhesion/cell adhesion domain

and a transglutaminase-like protease. ChW domain-con-

taining proteins appear to be almost exclusively limited

to Clostridium acetobutylicum (Sullivan et al., 2007) and

have been implicated in the degradation of polysaccha-

rides and proteins (N€olling et al., 2001; Sullivan et al.,

2007). The N-terminus of the predicted protein product

of uc509_p8059 contains a C47 peptidase family domain,

which is commonly associated with proteolytic activities

encoded by Staphylococcus and Enterococcus species, and

is predicted to possess a single cleavage site. The C47

domain contains 24% identity across the full length of

the domain with the C47 domain of S. aureus virulence

factor staphopain A, which is a broad-specificity protease

(Dubin et al., 2001). Interestingly, a clear homolog of

uc509_p8059 is present on the chromosome of the plant-

derived L. lactis KF147.

Novel plasmid traits

Current industrial L. lactis strains that genotypically or

phenotypically belong to the subspecies cremoris are

thought to be the descendants of a relatively small num-

ber or genetically related lineages, which were widely dis-

seminated around the globe during the early 20th century

with the advent of industrial fermentations (Lawrence

et al., 1978; Kelly et al., 2010). While these related strains

may present distinct phenotypes, such as bacteriophage

resistance (Ward et al., 2004), it has been suggested that

large redundancies exist in academic and industrial collec-

tions (Kelly et al., 2010). Evidence for this low diversity is

obvious from the high level of chromosomal homogeneity

amongst industrial lactococcal dairy strains as compared

to the more genetically diverse artisanal and plant strains

(Rademaker et al., 2007; Kelly et al., 2010). Constant

selective pressure as applied in dairy fermentation may

nevertheless have caused industrial strains to develop dis-

tinctive traits, in terms of mobile elements, prophages

and possibly plasmid complements (Le Bourgeois et al.,

2000).

Due to the presumed low diversity of current starter

strain collections (Kelly et al., 2010), new environmental

strains with desirable traits, such as higher stress tolerance

and new flavour or aroma development, are eagerly

sought to be exploited for particular industrial processes

(21, 22). Plant-derived and artisanal isolates exhibit phe-

notypes that may be suitable for industrial exploitation,

such as increased stress tolerance (Nomura et al., 2006),

novel flavour compound formation, food-grade markers

and production of broad-spectrum bacteriocins (Kelly

et al., 1998; Tanous et al., 2007; Fallico et al., 2011).

Many of these traits are plasmid-encoded and therefore

have the potential to be transferred to dairy strains (Fal-

lico et al., 2011). Research investigating lactococci from

diverse environments has also uncovered some interest-

ing, although in cases less desirable traits, such as antibi-

otic resistance. Furthermore, sequencing has revealed

potentially beneficial dairy adaptations, including stress

resistance mechanisms and supplementary mineral uptake

systems, such as Mg2+ and Mn2+ transporters (Seegers

et al., 2000; Mills et al., 2006), which were previously

unknown due to the absence of an easily attributable

phenotype (Siezen et al., 2005).

Flavour development

New flavour and aroma development is a highly desirable

trait in industry. Traditionally, L. lactis subsp. lactis

biovar diacetalyactis strains have been exploited in dairy

fermentations for their typical ability to metabolise

citrate, which leads to desirable aroma and flavour

FEMS Microbiol Rev && (2014) 1–23ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

6 S. Ainsworth et al.

development through the synthesis of diacetyl (Starren-

burg & Hugenholtz, 1991). Citrate uptake is possible due

to the presence of citP, encoding a citrate permease

(Sesma et al., 1990), which is often a plasmid-encoded

trait (Mills et al., 2006). More recently, glutamate dehy-

drogenase (GDH)-encoding genes have been observed on

plasmid sequences derived from plant and raw milk iso-

late strains (Tanous et al., 2005; Fallico et al., 2011).

GDH catalyses the reversible oxidative deamination of

glutamate to 2-oxoglutarate and ammonia using NADP

as a cofactor. As a result, amino acid to aroma com-

pound conversion is stimulated through the supply of

2-oxoglutarate which is required for transamination

(Tanous et al., 2002, 2007). In a plant environment, this

ability is hypothesised to enable the (lactococcal) carrier

to assimilate ammonia in an amino acid-deprived envi-

ronment (Tanous et al., 2005).

Stress response

Dairy fermentations and cheese ripening impart many

stresses upon lactococcal cells. These stresses include

changes in pH, temperature and osmolarity (Duwat et al.,

2000). Plasmid (complement) sequencing has revealed

that L. lactis plasmids can encode multiple stress-associ-

ated genes, which could aid in obtaining strain robust-

ness. Many of the identified plasmid-encoded stress

proteins appear to be duplicates of genes encoded on the

L. lactis chromosome (Siezen et al., 2005) and therefore

are presumed to enhance survival through a gene-dosage

effect.

The gene encoding the universal stress protein, uspA,

has been detected in all currently sequenced dairy plasmid

complements. The universal stress protein superfamily is

conserved in many domains of life and is thought to play

a role in protecting the cell from DNA-damaging agents

(Kvint et al., 2003). In Escherichia coli, UspA is expressed

in response to multiple environmental stress conditions,

such as carbon and nutrient starvation, heat exposure

and oxidation, amongst many others (Kvint et al., 2003).

The plasmid complement of L. lactis SK11 encodes, in

addition to a plasmid-specified UspA, carbon starvation

and cold-shock proteins (Siezen et al., 2005), both of

which may enhance strain survival during dairy fermenta-

tion. Two predicted cold-shock proteins are also encoded

on plasmid pNP40, and transcriptional fusions of pro-

moter regions from the two corresponding genes, cspC

and cspD, of pNP40 have indeed demonstrated induction

by cold shock (O’Driscoll et al., 2006). Furthermore, a

(small but) significantly increased level of survival was

observed for cells harbouring pNP40 undergoing freeze–thawing as compared to plasmid-free cells (O’Driscoll

et al., 2006).

Another example of a plasmid-encoded stress response

system is found as two predicted, individual copies of the

stress response-induced, low-fidelity polymerase, encoded

by umuC (Ainsworth et al., 2013) on L. lactis UC509.9

plasmid pCIS7. UmuC homologs in E. coli are known to

function as a lesion-bypass DNA repair system as part of

the SOS response (Maor-Shoshani et al., 2000). Predicted

UmuC proteins in L. lactis are encoded on several plas-

mids, for example plasmid pKF147A, found in a lactococ-

cal plant isolate, and plasmid pNP40, which originates

from a dairy isolate. Expression of the UmuC homolog

from pNP40 was monitored by transcriptional fusion

postexposure of the host strain with mitomycin C, which

resulted in a threefold transcriptional increase in umuC

promoter activity. In addition to UmuC, pNP40 is pre-

dicted to encode two further DNA-damage repair func-

tions. A homolog of RecA (Garvey et al., 1997; Lusetti &

Cox, 2002), which functions in DNA repair, the SOS

response and homologous recombination, is encoded by

orf18, whereas orf25 encodes a protein with a putative

UvrA excinuclease domain (Van Houten et al., 2005),

implicating this protein in nucleotide excision repair.

Finally, genes encoding hypothetical proteins with

BRCT (BRCA1 carboxyl-terminal) domains are present in

the plasmid complements of strains UC509.9, SK11,

IL594, A76 and DPC3901. BRCT domain proteins are

predicted to function in DNA-damage repair (Makiniemi

et al., 2001) and may act as a further stress response

mechanism.

Plasmid-encoded antimicrobial peptides and

corresponding immunity

Bacteriocins are small, ribosomally synthesised antimicro-

bial peptides which generally induce cell death through

disruption of membranes and cell wall biosynthesis. Their

action can be narrow (targeting bacteria of the same spe-

cies) or broad (targeting bacteria across genera; Cotter

et al., 2012). Many bacteriocins are produced by LAB,

although very few are commercially exploited in the food

industry in food preservation and safety (Cotter et al.,

2005). Bacteriocin production in L. lactis (and other

LAB) is very well investigated, and several lactococcal

bacteriocin production and immunity systems have been

shown to be plasmid encoded (for reviews see (Cotter

et al., 2005; Mills et al., 2006; Zendo et al., 2010; Cotter

et al., 2012).

Most lactococcal bacteriocins appear to possess a

narrow spectrum activity, thereby killing only closely

related bacteria. Furthermore, strains producing bacte-

riocins must also encode an immunity determinant for

self-protection. Immunity proteins are usually found in

operons encoding the bacteriocin they are providing

FEMS Microbiol Rev && (2014) 1–23 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

The lactococcal plasmidome 7

immunity against. However, plasmid sequencing has

revealed that several lactococcal plasmids harbour solitary

bacteriocin immunity genes. Examples include the nisin

resistance determinant nsr found on plasmids pAF65 (Fal-

lico et al., 2012) and pNP40 (O’Driscoll et al., 2006), har-

boured in L. lactis subsp. lactis biovar. diacetylactis strains

DPC3758 and DRC3, respectively. The Nsr protein confers

nisin resistance by proteolytic cleavage of the carboxy-ter-

minal of the nisin tail (Sun et al., 2009). Additionally,

located on pAF65 is lctFEG, which is responsible for syn-

thesis of the immunity determinant for lacticin 481 (Rince

et al., 1994; Fallico et al., 2012). Lactococcus lactis UC509.9

plasmid pCIS8 (Ainsworth et al., 2013), in addition to

encoding a complete lactococcin A gene cluster, also har-

bours a gene encoding a putative EntA_Immun (PF08951)

family protein, which is thought to confer broad-range

class II bacteriocin immunity (Johnsen et al., 2005).

Plant niche-specific adaptations

The sequence of L. lactis SK11 plasmid pSK11P revealed

the presence of a cluster encoding four genes, cscABCD,

thought to encode cell surface proteins (Siezen et al.,

2005). A subsequent bioinformatic investigation of these

genes found that they are conserved amongst the LAB

and apparently absent in many related pathogenic bacte-

ria, such as streptococci and staphylococci, which lead

these authors to suggest that they represent a niche-

specific trait (Siezen et al., 2006). The Csc proteins are

thought to be involved in acquisition of complex carbo-

hydrates. Transcriptomic analysis has demonstrated that

csc clusters are controlled by catabolite repression, which

also suggests functional links with sugar metabolism.

Additionally, some CscC proteins were found to contain

lectin/glucanase domains, known to interact with specific

complex carbohydrates.

In L. lactis, csc genes can be found both on the chro-

mosome and on plasmids (Siezen et al., 2006). The chro-

mosomally located csc loci are commonly flanked by IS

elements and are therefore thought to be horizontally

acquired and mobile. Their apparent confinement to the

LAB suggests a niche adaption for plant polysaccharide

utilisation. Their presence in dairy lactococci is perhaps a

relic of their plant ancestral heritage, although they may

still impart an as of yet unknown benefit upon their host

in the dairy environment.

Genes whose protein products may modify plant cell

wall polysaccharides have also been found on plasmid

pVF18 of the raw milk isolate L. lactis DPC3901 (Fallico

et al., 2011). This plasmid appears to harbour genes that

are beneficial for colonisation of a plant environment as

opposed to the dairy environment. Examples include

orf11 from pVF18, whose deduced gene product belongs

to protein families involved in hydrolysis of plant cell

walls, such as chito-oligosaccharide deacetylase from Rhi-

zobium, and orf25, which encodes a protein with a cupin

domain. Enzymes harbouring cupin domains are func-

tionally diverse and are associated with epimerases and

isomerases, which are involved in modification of cell

wall carbohydrates in bacteria and plants (Dunwell, 1998;

Dunwell et al., 2004; Fallico et al., 2011).

Host cell surface alterations

Various LAB species are considered probiotic (Clarke

et al., 2012), and muco-adhesive properties are thought

to play a major role in the efficacy of a probiotic species

(von Ossowski et al., 2010; Turroni et al., 2013a, b). Gas-

trointestinal adhesion aids maintenance of stable or

extended colonisation and therefore may, for example,

contribute to competitive exclusion of pathogenic species

(Saxelin et al., 2005).

Lactococcus lactis is not considered to represent a natu-

ral inhabitant of the human gastrointestinal tract (Luki�c

et al., 2012). As a result, investigations into the probiotic

potential of L. lactis have been sparse. Recently, investiga-

tions of plasmids from artisanal cheese and plant-derived

lactococcal isolates have revealed novel lactococcal muco-

adhesive traits (Le et al., 2013). The presence of such

traits on mobile elements opens up possibilities of manip-

ulation of these traits for functional food and pharmaceu-

tical applications.

A recent investigation of a unique auto-aggregation

characteristic displayed by an artisanal strain from semi-

hard cheese, L. lactis subsp. lactis BGKP1, revealed that

this phenotype was plasmid-encoded (Kojic et al., 2011).

A single gene, designated aggL, located on plasmid pKP1,

was predicted to encode a 200 kDa protein belonging to

the collagen-binding superfamily, and this gene was

shown to confer an aggregation phenotype upon various

lactococcal and enterococcal hosts. The presence of plas-

mids encoding AggL appeared to result in a fitness bur-

den to the cell, as a significantly increased doubling time

was observed for cells containing pKP1 compared with

pKP1-free strains. Subsequent investigation of AggL-

expressing cells revealed that this protein significantly

increases the hydrophobicity of the strain and that this

appeared to correlate with its binding properties to the

colonic mucus by means of nonspecific hydrophobic

interactions (Luki�c et al., 2012).

Additionally, bioinformatic analysis of pKP1 demon-

strated the presence of another novel lactococcal plasmid-

borne gene, named mbpL. The deduced MbpL protein is

predicted to contain a MucBP-like (mucin-binding pro-

tein) domain, which led the authors to speculate a poten-

tial role for this protein in mucin interaction (Kojic

FEMS Microbiol Rev && (2014) 1–23ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

8 S. Ainsworth et al.

et al., 2011). Further investigations determined that

(compared with a control strain not expressing MbpL)

strains expressing MbpL have a significantly enhanced

adhesion ability on HT29-MTZ intestinal ileum cells,

which predominantly excrete mucins MUC3 and

MUC5AC (Luki�c et al., 2012). These results therefore

suggest that two distinct adhesion mechanisms with alter-

native adhesion specificities are encoded by pKP1, indi-

cating that strains harbouring pKP1 are able to colonise

different parts of the gastrointestinal tract.

Interestingly, plasmid-borne genes predicted to encode

proteins required for pilus biogenesis have been identified

in a plant-derived L. lactis strain TIL448 (Meyrand et al.,

2013), allowing this lactococcal strain to bind to caco-2

intestinal epithelial cells. The plasmid, pTIL448 (Marie-

Pierre Chapot-Chartier, personal communication)

encodes the typical genetic biosynthetic machinery speci-

fying sortase-dependent heterotrimeric pilus biosynthesis,

including a major pilin, a class-C sortase which possesses

pilin polymerase activity, a minor pilin and a tip pilin.

Similar pili systems have been described in various gut

commensals such as lactobacilli (von Ossowski et al.,

2010, 2011; Lebeer et al., 2012) and bifidobacteria

(O’Connell Motherway et al., 2011; Turroni et al.,

2013a, b) and have been demonstrated to be involved in

adhesion to and immunomodulation of the host. The lac-

tococcal plasmid-encoded pili have been suggested to rep-

resent a niche adaption mechanism, as the tip of the

pilus is found to harbour a lectin-like domain, which

may function in adhesion of the strain to plant cell walls

through specific recognition of a particular plant poly/oli-

gosaccharide. Subsequent investigation into the mucin-

binding properties of TIL448 was examined using atomic

force microscopy. Results demonstrated a high number of

specific adhesive events between TIL448 and pig gastric

mucin (Le et al., 2013). Furthermore, blocking assays

with fractions of pig gastric mucin, including O-glycans,

demonstrated the role of neutral oligosaccharides in the

interaction of TIL448 and pig gastric mucin, which is in

agreement with the prediction of lectin-like domains

being present at the pilus tip (Meyrand et al., 2013).

Antibiotic resistance and food safety

Several recent reports have highlighted the presence of

antibiotic resistance in artisanal lactococcal isolates. A

study of 94 lactococcal isolates from traditional Italian

cheese found that 26 exhibited resistance to tetracycline,

while a further 17 were shown to be resistant to both tet-

racycline and erythromycin (Devirgiliis et al., 2010).

Southern blot analysis indicated that the genes encoding

these functions were plasmid-associated. Furthermore,

plasmid sequencing has demonstrated the presence of

antibiotic resistance genes on plasmids from strains that

had been isolated from raw milk (Florez et al., 2008; Fal-

lico et al., 2011); therefore, the suitability of newly iso-

lated plasmids for potential use in food production needs

to be carefully assessed so as to avoid unwanted spread of

such antibiotic resistance determinants.

Additionally, plasmid sequencing has revealed several

plasmids, such as pVF18 (Fallico et al., 2011), harbouring

genes encoding putative aminoglycoside 3-N-acety-

ltransferases (Siezen et al., 2005; Ainsworth et al., 2013),

which have been implicated in aminoglycoside antibiotic

resistance, amongst other functions. However, the pre-

dicted involvement of these genes in antibiotic resistance

has so far not been confirmed experimentally (Fallico

et al., 2011).

Bacteriophage resistance

Bacteriophages infecting industrial L. lactis strains can

lead to slow or failed fermentations and loss of product

(Kleppen et al., 2011) and are therefore of economic con-

cern. As such, bacteriophage resistance mechanisms of

lactococci, which appear to be predominantly a plasmid-

encoded feature, have been extensively studied (for

reviews see Chopin et al., 2005; Mills et al., 2006; Labrie

et al., 2010). Below we discuss recent discoveries and

advances regarding plasmid-borne phage resistance mech-

anisms in L. lactis.

CRISPR-Cas

CRISPR-Cas (clustered regularly interspaced short palin-

dromic repeats-CRISPR-associated) loci specify complex

bacteriophage defence systems, which provide acquired

immunity against phages through targeting invading

phage DNA in a sequence-specific manner (Horvath &

Barrangou, 2010). Despite being widespread in nature

and frequently found in other members of the LAB

(Horvath et al., 2009; Makarova et al., 2011), there is an

apparent absence of CRISPR-Cas bacteriophage defence

systems in L. lactis. Recently, a highly bacteriophage-resis-

tant industrial dairy isolate was isolated and was observed

to harbour a conjugation-transmissible plasmid, termed

pKLM encoding a novel type III CRISPR-Cas system

(Millen et al., 2012). Transfer of this plasmid to the plas-

mid-free strain IL1403 conferred resistance against infec-

tions by certain 949, 936 and P335 type phages, and loss

of the plasmid caused a reversion to the strain’s original

phage-sensitivity profile. Analysis of its spacer regions

revealed sequences with high homology to lactococcal

phages, suggesting that the system is active in Lactococcus.

However, the described system appeared unable to

acquire new spacers and therefore could only provide

FEMS Microbiol Rev && (2014) 1–23 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

The lactococcal plasmidome 9

protection against phages which shared homology with

existing spacers. CRISPR-Cas systems appear to be rare in

L. lactis as only four of more than 400 industrial isolates

screened by PCR were identified to contain the above sys-

tem (Millen et al., 2012). The authors suggest that the

paucity of CRISPR-Cas systems in L. lactis is due to the

plasmid dependency of industrial dairy lactococcal strains

and the ancillary activity of active CRISPR-Cas against

plasmids. Screening of plant isolates of L. lactis, which

appear to harbour a relatively small number of plasmids

(Kelly et al., 2010), may therefore increase the frequency

with which chromosomally encoded CRISPR-Cas systems

may be detected in this species.

Abortive infection systems

Abortive infection systems (Abi) are altruistic bacterio-

phage resistance systems (Chopin et al., 2005). Their

common defining feature is the prevention of phage pro-

liferation by interfering with some critical aspect of the

phage lytic cycle that also results in bacterial death. This

limits the number of progeny produced, thereby protect-

ing other members of a bacterial population susceptible

to the Abi-sensitive phage (Labrie et al., 2010). Abortive

infection systems are highly diverse and seem particularly

abundant in L. lactis (Chopin et al., 2005), where they

are often plasmid-encoded (Mills et al., 2006). There are

currently over 20 described lactococcal Abi systems which

affect different stages of phage multiplication (Table 2),

such as DNA replication (in the case of AbiA, encoded

on pTR2030; Hill et al., 1990), transcription (in the case

of AbiG, encoded on pCI750; O’Connor et al., 1999) and

major capsid protein production (in the case of AbiC,

encoded on pTN20; Klaenhammer & Sanozky, 1985).

Typically, they are encoded by a single gene, but multi-

gene Abi systems, such as AbiE (encoded on pNP40; Gar-

vey et al., 1995), AbiR (specified by pKR223; Twomey

et al., 2000) and AbiT (encoded on pED1; Bouchard

et al., 2002), have been described.

While discovery of novel Abi systems appears to have

slowed down in recent years (the only recently described

novel plasmid-encoded Abi is AbiZ (Durmaz & Klaen-

hammer, 2007; see Table 2), advances in determining

molecular triggers and modes of action of currently iden-

tified Abi systems have been made. Abi proteins display

low (if any) levels of similarity to other proteins; there-

fore, their mode of action is usually difficult to predict

(Chopin et al., 2005). The low cost and ease of genome

sequencing has allowed the identification of molecular

triggers, modes of action and possible interaction sites for

several Abi systems, mainly through the examination of

genomes of so-called Abi-escape mutants (Bidnenko

et al., 2009).

The most recently described lactococcal Abi, AbiZ, is

encoded on the conjugative plasmid pTR2030, which also

encodes another Abi system, AbiA (Hill et al., 1990), and

which is harboured by L. lactis ME2 (Durmaz & Klaen-

hammer, 2007). The AbiZ protein product, encoded by a

single ORF, confers a bacteriophage protective phenotype

against various P335 species phages, such as φ31. AbiZappears to exert its phage resistance by causing premature

lysis of the phage-infected host bacterium (Durmaz &

Klaenhammer, 2007).

AbiZ is predicted to possess two transmembrane heli-

ces, and experiments expressing the phage holin and lysin

suggest that AbiZ acts cooperatively with the holin to

increase the bacterial cell membrane permeability. How-

ever, examination of φ31 escape mutants suggests a link

between the phage’s early or middle genes and AbiZ.

These escape mutants of φ31 had undergone recombina-

tion with the host genome, exchanging an approximate

9-kb region which encompasses the presumed origin of

replication and early genes. The exact nature of the escape

mutations that confer insensitivity to AbiZ-mediated

phage resistance is not known. The authors suggest that

an as yet uncharacterised early or middle phage gene

product may act as a regulator of the phage holin, thus

explaining their observations (Durmaz & Klaenhammer,

2007).

Recently, AbiQ was reported to function as a toxin–antitoxin (TA) system, representing the first such system

described in Lactococcus (Samson et al., 2013a, b). TA

systems, first described in the 1980s as plasmid stability

systems (Jaffe et al., 1985), are typically comprised of a

stable toxin protein and an unstable antitoxin protein. As

plasmid stability systems, they ensure faithful plasmid

partitioning upon cell division, resulting in the death of

any cell which has not received a copy of the harbouring

plasmid. Since their identification, many systems have

been identified in bacteria, archaea and possibly in unicel-

lular fungi, and it has become clear that TA systems have

a much wider biological role than just being involved

plasmid stability (Yamaguchi et al., 2011; Schuster & Ber-

tram, 2013). Originally discovered on L. lactis W-37 plas-

mid pSRQ900 (Emond et al., 1998), AbiQ represents a

type III TA system, which induces cell death through the

accumulation of nonmature forms of viral DNA, and

which is effective against members of the so-called 936

and c2 phage species, as well as against certain rarely

encountered lactococcal phages (Emond et al., 1998; Sam-

son et al., 2013a, b). The AbiQ system is homologous to

the Pectobacterium atrosepticum Abi termed ToxIN (Fin-

eran et al., 2009). Both AbiQ toxin and antitoxin struc-

tures have been determined, representing the first Abi

system to be characterised at a structural level (Samson

et al., 2013a, b). AbiQ is transcribed and translated

FEMS Microbiol Rev && (2014) 1–23ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

10 S. Ainsworth et al.

Table 2. Currently known lactococcal abortive infection systems and related features

Abi

system

Discovered

on plasmid

Demonstrated

phage

protection* Proposed mode of action

Phage mutations

resulting in

Abi-escape

mutants Additional features References

AbiA pTR2030 c2 (c2); p2,

sk1 (936);

φ31, φP013,

φQ30, ul36,

φQ33 (P335)

Prevents DNA replication,

possibly through

preventing genome

circularisation

Virulent phages

acquire point

mutations in

single-stranded

DNA annealing

proteins

Homolog of AbiK;

heat sensitive;

copy number

effects level

of resistance

Hill et al. (1990),

Dinsmore et al.

(1998) and

Tangney &

Fitzgerald (2002)

AbiB pIL2101 bIL170 (936) Causes decay of phage

mRNA transcripts

Unknown – Parreira et al.

(1996)

AbiC pTN20 p2 (936); ul36

(P335)

Interferes with phage

protein production

Unknown – Durmaz et al.

(1992)

AbiD pBF61 c2 (c2); sk1

(936)

Unknown Unknown – McLandsborough

et al. (1995)

AbiD1 pIL105 bIL67 (c2); bIL66,

bIL170 (936)

Interferes with phage

DNA packaging

936, ORF1 regulator

of RuvC-like enzyme;

c2, unknown

Toxic to host;

activated at

level of translation

Anba et al.

(1995) and

Bidnenko

et al. (2009)

AbiE pNP40 712 (936) Unknown Unknown Two protein system,

AbiEi and AbiEii

Garvey et al.

(1995) and

Tangney &

Fitzgerald (2002)

AbiF pNP40 c2 (c2); 712

(936)

Affects DNA replication Unknown – Garvey et al.

(1995) and

Tangney &

Fitzgerald (2002)

AbiG pCI750 c2 (c2); 712 (936);

φQ30 (P335)

Affects RNA transcription Unknown Two protein system,

AbiGi and AbiGii

OConnor et al.

(1996),

O’Connor et al.

(1999) and

Tangney

& Fitzgerald (2002)

AbiH pLDP1 φ53 (c2); φ59

(936)

Unknown Unknown – Prevots et al. (1996)

AbiI pND852 c2 (c2); 712 (936) Unknown Unknown Activity at 37 °C Su et al. (1997)

AbiJ pND859 712 (936) Unknown Unknown Possible N-terminal

DNA-binding

helix-turn-helix

Deng et al. (1997)

AbiK pSRQ800 c2 (c2); p2, P008

(936); P335

(P335)

Prevents DNA replication

of P335 phages,

possibly through

preventing genome

circularisation

Virulent phages acquire

point mutations in

single-stranded

DNA-binding proteins;

temperate phages

recombine with host

prophage sequences

Homolog AbiA;

toxic to host;

copy number

effects level

of resistance

Bouchard

& Moineau

(2000),

Emond et al.

(1997),

Fortier et al.

(2005) and

Bouchard

& Moineau

(2000, 2004)

AbiL pND861 c2 (c2); 712 (936) Possibly translation Unknown Two protein system,

AbiLi and AbiLii

Deng et al. (1999)

AbiN Chromosomal φ53 (c2); φ59

(936)

Unknown Unknown Chromosomal

encoded; toxic

to host

Prevots et al.

(1998)

FEMS Microbiol Rev && (2014) 1–23 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

The lactococcal plasmidome 11

constitutively in noninfected cells, and the mechanism of

its activation is currently unknown, although it was sug-

gested that the antitoxin is sequestered by binding to a

phage product (Samson et al., 2013a, b). Examination of

AbiQ escape mutants of phage P008 suggests that orf210,

encoding a putative DNA polymerase, plays a role in its

activation.

AbiK was recently described as a novel reverse trans-

criptase (RT; Wang et al., 2011). AbiK is a self-priming

polymerase, generating long ‘random’ DNA sequences

> 500 nt. The N-terminal RT motif of AbiK is essential

for phage resistance (Fortier et al., 2005). Previous

mutational analysis of this DNA-binding, leucine-rich

repeat motif of the AbiK homolog, AbiA, was similarly

observed as being essential for conferring phage resistance

(Dinsmore et al., 1998). Despite the obtained information

as outlined above, the exact mechanism by which AbiA

and AbiK provide phage protection to a bacterial culture

remains unclear. However, as AbiK possesses RT activity

with which it produces long random DNAs, and as AbiK

escape mutants localise to saK, encoding a single-stranded

DNA annealing protein, which facilitates strand exchange,

the AbiA/K systems may inhibit specific homologous

recombination events such as genome circularisation. This

Table 2. Continued

Abi

system

Discovered

on plasmid

Demonstrated

phage

protection* Proposed mode of action

Phage mutations

resulting in

Abi-escape

mutants Additional features References

AbiO pPF144 φ53 (c2); φ59

(936)

Unknown Unknown Toxic to host Prevots &

Ritzenthaler (1998)

AbiP pIL2614 bIL66 (936) Prevents DNA replication,

affects temporal control

of early phage transcripts

Homologous

recombination

with resistant

936 phage,

exchange of gene eb7

Activity enhanced

by adjoining

upstream ORF

Domingues

et al. (2004a, b)

AbiQ pSRQ900 c21 (c2); p2 (936) Toxin-antitoxin system Heat stable; toxic

to host;

toxin-antitoxin

system

Emond et al. (1998)

AbiR pKR223 c2 (c2) Targets DNA replication Unknown Heat sensitive;

multicomponent

protein system

Twomey et al.

(2000)

AbiS pAW601 jj50, p2, sk1 (936) Unknown Unknown – Holubov�a

& Josephsen

(2007)

AbiT pED1 p2 (936); φQ30,

ul36 (P335)

Affects phage DNA

replication

Mutations in phage

bIL170 e14, phage

P008 orf41 and

phages P008 and

p2 major capsid

proteins

Two protein system,

AbiTi and AbiTii

Bouchard

et al. (2002)

AbiU pND001 c2 (c2); 712 (936);

ul36 (P335)

Delays RNA

transcription

Unknown Two protein system,

AbiU2 possibly

regulates AbiU1

Dai et al. (2001)

AbiV Chromosomal

(integrant

plasmid)

c2, bIL67, ml3, eb1

(c2); bIL170, jj50,

P008, p2, sk1

(936)

Inhibits phage early

transcripts

Mutations in the

SaV early gene

product

Dimeric protein;

can be expressed

at high levels;

forms AbiV2-Sav2complex

Haaber et al.

(2008, 2009,

2010)

AbiZ pTR2030 p2, sk1 (936); φ31,

φQ33, ul36 and

13 other P335

species phages

Premature lysis of

infected cells

P335 mutants arise

through homologous

recombination with

prophage sequences

encoding early and

middle phage genes

– Durmaz &

Klaenhammer

(2007)

*Specific phage names are supplied, with phage species indicated in brackets.

FEMS Microbiol Rev && (2014) 1–23ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

12 S. Ainsworth et al.

would explain the lack of DNA replication of sensitive

phages in AbiA/AbiK-containing cells, as phage genome

replication requires circularisation to produce concate-

meric DNA via a RCR-type mechanism (Scaltriti et al.,

2011; Wang et al., 2011).

AbiP is encoded on L. lactis IL420 plasmid pIL2617

and is an efficient Abi system that is active against partic-

ular members of the 936 species of lactococcal phages,

conferring high-level phage resistance. While the AbiP

phenotype is conferred by a single ORF (abiP), an adja-

cent upstream ORF (orf1) was identified that was shown

to enhance AbiP’s activity (Domingues et al., 2004a, b).

Further examination of AbiP has revealed that the protein

is associated with the bacterial membrane via an N-termi-

nal transmembrane domain and that it possesses a cyto-

solic nucleic acid-binding domain. The cellular

localisation of AbiP is important for its abortive infection

phenotype, as phage resistance was not observed in AbiP

mutants lacking the transmembrane domain. The nucleic

acid-binding domain of AbiP has a 10-fold binding pref-

erence for RNA relative to ssDNA. However, AbiP does

not appear to preferentially bind phage-originating

nucleic acids. Due to its lack of toxicity towards host

nucleic acids, the activation of AbiP is thought to be

dependent on (an) unknown phage-specific factor(s)

(Domingues et al., 2008).

Recently, the first abortive infection system activated at

the level of translation (Bidnenko et al., 2009) was eluci-

dated. AbiD1 was originally detected as being encoded on

L. lactis IL964 plasmid pIL105 (Gautier & Chopin, 1987)

and displays a resistance phenotype against 936 and c2

lactococcal phages. Experiments to understand the regula-

tion of the AbiD1 system showed that abiD1 is tran-

scribed in uninfected cells; however, abiD1-encompassing

transcripts are unstable and produced at low levels. In

addition, translation of AbiD1 is inefficient, which is

probably due to a stable inverted repeat that may pre-

clude the abiD1-associated translation initiation signals

from efficient ribosomal recognition. Infection of abiD1-

containing lactococcal cells by phage bIL66 activates

AbiD1 mRNA stabilisation and enhances its translation

through involvement of the N-terminus of phage bIL66

protein ORF1. ORF1 is believed to bind AbiD1 mRNA

and act as a cofactor to increase translation. The tightly

controlled and specific activation of the AbiD1 phage-

defence mechanism, by a phage component whose C-ter-

minus is absolutely required for infection, shows that

AbiD1 is highly evolved in its function.

AbiT is one of the few Abi systems that requires two

proteins, AbiTi and AbiTii, for its activity. The abiT

genes are cotranscribed and are thought to impart resis-

tance through inhibition of phage genomic DNA matu-

ration. AbiT is active against members of the 936 and

P335 phage species, causing efficiency of plaquing (EOP)

reductions that range from 10�5 to 10�7. Recently, pos-

sible phage-specific targets of AbiT activation have been

identified (Labrie et al., 2012). Examination of AbiT

escape mutants of multiple different 936 phages high-

lighted three individual genes harbouring mutations.

These were two separate early expressed genes (e14 and

orf41, for bIL170 and P008, respectively) and orf6 for

both P008 and P2, which was subsequently demon-

strated to encode the major capsid protein for phage p2

(Labrie et al., 2012). The AbiT resistance-causing muta-

tions in phage p2’s capsid protein were localised to its

C-terminus, which is incorporated into the phage virion.

The potential mechanism for phage resistance conferred

by AbiT is based on tentative similarities to the E. coli

antiphage system PifA (Labrie et al., 2012). Escherichia

coli phage mutants resistant to PifA were shown to

require a mutation in a gene necessary for replication,

or a double mutation in the gene specifying a capsid

protein (Molineux, 1991; Cheng et al., 2004). Similar to

PifA, AbiTi possesses a C-terminal hydrophobic region,

likely resulting in its localisation to the bacterial mem-

brane (Bouchard et al., 2002). AbiT, like PifA, also

reduces phage replication and prevents genomic matura-

tion. This is possibly due to the finding that AbiT alters

bacterial membrane permeability, which results in the

leakage of ATP and/or other molecules (Labrie et al.,

2012).

Plasmid metabolic impact

Lactococcal plasmid exploration efforts have been justified

because of their importance in developing new genetic

tools and discovery of biotechnological relevant traits

which can, for example, improve the robustness of dairy

starter cultures. However, several studies have noted that

while some plasmids may encode technologically desirable

traits, such as bacteriophage resistance, the same plasmids

may actually impede cellular fitness for two main reasons.

Firstly, addition of new plasmid material will increase

competition for the host’s replication, transcription and

translational machinery, thereby increasing the metabolic

burden on the host cell (Lee & Moon, 2003). Secondly,

plasmid-encoded factors may negatively impact upon host

cell metabolism. For example, conjugational transfer of a

60 kb plasmid pMRC01 to a new host promotes cell per-

meability and autolysis (Fallico et al., 2009). Transconju-

gants exhibited lower specific growth rates and higher

generation times as compared to their parental strains,

although the acidification ability of the strain appeared

unaffected. Extensive investigation has not yet been able

to reveal the mechanistic action of such phenotypes

(Fallico et al., 2009).

FEMS Microbiol Rev && (2014) 1–23 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

The lactococcal plasmidome 13

Plasmid pBL1 is a small (11 kb) plasmid which

encodes the synthetic machinery for bacteriocin Lcn972

(S�anchez et al., 2000). Strains harbouring this plasmid

grown under standard laboratory conditions are able to

produce Lcn972 without displaying any impact on

growth. However, a transcriptomics study of pBL1-con-

taining cells revealed apparent reduced expression of

the membrane transporter IIC of the cellobiose PTS,

celB (Campelo et al., 2011). Further physiological inves-

tigations demonstrated that the presence of pBL1

restricts cellobiose uptake, modulating fermentation

products towards that of a more mixed acid profile

and altering intracellular glycolytic intermediate levels

in L. lactis MG1363 cells grown on cellobiose as com-

pared to glucose. The authors noted that these findings

have practical consequences for strains being utilised

for bacteriocin production, with media rich in dextrins

or cellobiose to be avoided. Furthermore, they note

that CelB has been shown to take part in lactose

uptake in strains lacking lacEF; therefore, such strains

harbouring pBL1 would be seriously compromised

when used in dairy fermentations (Campelo et al.,

2011).

L. lactis plasmid evolution

As the number of available lactococcal plasmid sequences

has significantly increased in recent years, several authors

have remarked on the apparent mosaic nature of larger

lactococcal plasmids. For example, Gorecki et al. (2011)

noted that plasmids pIL1 and pIL2 are highly similar to

pCD4 and pCRL1127, respectively, whereas plasmid pIL6

appears to be a combination of particular genetic mod-

ules from plasmids pAH82 and pNP40. Also pLP712

appears to be a composite of modules present on

pGdh442, pSK11L, pSK11P and several other lactococcal

plasmids (Fig. 1; Wegmann et al., 2012). Furthermore,

the large lactose/proteinase plasmids that are typical of

the dairy L. lactis strains share extensive sequence identity

with each other. Plasmids pLP712 (Wegmann et al.,

2012), pSK11L, pSK11P (Siezen et al., 2005), pGdH442

(Tanous et al., 2007), pIL4, pIL5 (Gorecki et al., 2011),

pVF50 and pVF21 (Fallico et al., 2011), although repre-

senting unique plasmids, appear to be closely related

derivatives of each other. Interestingly, plasmid pGL6,

isolated from a clinical isolate of L. garviae (Aguado-Urda

et al., 2012), shares almost 50% of its coding sequence

Fig. 1. The mosaic nature of Lactococcus

lactis plasmid pLP712. Arrows in the inner

circle represent encoded open reading frames

(ORFs) present on pLP712. Homology of

discreet, coloured modules present on pLP712

is compared with homologous [amino acid

identify (%)] modules located on alternative

L. lactis plasmids. Blue: lac operon. Green:

pepF. Purple: prtP/M. Orange: repB, parAB.

Pink: module encoding a putative multicopper

oxidase and D-lactate dehydrogenase, along

with hypothetical ORFs. Red arrows:

transposase-encoding ORFs.

FEMS Microbiol Rev && (2014) 1–23ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

14 S. Ainsworth et al.

with the L. lactis IL594 plasmid pIL7, suggesting a hori-

zontal transfer event between (ancestors of) these two

species. Despite the apparent extensive similarity amongst

sequenced lactococcal plasmids, plasmid individuality is

also apparent, as 13% of thus far identified lactococcal

plasmid genes have currently no known lactococcal ho-

mologs.

The presence of multiple replicons and GC skew exam-

inations of large lactococcal plasmids (Siezen et al., 2005;

Gorecki et al., 2011) and the relative ease of isolation of

plasmid derivatives which have undergone physical rear-

rangements relative to plasmids present in parental strains

(Wegmann et al., 2012) suggests that the lactococcal

plasmidome (which is the overall plasmid content in a

given species or environment; Walker, 2012) is essentially

fluid and actively evolving. Wegmann et al. (2012) hy-

pothesised that it is plasmid-borne transposase-encoding

genes, commonly found flanking genetic modules, that

are directly involved in genetic reshuffling events that

cause the generation of alternative plasmid forms. Spon-

taneously generated deletion derivatives of pLP712 varied

in size and structure, which the authors suggest cannot

be accounted for on the basis of RecA-dependent homol-

ogous recombination alone. The widespread dissemina-

tion of uspA, discussed earlier, amongst L. lactis plasmids

is possibly due to its position within a predicted small

conjugative transposon, which also specifies a putative

Mn2+/Fe2+ cation transporter (Siezen et al., 2005). This

putative conjugative transposon is present in all currently

sequenced dairy plasmid complements and is also com-

mon amongst various Streptococcus and Enterococcus spe-

cies, suggesting its genetic spread by horizontal transfer.

Transposable elements, such as conjugative transposons

and insertion sequences, have long been known to reside

on lactococcal plasmids and have been associated with

important functional dairy phenotypes, such as bacterio-

cin production, carbohydrate metabolism and transport,

and bacteriophage resistance (for a review, see Mills et al.,

2006). Insertion sequence (IS) elements may comprise

significant proportions of the strain-specific chromosome

and can contribute to genome plasticity, pseudogenes

(Makarova et al., 2006) and in some cases activate gene

transcription (Bongers et al., 2003). Sequencing of lacto-

coccal genomes and plasmid complements have revealed

the extent of IS element abundance in Lactococcus. For

example, the genetic information of the combined IS ele-

ments in MG1363 encompasses over 66 kb of DNA, while

in L. lactis UC509.9, this increases to over 100 kb, in the

latter representing 5% of its corresponding chromosome

(Ainsworth et al., 2013). As previously noted (Mills et al.,

2006), their abundance in lactococcal plasmids is likely a

reflection of the importance of IS elements/transposons

in promoting evolution of lactococcal plasmids under

selective dairying conditions. Examination of all the pro-

teins currently encoded by the lactococcal plasmidome

suggests that copies of IS6, IS982 and ISLL6 are particu-

larly abundant (Fig. 2) and collectively constitute 1 in

every 10 genes encoded on lactococcal plasmids.

Evidence from lactococcal plasmid sequences and

experimental data suggests that lactococcal plasmid trans-

fer is predominantly driven by conjugation and transduc-

tion. It has long been ascertained that many lactococcal

plasmids are mobilisable (Coakley et al., 1997; O’Driscoll

et al., 2006). As the process of conjugation is considered

a natural DNA transfer system, representing a food-grade

process, it has been exploited for the dissemination of

technologically desirable phenotypes, such as bacterio-

phage resistance and bacteriocin production, to a wide

range of industrial strains (Mills et al., 2006).

Conjugation is a process whereby plasmid material is

passed from a donor cell to a recipient cell via a channel

or pilus also known as the conjugation apparatus (Groh-

mann et al., 2003). Mobile plasmids are transmitted in a

single-stranded conformation after it has been nicked at

the AT-rich origin of transfer (oriT) region by a nicking

enzyme. Molecular mechanisms of conjugation have been

described in detail for many Gram-positive and Gram-

negative species (Grohmann et al., 2003). Several

sequenced lactococcal plasmids are mobilisable or appear

to encode the apparatus to enable conjugal transfer (Mills

et al. 2006; O’Driscoll et al., 2006; Millen et al., 2012);

however, the majority of sequenced plasmids to date do

not appear to be transmissible, and the precise molecular

mechanisms of conjugation in L. lactis remain unclear.

Sequence analysis suggests that there are two types of

nicking enzymes present amongst L. lactis plasmids,

MobD and MobA. Both are members of the relaxase

(PF03432) family of enzymes. However, MobD and MobA

share only 24% amino acid identity across 74% of the

length of the enzyme. Additionally, mobA is usually associ-

ated with mobB and mobC, which specify three proteins

that are thought to form a relaxosome at the oriT (Hofre-

uter & Haas, 2002), while mobA is usually not associated

with Tra-encoding genes (below), as is the case, for exam-

ple, on plasmids pCIS8 and pSK11P (Ainsworth et al.,

2013; Siezen et al., 2005). Alternatively, mobD seems to be

associated with mobC and not with mobB, and this gene is

often located in a large gene cluster encoding other (pre-

sumed) conjugation functions (below).

The mobile lactococcal plasmid pNP40 (O’Driscoll et al.,

2006) and the apparently nonmobile pIL6 (Gorecki et al.,

2011), which encodes a MobD-type nickase, both contain a

17–18 kb gene cluster for (presumed) conjugal transfer,

encompassing approximately 20 open reading frames,

which are fairly well conserved between the two plasmids.

Human isolate L. lactis CV56 (Gao et al. 2011) harbours

FEMS Microbiol Rev && (2014) 1–23 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

The lactococcal plasmidome 15

two similar gene clusters for putative conjugal transfer on

separate plasmids, pCV56A and pCV56C, both of which

appear dysfunctional due to transposon insertions. The

majority of the open reading frames found in such operons

are hypothetical, and it is not apparent if they have an

essential or any role in the conjugation process. However,

based on sequence similarity, several genes have been iden-

tified which are predicted to be involved in transfer and

physical entry of conjugated DNA from the donor cell to

recipient. A pNP40 open reading frame, termed traF, is a

putative membrane-spanning protein, which O’Driscoll

et al. (2006) suggested to be involved in channel formation.

A TraF homolog is also encoded on pIL6. Genes termed

traG and traE are well conserved across sequenced lacto-

coccal conjugative operons and contain domains which

suggest that they are involved in the formation of a conju-

gal pilus structure, similar to type IV secretion systems

(O’Driscoll et al., 2006; Gorecki et al., 2011). Hypothetical

proteins are encoded by the two conjugal transfer

gene clusters, some of which contain potential CHAP and

muramidase domains. Both O’Driscoll et al. (2006) and

Gorecki et al. (2011) speculate that these proteins may be

involved in localised cell wall degradation to aid cell-to-cell

contact and promote plasmid delivery to the donor cell.

(a) (d)

(b)

(c)

Fig. 2. Comparative analysis conducted on seven complete Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris plasmid complements

(IL594, UC509.9, SK11, DPC3901, A76, CV56 and KF147). (a) Accumulated number of distinct genes in the generated pan-plasmidome, plotted

against the number of employed strains. (b) Accumulated number of genes in core-plasmidome, plotted against the number of employed strains.

(c) Total number of genes in the plasmidome, plotted against the number of employed strains. (d) Heatmap resulting from the hierarchical

clustering analysis based on the presence/absence of gene families in plasmidome. Highlighted in red are the common plasmid genes with their

predicted functions indicated.

FEMS Microbiol Rev && (2014) 1–23ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

16 S. Ainsworth et al.

Despite many plasmids apparently being nontransmiss-

ible, they frequently contain (remnant) features of a plas-

mid transmission machinery. A clear example of this is

the multicopy presence of the mobility protein MobC-

encoding gene (23 copies being present amongst

sequenced lactococcal plasmids). MobC is thought to act

as a molecular wedge, aiding in the separation of comple-

mentary DNA strands at the oriT locus (Zhang & Meyer,

1997). The high frequency of mobC presence and other

associated ‘orphan’ mobilisation genes across lactococcal

plasmids may be a reflection of plasmid acquisition/trans-

fer events by mobilisation.

Plasmid transfer via conjugation has been observed to

lead to rearrangements and deletions in existing and

introduced plasmids through homologous recombination

events (Wegmann et al., 2012). Conjugational transfer of

plasmids between L. lactis and members of several LAB

genera, such as Lactobacillus (Langella & Chopin, 1989;

Toomey et al., 2010) and Enterococcus spp. (Tuohy et al.,

2002; Devirgiliis et al., 2010) has been demonstrated in in

vitro and in vivo models (Toomey et al., 2009). These

studies suggest that plasmid transfer via conjugation is a

relatively common phenomenon in various naturally

occurring environmental matrices, thereby ensuring a

constant influx of genetic material for L. lactis and other

LAB plasmidome augmentation. High frequency plasmid

transfer by transduction in L. lactis has also been

observed. In L. lactis NCDO712, transduction of small

(< 5 kb) plasmids has been reported to occur at a fre-

quency of 2.1 9 10�3 to 2 9 10�4 transductants per pla-

que-forming unit (PFU; Wegmann et al., 2012), whereas

transduction of large (> 50 kb) plasmids has also been

observed to occur at lower frequencies. However, the size

of transferred plasmids appears to be limited by the phys-

ical packing space of the transducing bacteriophage. For

example, Wegmann et al. (2012) observed phage-medi-

ated transfer of the lactose-metabolising phenotype speci-

fied by plasmid pLP712; however, when examined, the

transduced plasmids were similar in size to the transduc-

ing phage genome and smaller than pLP712. Recently,

plasmid transduction between LAB species was observed

to occur from Streptococcus thermophilus to L. lactis (Am-

mann et al., 2008), adding a further mechanism by which

new genetic material can be incorporated into the L. lactis

plasmidome from a nonlactococcal donor.

Bioinformatic analysis (Fig. 2), using reciprocal best-

BLAST hits and MCL clustering algorithms, of the current

lactococcal plasmidome corroborates the notion that the

lactococcal plasmidome is fluid in nature and actively

evolving. Extrapolation of the results, performed using a

similar approach employed for analysing bacterial pan-

genomes, further suggests that the lactococcal pan-plasmi-

dome (defined as the collection of all distinct genes

residing on currently available lactococcal plasmid

sequences) is currently not yet fully defined (Fig. 2a and

c), thus offering a high potential for the discovery of

novel lactococcal plasmid-borne genes. This analysis also

reveals that genes commonly present in the combined

plasmid content of a given strain, excluding replication

proteins, principally consist of mobilisation proteins,

transposases and site-specific recombinases, further add-

ing to the notion that their innate variability is driven by

rapid evolution through mechanisms of module exchange

and genetic rearrangements. Despite the apparent fluid

nature of the lactococcal plasmidome, analysis by hierar-

chical clustering performed on plasmid complements of

seven strains IL594 (Gorecki et al., 2011), UC509.9 (Ains-

worth et al., 2013), SK11 (Siezen et al., 2005), DPC3901

(Fallico et al., 2011), A12 (Passerini et al., 2013), CV56

(Gao et al., 2011) and KF147 (Siezen et al., 2010) indi-

cates a clear separation of the two L. lactis subspecies

based on the presence/absence of plasmid genes. This

result is may be simply due to the evolutionary related-

ness amongst particular subspecies, yet may explain some

of the phenotypic differences between the two subspecies

(Fig. 2d).

The relatively low abundance of plasmids in plant iso-

lates of L. lactis coupled with the rarity of lactose in the

plant environment and extensive genome decay in dairy

isolates suggest that L. lactis strains obtained the majority

of its plasmids with dairy-associated phenotypes by hori-

zontal acquisition to adapt to the dairy environment. The

ancestral origins of these phenotypes are currently

unclear. Lactose is a sugar primarily found in milk and is

rare in the plant niche (Mills et al., 2006). This is consis-

tent with the notion that lactose utilisation genes have

not been detected in lactococcal plant isolates (Siezen

et al., 2010). It is possible that the genetic material sup-

porting this phenotype was first acquired from a mam-

malian commensal contaminant of milk, such as a

member of the Lactobacilli or pathogenic Streptococci.

The horizontal acquisition of such elements is consistent

with the differential modal codon usage (i.e. the codon

usage which matches most of the genes of the genome as

opposed to the genome wide average (Davis & Olsen,

2010; Wegmann et al., 2012) observed between pLP712

and its host, L. lactis NCDO712 (the parental strain of

L. lactis MG1363). In fact, lactococcal plasmid codon

usage is closer to that of the Streptococcus agalactiae chro-

mosome than that of the chromosome of the lactococcal

host. The ability to degrade casein may not have been

acquired by horizontal transfer, but through adaption of

already present proteases, as PrtP-like proteases are found

in both plant and dairy lactococcal isolates (Liu et al.,

2010; Price et al., 2011). Price et al. (2011) have sug-

gested that alterations in the substrate binding site of

FEMS Microbiol Rev && (2014) 1–23 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

The lactococcal plasmidome 17

PrtP-like proteases may have allowed adaption of these

proteins towards casein degradation.

Prospects for future research

Despite the recent increase in lactococcal genome

sequencing, a significant proportion of lactococcal plas-

mid genes remain uncharacterised. For example, it was

recently shown that the presence of a cryptic plasmid can

modify the cell surface of L. lactis, increasing cellular

adhesion properties through electron-donor/electron-

acceptor interaction (Cavin et al., 2007), although the

precise genetic cause of this phenomenon remains

unknown. We expect that the continued pace of lactococ-

cal genome sequencing and continuing interest in explo-

ration of environmental L. lactis strains for industrial

applications will undoubtedly result in the discovery of

novel plasmid-encoded functions. We also expect that as

a result of the fluidity of the lactococcal plasmidome, the

discovery and/or deliberate construction by conjugation,

mobilisation or transduction of L. lactis strains with novel

plasmid combinations will create new scientific and com-

mercial challenges and opportunities.

Acknowledgements

We would like to thank Marie-Pierre Chapot-Chartier for

providing unpublished data. This research was funded by

a Science Foundation Ireland (SFI) Principal Investigator-

ship award (Ref. No. 08/IN.1/B1909) to DvS.

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FEMS Microbiol Rev && (2014) 1–23 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

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