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Previous Article | Next Article 
Applied and Environmental Microbiology, January 1999, p. 330-335, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Expression, Regulation, and Mode of Action of the
AbiG Abortive Infection System of Lactococcus lactis
subsp. cremoris UC653
Lisa
O'Connor,
Mark
Tangney, and
Gerald F.
Fitzgerald*
Department of Microbiology and National Food
Biotechnology Centre, University College, Cork, Ireland
Received 4 June 1998/Accepted 30 September 1998
 |
ABSTRACT |
The abortive infection system AbiG is encoded by the lactococcal
plasmid pCI750. The abiG locus (consisting of two genes, abiGi and abiGii) was examined by Northern blot
analysis, revealing two transcripts of approximately 2.8 and 1.5 kb
which were homologous to the two gene-specific probes. A
transcriptional start site was mapped upstream of abiGi,
and it appeared that the two genes were cotranscribed, resulting in the
2.8-kb transcript. The smaller transcript may be the result of
independent transcription of abiGii within
abiGi or of the presence of a weak terminator within
abiGii. The locus was shown to be constitutively expressed.
Evidence is presented for the possible existence of a second Abi
mechanism on pCI750. Examination of phage sk1 RNA synthesis
demonstrated that both the subcloned AbiG and, to a greater extent,
pCI750 inhibited this process. pCI750 also severely inhibited synthesis of both early and late phage c2 transcripts, while the presence of the
subclone resulted in a reduction in late transcript synthesis only.
| |
TEXT |
The ongoing demand of the dairy
industry for lactococcal starter cultures with high levels of phage
resistance has stimulated the search for natural resistance systems
which are abundant in these hosts (for a recent review, see reference
9). This has resulted in the identification of a
myriad of abortive infection (Abi) systems, 13 of which have been
sequenced to date. It has become apparent that the genetic flexibility
of phages has allowed them to evolve in response to the selective
pressure of resistance mechanisms (6, 13, 19, 21), and it
has been proposed that the best defensive approach is that of rotating
a number of different mechanisms within an isogenic strain
(12). To optimize this strategy, it is useful to combine
systems targeting different stages of the phage life cycle. Therefore,
there is a need not only to accumulate a large number of resistance
systems but also to characterize them so that they may be used
optimally. In addition, it is of interest to understand how these
systems are controlled. The aim of this study was to examine the
expression and regulation of the two genes of the lactococcal AbiG
system (23), abiGi and abiGii, and to
determine the effect of this defense mechanism on intracellular phage development.
Bacterial strains, plasmids, and culture conditions.
The
strains and plasmids used in this study are listed in Table
1. Lactococcus lactis was
grown at 30°C in M17 medium (28) supplemented with 0.5%
glucose (GM17). Chloramphenicol was added at 5 µg/ml where necessary.
Phages were propagated by the method of Terzaghi and Sandine
(28).
Northern blot analysis of abiG.
The organization of the
abiG locus is diagrammed in Fig.
1. To determine if the two
abiG genes were coordinately or discoordinately transcribed,
RNA isolated (2) from Lactococcus lactis subsp. cremoris LOC758 (containing an abiG subclone,
pCI758) was probed with PCR products Gi-GSP (generated by using primers
GiL [5' TATATTCCTATGACCTG 3'] and GiR [5'
AATTCAAAAGACCATTC 3']) and Gii-GSP (generated by using primers
GiiL [5' TTGTATTAGATGAAACC 3'] and GiiR [5'
ACTCTATCTTGTAAATCC 3']), specific for abiGi and
abiGii, respectively (Fig. 1a) (Taq polymerase
was supplied by Promega, Madison, Wis.). In order to eliminate any
difficulties in comparing the estimated transcript sizes obtained,
duplicate RNA samples were electrophoresed on the same gel and then
transferred separately by capillary blotting to nylon membranes. These
were probed with the two different gene-specific probes. Analysis with
Gi-GSP identified three transcripts of approximately 2.8, 1.5, and 1.3 kb based on the Promega RNA markers (Fig.
2a). The 1.3-kb transcript was within a
smear, and in subsequent analysis of RNA isolated from LOC735
(harboring the smallest functional subclone, pCI735 [Fig. 1b]), only
a region of smearing with no distinctive band was observed (see Fig.
3a). The Gii-GSP probe hybridized to the same two transcripts
identified by using Gi-GSP, i.e., the 2.8- and 1.5-kb bands (Fig. 2b).
It therefore appears that the two genes are cotranscribed, resulting in
the 2.8-kb transcript (abiGi is 750 bp and is separated by 2 bp from the 1,194-bp abiGii). The smaller transcript may be
the result of independent transcription of abiGii from
within abiGi or of the presence of a weak terminator
structure within abiGii.
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FIG. 1.
(a) Molecular organization of the abiG locus
on a 5.8-kb segment of pCI758 (adapted from reference
23). The two open reading frames of abiG
and orfX (of unknown function) are indicated by horizontal
arrows.  indicates a 309-bp
direct repeat with 5 mismatches.
 and
 represent putative promoter
sequences and terminator structures, respectively. Gi-GSP and Gii-GSP
are PCR-generated probes specific for abiGi and
abiGii, respectively. Lowercase letters a through d
represent primers. (b) A 3.5-kb insert of pCI735. (c) The mapped
promoter and transcriptional start site (+1) upstream of
abiGi.
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FIG. 2.
Northern analysis of abiGi and
abiGii. RNA isolated from L. lactis subsp.
cremoris LOC758 (lanes 2) or pCI784 DNA used as a positive
control (lanes 1) was hybridized with 32P-labelled Gi-GSP
(a) or 32P-labelled Gii-GSP (b). Arrows indicate the sizes
of the transcripts, estimated by using RNA size standards.
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|
Mapping of the 5' ends of the abiG transcripts.
As
sequence analysis had identified a putative promoter upstream of
abiGi (23) (Fig. 1a), this region was first
examined in order to determine the 5' end of the abiG mRNA
by using the 5'/3' RACE (rapid amplification of cDNA ends) kit
(Boehringer Corporation Ltd., East Sussex, United Kingdom). Primer a
(5' ATATCGTGGTAAGAAGGC 3') (Fig. 1a) was used as outlined by
the manufacturers to make cDNA from LOC758 total RNA, while the nested
primer b (5' TTAGCACTAAGCTCCGAA 3') and primers from the kit
were used to generate a PCR fragment. A PCR product of approximately
100 bp was obtained. Sequence analysis of this product demonstrated its
homology to the region upstream of abiGi and identified a G
as the first base of the original cDNA (Fig. 1c). Due to the close
proximity (i.e., separation by 7 bp) of this base to the 
10 box of
the previously specified putative promoter, it was concluded that this
G was the transcriptional start site. This site and the accompanying
promoter were typical of those found in lactococci by Van de Guchte et
al. (29), who most frequently observed an A residue at the
start point, with G residues being the next most commonly encountered.
The 10 box did not possess the TGy extension (reported to be a
feature of strong promoters [20]) which has been
observed in the abiD1 (1) promoter, the putative
promoters of abiF (16) and abiI (27), and other lactococcal promoters.
In order to determine if the smaller transcript (1.5 kb) started
upstream of abiGii (within abiGi), even though no
putative promoter was evident based on sequence analysis
(20), attempts were made to map the 5' end of an
abiGii-specific message by using primer c (5'
TGTTGCTTTATCATTAG 3') and the nested primer d (5' TCTTCCTTTATTTCATCG 3') (Fig. 1a). While PCR products of
approximately 310 and 400 bp were obtained, sequence analysis
demonstrated no homology with the abiG locus, suggesting
that they were nonspecific products. The distance between primer c and
the transcriptional start site upstream of abiGi is 954 bp;
assuming that this is also the transcriptional start site for
abiGii, giving rise to the 2.8-kb transcript, it is likely
that the cDNA reaction of the 5' RACE protocol did not successfully
extend the 954 bp.
Previously, examination of the DNA sequence revealed a putative
terminator downstream of abiGii (23). The
distance between this and the mapped transcriptional start site is
2.539 kb, which is in reasonable agreement with the estimated size of
the larger transcript (2.8 kb). Given that the smaller transcript was
estimated at 1.5 kb and is recognized by both the Gi-GSP and Gii-GSP
probes, it seems most likely that it begins at the same site as the
larger transcript and results from the presence of a weak terminator within abiGii (although no obvious candidate was detected in
the sequence).
Transcriptional regulation of the abiG genes.
To
determine whether the abiG genes were constitutively
transcribed or induced upon phage infection, we used the AbiG-sensitive phage sk1, which is unable to plaque in the presence of AbiG. Total RNA
was isolated from phage sk1-infected cells of LM0230 (plasmid free),
LOC735 (containing a 3.5-kb subclone), and AB002 (containing the native
plasmid) and was probed with Gi-GSP and Gii-GSP (Fig. 1a).
Eighty-milliliter volumes of culture were grown to an optical density
at 600 nm of 0.3. A 20-ml sample was removed and harvested by
centrifugation in a Beckman JA-14 rotor at 10,000 rpm for 1 min, after
which the pellet was resuspended in ice-cold RNA extraction buffer
(2) and stored on ice until all the samples were collected.
CaCl2 was added to a final concentration of 5 mM, and phage
was added at a multiplicity of infection of 5 to 10; samples were
harvested as before. Total RNA was extracted as described by Arnau et
al. (2); samples were DNase treated and run on a 1.1%
formaldehyde gel.
The Gi-GSP probe recognized the previously identified 2.8- and 1.5-kb
transcripts in both LOC735 and AB002 (Fig.
3a). Surprisingly, an additional faint
band of approximately 7 kb was seen in the subclone containing LOC735,
and after extended exposure it was also visible in AB002 (data not
shown). This band may represent plasmid DNA contamination, even though
DNase treatment was performed, or it may be the result of low-level
read-through of the terminator of the abiG locus. The Gi-GSP
probe demonstrated that abiGi was not induced by phage
infection (Fig. 3a). While the 2.8-kb transcript was very concentrated
and thus a slight increase in transcription might be difficult to
detect, examination of the 1.5-kb transcript indicated clearly that it
was not induced. Similarly, the Gii-GSP probe hybridized to the 2.8- and 1.5-kb transcripts and, as expected, demonstrated that
abiGii also was not induced (Fig. 3b).
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FIG. 3.
Hybridization of total RNA isolated from phage
sk1-infected L. lactis subsp. cremoris LM0230,
LOC735, or AB002 at 0, 5, 15, and 30 min postinfection with
32P-labelled Gi-GSP (a) or 32P-labelled Gii-GSP
(b). The last lanes contain pCI784 DNA used as a positive control.
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|
Two other abi genes have been examined for induction. In the
case of abiA, the ability of its promoter to confer
chloramphenicol resistance when cloned upstream of a promoterless
cat68 gene suggested that it was constitutively expressed
(18), while Anba et al. (1) observed no
alteration in the level of transcription of abiD1 upon phage
infection. Northern analysis of the latter abi gene revealed
that transcription was initiated at a strong promoter but stopped at a
terminator located 48 bp downstream. This observation, together with
expression studies of the AbiD1 protein, strongly suggested that AbiD1
overproduction was toxic to the lactococcal cells. No such stringent
control was observed for abiG transcription, although it is
interesting that cloning of intact abiGi was lethal in
Escherichia coli cells (data not shown).
It was noted that a much higher level of transcription was observed in
LOC735- than in AB002-containing cells, by using either Gi-GSP or
Gii-GSP. As care was taken to load the same amount of RNA in each well,
it is most improbable that such a concentration effect could be
attributed to experimental error. Moreover, the samples used in Fig. 3a
and b were isolated on separate occasions, further supporting the
reproducibility of this observation. A possible explanation is that the
copy number of the subclone is higher than that of the original
plasmid, and indeed this subclone is based on pAM401, a
high-copy-number plasmid (31). It is widely accepted that
increasing the copy number of an abi gene results in
increased phage resistance (24, 10); however, in this
instance the opposite would appear to be the case, given that pCI750
offers more protection to cells against phage infection than pCI735
(23). It therefore seems very plausible that pCI750 encodes
a second Abi mechanism.
Influence of AbiG on bacteriophage RNA synthesis.
Previously,
we reported that AbiG-containing cells did not inhibit intracellular
phage DNA replication (23). In this study we examined the
ability of AbiG to interfere with phage RNA synthesis, using pCI750 and
the 3.5-kb subclone pCI735. Transcription of phages c2 and sk1 was
investigated as restriction data, and temporal transcription maps,
describing the order in which parts of the phage genome are expressed,
were available for both phages (5, 8). Phage sk1 is unable
to plaque in the presence of either the original plasmid or the
subclone, while c2 forms plaques of reduced size in the presence of
both and has an efficiency of plating (EOP) of 2.5 × 10
3 on AB002 but an EOP of 1.0 in the presence of AbiG
subclones (23).
The results obtained following probing of total-RNA extracts from
sk1-infected cells (obtained as described in the previous section) with
sk1 total DNA are presented in Fig. 4a.
The bands visible at 0 and 5 min after phage infection
(T0 and T5, respectively) for all three strains appear to represent nonspecific hybridization to
two host transcripts in addition to the 23S and 16S rRNA bands. A
comparison of the signal from the T15 samples
clearly demonstrated that less RNA is being synthesized in the pCI750-
and pCI735-containing cells. A comparison of the
T30 values suggests that virtually no phage RNA
synthesis occurred in the presence of pCI750 and only a very limited
amount occurred in the presence of the subcloned abiG. These
findings are substantiated by a 3.9-kb EcoRV probe specific
for the late-expressed region of phage sk1 (8). This probe
revealed considerably reduced synthesis of late-expressed RNA in the
presence of pCI735 and an apparent complete inhibition of synthesis in
cells containing the original plasmid (Fig. 4b). Whether early and
middle sk1 transcripts are also affected is difficult to determine from
experiments using the total phage DNA probe (Fig. 4a). The sk1-specific
signal (obtained by using either the total-DNA probe or the
late-specific probe) was a smear rather than discrete bands. Chandry et
al. (8) also observed this smearing and suggested that it
may be the result of nonspecific processing of transcripts, transcript
degradation, the presence of fragments of increasing size with a common
5' end, or a combination of these factors.
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FIG. 4.
Hybridization of total RNA isolated from phage
sk1-infected L. lactis subsp. cremoris LM0230,
LOC735, or AB002 at 0, 5, 15, and 30 min postinfection with
32P-labelled sk1 total DNA digested with EcoRV
(a) or a 32P-labelled 3.9-kb EcoRV fragment from
the late-expressed region (b). The first lanes contain
EcoRV-digested phage sk1 DNA used as a positive control.
Arrows indicate the 16S and 23S rRNA bands. (c) Temporal transcription
map of phage sk1 with early-, middle-, and late-expressed regions
indicated; adapted from Chandry et al. (8) with permission
from the publisher.
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In the case of early phage c2 RNA synthesis, the signal obtained was
more specific, with no signal observed prior to phage infection (i.e.,
T0 [Fig. 5a]).
The probe used was a 4.1-kb HaeIII fragment specific for the
early-expressed region of c2 (5). A comparison of the amount
of RNA synthesized in plasmid-free and pCI735-containing cells
suggested that early transcription was normal in the presence of the
abiG subclone. Indeed, there appeared to be slightly more
RNA present at T15 and
T30 in the presence of Abi. This presumably
reflects the experimental conditions, although care was taken to
harvest RNA from cells at the same optical density and to subsequently
load the same total amount of RNA in each well. In contrast to the
influence of the subclone, the presence of pCI750 dramatically
inhibited phage c2 early transcription. Late c2 transcription was
examined by using a 1.67-kb EcoRV fragment as a probe (Fig.
5b). The signal obtained was similar to late sk1 in that no discrete
bands were visible (see above). In this instance the abiG
subclone resulted in reduced levels of phage transcription, while the
presence of pCI750 severely inhibited the transcription process. The
latter result was expected, given the previously observed effect of
pCI750 on early c2 transcription.
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FIG. 5.
(a) Hybridization of total RNA isolated from phage
c2-infected L. lactis subsp. cremoris LM0230,
LOC735, or AB002 at 0, 5, 15, and 30 min postinfection with a
32P-labelled 4.1-kb HaeIII fragment from the
early-expressed region. (b) Hybridization of total RNA isolated from
phage c2-infected L. lactis subsp. cremoris
LM0230, LOC735, or AB002 at 0, 10, 15, 20, and 25 min postinfection
with a 32P-labelled 1.67-kb EcoRV fragment from
the late-expressed region. In both panels, lanes labeled c2 contain
phage c2 DNA used as a positive control. (c) Temporal transcription map
of phage c2 with early- and late-expressed regions indicated; adapted
from Beresford et al. (5).
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|
Notably, the level of interference in sk1 RNA synthesis was similar for
both pCI750 and pCI735 when total and late-specific transcription was
examined. In the case of c2 RNA synthesis, the subclone had no effect
on early transcription, while pCI750 did. Thus, it may be concluded
that abiG does not interfere with phage c2 early
transcription. However, when the effect of pCI750 on late c2
transcription was determined, there was clearly a degree of inhibition
greater than that mediated by the subclone. This raises the question
whether there is another mechanism(s) on pCI750, which is active
against c2, particularly affecting early transcription, while having no
significant effect on sk1.
Interestingly, since c2 DNA replication appeared to be unaffected by
the presence of pCI750 and yet early c2 transcription was inhibited, it
seems possible that early c2 genes are not necessary for c2 DNA
replication to occur. This concurs with the findings of Waterfield et
al. (30), who had previously found that a noncoding fragment
containing the ori was capable of directing DNA replication in Lactococcus but not in E. coli and concluded
that host factors alone were sufficient to initiate DNA replication at
ori.
In conclusion, AbiG inhibits phage sk1 and late c2 RNA synthesis;
however, whether this process of transcription is the primary target of
the system or a secondary effect remains to be determined. The AbiB
mechanism also interferes with phage RNA but has been shown to result
in rapid degradation of sensitive phage transcripts 10 to 15 min after
infection (25). Geis et al. (17) reported that
the presence of the Abi-encoding lactococcal plasmids pBU1-8 and 1149-3 resulted in complete inhibition of RNA synthesis and proposed that
phage transcription was the primary target of these systems. However,
it should be noted that these native lactococcal plasmids may encode
other resistance mechanisms, which together may have contributed to
this observed inhibition. Deficiencies in transcription have been
observed in some of the better-characterized Abi systems of
gram-negative bacteria, such as the 
-encoded Rex system
(15), the ColIb-encoded Abi (11), and the
F-encoded Pif (4); however, all these systems also severely
inhibited DNA replication, a feature not observed for AbiG.
Deficiencies in 
29 transcription and translation in the course of
its aborted infection of Vibrio cholerae biotype el tor
cells were shown to be due to destabilization of phage DNA concatamer
intermediates (7). AbiG most closely resembles the el
tor-encoded resistance in terms of its effect on phage DNA replication
and RNA transcription.
| |
ACKNOWLEDGMENTS |
This work was supported by European Community BRIDGE and
BIOTECHNOLOGY grants to G. F. Fitzgerald (contracts BIO-CT91-0263 and BIO4-CT96-0402, respectively).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and National Food Biotechnology Centre, University
College, Cork, Ireland. Phone: 353-21-902730. Fax: 353-21-276318. E-mail: g.fitzgerald{at}ucc.ie.
| |
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Applied and Environmental Microbiology, January 1999, p. 330-335, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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