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Applied and Environmental Microbiology, November 2001, p. 5225-5232, Vol. 67, No. 11
Department of Biotechnology, The University
of New South Wales, Sydney, New South Wales
2052,1 and DSM Food Specialties,
Moorebank, New South Wales 1875,2 Australia, and
Department of Microbiology, Oregon State University,
Corvallis, Oregon 97331-38043
Received 28 February 2001/Accepted 9 August 2001
This study reports on the identification and characterization of a
novel abortive infection system, AbiU, from Lactococcus lactis. AbiU confers resistance to phages from the three main industrially relevant lactococcal phage species: c2, 936, and P335. The
presence of AbiU reduced the efficiency of plaquing against specific
phage from each species as follows: 3.7 × 10 Lactococcus lactis is the
main bacterial species used as starter cultures in dairy fermentations.
However, phage infection of this species is a problem that causes
significant economic loss. Research on natural phage resistance systems
encoded by Lactococcus led to the identification of four
different mechanisms: adsorption inhibition, DNA injection blocking,
restriction-modification (R/M), and abortive infection (abi) (for a
recent review, see reference 12). The first three systems
operate at steps early in the phage infection process, whereas abortive
infection can target various different phases of phage development,
including DNA replication, RNA transcription, protein synthesis,
packaging, and morphogenesis.
To date, 18 abortive infection systems have been sequenced and are
designated AbiA through AbiR (7, 12). DNA and protein sequence analysis indicates that none of the abi systems has any significant homology with other genes and proteins in the databanks. In
addition, few motifs have been identified, making it difficult to
hypothesize the function(s) of each protein (12). While
comparison of the different abi systems revealed that AbiD, AbiD1, and
AbiK proteins are related, they have been reported to have very
different modes of action (1, 11, 22). Fourteen of the
eighteen abi systems sequenced are encoded by single genes. Four abi
systems The mode of action of various different abi systems have been studied.
AbiA, AbiF, and AbiK were found to interfere with phage DNA replication
(11, 13, 19). AbiQ was found to cause accumulation of the
replicative form of phage DNA so that it could not be cleaved into the
mature form. AbiB and AbiG were demonstrated to interfere with phage
RNA transcription (26, 27). AbiD1 has been shown to
interact with a phage operon consisting of four genes (1). AbiD1 appears to act in cooperation with Orf1 to decrease the level of
Orf3 to below that required for proper phage development (1). In the presence of AbiA, the appearance of major
capsid protein (MCP) of phage ul36 was delayed compared to that
produced in the sensitive host (24). AbiC reduces the
amount of MCP production (9). Like AbiA, AbiK also
inhibited phage ul36 DNA replication. As a consequence, MCP of phage
ul36 could not be detected in AbiK+ cells. It is
believed that AbiK acts prior to phage DNA replication (11). MCP was produced normally in
AbiQ+ cells, but the lytic cycle of the phage was
blocked after MCP synthesis (12). The mechanistic studies
clearly indicate that the different abi systems act on different phage
targets at various stages of development.
The various abi systems affect the three main species of lactococcal
phages (c2, 936, and P335) differently. Some Abis, such as AbiB, AbiE,
AbiH, and AbiJ, are effective against only one of the three main
species of lactococcal phages. Others, such as AbiC, AbiD, AbiD1, AbiF,
AbiG, AbiI, and AbiL, inhibit propagation of two species of phage. For
AbiA and AbiK, representatives of all three phage species are inhibited.
This study describes the isolation and molecular characterization of a
novel abortive infection phage resistance system, AbiU, from a native
plasmid that was isolated from a phage-resistant industrial strain,
L. lactis LL51-1.
Bacterial strains, plasmids, phages, and media.
The strains
and plasmids used in this study are listed in Table
1. Escherichia coli was grown
at 37°C in Luria-Bertani medium (30). L. lactis was grown at 30°C in M17 medium (31)
supplemented with 0.5% glucose (M17G). For propagation of phages,
calcium chloride (10 mM) was added to M17G medium. For selection and
plasmid maintenance, antibiotics were added to the medium as follows:
for E. coli, 100 µg of spectinomycin per ml; for L. lactis, 500 µg of spectinomycin, 300 IU of nisin, and 500 µg
of streptomycin per ml.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5225-5232.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Molecular Characterization of a New Abortive
Infection System (AbiU) from Lactococcus lactis
LL51-1
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1,
1.0 × 102, and 1.0 × 101,
respectively. abiU involves two open reading frames,
abiU1 (1,772 bp) and abiU2 (1,019 bp).
Evidence indicates that AbiU1 is responsible for phage resistance and
that AbiU2 may downregulate phage resistance against 936 and P335 type
phages but not c2 type phage. AbiU appeared to delay transcription of
both phage 712 and c2, with the effect being more marked on phage c2.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
AbiE, AbiG, AbiL, and AbiRinvolve two genes (6, 7, 13,
26). DNA sequence analysis, Northern hybridization, and reverse
transcription-PCR demonstrated that the two genes of AbiE, AbiG, and
AbiL are cotranscribed (6, 13, 26). AbiR is encoded by two
separate genetic loci (7).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains, plasmids, and phages
Conjugation and transformation. Conjugation was carried out by filter mating (15). L. lactis was transformed by electroporation as described by Powell et al. (28). For E. coli, the CaCl2 transformation method was used as described by Sambrook et al. (30).
Phage techniques. Cross-streaking was employed for the initial screening of phage-resistant and phage-sensitive isolates. Phage preparations were titered by a standard plaque assay (20). The efficiency of plaquing (EOP) was calculated by dividing the phage titer on test strains by that on the sensitive host LM0230. The efficiency of center of infection (ECOI) assays were conducted as described by Deng et al. (6). By convention, it is assumed that 100% of the infected sensitive cells result in progeny phage (ECOI = 1). Cell survival was determined as described by Behnke and Malke (3). The cell survival rate was calculated as the colony count of infected culture divided by colony count of uninfected control. One-step growth curves were carried out by the method of Klaenhammer and Sanozky (20).
Plasmid DNA techniques. Lactococcal plasmid DNA was isolated by the method of Anderson and McKay (2). Plasmid was isolated from E. coli by alkaline lysis and purified by cesium chloride-ethidium bromide density gradient centrifugation (30). Molecular cloning was essentially carried out as described by Sambrook at al. (30). Restriction endonucleases and T4 DNA ligase were purchased from Roche Molecular Biochemicals (Roche Diagnostics Australia Pty., Ltd.) or New England BioLabs (Genesearch, Pty., Ltd.) and used as recommended by the manufacturers.
DNA cloning. Restriction endonuclease, calf intestinal alkaline phosphatase, and T4 DNA ligase were purchased from Roche and used in cloning work according to the manufacturer's instructions. DNA cloning procedures were as described by Sambrook et al. (30). Plasmid pND002 was obtained by shotgun cloning a HindIII digest of pND001 into the HindIII site of pDL278.
Inactivating abiU1 was achieved by digesting pND002 with BamHI, gel purifying the largest fragment, and religation to produce pND003 (see Fig. 3). Plasmid pND002 was double digested with HindIII and ScaI, and the 2.9-kb fragment was gel purified and cloned into the HindIII/ScaI sites of pDL278 to create pND006 (see Fig. 3). The constructs were confirmed by restriction enzyme mapping.Nucleotide sequencing and analysis. Both DNA strands were sequenced by using an Applied Biosystems 377 DNA sequencer according to the manufacturer's protocol. Sequencing of the phage resistance determinant was initiated by using the M13mp19 primers (New England Biolabs). Based on the sequences obtained, 17- to 21-mer oligonucleotide primers were synthesized and used to "walk" along the DNA template. Recording and analysis of the nucleotide sequence were carried out by using AutoAssembler DNA sequence assembly software (Applied Biosystems) and the Australian National Genomic Information Service (ANGIS) Software System operated by ANGIS at the University of Sydney.
Northern blot hybridization. Total RNA was isolated from L. lactis at various times during the phage infection cycle by using the RNeasy Mini Kit (Qiagen, Pty., Ltd.) according to the manufacturer's instructions. RNA preparations were digested with RQ1 DNase (Promega Corp., Sydney, Australia) prior to use. RNA concentrations were determined spectrophotometrically at 260 nm. Northern hybridization was carried out by using the ECL Direct Nucleic Acid Labeling and Detection System (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Total RNA (12 µg) from each sample was loaded onto a 1.2% denaturing gel. Phage DNA probes were isolated by the method of Grosserberger (18) and purified by using the QIAEX II gel extraction kit (Qiagen).
Nucleotide sequence accession number. The GenBank accession number for the DNA sequence of the AbiU abortive infection system from L. Lactis LL51-1 is AF188839.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolation and cloning of the phage resistance determinant.
L. lactis LL51-1 is an industrial cheese starter strain that
shows good phage resistance (
r).
Characterization of this strain also revealed that it contains nine
plasmids (of ca. 75, 60, 50, 45, 27, 10, 9, 8, and 3 kb) and is
sensitive to streptomysin (Sms) and resistant to
nisin (Nisr).
r
Sms) and LM0230Smr
(Niss s
Smr), and transconjugants were obtained at
107 per donor on M17G plates containing
streptomycin and nisin. Nine transconjugants were resistant to phages
c2 and 712, which are representatives of the c2 and 936 lactococcal phage species, respectively. Two of the nine
transconjugants contained a single plasmid of ca. 75 kb, and
HindIII digests of plasmid DNA from both transconjugants produced identical restriction patterns. This 75-kb plasmid was designated pND001. When LM0230(pND001) was infected separately with
c2 and 712, reductions in EOP were observed (3 × 101 and 2 × 104,
respectively), and plaque sizes were reduced relative to LM0230 (Table
2).
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Characterization of the phage resistance mechanism in pND002.
Various assays were conducted to characterize the phage resistance
mechanism encoded by pND002. To investigate whether the phage
resistance phenotype encoded by pND002 was due to the inability of
phage to adsorb to the host cells, adsorption inhibition assays were
carried out by using 712. The results are listed in Table 2. Phage
712 adsorbed efficiently to the sensitive control strain LM0230(pDL278) and to the resistant strains LM0230(pND001) and LM0230(pND002), indicating that the phage resistance encoded by pND001 and pND002 did not affect 712 adsorption.
712 in LM0230(pDL278) and LM0230(pND002) were
conducted at 30°C (Fig. 1). Compared to
the sensitive control strain, LM0230(pDL278), the latent period for
LM0230(pND002) was extended by 5 min (30 versus 25 min). More
importantly, the burst size obtained with LM0230(pND002) was
significantly reduced relative to the control. Furthermore, the size of
the plaques obtained on LM0230(pND002) were much smaller than those
obtained on LM0230(pDL278) (0.3 versus 1.5 mm; Table 2). These results
indicate that pND002 may encode an abortive infection system.
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712 infection was examined (Table 2). Compared to the sensitive
host, the presence of pND002 in LM0230 did not improve the cell
survival rate after 712 infection. The LM0230(pND002) culture,
however, did not lyse, whereas the LM0230(pDL278) culture did. Similar
results were observed after c2 infection (Table 2).
ECOI assays with 712 were performed on both sensitive LM0230(pDL278)
and resistant LM0230(pND002). It was shown that 3.2 × 108 infectious centers were formed on
LM0230(pDL278), while only 5.0 × 106
infectious centers were formed on LM0230(pND002) under the same conditions. Therefore, the ECOI of LM0230(pND002) was 1.6%. This extremely low efficiency of infectious center formation is another typical characteristic of abortive infection systems.
Sequence analysis of the 5.2-kb insert in pND002.
Sequence
data showed that the exact length of the insert in pND002 was 5,213 bp.
Use of the GCG programs Frame and Mapping, available through ANGIS,
predicted the presence of three complete open reading frames (ORFs):
orf1, orf2 (designated abiU1 and
abiU2 after characterization), and orf3 and one
truncated ORF, orf4 (Fig. 3). The sequence of all ORFs,
together with the flanking control regions, is given in Fig.
2. All three complete
ORFs are read in the same direction, while orf4 is in the
opposite direction. abiU1 is 1,766 bp long, starting at bp
1078 with AUG as the start codon and ending at bp 2844, followed by two
stop codons, UAG and UAA. It has a G+C content of 26% and encodes a
predicted 589-amino-acid protein of 67.9 kDa. abiU1 is
preceded by a putative promoter (
35 box [TTGATT], 16-bp spacer,
10 box [ATAAAC]) and a putative ribosome-binding site (GGA;
calculated free energy [
G] = 9.4 kcal) located 5 bp
ahead of the start codon. In addition to the putative transcriptional
signals, two other promoter-like structures (35 and 10 box) were
identified upstream of abiU1. abiU2 is 1,023 bp in
length, starting at bp 3105 with AUG as the start codon and
ending at bp 4127, followed by one stop codon (UAA). It has a G+C
content of 25% and encodes a predicted protein of 40.6 kDa. Upstream
of abiU2 are a consensus 10 box (TATAAT) and a partial
consensus 35 box (TTGAAA) separated by 20 bp. A putative ribosome-binding site (AGGAG) with a G of 12.8 kcal is
located 6 bp upstream of the abiU2 start codon.
orf3 starts at bp 4275 and ends at bp 5012. It is 737 bp
long and has a G+C content of 37%. A consensus 10 region (TATAAT)
was identified 35 bp upstream of this ORF. At 17 bp upstream of this
10 region there is a typical 35 region (TTGAAT). A typical
ribosome-binding site was also identified 4 bp ahead of the ATG start
codon (AGGAGG). No 
-dependent or -independent terminator was
identified between abiU1 and abiU2 or downstream
of abiU2. No significant homology with abiU1 was found at the protein level when searched in NR proteins by BLASTP and
FASTA. No transmembrane regions were detected by using the DAS program
(5), thus suggesting that AbiU1 is a cytoplasmic protein.
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Identification of the ORF(s) that encodes phage resistance in
pND002.
To determine what is responsible for the phage-resistant
phenotype, two deletion plasmids were constructed. abiU1 and
orf4 were deleted, resulting in the pND003 (Fig.
3). To study the phenotype of
abiU1, abiU2 and orf3 were removed by
constructing pND006 (Fig. 3). Cross-streaking with 712 and c2
showed that LM0230(pND003) was sensitive to both phages, but
LM0230(pND006) was resistant to both (Table 2). These results suggest
that abiU1 is the primary phage resistance determinant in
pND002.
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Possible downregulation function of AbiU2 on AbiU1.
The degree
of phage resistance obtained with pND002 and pND006 was compared (Table
2). With 712, the EOP obtained on LM0230(pND006) was much lower than
that obtained with LM0230(pND002) (104 versus
102). When the same
experiment was repeated with c2, however, there was very little
difference observed between the EOPs obtained with LM0230(pND006) and
LM0230(pND002) (Table 2).
ul36. Plasmids pND002 and pND006, as
well as the vector pDL278, were electroporated into L. lactis UL8, which is the host for ul36. A similar phenomenon to
that reported with 712 was observed: the EOP obtained with UL8(pND006) was 101 lower than that
obtained with UL8(pND002) (Table 2). It should also be noted that
the sizes of the ul36 plaques on UL8(pND006) were smaller than those
obtained on UL8(pND002). The cell survival rate after ul36
infection of the two hosts UL8(pND002) and UL8(pND006) was similar to
the sensitive host UL8(pDL278), further indicating that abiU
is an abortive infection system.
The data suggest that this abortive infection system acts differently
on phage from different species. The presence of abiU1 in
pND006 confers a low EOP against all phage species. In comparison to
pND006, the presence of the complete 5.2-kb fragment in pND002 actually
reduces phage resistance against 936 and P335 species but does not
affect the phage resistance observed with c2 (Table 2). The presence
of the sequence downstream of abiU1 appears to downregulate
phage resistance in pND002. Given the homology of the AbiU2 protein to
AbiGii, it is tempting to speculate that this protein may be
responsible for this effect.
Effect of pND002 on phage DNA transcription.
To study the
effect on phage 712 transcription, total RNA was isolated from
LM0230(pDL278), LM0230(pND002) and LM0230(pND006) at 10-min
intervals after 712 infection and Northern blotted. The blot was
probed with labeled genomic DNA from 712 (Fig.
4). Transcripts of 712 first appeared
10 min after infection of strains LM0230(pDL278) and
LM0230(pND002). Transcripts were first detected in
LM0230(pND006) 30 min after infection, and the degree of
transcription increased by 40 min. Plasmids pND002 and pND006 appear to
delay phage transcription relative to the control strain.
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c2 RNA transcription was also investigated.
Samples were withdrawn 0, 45, and 75 min after c2 infection and
analyzed by Northern hybridization by using labeled genomic DNA of
c2 (Fig. 5). Phage c2 transcripts were
detected 45 and 75 min after infection in the sensitive host
LM0230(pDL278) but not in the resistant host LM0230(pND006).
Apparently, the presence of pND006 significantly reduced or delayed
c2 RNA synthesis.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, aspects of the molecular characterization of a
lactococcal phage abortive infection system (AbiU) have been described.
AbiU was isolated from the plasmid pND001 found in the industrial
strain L. lactis LL51-1. pND001 conferred resistance to
small isometric-headed 712 (936 species) and prolate-headed c2
(c2 species). EOP tests of LM0230(pND001) and LM0230(pND002) on
c2 showed a similar level of resistance from both plasmids. However,
EOP values of LM0230(pND001) to 712 were 2 orders of magnitude less
than that of LM0230(pND002) (Table 2). This suggests that another
mechanism of phage resistance against 712 exists or that the
existing mechanism is enhanced on pND001 but not on pND002.
The phage resistance phenotype conferred by pND002 was characterized.
Plasmid pND002 conferred reduced plaque size (Table 2), EOP (Table 2),
burst size (Fig. 1), and ECOI against 712. Plasmid pND002 conferred
reduced plaque size and EOP against c2 and ul36; this plasmid
prevented lysis of the host but conferred a very low survival rate
against all three phage tested. These observations are typical of those
observed with abortive infection systems.
Analysis of the 5.2-kb fragment in pND002 revealed the presence of
three complete ORFs and one incomplete ORF. Cloning experiment were
used to determine that abiU1 is directly involved in phage resistance. Comparison of the EOPs obtained with pND002 and pND006 against phages 712 and ul36 suggest that sequences downstream of
abiU1 interfere with or downregulate phage resistance. Like other abi genes (12), both abiU1 and
abiU2 have low G+C contents of 26 and 25%, respectively. In
contrast, the G+C content of the truncated orf4 is 32%, and
that of orf3 is 37%, values which are close to the average
G+C content of lactococcal chromosome. The homology of the AbiU2 and
AbiGii proteins adds support to the hypothesis that abiU2 is
involved in phage resistance. Stronger resistance to 712 and ul36
was conferred by pND006 relative to pND002, suggesting that
abiU2 or its protein might be involved in abiU1
or AbiU1 downregulation (Table 2). This downregulation function of
abiU2 on abiU1 seems to affect resistance against only isometric-headed phage from the 936 and P335 species and has no
effect on prolate-headed phage from the c2 species.
The possible downregulation function of AbiU2 on AbiU1 resembles the
negative control of PifC on pif gene expression in F exclusion of phage T7 in E. coli cells (23).
The Pif system, located on the F plasmid in E. coli, is one
of the abi systems identified in E. coli and confers host
resistance to phage T7 infection. The pif region contains at
least three genespifC, pifA, and
pifB (29)in which pifA encodes
phage resistance. It is known that pifA and pifC
lie within a polycistronic operon (4) and that the
promoter is upstream of pifC. Construction of fusion
proteins of PifA-LacZ, PifB-LacZ, and PifC-LacZ showed that
pifC expression is autoregulated. PifC, in trans,
significantly decreases the level of 
-galactosidase activity
produced by PifA-LacZ, PifB-LacZ, and PifC-LacZ. In addition,
inactivating pifC in cis dramatically increased
the resistance to T7 conferred by PifA, just as deleting
abiU2 increases the phage resistance conferred by AbiU1.
Interestingly, PifC is a 40-kDa protein (23), which is
similar in size to the predicted 40.6 kDa of AbiU2, whereas PifA is a
70-kDa protein (29), a size similar to the predicted 67.9 kDa of AbiU1. The DNA of the pif region has not been
sequenced to allow a more detailed comparison.
One important step in understanding the mechanism of an abortive
infection system is to locate the stage at which it inhibits the phage
life cycle. Detecting phage mRNA transcription by Northern hybridization demonstrated that AbiU appeared to delay transcription of
both c2 and 712. The effect was more extreme for c2 (Fig. 4
and 5). However, the Northern hybridization signals that were observed
were not discrete. Likewise, O'Connor et al. also observed similar
signals when studying AbiG and sk1 (26). These authors tentatively explained it as a result of nonspecific processing of
transcripts, transcript degradation, the presence of fragments of
increasing size with a common 5' end, or some combination of these factors.
Among the three lactococcal mechanisms reported to inhibit or delay phage transcription (16, 26, 27), AbiU resembles most closely pBu1-8 (16). However, since pBu1-8 is a native plasmid and the phage resistance determinant was not subcloned, it is not known whether other phage resistance systems exist on pBu1-8 that may provide additional phage resistance. On the basis of sequence homology, AbiU appears related to AbiG, which is part of another mechanism that interferes with phage transcription.
The phage resistance spectrum of an abi system is varied. Most abi systems confer resistance to one or two phage species, whereas only AbiA and AbiK have been reported to confer resistance to three species of phage: c2, 936, and p335 (11, 19). AbiU is the third abi system that encodes resistance against representative phages from all three of these phage species.
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ACKNOWLEDGMENTS |
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This work was supported by the Australian Cooperative Research Center for Food Industry Innovation and by DSM Food Specialties, Australia.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biotechnology, University of New South Wales, Sydney, NSW 2052, Australia. Phone: 61-2-93853868. Fax: 61-2-93136710. E-mail: p.su{at}unsw.edu.au.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, November 2001, p. 5225-5232, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5225-5232.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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