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Applied and Environmental Microbiology, August 2001, p. 3564-3576, Vol. 67, No. 8
Russell Grimwade School of
Biochemistry and Molecular Biology, The University of
Melbourne, Parkville, Victoria 3010,1 and
Food Science Australia2 and
Department of Food Science and Agribusiness, The University
of Melbourne,3 Werribee, Victoria 3030, Australia
Received 8 December 2000/Accepted 20 April 2001
The Lactococcus lactis temperate bacteriophage
BK5-T is one of twelve type phages that define L. lactis phage species. This paper describes the nucleotide
sequence and analysis of a 21-kbp region of the BK5-T genome and
completes the nucleotide sequence of the genome of this phage. The
40,003-nucleotide linear genome encodes 63 open reading frames.
Sequence runoff experiments showed that the cohesive ends of the BK5-T
genome contained a 12-bp 3' single-stranded overhang with the
sequence 5'-CACACACATAGG-3'. Two major BK5-T
structural proteins, of approximately 30 and 20 kDa, were
identified, and N-terminal sequence analysis determined that they were
encoded by orf7 and orf12, respectively. A
169-bp fragment containing a 37-bp direct repeat and several smaller repeat sequences conferred resistance to BK5-T infection when introduced in trans to the host cell and is likely a part
of the BK5-T origin of replication (ori).
Lactic acid bacteria are used
extensively as starter cultures in the dairy industry. The bacteria
ferment lactose to lactic acid, a crucial process in cheese
manufacture. One of the major microbiological problems faced by
the dairy fermentation industry is the susceptibility of the starter
bacteria to bacteriophage infection. Such infections result in lysis of
starter cultures and failure of the fermentation. Since reports
of bacteriophage infection of starter strains as early as 1935 (64), an increasing number of bacteriophages have been
identified and considerable research has been conducted to improve our
understanding of these viruses and their interaction with their hosts.
BK5-T is a temperate bacteriophage with a small isometric head and a
noncontractile tail of 232 nm in length (24) first isolated from Lactococcus lactis subsp. cremoris
BK5 (23). Boyce et al. (8) determined the
nucleotide sequence of approximately 19 kbp of the BK5-T genome, which
defined the EcoRI restriction fragments
EcoRI-a and EcoRI-b (32). Thirty-two
open reading frames (ORFs) were identified, and the predicted
amino acid sequences encoded by several of these ORFs demonstrated
significant homology with sequences available in protein databases.
Analysis of the partial genome sequence also identified a putative
BK5-T immunity region containing regulatory elements involved in
determination of the phage lysis-or-lysogeny decision (7).
This report describes the nucleotide sequence of the remaining 21 kbp
of the BK5-T genome and characterizes the phage termini, major
structural proteins, and the putative origin of replication.
Bacterial strains and media.
The strains and plasmids used
in this investigation are listed in Table
1. Escherichia coli strain
JM107 was incubated at 37°C with shaking in 2TY medium
(42). The BK5-T indicator strain L. lactis
subsp. cremoris H2 was grown at 30°C for 16 h in M17 medium supplemented with 0.5% (wt/vol) glucose (M17G)
(57). When necessary, the antibiotic erythromycin (2.5 µg/ml for L. lactis or 200 µg/ml for E. coli)
or ampicillin (150 µg/ml for E. coli) was included in the
media.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3564-3576.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Sequence Analysis and Molecular
Characterization of the Lactococcus lactis Temperate
Bacteriophage BK5-T
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Strains and plasmids used in this investigation
Phage preparation.
Bacteriophage BK5-T was propagated by
lytic infection of L. lactis H2 in M17G medium with 10 mM
CaCl2. The bacteriophage were precipitated with 1 M NaCl
and 10% (wt/vol) polyethylene glycol (PEG), and when necessary,
further purified and concentrated by CsCl density gradient
centrifugation (49). Phage DNA was isolated from the
PEG-precipitated phage by the procedure described for coliphage 
by
Qiagen (Qiagen, GmbH, Hilden, Germany).
N-terminal amino acid sequencing of phage proteins.
BK5-T
phage purified by CsCl density gradient centrifugation
(49) were heated at 95°C for 15 min in cracking buffer
(50 mM Tris-HCl [pH 6.8], 1% [wt/vol] sodium dodocyl sulfate
[SDS], 2 mM EDTA, 10% [vol/vol] glycerol, 1% [vol/vol]
-mercaptoethanol) and separated by SDS-polyacrylamide gel
electrophoresis (PAGE) on a precast Novex (Amrad Biotech, Victoria,
Australia) 4 to 20% acrylamide Tris-glycine gel. The denatured
proteins were transferred by electroblotting to a Bio-Rad
polyvinylidene difluoride membrane in a buffer containing 10 mM
3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) and 20%
(vol/vol) methanol. The membrane was stained with 0.1% (wt/vol)
Ponceau S containing 1% (vol/vol) glacial acetic acid, the phage
protein bands were excised, and their N-terminal amino acid sequences
were determined by the Australian Proteome Analysis Facility (Macquarie
University, Sydney, Australia).
Nucleotide sequencing. The BK5-T EcoRI restriction fragments EcoRI-f, EcoRI-d, and EcoRI-g, which had previously been cloned in pACYC184 by Lakshmidevi et al. (33), were subcloned into the E. coli vector pGEM-3Zf (+) (Table 1). EcoRI-f and EcoRI-d were subcloned directly in both orientations and EcoRI-g was digested with HindIII and the five fragments generated were individually cloned in both orientations into pGEM-3Zf (+). All the clones were subjected to exonuclease III treatment and a series of subclones containing deleted fragments was used for sequencing templates. Plasmid DNA for sequencing was purified by the Qiagen miniprep procedure (Qiagen), followed by PEG precipitation as described by the Applied Biosystems (ABI) Taq DyeDeoxy terminator cycle sequencing kit protocol. Determination of the nucleotide sequence was conducted as outlined in the ABI sequencing manual. The sequencing reaction products were analyzed on an ABI model 373A automated sequencer.
The nucleotide sequence across junction points between clones was determined by chromosome walking, using synthesized oligonucleotides and purified BK5-T phage DNA as template. Assembly of the nucleotide sequence was conducted using Sequencher software (Sequencher 3.0, Gene Codes Corporation, Ann Arbor, Mich.). Computer analyses and database searches were conducted using programs available at the Australian National Genomic Information Service.Sequence runoff experiments. Two oligonucleotides, COS1 (5'-GACCATCATGGATAACTTGGC-3') and COS2 (5'-CGCCAACAAGCACTTGCGAG-3'), were designed approximately 200 bp from either end of the linear BK5-T genome. These oligonucleotides were used for linear amplification of DNA using three different preparations of purified unligated BK5-T phage DNA as sequencing template, and the nucleotide sequences of the amplicons were determined as described above.
Nucleotide sequence accession number. The sequence reported here is available in GenBank under accession no. AF176025.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Complete nucleotide sequence of BK5-T. Boyce et al. (8) determined the nucleotide sequence of 18,935 bp of the BK5-T genome, and the remaining 21 kbp is reported here. The total circular length of the BK5-T genome was determined to be 40,003 bp. The codon usage of the BK5-T ORFs was determined and found to correlate with the codon usage of the host lactococcal genome.
Analysis of BK5-T ORFs.
A schematic diagram of the BK5-T ORFs
is shown in Fig. 1. The BK5-T ORFs were
numbered sequentially along the genome, as described for other phages
(10, 26, 27, 55), in contrast to the previous nomenclature
of Boyce et al. (8), which was based on the number of
codons in the ORF (Table
2).
A total of 63 ORFs which met the following criteria were identified:
(i) the ORF contained greater than 40 codons; (ii) the ORF was
preceded by an identifiable ribosomal binding site (RBS) (4 to 12 nucleotides from the putative start codon) or was likely to be
translationally coupled to the preceding ORF; and (iii) the ORF began
with either an AUG, GUG, AUA, or UUG codon. In addition, several
ORFs that had previously been identified by Boyce et al.
(8) that did not meet all of the above criteria were
included for consistency.
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(i) ORF1 and ORF2.
The predicted size of the gene products
encoded by orf1 and orf2 and the location of
these genes immediately downstream from the cos site are
indicative of the terminase subunits of other phages, including
coliphage (4), Streptococcus thermophilus phages 
7201, DT1, Sfi19, and Sfi21 (16), and
L. lactis phage sk1 (10). Terminase subunits
form a hetero-oligomeric complex which functions in concert to bind and
nick DNA at the cos site prior to packaging of the DNA
concatamers into phage heads. Terminase activity is an ATP-dependent
process, and an A-type Walker nucleoside triphosphate-binding motif
(62) was identified between residues 49 and 53 in the
putative BK5-T large terminase subunit ORF2. No such motif was detected
in ORF1. In addition to homology with streptococcal phages (Table 2),
BK5-T ORF1 exhibited homology to putative packaging protein GP3 found
in three Salmonella enterica serovar Typhimurium
bacteriophages belonging to the Podoviridae family. Although the
homology between the two proteins is low (23% over 163 amino acids),
it suggests an evolutionary link between two quite distantly related
phage species.
(ii) ORF5.
The amino acid sequence of ORF5 showed 56 to 57%
identity to four uncharacterized streptococcal phage proteins
(16, 39, 58), 24% identity to ORF4 of
Staphylococcus aureus PVL (26), and 21%
identity with GP34 of Streptomyces actinophage phage C31 (54). The last two proteins showed sequence homology to
the experimentally determined portal protein of coliphage HK97
(19). Portal proteins create a multisubunit ring structure
that serves as an entry point for the translocation of DNA into the
phage head (3).
(iii) ORF6. ORF6 shares homology with phage proteins of unknown function from S. thermophilus, Lactobacillus gasseri, and Pseudomonas aeruginosa (Table 2) and exhibits homology to a series of endopeptidase ClpP proteins encoded in the genome of E. coli, plants, and higher eukaryotes, but not to the ClpP protein from L. lactis (22). The highly conserved Ser85 and His108 and surrounding residues, which form the proteolytic cleavage site in ClpP protein sequences, were conserved in both BK5-T ORF6 and the L. lactis ClpP protein.
(iv) ORF7.
The deduced amino acid sequence of ORF7
exhibited significant homology to major structural proteins from
four S. thermophilus phages, Bacillus subtilis
phage 105 (ORF27), Lactobacillus casei A2, P. aeruginosa D3, and S. aureus phage PVL (ORF7)
(Table 2). The BK5-T orf7 encodes a major structural
protein (see below).
(v) ORF12.
The predicted amino acid sequence of ORF12 showed
approximately 45% identity with ORFs of unknown function from S. thermophilus phages Sfi19 (ORF203) and Sfi21 (ORF202)
(15) and with an experimentally determined small major
structural protein from S. thermophilus phage 7201
(34). The gene product of BK5-T orf12 is a
major structural protein (see below).
(vi) ORF25, ORF26, and ORF27.
The topological positions of
orf25, orf26, and orf27 are similar to the
arrangement of the holin and lysin cassette observed in prophage-like
elements found in the chromosome of many Bacillus strains
(29) and in bacteriophages infecting S. thermophilus (15, 40, 52). The predicted amino acid
sequence of ORF25 demonstrates 28% identity to the Clostridium
acetobutylicum ORF2 of unknown function, which is found upstream
from the lyc gene, whose gene product exhibits sequence
homology to a number of autolytic lysozymes (13). The
amino acid sequence of ORF26 shows homology to the putative holin
proteins from S. aureus phage PVL (26), B. subtilis phage SPP1, and experimentally determined holin
proteins from Bacillus licheniformis (30, 44)
(Table 2). The BK5-T ORF25 and ORF26 are therefore likely to encode a
two-component holin system similar to that suggested for phages
infecting S. thermophilus (15, 40, 52). Holin
proteins function to disrupt the cell membrane to allow the lysin
protein access to the cell wall (67).
31, 73% homology to the
N-acetylmuramoyl-L-alanine amidase (Pal) of
pneumococcal phage Dp-1 (51), and 30% identity to
putative lysin proteins of S. thermophilus phages (Table 2), suggesting that ORF27 encodes the BK5-T lysin.
(vii) ORF32. Boyce et al. (9) showed that the gene product encoded by orf32 exhibited significant homology to a number of lactococcal temperate phage integrase proteins and that it was essential for the establishment and/or maintenance of lysogeny in BK5-T.
(viii) ORF35.
Boyce et al. (7) identified a
helix-turn-helix DNA binding motif (amino acid positions 33 to 54) as
predicted by the Dodd and Egan algorithm (18) and a
putative RecA Ala-Gly cleavage site in ORF35. ORF35 showed homology to
two putative cI repressor proteins from the lactococcal phages Tuc2009
and LC3 and to an experimentally determined cI homologue
from phage rlt (Table 2), suggesting that orf35 encoded the
BK5-T cI repressor homologue. It was predicted that ORF35
facilitated lysogenic development by repressing a putative BK5-T
promoter, P2, found in the putative BK5-T immunity region
(7).
(ix) ORF36.
orf36 was previously proposed to encode
the BK5-T Cro protein homologue, based on its size and position
relative to the putative BK5-T immunity region (7).
However, no helix-turn-helix DNA binding motif was identified in ORF36,
and preliminary data (Mahanivong, unpublished data) suggested that
ORF36 did not bind to the putative BK5-T immunity region. ORF36 shares
strong identity (93% over 55 amino acids) with lactococcal phage TPW22
ORF9 (47) and 52% identity over 54 amino acids with the
uncharacterized ORF8 from lactococcal phage rlt (60),
ORF57 from 31.1 (20), and ORF9 from TP901-1. It is
interesting that the relative position of BK5-T ORF36 is dissimilar to
the homologues from rlt and TP901-1 (Fig.
2), suggesting a horizontal introduction
of the ORF into the genome or a genetic rearrangement event.
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(x) ORF37. orf37 is located 231 bp downstream of orf36. Analysis of the predicted amino acid sequence of ORF37 identified a putative helix-turn-helix DNA binding motif at amino acid positions 23 to 42. The predicted amino acid sequence of ORF37 demonstrates 30% identity to the Cro homologue from temperate streptococcal phage TPJ34 (ORF67) and ORF39 from lytic streptococcal phage DT1 (Table 2). Preliminary DNA binding studies (Mahanivong, unpublished data) suggest that ORF37 binds to the putative BK5-T immunity region and that it is likely to be the BK5-T Cro homologue.
(xi) ORF38. The identification of a putative helix-turn-helix DNA binding motif (18) at amino acid positions 174 to 193 suggests that orf38 encodes a DNA binding protein. The predicted amino acid sequence of ORF38 shows 97% identity to ORF5 of L. lactis phage rlt (60) and both genes are located in the same relative position immediately downstream of their Cro protein homologues (Fig. 2). BK5-T ORF38 also shows 50% identity to L. casei phage A2 ORFA, which is located immediately downstream of its cI homologue (31).
(xii) ORF48.
The predicted amino acid sequence of ORF48
exhibits limited homology with the putative single-stranded DNA binding
(SSB) proteins encoded by B. subtilis, S. aureus phage
PVL, and B. subtilis phage SPP1 and with ORFs of unknown
function from L. lactis phages sk1 and bIL170 (Table 2). SSB
proteins are a ubiquitous class of proteins identified in prokaryotic
and eukaryotic organisms. They function primarily to bind
single-stranded DNA and play important roles in DNA replication,
recombination, and repair. A number of SSB proteins have been
characterized and some functionally relevant structural features have
been identified. There are aromatic amino acid residues in the N
terminus of E. coli and mitochondrial SSB proteins
(36) that are important for binding single-stranded DNA,
but these residues are not observed in BK5-T ORF48. The greatest region of homology between BK5-T ORF48 and the SSB protein
sequences occurs at the acidic C-terminal end of the proteins. The
C-terminal six amino acids (DEDLPF) of BK5-T ORF48 were compared with
protein databases and showed matches to other putative SSB proteins
of bacterial (B. subtilis, E. coli, L. lactis, and
Thermus aquaticus) or phage (including Tuc2009,
adh, SPP1, and 105) origin (data not shown). The functional
relevance of this acidic domain is unknown; however, the
loss of this domain from the E. coli SSB protein
(14) results in a nonfunctional protein.
(xiii) ORF49.
ORF49 contains a helix-turn-helix region (Fig.
3A), suggesting that it is a DNA binding
protein, and its nucleotide sequence contains a series of direct and
indirect repeats, which are often indicative of a phage origin of
replication (10, 21). The amino acid sequence of BK5-T
ORF49 shows 82% identity to ORF269 in the recombinant lytic
lactococcal phage 31.1 (20) and 80% identity to ORF235
of lactococcal phage ul36.1 (6) (Table 2). Mutant phage
31.1 was isolated after 31 infection of cells expressing a phage
resistance phenotype (Per) due to a plasmid-encoded 31 ori. Phage 31.1 overcame the resistance by
acquiring 7.8 kbp of DNA from the host chromosome, which
contained an alternative phage ori. The ori
was postulated to be contained within ORF269. Mutant phage ul36.1 arose
as a variant resistant to the phage resistance mechanism AbiK and had
incorporated noncontiguous sections of the host chromosome
DNA, one of which contained ORF235, also postulated to contain a phage
ori.
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(xiv) ORF53.
The deduced amino acid sequence of ORF53
demonstrates significant homology with ORF20 and ORF139 of L. lactis phages rlt (60) and 31.1 (20),
respectively. Furthermore, it shows homology to dUTPase enzymes of
bacterial and eukaryotic origin. The function of this enzyme,
which hydrolyzes dUTP to dUMP and pyrophosphate, is to regulate
intracellular levels of dUTP and to prevent incorporation of dUTP into
DNA (53). The absence of dUTPase in E. coli
leads to an increased recombination and mutation rate during DNA
replication (59).
(xv) ORF55.
ORF55 did not show any significant homology with
other protein sequences in the databases and its function is unknown.
Interestingly, orf55 is located in the opposite orientation
to the surrounding ORFs and is preceded by a putative RBS
(AGAGGAA) and promoter (
10 [TATAAT] and 35 [TTCAAT])
located 11 and 25 nucleotides upstream from the ATG start codon,
respectively. The codon usage of ORF55 is similar to that of the
rest of the phage. A region of dyad symmetry is located 9 bp downstream
of the stop codon of orf55 but is surrounded by a string
of A nucleotides rather than the T nucleotides indicative of a
rho-independent transcription terminator. The location and orientation
of this ORF have not been reported in the genomes of other
characterized lactococcal temperate phages (26, 27, 60).
Located within ORF55, but oriented in the opposite direction, is
another putative promoter (10 [TATAAT] and 35 [ATGTTC]),
which could direct transcription through orf56 and
downstream genes. The arrangement of oppositely oriented putative
promoters within and upstream of orf55 would generate
transcripts with overlapping and complementary 5' ends. Similar
overlapping transcripts are observed in the oop region (28), and this region may represent a control point in the
BK5-T gene expression.
Determination of the sequence of the BK5-T cos termini. BK5-T had previously been demonstrated to contain cohesive termini (8). Sequence runoff experiments were conducted on purified phage DNA to determine the DNA sequence of cosN, which defines the single-stranded overhang. COS1 and COS2 were used as primers to determine the sequence of ligated and unligated cos ends. The nucleotide sequences obtained from unligated phage DNA and ligated DNA were compared, and the absence of a 12-bp sequence in the unligated preparations indicated that BK5-T contained a 3' overhang of 12 bp with the sequence 5'-CACACACATAGG-3'. Thus, the BK5-T cos site, like those of all other phages infecting gram-positive organisms studied to date, possesses a single-stranded 3' overhang (11, 26, 35, 60).
N-terminal sequence of phage structural proteins.
BK5-T phage
was purified by CsCl density gradient centrifugation, and the proteins
were analyzed by SDS-PAGE. Two major bands, with molecular masses of 20 and 30 kDa, and several bands of lower molecular mass were observed
(Fig. 4). The proteins were transferred to a polyvinylidene difluoride membrane and the N-terminal sequence of
the 20- and 30-kDa proteins was determined. The first six amino acids
of the 30-kDa protein were TVSSKT. This sequence was identical to
residues 110 to 115 of the BK5-T ORF7. The absence of the first 109 amino acids suggests that ORF7 is cleaved prior to the formation of the
mature protein. The estimated molecular mass of the full length ORF7 is
45 kDa, whereas a 32-kDa protein is predicted for the mature ORF7
protein, which is consistent with observed experimental results (Fig.
4).
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adh (Table 2).
Identification of the putative BK5-T origin of replication.
Comparison of the genome organization of BK5-T with that of other
lactococcal bacteriophages (41) suggested that the BK5-T ori was likely to be located in orf49. ORF49
contains a helix-turn-helix DNA binding motif (amino acid positions 47 to 66) (Fig. 3A) and may bind to the various repeat sequences within
orf49 as has been observed in coliphage (48) and phage Tuc2009 (41). The genetic arrangement of the BK5-T orf48 and orf49,
encoding a putative SSB protein and a replication protein,
respectively, is similar to that of phage Tuc2009 (41),
but there is no sequence similarity between the analogous proteins.
5). Control strains containing pTRKH2 with no insert or
an insert of BK5-T DNA from the structural region were sensitive to
phage infection (EOP = 1).
A number of direct and inverted repeat sequences were identified within
orf49 (Fig. 3B) and these sequences are likely to define the
BK5-T ori. A 306-bp PCR product containing this region was
cloned into pTRKH2 in both orientations (plasmids pCM30 and pCM31) and
shown to confer a similar level of phage sensitivity to the clone
containing the complete orf49 sequence (Fig.
5). Exonuclease III deletion constructs
were generated to further define the region required to confer
resistance to BK5-T infection. Removal of the first 10 bp of the 5' end
of the DR1 sequence in clone pCM30.4 caused a 4-log decrease in phage
resistance, indicating that the DR1 sequence was necessary to effect
phage resistance.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The nucleotide sequence of the temperate lactococcal bacteriophage BK5-T was completely determined and enabled comparative genomic sequence analysis. Molecular characterization of the phage structural proteins, cos termini, and replication functions were conducted.
The BK5-T 12-nucleotide single-stranded cosN sequence was
determined. In numerous other phages, the region surrounding
cosN contains recognition sequences for the binding of
terminase (cosB), integration host factor, and other host-
or phage-encoded proteins. In phage , there are three direct repeat
sequences in cosB, referred to as R sites, which have been
shown to be important for terminase binding (4).
Similar repeat sequences were identified in phage sk1
(11) and P. aeruginosa phage D3
(50). Although no such repeat sequences were found in the
BK5-T genome, it does contain the sequence
C3TC5 located 20 nucleotides 5' of
cosN and a string of 7 consecutive G nucleotides occurs 43 nucleotides 3' of cosN (Fig.
6). S. thermophilus phage
7201 contains the sequence C5GC5 16 bases 3'
of cosN, and Sfi19 and Sfi21 both contain the sequence C5GC4 21 nucleotides 5' of cosN
(39). L. lactis phage sk1 also contains a
string of 8 consecutive C nucleotides found 30 bases 5' of
cosN (11). The occurrence of such sequences in
these low-GC organisms is unusual and these sequences may constitute
recognition sites for terminase binding and activity, although this
remains to be determined. A 130-bp region surrounding BK5-T
cosN contains a number of runs of four to seven identical
bases, primarily A or T (Fig. 6). Similarly, the cos site of
coliphage contains runs of adenines and thymines that are important
to DNA bending which occurs upon integration host factor binding
(66).
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BK5-T orf7 and orf12, which encode the putative
major head and tail proteins, were identified. N-terminal amino acid
sequence analysis indicated that the BK5-T putative major capsid
protein was processed to generate the mature protein. Similar
proteolytic cleavage was observed in the mature capsid proteins
of a number of phages, including PVL (26) and
L. gasseri adh (1). BK5-T orf5, orf6, and orf7 encode gene products that
are involved with phage head structure and assembly and are likely to
have been acquired as a functional unit from a common ancestral phage
(54). They encode the putative portal, ClpP protease, and
major capsid proteins, respectively, and show identical arrangement to
three functionally equivalent genes found in four phages infecting
P. aeruginosa (50), S. aureus
(26), S. actinophage (54), and E. coli (19). It is tempting to speculate that
the putative BK5-T ClpP protease (ORF6) may be involved in the cleavage
of ORF7, as observed in coliphage HK97, where the phage-encoded GP4 is
a protease that cleaves the N-terminal 102 amino acids from the major
capsid protein encoded by the adjacent downstream gene (19).
Investigations of the BK5-T replication functions identified a
repeat-rich region within orf49 which conferred phage
resistance in a manner suggestive of a phage replication origin.
However, the specific function of the repeat sequences in the
replication process remains to be determined. ORF49 showed significant
homology with ORF269 and ORF235 from lactococcal phages 31.1
(20) and ul36.1 (6). Cloned DNA fragments
containing these latter ORFs also conferred resistance to their
respective phages (6). These ORF homologues are likely to
encode replication proteins which bind to the repeat regions in their
coding sequences to initiate phage DNA replication in a similar manner
to the coliphage O replication protein (56). The
greatest region of sequence diversity between BK5-T and its homologues
occurs at amino acid positions 40 to 120 (Fig. 3A). This region
contains the putative helix-turn-helix DNA binding motif (amino acid
positions 45 to 66) and DR1, a possible binding site on the DNA.
Interestingly, the nucleotide sequences of the other repeat structures
downstream from DR1 were identical (Fig. 3B). The variation in amino
acid sequence in the helix-turn-helix motif could indicate different
DNA targets for the homologues, as represented by the differences in
nucleotide sequence in the region of DR1.
The organization and orientation of the ORFs in BK5-T were similar to those observed in other temperate phages infecting lactic acid bacteria (1, 27, 39, 55, 60). The majority of the ORFs are oriented in one direction, while ORFs involved in lysogeny (ORFs 32, 33, and 35) and possibly regulation (ORF43 and ORF55) are oriented in the opposite direction. Moreover, organization of functional modules within BK5-T revealed a striking correlation with the pattern of functional modules observed in the genomes of many Siphoviridae phages (26, 38, 39, 58, 61), viz. packaging, structure and morphogenesis, lysis, integration, lysogeny (in temperate phages), and replication. A detailed comparison of the genetic organization of BK5-T compared with other phages is presented elsewhere (17).
Examination of the BK5-T genome allowed comparative analyses of genomic exchange at three levels: functional modules, individual genes, and gene segments. The strong conservation of the genetic organization, ORF sequence, and gene sequence of the packaging and morphogenesis modules (ORF63 to ORF18) observed between BK5-T and the streptococcal phages (15, 17) suggests recent divergence of these modules from a common ancestor. In contrast, ORF30 to ORF61 showed greater identity with phages infecting lactococci. This latter region encompasses the integration, lysogeny, and replication modules. This structure suggests that BK5-T is a recently evolved chimeric phage containing modules that are derived from two distinct ancestral phages. Within modules, differences were also observed in the relative location of homologous ORFs. The BK5-T orf36 was located upstream of the putative BK5-T orf37 cro homologue (Fig. 2). In contrast, the ORF36 homologues from rlt and TP901-1 were both located four ORFs downstream from their corresponding cro gene homologues. Also the BK5-T orf63 was located 5' of the cos site, whereas the homologous ORFs from the skI and bIL170 were located 3' of their respective large terminase subunits.
The acquisition or deletion of entire ORFs was also evident. The BK5-T ORF14 homologue was absent in the streptococcal phages, indicating that it was either introduced into the BK5-T genome via a nonhomologous event or lost from the streptococcal phage genomes since their divergence. The BK5-T orf56 is surrounded by a 30-bp direct repeat sequence, which could facilitate its acquisition or deletion by a specific recombination event. ORF56 showed significant homology to ORFs from lactococcal phages sk1 and bIL170 (Table 2); however, no repeat sequences were found surrounding the ORFs in these phages. A single copy of the 30-bp direct repeat in rlt may be the remnant of an ORF deletion by recombination occurring between the repeat sequences. Examples of ORF deletions within flanking direct repeat sequences were also observed between phage bIL67 and c2 (37).
Evolution within ORF sequences was evident, as indicated by the conservation of functional domains linked to regions that show no apparent homology. Comparison of the amino acid sequence of the BK5-T cI homologue ORF35 indicates that the C-terminal domain had greater than 94% identity with the homologous proteins from lactococcal phages Tuc2009 and rlt. In contrast, there was negligible homology in the N-terminal domain of these proteins. The N-terminal domain contains the putative DNA binding motif and this divergence presumably accommodates phage-specific DNA recognition sequences.
As more sequence data on a variety of bacteriophage genomes becomes available, there is much focus on the genomic evolution and relationships between phages. These data suggest that phages have evolved by exchange of functional modules, individual genes, or gene segments by various genetic recombination events (5, 38).
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ACKNOWLEDGMENTS |
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We acknowledge Sean Moore for his technical assistance and Scott Chandry for his helpful advice and discussions.
C.M. gratefully acknowledges receipt of a Melbourne Research Scholarship.
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FOOTNOTES |
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* Corresponding author. Mailing address: Food Science Australia, Private Bag 16, Werribee, Victoria 3030, Australia. Phone: 61 3 9731 3268. Fax: 61 3 9731 3254. E-mail: alan.hillier{at}foodscience.afisc.csiro.au.
This report is dedicated to the memory of Barrie E. Davidson, who
passed away in July 2000.
Present address: Department of Microbiology, Monash University,
Clayton, Victoria 3800, Australia.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, August 2001, p. 3564-3576, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3564-3576.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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