Transcriptional regulation and immunity in mycobacteriophage Bxb1: Mycobacteriophage Bxb1 gene...

Molecular Microbiology (2000) 38(5), 971±985

Transcriptional regulation and immunity inmycobacteriophage Bxb1

Shruti Jain and Graham F. Hatfull*

Department of Biological Sciences, University of

Pittsburgh, Pittsburgh, PA 15260, USA.

Summary

Mycobacteriophage Bxb1 is a temperate phage of

Mycobacterium smegmatis that shares a similar

genome organization to mycobacteriophage L5,

although the two phages are heteroimmune. We

have investigated the regulatory circuitry of Bxb1

and found that it encodes a repressor, gp69, which

regulates at least two promoters, an early lytic

promoter, Pleft, and the divergent promoter, Pright.

Bxb1 gp69 is 41% identical to the L5 repressor (gp71)

and binds to repressor binding sites that conform to

a similar, but distinct, 13 bp asymmetric consensus

sequence to that for the L5 gp71 binding sites. The

two phage repressors have a strong preference for

their cognate binding sites, thus accounting for their

immunity phenotypes. The Bxb1 genome contains 34

putative repressor binding sites located throughout

the genome, but situated within short intergenic

spaces and orientated in only one direction relative

to the direction of transcription. Comparison with the

locations of repressor binding sites within the L5

genome provides insights into how these unusual

regulatory systems evolve.

Introduction

Temperate bacteriophages can pursue two alternative

developmental routes upon infection of a bacterial host

cell: lytic growth, which results in the generation and

release of progeny phage particles and cell death; and

lysogeny, in which the lytic functions are switched off and

the phage DNA (usually, although not always) integrates

into the host chromosome. In the well-studied group of

lambda-like phages, maintenance of the lysogenic state

requires the expression of a repressor that binds to two

tripartite operator sites to downregulate the activity of the

early lytic promoters PL and PR (Ptashne, 1987) and to

regulate its own synthesis (Ptashne et al., 1976). The

repressor also acts to confer immunity to superinfection

by homoimmune phage particles by binding to cognate

operator sites present on the genome of superinfecting

phages. However, the regulation of lysogenic mainte-

nance and superinfection immunity by repressors is a

common theme among a diverse array of temperate

bacteriophages (Scott et al., 1978; Dhaese et al., 1985;

Ladero et al., 1998; Salmi et al., 1998; Kameyama et al.,

1999; Nesper et al., 1999).

Mycobacteriophage L5 is a temperate phage of the

mycobacteria that uses a very different immunity system

from that of the lambda-like phages (Donnelly-Wu et al.,

1993; Hatfull, 1994; 1999; Nesbit et al., 1995; Brown et al.,

1997). The L5 repressor (gp71) is a 183-amino-acid

protein that contains a putative helix±turn±helix DNA

binding motif near its N-terminus and recognizes a 13 bp

asymmetric binding site (Donnelly-Wu et al., 1993; Brown

et al., 1997). Only one early lytic promoter has been

identified in L5 (Pleft), and this is under direct negative

regulation by gp71 (Nesbit et al., 1995). However, there is

just a single gp71 binding site overlapping the 235 region

of this promoter, and this operator appears to be sufficient

for regulation of Pleft by L5 gp71 (Brown et al., 1997).

An unusual feature of the regulatory system in L5 is that

the genome contains a large number of repressor binding

sites (Brown et al., 1997). There are at least 29 additional

sites with close sequence similarity to the Pleft operator,

and gp71 binds to at least 24 of the 30 sites. These sites

conform closely to a 13 bp consensus sequence (5 0-GGTGGc/aTGTCAAG), and all the 24 sites contain the

consensus nucleotide in eight of the 13 positions (Brown

et al., 1997). These sites are found throughout the L5

genome, are located within short intergenic regions and

are situated in only one orientation relative to the direction

of transcription (Brown et al., 1997). They are referred

to as `stoperators' because, when the gp71 repressor

is bound, the elongation of transcription complexes is

inhibited; the strong orientation dependence of this effect

and the rather modest affinity of L5 gp71 for its binding

sites (Kd � < 5 � 1028 M) suggests an active mechan-

ism rather than a simple passive block. Mycobacterioph-

age D29, a close and homoimmune relative of L5,

contains a similar set of sites, which conform to the

same consensus sequence and appear to be functionally

identical (Ford et al., 1998). The Streptomyces phage

fC31 also has multiple repressor binding sites, most of

which are also present in intergenic regions, although

their specific role in lysogenic maintenance is not clear

Q 2000 Blackwell Science Ltd

Accepted 8 September, 2000. *For correspondence. E-mail [email protected]; Tel. (11) 412 624 6975; Fax (11) 412 624 4870.

(Ingham et al., 1994; Wilson et al., 1995; Smith et al.,

1999).

The biological role of the stoperator sites is uncertain,

although we presume that they act to silence the

prophage genome in lysogeny. In this respect, we note

that lambda and its relatives contain a large number of

rho-dependent and rho-independent terminators that are

not only present in the regulatory regions, but are found

throughout the genome (Juhala et al., 2000). These

terminators presumably act, at least in part, to maintain a

low transcriptional state of the prophage genome,

although they do not present an impediment to lytic

growth because of the N- and Q-mediated antitermination

systems (Ptashne, 1987). Although the L5 genome does

contain some recognizable factor-independent termina-

tors (see Mediavilla et al., 2000), these are all located

around the ends of the early and late operons and are not

found within them (Hatfull and Sarkis, 1993). Mycobacter-

iophage antitermination systems have not been studied,

but there is no obvious regulatory necessity for their

inclusion in these genomes. It therefore seems likely that

many of the lambda terminators act like L5 stoperators to

confer a low level of expression of phage genes from

prophage genomes.

In this paper, we describe the immunity system of

mycobacteriophage Bxb1, a phage that is related to L5

but shares little DNA sequence similarity (Barletta et al.,

1992). Bxb1 and L5 are heteroimmune as a result of the

specificity of their respective repressors, which bind with a

strong preference to their cognate binding sites. Bxb1

contains even more putative binding sites than L5 but, as

in L5, they are located within intergenic regions and in

only one orientation relative to the direction of transcrip-

tion. A comparison of the Bxb1 regulatory circuitry and the

location and nature of the repressor binding sites relative

to those in L5 provides insights into these regulatory

systems and how they may evolve.

Results

Identification of the Bxb1 repressor protein

From the analysis of the Bxb1 genome sequence, it was

found that gene 69 encodes a protein that shares 41%

identity with the L5 repressor gp69 (Mediavilla et al.,

2000). Bxb1 gp69 is 170 amino acids long and can be

aligned with L5 gp71 without gaps except for the extreme

N- and C-termini (Fig. 1). In L5 gp71, there is a putative

helix±turn±helix motif close to the N-terminus, which is

presumably involved in DNA recognition, as amino acid

substitutions in it lead to a loss of DNA binding (K. Brown

and G. F. Hatfull, unpublished observations). A related

sequence is present in Bxb1 gp69, with over half the

residues being identical, including the three amino acids

in the `turn'. The most notable difference between the two

motifs is the first position of the second helix, which is

usually associated with making direct contact with DNA at

the recognition site (Harrison and Aggarwal, 1990). In L5

gp71, this position is an arginine, whereas in Bxb1 gp69, it

is a proline residue, an unusual amino acid to find in this

position, although its presence is not unprecedented

(Laughon and Scott, 1984; Harrison and Aggarwal, 1990).

These differences are likely to contribute to changes in

sequence recognition by these proteins. We noted

previously that the C-terminal part of L5 gp71 has an

abundance of acidic amino acids (Donnelly-Wu et al.,

1993), but this feature is much less evident in Bxb1 gp69,

suggesting that this may not be an important aspect of the

function of these repressors.

Bxb1 gene 69 was inserted into the T7 overexpression

vector pET21a, and the resulting plasmid (pSJ6) was

used to overexpress Bxb1 gp69. Good levels of gp69

synthesis were observed after induction with IPTG,

although the protein migrates with an apparent molecular

mass of 27 kDa, somewhat greater than its predicted

mass of 18.7 kDa, a property that is shared with L5 gp71

(K. Brown and G. F. Hatfull, unpublished observations).

Bxb1 gp69 was purified to . 90% homogeneity by

chromatography using a BioCAD SPRINT system (data

not shown).

Identification of the Bxb1 Pleft promoter

In L5, there is one major promoter, Pleft, which is regulated

directly by the gp71 repressor (Nesbit et al., 1995), and it

seems likely that Bxb1 would have a similar promoter that

is responsible for early lytic gene expression. In L5, the

Pleft promoter is located upstream of gene 89, the first

open reading frame (ORF) in the leftwards operon, at co-

ordinate 51, 672 (Fig. 2A). However, an analogous Bxb1

promoter cannot easily be identified by sequence com-

parison of the genomes, as these segments are not

closely related and the genome organizations of L5 and

Bxb1 are somewhat different at the righthand ends

(Fig. 2A; see Mediavilla et al., 2000). In particular, there

are three small genes transcribed in the rightwards

direction at the extreme right end of the Bxb1 genome

for which there are no obvious counterparts in L5.

Although these genes are small (encoding putative

protein products of 7.2 kDa, 5.6 kDa and 6.8 kDa), the

veracity of the assignments is supported by the observa-

tion that Bxb1 gp84 has significant sequence similarity

(76% identity) to L5 gp79. It therefore seems probable

that a promoter for leftwards transcription of the early

genes (analogous to L5 Pleft) is located between the

divergently transcribed genes 83 and 84.

A DNA segment containing this intergenic region (co-

ordinates 48 657±48 888) was amplified by polymerase

972 S. Jain and G. F. Hatfull

Q 2000 Blackwell Science Ltd, Molecular Microbiology, 38, 971±985

chain reaction (PCR) and inserted into the integration-

proficient shuttle vector pDK16 (Table 1), which contains

a promoterless lacZ reporter gene (Fig. 2B). The resulting

plasmid (pSJ9) efficiently transforms Mycobacterium

smegmatis to give colonies with a blue colour on Xgal

plates, and b-galactosidase assays indicate that it has a

modest level of promoter activity (Fig. 2B). However,

when pSJ9 was introduced into a Bxb1 lysogen, the

colonies were only pale blue on Xgal plates and had . 30-

fold less b-galactosidase activity than the non-lysogen

(Fig. 2B). To determine whether this reduction in activity

was caused by the action of the Bxb1 repressor, we

inserted a copy of Bxb1 gene 69 into pSJ9 (to give pSJ15)

and introduced this plasmid into M. smegmatis. The

resulting colonies were also pale blue on Xgal plates

and have only low levels of b-galactosidase activity. We

conclude that this 232 bp segment of the Bxb1 genome

contains a leftwards-facing promoter that is directly

regulated by the Bxb1 repressor.

The same segment of the Bxb1 genome was also

Fig. 2. Identification of the Bxb1 Pleft

promoter.A. Organization of the right ends of the L5and Bxb1 genomes. Segments of < 4 kbp ofthe L5 and Bxb1 genomes are shown with co-ordinates above each genome. The positionsof putative genes are shown as grey boxes.Lightly shaded genes below the genome aretranscribed leftwards, and darkly shaded onesabove are transcribed rightwards. Thepositions of the Pleft promoters are shown.B. Regulation of the Bxb1 Pleft promoter.Plasmids containing various segments ofBxb1 DNA were introduced into eitherM. smegmatis mc2155 or a Bxb1 lysogen[mc2155 (Bxb1)] and the b-galactosidaseactivities determined. Plasmids pSJ23 andpSJ22 contain 232 bp and 126 bp fragmentsof Bxb1 DNA, respectively, fused to lacZ inthe extrachromosomal vector pSD5B. PlasmidpSJ9 contains the same 232 bp fragment asin pSJ23 but fused to lacZ in the integratingvector pDK16. Plasmids pSJ24 and pSJ15are similar to pSJ23 and pSJ9, respectively,but also contain a copy of the Bxb1 repressorgene 69. The b-galactosidase activities areaverages from three individual transformants,except for those indicated (*), in which therange of activities is reported. b-Galactosidase activities were determined asdescribed in Experimental procedures and areexpressed as nmol min21 mg21 total protein.ND denotes the assays not done.

Fig. 1. Sequence alignment of the repressorproteins of mycobacteriophage Bxb1 (gp69)and L5 (gp71). Amino acid identities andsimilarities are indicated by colons and dotsrespectively. Dashes indicate gaps insertedfor optimal alignment of the sequences. Thetwo helices of the putative helix±turn±helixDNA-binding motifs are underlined.

Mycobacteriophage Bxb1 gene expression 973


Table 1. Plasmids used in this study.

Plasmid Description Source or reference

pET21a E. coli expression vector for inducible expression of genes NovagenpSD5B lacZ-based promoter probe shuttle vector for mycobacteria with XbaI and SphI as promoter cloning sites upstream of lacZ Jain et al. (1997)pDK10 pGEM5Zf(1) containing the mycobacteriophage L5 `attP±int' DNA fragment DasGupta et al. (1998)pDK16 lacZ-based promoter probe integrative vector for mycobacteria with NcoI, SpeI and NotI as promoter cloning sites upstream of lacZ Kaushal and Tyagi (unpublished)pSJ6 Bxb1 gene 69 in NdeI±NotI sites of pET21a This studypSJ7 232 bp Pright amplicon in XbaI upstream of lacZ in pSD5B This studypSJ8 232 bp Pleft/right in SmaI site of pUC118 This studypSJ9 232 bp Pleft in NcoI site upstream of lacZ in pDK16 This studypSJ10 735 bp amplicon with gene 69 and its upstream region containing the promoter in NheI site of pSJ7 This studypSJ11 735 bp amplicon (as in pSJ10) in XbaI upstream of lacZ in pSD5B This studypSJ12B 735 bp amplicon (as in pSJ10) in SmaI site of pUC118 This studypSJ14 293 bp KpnI±EcoRV fragment from pSJ12B containing promoter of gene 69 in XbaI upstream of lacZ in pSD5B This studypSJ15 735 bp amplicon (as in pSJ10) in ScaI site of pSJ9 This studypSJ18 401 bp KpnI fragment from pSJ12B (as in pSJ16) in ScaI site of pSJ9 This studypSJ19 293 bp KpnI±EcoRV fragment from pSJ12B (as in pSJ14) in ScaI of pSJ9 This studypSJ21 126 bp amplicon with Bxb1 Pright in XbaI upstream of lacZ in pSD5B This studypSJ22 126 bp amplicon with Bxb1 Pleft in XbaI upstream of lacZ in pSD5B This studypSJ23 232 bp amplicon with Pleft in XbaI upstream of lacZ in pSD5B This studypSJ24 735 bp amplicon (as in pSJ10) in NheI site of pSJ23 This studypSJ26 232 bp Bxb1 Pright upstream of the promoterless lacZ in the integration vector This study

The details of the primers used for obtaining the PCR amplicons are mentioned in Experimental procedures. Pleft and Pright are the divergently placed promoters within the 232 bp and 126 bpamplicons obtained from the right end of the Bxb1 genome. pET21a, pDK10 and pSJ6 carry an ampicillin resistance gene marker for selection of transformants. All other plasmids carry the gene forresistance to kanamycin.

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inserted into the extrachromosomal lacZ reporter plasmid

pSD5B (Table 1). This plasmid (pSJ23) efficiently trans-

forms a Bxb1 lysogen to give pale blue colonies and

confers low levels of reporter gene activity. However,

when attempting to introduce pSJ23 into a non-lysogen,

transformants were obtained at a low frequency, grew

very slowly and had b-galactosidase activities that varied

greatly. A plasmid (pSJ22) containing a smaller segment

of the Bxb1 genome (co-ordinates 48 763±48 888) has a

similar phenotype (Fig. 2B). A simple explanation for this

phenomenon is that the wild-type Pleft±lacZ fusion is not

tolerated because of a high level of promoter activity, and

those transformants that do arise have mutations that

reduce the level of expression or lower the plasmid copy

number. Introduction of the repressor gene into pSJ23

(to give pSJ24) enabled efficient transformation into an

M. smegmatis non-lysogen with normal growth rates and

a consistently low level of b-galactosidase activity.

Presumably, the inability of pSJ23 to transform non-

lysogenic M. smegmatis efficiently results from the high

activity of a repressor-regulated promoter even though the

level of reporter gene activity in the integration-proficient

vector seems relatively modest. We have also observed

that the Pleft promoter of L5, which is very active when

fused to the FFlux reporter gene in an integrating vector

(Brown et al., 1997), also gives only modest activity when

fused to lacZ (L. Bibb and G. F. Hatfull, unpublished

observations). The mediocre levels of b-galactosidase

activity may therefore reflect, at least in part, a feature of

the reporter gene rather than the promoter per se.

The transcriptional start site of the promoter in pSJ9

was identified by primer extension analysis, which

revealed a major start site at co-ordinate 48 802

(Fig. 3), within the smaller fragment inserted into the

lacZ vector pSJ22 (Fig. 2B). Immediately upstream of the

start site are putative 210 and 235 regions, and this

small region exhibits a reasonable similarity to the Pleft

promoter of L5 with about 50% nucleotide identity

(Fig. 3B). The 235 region is identical to that in L5 (5 0-TTGACA), and the 210 segment differs by a single

nucleotide (5 0-CATACT in Bxb1, 5 0-CATTCT in L5); the

spacing between the 210 and 235 regions is also

different (18 bp in L5 and 17 bp in Bxb1). By analogy

with L5, we propose to refer to this as the Bxb1 Pleft

promoter.

The Pleft promoter in L5 is known to be regulated by the

L5 repressor, and a 13 bp repressor binding site overlaps

the 235 region (Fig. 3B). In Bxb1, a related sequence is

present that also overlaps the 235 region (where it is

identical) but differs from the L5 sequence in four

positions (Fig. 3B). The previous analysis of repressor

binding sites in L5 suggests strongly that this Bxb1

sequence would not be bound by the L5 repressor (Brown

et al., 1997). This therefore raises the question as to

whether there are multiple occurrences of this sequence

in the Bxb1 genome (as there are L5 repressor binding

sites; Brown et al., 1997) and whether this represents a

recognition sequence for the Bxb1 repressor.

Repressor binding sites in the Bxb1 genome

The Bxb1 genome was searched for the presence of

sequences closely related to the 13 bp segment that

overlaps the 235 region of the Bxb1 Pleft promoter. This

search revealed the presence of at least 34, 13 bp

segments that correspond to a consensus sequence 5 0-GTTACGt/ag/aTCAAG, which, like the L5 consensus

sequence is quite asymmetric (Fig. 4). The assignation

of these as repressor binding sites is strengthened by two

other properties that are shared with the analogous sites

Fig. 3. Mapping the Pleft transcription start site.A. Total RNA was isolated from M. smegmatis mc2155 transformedwith pSJ9 and reverse transcribed with the end-labelled primerSJ98-4 as described in Experimental procedures. Dideoxysequencing reactions (lanes G, A, T and C) were carried out withpSJ9 DNA using the same primer. The primer extension productcorresponding to the transcription start site is indicated by anarrow.B. Alignment of the Pleft promoters of L5 and Bxb1. The putative210 and 235 promoter regions are underlined, and the start sitesare indicated by arrows. The putative repressor binding sites areboxed.



in L5. First, they are predominantly located within non-

coding regions, short intergenic spaces or very close to

initiation or termination codons (Fig. 4; see Mediavilla

et al., 2000). Only two of the 34 putative sites are within

the middles of genes (in genes 12 and 49), whereas eight

overlap start or stop codons. Secondly, the orientation of

the sites correlates tightly with the direction of transcrip-

tion. For example, in the rightwards-transcribed structural

operon, there are 11 sites, all of which are in the

designated `±' orientation. In contrast, within the leftwards

transcribed genes (36±83), there are 18 sites, all of which

lie in the `1' orientation. There are also three sites at the

extreme right end of the genome that are in the `±'

orientation, but these are positioned around the three

rightwards-transcribed genes, 84±86. The only site that

does not appear to follow this pattern is one of the two

sites located in the 33±34 intergenic region, which is in

the `1' orientation (normally associated with leftwards

transcription).

The Bxb1 consensus sequence is similar to, but distinct

from, that of the L5 binding sites (Fig. 4). The sites are

similar in that they share seven positions, in which the

consensus base is the same and that are very highly

conserved in both genomes (positions 1, 3 and 9±13).

However, there are three positions that are very highly

conserved in both but with different consensus nucleotides

Fig. 4. Repressor binding sites in the Bxb1genome. Putative Bxb1 repressor bindingsites were identified by searching the Bxb1genome for sequences similar to the 13 bpsequence overlapping the 235 promoter(Fig. 3). A total of 34 sites was found andthey are aligned (shown in red) with therepresentation of bases in each positionshown below, along with a consensussequence. The base representation andconsensus sequence for the L5 gp71repressor binding sites are also shown. Theorientation of each site in the Bxb1 genome isindicated as well as its position relative toBxb1 genes. See Mediavilla et al. (2000) for arepresentation of sites in the Bxb1 genome.



(positions 2, 4 and 5). The bases in these positions are

therefore excellent candidates for conferring specificity

of repressor binding and, ultimately, heteroimmunity

between L5 and Bxb1. Three positions (6±8) are more

complex, as each of these is well conserved in one phage,

but much less so in the other. However, in positions 7 and

8, the bases that are highly conserved in L5 represent the

preferred bases in Bxb1, which could be accounted for by

supposing that these positions are important for the

evolutionary transition of specificity through relaxed as

opposed to switched specificity.

A comparison of the specific locations of the repressor

binding sites in the L5 and Bxb1 genomes also provides

some insights into how these may evolve. For example,

there are three locations in the left arms of these genomes

where a site is present in Bxb1, but absent from L5, even

though the flanking genes are homologous (but enjoy only

low levels of DNA sequence similarity; see Mediavilla

et al., 2000). In each of these examples, the intergenic

spacing is larger in Bxb1 than in L5, suggesting that the

acquisition of these sites was achieved through DNA

insertion, rather than a series of base substitutions. The

specific intergenic spacings are: 17 bp between L5 genes

28 and 29 compared with 37 bp between Bxb1 genes 25

and 26; 13 bp between L5 genes 12 and 13 compared

with 19 bp between Bxb1 genes 9 and10; 51 bp between

L5 genes 15 and 16 compared with 60 bp between Bxb1

genes 12 and 13. One of these is illustrated in Fig. 5A. In

contrast, there are other examples in which both genomes

contain cognate repressor binding sites located between

pairs of homologous genes, but the intergenic spacing is

very similar (an example is shown in Fig. 5B). In these

cases, the change from one binding site specificity to the

other could be accomplished through a series of base

substitutions (five in the example given in Fig. 5B).

Binding of gp69 to Bxb1 Pleft

To determine whether Bxb1 gp69 does indeed recognize

and bind to these putative DNA sites, the binding of

purified gp69 to Bxb1 fragments was studied in vitro.

First, we examined the binding of gp69 to a 232 bp DNA

fragment that contains the Bxb1 Pleft promoter, which is

predicted to contain three gp69 binding sites (nos 1, 2 and

3; Fig. 4). Upon the addition of increasing amounts of

gp69 to Pleft DNA, four electrophoretically separable

complexes were observed (Fig. 6A), with the slower

migrating complexes being more abundant at higher

concentrations of gp69. The identity of each of these

complexes is unclear, although the binding pattern is

consistent with the presence of multiple binding sites to

which gp69 has different affinities.

The binding of g69 to these sites was further dissected

by separating the sites onto two smaller DNA fragments,

one of which contains sites 1 and 3, and the other has site

2. The addition of gp69 to the site 2-containing fragment

generates just a single DNA complex, whereas the other

fragment forms three separable complexes (Fig. 6B).

There are several possible explanations for the formation

of three complexes from the two-site fragment. First, there

may be a third gp69 binding site on this fragment, in

addition to the two predicted sites (1 and 3). Secondly, the

two fastest migrating complexes may have the same

protein±DNA ratio, but with gp69 occupying one site in

one complex and the other site in the other complex,

these complexes could exhibit different mobilities, espe-

cially if the DNA is somewhat bent in these complexes.

The slowest migrating complex would then have both

sites occupied. Thirdly, two of the complexes could

have the same stoichiometry but adopt different shapes

depending on additional protein±protein interactions.

Although we do not yet know which explanation accounts

for the observed complexes, we favour the second of the

above explanations. DNase I footprinting experiments

reveal only three regions of protection within this region,

corresponding to sites 1, 2 and 3 (Fig. 6C). The affinity of

gp69 appears to be slightly higher for site 2 than for site 1

(Kd � < 5 � 1028 M) and is substantially lower for site 3

(Fig. 6C). Sites 1 and 2 differ in the bases at positions 7

and 8 of the consensus (Figs 4 and 6D), suggesting that,

although these positions are less strongly conserved, the

specific bases do play a role in gp69 recognition and

binding affinity; however, flanking sequences could also

influence the binding affinity of Bxb1 gp69. Interestingly,

the bases associated with the highest affinity site (site 2; A

at both positions 7 and 8) represent significant departures

from the L5 consensus (Fig. 4), and these positions may

therefore contribute to immune specificity.

Although the sequences surrounding the Pleft promoters

in L5 and Bxb1 are not closely related, there are

interesting similarities in the organizations. In particular,

both L5 and Bxb1 have both sites 1 and 2, and they are

spaced at precisely the same relative positions, with a

site±site distance of 116 bp (i.e. the distance from

position 1 of site 2 to position 1 of site 1). As noted

previously for L5, this places sites 1 and 2 almost exactly

11 integral turns of DNA apart, such that two bound

repressor molecules would be on the same face of the

DNA helix, where they would have the potential to interact

to form a DNA loop. However, the occupancy of the L5

sites is not affected by placing them on different DNA

fragments or altered by changing the spacing between the

sites, which argues against such an interaction (Brown

et al., 1997). This also appears to be the case for Bxb1

sites 1 and 2, in that there are no complexes observed

with a DNA fragment containing both sites that cannot be

accounted for by occupancy of the sites on separate DNA

fragments (Fig. 6). However, the conservation of this site



spacing, regardless of the low degree of DNA sequence

similarity between these regions, raises the possibility that

such protein±protein interactions may be of importance,

even though they are not detected with the assays and

DNA substrates that have been used.

The gp69 binding site designated as site 3 is situated in

the opposite orientation to sites 1 and 2 and has no

counterpart in L5. In L5, there are two binding sites to the

right of site 1, one is 59 bp upstream (to the right) of site 1

and is in the same orientation as it, whereas the second

site is 233 bp to the right of site 1 and is in the opposite

orientation. No ORFs have been identified between L5

Pleft and the right end of the genome, although we cannot

rule out a rightwards-facing promoter in this part of the

genome. In contrast, in Bxb1, there are three rightwards-

transcribed genes (84±86), and a promoter must be

located between genes 83 and 84. Site 3 is thus a

candidate operator site for such a promoter.

Identification of the Pright promoter

To determine whether rightwards transcription initiates

from within the gene 83±84 region, total RNA isolated

from a Bxb1 lysogen was used as a template for primer

extension (Fig. 7A). A primer extension product was

observed that corresponds to transcription initiation at

position 48 875, although only a weak signal could be

detected; some additional primer extension end-points

were also observed, but these all mapped within the

ORF for gene 84. Immediately upstream of this start site

are putative 210 (5 0-TAACCT) and 235 (5 0-TTGATC)

recognition sequences, separated by a 17 bp spacer

(see Fig. 6D). We have designated this putative promoter

as Pright. The gp69 binding site 3 coincides with this

promoter, but overlaps the 210 region rather than the

235 motif observed with site 1 and the Pleft promoter.

The observation that gp69 binding site 3 overlaps the

putative 210 region of the Pright promoter suggests that

transcription from Pright is under repressor control. To test

this, we constructed reporter gene fusion plasmids in

which the Pright promoter is fused to the lacZ gene, using

both extrachromosomal and integration-proficient vectors.

However, the extrachromosomal version of the recombi-

nant plasmid failed to transform M. smegmatis, unless the

strain was lysogenic for Bxb1. This could the result of poor

tolerance of either the Pright promoter or the Pleft promoter,

which is also present on this plasmid; as reported above,

plasmids with the Pleft promoter fused to lacZ also

transform non-lysogen strains poorly. However, the

integration-proficient plasmids did transform, and b-

galactosidase assays indicated that the promoter was

downregulated about 10-fold in the Bxb1 lysogen, most

Fig. 5. Origins of repressor binding sites.A. Acquisition of sites by insertion. Comparison of the L5 and Bxb1 genomes reveals at least three positions in which a repressor binding siteis located in one genome, but absent from the homologous location in the other genome (see text for further details). In all three cases, theintergenic distance is significantly larger in the genome that contains the site, consistent with the site being acquired by DNA insertion ratherthan by base substitution. The sequences of the intergenic spaces in one example are shown, in which a repressor binding site is locatedbetween Bxb1 genes 9 and 10, but is absent from the space between the homologous L5 genes 12 and 13.B. Acquisition of sites by substitution. There are also several examples in which the spacing between flanking genes is similar, and both L5and Bxb1 contain repressor binding sites. The example shown compares the junctions of Bxb1 genes 22 and 23 and L5 genes 26 and 27. Therepressor binding sites are in similar locations, and the switch in repressor binding specificity presumably arises from base substitutions. In (A)and (B), the 13 bp repressor binding sites are boxed, and nucleotide and amino acid residues that are identical in the two phages are shownin red. Ribosome binding sites are underlined. The co-ordinates of the leftmost nucleotides are shown.



probably because of the action of the gp69 repressor

(Fig. 7B). The level of gp69 regulation is less than that

seen for the Pleft promoter and presumably reflects the

differences in the affinity of the gp69 repressor for binding

sites 1 and 3 (Fig. 6).

Expression of the Bxb1 repressor gene

Examination of the Bxb1 genome map (see Fig. 4; Mediavilla

et al., 2000) shows that there is a small non-coding gap of

261 bp immediately upstream (i.e. to the right) of gene 69.

Presumably, there is at least one promoter in this region

that is responsible for the expression of gene 69. We note

that, in L5, there is a similar non-coding space upstream

of gene 71, within which there are three promoters

(designated P1, P2 and P3). However, there is no obvious

DNA sequence similarity between the two intergenic

regions, and it is not possible to predict the locations of

putative promoters.

To determine whether the Bxb1 region upstream of

Fig. 6. DNA binding of the Bxb1 gp69 repressor to Pleft DNA.A. Mobility shift assays for the binding of Bxb1 gp69 and L5 gp71 repressor proteins to Bxb1 and L5 Pleft promoters. Bxb1 gp69 or L5 gp71was added at decreasing concentrations (threefold serial dilutions; as indicated) to a 203 bp DNA fragment containing the Bxb1 Pleft promoter(top) or a 219 bp fragment containing the L5 Pleft promoter (bottom), and the protein±DNA complexes were separated from free DNA byelectrophoresis through a native 4% polyacrylamide gel. The amounts of Bxb1 gp69 present in each reaction (from left to right) areapproximately 26, 8, 2.6, 0.8, 0.26 and 0.08 pmol.B. Bxb1 gp69 binding to DNA subfragments. The DNA present in the Bxb1 Pleft fragment shown in (A) was amplified as two separate DNAfragments, one of 95 bp containing site 2 (probe 2) and the other of 107 bp containing sites 1 and 3 (probe 1). Binding of gp69 was as in (A).The amounts of Bxb1 gp69 in each reaction (from right to left) are approximately 8, 2.6, 0.8, 0.26 and 0.08pmol. The fastest moving complexwith probe 1 is likely to be a site 1 complex, as this is a higher affinity site than site 3; see (C).C. DNase I footprinting of Bxb1 Pleft DNA±gp69 complexes. Bxb1 gp69±DNA complexes were formed as in (A), cleaved with DNase I and theproducts separated by denaturing PAGE. Bxb1 gp69 was added in threefold serial dilutions, and lane `D' contained no gp69. The positions ofprotection at sites 1, 2 and 3 are indicated. The amounts of Bxb1 gp69 added to each reaction are (from right to left) 26, 8, 2.6, 0.8 and0.26 pmol.D. Sequence of the Bxb1 gene 83±84 intergenic region. The locations of repressor binding sites 1, 2 and 3 are boxed, and the 235, 210 andstart site positions of the Pleft and Pright promoters are shown. The sequences to the left of the mark (I) are present in the 95 bp fragment,probe 2 (B), and those to the right are in the 107 bp fragment, probe 1 (B). Sequences not present in the probes are shown in italics.



gene 69 is promoter active, a 293 bp fragment (containing

Bxb1 DNA corresponding to co-ordinates 44 679±

44 969) was inserted upstream of the lacZ reporter

gene in a shuttle plasmid vector and introduced into M.

smegmatis (Fig. 8A). This plasmid exhibited considerable

b-galactosidase activity of a magnitude similar to that of

the reporter for a similar fusion with the region upstream

of L5 gene 71 (Nesbit et al., 1995). When the Bxb1

repressor gene was also present (plasmid pSJ11), there

was a modest downregulation (< twofold) of the activity,

suggesting that Bxb1 gp69 regulates its own synthesis (as

also seen in L5).

The positions of transcription initiation from this

segment of the Bxb1 genome were determined by primer

extension, using RNA isolated from an M. smegmatis

strain carrying either pSJ11 or pSJ14 (Fig. 8B). Two

initiation sites were observed (at co-ordinates 44 778 and

44 886), suggesting that there are at least two promoters

in this region; we will refer to these as P1 and P2. Putative

210 and 235 regions for both promoters can be identified

and are shown in Fig. 8C. However, there is only one

possible gp69 binding site in this region, site 28 (Fig. 4),

which overlaps the putative 235 region of the P1

promoter. DNA-binding studies show that gp69 does

bind to this DNA fragment but predominantly forms just a

single complex, consistent with the presence of a single

binding site (Fig. 8D). The affinity for this site is con-

siderably lower than that for sites 1 and 2 at Pleft (Fig. 8D),

which presumably accounts for the low level of gp69

repression.

Discussion

We have described here some central aspects of

immunity regulation in Bxb1 and how they compare with

the related system in L5. As phages for comparative

studies, L5 and Bxb1 are a convenient pair, as they are

not particularly closely related at the nucleotide level and

yet share similar genome architectures, virion structures

and systems for genetic regulation. In other aspects, such

as host range and halo formation, they are clearly different

(Mediavilla et al., 2000). A comparative approach is

particularly useful for understanding the genetic circuitry in

these phages, in part because of the novel immunity system

in phage L5 that involves an unusual repressor and multiple

binding sites. The study of Bxb1 immunity regulation not

only validates the interpretations of the L5 studies but

offers insights into how these systems have evolved.

The observation that L5 and Bxb1 are heteroimmune

can be readily explained in view of the differences

between the phage repressors and their binding sites.

L5 gp71 and Bxb1 gp69 are only 41% identical, and the

amino acid difference at the first position of the second

helix of the putative helix±turn±helix motif is an obvious

candidate for being more directly involved in determining

DNA-binding specificity. The proline at this position in

Bxb1 gp69 is unusual, but not unprecedented (Harrison

and Aggarwal, 1990). The consensus sequences for the

sites are also quite distinct, in that three of the consensus

positions (positions 2, 4 and 5) that are tightly conserved

are different in the two phages (T, A and C in Bxb1 versus

G at all three positions in L5). Nevertheless, there is also a

lot of similarity between the Bxb1 and L5 consensus

sequences, and seven of the positions are both highly

conserved and identical in the two phages. Moreover, the

DNA-binding studies show that the Bxb1 repressor binds

with a much greater preference to its cognate sites than

those in L5, and the reciprocal behaviour is seen with L5

gp71. The difference in affinities is < 1000-fold in each

case.

Fig. 7. Identification of the Bxb1 Pright promoter.A. Identification of the Pright transcription start site by primer extension. Total RNA extracted from a Bxb1 lysogen of M. smegmatis mc2155was used as template for extension from primer SJ99-5. The same primer was used for sequencing reactions. The position of the majorprimer extension product is shown by an arrow.B. Activities of a Pright±lacZ fusion plasmid. Plasmid pSJ26 was constructed by inserting a Pright promoter fragment into the integrating lacZreporter vector and transformed into both M. smegmatis mc2155 and a Bxb1 lysogen. The b-galactosidase activities are presented asnmol min21 mg21 total protein.



A closer examination of the L5 and Bxb1 repressor

binding site sequences is very informative. In L5 positions

7±12, the sequence 5 0-TGTCAA is very well conserved,

and this segment corresponds to the location of the 235

motif (i.e. 5 0-TTGACA on the other strand) within the Pleft

promoter (Fig. 4). This would indicate that this particular

235 sequence is present at each of the stoperator sites in

the L5 genome. However, the Bxb1 sites show that this

may not be an important feature, as positions 7 and 8 are

not as well conserved and, at both positions, there is

considerable variation. In Bxb1, the Pleft operator (site 1)

is only one of 12 sites that has the 5 0-TGTCAA motif,

whereas 23 of the 24 l5 binding sites have this sequence.

Nevertheless, it is noteworthy that the major differences in

the sites (those that are expected to alter repressor

binding specificity) are located away from this 235 region

at positions 2, 4 and 5.

The extreme right end of the Bxb1 genome represents

one of the greatest departures from the organization of

the L5 genome. Here, there is divergent transcription

originating from a 213 bp intergenic region that contains

the Pleft and Pright promoters, both of which are under

repressor control. The Bxb1 Pleft promoter is analogous to

the L5 Pleft promoter, being located upstream of the first

of the identified ORFs in the leftwards operons in the

right arm and with almost identical 210 and 235 RNA

polymerase recognition sequences. No counterpart to the

Bxb1 Pright promoter has been identified in L5, but nor

Fig. 8. Expression and autoregulation of the Bxb1 repressor gene 69.A. b-Galactosidase activities of gene 69± lacZ reporter plasmids. Plasmid pSJ14, containing a DNA fragment with the putative gene 69promoter fused to lacZ, and pSJ11, which contains a similar fragment but with the entire gene 69 ORF, were introduced into M. smegmatismc2155, and b-galactosidase activities were determined as described.B. Primer extension analysis of the Bxb1 gene 69 promoter. Total RNA isolated from M. smegmatis carrying either plasmid pSJ14 or plasmidpSJ11 (shown in lanes P14 and P11 respectively) was used as template for extension with primer SJ99-4. The major transcription start sites(P1 and P2) are shown by arrows.C. Location of the gene 69 promoters. The sequence from the region upstream of gene 69 (co-ordinates 44 928±44 741) is shown with the P1and P2 transcription start sites as indicated. Putative 210 and 235 sequence motifs are underlined by a dashed line. The amino acidsequence of the start of gp69 and the gene 69 ribosome binding site are indicated. The position of a putative repressor binding site (28) isboxed.D. Binding of Bxb1 gp69 to the gene 69 promoter region. Serial dilutions of Bxb1 gp69 were incubated with a 309 bp DNA fragment containingthe gene 69 upstream region and a 203 bp fragment containing the Pleft promoter. Protein±DNA complexes were separated from free DNA byelectrophoresis through a 4% native polyacrylamide gel. The amounts of Bxb1 gp69 added to each reaction (from right to left) areapproximately 2.6, 0.8, 0.26 and 0.08 pmol.



have any rightwards-transcribed ORFs at the right end of

the genome. We note that there is a repressor binding site

at the extreme right end of the L5 genome that is oriented

in such a way as to regulate rightwards transcription, and

the possibility that there is a rightwards promoter located

near this site cannot be eliminated.

The function of the rightwards-facing genes 84, 85 and

86 in Bxb1 is unclear. The Pright promoter located

immediately upstream of these is only rather modestly

downregulated (<10-fold), and transcription initiation

could be detected using RNA isolated from a Bxb1

lysogen. As there is also a putative terminator located

between genes 85 and 86, the segment containing the

promoter, genes 84 and 85 and the terminator are

reminiscent of the morons described in the lambda-like

phages (Juhala et al., 2000). However, we also cannot

rule out the possibility that Pright plays a role in late lytic

growth, either as a result of inefficient termination or

perhaps through the use of an antiterminator, and could

thus be responsible for synthesis of the late genes. It is

also unclear how gene 86 is expressed and whether there

is an additional promoter between genes 85 and 86.

Autoregulation of repressor synthesis appears to be a

common theme among temperate bacteriophages. In

phage lambda, this is accomplished through the action of

the tripartite operator OR, in which the binding of the cI

repressor to the highest affinity site, OR1, results in the

activation of cI synthesis (Meyer et al., 1975); at higher

concentrations, the repressor binds to the lower affinity

site OR3, resulting in downregulation of cI expression

(Hochschild et al., 1986; Ptashne, 1987). In Bxb1 and L5,

the repressor gene promoters do not appear to require an

activator, but both are modestly downregulated by their

repressor (two- to threefold). Presumably, this acts to

maintain the required intracellular concentration of the

repressor.

It is very common, especially within phage genomes, to

find genes encoding DNA-binding proteins situated close

to the site of action of the gene product. This is seen

for phage recombination systems (Campbell, 1981),

packaging systems (Catalano et al., 1995) as well as for

most phage immunity systems, and is presumed to result

from the evolutionary advantage of the cis- and trans-

acting components to co-evolve (e.g. by moving together

from one genome to another). However, evolution of

the mycobacteriophage immunity systems described here

must be more complex, as the binding sites are both

numerous and distributed across the phage genomes.

Comparison of the Bxb1 and L5 genomes provides some

insights into how this may occur. First, it is noteworthy that

the switch in immune specificity appears to be quite

complete, and the Bxb1 genome is devoid of L5 repressor

binding sites and vice versa. Secondly, the change in

specificity appears to be achieved through mutations in

the repressor gene and acquisition of cognate repressor

binding sites. Comparison of the genomes suggests that

they can be acquired de novo through DNA insertion or by

base substitution of existing binding sites. However, there

are presumably quite strong evolutionary pressures to

obtain these sites, as a large number of them are present

and they generally conform tightly to a consensus

sequence.

Experimental procedures

Bacterial strains and plasmids

Escherichia coli DH5a was used for all cloning purposes.M. smegmatis mc2155 is a high-efficiency transformationstrain (Snapper et al., 1990). mc2155(L5) and mc2155(Bxb1)are the mycobacteriophage L5 and Bxb1 lysogens of M.smegmatis mc2155 respectively. M. smegmatis mc2155 andmc2155(L5) were grown in Middlebrook 7H9 broth (Difco)supplemented with ADC enrichment and 0.2% Tween 80 withshaking at 378C. mc2155(Bxb1) was grown similarly at 308C.Plasmids were introduced into M. smegmatis strains byelectroporation using a Bio-Rad gene pulser with minormodifications of the method described previously (Snapperet al., 1990). Plasmid pET21a was procured from Novagen.Plasmids pSD5B, pDK10 and pDK16 were kind gifts from DrAnil K. Tyagi, University of Delhi South Campus, India. DNAmanipulations were carried out as described previously(Sambrook et al., 1989). Mycobacteriophage Bxb1 infections,purification of phage and isolation of its genomic DNA werecarried out as described for L5 (Sarkis and Hatfull, 1998).

Cloning, expression and purification of Bxb1 repressor

protein (gp69)

Bxb1 gene 69 coding for the repressor protein gp69 wasamplified by PCR using primers SJ98-2 (5 0-CAAGGAGGACATATGAGAACCACC; co-ordinates 44 774±44 751) andSJ98-3 (5 0-AGGGGCGGGTGGCCATCAGGG; co-ordinates44 235±44 255) to obtain a product of 540 bp (the nucleo-tides that were modified to create restriction enzyme sites areunderlined in the primer sequences). The amplicon wasdigested with NdeI and EaeI and cloned into NdeI±NotI-digested pET21a vector to obtain pSJ6. E. coli BL21(DE3)-pLysS transformed with pSJ6 was grown in Luria±Bertani(LB) medium containing carbenicillin (50 mg ml21) andchloramphenicol (34 mg ml21) to an A600 of 0.8±1.0 at 378Cwith shaking. Expression of gene 69 was induced by theaddition of IPTG to a final concentration of 0.3 mM, and theincubation was continued for an additional 90 min. Next,the culture was chilled on ice, and cells were harvested bycentrifugation at maximum speed at 48C for 10 min. For theanalysis of gene expression, the cell pellet from a 3 ml culturewas boiled in 200 ml of 1 � SDS buffer (60 mM Tris-Cl,pH 6.8, 1% SDS, 350 mM b-mercaptoethanol, 10% glycerol)for 10 min, and proteins were resolved by 10% SDS±PAGEanalysis. For purification of gp69, cells from a 250 ml culturewere suspended in 5 ml of buffer containing 12.5 mMHEPES-MES buffer, pH 7.94, 1 mM dithiothreitol (DTT),



1 mM EDTA, 1 mM phenylmethylsulphonyl fluoride (PMSF)and lysed by freeze thawing in liquid nitrogen. Polyethyle-neimine (PEI) at pH 8.0 was added to the cell-free extract toa final concentration of 0.7% and left on ice for 45 min. Afterclarification, ammonium sulphate was added to the super-natant to a final concentration of 40%. The suspension wascentrifuged at 10 000 g for 15 min at 48C, and the super-natant was loaded on a POROS-PE column using a BioCADSPRINT system (PE Biosystems). Proteins were eluted fromthe column using a gradient of 40±0% ammonium sulphate in12.5 mM HEPES-MES buffer, pH 7.94. Various fractionswere analysed by 10% SDS±PAGE, and those containingpurified gp69 were pooled and dialysed against 4 l of12.5 mM HEPES-MES buffer, pH 7.94, at 48C with fourchanges. Bxb1 gp69 precipitated out of solution and wassolubilized in buffer containing 10 mM Tris-Cl, pH 8.0, 1 mMEDTA, 200 mM NaCl and 50% glycerol for future studies.

DNA-binding studies

The DNA fragments were PCR amplified using Bxb1 genomicDNA as a template with Pfu DNA polymerase (Stratagene).A 232 bp fragment containing the Bxb1 Pleft promoter regionand encompassing the gp69 binding sites 1 and 2 wasamplified using primers SJ98-4 (5 0-CCGGACCGAGTCGACGATGCG; co-ordinates 48 657±48 677) and SJ98-5 (5 0-CGGTGTACGTGTGCTCTAGAGG; co-ordinates 48 888±48 867)(underlined nucleotides indicate the base positions that weremodified to create restriction enzyme sites). Primers SJ98-5and SJ99-2 (5 0-CCACCGGGATGAAGCTGATGATG, position48 763±48 785) amplified the 126 bp product containing thegp69 binding site 1 that overlaps with the Pleft promoter. The106 bp amplicon containing gp69 binding site 2 was obtainedwith primers SJ98-4 and SJ99-1 (5 0-TGCTCGCGGTGATCGCGGGC; co-ordinates 48 762±48 743). Purified ampliconswere digested with appropriate restriction enzymes (232 bpwith SalI and XbaI; 126 bp with XbaI and 106 bp with Sal I)and radiolabelled using [a-32P]-dATP (NEN Life ScienceProducts) with Klenow fragment. Bxb1 gp69-binding assayswere performed with the purified protein essentially asdescribed for L5 gp71 (Brown et al., 1997). In brief, dilutionsof purified gp69 were incubated with the radiolabelled DNAfragments in a 10 ml reaction volume comprising 2 mg of calfthymus DNA, 10 mg of bovine serum albumin (BSA), 30 mMNaCl, 1 mM Tris-Cl, pH 7.9, and 3 mM EDTA for 15 min onice. Ten � sucrose loading dye (3 ml; 60% sucrose,bromophenol blue, xylene cyanol) was added to eachreaction, and the sample was immediately loaded on aprerun native polyacrylamide gel (4% acrylamide, 2.5%glycerol, 3.3 mM NaOAc, pH 7, 6.75 mM Tris-Cl, pH 8,1 mM EDTA) in TEA buffer (6.7 mM Tris-Cl, pH 7.9,3.3 mM NaOAc, pH 7, 1 mM EDTA). Electrophoresis wascarried out at 58C at 200 V.

For DNase I footprinting, pSJ8 (Table 1) was linearizedwith EcoRI and radiolabelled as described above. It wasdigested further with HindIII, and the DNA fragments wereresolved on a 1.5% agarose gel. The gel piece containing the287 bp fragment of the Bxb1 Pleft promoter was cut out, andthe DNA was eluted using a QiaexII gel extraction kit(Qiagen). Binding of gp69 to this DNA fragment wasperformed as mentioned above except that the reaction

was incubated at 378C. Two units of DNase I (10 U ml21;Stratagene) were added to each reaction, and incubation wascontinued at 378C for 12 s. The reaction was stopped byadding 100 ml of DNase I stop buffer (30 mg of tRNA, 0.3 mMNaOAc, pH 7, 10 mM EDTA, 0.01% SDS). Next, thereactions were extracted with buffer-saturated phenol(pH 8.0) once and precipitated with three volumes of chilledethanol. The samples were suspended in 1.2 ml of water and1.8 ml of sequencing stop dye (95% formamide, bromophenolblue, xylene cyanol) and heated at 908C for 5 min. DNAbands were resolved on a 6% denaturing polyacrylamide gel.A G1A sequencing ladder was prepared by Maxam±Gilbertchemical sequencing reactions as described previously(Ausubel et al., 1996).

Construction of reporter fusion plasmids

To investigate the transcriptional capabilities of the diver-gently located Pleft and Pright promoters of mycobacterioph-age Bxb1, a 232 bp DNA fragment carrying these promoterswas amplified using primers SJ98-4 and SJ98-5 (described inthe previous section) and kinased at its 5 0 ends using T4polynucleotide kinase (Boehringer Mannheim). It was thencloned upstream of the promoterless lacZ gene in bothorientations in the promoter probe vector pSD5B (Jain et al.,1997), which had been digested with XbaI and end repairedwith T4 DNA polymerase (New England Biolabs) to obtainpSJ23 and pSJ7. In these plasmids, lacZ was transcribed byPleft in pSJ23 and by Pright in pSJ7. In addition, the 232 bppromoter fragment was cloned in the NcoI site of theintegration-proficient vector pDK16 after filling its stickyends with Klenow fragment (New England Biolabs) and inthe SmaI site of pUC118. The recombinants resulting fromthese ligations were termed pSJ9 and pSJ8, respectively,and were selected such that the Pleft promoter expressed thedownstream lacZ gene in each case. To construct pSJ21 andpSJ22, the 126 bp amplification product containing the gp69binding sites 1 and 3 was obtained with the primers SJ98-5and SJ99-2 (described in the previous section), kinased andcloned in both orientations in pSD5B at its XbaI site after endrepair with Klenow fragment. The orientation of the clonedDNA fragment in the recombinants in which Pleft transcribedthe downstream lacZ reporter gene was designated pSJ22,and the opposite orientation in which Pright expressed lacZwas termed pSJ21. Finally, to construct pSJ26, pDK10(DasGupta et al., 1998) was linearized with EcoRV. It wasthen cloned into the large fragment obtained after thedigestion of pSJ7 with NheI and MscI followed by end repairwith T4 DNA polymerase. This manipulation replaced themycobacterial origin of replication in pSJ7 with mycobacter-iophage L5-based integration signals derived from pDK10.

To study the gene regulation in Bxb1 by the product ofgene 69 encoding the repressor protein, the 735 bp DNAfragment containing the Bxb1 gene 69 along with itsupstream sequences was amplified using primers SJ98-3(5 0-AGGGGCGGGTGGCCATCAGGG; co-ordinates 44 235±44 255) and SJ99-3 (5 0-GGTTGTGACGATGCCGGTTCAGC;co-ordinates 44 969±44 947) and kinased at its 5 0 end.Subsequently, it was cloned into pSJ7 and pSJ23 at theirNheI sites, which were end repaired with Klenow fragment toobtain pSJ10 and pSJ24, respectively, and also in the ScaI



site of pSJ9 to construct pSJ15. pSJ12B was made byinserting the 735 bp amplicon in the SmaI site of pUC118.To identify the promoter region of gene 69, the 735 bpamplicon and the T4 DNA polymerase-treated 293 bpKpnI±EcoRV restriction fragment from pSJ12B (containingthe gene 69 upstream sequences only) were cloned into theend-repaired XbaI site of pSD5B in the correct orientation toobtain pSJ11 and pSJ14 respectively. Finally, pSJ18 andpSJ19 were constructed by inserting the 401 bp KpnI and293 bp KpnI±EcoRV fragments from pSJ12B, after theirtreatment with T4 DNA polymerase into the ScaI site ofpSJ9 respectively.

b -Galactosidase assays

M. smegmatis transformed with the individual plasmids wasgrown in Middlebrook 7H9 medium supplemented with ADC,Tween 80 and kanamycin (20 mg ml21) to an A600 of 2±2.5.The cells were harvested by centrifugation and suspended inone-fifth of the culture volume of buffer containing 100 mMTris-Cl, pH 8.0, 1 mM EDTA, 1 mM DTT and 1 mM PMSF.Cells were disrupted by sonication and centrifuged at12 000 g at 48C for 15 min. The cell-free extracts wereused for b-galactosidase assays as described previously(Miller, 1972; Jain et al., 1997) using o-nitrophenyl b-D-galactopyranoside (ONPG) as substrate. The enzyme activitywas expressed as nmol of ONPG converted to o-nitrophenolmin21 mg21 protein.

RNA isolation and primer extension analysis

M. smegmatis transformed with the appropriate plasmid wasgrown to an A600 of 2±2.5 in 10 ml of Middlebrook 7H9 mediumsupplemented with ADC and kanamycin (20 mg ml21). Thecells were suspended in 100 ml of TE containing 16 mg ml21

lysozyme and incubated at 378C for 30 min. The cell suspen-sion was transferred to a 1.5 ml microfuge tube containing400 ml of 0.5 mm Zirconia beads (Biospec Products) and350 ml of RLT buffer (RNeasy kit; Qiagen). The tube wasvortexed five times by giving 30 s pulses with chilling in betweenthe pulses. This was followed by centrifugation in the cold for5 min. The supernatant was collected in a fresh tube, andtotal RNA was purified with the RNeasy kit according to themanufacturer's instructions. RNA (10 mg) was digested with10 U of DNase I (RNase free; Stratagene) at 378C for 15 minand purified using the RNeasy kit.

For primer extension reactions, primers were end labelledwith [a32P]-ATP (NEN Life Science Products), and 10 mg ofthe total RNA was reverse transcribed using Superscript II(Life Technologies) according to the manufacturer's instruc-tions. Sequencing reactions were carried out using aThermoSequenase cycle sequencing kit (Amersham Phar-macia Biotech). Reactions were resolved on a 6% denatur-ing polyacrylamide gel before autoradiography. PrimersSJ98-4 (described in the previous section), SJ99-4 (5 0-GGGGAGCTGTTCTCTGGTGG; co-ordinates 44 736±44 755) and SJ99-5 (5 0-GAACGACGACTGATCGAACG;co-ordinates 49 025±49 006) were used for the determina-tion of transcription start sites of Pleft, gene 69 promoter andPright respectively.

Acknowledgements

We are grateful to Dr Anil K. Tyagi, India, for providing themycobacterial promoter probe vectors pSD5B, pDK10 andpDK16. We thank Aisha Mitchell for excellent technicalassistance, and Lori Bibb for valuable comments on themanuscript. This work was supported by grant AI28927 fromthe National Institutes of Health.

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Transcriptional regulation and immunity in mycobacteriophage Bxb1: Mycobacteriophage Bxb1 gene...

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