Microbiology (2011), 157, 2841–2853
DOI 10.1099/mic.0.046987-0
Regulation of the integrase and cassette promoters
of the class 1 integron by nucleoid-associated
proteins
Caran A. Cagle,3 Julia E. S. Shearer and Anne O. Summers
Correspondence
Department of Microbiology, The University of Georgia, Athens, GA 30602-2605, USA
Anne O. Summers
[email protected]
Received 19 November 2010
Revised 14 April 2011
Accepted 16 July 2011
The integrase IntI1 catalyses recombination of antibiotic-resistance gene cassettes in the
integron, a widely found bacterial mobile element active in spreading antibiotic multi-resistance.
We have previously shown that resistance cassette recombination rate and specificity depend on
the amount of intracellular integrase. Here, we used in vivo and in vitro methods to examine
convergent expression of the integrase promoter (Pint) and of the cassette promoters (Pc and P2)
in the prototypical plasmid-borne class 1 integron, In2. Highly conserved Pint has near consensus
”10 and ”35 hexamers for s70 RNA polymerase, but there are 11 naturally occurring
arrangements of Pc alone or combinations of the Pc+P2 cassette promoters (note that P2 occurs
with a 14 or 17 bp spacer). Using a bi-directional reporter vector, we found that Pint is a strong
promoter in vivo, but its expression is reduced by converging transcription from Pc and P2. In
addition to cis-acting convergence control of integrase expression, the regulator site prediction
program, PRODORIC 8.9, identified sites for global regulators FIS, LexA, IHF and H-NS in and near
the integron promoters. In strains mutated in each global regulator, we found that: (1) FIS
repressed integrase and cassette expression; (2) LexA repressed Pint and P2 with the 14 bp
spacer version of P2 and FIS was necessary for maximum LexA repression; (3) IHF activated Pint
when it faced the strong 17 bp spacer P2 but did not elevate its expression versus LexArepressed P2 with the 14 bp spacer; and (4) H-NS repressed both Pint and the 14 bp P2 but
activated the 17 bp P2 cassette promoters. Mobility shift assays showed that FIS and IHF interact
directly with the promoter regions and DNase I footprinting confirmed extensive protection by FIS
of wild-type In2 integron promoter sequence. Thus, nucleoid-associated proteins, known to act
directly in site-specific recombination, also control integron gene expression directly and possibly
indirectly.
INTRODUCTION
The class 1 integron is very important in disseminating
multi-drug resistance in bacteria (Hall & Collis, 1995;
Stokes & Hall, 1989). It consists of two conserved regions
flanking a variable region (Fig. 1a). The central variable
region comprises tandemly arrayed gene cassettes which
are mobile, non-self-replicating DNA elements encoding
an ORF and an integrase-specific recombination site called
attC (Hall & Collis, 1995; Hansson et al., 1997).
Historically, the two canonical integron components were
defined as the conserved region 59 to the resistance gene
cassettes, which encodes the integrase gene (intI1), a unique
site-specific tyrosine recombinase found in chromosomal
3Present address: South-east Poultry Research Laboratory, USDA/
ARS, 934 College Station Road, Athens, GA 30605, USA.
Abbreviation: EMSA, electrophoretic mobility shift assay.
Two supplementary figures and a supplementary table are available with
the online version of this paper.
046987 G 2011 SGM
islands and on large conjugative plasmids, and an adjacent
recombination site, attI, where cassettes are typically
inserted (Hall & Stokes, 1993). The promoters we describe
here are all contained within this so-called ‘59 conserved
region’ of the integron, In2 (Fig. 1b). The 39 conserved
region in class 1 integrons lies beyond any mobile
resistance gene cassettes and consists of a sulfadiazine
resistance gene (sul), a slightly truncated but functional
gene for resistance to quaternary ammonium disinfectants
(qacED) and a short ORF of unknown function (ORF5). It
is thought that these 39 conserved genes are cassettes
which have lost their attC sites and so are no longer
mobile. The 39 conserved region is not a subject of this
work and is not shown in Fig. 1(b) where it would lie to
the far left.
Most gene cassettes lack their own promoters (Stokes &
Hall, 1991; Tolmasky & Crosa, 1993) and are transcribed as
one transcript by a promoter (Pc) located within the
divergently transcribed intI1 gene over 200 bp away from
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C. A. Cagle, J. E. S. Shearer and A. O. Summers
Fig. 1. Promoter sequences and predicted transcriptional regulator binding sites of the ‘59 conserved region’ of integron In2. (a)
A schematic overview of In2 arrangement as it occurs naturally. Only the indicated parts of the 59 conserved region depicted in (a)
were inserted into the vector pCB267. LacZ reports Pint expression and PhoA reports expression of the cassette promoters Pc and
P2 with either of the alternative 17 bp and 14 bp spacers between the ”10 and ”35 hexamers of P2. (b) Promoter constructs
used in this work were Pi217c, Pi217, Pi214 and Pi. The subscripts in the construct names refer to the specific promoters present,
i5Pint, 2175P2 promoter with 17 bp spacer, 2145P2 promoter with 14 bp spacer and c5Pc. The ”35 and ”10 hexamers of Pint
are boxed, and the integrase IntI1 initiation codon is in lower-case bold type. The recognition hexamers (bold type) of the cassette
promoters (P2 and Pc) are on the opposite strand (not shown to reduce the complexity of the figure); they converge with Pint
resulting in a 3 bp overlap with P2. Predicted binding sites (Supplementary Table S1) for FIS (blue), H-NS (purple), IHF (red) and
LexA (green) are numbered. Sites on the top strand are indicated by solid lines with odd numbers; sites on the bottom strand are
indicated by dotted lines with even numbers. The three-cytosine (3C) polymorphism in P214 is indicated by dashes. In Pi217c dots
indicate continuous sequence wrapped from the first line to its continuation as the fifth line. Numbers at the ends of all constructs
indicate the number of nucleotides present in the construct but not shown in the figure. The cassette insertion point would lie 200
bases on the 39 side of the last base in the ”10 hexamer of P2, but attI is not present in these constructs.
the cassette insertion point attI (Fig. 1, legend) (Hall &
Collis, 1995; Recchia & Hall, 1995; Stokes & Hall, 1991).
Cassettes closest to attI have higher expression than those
situated farther from it (Collis & Hall, 1992). A rare
additional cassette promoter (P2) occurs in In2 and a few
other integrons due to a 3 bp insertion that generates a
17 bp spacer fortuitously placed between good 235 and
210 RNA polymerase recognition hexamers (Fig. 1).
Without this insertion P2 with only a 14 bp spacer is
thought to be non-functional; here, we call this promoter
P214 and for consistency in this context refer to P2 as P217.
The integrase promoter Pint reads convergently towards Pc ,
and its 210 hexamer overlaps with the 210 hexamer of P217 /
P214. More research has been done on the transcription of
gene cassette arrays than on that of the integrase, but a recent
2842
comparison of intI transcription in the prototypical
chromosomal Vibrio cholerae integron and the plasmidborne class 1 integron of Escherichia coli showed that LexA
regulates both (Guerin et al., 2009).
Pint is highly conserved in all class 1 integron variants
(Zhang et al., 2000), but the cassette promoters are quite
varied. There are currently eight Pc variants, three of which
sometimes contain P217 resulting in 11 total Pc or Pc+P217
combinations (Jové et al., 2010). The initial variants
discovered were named according to their strength relative
to the Ptac promoter (Lévesque et al., 1994) and the names
of subsequent variants were compared with the same
benchmark by quantitative reverse transcription PCR
(qRT-PCR) (Papagiannitsis et al., 2009) or lacZ fusions
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Microbiology 157
Class 1 integrase and cassette promoters regulation
(Jové et al., 2010). In addition, single base changes 2 bp
upstream of the Pc 210 hexamer affect promoter strength
to varying extents (Burr et al., 2000; Nesvera et al., 1998).
Recently, two novel forms of P217 were designated P2m1
and P2m2 but appear to be inactive (Jové et al., 2010). The
only current evidence for involvement of trans-acting
proteins in cassette promoter(s) regulation comes from
transcriptome analysis of the chromosomal superintegron
of V. cholerae strain N16961; cassette expression was
activated by HapR (a regulator of virulence factor TcpP)
and RpoS (stationary phase sigma factor) and repressed by
RpoN (nitrogen stress sigma factor) suggesting that cassette
expression increases in crowded, stressed or non-replicating bacteria (Yildiz et al., 2004) but whether activation
was direct or indirect was not determined.
Here we used bidirectional transcriptional fusions of the
convergent promoters Pint (lacZ reporter) and Pc+P2 or P2
(phoA reporter) of the Tn21 integron (In2) to ask: (i) what
is the activity of wild-type Pint after deletion of one or both
competing cassette promoters; (ii) what is the activity of P2
with 14 or 17 bp spacers; (iii) are there regulators other
than LexA for any of these promoters and, if so, (iv) are
their effects direct or indirect?
METHODS
Bacterial strains, plasmids and culture conditions. Bacterial
strains and promoter constructs are described in Table 1. E. coli
strains were grown in Luria–Bertani (LB) broth at 37 uC and
250 r.p.m. and supplemented with 100 mg ampicillin (Ap) ml21 and
50 mg kanamycin (Km) ml21, the latter only for chromosomal
knockout mutants. Plasmid constructs carrying Pint, P2 and Pc
(Pi217c), Pint and P2 (Pi217) and Pint only (Pi) were constructed by PCR
amplification of the promoter region of In2 in Tn21 of plasmid NR1
incorporating BamHI and HindIII restriction sites using primer pairs
IP212U and IP212L, IPU and IP134L and IPU and IP97L (Table 1).
PCRs (50 ml) were run for 5 min at 95 uC followed by 30 cycles of
30 s at 95 uC, 30 s at 57.4 uC, 30 s at 72 uC and a final elongation step
of 5 min at 72 uC. Amplicons were digested with BamHI and HindIII
and ligated to BamHI- and HindIII-digested pCB267 (Schneider &
Beck, 1986) which contains translational stop codons in all three
reading frames upstream of the lacZ and PhoA initiation codons. The
Pi214 construct carrying Pint and P2 with 14 bp spacer was generated
from the Pi217 construct by PCR amplification with primers IP131U
and IP131L using a QuikChange Site-Directed Mutagenesis kit
(Stratagene).
b-Galactosidase assay. In initial development work we found that
the integrase and cassette promoters were all optimally detectable in
late exponential to early stationary phase. Overnight cultures in LB
broth were diluted 1 : 20 with fresh LB containing 100 mg Ap ml21.
The cells were grown for an additional 6 h, 1 ml aliquots were taken
and cells were pelleted, washed and resuspended (Miller, 1972). Cells
were lysed in 100 mM Na2HPO4.7H2O, 20 mM KCl, 2 mM MgSO4,
0.8 mg CTAB ml21, 0.4 mg sodium deoxycholate ml21 and 5.4 ml 2mercaptoethanol ml21 according to Zhang & Bremer (1995), except
lysis was carried out at 25 uC for 30 min in a 96-well microtitre plate.
ONPG (100 ml of 5 mg ml21) was added to each 200 ml reaction,
which was incubated at 25 uC for 60 min. During incubation, A405
(ONPG) and OD550 (cell debris) readings of sample wells were carried
out 0, 15, 30, 45 and 60 min after addition of ONPG to determine the
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rate of galactoside hydrolysis in each culture aliquot. Each reported
LacZ activity is the average of three independent experiments done in
duplicate. Host strain background activity done at the same time was
subtracted in each experiment. The t-test was used (P,0.05) to assess
the significance of the difference in expression by a specific promoter
in the wild-type host compared with a host strain mutant in a DNAbinding protein.
Alkaline phosphatase assay. Cells were prepared as for the b-
galactosidase assay except that they were washed and resuspended in
1 mM Tris/HCl, pH 8.0. Cells were lysed with 1 mM Tris/HCl,
pH 8.0, 0.8 mg CTAB ml21 and 0.4 mg sodium deoxycholate ml21
at 25 uC for 30 min in a 96-well microtitre plate. A 100 ml volume of
(SO942)104 phosphatase substrate 5 mg ml21 (Sigma-Aldrich) was
added to each 200 ml reaction which was then incubated at 25 uC for
60 min. During incubation, A 405 and OD550 readings of sample wells
were carried out 0, 15, 30, 45 and 60 min after addition of phosphatase
to determine alkaline phosphatase activity. Each reported activity is the
average of three independent experiments done in duplicate and host
strain background activity measured at the same time was subtracted in
each experiment. The t-test was used (P,0.05) to assess the significance
of the difference in expression from a specific promoter in the wildtype host to that same promoter’s expression in a host strain mutant in
a DNA-binding protein.
Electrophoretic mobility shift assay (EMSA). PCR amplicons
were amplified from plasmids carrying promoters (see above) and
cleaned with a QIAquick PCR purification kit (Qiagen). The cleaned
Pi214 PCR product was separated and extracted from the gel with a
QIAquick gel extraction kit (Qiagen) to remove plasmid template.
FIS, IHF and H-NS at 0, 5, 50, 500, 2500 and 5000 nM were mixed
with 5 nM DNA in 10 ml binding buffer 1 [20 mM HEPES (pH 7.5),
100 mM NaCl, 5 % (v/v) glycerol, 100 mg BSA ml21, 2 mM DTT and
1 mM EDTA] for 30 min at 25 uC. Reactions with LexA were carried
out at 0, 20, 100, 200, 1000 and 2000 nM with 5 nM DNA in 10 ml
binding buffer 2 [10 mM HEPES (pH 7.9), 10 mM Tris (pH 7.9), 5 %
(v/v) glycerol, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 50 mg
BSA ml21] (Mazón et al., 2004). Mixed-protein EMSAs were carried
out in buffer 1 with pre-determined minimum protein binding
concentration (5 or 500 nM) for one protein and incubated with 5, 50
or 500 nM concentrations of the other protein (IHF, FIS, LexA or HNS) present at the start of the reaction. All 10 ml of each binding
reaction was immediately loaded onto a 5 % nondenaturing
polyacrylamide ready gel (Bio-Rad) and run at 25 uC in 16 TBE
buffer for 5 min at 120 V, then 40 min at 80 V and stained with
SYBR Green (Invitrogen).
DNase I footprinting. DNase I footprinting by capillary electro-
phoresis was performed as described previously (Yindeeyoungyeon &
Schell, 2000) with slight modifications. The 318 bp PCR amplicon
(318amp) was amplified by PCR from NR1 using IPwtU and IPwtL
fluorescent primers (Table 1) and, thus, encodes the wild-type Tn21
In2 integron promoter region including Pint, Pc and P2 with 17 bp
spacer (Figs 1 and 6). This amplicon was produced by PCR in a 100 ml
reaction by incubation at 95 uC for 1 min, followed by 30 cycles at
95 uC for 1 min, 59 uC for 1 min and 72 uC for 1 min, with a final
extension at 72 uC for 10 min. The 318amp was cleaned by using the
QIAquick PCR Purification kit (Qiagen). The 15 ml protein–DNA
binding reaction was carried out in binding buffer 1 with 2.4 pmol
cleaned 318amp and 6.8 mg total protein (0–5.22 mg DNA-binding
protein plus BSA to total 6.8 mg) and the binding reaction was
incubated at 25–26 uC for 30 min. Then, 7.5 ml DNase I (Roche)
diluted 1 to 10 000 in D buffer [10 mM Tris/HCl (pH 7.5), 10 mM
MgCl2, 5 mM CaCl2, 0.1 mg BSA ml21] was added to the 15 ml
binding reaction and incubated at 25–26 uC for 30 or 35 min. The
reaction was stopped with 2.5 ml 0.5 M EDTA (pH 8) and placed at
4 uC. DNA-binding proteins used for footprinting were: FIS at 2.4,
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C. A. Cagle, J. E. S. Shearer and A. O. Summers
Table 1. Strains, plasmids and primers
Strain, plasmid or primer
Strains
MG1656-197
MG1656-430
BW25113
JW3229-1
JW1702-1
JW1225-2
Plasmids
pNR1
pCB267
pWT
pI2-17
pI2-14
pI
Primers
IP97L
IP134L
IPU
IP212U
IP212L
IP131U
IP131L
IPwtU
IPwtL
Genotype or sequence
lacZ derivative of MG1655
lacZ, DsulA, DlexA
D(araD–araB)567, DlacZ4787( : : rrnB-3),
D(araD–araB)567, DlacZ4787( : : rrnB-3),
D(rhaD–rhaB)568, hsdR514
D(araD–araB)567, DlacZ4787( : : rrnB-3),
D(rhaD-rhaB)568, hsdR514
D(araD–araB)567, DlacZ4787( : : rrnB-3),
D(rhaD–rhaB)568, hsdR514
Source or reference
rph1, D(rhaD–rhaB)568, hsdR514
Dfis779 : : kan, rph1,
Guerin et al. (2009)
Guerin et al. (2009)
CGSC*
CGSC
DihfA786 : : kan, rph1,
CGSC
Dhns746 : : kan, rph-1,
CGSC
Carrying promoter region of In2 in Tn21
Bi-directional (phoA and lacZ) transcriptional reporter vector, ApR
Derivative of pCB267 carrying all three promoters: Pint, P2 and Pc;
referred to as Pi217c
Derivative of pCB267 carrying the Pint integrase promoter and P2 with
17 bp spacer; referred to as Pi217
Derivative of pCB267 carrying the Pint integrase promoter and P2 with
14 bp spacer; referred to as Pi214
Derivative of pCB267 carrying only the Pint integrase promoter;
referred to as Pi
R. Rownd, University of
Wisconsin
Schneider & Beck (1986)
This study
This study
This study
This study
59-ATGACTAAGCTTTTGGGGTACAGTCTAT-39
59-GGTGGTAAGCTTGCAGTGGCGGTTTT-39
59-TCGTTGGATCCCCATAACATCAAACAT-39
59-GGATCCCCACGGCGTAACG-39
59-AAGCTTACGAACCCAGTGGACAT-39
59-GGCATAGACTGTACAAAAAAACAG-39
59-CTGTTTTTTTGTACAGTCTATGCC-39
59-(NED)-AACATCGTTGCTGCTCCATAA-39
59-(6-FAM)-TTCGCCAGCCAGGACAGAAAT-39
*CGSC, E. coli Genetic Stock Center; http://cgsc.biology.yale.edu/index.php.
4.8, 12.0 and 24.0 pmol; IHF at 12, 24, 30, 60, 120 and 240 pmol; HNS at 4.8, 24 and 48 pmol. Footprinting with combinations of DNAbinding proteins was performed with: 24 pmol FIS with 60 pmol IHF;
24 pmol FIS with 24 pmol H-NS; and 24 pmol H-NS with 60 pmol
IHF. Footprinting of 318amp was also carried out with LexA and
MerR (each at 4.8, 24 and 48 pmol) as negative controls.
A 2 ml sample of the 25 ml footprinting reaction was checked by
agarose gel electrophoresis for evidence of successful digestion (results
not shown); then 7 ml HPLC-grade water was added, the resulting
30 ml was extracted with phenol : chloroform : isoamyl alcohol
(25 : 24 : 1), and the aqueous phase was cleaned by Centri-Sep column
(Princeton Separations). Aliquots (2 or 3 ml) of cleaned footprinting
reaction (column eluate) were each added to 10 ml deionized
formamide and 1 ml 6-carboxyl-X-rhodamine (ROX)-labelled DNA
ladder (Georgia Genomics Facility) in a 96-well plate for capillary
electrophoresis using an ABI3730 sequencer. GeneMapper 4.0 (Applied
Biosystems) was used for data analysis. Capillary electrophoresis of
a 318amp BbvI partial digest revealed that the fragment sizes estimated
by GeneMapper based on the ROX ladder were within 1–4 nt for
the top strand and within 0–1 nt for the bottom strand
due to the differing mobility of the 2,79,89-benzo-59-fluoro-29,
4,7-trichloro-5-carboxyfluorescein (NED) and 6-carboxyfluorescein
2844
(6-FAM) fluorescent markers. The positions for the protected
nucleotides on the top strand (Fig. 6) were assigned based on a
combination of the GeneMapper position estimates, bottom strand
protected positions and the predicted FIS binding sites (Figs 1 and 6,
Supplementary Table S1, available with the online version of this
paper).
RESULTS
Promoter strength of Pint, Pc and/or P2 when
alone or in competition
Variation in cassette promoters (Lévesque et al., 1994;
Papagiannitsis et al., 2009) represents only part of the
integron transcription mechanics because Pc and P2
converge on, and the latter partially overlaps, the integrase
promoter (Pint) by 3 bp. Owing to the strong predictions
(Supplementary Table S1 and see below) of multiple
overlapping binding sites for four distinct nucleoidassociated proteins we chose traditional deletion analysis,
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Class 1 integrase and cassette promoters regulation
*
250
1400
(a)
1200
200
*
LacZ activity
LacZ activity
1000
150
*
100
*
*
*
*
50
*
Pi217c
90 000
80 000
*
*
1600
1400
40 000
Pi
(d) *
1200
*
*
*
*
30 000
20 000
PhoA activity
50 000
*
400
0
70 000
60 000
600
Pi214
*
(c) *
*
800
200
*
*
Pi217
0
PhoA activity
(b) *
1000
800
*
600
400
10 000
200
0
Pi217c
Pi217
0
Pi214
Fig. 2. Expression from Pint and the cassette promoters in global regulator mutant strains. (a, b) Expression of the integrase
promoter Pint assayed by LacZ activity. (a) In the Pi217c construct Pint converges with both cassette promoters Pc and P2; in the
Pi217 construct Pint converges with P217 only; and in the Pi214 construct Pint converges with the disabled P214 promoter. (b) In
the Pi construct Pint does not converge with either cassette promoter. Host strains are wild-type (black), fis (dark grey), ihf
(medium grey), hns (light grey) or lex (white). (c, d) Expression of the cassette promoters assayed by PhoA activity. (c) The
Pi217c construct reports the combined activity of Pc and P217 converging with Pint and the Pi217 construct reports the activity of
P217 alone converging with Pint. (d) The Pi214 construct reports the activity of P214 alone converging with Pint. Host strains are
as in (a) and (b). Error bars represent SD and asterisks indicate P-values ,0.05 for comparison of the mutant with its wild-type
parent. See construct details in Fig. 1 and summary in Fig. 7.
rather than point mutations, as a first step in dissecting
regulation of these three promoters, since single mutations
in any of them could also have affected one or more of the
others (Fig. 1b). We found that Pint expressed best alone (in
the Pi construct), increasing 6.5-fold in the wild-type host
when both competing cassette promoters Pc and P217 were
removed (Fig. 2a, b, black bars). Surprisingly, deletion of Pc
(see the Pi217 construct) dramatically weakened Pint
expression (Fig. 2a, black bar) making it almost 12-fold
less active than when alone (Fig. 2b, black bar); the possible
bases for this result will be discussed below. Moreover,
without Pc, additional removal of three CCCs to make the
construct with a 14 bp spacer, Pi214, further diminished Pint
expression (Fig. 2a, black bars) likely due to formation of a
LexA site by the three-base deletion (Fig. 1, green line; and
see below) (Guerin et al., 2009).
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Previous tests of cassette expression in Tn2603 established
that P217 accounted for approximately 90 % of total cassette
expression (Papagiannitsis et al., 2009). Our phoA transcriptional fusions to P217 alone (construct Pi217) and to
P217+Pc (Pi217c construct) confirmed that Pc contributed
little to total cassette expression (Fig. 2c, black bars). It has
been assumed that P2 with a 14 bp spacer is non-functional.
Indeed, although not completely inactive, construct Pi214
had less than 1 % of P217 function (Fig. 2d, black bars).
Predicted FIS, IHF, H-NS and LexA transcriptional
regulator binding sites
To identify possible regulators of the integron promoters
we used the virtual footprint tool PRODORIC (http://www.
prodoric.de) (Münch et al., 2003) to search the entire In2
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C. A. Cagle, J. E. S. Shearer and A. O. Summers
integron promoter region for all known regulatory protein
binding sites. PRODORIC predicted several potential binding
sites for FIS, H-NS and IHF (Fig. 1b), multifunctional
proteins that can serve as recombination accessory
elements, nucleoid compaction proteins and global transcriptional regulators of many distinct metabolic pathways
(Dame et al., 2001; Dorman, 2009; Gottesman, 1984;
Luijsterburg et al., 2006; Navarre et al., 2006). All such
proteins have highly degenerate 10–18 bp sites, so their
identification is based on a probability score (Supplementary
Table S1; Fig. 1b) (Münch et al., 2003). Recent research has
shown LexA repression of Pint in the V. cholerae superintegron (Guerin et al., 2009) and PRODORIC predicted strong
LexA sites on the top and bottom strands of P214 (construct
Pi214; Supplementary Table S1; Fig. 1b) occurring by virtue of
a CCC-deletion in four of the 11 currently known natural
promoter variants.
FIS and LexA repress integron promoters
To assess in vivo a possible role for each of these proteins
we obtained E. coli strains with a single mutation in each,
as well as their respective parental strains. Compared with
expression in the wild-type background, when FIS was
absent, expression of the integrase promoter Pint increased
(Fig. 2a, dark grey bars) by 20 % when competing against
cassette promoters with or without Pc (constructs Pi217c
and Pi217). When both cassette promoters were absent
(construct Pi; Fig. 2b, dark grey bar) the already higher
Pint expression increased another 2.5-fold without FIS.
Interestingly, lack of FIS strongly derepressed Pi facing the
P214 promoter (construct Pi214; Fig. 2a, dark grey bar)
although in the fis host LexA should be bound there. For
the cassette promoters, the increase in the already high
expression of P217 (constructs Pi217c and Pi217) averaged
30 % without FIS (Fig. 2c, dark grey bars). Surprisingly,
even the weak expression of P214 increased 3.5-fold in the
absence of FIS (construct Pi214; Fig. 2d, dark grey bar)
despite its non-ideal shorter spacer region and the expected
strong repression by LexA (Guerin et al., 2009). All of these
observations are consistent with stabilization by FIS of
LexA repression at Pi214; see also below. The alternate
possibility, that lack of FIS simply increased the copy
number of all plasmids, is less likely because the opposing
promoters on the same vector do not increase to the same
magnitude. This is particularly striking in construct Pi214
where expression from integrase promoter Pi increased
over 10-fold but that from the cassette P214 increased only
3.5-fold. Moreover, the size differences in these plasmids
are minuscule and would not lead to copy number changes
simply on the basis of replication time in this ColE1-type
replicon; the entire promoter insert is only 212 bp of a
total 7858 bp for the full-length reporter vector and in its
shortest form, the construct Pi is only 115 bp shorter.
Lack of LexA very modestly increased (~5–10 %) expression of Pint facing P217 with or without Pc (constructs Pi217c
and Pi217; Fig. 2a, white bars) compared with wild-type
2846
since there is no LexA site in these promoters. But, as
expected, with P214 absence of LexA resulted in a 23-fold
derepression of Pint (construct Pi214; Fig. 2a, white bar),
much greater than in the absence of FIS alone (note that
FIS is still present in the lex host). Interestingly, Pint alone
(construct Pi) was derepressed twofold without LexA,
probably because this construct retained half each of its
two LexA sites (top and bottom strands), including the
more conserved 59-CTG of the 16 bp LexA (top strand)
binding site (Münch et al., 2003). For the cassette
promoters, absence of LexA consistently derepressed the
integrase promoters P217+Pc (construct Pi217c), P217 alone
(construct Pi217) (Fig. 2c, white bars) and even the weak
expression of P214 (Fig. 2d, white bar) but none of these
differed significantly from the values for the wild-type host.
Thus, within the limits assayable by transcriptional fusions,
the unexpected observations that lack of FIS compromised
LexA repression of both promoters P214 and Pi suggests
either that FIS stabilizes LexA occupancy via their closely
adjacent sites in the integron promoters region (Fig. 1b) or
that absence of FIS lowers cellular abundance of LexA.
Again, altered copy number of the reporter vector itself is
an unlikely explanation of these results because in the
construct Pi214, the integrase promoter increased 23-fold
but the cassette promoter P214 increased insignificantly
compared with the wild-type host.
IHF may activate integron promoters
Two sites for IHF binding were predicted in the integron
promoters region (Fig. 1). In the absence of IHF, Pint
expression decreased 30 % compared with the wild-type
when it was facing P2 with or without Pc (constructs Pi217c
and Pi217; Fig. 2a, medium grey bars). The low LexArepressed expression of Pint facing P214 decreased to
practically nothing without IHF (construct Pi214; Fig. 2a,
medium grey bar). When unencumbered by convergent
promoters or repressors, Pint expression still declined by
approximately 20 % when IHF was absent (construct Pi; Fig.
2b, medium grey bar). The cassette promoters also decreased
15–25 % without IHF (constructs Pi217c and Pi217; Fig. 2c,
medium grey bars), but there was no change in LexArepressed P214 (construct Pi214; Fig. 2d, medium grey bars).
Thus, within the limits assayable by transcriptional fusions,
IHF may be a weak activator of Pint and of the cassette
promoters not subject to LexA repression. Alternatively, loss
of IHF might cause a slight decrease in copy number of all
plasmids relative to the wild-type parental hosts.
H-NS represses the integrase promoter and
activates cassette promoters not repressed by
LexA
Between one and three H-NS binding sites were predicted
for both forms of P2 (Fig. 1), so we examined the effect of
eliminating H-NS, which binds A/T-rich curved DNA
regions (Navarre et al., 2006; Shao et al., 2008). Without H-NS, Pint expression increased compared with the
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Class 1 integrase and cassette promoters regulation
wild-type host in all promoter constructs, except in Pi217
where it was, surprisingly, undetectable (Fig. 2a, b, light
grey bars; this strain grew very slowly). The extent of hns
derepression of Pint varied widely in the other promoter
constructs; without H-NS Pint increased fivefold in Pi214, but
only 40 % in Pi and ,10 % in the wild-type construct Pi217c
(Fig. 2a, b, light grey bars). The H-NS effect on Pi was
unexpected as this construct lacked any predicted H-NS
binding site; its 39 junction with the vector adds a single
adenine, not enough to be predicted as an H-NS site. In
contrast, expression from the cassette promoters P217 +Pc
and P217 alone (constructs Pi217c and Pi217; Fig. 2c, light grey
bars) decreased 35–40 % in the absence of H-NS, consistent
with its having a modest activating effect on these promoters
not subject to LexA repression. However, P214 expression
increased 60 % without H-NS (construct Pi214; Fig. 2d, light
grey bars). This is surprising as the LexA sites of P214 are
superimposed on one of its two putative H-NS sites,
suggesting that they might compete for occupancy.
However, derepression of P214 in the absence of H-NS
suggests that, as with FIS above, LexA repression may be
stabilized here by H-NS. Here, again plasmid copy number
changes cannot explain expression differences in these
promoters. With construct Pi217c expression of Pint increases
in the hns host while cassette expression decreases. For
construct Pi217c expression of Pint declines to almost nothing
but cassette expression drops by only 40 % and in construct
Pi214 expression of Pint increases fivefold but that of P214 is
only 60 % higher than in the parental host.
physical interaction of the promoters with the corresponding proteins or indirectly via the regulation of some other
necessary transcription factor by these proteins. We first
used EMSAs of each promoter amplicon with purified
LexA, FIS, IHF and H-NS to examine this point. As
expected, at a 1 : 1 molar ratio, LexA readily formed a single
complex with amplicon Pi214; however, it did not retard all
of the DNA encoding Pint+P217 at 100-fold molar excess
(amplicon Pi217; Fig. 3a, b, lanes 2–4). Even at 1000-fold
excess (Fig. 3b, lane 5) some free DNA remained. In
contrast, equimolar FIS gave five conformers with the wildtype promoter amplicon, Pi217c (Fig. 3c, lane 2), leaving
very little free DNA. This electrophoresis pattern suggests
that a single FIS protein binding to any of the several
binding sites can give rise to at least five electrophoretically
distinct conformers of the amplicon. The conformer
distribution became more homogeneous at higher protein
excess (Fig. 3c, lanes 3–5) and no free DNA was visible even
at just a 10-fold molar excess of FIS (lane 3). FIS had
similarly high affinities for the other three promoter
amplicons (Fig. 3d), forming fewer conformers consistent
with the removal of predicted FIS sites in the smaller
amplicons. However, for even the smallest promoter
amplicon (Pi, lane 2), .90 % of the amplicon is retarded
at a 1 : 1 protein : DNA ratio, consistent with a high affinity
for FIS. Thus, LexA and FIS act directly in this promoter
region and, in this assay, FIS has a higher affinity for any
one of its sites than LexA does for its two overlapping sites.
To further examine the in vitro interactions of FIS, IHF and
H-NS with the promoter region of Pi217c, DNase I
footprinting was performed using a 318 bp amplicon
(318amp) including the wild-type In2 integron promoter
region, in which the first position shown for Pi217c (Fig. 1)
corresponds to position 50 of the 318amp (Fig. 4). FIS
In vitro binding of LexA, FIS, IHF and H-NS to the
integron promoter region
The behaviour of integrase and cassette promoters in lex,
fis, ihf and hns mutants might arise directly from the lack of
(a)
(b)
l2
l2
l1
F
F
1
2
3
4
5
(c)
1
2
3
4
5
(d)
f8
f7
f6
f1-f5
F
F
1
2
3
http://mic.sgmjournals.org
4
5
1
2
3
4
5
6
7
8
Fig. 3. Binding of LexA or FIS to amplicons of
the integron promoter constructs. (a, b) LexA
(0, 5, 50, 500 or 5000 nM, lanes 1–5,
respectively) bound to (a) amplicon Pi214
(5 nM; lanes 2–5) or (b) amplicon Pi217
(5 nM; lanes 2–5). (c) FIS (0, 5, 50, 500 or
5000 nM, lanes 1–5, respectively) bound to
amplicon Pi217c (5 nM; lanes 2–5). (d) FIS
(0 nM, odd-numbered lanes; 5 nM, evennumbered lanes) bound separately to each
amplicon Pi (lanes 1, 2), Pi214 (3, 4), Pi217 (5,
6) and Pi217c (7, 8). Free (F) DNA is indicated
by a line. LexA conformers l1 and l2 are
indicated by lines (a, b); FIS conformers f1, f2,
f3, f4 and f5 are indicated by a bracket and FIS
conformers f6, f7 and f8 are indicated by lines
(c). Details of each construct are shown in Fig.
1 and in the summary in Fig. 7.
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2847
C. A. Cagle, J. E. S. Shearer and A. O. Summers
Fig. 4. Regions protected from DNase I cleavage by FIS. The amplicon (318amp; see Methods) has the same sequence as
Pi217c (Fig. 1). Top strand FIS protections (Supplementary Fig. S1) are highlighted in yellow (positions 54–126 and 193–219).
Bottom strand protections (Supplementary Fig. S2) are highlighted in blue (positions 59–89, 99–126 and 181–218) and
sequences protected on both strands are highlighted in green. Arrowheads indicate DNase I hypersensitive sites, black for the top
strand and red for the bottom strand. Predicted FIS binding sites (Supplementary Table S1) and promoters are as shown in Fig. 1.
showed clear regions of DNase I protection for both the
top and bottom strands at 12 pmol (results not shown)
and 24 pmol (see Supplementary Figs. S1 and S2 available
with the online version of this paper), 5 : 1 and 10 : 1 molar
excess over the DNA, respectively. The large cassetteproximal (left-side of Figs 1 and 4) protected region covers
from the Pint 235 hexamer to well beyond the P2 235
hexamer (positions 54–126, Fig. 4). The integrase-proximal
(right side of Figs 1 and 4) FIS-protected region covers the
210 hexamer and spacer of Pc just up to the Pc 235
hexamer (positions 193–219, Fig. 4). The seven predicted
FIS binding sites on the top strand match well to the FISprotected regions (Supplementary Fig. S1). Only five FIS
binding sites were predicted for the bottom strand, but the
protected nucleotides (positions 59–89, 99–126, 181–218,
Fig. 4) match well to those protected on the top strand and
indicate that FIS binding to the top strand also protects the
bottom strand from DNase I cleavage (Supplementary Fig.
S2). Hypersensitive sites were widely distributed with four
(one purine and three pyrimidines) on the top strand (Fig.
4, black arrows) and ten (three purines and seven
pyrimidines) on the bottom strand (Fig. 4, red arrows).
IHF formed one to three discrete complexes in EMSAs with
all promoter amplicons but only at 100- and 1000-fold
molar excess (Fig. 5a–d), typical of other IHF binding
interactions which are of relatively low affinity (Ali et al.,
2001; Arfin et al., 2000; Yang & Nash, 1995). The largest
amplicon Pi217c contained two predicted IHF sites (I1 and
I4) and was the most effective in forming electrophoretically stable conformers, as evidenced by its leaving the
smallest amount of free DNA (Fig. 5a, lane 4). The Pi217
amplicon with one predicted IHF site showed diffuse
retardation by IHF at 10 : 1 and 100 : 1 protein molar
excesses (Fig. 5b, lanes 3 and 4). Although only amplicons
Pi217c, Pi217 and Pi214 have IHF sites detectable by
PRODORIC, these EMSA observations suggested that the in
vivo activator role for IHF seen with all promoter
configurations might involve direct interaction. However,
DNase I footprinting with IHF showed no protection until
60 pmol, a 25 : 1 ratio of IHF to amplicon, also consistent
with low-affinity binding (data not shown). IHF can affect
2848
gene expression indirectly by interacting with other
transcriptional regulators (Freundlich et al., 1992); thus,
IHF may affect int and/or integron gene cassette in vivo
transcription without binding the In2 promoters.
H-NS bound to the amplicons Pi217c, Pi217 and Pi214, (Fig.
5e–g) but not to Pi (Fig. 5h), the only promoter lacking a
predicted H-NS site (Fig. 1). H-NS bound in a range of 1to 100-fold protein molar excess producing one to three
conformers, but the patterns shown occurred only half of
the time for undetermined reasons. DNase I footprinting
with 48 pmol H-NS, a 20 : 1 H-NS to amplicon ratio, gave
a digest fragment pattern nearly identical to the DNase I
protection pattern observed with the negative control
proteins LexA and MerR at the same molarity (data not
shown). Since two standard in vitro methods gave
equivocal results we did not resolve whether the in vivo
affects of H-NS on this system are direct or indirect. H-NS
binding requires curved DNA (Schröder & Wagner, 2002)
whose formation in vitro may be affected by the length and
overall composition of the target DNA.
Competition and cooperation among the
regulatory proteins
The maximum copy numbers of FIS and IHF occur at very
different periods of the growth cycle: in very early
exponential phase there are 30 000 FIS dimers per cell
and only 6000 dimers of IHF (Ali Azam et al., 1999).
However, in late exponential phase, IHF peaks at 27 500
dimers per cell, when FIS has decreased to less than 1000
(Ali Azam et al., 1999). H-NS has a pattern similar to IHF
and LexA’s cellular concentration does not vary so
dramatically with growth phase but increases when DNA
is damaged (Ali Azam et al., 1999; Kelley, 2006). So, we
used competitive EMSA to determine whether these
proteins affect each other’s binding to the integron
promoter region (Fig. 6) and we used combinations of
FIS, IHF and H-NS in DNase I footprinting to determine
the effect of two proteins on wild-type promoter region
binding. FIS+IHF, LexA+IHF and FIS+LexA were
incubated with the wild-type promoter amplicon Pi217c
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Class 1 integrase and cassette promoters regulation
(a) i3
(e)
i2
h3
h2
F
F
1
2
3
4
5
(b) i3
1
2
3
4
5
(f)
h3
h2
i2
i1
F
h1
F
1
2
3
4
1
5
(c)i3
2
3
4
5
(g)
h3
h2
i2
i1
h1
F
F
1
2
3
4
1
5
(d)i3
2
3
4
5
(h)
i1
F
F
1
2
3
4
5
1
2
3
4
5
Fig. 5. Binding of IHF and H-NS to amplicons of the integron promoter constructs. IHF (0, 5, 50, 500 or 5000 nM, lanes 1–5,
respectively) bound to amplicons Pi217c (a), Pi217 (b), Pi214 (c) and Pi (d). IHF conformers i1, i2 and i3 are indicated by lines. HNS (0, 5, 50, 500 or 5000 nM, lanes 1–5, respectively) bound to constructs Pi217c (e), Pi217 (f), Pi214 (g) and Pi (h). All
constructs were at 5 nM. Free (F) DNA is indicated by lines. IHF conformers h1, h2 and h3 are indicated by lines. Details of
each construct are shown in Fig. 1 and in the summary in Fig. 7.
(Fig. 6a) or with short spacer amplicon Pi214 (Fig. 6b, c)
where an IHF site is predicted to overlap with the LexA site
(Fig. 1b). IHF only competed with FIS or LexA binding at
very high excesses such as could occur in late exponential
phase (Fig. 6a, b, lanes 7 and 8). FIS and LexA were
incubated with the amplicon of short spacer Pi214 containing adjacent FIS and LexA sites. LexA clearly altered the
distribution of FIS conformers at lower molar ratios of FIS
(Fig. 6c, compare lanes 3 and 4) but not at higher FIS
ratios. In footprinting with FIS+IHF or FIS+H-NS, the
protection patterns were indistinguishable from the pattern
seen with FIS alone (results not shown). The DNase I
http://mic.sgmjournals.org
protection observed with IHF+H-NS was identical to that
observed with 60 pmol IHF alone. Thus, H-NS did not
increase the non-specific binding of IHF to the 318amp
wild-type integron promoter region.
DISCUSSION
The class 1 integron family has many well-studied cassette
promoter variants, but the integrase promoter (Pint) is
highly conserved and until recently there was no information on Pint expression, or any on regulators affecting Pint
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2849
C. A. Cagle, J. E. S. Shearer and A. O. Summers
(a)
i3
f1-f5
F
1
(b)
2
3
4
5
6
7
8
i2
l1
i1
F
(c)
1
2
3
4
5
6
7
8
f8
f1-f5
l1
F
1
2
3
4
5
6
7
8
and/or the cassette promoters. Here we established that
alone Pint is a relatively strong promoter and its 6.5-fold
weaker expression in its natural configuration in In2 can be
ascribed to direct competition with convergent Pc and P2.
Note that the leader mRNA for the lacZ gene is 109 bp
longer in the Pi217c construct than it is in the two
constructs lacking Pc (Pi214 and Pi217). Counterintuitively,
bringing the LacZ reading frame 145 bp closer to Pint
decreases the absolute LacZ activity of the two Pint
reporters, Pi217 and Pi214 (Figs 1 and 2a, black bars). In
Pi217 the 50 % decrease in LacZ activity suggests that the
secondary structure in the integrase mRNA might delay
turnover of the LacZ mRNA. The further drop in LacZ
activity with construct Pi214 more likely results simply from
the formation of a LexA site immediately downstream of
the Pint 210 hexamer (Fig. 1b). Finally, in construct Pi the
highest absolute activity of Pi (Fig. 2b) is achieved by
removing most of that LexA site. In contrast, the removal
of cassette promoter Pc modestly strengthens PhoA activity
driven by the 17 bp Pi217 promoter (Fig. 2c, black bars);
this might also suggest a protective effect of the secondary
structure in the long cassette mRNA leader which traverses
the same region as the integrase mRNA. Of course, the
weak 14 bp spacer form of P214 is most profoundly affected
2850
Fig. 6. Non-competitive binding of IHF with
FIS and LexA and LexA modulation of FIS
binding. FIS (a) or LexA (b, c) (0 nM, oddnumbered lanes; 5 nM, even-numbered lanes)
incubated with amplicon (all 5 nM) Pi217c (a) or
Pi214 (b, c) and increasing IHF (a, b) or FIS (c)
[0 (lanes 1, 2), 5 (3, 4), 50 (5, 6) or 500 (7,
8) nM). Free (F) DNA is indicated by a line. IHF
conformers i1 and i2, LexA conformer l1 and
FIS conformer f8 are indicated by lines and FIS
conformers f1, f2, f3 and f4 are indicated by a
bracket. Details of each construct are shown in
Fig. 1 and in the summary in Fig. 7.
by its spacer defect (Fig. 2d) as also noted in other recent
work (Guerin et al., 2009). The remaining scant expression
of cassette promoter P214 may only be read through from
an unidentified vector promoter, although its 3.5-fold
derepression in the absence of FIS (which strongly binds
the Pi214 amplicon) suggests that this might be residual
intrinsic activity of this promoter. Note that the derepressed MerR promoter with a 15 bp spacer can produce
.1500 PhoA Miller units with this same vector (Ross et al.,
1989).
Here we show for the first time, to our knowledge, in lateexponential-phase cells, that FIS directly represses the
integrase and the cassette promoters, apparently assisting
LexA, and that IHF may indirectly activate Pint and the
cassette promoters. Since both EMSA and footprinting
show weak or no interaction of IHF with amplicons
containing these promoters, its direct involvement seems
unlikely, and neither LexA nor FIS affects IHF binding. The
small expression decrease from Pint and the cassette
promoters in ihf mutants might arise from a decrease in
plasmid copy number. Our work also shows that in vivo HNS activates Pint in settings where it converges with only
P217 (Fig. 2a) or is repressed by LexA (in P214, Fig. 2a).
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Class 1 integrase and cassette promoters regulation
Although EMSA data tentatively suggest direct H-NS
interaction, this conclusion is tempered by the reproducibility of EMSA and the absence of DNase I protection by HNS on the In2 integron promoter region.
This work brings to light a previously unknown dimension
in the control of integron behaviour, i.e. the involvement
of three global regulators, FIS, IHF and H-NS with
implications for growth cycle effects in addition to the
recently noted LexA-mediated stress response (Guerin
et al., 2009). In other recent work we have shown an
increase in integron recombination products during the
transition from exponential to stationary phase and also in
cells artificially overexpressing the integrase (Shearer &
Summers, 2009); these phenomena likely involve modulation of cellular integrase concentrations by the proteins
we have examined here.
We summarize our observations of these interactions in
late exponential phase cells in Fig. 7. Our model system,
In2, expresses cassettes with the strong P217 (Fig. 7, left
panel) which we examined with (upper) and without
(lower) the ‘weak’ version of the distant Pc promoter.
Removal of Pc results in only a slight increase in cassette
expression but even a small increase in the strong P2
promoter might explain the 50 % decrease in converging
Pint expression in this construct. Some or all of the
remaining FIS and IHF sites function in vivo and in vitro in
both constructs depicted in the left panel. Curiously, loss of
H-NS affected Pint expression differently in these constructs
and varied with cassette promoters as indicated by the slow
growth rate (not shown) and low LacZ production in the
hns mutant strain carrying cassette promoter P217 in
construct Pi217. The decline in cassette expression is not
severe in construct Pi217 when compared with wild-type
Pi217c, so an H-NS effect on integrase expression cannot
simply be accounted for by possible plasmid instability.
The most widely found variant of P2 is P214 (Jové et al.,
2010) in which cassette expression depends entirely on
various constitutive Pc promoters and is strongly repressed,
as is Pi, by LexA, aided by FIS. LexA repression is released
by SOS-inducing stresses including sublethal doses of
antibiotics like trimethoprim, ciprofloxacin and b-lactams
(Butala et al., 2009; Drlica & Zhao, 1997; Guerin et al.,
2009; Lewin & Amyes, 1991; Miller et al., 2004). Integrons
with the rare very strong P217 cassette promoter have
traded LexA repression for promoter competition to
control integrase expression from Pi but have retained
some measure of repression by FIS, which is abundant in
very early exponential phase. Overexpression of recombinases can be dangerous to the cell, and expression of
antibiotic resistance genes when they are not needed is
metabolically costly. Thus, it is clear why the P217 variant
promoter occurs in only 8.7 % of the currently known class
1 integron population (Jové et al., 2010). All of the
promoter variants have the same predicted number of each
type of global regulator sites as the Pi217c construct and its
derivatives examined here, although there are slight
variations in the positions (data not shown). Thus,
involvement of nucleoid-binding proteins is an even more
conserved feature of gene expression in class 1 integrons
than any single variant of the highly variable Pc promoter
itself. The mechanistic details of their contributions to the
evolutionary success of these formidable agents of antibiotic multi-resistance will be the subject of future work.
In promoter constructs with P214 (Fig. 7, middle panel), the
extent to which IHF or H-NS might compete with LexA
binding if a direct interaction occurs would depend on the
affinity of LexA for its integron binding sites relative to its
chromosomal binding sites. Of course, when the SOS
response is enabled by DNA damage, LexA repression of
integrase and cassette promoters would be relieved, but
through most of the period of active growth, promoters
could still be modulated by H-NS and IHF. The apparent
cooperation between LexA and FIS at this promoter
warrants further examination; FIS may alter H-NS or
IHF binding in or near the LexA site, facilitating LexA
binding, or it might simply interact with LexA. Lastly,
without converging cassette promoters, the integrase
promoter alone (Fig. 7, right panel) is still controlled to
a great degree by FIS which will be largely absent during
most of exponential growth and into stationary phase.
Pi217c
Pi214
Pi
P217
Pint
Pint
Pint
Pc
stress
P214
Pi217
Pint
P217
http://mic.sgmjournals.org
Pint
P214
FIS
IHF
LexA
H-NS
Fig. 7. Summary of integron gene expression
influenced by global regulators. Potential
binding by LexA and global regulators to
promoter constructs in late exponential to early
stationary phase cells. Although FIS and IHF
bend DNA 40–906 (Kostrewa et al., 1991)
and 1406 (Rice et al., 1996), respectively, we
do not represent this here since the bend
directions are not known.
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C. A. Cagle, J. E. S. Shearer and A. O. Summers
ACKNOWLEDGEMENTS
Hall, R. M. & Stokes, H. W. (1993). Integrons: novel DNA elements which
We thank Anna Karls (University of Georgia) for purified FIS and
IHF, helpful advice regarding this work and comments on the
manuscript, Didier Mazel (Institut Pasteur) for the lex strain and its
parent and Sylvie Rimsky and Colin Corcoran (École Normale
Supérieure de Cachan, France) for purified H-NS and an H-NS
overexpression plasmid. We also thank Natalie Strynadka (University
of British Columbia) for providing the LexA overexpression plasmid
and John Little (University of Arizona) and Ishita Mukerji (Wesleyan
University) for purified wild-type LexA and IHF, respectively. We
thank Ken Jones (Georgia Genomics Facility, University of Georgia)
for his aid in footprinting and we also thank Georgia colleagues
Sidney Kushner and John Maurer for valuable comments on the
manuscript. This work was supported in part by United States
Department of Agriculture (USDA) grant 99-35212-8680 and
National Science Foundation (NSF) grant 06-26940 to A. O. S. and
was submitted in partial fulfilment of the requirements for the PhD
degree by C. A. C.
Hansson, K., Sköld, O. & Sundström, L. (1997). Non-palindromic
attl sites of integrons are capable of site-specific recombination with
one another and with secondary targets. Mol Microbiol 26, 441–453.
capture genes by site-specific recombination. Genetica 90, 115–132.
Jové, T., Da Re, S., Denis, F., Mazel, D. & Ploy, M. C. (2010). Inverse
correlation between promoter strength and excision activity in class 1
integrons. PLoS Genet 6, e1000793.
Kelley, W. L. (2006). Lex marks the spot: the virulent side of SOS and
a closer look at the LexA regulon. Mol Microbiol 62, 1228–1238.
Kostrewa, D., Granzin, J., Koch, C., Choe, H. W., Raghunathan, S.,
Wolf, W., Labahn, J., Kahmann, R. & Saenger, W. (1991). Three-
dimensional structure of the E. coli DNA-binding protein FIS. Nature
349, 178–180.
Lévesque, C., Brassard, S., Lapointe, J. & Roy, P. H. (1994). Diversity
and relative strength of tandem promoters for the antibioticresistance genes of several integrons. Gene 142, 49–54.
Lewin, C. S. & Amyes, S. G. (1991). The role of the SOS response in
bacteria exposed to zidovudine or trimethoprim. J Med Microbiol 34,
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