Journal of Bacteriology, April 1999, p. 2261-2266, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Mutational Analysis of the vacA Promoter
Provides Insight into Gene Transcription in Helicobacter
pylori
Mark H.
Forsyth1 and
Timothy L.
Cover1,2,*
Departments of Medicine and Microbiology and
Immunology, Vanderbilt University School of
Medicine,1 and Veterans Affairs Medical
Center,2 Nashville, Tennessee
Received 29 September 1998/Accepted 20 January 1999
 |
ABSTRACT |
Analysis of 12 Helicobacter pylori promoters indicates
the existence of a consensus 
10 hexamer (TAtaaT) but
little conservation of 35 sequences. In this study, mutations in
either the H. pylori vacA 10 region or the 35 region
resulted in decreased vacA transcription and suggested that
an extended 10 motif is utilized. Thus, despite the lack of a 35
consensus sequence for H. pylori promoters, the 35 region
plays a functional role in vacA transcription.
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TEXT |
Helicobacter pylori
bacteria are curved gram-negative organisms that persistently colonize
the gastric epithelium of humans and other primates. Gastric mucosal
inflammation occurs in all H. pylori-infected persons and
usually does not cause any symptoms. However, H. pylori
infection is a risk factor for the development of peptic ulcer disease
and gastric adenocarcinoma (7).
At present, very little is known about the basic processes of gene
transcription and transcriptional regulation in H. pylori. Our current knowledge of gene transcription in prokaryotes is based
primarily on extensive studies that have been done with Escherichia coli and related organisms (5, 14,
15). The DNA-dependent RNA polymerase holoenzyme of E. coli is composed of a core enzyme with an 
2
'
structure, along with one of several possible 
subunits. The core
enzyme is capable of RNA synthesis and nonspecific DNA binding, and the
individual factors mediate specific binding of the holoenzyme to
promoter elements (for reviews, see references 5,
14, and 15). The general factor used for the transcription of most genes in E. coli is
70 (RpoD) (12), which binds to two hexameric
DNA motifs centered approximately 10 and 35 bp upstream from
transcriptional start points (TSP). Consensus DNA sequences for both of
these hexamers (TATAAT and TTGACA) have been
determined in E. coli, and many other genera of prokaryotes
utilize similar sequences to facilitate binding of RNA polymerase
(10, 11, 18, 27).
Analysis of consensus promoter sequences and RpoD in H. pylori.
To identify consensus promoter sequences in H. pylori, we analyzed 11 different genes for which primer extension
data were available (Fig. 1). A consensus
10 hexamer (TAtaaT), which closely resembles the 10
consensus sequence in E. coli (TATAAT), was identified. However, we were unable to identify an obvious 35 consensus sequence for this set of H. pylori genes.
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FIG. 1.
Alignment of putative H. pylori promoter
sequences. Putative promoter sequences deduced from primer extension
analyses of 11 different H. pylori genes were aligned based
on their TSP, designated as +1. Sequences similar to the consensus
E. coli 10 hexamer (TATAAT) are boxed. The
consensus sequence above the alignment shows highly conserved positions
(capital letters) and weakly conserved positions (lowercase letters).
In a subset of these promoter sequences, there is a 15/13 TGN motif
(indicated with a box) that is similar to a corresponding motif in
E. coli extended 10 hexamers (1, 17). Asterisks
denote the degree of conservation at each position among these
promoters, as follows: *****, 11 of 12; ****, 10 of
12; ***, 9 of 12; **, 8 of 12; and *, 7 of 12. Genes
analyzed are vacA (vacuolating toxin), katA
(catalase), cagA and cagB (genes located in the
cag pathogenicity island), cheY (chemotaxis
response regulator), ureA (urease), hspA (GroES
heat shock protein), hpaA (flagellar sheath protein),
copA (copper-transporting ATPase), repA (plasmid
replication), and sodB (superoxide dismutase) (2, 8, 9,
13, 16, 19, 20, 23, 24, 25).
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|
The absence of a consensus H. pylori 35 sequence led us to
speculate that there may be structural differences between H. pylori RpoD (22) and orthologous proteins in other
bacterial species. To investigate this possibility, we aligned the DNA
binding domains of RpoD from H. pylori 26695 (26)
with the corresponding domains of RpoD from 10 other bacterial genera.
Alignment of the various 2.4 domains, responsible for binding to 10
hexamers, indicates that the H. pylori RpoD sequence differs
from the consensus sequence at four positions (Fig.
2A). More striking is the degree to which
divergence has occurred in the 4.2 domain of H. pylori RpoD,
which typically mediates binding to 35 promoter elements (Fig. 2B).
The high degree of degeneracy in domain 4.2 of H. pylori RpoD is consistent with the presence of 35 promoter elements in
H. pylori that are quite different from those found in
E. coli.
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FIG. 2.
Alignment of 10 and 35 binding domains of bacterial
RpoD proteins. Domains of RpoD responsible for binding 10 and 35
promoter elements (domains 2.4 and 4.2, shown in panels A and B,
respectively) were aligned with the ClustalW algorithm. Domain 2.5, which mediates binding to extended 10 sequences (1), is
found at positions 22 to 39 in panel A. Conservative substitutions are
shown in light gray boxes, and nonconservative amino acid substitutions
are in white boxes. A consensus sequence for each domain is shown below
each alignment. The Swiss-Prot accession numbers are as follows:
H. pylori, P55993; Campylobacter jejuni, AJ
002379; E. coli, P00579; Salmonella typhimurium,
P07336; Pseudomonas fluorescens, P52326; Haemophilus
influenzae, P43766; Bacillus subtilis, P06224;
Xanthomonas campestris, U82763; Streptococcus
pneumoniae, Y11463; Rhodobacter capsulatus, P46400; and
Neisseria gonorrhoeae, P52325.
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Mutational analysis of the H. pylori vacA
promoter.
To experimentally investigate the promoter sites
required for binding of H. pylori RNA polymerase, we
selected vacA (encoding a vacuolating cytotoxin)
(3) as a model and introduced a series of mutations into the
promoter region of this chromosomal gene. Previous mapping of the 5'
end of the vacA transcript in H. pylori by primer
extension analysis has demonstrated that there is a single conserved
TSP located 119 nucleotides upstream of the AUG start codon, which
suggests that vacA transcription is initiated by a single
promoter (8, 21). Alignment of the vacA 10
regions of 12 different H. pylori strains (Fig.
3A) reveals a TAAAAA consensus sequence, which matches the consensus E. coli 10 hexamer
(TATAAT) at four of six positions. Alignment of the
vacA 35 regions reveals a consensus sequence (TTTATG)
that matches the consensus E. coli 35 hexamer
(TTGACA) at three of six positions (Fig. 3B).
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FIG. 3.
Alignment of vacA 10 and 35 promoter
elements. The putative vacA 10 (A) and 35 (B) promoter
elements from 12 different H. pylori strains were aligned
and compared. Solid bars indicate the predicted sites of RNA polymerase
contact, and consensus sequences are indicated below the alignments.
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pCTB2CAT (8), which contains 567 bp of cysS, the
cysS-vacA intergenic region, 274 bp of vacA
coding sequence from H. pylori 60190, and a chloramphenicol
acetyltransferase (CAT) gene inserted at the 3' terminus of
cysS, was used as the template for all site-directed mutagenesis reactions. Inverse PCR for substitution mutagenesis of the
10 and 35 sequences and deletion of 108/67, 66/40, and
37/32 sequences was performed by previously described methods (8). Briefly, oppositely oriented primers with
BglII restriction sites incorporated at their 5' ends were
used to generate substitution mutations at the sites of the 10 and
the 35 hexamers. After completion of 12 cycles of thermal cycling,
template DNA was eliminated by DpnI (New England Biolabs
[NEB], Beverly, Mass.) digestion, and PCR products were then digested
with BglII (NEB), purified by phenol-chloroform extraction
and ethanol precipitation, and recircularized with T4 DNA ligase (NEB).
To generate deletion mutations, oppositely oriented primers were
designed such that the 5' nucleotides of each of the two primers
defined the region to be deleted. PCR products amplified with these
primers were end polished with Pfu DNA polymerase
(Stratagene) and recircularized with T4 DNA ligase. Religated plasmids
(Table 1) were transformed into E. coli DH5. Point mutations in the 10 region as well as substitution and insertion mutations in the 10 to 35 spacer region
were introduced into pCB2CAT by using the GeneEditor system (Promega),
a positive selection method, following the manufacturer's protocols.
Mutation-bearing plasmids were introduced into the chromosome of
H. pylori 60190 VX-1 (a reporter strain that contains a
vacA::xylE transcriptional fusion) by
natural transformation and allelic exchange, as described previously
(4, 8). Mutants were selected on brucella agar plates
containing kanamycin (25 µg/ml) and chloramphenicol (10 µg/ml). As
a control, pCTB2CAT was introduced into H. pylori 60190 VX-1
to generate strain 60190 VX-1 CAT control (8).
To determine whether the putative vacA 10 hexamer is
essential for transcription, we substituted the sequence AGATCT
for the native 10 vacA sequence (TAAAAG)
found in H. pylori 60190. Introduction of this
mutation into the chromosome of H. pylori vacA reporter
strain 60190 VX-1 resulted in a mutant strain, 60190 VPC1/VX-1, with
about 15-fold less XylE activity than the control strain (60190 VX-1
CAT control) (Fig. 4). This indicates
that the native 10 sequence is essential for vacA
transcription, as expected.
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FIG. 4.
Mutations in the vacA 10 promoter element.
Mutations were introduced into the vacA 10 region of a
reporter strain (H. pylori 60190 VX-1), which contains a
vacA::xylE transcriptional fusion.
Strain 60190 VX-1 CAT control is a
vacA::xylE reporter strain that
contains a CAT gene at the 3' end of cysS but no changes in
vacA promoter sequences (8). Strain 60190 VPC1/VX-1 contains a 6-bp substitution for the native 10
vacA hexamer. Other strains contain single nucleotide
substitutions at the 15, 14, 13, and 12 positions of the
vacA promoter. Sites of nucleotide substitutions are
indicated by asterisks. The XylE specific activity of each strain is
shown on the right. These values represent the means ± standard
deviations for triplicate independent assays. P values
represent comparisons with the XylE specific activity of the control
strain (60190 VX-1 CAT control). OD600, optical density at
600 nm.
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Five of the promoters shown in Fig. 1, including vacA,
contain a TGN motif located at positions 15 to 13. To test whether this region might be involved in vacA transcription, point
mutations were introduced at the 15, 14, 13, and 12 positions
of the vacA promoter region in the chromosome of H. pylori 60190 VX-1 (Fig. 4). A transversion mutation at 15 (T to
A) had no effect on the level of vacA transcription, whereas
a transversion mutation at 14 (G to T) reduced vacA
transcription more than twofold. Mutation of the 13 nucleotide had no
effect on vacA transcription, but a mutation at the 12
position (T to G) resulted in a fourfold decrease. Taken together,
these data indicate that the 10 region of the vacA
promoter is essential for vacA transcription; they also
suggest that the 14 position, located outside the consensus 10
hexamer, may contribute to H. pylori RNA polymerase binding.
To determine whether sequences in the 35 region play a functional
role in vacA transcription, we deleted six nucleotides in
this region (TTTATG) from the chromosome of H. pylori 60190 VX-1 (yielding strain 60190 VPC6/VX-1) (Fig.
5). Deletion of this region (37 to
32) resulted in an approximately sixfold decrease in XylE activity.
We also constructed a second mutant strain in which the native
37/32 (TTTATG) sequence was replaced with a heterologous
6-bp sequence (AGATCT), thereby altering six positions within the 35 region. The XylE activity of the 37/32 substitution mutant (60190 VPC7/VX-1) was about fourfold less than the activity of
the control strain (Fig. 5), which suggests that this region is indeed
involved in binding RNA polymerase.
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FIG. 5.
Roles of the 35 hexamer, 10/35 spacing, and the
108 to 40 region in vacA transcription. Various deletion
mutations (open boxes), substitution mutations (vertically hatched
boxes), or an insertion mutation were introduced into the
vacA promoter region of an H. pylori reporter
strain that contains a vacA::xylE
transcriptional fusion (60190 VX-1). The vacA TSP is
represented as +1. The putative vacA 35 sequence
(TTTATG) and the 10 sequence (TAAAAG) are
indicated with arrows, and upstream cysS and CAT genes are
also indicated. Strain 60190 VX-1 CAT control is a
vacA::xylE reporter strain that
contains a CAT gene at the 3' end of cysS, but no changes in
vacA promoter sequences (8). The XylE specific
activity of each strain is shown on the right. These values represent
the means ± standard deviations for triplicate independent
assays. P values represent comparisons with the XylE
specific activity of the control strain (60190 VX-1 CAT control).
OD600, optical density at 600 nm.
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In most 70 promoters, the region between the 35 and
10 hexamers does not play a sequence-specific role in mRNA
initiation, but it is important for the maintenance of the proper
spacing between the two RNA polymerase contact sites (6). To
test, by an alternate approach, whether specific 35 sequences are
important in vacA transcription, we introduced two separate
mutations into the spacer region of the vacA promoter.
First, a 5-bp heterologous substitution was made from 23 to 19
(CCTTA to GATCT). XylE specific activity in this mutant strain (60190 VPC10/VX-1) was similar to that of the control strain (Fig. 5), which
indicates that specific sequences are not required in this spacer
region. Next, a 10-bp heterologous insertion was introduced into the
spacer region between 24 and 23, so that the native 35 and 10
sequences remained intact on the appropriate faces of the DNA helix. In
this mutant strain (60190 VPC11/VX-1), the original 35 and 10 sites
are now further apart and contact with RpoD at both sites
simultaneously would be unlikely. XylE levels in mutant strain 60190 VPC11/VX-1 were markedly reduced, which provides further evidence that
the native 35 sequence is involved in H. pylori RNA
polymerase binding.
To examine the possibility that vacA transcription may
depend on sequences upstream from the 35 hexamer, we deleted a 27-bp region from 66 to 40, as well as a 42-bp region from 108 to 67,
and introduced each of these deletions into the chromosome of H. pylori 60190 VX-1. These sequences span the entire region between
the vacA promoter and the upstream gene (cysS).
XylE levels in these deletion mutants, under standard in vitro culture
conditions, were essentially the same as in the control strain (60190 VX-1 CAT control) (Fig. 5). This demonstrates that all sequences
necessary for basal levels of vacA transcription lie between
39 and 7.
The construction of various mutations in the H. pylori vacA
promoter region allows us to reach several conclusions regarding transcription of this gene. First, as expected, at least a portion of
the predicted 10 hexamer (TAAAAG) is essential for
vacA transcription. Second, mutation of the 14 position
(the G position of the TGN motif) results in a significant decrease in
vacA transcription. This suggests that H. pylori
RpoD may utilize an extended 10 promoter, perhaps analogous to a set
of E. coli 70 promoters that contain a TGN
motif immediately upstream of the canonical 10 hexamer (1, 17,
23). Moreover, we speculate that the 14 vacA
mutation in this study might underestimate the importance of this
position, as it could lead to an alternative promoter (TATAAA).
Finally, despite a lack of consensus 35 sequences among
H. pylori promoters, mutation of this region results in decreased levels of vacA transcription. Thus, transcription
of H. pylori vacA seems to involve the use of both an
extended 10 sequence and a 35 binding site. More than half of the
H. pylori promoters examined in Fig. 1 have a G nucleotide
at position 14, and therefore, we speculate that this nucleotide
might also be important in the transcription of other genes. It will be
important in future studies to experimentally examine the potential use of 35 sequences in transcription of multiple H. pylori
genes and also to determine whether extended 10 sequences are
commonly utilized.
The lack of conservation among H. pylori promoters outside
the 10 hexamer suggests that there may be considerable variation in
the avidity of RpoD binding to different promoters. We speculate that
this may represent a mechanism for determining the levels at which
individual genes are constitutively transcribed. Further study of these
interactions may provide insight into the fundamental process of gene
transcription in H. pylori and may be relevant to
understanding how this organism persists in the human stomach for many decades.
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ACKNOWLEDGMENTS |
This work was supported by grant AI 39657 from the National
Institutes of Health and by the Medical Research Service of the Department of Veterans Affairs. Sequencing facilities used in this
study are supported by NIH grant CA68485.
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FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Infectious Diseases, A3310 Medical Center North, Vanderbilt University School of Medicine, Nashville TN 37232-2605. Phone: (615) 322-2035. Fax: (615) 343-6160.
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Journal of Bacteriology, April 1999, p. 2261-2266, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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