From the Waksman Institute, Piscataway, New Jersey
08854, the § Department of Microbiology and Immunology,
Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada, the
¶ Departments of Molecular Microbiology and of Genetics,
Washington University School of Medicine, St. Louis, Missouri 63110, and the
Department of Genetics, Rutgers, The State University of
New Jersey, Piscataway, New Jersey 08854
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The rpoB and rpoC genes
of eubacteria and archaea, coding respectively for the - and
'-like subunits of DNA-dependent RNA polymerase, are
organized in an operon with rpoB always preceding rpoC. The genome sequence of the gastric pathogen
Helicobacter pylori (strain 26695) revealed homologs of two
genes in one continuous open reading frame that potentially could
encode one 2890-amino acid-long
-
' fusion protein. Here, we show
that this open reading frame does in fact encode a fused
-
'
polypeptide. In addition, we establish by DNA sequencing that
rpoB and rpoC are also fused in each of four
other unrelated strains of H. pylori, as well as in
Helicobacter felis, another member of the same genus. In contrast, the rpoB and rpoC genes are separate
in two members of the related genus Campylobacter
(Campylobacter jejuni and Campylobacter fetus)
and encode separate RNA polymerase subunits. The
Campylobacter genes are also unusual in overlapping one
another rather than being separated by a spacer as in other
Gram-negative bacteria. We propose that the unique organization of
rpoB and rpoC in H. pylori may
contribute to its ability to colonize the human gastric mucosa.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DNA-dependent RNA polymerase
(RNAP)1 is the central enzyme
of gene expression and a major target for regulation. RNAPs are large,
multisubunit protein complexes. The best studied RNAP, from
Escherichia coli (>400 kDa), contains four core
polypeptides: ' (155 kDa),
(150 kDa), a dimer of
(37 kDa),
and one of several possible
(specificity) subunits. RNAPs from
other bacteria have similar subunit composition and exhibit striking
and co-linear sequence similarities with the E. coli enzyme
(1). The two largest RNAP core subunits comprise 60% of the RNAP mass
and appear to be responsible for most of the functions of the
enzyme.
The synthesis of RNAP subunits is coordinately regulated (2), but the
exact mechanisms at play are unknown. In most eubacteria and archaea,
genes encoding the - and
'-like subunits are organized in an
operon with the gene for the
-like subunit (rpoB) always preceding that for the
'-like subunit (rpoC) (3, 4). The two genes are separated by a short, untranslated linker (3, 5,
6),2 whereas in archaea they
overlap by several codons (4).
The genome sequence of the gastric pathogen Helicobacter
pylori (strain 26695) revealed one continuous open reading frame containing the homologs of rpoB and rpoC,
potentially encoding one fused 2890-amino acid-long -
'
polypeptide (8). Our previous analysis using E. coli RNAP
showed that such a
-
' fusion is compatible with RNAP function:
(i) the product of artificially fused rpoB and
rpoC genes of E. coli could assemble into a
functional RNAP in vivo and in vitro and (ii) an
E. coli strain containing the fused rpoBC gene as
its only source for RNAP was viable and contained RNAP of the expected
(
-
')
2 subunit composition (9). This tethering of
E. coli
and
' increased the efficiency of RNAP
assembly in vitro and suppressed an
rpoCts assembly mutation in
vivo.3 It thus seemed
that natural tethering could be advantageous for an organism like
H. pylori that needs to colonize the stomach, an
intrinsically acid-rich and putatively hostile environment (10).
However, RNAP had never been purified from H. pylori, and
therefore post-translational proteolysis of fused
-
' protein followed by assembly into "normal" RNAP with a
'
2 subunit composition could not be ruled out
a priori.
H. pylori belongs to the group of proteobacteria (10).
With the exception of H. pylori, no rpoBC gene
sequences from this group of bacteria were known. Thus, it seemed
possible that rpoBC fusion could be (i) characteristic of
proteobacteria in general; (ii) a specific feature of the
Helicobacter genus; (iii) an accidental feature of the
H. pylori species; or (iv) an accidental feature of the
particular H. pylori strain that was sequenced. To assess these possibilities, we sequenced the rpoB-rpoC junction in
four different strains of H. pylori, in an isolate of
Helicobacter felis (11), and in two species of the related
genus Campylobacter. In addition, we purifed and
characterized the product of rpoBC gene from H. pylori 26695.
Our results establish that in H. pylori 26695 the
rpoB-rpoC gene does in fact encode a fused -
'
polypeptide. In addition, we find that translational fusion of
rpoBC genes is characteristic of two gastric
Helicobacters but not of Campylobacter jejuni and Campylobacter fetus, members of a related genus that
colonize nongastric sites. We suggest that the
-
' tethering in
Helicobacter might (i) be an accident of evolution because
of a frameshift mutation in an ancestor that, like current
Campylobacters, contained overlapping but separate
rpoB and rpoC genes or (ii) help gastric organisms cope with their acid- and urea-rich niche.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacterial Growth and DNA Preparation--
H. pylori
were grown under microaerobic conditions (5% O2, 10%
CO2, 85% N2) on Brucella agar medium
supplemented with 5% horse blood, 1% Isovitalex, amphotericin B (8 mg/liter), trimethoprim (5 mg/liter), vancomycin (6 mg/liter),
essentially as in Ref. 12. For biochemical purification of RNAP
H. pylori 26695 liquid cultures were grown in Brucella broth
with 10% fetal calf serum in 500-ml screw capped flasks; the medium
was equilibrated with 7% O2, 5% CO2 in the
microaerobic incubator for 1 h prior to inoculation, and then the
bacteria were added, and the flasks were sealed and placed on a rotary
shaker at 150 rpm. The bacteria were harvested in late log phase
(OD660 = ~0.8), and the cell pellets were stored at
70 °C. C. jejuni strain H840 and C. fetus
strain have been previously described (13) and were grown at 37 °C
on Brucella agar plates in a microaerobic incubator maintained at 7%
O2, 5% CO2.
Molecular Biology--
The following primers were used for PCR:
the upstream primer (GGGGGTCAAAGGTTTGGGGAAATGGAAGTGTGGGC)
corresponds to H. pylori 26695 rpoBC
positions 3893-3926 ( conserved segment I, see Ref. 1 for
nomenclature); the downstream primer,
TTTGGAGTGCGTGATCGCCACGCCGCATTTTTCGCA is complimentary to
rpoBC positions 4393-4428 (
' conserved segment A).
Standard PCR reactions contained in 100 µl 200 ng of genomic DNA, 4 pmol of each primer, 1 mM dNTPs, and 5 units of
Taq DNA polymerase in the standard PCR II buffer
(Perkin-Elmer) supplied with 2. 5 mM MgCl2. 30 amplification cycles (1 min at 94 °C, 1 min at 48 °C, and 1.5 min
at 72 °C) were performed. PCR fragments (~500 bp) were cloned in
pT7blue blunt vector (Novagene, Inc., Madison, WI) and sequenced using
T7 promoter and U-19 sequencing primers (Novagene, Inc.) primers on
both strands at the Rockefeller University Protein-DNA Technology
Center.
Protein Purification and Sequencing--
4 g of H. pylori (strain 26695) cells were resuspended in 15 ml of lysis
buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, pH 7.9, 1 mM -mercaptoethanol) and
lysed by passage through an Emulsiflex C-5 homogenizer (Avestin). The
lysate was cleared by low speed centrifugation, and PEI was added to a
final concentration of 0.8%. The PEI pellet was collected by low speed
centrifugation, washed by 20 ml of lysis buffer, and extracted with 20 ml of lysis buffer containing 1 M NaCl. Proteins in 1 M NaCl extract were precipitated with ammonium sulfate (0.7 g/ml extract), and the pellet was recovered by centrifugation,
dissolved in 20 ml of lysis buffer, and loaded on a 1-ml heparin HiTrap
cartridge (Amersham Pharmacia Biotech) equilibrated in the same buffer
and attached to a Waters 650 chromatographer. The column was washed
with the buffer + 0.3 M NaCl and eluted with buffer + 0.6 M NaCl. Fractions containing 300-kDa band (monitored by
SDS-PAGE) were pooled concentrated on a C-100 concentrator (Amicon) to
~1 mg/ml, diluted 2-fold with glycerol, and stored at
20 °C.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
rpoBC Genes Are Fused in Helicobacter but Not in Campylobacter-- Two primers that target the rpoB-rpoC junction and that are complimentary to highly conserved sequences in the 3' end of the rpoB portion and the 5' end of the rpoC portion of the H. pylori 26695 rpoBC gene were used for PCR amplification with genomic DNA from the following organisms: H. pylori strains Hp1 (14), J-166 (15), SS1 (16), and NCTC11638 (17); an isolate of Helicobacter felis; and isolates of two different Campylobacter species: C. jejuni, strain H840, and C. fetus (13). In all cases, a single major PCR fragment ~500 bp in length was amplified. The fragment was cloned, and its sequence was determined. Alignment of sequences at and around the rpoB-rpoC junction site is shown on Fig. 1. Each H. pylori strain differed from the 26695 sequence in ~10 of 474 positions (italicized in Fig. 1). This relatively high (~2%) level of DNA polymorphism between different H. pylori strains is consistent with published data on the extent of polymorphism within H. pylori (18, 19). Most of these differences involved third codon positions, and none resulted in changes in the deduced amino acid sequence of the protein. Thus, the rpoB-rpoC fusion is maintained in all four strains of H. pylori.
|
Helicobacter rpoBC Encodes a Fused -
' RNAP Subunit--
The
results of DNA sequencing experiments presented above do not test
directly whether Helicobacter actually produces a fused RNAP
subunit. To critically test this issue, we purified RNAP from H. pylori strain 26695.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The principal conclusion of this work is that a single fused RNAP
rpoB-rpoC gene is a regular feature of at least two species of gastric helicobacters, H. pylori and H. felis,
and that the gene product, a 300-kDa -
' fusion protein is the
predominant or only form of this gene product in vivo.
Because no proteins in H. pylori lysates that would
comigrate with separate RNAP
and
' subunits were detected, the
fused subunit is probably not extensively proteolyzed; it is likely to
be the only source of RNAP
and
' in the cell, as is also the
case for an E. coli strain with a
-
' fusion RNAP that
we had engineered to study holoenzyme topology and assembly (9).
No transcriptional activity was found in fractions of H. pylori extract containing the -
' fused protein under
standard conditions that had been optimized for E. coli RNAP
transcription in vitro, although similarly prepared
RNAP-containing fractions from extracts of E. coli and also
of C. jejuni were active under our assay conditions. It is
known that RNAPs from different eubacterial species can have markedly
different requirements for efficient transcription in vitro
(21-23), and hence, the inactivity of H. pylori RNAP under our present conditions may reflect an unusual buffer or salt
requirement and conditions in the gastric environment in which it
grows. Because H. pylori is a fastidious microbe and rather
difficult to grow in large quantities, we have not yet purified the
large amounts of H. pylori RNAP that should facilitate
establishing a system for transcription by this RNAP in
vitro.
Although H. pylori is extremely diverse as a species, our DNA sequencing results establish that the rpoBC fusion is not just a peculiar feature of the one H. pylori strain that was chosen for the genome project (26695), but rather it is a common feature of H. pylori in general and indeed of at least one other gastric helicobacter. In contrast, rpoB and rpoC are separate genes in the closely related genus, Campylobacter. The Campylobacter genus is also unusual among Gram-negative bacteria, however, because its rpoB and rpoC genes overlap by two codons. A frameshift mutation at or shortly before the overlap area might have created the continuous open reading frame found in present day helicobacters. Alternatively, a frameshift mutation in a Helicobacter-like ancestral rpoBC gene might have created separate but overlapping genes of present day campylobacters. A number of nongastric helicobacters and also members of closely related genera that colonize gastric and nongastric sites have been discovered recently (10, 24), and DNA sequence analyses similar to those carried out here should help us learn how these unusual arrangements of RNAP subunit genes have evolved.
The functional significance of the rpoBC fusion is not
known. In organisms with transcriptional-translational coupling, the rpoB and rpoC genes are always found in the same
operon, suggesting that RNAP assembly in the cell may occur
contranslationally. Fusion of the two genes may further increase RNAP
assembly efficiency. In E. coli, the -
' fusion appears
to stabilize RNAP in vitro and in
vivo.3 Thus the fusion could be advantageous for
gastric helicobacters, which must grow in the putatively hostile,
acid-rich stomach environment. On the other hand, many acidophilic
archaea have separate rpoB- and rpoC-like
subunits and in fact contain natural splits in their
- and
'-like
subunits (7). Experiments aimed at directly testing the importance of
the rpoBC fusion in H. pylori are in progress.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Katherine M. Eaton and Ausra Raudonikiene for generously providing H. felis and H. pylori DNAs.
![]() |
FOOTNOTES |
---|
* This work was supported by a Burroughs Wellcome Fund Career Award in the Biomedical Sciences (to K. S.), grants from Astra Pharma, Canada (to P. H.), Medical Research Council of Canada Grant R-14292 (to P. H.), National Institutes of Health Grants DK48029, AI138166, and HG00820 (to D. B.), and American Cancer Society Grant VM-121 (to D. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence shall be addressed: Waksman Inst., State University of New Jersey, Rutgers, Piscataway, NJ 08854. Tel.: 732-445-6095; Fax: 732-445-5735; E-mail: severik{at}waksman.rutgers.edu.
1 The abbreviations used are: RNAP, RNA polymerase; PEI, polyethyleneimine; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; bp, base pairs.
2 O. J. Nolte (1995) GenBankTM accession number Z54353.
3 T. Naryshkina and K. Severinov, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|