Department of Zoology, University of British
Columbia, Vancouver, British Columbia, Canada
Changes in intracellular 3',5' cyclic AMP (cAMP) concentration
regulate the development of natural competence in
Haemophilus influenzae. In Escherichia coli,
cAMP levels are modulated by a cAMP phosphodiesterase encoded
by the cpdA gene. We have used several approaches to
demonstrate that the homologous icc gene of H. influenzae encodes a functional cAMP phosphodiesterase and that
this gene limits intracellular cAMP and thereby influences competence
and other cAMP-dependent processes. In E. coli, expression of cloned icc reduced both cAMP-dependent sugar
fermentation and 
-galactosidase expression, as has been shown for
cpdA. In H. influenzae, an icc null
mutation increased cAMP-dependent sugar fermentation and competence
development in strains where these processes are limited by mutations
reducing cAMP synthesis. When endogenous production of cAMP was
eliminated by a cya mutation, an icc strain was
10,000-fold more sensitive to exogenous cAMP than an
icc+ strain. The icc strain showed
moderately elevated competence under noninducing conditions, as
expected, but had subnormal competence increases at onset of stationary
phase in rich medium, and on transfer to a nutrient-limited medium,
suggesting that excessive cAMP may interfere with induction. Consistent
with this finding, a cya strain cultured in 1 mM cAMP
failed to develop maximal competence on transfer to
inducing conditions. Thus, by limiting cAMP levels, the H. influenzae cAMP phosphodiesterase may coordinate its
responses to nutritional stress, ensuring optimal competence
development.
 |
INTRODUCTION |
The cyclic nucleotide 3',5' cyclic
AMP (cAMP) is a central mediator of the responses of enteric bacteria
to changes in the nutritional environment. Expression of a large number
of cAMP-controlled genes is elevated when intracellular cAMP levels
peak at the onset of starvation (24) or stationary phase
(38).
In enteric bacteria, intracellular levels of cAMP are determined by the
relative rates of its synthesis, excretion, and degradation (4). Adenylate cyclase, which synthesizes cAMP, has only
basal activity during exponential growth on preferred sugars and is stimulated by the phosphoenolpyruvate:sugar phosphotransferase system
(PTS) when these sugars are depleted from the medium. cAMP is
excreted when preferred sugars are restored (7), but the regulatory significance of excretion is unclear and awaits isolation of
excretion-defective mutants. Finally, cAMP levels can be reduced by cAMP-specific phosphodiesterases, which catalyze cleavage of 3',5'-cAMP to 5'-cAMP. Although the regulatory role of
cAMP phosphodiesterases has been unclear (9), Imamura et al.
recently demonstrated that the cAMP phosphodiesterase produced by the
Escherichia coli cpdA gene modulates intracellular cAMP
levels and proposed that such phosphodiesterases may regulate
expression of cAMP-dependent genes (20).
In Haemophilus influenzae, cAMP regulates development of
natural competence, and cells mutant in the adenylate cyclase
gene (cya) or the cAMP receptor protein (CRP) gene
(crp) cannot become competent (11, 14).
Although direct measurement of cAMP concentrations in H. influenzae has not been achieved (14), competence is
undetectable during exponential growth, when cAMP levels are expected
to be low. It develops spontaneously when exogenous cAMP is added or when endogenous cAMP levels are expected to be high, for example, at
the onset of stationary phase in rich medium. The highest levels of competence are seen when exponential-phase cells are transferred to
the nutrient-limited medium MIV (18), when intracellular cAMP levels are expected to peak.
Competent cells can take up several hundred kilobases of DNA from their
environment. Some of this DNA may be homologously recombined into
the chromosome; nonrecombined DNA is degraded, and the nucleotides are
reused (35). The requirement of cAMP for
competence induction suggests that H. influenzae's
ability to take up DNA from the environment may be a response to
nutritional stress, with the degraded DNA serving as a source of
nucleotides. To explore this possibility, we are investigating the
regulation of intracellular cAMP levels in H. influenzae and their relationship to competence.
We have previously shown that the synthesis of cAMP by adenylate
cyclase is regulated by a simple fructose-specific PTS and that this in
turn regulates competence development (22). Efflux of cAMP
has not been studied. H. influenzae does, however, possess a
cpdA homolog, icc (15, 20). Below, we
show that icc encodes a cAMP phosphodiesterase which
functions together with adenylate cyclase to fine-tune intracellular
cAMP levels and to modulate competence development (and
other cAMP-dependent processes) according to growth rate and/or
nutrient availability.
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids.
Strains and plasmids used in
this study are listed in Tables 1 and
2. Strain RR801 was derived from a
ptsI::kan crr::cat strain obtained from M. Gwinn (17). Double-mutant strains
RR819, RR820, and RR821 were constructed by transforming strains RR668, RR745, and RR801 to spectinomycin resistance with limiting RR812 chromosomal DNA.
Culture conditions.
H. influenzae cells were grown
aerobically at 37°C in brain heart infusion broth (BHI; Difco)
supplemented with hemin and NAD (sBHI), with the recommended
concentrations of antibiotics (6). Additional hemin was
applied to sBHI plates greater than 24 h old. E. coli
strains were routinely grown aerobically in Luria-Bertani broth at
37°C, with the recommended concentrations of antibiotics
(5).
Competence and transformation.
Spontaneous competence
development by H. influenzae was followed during growth in
sBHI, while maximal competence was induced by transfer of
exponential-phase cells to MIV starvation medium (18).
Competence was assessed as transformation frequency. E. coli cells were made competent by transfer to cold
CaCl2 and transformed by standard procedures
(5).
Sugar fermentation assays.
Sugar fermentation by H. influenzae was assayed in Difco phenol red broth (PRB)
supplemented with NAD, hemin, 10% BHI, and 1% sugar to be tested.
After overnight growth in loosely capped tubes, results were scored by
measurement of culture pH. Sugar fermentation by E. coli was scored on Difco MacConkey agar plates containing 1%
lactose or maltose.
Cloning and mutagenesis of icc.
The availability of
the full H. influenzae genome sequence (36) has
simplified the construction of directed mutations in genes of interest.
A 3-kb region of KW20 chromosomal DNA containing the icc
operon (genes HI 0398 and HI 0399) (15) was amplified by PCR
using the following forward and reverse primers (H. influenzae genome positions are given in parentheses):
5'-GCGTAAATCGCCAAGTGACGG-3' (bp 419090 to 419111) and
5'-GCATTCCGTTCAACTTGGGC-3' (bp 422142 to 422122). The
resulting fragment was cloned into the vector pGEM-T (Promega) to
generate pCMP. A gel-purified spectinomycin resistance gene
(spc) cassette from BamHI-cut plasmid pKRP13 was ligated into BclI-cut pCMP (prepared from the dam
E. coli strain GM2123) (Fig.
1), giving pCMP::spc.
MIV-competent KW20 cells were transformed to spectinomycin resistance
with ApaI-linearized pCMP::spc. Southern blotting
of genomic DNA from the new icc::spc
strain RR812 with spc and icc PCR product probes
confirmed that it contained a single cassette insertion in the
icc gene.
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FIG. 1.
The 3-kb H. influenzae icc operon
fragment cloned in pCMP. xseA, exonuclease VII, large
subunit; ompP1, outer membrane protein; hyp., hypothetical
protein; P1 and P2, putative promoter sites; open circle, putative
CRP-binding site; inverted triangle, location of spc
cassette insertion at bp 1573 (a BclI site) of
pCMP::spc.
|
|
-Galactosidase assay.
Cells were grown in M9 minimal
glucose medium (5) supplemented with 0.5% Casamino Acids
and 1 mM isopropyl--D-thiogalactopyranoside. -Galactosidase activity was assayed by using a modification
(33) of the method described by Miller (25).
| |
RESULTS |
Analysis of the icc gene.
The gene now known to
encode the E. coli cAMP-specific phosphodiesterase was
originally characterized as affecting only the expression of the
lacZ gene (19), and the gene sequence was submitted to GenBank under the name icc. Imamura et al.
subsequently demonstrated that this gene encodes a 3',5'-cAMP-specific
phosphodiesterase (20) and renamed it cpdA. The
existence of an H. influenzae icc homolog was noted
(15, 20).
The predicted 274-amino-acid Icc protein shows 53.3% identity and
71.3% similarity to its E. coli homolog, CpdA
(36). Gapped TBLASTN (3) searching of the GenBank
databases and available bacterial genome sequences detected putative
Icc homologs with extensive amino acid sequence identities (E [expect
value] < 1e-30) in only three other bacterial species, all members of
the gamma subdivision of the proteobacteria (31) (Table
3). In addition, cAMP-specific
phosphodiesterases have been purified from the proteobacteria Serratia marcescens (30), Salmonella
typhimurium (2), and Klebsiella aerogenes
(10). As expected, the icc-containing organisms with complete genome sequences available also contain adenylate cyclase
(cya) homologs: H. influenzae, E. coli, Pseudomonas aeruginosa, and S. typhimurium. These findings also suggest that 3',5'-cAMP phosphodiesterases may be limited to the proteobacteria.
The icc gene may belong to a conserved nucleotide-processing
operon. The upstream gene HI 0398 is separated from icc (HI
0399) by only 13 bp (Fig. 1). Similarly, an HI 0398 homolog is found immediately upstream of the icc homologs of P. aeruginosa and Actinobacillus actinomycetemcomitans
and is separated by one gene from the E. coli icc
homolog cpdA (36). HI 0398 and its homologs all
contain a signature sequence common to members of the MutT family of
proteins, whose members hydrolyze nucleoside diphosphate linkages
in various substrates (29). Although we found no putative icc-specific promoters (by sequence comparison with
the E. coli promoter consensus), we identified two
potential promoters upstream of the HI 0398 initiation codon: P1 at bp
419321 to 419351 and P2 at bp 419417 to 419445.
We identified a putative binding site for CRP immediately 5' to P2
(5'-TTTTGTGACTCACTTCAAACTC-3' at bp 419394 to 419416) by comparison with the E. coli CRP-binding consensus
(7) (Fig. 1). This finding suggests that expression of
icc is not constitutive but may be induced as cAMP levels
rise.
Demonstration of a functional icc-encoded
phosphodiesterase.
To determine whether the E. coli and H. influenzae cAMP phosphodiesterase
homologs have similar activities, we compared the effects of
overexpression of each on cAMP-dependent processes in E. coli. Expression of the E. coli lactose and
maltose utilization operons is cAMP dependent, and Imamura et al. have
shown that overexpression of cpdA reduces
transcription of lacZ and catabolism of lactose
analogs (20). As judged by colony color on MacConkey agar
plates supplemented with 1% lactose or maltose, plasmids carrying the
E. coli and H. influenzae homologs of
cpdA reduced fermentation of both sugars by E. coli W3110 to similar degrees. Disruption of the icc
gene by gene cassette insertion in pCMP::spc abolished this
effect, while vector controls pACYC184 and pGEMsxy had no
effect on sugar fermentation (23).
-Galactosidase activity is a more sensitive indicator of
intracellular cAMP concentration in cells expressing different
phosphodiesterase homologs. We found that pCMP, like pAX923
(20), reduced -galactosidase activity (presented as
mean ± standard error of the mean [SEM]) by at least 75% (Fig.
2). These data confirm that the
H. influenzae gene icc encodes a cAMP
phosphodiesterase.
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FIG. 2.
-Galactosidase activity in E. coli
cells carrying cpdA/icc plasmids. Activity is in Miller
units (25). Mean values from three replicates are shown;
error bars represent SEM. (SEM for pAX923 is too small to be visible on
this scale.)
|
|
Regulation of H. influenzae cAMP levels by
icc.
To determine whether the icc gene limits
intracellular cAMP levels in H. influenzae, we
constructed an icc mutant strain and assessed the effects on
known cAMP-dependent processes: ribose fermentation and competence
development.
We have previously shown that utilization of ribose by H. influenzae is cAMP dependent. PRB assays demonstrated that
icc disruption has little effect on ribose fermentation in a
wild-type background (Fig. 3). We
predicted, however, that if the icc mutation prevents degradation of cellular cAMP, it should restore ribose fermentation to
strains with low cAMP (ptsI or crr) but not to a
strain totally lacking cAMP (cya). As expected, an icc
cya strain remained unable to ferment ribose, but icc
ptsI and icc crr strains fermented ribose at almost
wild-type levels, presumably because they cannot degrade the small
amounts of cAMP that they synthesize (Fig. 3).
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FIG. 3.
Effect of icc disruption on ribose
fermentation in wild-type (WT), cya, and PTS-disrupted
backgrounds. A 100-µl aliquot of exponentially growing wild-type or
mutant strains was inoculated into 5 ml of supplemented PRB plus 1%
ribose. After rotation of the cultures overnight, we measured the pH of
each culture and calculated the pH change ( pH). The degree of ribose
fermentation of each mutants strain was expressed as a percentage of
wild-type pH (wild-type H. influenzae cultures in
1% ribose showed an average pH drop from 7.47 to 5.43). Mean values
from three replicates are shown. Error bars represent SEM. (SEM values
for cya, icc cya, ptsI, and icc
crr strains are too small to be visible on this scale.)
|
|
Development of competence for natural transformation is also cAMP
dependent and is our most sensitive indicator of changes in
intracellular cAMP levels in H. influenzae (e.g., Fig.
4). Wild-type H. influenzae strain KW20 reaches transformation frequencies (TF) as
high as 10
2. The cya mutation completely
abolishes competence development (TF < 107)
(14), while ptsI strains show a ~200-fold
reduction in competence (TF = 7 × 105)
(22). As expected, competence remained undetectable (TF < 107) in an icc cya strain, but disruption
of the phosphodiesterase gene in a ptsI background boosted
competence 10-fold (TF = 7.6 × 104)
(23). These data confirm that H. influenzae
cells lacking the icc-encoded phosphodiesterase have
increased intracellular cAMP levels.
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FIG. 4.
cAMP sensitivity of icc cya ( ) and
icc+ cya ( ) H. influenzae
strains. Strains were grown to mid-exponential phase in sBHI, and
competence was induced by transfer of cells to MIV medium containing 0 to 5 mM cAMP, as described. Cells were incubated with MAP7 chromosomal
DNA (1 µg ml1) for 30 min; transformation frequency was
calculated as the number of novobiocin-resistant (Novr)
transformants divided by the total number of cells. Representative
results are shown.
|
|
Sensitivity of H. influenzae icc strains to
exogenous cAMP.
Alper and Ames (2) found that the
presence of a cpd mutation in S. typhimurium strains lacking endogenous cAMP (cya
mutants) reduced 10-fold the exogenous cAMP required for
expression of catabolic operons. We examined the cAMP sensitivity of
H. influenzae cya strains carrying
icc+ and icc alleles by assaying the
sensitivity of competence development to exogenous cAMP. As
expected, the icc strain was much more sensitive to
exogenous cAMP: the lowest concentration tested (10 nM) increased competence 10,000-fold in this strain but only 3-fold in the
icc+ strain (Fig. 4). These data imply that the
H. influenzae icc strain is unable to degrade
exogenously supplied cAMP.
Regulation of competence development by a cAMP
phosphodiesterase.
We found that RR812 developed spontaneous
competence unusually early during exponential growth in rich medium
(Fig. 5). This observation is consistent
with a model in which Icc regulates intracellular cAMP
concentrations throughout growth, not merely when cAMP levels
are high.
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FIG. 5.
Spontaneous competence of RR812 (icc) ()
and KW20 () in sBHI. Transformation frequencies were assayed as
described in the legend to Fig. 4. Representative results are shown.
Novr, novobiocin resistant.
|
|
Our simplest model predicted that cAMP phosphodiesterase inactivation
should increase intracellular cAMP concentrations and that an
icc strain should achieve at least wild-type levels of competence. It was therefore surprising to discover that the
icc strain failed to reach wild-type levels of competence in
both rich and nutrient-limited media (Fig. 5 and
6). We subsequently determined that this
was not due to a simple toxic effect of high intracellular cAMP, since
up to 10 mM exogenous cAMP did not significantly affect H. influenzae viability. Moreover, icc disruption itself does not reduce viability of this strain
it grows with a normal doubling time in batch culture. Instead, we reasoned that the timing of
an increase in intracellular cAMP relative to growth rate or nutrient
availability might be significant in the induction of maximal
competence. To investigate this, we assessed competence of a
cya strain provided with 1 mM exogenous cAMP either
continuously or only after transfer of cells to starvation medium. We
found that cya cells that encounter a dramatic increase in
cAMP coincident with nutrient limitation develop wild-type competence
levels. However, cells cultured in 1 mM cAMP prior to transfer to MIV show delayed and reduced competence, similarly to the icc
strain (Fig. 6). These data suggest that the timing of a cAMP increase is critical in competence induction and that Icc regulates this timing
in H. influenzae.
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FIG. 6.
MIV-induced competence of RR812 (icc) and
KW20 compared with RR668 (cya) precultured with or without 1 mM cAMP. Transformation frequencies were assayed as described in
the legend to Fig. 4. Filled squares, KW20; open circles, RR812
(icc); open triangles, RR668 transferred to MIV plus 1 mM
cAMP; filled triangles, RR668 (cya) cultured in sBHI plus 1 mM cAMP before transfer to MIV plus 1 mM cAMP. Representative
results are shown. Novr, novobiocin resistant.
|
|
| |
DISCUSSION |
Why bacteria limit increases in cellular cAMP.
To
optimize transcription of cAMP-dependent genes involved in nutrient
transport and catabolism, bacterial cells must closely regulate cAMP levels throughout growth. Because cAMP is a
very stable molecule, active mechanisms for cAMP elimination
are needed to quickly reduce cAMP concentrations
whenever conditions improve. It has also been suggested that cAMP
phosphodiesterases may protect cells from the transcriptional
repression that can result from excessive cAMP (2). Such
repression may be caused by the cAMP-CRP complex directly: although
this complex is better known as a transcriptional activator, it
can also function as a transcriptional repressor (7). In
addition, excessive cAMP may prevent efficient transcription of
cAMP-dependent genes by overloading the CRP complex: the CRP protein
has two identical subunits, each of which binds one molecule of
cAMP, and the CRP-cAMP2 complex has much lower
affinity for CRP-binding sites than the CRP-cAMP1
complex (7).
Uncontrolled cAMP increases may repress transcription of competence
genes.
It was puzzling that disruption of the H. influenzae phosphodiesterase gene reduced maximal competence
levels in both stationary phase and MIV (Fig. 5 and 6), even though
ribose fermentation data (Fig. 3) implied that intracellular cAMP
concentrations were raised. However, competence is also thought
to require at least one cAMP-independent regulatory event
(14), and the uncontrolled increase in intracellular cAMP in
the icc strain may interfere with necessary coordination
between the cAMP-dependent and cAMP-independent signals. Thus, one role
of the cAMP phosphodiesterase may be to regulate the timing of
increases in intracellular cAMP.
The biological function of competence.
In H. influenzae, competence is induced by conditions of nutrient
limitation and absolutely requires an increase in cAMP, an established
indicator of nutritional stress. Ferenci and colleagues have
demonstrated that E. coli cAMP levels peak during the
transition to starvation, allowing transient hunger-response expression
of cAMP-dependent genes (13, 26-28). The pattern of
competence development by H. influenzae suggests that
it may also be a hunger response. We have previously shown that
adenylate cyclase activity is influenced by the PTS, which responds to
changes in carbon source availability. We have now shown that an
H. influenzae cAMP phosphodiesterase participates in
regulation of cAMP levels and maximal competence development.
This work was supported by an operating grant to R.J.R. from the
Medical Research Council of Canada. L.P.M. was supported by a
studentship from the Canadian Cystic Fibrosis Foundation.
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Abstract
Introduction
