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Journal of Bacteriology, January 2001, p. 768-772, Vol. 183, No. 2
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.2.768-772.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Competence Modulation by the NADH Oxidase of
Streptococcus pneumoniae Involves Signal
Transduction
José R.
Echenique and
Marie C.
Trombe*
Laboratoire de Genétique et Physiologie
Bactérienne, Centre Hospitalo Universitaire de
Rangueil, Université Paul Sabatier, 31403 Toulouse Cedex,
France
Received 27 June 2000/Accepted 16 October 2000
 |
ABSTRACT |
Oxygen controls competence development in Streptococcus
pneumoniae. Oxygen signaling involves the two-component signal
transduction systems CiaRH and ComDE and the competence-stimulating
peptide encoded by comC and processed by ComAB. We found
that NADH oxidase (Nox) was required for optimal competence.
Transcriptional analysis and genetic dissection showed that Nox was
involved in post-transcriptional activation of the response regulator
ComE and in the transcriptional control of ciaRH and
comCDE. Thus, in S. pneumoniae, Nox, with O2 as its secondary substrate, is part of the
O2-signaling pathway.
| |
TEXT |
Competence development in
Streptococcus pneumoniae is strongly dependent on the
availability of oxygen in cultures. The comCDE operon, which
encodes ComC, the precursor of the competence-stimulating peptide, and
its dedicated two-component system (TCS), ComDE (4, 5), is
the major target of this regulation. A correlation has been found
between the level of comCDE transcripts and developmental transformability in culture. Another TCS, encoded by
ciaRH, is involved in this regulation (3).
Loss-of-function mutations in ciaRH derepress competence,
suggesting that ciaRH negatively regulates the level of
comCDE transcripts and competence development (2). The NADH oxidase (Nox) is required for optimal
competence expression in aerobic cultures. Loss-of-function mutations
in nox reduce bacterial transformability by 50% and alter
its expression pattern (1). The role of Nox in signaling
the O2 status of the cultures has been
investigated. We assessed transformability and the levels of
comCDE and ciaRH mRNA in strains carrying a nox loss-of-function mutation alone or in addition to the
comE38KE gain-of-function missense mutation and the
ciaR loss-of-function mutation. The results obtained show
that Nox, whose enzymatic activity relies on oxygen availability,
belongs to the O2 signaling network.
Influence of Nox on levels of ciaRH and
comCDE mRNA.
It has been suggested that the
O2 reductase/NADH oxidase, Nox, of S. pneumoniae is involved in O2 sensing
(1). We therefore compared the levels of comCDE
and ciaRH mRNA in Nox null mutant (Nox0) and wild-type bacteria to determine
whether Nox affected the transcription of these TCSs involved in the
O2 response (2).
Cp8056 (nox71K stop codon mutation) and Cp1015
(nox+) (Table
1) were subjected to a transformation
test, and total RNA from these strains was subjected to Northern
blotting with comE- and ciaR-specific DNA probes,
as previously described (2). In Cp8056, we observed an
early and narrow transformability window, not exceeding an optical
density at 400 nm (OD400) of 0.1 to 0.2, typical
of Nox-defective strains, with an early competence peak and a rapid loss of transformability during exponential growth (1).
The amount of comCDE mRNA paralleled
transformability. The inverse pattern was observed with
ciaRH transcripts. Four to five times more ciaRH
mRNA was detected in cultures in which competence was abolished than in
the wild-type cultures. In cultures grown for 2 h, in which
competence was maximal, the level of comCDE
transcripts increased as the level of ciaRH decreased to
wild-type levels (Fig. 1B). This suggests
that opposite regulatory events control comCDE and
ciaRH transcript levels in nox strains.
Therefore, Nox influences the expression of both ciaRH
and comCDE. It should be stressed that nox
expression was not controlled by Nox activity, regardless of genetic
background and growth status (Fig. 1), as previously suggested by
Western blot analysis and specific activity measurements
(1).
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FIG. 1.
Effect of the nox71K stop codon mutation
on levels of ciaRH and comCDE mRNA and
competence development. Aliquots of cultures of Cp1015 (A) and Cp8056
(nox) (B) growing under aerobiosis in CTM medium
(1) were withdrawn at 30-min intervals. The change in
biomass was measured as OD400 (black bars), and cultures
were subjected to the transformation test (grey bars) or Northern
blotting (2). For transformation tests, chromosomal DNA (1 µg/ml) carrying the rif-23 allele,
conferring resistance to rifampin (2 µg/ml), was used.
Rifr transformants were selected on plates, and their
frequency in the population was calculated as described by Auzat et al.
(1). For Northern blotting, probes specific for comE,
nox, and ciaR were used to detect the
corresponding 2.4-, 1.4-, and 2.1-kb mRNAs, with 16S rRNA used as a
qualitative and quantitative internal control. Quantitative
densitometry of 16S rRNA showed differences of <18% between samples
taken throughout the period of exponential growth from a given culture.
The experiment was repeated with independent cultures to test
reproducibility. The columns are aligned with the corresponding signal
in the Northern blots.
|
|
Role of Nox in signal transduction.
We used genetic dissection
to investigate the correlation between the specific regulation of
comCDE and ciaRH mRNA levels and the
transformability of nox strains. Strain Cp6656
(nox71K stop codon, comE38KE) had a
transformability profile and level of competence
development similar to those of strain Cp6600
(comE38KE) (Fig. 2),
indicating that the gain-of-function mutation comE38KE is
epistatic to nox. Therefore, there was enough active
ComE38KE in this Nox-defective strain for full transformability
throughout the growth cycle. This demonstrates that Nox activity
intervenes in maintaining threshold cellular levels of active ComE for
competence or in the ComE activation step.
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FIG. 2.
Effect of the nox71K stop codon mutation
on competence development in the comE38KE (Cp6656)
genetic background. Strains Cp6600 (comE38KE) (A) and
Cp6656 (nox comE38KE) (B) were grown under aerobiosis
and analyzed as described in the legend to Fig. 1. Blots were
hybridized separately with probes for 16S rRNA, comE,
and nox. 16S rRNA signals differed by <20% between
wells. The experiment was performed three times with independent
cultures to test reproducibility.
|
|
Cultures of strain Cp1856 (
nox71K stop codon
,
ciaR) were also subjected to the competence test, and RNA from
this strain was
subjected to Northern blotting with
comE-specific DNA. The results
obtained were compared to
those for Cp1800 (
ciaR). In contrast
to Cp1800, which is
transformable throughout the growth cycle
(Fig.
3A), the transformability pattern of
Cp1856 (Fig.
3B) was
similar to that of Cp8056 (Fig.
1B), with a peak
early in exponential
growth and a rapid decrease. However, the
ciaR insertion mutation
resulted in high levels of
comCDE transcripts being present throughout
the period of
exponential growth, whatever the genetic background
(
nox+ and
nox). This
suggests that the absence of CiaR is sufficient
for high levels of
comCDE mRNA to be maintained in cells. It is
possible that
both CiaR and ComE control
comCDE transcription
independently. A negative control of CiaR in addition to the positive
control of ComE might adjust the cellular levels of
comCDE
mRNA
to cell needs. In line with this proposal it was shown that a
ciaR loss-of-function mutation and a
comE38KE
gain-of-function
mutation exhibit additive effects on competence
derepression,
leading to transformant yields of 25 to 30% in strains
carrying
both mutations (data not shown). For strain Cp8456
(ciaR
nox),
in which
comCDE transcription was increased due
to a plasmid insertion
mutation in
ciaR, the level of
comCDE mRNA was not related to
transformability in cultures
at ODs higher than 0.1. Nox deficiency
prevented further steps of
signal transduction, resulting in the
abortive transcription of
comCDE in bacteria from cultures with
an
OD
400 greater than 0.1. This effect of Nox was
abolished by
the
comE38KE mutation (Fig.
2). Thus, Nox is
specifically involved
in the control of active ComE, which is essential
for the expression
of late competence genes.
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FIG. 3.
Effect of the nox71K stop codon mutation
on competence development in the ciaR genetic
background. Strains Cp1800 (ciaR) (A) and Cp 1856 (nox ciaR) (B) were grown under aerobiosis and analyzed
as described in the legend to Fig. 1. Blots were hybridized separately
with probes for 16S rRNA, comE, and nox.
16S rRNA signals differed by <20% between wells. The experiment was
performed three times with independent cultures to test
reproducibility.
|
|
These results suggest that Nox exerts transcriptional control over
ciaRH and
comCDE and is required for the
maintenance of
threshold levels of active ComE during exponential
growth for
the positive regulation of late competence genes.
Both effects
culminate in the high yield and specific pattern of
competence
expression in cultures growing exponentially (see Fig.
4 for a
possible regulatory scheme).
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FIG. 4.
Possible regulatory scheme involving Nox and the CiaRH
and ComDE TCSs, as deduced from genetic dissection.
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|
Nox is involved in the recycling of NADH, thereby controlling the redox
status of the cells. O
2 is a secondary substrate
of
Nox, and the activity of Nox therefore depends directly on
O
2 availability. The development of competence in
cultures probably
involves metabolic signals produced by Nox and
transduced via
ciaRH and
comDE, with the
competence-stimulating peptide as an
intercellular mediator.
Loss-of-function mutation in the
nox gene
results in
residual competence levels, while in wild-type bacteria
grown under
microaerobic conditions competence is below the detection
level
(
2). Thus, another route in addition to that involving
Nox
must be involved in O
2 signaling in
S. pneumoniae. Nox activity
also determines the virulence of this
pathogen (
1). It has
recently been reported that the
ciaR insertion mutation greatly
reduces virulence
(
8) and facilitates competence development
in bacteria
grown under microaerobiosis (
2). Steps in the cellular
response to metabolic "messengers" produced by the
O
2-dependent
Nox and transduced by the transfer
of phosphate groups may be
common to virulence and competence. This is
the first time that
such a mechanism, mediating
O
2 signaling via the activity of an
O
2-dependent Nox and related to TCS-mediated
signal transduction,
has been described. The high conservation of Nox
among pathogens,
notably
Streptococcus faecalis and
Streptococcus pyogenes (
1,
6), raises the
question as to whether similar mechanisms for
O
2
adaptation exist in other pathogenic
species.
| |
ACKNOWLEDGMENTS |
This work was supported by Université Paul Sabatier Toulouse
and Rhône-Poulenc Rorer, France. J.R.E was supported by an RPR
postdoctoral fellowship.
We thank Delphine Dos Santos and Suzanne Eychenne for technical
assistance. We also thank the Technical Department of Institut Louis
Bugnard/INSERM, Toulouse, for providing access to certain pieces of apparatus.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Genétique et Physiologie Bactérienne, Centre
Hospitalo Universitaire de Rangueil, Université Paul
Sabatier, 31403 Toulouse Cedex, France. Phone: (33)561322974. Fax:
(33)561322620. E-mail: trombe{at}cict.fr.
| |
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Journal of Bacteriology, January 2001, p. 768-772, Vol. 183, No. 2
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.2.768-772.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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