![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Journal of Bacteriology, May 2001, p. 2957-2962, Vol. 183, No. 9
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.9.2957-2962.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Competence Regulation by Oxygen Availability and by Nox Is
Not Related to Specific Adjustment of Central Metabolism in
Streptococcus pneumoniae
Sabine
Chapuy-Regaud,1
Frédérique
Duthoit,1
Laurence
Malfroy-Mastrorillo,1
Pierre
Gourdon,2
Nic D.
Lindley,2 and
Marie-Claude
Trombe1,*
Laboratoire de Génétique et
Physiologie Bactérienne EA 3036, Centre Hospitalo-Universitaire
de Rangueil, Université Paul Sabatier, 31403 Toulouse
Cedex,1 and Centre de
Bioingénierie Gilbert Durand, UMR INSA/CNRS and INSA/INRA,
Institut National des Sciences Appliquées, 31077 Toulouse Cedex
4,2 France
Received 17 November 2000/Accepted 6 February 2001
 |
ABSTRACT |
In Streptococcus pneumoniae oxygen availability is a
major determinant for competence development in exponentially growing cultures. NADH oxidase activity is required for optimal competence in
cultures grown aerobically. The implication of oxidative metabolism and
more specifically of Nox on central metabolism has been examined. Glycolytic flux throughout exponential growth revealed homolactic fermentation with a lactate production/glucose utilization ratio close
to 2, whatever the aerobiosis level of the culture. Loss-of-function mutations in nox, which encodes NADH oxidase, did not
change this trait. Consistently, mRNA levels of
glyceraldehyde-3-phosphate dehydrogenase, L-lactate
dehydrogenase, pyruvate oxidase, and NADH oxidase remained comparable
to wild-type levels, as did the specific activities of key enzymes
which control central metabolism. Competence regulation by oxygen
involving the NADH oxidase activity is not due to significant
modification of carbon flux through glycolysis. Failure to obtain
loss-of-function mutation in L-ldh, which encodes the
L-lactate dehydrogenase, indicates its essential role in
pneumococci whatever their growth status.
| |
TEXT |
The aerotolerant anaerobe
Streptococcus pneumoniae is a human pathogen showing high
transformability by soluble DNA. Central metabolism in these bacteria
is classically described as homolactic and genome analysis revealed the
absence of most of the genes involved in aerobic respiration (29 genes
searched for), the tricarboxylic acid (TCA) cycle, and gluconeogenesis
(2), suggesting that fermentative metabolism is the major
catabolic pathway. However, mutational analysis revealed the role of
oxidases having O2 as the substrate in both virulence and
competence. Notably, insertion mutations in spxB (GenBank
accession number L39074), which encodes a putative pyruvate oxidase,
and in nox (GenBank accession number AF014458), which
encodes the H2O-producing NADH oxidase (Nox), severely
reduce experimental virulence and bacterial persistence in
tissues of infected mice (1, 15). Interestingly, the NADH oxidase activity is also required for optimal competence in laboratory strains (1). Oxidative metabolism is thus involved in the
physiological specialization of S. pneumoniae in animals and
in fermentative conditions in the laboratory. Indeed, O2
availability and Nox activity are major determinants for
transcriptional regulation of the early competence genes
comCDE (5). The operon comCDE encodes the procompetence activator and its dedicated two-component signaling system ComDE. Maturation and export of ComC to the
competence-stimulating peptide CSP requires the ComAB ATP-binding
cassette (ABC) transporter. CSP activates the autokinase ComD, with
subsequent activation of the response regulator ComE (9).
This signaling pathway is involved in the transduction of putative
signals produced when bacteria are grown aerobically (5).
In lactic acid bacteria, the presence of oxygen in strains possessing
oxidase activities has a distinct effect on carbon metabolism. Pyruvate
metabolism can shift towards mixed-acid fermentation, thereby
increasing ATP production via acetate generation under various
environmental conditions. Induction of alternative pyruvate metabolic
pathways has been attributed to the antagonistic control of the
L-lactate dehydrogenase (L-Ldh) (GenBank
accession number AJ000336) and of the other pyruvate-consuming
reactions, catalyzed either by pyruvate formate lyase or by pyruvate
dehydrogenase, by the NADH/NAD+ ratio (7, 13).
Constraints imposed on glyceraldehyde-3-phosphate dehydrogenase (Gap)
activity (6) by the NADH/NAD+ ratio trigger
this metabolic shift, leading to significant reorientation of pyruvate
metabolism toward acetate synthesis with the associated gain in energy
generation, plus deviation of carbon flux toward alternative pathways
of pyruvate metabolism, notably the acetoin pathway. Metabolic
reorientation is accentuated in the presence of oxygen
(14), and metabolic engineering strategies have exploited this, by overexpression of the nox gene, which encodes NADH
oxidase (11). However, the effects on central metabolism
of a loss-of-function mutation in nox have not been
addressed. In view of the correlation between Nox activity and
functional characteristics of S. pneumoniae such as
competence and virulence, the possibility that these traits can be
related to changes in energy metabolism due to modified carbon flux
through glycolysis was investigated. The data obtained did not reveal a
shift in central metabolism when the oxidative status of the cultures
was changed or when the NADH oxidase activity of the bacteria was
abolished by mutation. This suggests that the implication of Nox in
competence involves functions other than the direct oxidation of NADH
associated with the central metabolic pathways.
Influence of O2 availability on competence and glucose
fermentation in the nox+ and nox
mutant genetic backgrounds.
Metabolic flux in bacteria was
measured by quantifying the fermentative metabolites produced
throughout exponential growth using high-performance liquid
chromatography (HPLC). Competence development was determined in
parallel in the same cultures by the measurement of transformant
recovery throughout growth (Fig. 1D and
E) in a medium where O2
availability is the single parameter determining competence development
in the wild-type strain Cp1015 (5). Strain Cp1015 showed a
similar pattern of glucose utilization and lactate production during
growth whatever the oxygen status of the culture (Fig. 1A and B), and
this trait was comparable to that of the nox mutant strain
Cp8056 growing in the presence of oxygen (Fig. 1C). In an
OD400 (optical density at 400 nm) window of 0.2 to 0.8, lactate production/glucose utilization ratios were close to 2, reflecting an essentially homolactic metabolism, despite minor trace
amounts of both acetate and ethanol in the medium. In order to
investigate the mechanism of such a strong regulation, the transcript
levels of genes identified as having an important role in controlling
carbon flux through energy metabolism, i.e., genes encoding
L-Ldh, Nox, SpxB, and putative Gap (see Table 1), were
compared by Northern blotting of total RNA with specific probes and
also by measurements of the specific activities of the corresponding
enzymes in crude extracts of nox+ and
nox mutant strains.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 1.
Kinetics of glucose utilization and lactate production
during growth in CTM medium (1) under aerobiosis (A) and
microaerobiosis (B) of the wild-type strain Cp1015, compared to that of
the nox mutant strain Cp8056, grown under aerobiosis (C).
Growth curves (D) were obtained by OD400 measurements.
Profiles of competence development throughout aerobic growth of Cp1015
and Cp8056 are also presented (E). For transformation tests,
chromosomal DNA (1 µg/ml) carrying the rif23
allele allowing resistance to rifampin (2 µg/ml) was used.
Rifr transformants were selected on plates, and their
frequency in the population was calculated as described previously
(1). For HPLC analysis, supernatants of cultures were
filtered through 0.2-µm-pore-size Millipore filters.
|
|
Expression of key enzymes controlling the carbohydrate flux.
Northern blot analysis of total-RNA preparations from cultures
(OD400, 0.1) of the wild-type strain Cp1015 and the
Nox0 mutant Cp8056 was performed using as specific probes a
1,124-bp nox fragment from pNOX2 (GenBank accession number
AF014458, positions 10 to 1133), an 847-bp L-ldh fragment
from pTSS7 (GenBank accession number AF000336, positions 267 to 1103),
a 1,206-bp spxB fragment from pTSS13 (GenBank accession
number L39074, positions 475 to 1680), a 650-bp fragment from pP16
(GenBank accession number X58312, positions 166 to 816), and a 699-bp
GAPD1-GAPD2 PCR fragment (Table 1).
Although it has been extensively shown that in nox mutants
competence peaks at an OD400 of 0.1, in contrast to the
wild-type strain, which becomes competent at an OD400 of 0.3, both strains showed essentially identical levels for the 1-kb
L-ldh, 1.4-kb nox, 1.8-kb spxB, and
1.3-kb gap mRNAs in bacteria from cultures at an
OD400 of 0.1 (Fig. 2). It
should be noted that the calculated sizes of the mRNAs are close to the
sizes of their corresponding open reading frames, indicating that
monocistronic operons encode these different proteins.
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 2.
Effect of nox loss-of-function mutation and
oxygen limitation on nox, L-ldh, spxB, and
gap mRNA levels in cultures at an OD400 of 0.1. Total-RNA preparations from competent cultures of the nox
strain Cp8056 were subjected to Northern blot analysis with probes
specific for ldh, nox, gap, and spxB. 16S rRNA
was taken as a qualitative and quantitative internal control (see Table
1). A parallel analysis was performed on noncompetent cultures of the
wild-type strain Cp1015 grown aerobically or microaerobically to an
OD400 of 0.1. Signals were quantified by densitometry, and
ratios of specific mRNA to 16S rRNA are given. The experiments were
repeated with independent cultures to test reproducibility.
|
|
At this point in growth, no measurable difference was found among the
specific activities of the encoded Gap, L-Ldh, and Nox. In
order to assess other checkpoints in the carbon flux, the specific activities of the fructose diphosphate aldolase and phosphoglycerate isomerase were also measured, confirming that the cellular glycolytic potential was not changed by the nox loss-of-function
mutation (Table 2).
Another trait that characterized the Nox0 strains was the
rapid loss of competence when cultures grew to an OD400
greater than 0.1 (Fig. 1E). To assess whether such a trait involved
specific regulation of carbohydrate metabolism, the levels of the
previously mentioned mRNAs in cells were compared throughout growth up
to an OD400 of 0.8. For both the wild-type and
nox mutant strains, cellular mRNA levels were constant for
L-ldh, spxB, and nox (data not shown); cellular
gap mRNA levels increased slightly (relative value, 1 to 2)
when cultures reached the late-exponential-growth phase (Fig.
3). The physiological importance of
growth phase-related variation in gap transcript levels is
not yet known but is probably related to medium acidification
phenomena. The data obtained indicate that expression of the enzymes
considered in this study did not vary during competence development
and, furthermore, was not influenced by Nox activity.
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 3.
Evolution of gap mRNA levels throughout
growth in cultures from the wild-type strain Cp1015 and the
Nox0 mutant Cp8056. Aliquots of cultures of Cp1015 (A) and
Cp8056 (nox) (B) growing under aerobiosis in CTM medium
(1) were withdrawn at 30-min intervals. Total-RNA
preparations from these cultures were subjected to Northern blotting
with a gap-specific probe. 16S rRNA was used as a
qualitative and quantitative internal control. Signals were quantified
by densitometry, and the 16S rRNA/gap mRNA ratio was
calculated. For each strain, results were expressed relative to data
obtained for cultures grown for 1 h 30 min, taken as 1. The
experiments were repeated with independent cultures to test
reproducibility.
|
|
Although it has been clearly demonstrated that competence is dependent
upon both the presence of a functional Nox (1) and adequate aeration (5), no significant reorientation of
pyruvate metabolism from normal homolactic characteristics could be
demonstrated under competence conditions and in nox strains.
The consequence of a forced reorientation of the glycolytic flux on
competence expression was assessed further by a mutational strategy.
Mutagenesis of L-ldh.
The enzymes providing an
alternative pathway of pyruvate metabolism are present in the cell, as
seen by the presence of trace amounts of mixed-acid fermentation
products. Since this potential is not exploited to increase ATP
generation in the presence of oxygen, at least in competence medium
(1), an attempt was made to force the metabolic flux
through this pathway, thereby revealing a putative "oxidative
phenotype," by mutational blocking of L-Ldh activity.
Attempts to introduce a stop codon mutation by mutating the nucleotides
at positions 241 and 243 (GenBank accession number AJ000336) in the 5'
region of the L-ldh gene (10) using mutational strategies routinely used in the laboratory (1, 5) were unsuccessful (Table 1 and data not shown). Furthermore, insertional mutation by single-crossover integration of the nonreplicative plasmid
pAM239 carrying an L-ldh fragment in the resident chromosome was tried. Both an internal L-ldh fragment (positions 434 to
1103) and a fragment containing the 3' end of L-ldh
(positions 720 to 1173) were cloned into pAM239, and the recombinant
plasmids pLDH1 and pLDH2 were used to transform Cp1015. Recombinant
colonies carrying the inserted plasmid and expressing the spectinomycin resistance gene present on the plasmid were screened for their growth
in spectinomycin-containing medium. Recombinant clones were obtained
only when plasmid pLDH2 was used as donor DNA (10 independent
experiments). Insertion in L-ldh in these clones was verified by PCR amplification of a 650-nucleotide fragment with primers
LDH11 and FPAM (Fig. 4). Transformation of Cp1015 with the
mutagenic plasmids under microaerobic conditions in GasPak jars
(5) gave identical results (data not shown). Plasmid
insertion was tolerated only with plasmid pLDH2, since it probably
allowed the reconstitution of a wild-type copy of L-ldh
(Fig. 4). This suggests that disruption
of L-ldh cannot be obtained and therefore the
L-Ldh pathway is essential in S. pneumoniae,
despite the presence of pyruvate oxidase/NADH oxidase and the
theoretically better energetic yield of the mixed-acid pathway compared
to homolactic fermentation (4). This finding is
reminiscent of work with Streptococcus mutans in which a
loss-of-function mutation in L-ldh was not obtained
(3).
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 4.
L-ldh mutagenesis by plasmid insertion
duplication. The wild-type strain Cp1015 was transformed with the
nonreplicative plasmids pLDH1, containing an internal fragment of
L-ldh (A) and pLDH2, containing a fragment corresponding to
the part of L-ldh encoding the C terminus of the enzyme (B).
(C) Recombinant colonies were selected for resistance to spectinomycin
(spcR) and verified by PCR amplification of the chromosome
with oligonucleotides LDH11 and FPAM (lane 1, Eurogentec SmartLadder
molecular weight marker, lane 2, PCR product of the pLDH2 plasmid,
taken as a control; lanes 3 to 8, PCR products of transformed
colonies).
|
|
In S. pneumoniae expression of the key enzymes Gap and
L-Ldh, and also of SpxB, is strictly regulated, as shown by
constant levels of the corresponding cellular mRNAs, regardless of the oxygen status of the cultures and the presence or absence of functional Nox. Furthermore, Nox has no impact on the specific activity of Gap,
L-Ldh, fructose diphosphate aldolase, or phosphoglycerate isomerase. Neither variations in O2 availability nor in
NADH oxidase activity influence the carbon flux under the conditions
tested, which remained essentially homolactic. In agreement with these data, attempts to obtain mutants devoid of L-Ldh activity
by plasmid insertion mutagenesis were unsuccessful. Despite the
requirements of oxygen and Nox for optimal competence expression under
the control of the two-component signal transducing systems CiaRH and
ComDE (5), no change in the glycolytic flux was related to
the pattern of competence expression in response to oxygen availability. The present work shows that these regulations,
culminating in competence, are not related to stimulation or
reorientation of central metabolism, resulting in increased
production of phosphodonors and subsequent activation of signal
transduction by phosphotransfer through two-component signalling
systems. The role of oxidative metabolism in competence regulation
remains under investigation.
| |
ACKNOWLEDGMENTS |
This work was supported by Université Paul Sabatier, EA 3036, and Rhône-Poulenc Rorer (RPR), Vitry, France. L.M.-M. was supported by an RPR postdoctoral fellowship.
We thank Delphine Dos Santos and Ghislain Fournous for preparing the
bacterial cultures for different analyses and Suzanne Eychenne for
technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Génétique et Physiologie Bactérienne EA 3036, Centre
Hospitalo-Universitaire de Rangueil, Université Paul Sabatier,
31403 Toulouse Cedex, France. Phone: 61 32 29 74. Fax: 61 32 26 20. E-mail: Trombe{at}CICT.FR.
| |
REFERENCES |
| 1.
|
Auzat, I.,
S. Chapuy-Regaud,
G. Le Bras,
D. Dos Santos,
A. D. Ogunniyi,
I. Le Thomas,
J.-R. Garel,
J. C. Paton, and M.-C. Trombe.
1999.
The NADH oxidase of Streptococcus pneumoniae: its involvement in competence and virulence.
Mol. Microbiol.
34:1018-1028[CrossRef][Medline].
|
| 2.
|
Baltz, R. H.,
F. H. Norris,
P. Matsushima,
B. S. Dehoff,
P. Rockey,
G. Porter,
S. Burgett,
R. Peery,
J. Hoskins,
L. Braverman,
I. Jenkins,
P. Solenberg,
M. Young,
M. A. McHenney,
P. R. Rosteck, and P. L. Skatrud.
2000.
DNA sequence sampling and gene disruption for identification of new antibacterial targets in Streptococcus pneumoniae, p. 33-44.
In
A. Tomasz (ed.), Streptococcus pneumoniae: molecular biology and mechanisms of disease. Mary Ann Liebert, Inc., Larchmont, N.Y.
|
| 3.
|
Chen, A.,
J. D. Hillman, and M. Duncan.
1994.
L-(+)-Lactate dehydrogenase deficiency is lethal in Streptococcus mutans.
J. Bacteriol.
176:1542-1545[Abstract/Free Full Text].
|
| 4.
|
Condon, S.
1987.
Responses of lactic acid bacteria to oxygen.
FEMS Microbiol. Rev.
46:269-280[CrossRef].
|
| 5.
|
Echenique, J. R.,
S. Chapuy-Regaud, and M.-C. Trombe.
2000.
Competence regulation by oxygen in Streptococcus pneumoniae: involvement of ciaRH and comCDE.
Mol. Microbiol.
36:688-696[CrossRef][Medline].
|
| 6.
|
Even, S.,
C. Garrigues,
P. Loubière,
N. D. Lindley, and M. Cocaign-Bousquet.
1999.
Pyruvate metabolism in Lactococcus lactis is dependent upon glyceraldehyde-3-phosphate dehydrogenase activity.
Metab. Eng.
1:198-205[CrossRef][Medline].
|
| 7.
|
Garrigues, C.,
P. Loubière,
N. D. Lindley, and M. Cocaign-Bousquet.
1997.
Control of the shift from homolactic-acid to mixed-acid fermentation in Lactococcus lactis: predominent role of NADH/NAD+ ratio.
J. Bacteriol.
179:5282-5287[Abstract/Free Full Text].
|
| 8.
|
Gil, D.
1990.
Elaboration et caractérisation d'un nouveau type de vecteur de clonage à nombre de copies régulable. Thèse de l'Université.
Université Paul Sabatier, Toulouse, France.
|
| 9.
|
Håvarstein, L. S.
1998.
Identification of a competence regulon in Streptococcus pneumoniae by genomic analysis.
Trends Microbiol.
6:297-299[CrossRef][Medline].
|
| 10.
|
Jado, I.,
A. Fenoll,
J. Casal, and A. Perez.
1998.
Nucleotide sequence and chromosomal location of L-lactate dehydrogenase gene from Streptococcus pneumoniae.
Curr. Microbiol.
37:77-79[CrossRef][Medline].
|
| 11.
|
Lopez de Felipe, F.,
M. Kleerebezem,
W. M. de Vos, and J. Hugenholtz.
1998.
Cofactor engineering: a novel approach to metabolic engineering in Lactococcus lactis by controlled expression of NADH oxidase.
J. Bacteriol.
180:3804-3808[Abstract/Free Full Text].
|
| 12.
|
Morrison, D. A.,
M.-C. Trombe,
M. K. Hayden,
G. A. Waszak, and J. D. Chen.
1984.
Isolation of transformation-deficient Streptococcus pneumoniae mutants defective in control of competence, using insertion-duplication mutagenesis with the erythromycin resistance determinant of pAM 1.
J. Bacteriol.
159:870-876[Abstract/Free Full Text].
|
| 12a.
|
Short, J. H.,
J. H. Fernandez,
J. A. Sorge, and W.D. Huge.
1988.
Lambda ZAP: a bacteriophage lambda expression vector with in vitro excision properties.
Nucleic Acids Res.
16:7583-7600[Abstract/Free Full Text].
|
| 13.
|
Snoep, J. L.,
M. J. Teixeira de Mattos, and O. M. Neijssel.
1991.
Effect of the energy source on the NADH/NAD+ ratio and on pyruvate catabolism in anaerobic chemostat cultures of Enterococcus faecalis NCTC 775.
FEMS Microbiol. Lett.
81:63-66.
|
| 14.
|
Snoep, J. L.,
M. R. de Graef,
M. J. Teixeira de Mattos, and O. M. Neijssel.
1992.
Pyruvate catabolism during transient state conditions in chemostat cultures of Enterococcus faecalis NCTC775: importance of internal pyruvate concentration and NADH/NAD+ ratio.
J. Gen. Microbiol.
138:2015-2020[Medline].
|
| 15.
|
Spellerberg, B.,
D. R. Cundell,
J. Sandros,
B. J. Pearce,
I. Idänpäan-Heikkilä,
C. Rosenow, and H. R. Masure.
1996.
Pyruvate oxidase as a determinant of virulence in Streptococcus pneumoniae.
Mol. Microbiol.
19:803-813[CrossRef][Medline].
|
Journal of Bacteriology, May 2001, p. 2957-2962, Vol. 183, No. 9
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.9.2957-2962.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Battig, P., Muhlemann, K.
(2008). Influence of the spxB Gene on Competence in Streptococcus pneumoniae. J. Bacteriol.
190: 1184-1189
[Abstract]
[Full Text]
-
Pericone, C. D., Park, S., Imlay, J. A., Weiser, J. N.
(2003). Factors Contributing to Hydrogen Peroxide Resistance in Streptococcus pneumoniae Include Pyruvate Oxidase (SpxB) and Avoidance of the Toxic Effects of the Fenton Reaction. J. Bacteriol.
185: 6815-6825
[Abstract]
[Full Text]
-
Kadioglu, A., Echenique, J., Manco, S., Trombe, M.-C., Andrew, P. W.
(2003). The MicAB Two-Component Signaling System Is Involved in Virulence of Streptococcus pneumoniae. Infect. Immun.
71: 6676-6679
[Abstract]
[Full Text]
Top
Abstract
Text
