JOURNAL OF BACTERIOLOGY, Mar. 2007, p. 1582–1588
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 189, No. 5
Physiologic Effects of Forced Down-Regulation of dnaK and groEL
Expression in Streptococcus mutans?
Jose ´ A. Lemos, Yaima Luzardo, and Robert A. Burne*
Department of Oral Biology, University of Florida College of Dentistry, Gainesville, Florida 32610-0424
Received 25 October 2006/Accepted 5 December 2006
Strains of Streptococcus mutans lacking DnaK or GroEL appear not to be isolable. To better distinguish the
roles played by these chaperones/chaperonins in the physiology of S. mutans, we created a knockdown strategy
to lower the levels of DnaK by over 95% in strain SM12 and the level of GroEL about 80% in strain SM13.
Interestingly, GroEL levels were approximately twofold higher in SM12 than in the parent strain, but the levels
of DnaK were not altered in the GroEL knockdown strain. Both SM12 and SM13 grew slower than the parent
strain, had a strong tendency to aggregate in broth culture, and showed major changes in their proteomes.
Compared with the wild-type strain, SM12 and SM13 had impaired biofilm-forming capacities when grown in
the presence of glucose. The SM12 strain was impaired in its capacity to grow at 44°C or at pH 5.0 and was
more susceptible to H2O2, whereas SM13 behaved like the wild-type strain under these conditions. Phenotyp-
ical reversions were noted for both mutants when cells were grown in continuous culture at a low pH,
suggesting the occurrence of compensatory mutations. These results demonstrate that DnaK and GroEL
differentially affect the expression of key virulence traits, including biofilm formation and acid tolerance, and
support that these chaperones have evolved to accommodate unique roles in the context of this organism and
Streptococcus mutans, a bacterial pathogen associated with
human dental caries, thrives in multispecies biofilms on tooth
surfaces, where it is subjected to a continuous assault by host
defenses and to rapid and dramatic fluctuations in nutrient
availability, carbohydrate source, and pH. The organism has
evolved multiple physiologic and genetic adaptations to opti-
mize growth under these dynamic conditions, including the
capacity to scavenge and metabolize a wide variety of carbo-
hydrates and to adapt to a wide range of stresses, especially low
pH (15). The rapid and efficient responses to environmental
stimuli are considered to be critical to the persistence and
virulence of this organism, so efforts have been focused on
dissecting the molecular control of responses to low pHs, other
stresses, and nutrient flux. Through such studies, it has
emerged that the expression of the class I heat shock proteins
of S. mutans is highly responsive to intermittent and sustained
exposure to relevant environmental stresses (13, 18).
The ubiquitously distributed class I stress proteins, the
DnaK and GroEL molecular chaperones, are central to the
tolerance to environmental stresses and participate in a variety
of cellular processes including protein folding, protein trans-
location, and assembly and disassembly of protein complexes
(8, 11, 26). The GroEL and DnaK complexes, which include
GroES and DnaJ-GrpE, respectively, also regulate signal
transduction pathways by controlling the stability and activities
of transcriptional regulators and protein kinases (8, 11). In
many gram-positive bacteria, transcription of the groE (groES-
groEL) and dnaK (hrcA-grpE-dnaK-dnaJ) operons is negatively
controlled by HrcA, which binds to a highly conserved cis-
acting element (CIRCE) located in the regulatory regions of
these operons (28, 30). In some cases, the groE and dnaK
operons can also be under the negative control of CtsR, a
repressor that binds to a conserved direct-repeat sequence and
that was initially identified for its role in regulating clp gene
expression (5, 7).
Previously, we demonstrated that the transcription of the
dnaK operon in S. mutans is tightly controlled by HrcA (13,
18), which binds to two CIRCE elements located in the dnaK
promoter region. The expression of groE in S. mutans is under
the dual control of the HrcA and CtsR repressors, although the
repression by CtsR is not as strong as the repression by HrcA
(16). It was also demonstrated that the transcription of both
operons is rapidly induced by acid shock and other stresses and
that elevated levels of DnaK are maintained under acidic con-
ditions (13, 18). In Escherichia coli, GroEL is essential for
growth at all temperatures, while DnaK is essential only at
temperatures above 37°C and below 15°C (9, 10, 12). In the
gram-positive paradigm Bacillus subtilis, DnaK is essential only
at temperatures above 52°C (27). In S. mutans, attempts to
inactivate hrcA by inserting the strongly polar ?Km cassette
resulted in the isolation of only single-crossover insertions,
even at lower temperatures or in buffered medium, indicating
that the transcription of the downstream grpE-dnaK-dnaJ
genes, coding for the DnaK machinery, was essential for cell
viability. Similarly, strains lacking GroEL in S. mutans could
not be isolated.
To evaluate the role of HrcA as a repressor protein in
chaperone expression, an HrcA-deficient strain, SM11, was
constructed by allelic replacement of the 5? portion of the gene
with a polar kanamycin cassette (?Km) followed by the Strep-
tococcus salivarius urease promoter (PureI) (6, 18). The HrcA
mutant strain, which had constitutively elevated levels of
* Corresponding author. Mailing address: Department of Oral Bi-
ology, University of Florida College of Dentistry, P.O. Box 100424,
1600 SW Archer Road, Gainesville, FL 32610-0424. Phone: (352)
392-4370. Fax: (352) 392-7357. E-mail: email@example.com.
?Published ahead of print on 15 December 2006.
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