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. 2010 Sep;192(17):4395-402.
doi: 10.1128/JB.00574-10. Epub 2010 Jun 25.

The pdh operon is expressed in a subpopulation of stationary-phase bacteria and is important for survival of sugar-starved Streptococcus mutans

Monica Busuioc  1 , Bettina A Buttaro, Patrick J Piggot
Affiliations

The pdh operon is expressed in a subpopulation of stationary-phase bacteria and is important for survival of sugar-starved Streptococcus mutans

Monica Busuioc et al. J Bacteriol. 2010 Sep.

Abstract

Streptococcus mutans is a facultative member of the oral plaque and is associated with dental caries. It is able to survive long periods of sugar starvation. We show here that inactivation of pdhD, putatively encoding a subunit of the pyruvate dehydrogenase complex, impairs survival of both batch cultures and biofilms. We show that pdhD and the downstream genes pdhA, pdhB, and pdhC form an operon that is predominantly transcribed in stationary phase. Analysis with fluorescent reporters revealed a bimodal expression pattern for the pdh promoter, with less than 1% of stationary-phase populations expressing pdh. When it was first detected, after 1 day of sugar starvation in batch culture, expression was mostly in individual bacteria. At later times, expressing bacteria were often in chains. The lengths of the chains increased with time. We infer that the pdh-expressing subpopulation is able grow and divide and to persist for extended times in stationary phase.

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Figures

FIG. 1.
FIG. 1.
Schematic representation of the pdh region of the S. mutans genome. Genes are indicated with arrows. IGR indicates an intergenic region. The putative promoter region used for promoter studies is shown at the top of the figure. The region of pdhD replaced with a kan cassette is indicated at the bottom of the figure. The regions used as probes for Northern blots are shown as thick lines.
FIG. 2.
FIG. 2.
Northern blot analysis of pdh expression. Analysis of RNA isolated from batch cultures. (A) RNA isolated from S. mutans UA159 during exponential growth (lanes i) and 20 h into stationary phase (lanes ii). Ten micrograms of RNA was loaded for each sample. Ethidium bromide staining (left side) was used to confirm equal loading of the genes. The blots (right side) are on a different scale. The probes used for each blot were for internal portions of the pdh operon genes (Fig. 1) and are indicated to the right of each blot. A probe for an exponential-phase gene bound to the RNA prepared from the parental strain confirmed the quality of the RNA (data not shown). (B) Comparison of RNA isolated from 20-h stationary-phase cultures of strain UA159 (i) and ΔpdhD mutant SL14043 (ii) and probed for pdhA. The positions of size markers (in kb) are indicated on the left side of the Northern blots.
FIG. 3.
FIG. 3.
Expression of gfp from the pdh promoter is limited to a subpopulation in sugar-starved batch cultures. Strain SL15013 was grown in CDM with 6 mM glucose at 37°C in a 5% CO2 incubator. Samples were removed at the time indicated to the left, given as days in stationary phase. (Left) Differential interference contrast image; (center) GFP fluorescence; (right) merged image. Bars, 5 μm.
FIG. 4.
FIG. 4.
Bacteria expressing pdh in older cultures are often present in long chains. Representative images of strain SL15103 taken from cultures that have been in stationary phase for 20 or 30 days are shown. An overlay of GFP fluorescence on differential interference contrast images is shown. Note that the images have different magnifications. Bars, 5 μm.
FIG. 5.
FIG. 5.
Expression of yfp from the pdh promoter is limited to a subpopulation in sugar-starved batch cultures. Strain SL13603 was grown in CDM with 6 mM glucose at 37°C in a 5% CO2 incubator. Samples were removed at the time indicated at the top of each panel, given as days in stationary phase. An overlay of YFP fluorescence on differential interference contrast images is shown. Bars, 5 μm.
FIG. 6.
FIG. 6.
Effect of mild sonication on the culturable count of batch cultures of strain SL15103 after different times in sugar-starved stationary phase. At the indicated times in stationary phase, bacteria were diluted and plated on TH agar. Three parallel cultures were followed, and samples were removed from each culture and plated in duplicate. Bars represent standard deviations.
FIG. 7.
FIG. 7.
Expression of gfp from the pdh promoter in biofilms. Representative images of static biofilms of S. mutans SL15013 established with CDM containing 3 mM sucrose are shown. Cultures were inoculated in 24-well plates containing sterile 12-mm-diameter glass coverslips. The plates were incubated at 37°C in 5% CO2. At the indicated times, the coverslips were washed with PBS and imaged. An overlay of GFP fluorescence on differential interference contrast images is shown. Bars, 5 μm.
FIG. 8.
FIG. 8.
Effect of a ΔpdhD mutation on the survival of S. mutans. (A) Batch cultures grown in CDM plus 6 mM glucose; (B) batch cultures grown in CDM plus 3 mM sucrose; (C) static biofilms grown in CDM plus 3 mM sucrose. At the indicated times in stationary phase, bacteria were diluted and plated on TH agar. The limit of detection was 10 CFU/ml; samples with counts below that limit of detection are arbitrarily indicated as having a log number of CFU/ml of 1. Filled squares, S. mutans UA159; open squares, Δpdh mutant SL14043. In each case, the results of a representative experiment of at least three experiments are shown.
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References

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