APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2003, p. 1287–1289 0099-2240/03/$08.00⫹0 DOI: 10.1128/AEM.69.2.1287–1289.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 69, No. 2
Impaired Temperature Stress Response of a Streptococcus thermophilus deoD Mutant Mario Varcamonti,1* Maria R. Graziano,1 Romilde Pezzopane,1 Gino Naclerio,2 Slavica Arsenijevic,1 and Maurilio De Felice1 Section of Microbiology, Department of General and Environmental Physiology, University “Federico II,” 80134 Naples,1 and Department of Environmental Sciences and Technologies, University of Molise, 86170 Isernia,2 Italy Received 31 July 2002/Accepted 18 November 2002
An insertional deoD mutant of Streptococcus thermophilus strain SFi39 had a reduced growth rate at 20°C and an enhanced survival capacity to heat shock compared to the wild type, indicating that the deoD product is involved in temperature shock adaptation. We report evidence that ppGpp is implicated in this dual response. accession number AF320011) revealed the presence of the complete deoD gene of S. thermophilus SFi39, encoding a putative 236-amino-acid peptide having 69% identity with the L. lactis PNP. In order to look for a putative function of PNP in S. thermophilus temperature adaptation, we constructed a deoD insertional knockout mutant by using the pGh⫹host9 plasmid (8). A 600-bp fragment internal to deoD was cloned in pGh⫹host9, and the recombinant plasmid (pMRG1) was transformed into S. thermophilus SFi39. Transformants were subjected to a chromosomal integration procedure (1) to obtain two incomplete copies of deoD. Integration was confirmed by PCR amplification and Southern blot analysis (data not shown). An enzyme assay (6) performed on the total proteins extracted from cells of the wild type and the deoD mutant showed that the latter strain is devoid of PNP activity (Fig. 1), which further confirmed that the deleted gene encodes PNP. Strain SFi39 grew optimally in M17L medium at 42°C (generation time, 25 min) and had a minimal growth temperature of 20°C; at this temperature, the generation time was 4 h. To study the deoD function and its role in cold adaptation, cells of strains SFi39 and of the isogenic deoD mutant were first grown to an optical density at 600 nm of 0.5 at 42°C and then pelleted, resuspended in two flasks containing the same medium, and preincubated at 42 and 20°C, respectively; growth of each culture was then followed by an increase of optical density at the corresponding temperatures. As shown in Fig. 2, at 42°C, growth was independent of the genotype while at 20°C, the deoD mutant definitely grew more slowly than strain SFi39 (generation time, 8 versus 4 h). Both strains reached the same maximum optical density at 600 nm at 20°C (data not shown). This result points to a definite requirement of the deoD-encoded function in cold adaptation. Since survival to heat shock in an L. lactis strain with an insertional mutation in deoB (which may have a polar effect on the downstream deoD gene) is enhanced compared to that in the wild type (16), heat shock tolerance was tested in our deoD mutant to check whether the involvement of deoD in the same phenomenon occurs in S. thermophilus. Cells from the wild type and the deoD mutant were grown to middle exponential phase at 42°C and then heat shocked at 60°C for 15 min. As
Lactic acid bacteria (LAB) are widely used as starter strains in food fermentations, during which bacterial cells have to match various types of temperature shifts, and therefore, the study of adaptation mechanisms is very important in order to optimize the choice of strains and fermentation protocols (2). Among LAB, Streptococcus thermophilus is the most important in the manufacture of many European cheeses and yogurt. The stress response of S. thermophilus has thus far been the object of a very limited number of studies (4, 15, 17), and only recently has the adaptation to temperature shifts been preliminarily investigated (9, 21). Insertional mutants of Lactococcus lactis showing multistress (pH, stationary phase, heat, and oxidative shock) resistance are altered in the genes involved in purine metabolism (16). One of these, deoB, is part of the deoB-orfC-deoD operon in this organism (3) which, according to the authors, is probably also impaired in the expression of the other two genes (orfC and deoD) because of a polar effect of the transposon insertion in deoB. This mutant showed improved survival to heat shock compared to the wild type. To investigate whether purine metabolism is involved in the temperature stress response in the S. thermophilus strain SFi39 (7) as well, we decided to clone and mutagenize the deoD gene, which encodes purine nucleoside phosphorylase (PNP). PNP catalyzes the phosphorolytic cleavage of nucleosides, thereby forming the free purine and (deoxy)ribose-1-phosphate (22). This enzyme is involved in the purine salvage pathway and in the degradation of nucleosides and is necessary for the assimilation of exogenous free bases or nucleosides from the environment and for the reutilization of the bases and nucleosides provided by nucleotide turnover. Two primers were designed based on a partial deoD sequence present in the GenBank data bank (accession number AF373595) (18) to amplify a probe that was then used in a Southern blot experiment on the S. thermophilus SFi39 chromosome digested with EcoRV. A 2-kbp fragment was purified, cloned in pUC18, and sequenced. The sequence (GenBank
* Corresponding author. Mailing address: Section of Microbiology, Department of General and Environmental Physiology, Via Mezzocannone 16, University “Federico II,” 80134 Naples, Italy. Phone: (39) 081-2534634. Fax: (39) 081-5514437. E-mail:
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FIG. 1. PNP enzyme activity of S. thermophilus strain SFi39 (open circles) and the isogenic deoD mutant (open squares) at optical densities at 293 nm (O.D.293).
shown in Fig. 3, deoD mutant cells have a higher tolerance to heat shock (70 versus 25% survival). Due to the function of PNP, the deoD mutation could alter the intracellular levels of metabolites like IMP, GMP, or its derivatives. We therefore studied the (p)ppGpp (the stringent response alarmone) intracellular levels after heat shock. We analyzed the levels of (p)ppGpp nucleotides in the wild type and the deoD mutant by thin-layer chromatography after growth in chemically defined medium as previously reported (16). In the wild type grown at 42°C, both the tetra- and pentaguanosine nucleotides were present (Fig. 4) and the amount of pppGpp was threefold higher than that of ppGpp (quantitative analysis was performed with the Bio-Rad GelDoc 2000 photographic apparatus equipped with Bio-Rad Multy Analyst software) whereas a decrease in the pppGpp/ppGpp ratio was observed after heat shock. In the deoD mutant, the pppGpp/ppGpp ratio at 42°C was instead 1:1 (Fig. 4) and after heat shock, the levels were comparable to those of the wild
FIG. 2. Growth of S. thermophilus strain SFi39 and the isogenic deoD mutant at 42°C (squares and circles, respectively) and 20°C (diamonds and triangles, respectively) at optical densities at 600 nm (O.D.600).
APPL. ENVIRON. MICROBIOL.
FIG. 3. Survival capacity of S. thermophilus strain SFi39 (shaded bars) and the isogenic deoD mutant (hatched bars) after heat shock (15 min at 60°C). Survival is reported as the percentage of colony-forming cells after the treatment compared to that after growth at 42°C (100%).
type. This result suggests that, if the ppGpp level is important to perform an optimal heat-shock response, increased heatshock survival of the deoD mutant may have resulted from the high level of ppGpp already present at 42°C. The involvement of the stringent response in starvation and stress tolerance has been shown to occur in Escherichia coli (10, 11), Salmonella enterica serovar Typhimurium (10), Vibrio cholerae (13), Bacillus subtilis (20), L. lactis (16), and Listeria monocytogenes (12). Our results show that an increase in ppGpp is implicated in heat-stress tolerance in S. thermophilus. The high level of this alarmone in the deoD mutant at 42°C suggests that this strain is in a preadaptation constitutive state, which may account for its enhanced heat survival. It is tempting to discuss these results in the context of the model of Van Bogelen and Neidhardt (19), as reviewed by Graumann and Marahiel (5). These authors suggested that the ribosome may be the temperature sensor in bacteria and that the concentration of charged tRNA within the cell could be the sensing signal. After a sudden increase in temperature, the speed of translation exceeds that of the instantaneous supply of charged tRNA and the ribosome’s A-site may become empty. Conversely, cold shock results in a reduced translational capacity and a high concentration of charged tRNA may block the A-site. This mechanism could explain why the concentration of ppGpp, whose production is associated to the ribosomal state, increases at high temperatures and decreases after a temperature drop (14). An alteration of the purine metabolism in our deoD mutant led to an increase in the ppGpp basal level,
FIG. 4. (p)ppGpp levels in the wild type and the isogenic deoD mutant at 50 and 42°C.
S. THERMOPHILUS TEMPERATURE ADAPTATION
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which caused a defect in the cold-shock response and, conversely, an increase in heat-shock survival. The data reported here show for the first time that the lack of PNP modifies the basal level of ppGpp and affects both the cold- and heat-shock responses. Nucleotide sequence accession number. The deoD sequence determined in this study was submitted to the GenBank database and assigned accession no. AF320011.
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This work was supported by the CNR Targeted Project “Biotecnologie,” MURST-CNR “Valorizzazione dei prodotti alimentari tipici mediterranei,” and MURST-PRIN 1999 and 2000. We thank Merck & Co. for generously providing thin-layer chromatography plates.
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