HtrA is a key factor in the response to specific stress conditions in Lactococcus lactis

FEMS Microbiology Letters 224 (2003) 53^59

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HtrA is a key factor in the response to speci¢c stress conditions in Lactococcus lactis Catherine Foucaud-Scheunemann
, Isabelle Poquet INRA, Unite¤ de Recherches Laitie'res et Ge¤ne¤tique Applique¤e, 78352 Jouy en Josas Cedex, France Received 6 January 2003; received in revised form 10 April 2003; accepted 7 May 2003 First published online 10 June 2003

Abstract We investigated the physiological role of Lactococcus lactis housekeeping surface protease HtrA. It is involved in surface properties under regular growth conditions, as the htrA mutant strain forms longer chains in liquid medium. It participates in cellular defence against environmental stress conditions: compared to the wild-type strain, the htrA mutant strain exhibited increased sensitivity to heat, ethanol, puromycin, and NaCl, but not to pH, H2 O2 , bile salts or to carbon or nitrogen starvation. htrA transcription in the wild-type strain showed a transient increase under stress conditions determined as requiring htrA, but not under overexpression of a secreted heterologous protein. Our results demonstrate that in L. lactis, htrA is a key factor in the response to specific stress conditions. 2 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : HtrA; Protease ; Stress; mRNA; Growth ; Lactococcus lactis

1. Introduction The Gram-positive lactic acid bacterium Lactococcus lactis is commonly isolated as natural micro£ora from plant materials and dairy products and is widely used as a starter in dairy technology. During this process as well as in its natural environment or in the intestinal tract, it is subject to a variety of adverse conditions, including acid, oxidation, heating and cooling, high osmolarity (i.e. dehydration), bile salts, ethanol and starvation. In response to these stress conditions, bacterial cells are able to rapidly and transiently induce speci¢c or general protection mechanisms [1], involving in particular chaperone proteins and proteolytic enzymes, which act in the cytoplasm or in the cell envelope to repair or degrade abnormal proteins [2,3]. The Escherichia coli DegP/HtrA (High temperature requirement) protein was the ¢rst envelope protease identi¢ed and has since been the most extensively characterized [4,5]. At elevated and normal temperature, this serine protease is involved in the housekeeping degradation of mis-

* Corresponding author. Tel. : +33 1 34 65 20 91; Fax : +33 1 34 65 20 65. E-mail address : [email protected] (C. Foucaud-Scheunemann).

folded proteins (e.g. PhoA fusion proteins and stress denatured proteins), whereas at lower temperature, it displays a chaperone activity [5,6]. Because abnormal proteins are presumably harmful when accumulated in the cell, E. coli HtrA is indispensable for bacterial survival at elevated temperature [6] or under certain oxidative stresses (ferrous ion and cumene hydroperoxide, but not H2 O2 or paraquat [7]), and it is induced under these conditions [4,7]. Recently, its crystal structure was solved and the molecular basis of the structural organization was investigated: it exists as a hexamer formed by staggered association of trimeric rings [8]. The E. coli DegP/HtrA protein belongs to the HtrA family of housekeeping proteases, members of which are found in most organisms, including bacteria, yeasts, plants and humans [4,5]. The majority of bacterial genomes encode more than one HtrA-like protein [4]. Common roles of bacterial HtrA include stress resistance and virulence in the case of pathogens [4]. However, beyond this simple view, HtrA appears to have speci¢c functions in di¡erent microorganisms. First, a regulatory function has been described for some HtrA homologs: DegS/HhoB of E. coli [9,10], YkdA/HtrA and YvtA/HtrB of Bacillus subtilis [11] and AlgW and MucD of Pseudomonas aeruginosa [12]. Second, in contrast to numerous HtrA-like proteins of pathogens [4,13^16], Brucella abortus HtrA is not required

0378-1097 / 03 / $22.00 2 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00419-1

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for virulence in mice [17], and htrA mutant strains of Brucella melitensis [18] or Yersinia pestis [19] are only partially attenuated. Third, although the majority of HtrA homologs are involved in both heat and H2 O2 stress resistance [4,12,15,16,20,21], E. coli HtrA/DegP and Salmonella typhimurium HtrA are speci¢cally involved in either heat [6] or H2 O2 stress survival, respectively. Furthermore, in B. subtilis, inactivation of either ykdA/htrA or yvtA/htrB unexpectedly improves survival to heat shock and H2 O2 exposure, due to overexpression of the other gene [11]. The above considerations make it di⁄cult to predict the functions of HtrA proteins in a given microorganism. The L. lactis HtrA protein has been recently identi¢ed [22]. This envelope protein was shown to display the housekeeping function previously described for other HtrA family members: it is responsible for the degradation of exported heterologous and fusion proteins, and it is essential for growth at elevated temperature [22,23]. However, it was also demonstrated to be original in two ways [22]. First, HtrA is the unique housekeeping surface protease in L. lactis. Second, HtrA performs a new role in the processing of native proteins, the endogenous autolysin AcmA and the Staphylococcus aureus nuclease Nuc. Both the ecological niche and the metabolism of L. lactis di¡er markedly from those of E. coli where htrA is most fully described. Furthermore, L. lactis HtrA shares only 34 and 47% identity with HtrA from E. coli [4] and the thermophilic lactic acid bacteria Lactobacillus helveticus [24], respectively, thus making it di⁄cult to make predictions as to the speci¢city and sensitivity of HtrA of L. lactis to environmental conditions. Considering that the htrA gene is of practical signi¢cance because of the use of L. lactis both as a starter in industrial fermentation and as a cell factory (for protein production) in biotechnological applications [23,25], our goal is to understand the contribution of htrA to cell physiology and environmental stress response of L. lactis and to examine its regulation with respect to environmental conditions. We provide evidence that L. lactis HtrA is involved in surface properties under regular growth conditions and in the resistance to speci¢c stress conditions (heat, NaCl, puromycin and ethanol).

2. Materials and methods 2.1. Bacterial strains and plasmids L. lactis subsp. lactis strains used were IL1403 (wildtype, plasmid free), VEL8702 (isogenic htrA mutant, inactivated by single crossover mutation, chloramphenicol resistant CmR ) and VEL8703 (CmR isogenic htrAþ control strain) [22]. Plasmids used were pSEC1 (CmR ) encoding the secreted staphylococcal nuclease (NucB form fused to Usp45 signal peptide) that is expressed under the nisininducible promoter [26], and pVE8021 (erythromycin re-

sistant EmR ) encoding the exported Exp5-vSP Nuc tripartite fusion protein (allowing the export of a cytoplasmic protein fragment) that is expressed under the ponA promoter, misfolded and highly degraded by HtrA [22,27]. 2.2. Growth conditions Cultures were propagated at 30‡C in M17 broth [28] containing 0.5% (v/v) glucose (referred to as GM17) and Cm (2.5 mg l31 for VEL8702 and VEL8703 or 5 mg l31 for IL1403(pSEC1)) or Em (5 mg l31 for IL1403(pVE8021) and VEL8702(pVE8021)). For sedimentation studies, bacteria were grown for 24 h in plastic 40-ml £asks. For stress studies, cultures were inoculated with 1% (v/v) exponential phase cells grown in the same medium, giving an initial optical density at 600 nm (OD600 ) of 0.02 Q 0.01 corresponding to (1.6 Q 0.3)U107 colony forming units (CFU) ml31 . To compare the e¡ect of stress conditions on both strains, cells grown to early exponential phase (OD600 of 0.10 Q 0.01 corresponding to (3.2 Q 0.5)U107 CFU ml31 , unless speci¢ed otherwise), were studied under the following conditions: temperature shift from 30‡C to 20, 39, 43 or 55‡C, or addition to the growth medium of 2.5^6.5% (w/v) NaCl, 2.5^10% (v/v) ethanol, 15^60 mg l31 puromycin, 0.15^1 mM H2 O2 , or 0.005^0.5% (v/v) bile salts composed of sodium cholate and sodium deoxycholate (1:1). For pH stress, cells were harvested by centrifugation (8000Ug, 5 min, 30‡C) and suspended in GM17 broth at pH ranging from 4.5 to 8.0, adjusted by addition of HCl or NaOH. For starvation, cells harvested by centrifugation were suspended in a chemically de¢ned medium [29] deprived of glucose (carbon starvation) or of amino acids (nitrogen starvation). For secretion stress, the staphylococcal nuclease was induced from pSEC1 as previously described using 10 ng ml31 nisin for 1 h [26] and Exp5-vSP Nuc was expressed from pVE8021. 2.3. Growth determination Bacterial populations were estimated using a spiral plater (Spiral System DS, Cincinnati, OH, USA) on GM17 agar supplemented with Cm or Em when necessary. To minimize errors in plate counting resulting from the varying chain lengths, blending of 100-fold culture dilution in 0.9% (w/v) NaCl for 30 s [30] reduced the CFU of both strains to diplococci and was applied routinely. 2.4. Bacterial morphology To estimate bacterial chain length, cells were colored with methylene blue (0.006% (w/v) in 95% (v/v) ethanol) and the number of cells per chain was determined by microscopic examination (1000U magni¢cation) of 100 chains.

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2.5. Transcriptional analyses Total RNA was extracted as described by Raya et al. [31] from IL1403 cultures that had been grown at 30‡C in GM17 to an OD600 of 0.10 Q 0.01 and subsequently submitted to stress. RNA electrophoresis and Northern experiments were carried out essentially as described by Sambrook et al. [32]. The 0.24^9.50 kb RNA marker (Invitrogen) was used for size determination. Samples were transferred by capillary blotting from the gel onto a Nytran-N nylon membrane (Schleicher and Schuell) and hybridized at 42‡C using the direct nucleic acid labeling and detection system by electrochemoluminescence (ECL) (Amersham Biosciences). An IL1403 htrA speci¢c probe was obtained by polymerase chain reaction (PCR) ampli¢cation using the oligonucleotides 5P-TTTACTTTTTTCCACTTTTCTGTGG-3P and 5P-TTTACTTCTAGACACTTTTCTGTGG-3P. Following hybridization, the membrane was scanned (Magic Scan32V4.3, Amersham Biosciences) and the signal quanti¢ed using Image QuaNT (version 5.0, Molecular Dynamics): a serial dilution of one RNA sample was used as a linear standard for quanti¢cation. The total amount of RNA present on the membrane was evaluated by hybridizing with a probe speci¢c for L. lactis 16S rRNA. 2.6. Statistical analysis of the results Mean values of experimental data with standard deviation were calculated on the basis of three independent experiments and compared using a two-way analysis of variance.

3. Results and discussion 3.1. L. lactis htrA is involved in cell surface properties On solid GM17 agar medium containing Cm, colonies of the htrA mutant VEL8702 were smaller in shape than those of the htrAþ control VEL8703, as previously observed [22]. In the absence of Cm selection, colonies of VEL8702 remained small, but revertant clones with a normal appearance emerged. The population in a colony of IL1403 or VEL8703 was higher ((10 Q 0.7)U109 CFU ml31 or (12.0 Q 1.5)U109 CFU ml31 , respectively) than that of VEL8702 ((3.44 Q 0.5)U109 CFU ml31 ). As no di¡erence in cell morphology could be detected by light microscopy, population levels could contribute to the colony sizes (approximately 2 mm for IL1403 and VEL8703 and 1 mm for VEL8702). In GM17 broth, bacterial sedimentation was greater for the htrA mutant than for the wild-type strain, suggesting either a di¡erent morphology or the inhibition of growth to approximately 1 cm from the surface, because of oxygen penetration into the medium. Signi¢cant di¡erences in

Fig. 1. Growth of L. lactis IL1403 (left) and VEL8702 (right) in the absence (E) or in the presence of 5% ethanol (¢lled symbols). Cells grown until early (F) or late (R) exponential phase were challenged with 5% ethanol (s) and the OD600 was measured.

bacterial chain length were observed. The average chain length of IL1403 was short, augmenting slightly from 2.2 Q 0.5 in the exponential growth phase to 3.2 Q 0.2 cells per chain in the late stationary growth phase. In contrast, that of the htrA mutant was longer, increasing over the same growth period from 3.5 Q 0.2 to 8.7 Q 0.5 cells per chain. We consider it likely that the formation of long chains in VEL8702 is a major cause of cellular sedimentation in liquid medium, rather than cell shape or oxygen sensitivity (see below), resulting from altered surface properties in the htrA mutant. Note that a Streptococcus mutans htrA mutant was recently described as producing clumps in liquid medium and having altered colony morphology, also leading authors to suggest that HtrA plays a role in surface properties [20]. As our previous results in L. lactis indicate that HtrA is involved in the processing of the autolysin AcmA [22], we propose that the alteration of surface properties in htrA mutant may be via HtrA role in processing of surface proteins. 3.2. htrA is required for ethanol resistance during exponential growth The capacity of IL1403 and VEL8702 to survive in the presence of ethanol was examined as a function of the growth phase. Growth values were determined by optical density measurements (OD600 , Fig. 1) and plating. Both strains grew similarly in the absence of ethanol (Fig. 1 and Table 1). However, growth values of the htrA mutant were more a¡ected in the presence of 5% ethanol than those of the wild-type strain, the role of htrA in ethanol resistance varying with the growth phase. Growth values of VEL8702 in the stationary phase were markedly reduced (OD600 of 0.6 Q 0.2, corresponding to (2.8 Q 0.2)U107 CFU ml31 ; Fig. 1 and Table 1) when ethanol was added to early exponentially growing cultures. However, they proceeded almost una¡ected (OD600 of 1.8 Q 0.3, correspond-

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ing to (3.2 Q 0.3)U108 CFU ml31 ) when addition was performed in the late exponential growth phase and unchanged when addition was done later (OD600 of 2.2 Q 0.3, corresponding to (1.5 Q 0.6)U109 CFU ml31 ). Our results indicate that early exponential phase cultures of both strains are highly sensitive to ethanol stress and that HtrA plays a prominent role in ethanol stress resistance during active exponential growth. 3.3. htrA is involved in the response to speci¢c stress conditions To determine the overall contribution of htrA in stress resistance, both wild-type and mutant strains were exposed to various stress conditions in the early exponential growth phase. Among these, the e¡ects of ethanol, bile salts, and starvation on htrA mutant physiology were studied here for the ¢rst time. Growth values were determined during the stationary phase by optical density measurements (ODmax600 ) and by plating (Nmax ) (Table 1). Lethal levels of each stress were de¢ned for the wildtype strain IL1403 as growth values that are similar to or lower than those in the initial conditions (OD600 of

0.10 Q 0.01, corresponding to (3.2 Q 0.5)U107 CFU ml31 ), showing that cells either did not grow or were killed by stress conditions. They were respectively: for temperature, 43‡C ; for ethanol, 10%; for NaCl, 5.5%; for bile salts, 0.02%; for H2 O2 , 1 mM; for puromycin, 60 mg l31 . For pH, no lethal level was determined. Growth of the htrA mutant was then compared to that of IL1403. Upon addition of H2 O2 or bile salts, or upon a pH shift (Table 1) or starvation (results not shown), growth of both strains was a¡ected to the same extent. In contrast, after heat shock, or addition of ethanol, puromycin or NaCl (Table 1), when stress levels were not lethal, growth of VEL8702 was a¡ected more than that of IL1403. Previous studies showed that HtrA is required in various bacterial species for heat shock and/or oxidative stress resistance [4,6,7,12^17,19^21,24]. HtrA is also essential for the growth of Yersinia enterocolitica in the presence of NaCl [21] and of S. mutans at extreme pH [20]. In L. lactis, htrA improves survival under several stress conditions: heat shock, ethanol, puromycin and NaCl, but it is not involved in H2 O2 and pH resistance. Concerning HtrA role in stress response, we propose that it depends

Table 1 Growth of L. lactis IL1403 and VEL8702 in GM17 Growth condition

L. lactis IL1403

30‡C, no addition Temperature (‡C) 20 39 43 +ethanol (%) 2.5 5 10 +NaCl (%) 3.5 4.5 5.5 +puromycin (mg l31 ) 15 30 60 pH 8.0 4.5 +bile salts (%) 0.005 0.020 +H2 O2 (mM) 0.15 1.00

VEL8702 ODmax600 a

Nmax (CFU ml31 )a

2.3

1.9U10

9

2.2

1.5U109

2.2 1.6 0.1

5.7U108 4.8U108 6 107

1.6 0.3 0.2

8.4U108 1.8U107 6 107

2.2 1.3 0.3

8.8U108 3.2U108 3.4U107

1.2 0.6 0.2

7.1U108 2.8U107 2.0U107

1.6 0.9 0.3

3.1U109 2.9U108 4.2U107

0.9 0.3 0.1

4.7U108 4.7U107 2.1U107

2.0 2.1 0.4

1.3U109 1.2U109 4.6U107

1.2 1.1 0.2

4.8U108 1.0U108 1.3U107

2.0 0.4

1.7U109 1.8U108

1.7 0.3

0.8U109 1.5U108

1.8 0.5

2.6U108 4.5U107

1.8 0.6

2.3U108 4.0U107

2.0 0.1

8.6U108 6 107

1.8 0.2

8.0U108 6 107

ODmax600 a

Nmax (CFU ml31 )a

Cells grown to early exponential phase (OD600 of 0.10 Q 0.01, corresponding to (3.2 Q 0.5)U107 CFU ml31 ) were studied under various stress conditions. Bacterial populations were determined during the stationary phase as the number of cells (Nmax ) and the optical density (ODmax600 ). a Mean values of experimental data with standard deviation were calculated on the basis of three independent experiments and compared using a twoway analysis of variance, values varied by less than 10%.

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Fig. 2. Northern blot analysis of (5%), puromycin (30 mg l31 ), or The mRNA induction ratio was scripts obtained under particular

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htrA transcripts under stress conditions as a function of time. Transcription was induced by heat shock (39‡C), ethanol NaCl (4.5%). Samples (25 Wg of total RNA) were taken before (0) and 15, 30, 45 and 60 min after initiation of stress. calculated by dividing the signals from RNA of stressed cells by those of the control (0). Northern blot of htrA transtress conditions is presented above the corresponding graph.

upon the microorganism, and that in L. lactis, HtrA acts as a key factor in the response to speci¢c stress conditions, i.e. heat shock, ethanol, puromycin and NaCl. 3.4. htrA expression is regulated at the transcriptional level To examine whether expression of htrA is induced by stress in L. lactis, we exposed early exponential phase cultures of IL1403 (i.e. initial conditions were OD600 of 0.10 Q 0.01) to di¡erent conditions. A major htrA-mRNA band of approximately 1.5 kb was detected in all samples of L. lactis IL1403 but not of VEL8702, demonstrating that the htrA gene is a monocistronic transcriptional unit as previously proposed [22]. Cells exposed to sublethal stress levels exhibited a transitory induction with a rapid decline of the amounts of htrA transcripts after 15^30 min. This resembles the kinetics pattern observed for htrA transcription in L. helveticus upon heat shock or addition of ethanol [24]. The strongest induction resulted from exposure to 30 mg l31 puromycin l31 for 15 min (Fig. 2), and gave rise to a fourfold increase in htrA speci¢c transcripts. A temperature upshift from 30 to 39‡C, or an exposure to 5% ethanol for 30 min, or the addition of 4.5% NaCl for 15 min, resulted in a two- to three-fold induction (Fig. 2). H2 O2 , bile salts, pH, starvation stresses or a temperature downshift from 30 to 20‡C had no e¡ect on htrA transcription (data not shown). Expression of htrA transcripts in L. lactis IL1403 was

further examined as a function of the stress level. Temperature was shifted from 30‡C down to 20‡C or up to 55‡C for 10 min, or cultures were administered 2.5^10% ethanol for 30 min, 15^60 mg l31 puromycin or 2.5^6.5% NaCl for 15 min (Fig. 3). Growth remained unchanged after shortterm exposure to 10% ethanol, 60 mg l31 puromycin, or 6.5% NaCl, whereas growth ceased after 10 min at 55‡C. In contrast, long-term exposure to any of these stress conditions was lethal (Table 1). Expression of htrA increased with augmenting levels of the stress, except for NaCl, which provoked the maximum induction at a moderate dose (4.5%). Addition of 10% ethanol to the growth medium induced the highest level of htrA transcripts, with a nine-fold induction. All these results show that L. lactis htrA is regulated at the transcriptional level, and that it is speci¢cally induced under the stress conditions determined as requiring it in this study. Increased expression of htrA-mRNA or HtrA protein was also reported in few bacteria upon heat shock [11,24,33^35], addition of ethanol [24,33], NaCl [24] or ferrous ions [7]. This indicates that the expression pattern of L. lactis htrA shared stress induction with most characterized htrA homologs. L. lactis htrA regulation requires further investigation. As recently evidenced for B. subtilis ykdA/htrA and yvtA/htrB [36], L. lactis htrA induction upon stress conditions could depend on octamer repeats present in the promoter region (data not shown), acting as binding sites for the regulator of one of the L. lactis twocomponent systems homologous to B. subtilis CssRS.

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Fig. 3. Northern blot analysis of htrA transcripts under stress conditions as a function of the stress agent level. Transcription was induced by temperature shock, ethanol, puromycin, or NaCl. Samples were taken before (0), and 15 min (puromycin, NaCl) or 30 min (temperature, ethanol) after initiation of stress. The mRNA induction ratio was calculated as indicated in Fig. 2. Northern blot of htrA transcripts obtained under particular stress conditions is presented above the corresponding graph.

Survival and gene expression were rarely examined in combination : where studied, htrA-mRNA induction correlated both with the degree of stress and bacterial sensitivity to a given stress condition (this study, [24]). However, whereas htrA transcription is induced in L. helveticus after exposure to heat and NaCl, htrA facilitated growth was observed only under heat shock conditions (but the e¡ect was very weak) [24]. In contrast, htrA in L. lactis is induced and required for growth under both stress conditions. We suggest that the e¡ects of HtrA on survival under stress conditions were overlooked in L. helveticus because experiments were performed on stress-resistant late exponential phase cultures (OD600 of 0.8). Possibly, htrA will prove to be required in L. helveticus for early exponential growth in the presence of both NaCl and heat [37]. To investigate whether it could be triggered by secretion stress, htrA expression was analyzed upon high-level secretion of the staphylococcal nuclease [26] or production of the highly HtrA-sensitive protein, Exp5-vSP Nuc [22,27]. Those conditions a¡ect neither the htrA-mRNA level in IL1403 nor the growth of VEL8702 (data not shown). This suggests that they could be overcome by the basal htrA expression level and do not constitute a severe secretion stress as previously described for B. subtilis [38]. In contrast, the presence of abnormally un¢nished pro-

teins generated by puromycin treatment [39] signi¢cantly reduced growth of htrA mutant strain of L. lactis (this study) or B. abortus [17], whereas the wild-type strains were not a¡ected. In L. lactis wild-type strain, the concomitant increased expression of htrA supports the involvement of HtrA as a major stress factor in the proteolysis of aberrant proteins, which might accumulate under several environmental stress conditions (e.g. heat shock, ethanol, and NaCl). However, the reason why HtrA responds only to speci¢c stress conditions in L. lactis (this study) and in other bacteria [7,12,14,19] remains to be explored. Results of this study will be useful in de¢ning adverse growth conditions of the L. lactis htrA mutant strain, which may be important for potential applications in industrial fermentation and in biotechnology.

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