The hsp 16 Gene of the Probiotic Lactobacillus acidophilus Is Differently Regulated by Salt, High Temperature and Acidic Stresses, as Revealed by Reverse Transcription Quantitative PCR (qRT-PCR) Analysis

Small heat shock proteins (sHsps) are ubiquitous conserved chaperone-like proteins involved in cellular proteins protection under stressful conditions. In this study, a reverse transcription quantitative PCR (RT-qPCR) procedure was developed and used to quantify the transcript level of a small heat shock gene (shs) in the probiotic bacterium Lactobacillus acidophilus NCFM, under stress conditions such as heat (45 °C and 53 °C), bile (0.3% w/v), hyperosmosis (1 M and 2.5 M NaCl), and low pH value (pH 4). The shs gene of L. acidophilus NCFM was induced by salt, high temperature and acidic stress, while repression was observed upon bile stress. Analysis of the 5′ noncoding region of the hsp16 gene reveals the presence of an inverted repeat (IR) sequence (TTAGCACTC-N9-GAGTGCTAA) homologue to the controlling IR of chaperone expression (CIRCE) elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidophilus might be transcriptionally controlled by HrcA. In addition, the alignment of several small heat shock proteins identified so far in lactic acid bacteria, reveals that the Hsp16 of L. acidophilus exhibits a strong evolutionary relationship with members of the Lactobacillus acidophilus group.

NCFM under abiotic stress such as high temperature, acidic, salt and bile stress. We have focused on these specific stress conditions as they are often encountered by LAB either during food fermentation or gastrointestinal transit.

Genomic Organization Lactobacillus Acidophilus hsp16
The genomic organization of the L. acidophilus NCFM hsp16 gene is reported in Figure 1. A putative transcription initiation site was mapped to position -90, relative to the translational start codon (ATG). A typical prokaryotic Shine-Dalgarno ribosome binding site (RBS), AAAGGA, is present, complementary to the 3'-end (TCCTTT) of L. acidophilus NCFM 16S rRNA. The -10 (TAAATA) and -35 (TTAGCA) boxes, separated by 18 nucleotides, were identified at an appropriate distance from the transcriptional start site. An inspection of the 3'-side noncoding region of the small heat shock gene revealed an inverted-repeat sequence that could form a stem-and-loop structure in the mRNA and it is likely to function as a transcriptional terminator. The proposed transcription start site is preceded by a sequence that shows 63% identity with the extended −10 box consensus sequence (TNTGNTATAAT) of the σA promoters of Gram-positive bacteria [17]. Analysis of the 5' noncoding region reveals the presence of an inverted repeat (IR) sequence (TTAGCACTC-N9-GAGTGCTAA) homologue to the controlling IR of chaperone expression (CIRCE) elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidophilus might be transcriptionally controlled by HrcA [18]. In order to assess the distribution of sHsp homologs across prokaryotics, especially LAB, we surveyed representative available sequenced genomes for the presence of sHsp-encoding genes ( Table 1). Alignment of the sHSPs amino acid sequences indicates moderate similarity among the -crystallin domains, whereas N and C terminal regions were found to be much more variable (Figure 2).
Hsp16 exhibits the highest level of sequence identity (90%) with Lactobacillus ultunensis sHsp, followed by Lactobacillus crispatus sHsp (87%), Lactobacillus helveticus sHsp (82%), Lactobacillus gasseri sHsp (63%), and Lactobacillus johnsonii sHsp (62%). -crystallin domain protein alignment was performed using ClustalW and resulted in an unrooted neighbor-joining phylogenetic tree ( Figure 3). Hsp16 branches together with the sHSP sequences of members of the Lactobacillus acidophilus group. More generally, as observed by Ventura et al. [14], the distribution of sHsp-encoding genes is expected to be a consequence of either a vertical or horizontal transfer mechanism.  The hsp16 locus in L. acidophilus and other bacteria is schematically reported in Figure 4. The comparative analysis was performed with loci of the most similar protein to the predicted L. acidophilus Hsp16. Generally, in contrast to high molecular weight chaperone members, sHsps show huge variation in sequence, polypeptide size, and oligomer subunit number [12]. To our surprise, in some cases we noticed a considerable homology coupled with a strongly conserved genomic organization of the shsp loci. Quite the opposite result was reported by Ventura et al. [14] who, studying hsp20 in Bifidobacterium breve, found significant DNA sequence homology detectable between the various Bifidobacterium sHsp-encoding genes. However, in contrast, they evidenced a variable organization of the flanking genes.

Relative Expression Levels of hsp16 Gene Under Different Abiotic Stresses
Expression of the hsp16 gene of L. acidophilus was monitored by quantitative Real Time (qRT) PCR in presence of abiotic stresses such as high temperature (45 °C and 53 °C), high salt content (NaCl 1 M and 2.5 M), acid stress (pH 4), presence of bile 0.3% (w/v), or ethanol (12%). All stress conditions were imposed for 5 min and 15 min, and stress intensities were chosen based on previous works [4]. We assessed the use of the ldhD gene as internal control for reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in L. acidophilus. The ldhD transcript level was partially affected by the stress conditions tested in our work ( Figure 5). In particular, significant difference was observed upon severe heat (53 °C) and bile stresses, suggesting that the identification of a gene as internal control requires further studies. As a consequence, relative gene expressions results were normalized to the quantity of total RNA. hsp16 expression was calculated relative to the control unstressed cells. An increased expression level was observed for the hsp16 gene of L. acidophilus either at 45 °C or at 53 °C ( Figure 6). A transcriptional induction was already evident after 5 min stress, with a stronger induction after 15 min temperature upshift to 45 °C or 53 °C (7-and 18-fold, respectively) ( Figure 6). hsp16 gene involvement in the response to heat stress is corroborated by the increased gene expression after exposure to 53 °C; indeed, this temperature, identified as sub-lethal, probably represents a significant obstacle to the growth of bacteria cells. The strong induction observed after 15 min at 53 °C may contributes to explain the thermotolerance enhancement revealed by Kim et al. [4] after exposure at the same temperature.
Kim et al. [4] previously identified NaCl at 2% and 18% w/v as a sublethal and lethal osmotic stress respectively, allowing growth of L. acidophilus cells. Therefore, we used NaCl at the concentration of 1 M (5.8% w/v) and 2.5 M (14.5% w/v), two different values of sublethal salt stress. Hyperosmotic stress transiently induced hsp16 gene expression (Figure 7). Indeed, a maximum increase in the amount of the corresponding mRNA was observed after 5 minutes salt stress with 3-and 2-fold induction when salt was added at the final concentration 1 M and 2.5 M, respectively. Thereafter, a decrease in mRNA level occurred after 15 min stresses were imposed. The adaptive response to NaCl stress was previously shown to provide cross-protection against heat stress [4], but not vice versa, suggesting that at least a subset of heat shock proteins might be as well induced by salt stress, but a temperature upshift may not necessarily induce NaCl stress response. Our results support the hypothesis that a cross-talk does exist between salt and heat response. Indeed, Kim et al. [4] observed that the cells pre-exposed to the NaCl stress were significantly more resistant when subjected to heat stress. Similar behavior was observed among LAB in Lactococcus lactis, where heat shock proteins DnaK, GroEL, and GroES were induced by salt stress [19]. Given that the liver secretes as much as a liter of bile into the intestinal tract each day, in order to emulsify and solubilize lipids, exposure to bile also represents a harsh challenge for bacteria [20]. Several studies indicate that the molecular chaperones DnaK and GroEL are induced by bile [21][22][23][24]. Kim et al. [4] also highlighted in previous studies that the cells pre-exposed to the bile stress gained greater resistance to heat stress; conversely, pre-exposure to heat stress could not increase resistance against lethal bile stress. Kim et al. [4] identified bile at 0.05% (w/v) as the sublethal level, since cells were still growing slowly at this level, and bile at 0.5% (w/v) as the lethal level, even if not all cells were killed at this level. Therefore, we performed our experiments using bile at 0.3% (w/v), a value that is halfway between the sublethal and lethal level. A low repression of hsp16 gene (0.5-fold) was detected in response to bile stresses, both after 5 and 15 minutes exposure (Figure 8). This result suggests that Hsp16 is not directly involved in response to stress induced by bile treatment and is not part of the hypothesized cross response to different stress types. Lactobacilli, including L. acidophilus, can compete for growth with other food-borne microbes by taking advantage from the environmental acidification, resulting from their metabolic activities. For this reason, the biological mechanisms allowing LAB to cope with acidic stress have been receiving growing attention. The survival of L. acidophilus in acidic environments has been studied, and this species was proven to be highly resistant to acid [25]. Lorca et al. [26] found that the heat shock proteins DnaK, DnaJ, GrpE, GroES and GroEL were among those proteins whose synthesis was induced in response to acid adaptation. We investigated the relative hsp16 gene expression during acid stresses (pH 4) obtained by addition of either hydrochloric acid or lactic acid. As shown in Figure 9, hsp16 gene expression increased when acidic stress was imposed. However, a different level of induction was observed for the two acidifying agents. The highest induction was reported after 15 min (9-fold) of exposure to medium acidified with lactic acid, while a less pronounced induction was detected in presence of hydrochloric acid after 5 (4-fold) and 15 min (5-fold) and in the presence of lactic acid after 5 minutes (6-fold) ( Figure 9). Interestingly, not only did we notice a remarkable hsp16 induction under acidic condition, but we also detected a different pattern in response to the same hydrogen ion concentration, achieved by hydrochloric acid and lactic acid. A possible explanation for these differences could be connected with biological mechanisms that have been postulated to explain the inhibitory effects of lactic acid: (i) toxicity arising from the dissipation of the membrane potential, (ii) acidification of the cytosol, or (iii) intracellular anion accumulation [27]. Given these results, it would be interesting to study hsp16 expression also upon simultaneous stresses and under conditions affecting the membrane fluidity. Indeed, it was demonstrated that changes in membrane fluidity control the expression of a subset of bacterial sHsps, which are localized in the membrane fraction and, most importantly, can affect membrane physical state and stress tolerance [12,28]. A relevant example of such a biological activity is given by the sHsps Lo18 of the lactic bacterium Oenococcus oeni [29][30][31].

Experimental Section
L. acidophilus NCFM was routinely grown at 28 °C in MRS broth at pH 6.8, without shaking. For heat stresses, mid-exponential (OD 600 = 0.6) cultures were transferred to water baths maintained at 45 °C and 53 °C. Acidic stress was imposed by transferring mid-exponential cultures into MRS broth adjusted to pH 4, with either lactic acid or hydrochloric acid. For bile and salt stresses, L. acidophilus cells were harvested by centrifugation (4,500 ×g, 10 min), and re-suspended in 30 mL of fresh MRS broth containing 0.3% (w/v) bile or NaCl (1 M and 2.5 M). Stresses were imposed for 5 min and 15 min.
For real time PCR analysis, total RNAs were extracted using UltraClean Microbial Isolation Kit (MoBio, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA quality was verified as by gel electrophoresis. About 1 µg of total RNA was retrotranscribed using Quantitect Reverse Trascription (Qiagen, Chatsworth, CA, USA) which includes DNase treatment. Real time PCR was performed on Applied Biosystems 7300 Real-Time PCR System using SYBR Green as fluorescent dye. 5 µL of 20-fold diluted cDNA, was added to 15 µL to real-time PCR Mix containing Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), and 100 nM of forward and reverse primers for ldhD amplification (ldhD forward 5'-GTCGGTGTTGTTGGTACTGG 3' and ldhD reverse 5'-TTAGCTGGAACGTCTGGTAC-3'), and 250 nM of forward and reverse primers for hsp16 amplification (hsp16 forward 5'-CGTGGCCGGTACTAGAAAAG-3' and hsp16 reverse 5'-TGCTTTGGTAGGGTGATGGT-3'). Primers sequences were designed using OligoPerfect Designer software (Invitrogen, Carlsbad, CA, USA), secondary structures and dimers formation were controlled using Oligo Analyzer 3.0 software (Integrated DNA Technologies, Coralville, IA, USA). The thermal conditions were as it follows: 95 °C for 10 min followed by 35 cycles of 95 °C for 20 s, 58 °C for 30 s, 72 °C for 30 s. Melting curve analyses were performed to verify the specificity of real-time PCR, by slowly increasing the temperature from 65 °C to 95 °C. All samples were performed in duplicate and a negative control (distilled water) was included in each run. The results were analyzed using the absolute quantification method. The amount of target RNA was determined by running a standard curve obtained with serial dilutions (ratio 1:10) of cloned target gene cDNA. Relative gene expressions were normalized to the quantity of total RNA. The use of ldhD gene of L. acidophilus as internal control was also assessed.
Sequence comparisons with the GenBank database were accomplished using the National Center for Biotechnology Information (NCBI) BLAST2 [32] network service, with the default parameter values provided. Multiple alignments were performed with the European Bioinformatics Institute (EBI) CLUSTALW2 program [33,34] and visualized using Jalview [35]. The neighbor-joining tree was constructed in ClustalX [33,34] and visualized using TreeView [36].

Conclusions
The biomedical relevance of L. acidophilus is testified by its natural occurrence in the human intestinal microbiota, its probiotic properties, and its possible use as a vaccine delivery system [37][38][39]. Given its use to drive dairy fermentations and in functional probiotic foods, this species is commonly exposed to multiple physiological stresses. Microbial sHsp not only detain a biotechnological potential in reason of their biochemical properties [40,41], but also find application as biomarkers for preliminary screening of LAB strain technological features [42,43]. Although stress response has been studied extensively in some microrganisms, only a limited number of works deal with L. acidophilus. Because of the biomedical and technological relevance of L. acidophilus, studies on the stress response mechanisms of this organism would be helpful. Here we characterize, for the first time, the expression pattern of L. acidophilus hsp16 in relation to stress conditions that this bacterium commonly encounters both in its natural niches and for its diverse biotechnological applications. L. acidophilus hps16 gene structure, genomic organization, and deduced amino acid sequence were analyzed and compared with other LAB, indicating a strong evolutionary relationship with members of the Lactobacillus acidophilus group.