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Comparative proteomic analysis of Lactobacillus plantarum WCFS1 and ΔctsR mutant strains under physiological and heat stress conditions.

Russo P, de la Luz Mohedano M, Capozzi V, de Palencia PF, López P, Spano G, Fiocco D - Int J Mol Sci (2012)

Bottom Line: The proteomic analysis of L. plantarum WCFS1 and ctsR mutant strains confirms at the translational level the CtsR-mediated regulation of some members of the Clp family, as well as the heat induction of typical stress response genes.Heat activation of the putative CtsR regulon genes at transcriptional and translational levels, in the ΔctsR mutant, suggests additional regulative mechanisms, as is the case of hsp1.Furthermore, isoforms of ClpE with different molecular mass were found, which might contribute to CtsR quality control.

View Article: PubMed Central - PubMed

Affiliation: Department of Agriculture, Food and Environment Sciences, Via Napoli 25, Foggia 71122, Italy; E-Mails: p.russo@unifg.it (P.R.); vittorio.capozzi@gmail.com (V.C.).

ABSTRACT
Among Gram-positive bacteria, CtsR (Class Three Stress gene Repressor) mainly regulates the expression of genes encoding the Clp ATPases and the ClpP protease. To gain a better understanding of the biological significance of the CtsR regulon in response to heat-shock conditions, we performed a global proteomic analysis of Lactobacillus plantarum WCFS1 and ΔctsR mutant strains under optimal or heat stress temperatures. Total protein extracts from bacterial cells were analyzed by two-dimensional gel fractionation. By comparing maps from different culture conditions and different L. plantarum strains, image analysis revealed 23 spots with altered levels of expression. The proteomic analysis of L. plantarum WCFS1 and ctsR mutant strains confirms at the translational level the CtsR-mediated regulation of some members of the Clp family, as well as the heat induction of typical stress response genes. Heat activation of the putative CtsR regulon genes at transcriptional and translational levels, in the ΔctsR mutant, suggests additional regulative mechanisms, as is the case of hsp1. Furthermore, isoforms of ClpE with different molecular mass were found, which might contribute to CtsR quality control. Our results could add new outlooks in order to determine the complex biological role of CtsR-mediated stress response in lactic acid bacteria.

No MeSH data available.


Related in: MedlinePlus

Transcriptional heat induction of putative CtsR regulon genes. Heat induction of putative CtsR regulon genes in L. plantarum WCFS1 wild type and ΔctsR mutant strains as revealed by qRT-PCR analysis. The mRNA levels in wild type (open bars) and ΔctsR mutant (grey bars) strains were calculated relative to the corresponding unstressed control cultures. RNA was extracted and analyzed from mid-exponentially growing cultures before (control) and after 10 min temperature up-shift to 42 °C (heat stress). ldhD was used as internal control gene. Data shown are mean ± SD of three independent experiments. For all genes except clpP, heat induction values were statistically significant with respect to control conditions (p ≤ 0.05).
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f4-ijms-13-10680: Transcriptional heat induction of putative CtsR regulon genes. Heat induction of putative CtsR regulon genes in L. plantarum WCFS1 wild type and ΔctsR mutant strains as revealed by qRT-PCR analysis. The mRNA levels in wild type (open bars) and ΔctsR mutant (grey bars) strains were calculated relative to the corresponding unstressed control cultures. RNA was extracted and analyzed from mid-exponentially growing cultures before (control) and after 10 min temperature up-shift to 42 °C (heat stress). ldhD was used as internal control gene. Data shown are mean ± SD of three independent experiments. For all genes except clpP, heat induction values were statistically significant with respect to control conditions (p ≤ 0.05).

Mentions: Changes in the expression of the genes encoding the proteins under investigation and/or under putative CtsR control were monitored by qRT-PCR transcriptional analysis. The mRNA levels in both wild type and mutant strains were analyzed for clpE, clpB, clpP and hsp1, identified in the protein map, as well as for the clpC gene, whose promoter, like that of hsp1, was previously shown to be specifically recognized and bound by the recombinant L. plantarum WCFS1 CtsR [14]. In order to evaluate heat stress induction at the transcriptional level, the mRNA pattern of these L. plantarum WCFS1 genes, presenting CtsR operators, was monitored prior to and after short heat exposure, in both wild type and mutant strains. Relative to control temperature condition, increased mRNA levels were detected after heat shock for all the analyzed genes in both strains (Figure 4). Overall, the transcriptional profile matched the proteomic data (stress induction factors of Table 1), except for clpC, whose protein could not be identified by the 2D-gel analysis, and clpP level in the wild type, whose mRNA was poorly heat-induced (1.3 fold), in contrast to the increased protein concentration (4.9 fold).


Comparative proteomic analysis of Lactobacillus plantarum WCFS1 and ΔctsR mutant strains under physiological and heat stress conditions.

Russo P, de la Luz Mohedano M, Capozzi V, de Palencia PF, López P, Spano G, Fiocco D - Int J Mol Sci (2012)

Transcriptional heat induction of putative CtsR regulon genes. Heat induction of putative CtsR regulon genes in L. plantarum WCFS1 wild type and ΔctsR mutant strains as revealed by qRT-PCR analysis. The mRNA levels in wild type (open bars) and ΔctsR mutant (grey bars) strains were calculated relative to the corresponding unstressed control cultures. RNA was extracted and analyzed from mid-exponentially growing cultures before (control) and after 10 min temperature up-shift to 42 °C (heat stress). ldhD was used as internal control gene. Data shown are mean ± SD of three independent experiments. For all genes except clpP, heat induction values were statistically significant with respect to control conditions (p ≤ 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472708&req=5

f4-ijms-13-10680: Transcriptional heat induction of putative CtsR regulon genes. Heat induction of putative CtsR regulon genes in L. plantarum WCFS1 wild type and ΔctsR mutant strains as revealed by qRT-PCR analysis. The mRNA levels in wild type (open bars) and ΔctsR mutant (grey bars) strains were calculated relative to the corresponding unstressed control cultures. RNA was extracted and analyzed from mid-exponentially growing cultures before (control) and after 10 min temperature up-shift to 42 °C (heat stress). ldhD was used as internal control gene. Data shown are mean ± SD of three independent experiments. For all genes except clpP, heat induction values were statistically significant with respect to control conditions (p ≤ 0.05).
Mentions: Changes in the expression of the genes encoding the proteins under investigation and/or under putative CtsR control were monitored by qRT-PCR transcriptional analysis. The mRNA levels in both wild type and mutant strains were analyzed for clpE, clpB, clpP and hsp1, identified in the protein map, as well as for the clpC gene, whose promoter, like that of hsp1, was previously shown to be specifically recognized and bound by the recombinant L. plantarum WCFS1 CtsR [14]. In order to evaluate heat stress induction at the transcriptional level, the mRNA pattern of these L. plantarum WCFS1 genes, presenting CtsR operators, was monitored prior to and after short heat exposure, in both wild type and mutant strains. Relative to control temperature condition, increased mRNA levels were detected after heat shock for all the analyzed genes in both strains (Figure 4). Overall, the transcriptional profile matched the proteomic data (stress induction factors of Table 1), except for clpC, whose protein could not be identified by the 2D-gel analysis, and clpP level in the wild type, whose mRNA was poorly heat-induced (1.3 fold), in contrast to the increased protein concentration (4.9 fold).

Bottom Line: The proteomic analysis of L. plantarum WCFS1 and ctsR mutant strains confirms at the translational level the CtsR-mediated regulation of some members of the Clp family, as well as the heat induction of typical stress response genes.Heat activation of the putative CtsR regulon genes at transcriptional and translational levels, in the ΔctsR mutant, suggests additional regulative mechanisms, as is the case of hsp1.Furthermore, isoforms of ClpE with different molecular mass were found, which might contribute to CtsR quality control.

View Article: PubMed Central - PubMed

Affiliation: Department of Agriculture, Food and Environment Sciences, Via Napoli 25, Foggia 71122, Italy; E-Mails: p.russo@unifg.it (P.R.); vittorio.capozzi@gmail.com (V.C.).

ABSTRACT
Among Gram-positive bacteria, CtsR (Class Three Stress gene Repressor) mainly regulates the expression of genes encoding the Clp ATPases and the ClpP protease. To gain a better understanding of the biological significance of the CtsR regulon in response to heat-shock conditions, we performed a global proteomic analysis of Lactobacillus plantarum WCFS1 and ΔctsR mutant strains under optimal or heat stress temperatures. Total protein extracts from bacterial cells were analyzed by two-dimensional gel fractionation. By comparing maps from different culture conditions and different L. plantarum strains, image analysis revealed 23 spots with altered levels of expression. The proteomic analysis of L. plantarum WCFS1 and ctsR mutant strains confirms at the translational level the CtsR-mediated regulation of some members of the Clp family, as well as the heat induction of typical stress response genes. Heat activation of the putative CtsR regulon genes at transcriptional and translational levels, in the ΔctsR mutant, suggests additional regulative mechanisms, as is the case of hsp1. Furthermore, isoforms of ClpE with different molecular mass were found, which might contribute to CtsR quality control. Our results could add new outlooks in order to determine the complex biological role of CtsR-mediated stress response in lactic acid bacteria.

No MeSH data available.


Related in: MedlinePlus