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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

Amino acid sequence of L. plantarum WCFS1 ClpE. The N-terminal zinc finger region is underlined. The peptides identified only after trypsin digestion of the four ClpE isoforms with heavier Mr in black font on grey background, those identified from trypsin digestion of the three lighter Mr isoforms are in white font on grey background. The peptides found after trypsin digestion of all isoforms are on black background.
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f3-ijms-13-10680: Amino acid sequence of L. plantarum WCFS1 ClpE. The N-terminal zinc finger region is underlined. The peptides identified only after trypsin digestion of the four ClpE isoforms with heavier Mr in black font on grey background, those identified from trypsin digestion of the three lighter Mr isoforms are in white font on grey background. The peptides found after trypsin digestion of all isoforms are on black background.

Mentions: We detected two putative isoforms (spots 12 and 13) of the 21.5 kDa proteolytic subunit ClpP. Spot 11 was also identified as ClpP, although its molecular mass (33 kDa) and isoelectric point did not correspond to the theoretically predicted ones. This particular form of ClpP was previously observed by Cohen et al. [15] in L. plantarum WCFS1 at stationary-phase, suggesting that this might be due to a complex with an unidentified small molecule. Three isoforms (spots 21–23) with a Mr of 95.6 kDa were identified as the ATP-binding subunit ClpB, varying slightly in their isoelectric points (Figure 2). Seven well-defined spots (14–20) were identified as being isoforms of the ATP-binding subunit ClpE. These peptides were resolved in the gel into two main groups (four, spots 17–20, and three, spots 14–16) with a Mr difference of 5.8 kDa (Figure 2). Recently, Elsholz et al. [7] demonstrated that the heat stress regulation of CtsR was regarded to be dependent on an intrinsic heat-sensing property of CtsR in all low GC, Gram-positive bacteria. Therefore, de-repression is considered as a physiological process, which is independent from CtsR degradation. It was reported that ClpE plays a role in the control of CtsR stability, but the mechanisms of how this occurs has not yet been clarified [7,22–24]. Two ClpE forms, differing by 6 kDa in their Mr, as revealed by western blot analysis, were previously reported in extracts from Lactococcus lactis strains subjected to either high temperature [22] or oxidative stress [24]. Based on these previous studies and on our data, we can therefore suggest that a variation of the relative concentration of these forms may be associated to a cellular heat stress condition. However, in contrast to what was observed by Elsholz et al. [24], and taking into account our proteomic analyses, it is the small subunit that seems to characterize physiological conditions in the wild type, whereas the heavier Mr isoforms appear only in response to heat shock. Accordingly, in the mutant strain, which constitutively exhibits a proteomic pattern that is typical of a stress condition, all ClpE forms are evident in extracts from both unstressed and heat stressed cells. The peptide mass fingerprint of the four heavier isoforms revealed the occurrence of a 15 amino acid fragment corresponding to the N-terminal zinc finger region (Figure 3). However, this peptide was never detected in the digestion pattern of the three small ClpE isoforms; instead, a different 13 amino acids N-terminus proximal fragment, starting from position 66, was found. Such a fragment was never observed after trypsin digestion of ClpE spots with heavier Mr (Figure 3).


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)

Amino acid sequence of L. plantarum WCFS1 ClpE. The N-terminal zinc finger region is underlined. The peptides identified only after trypsin digestion of the four ClpE isoforms with heavier Mr in black font on grey background, those identified from trypsin digestion of the three lighter Mr isoforms are in white font on grey background. The peptides found after trypsin digestion of all isoforms are on black background.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-ijms-13-10680: Amino acid sequence of L. plantarum WCFS1 ClpE. The N-terminal zinc finger region is underlined. The peptides identified only after trypsin digestion of the four ClpE isoforms with heavier Mr in black font on grey background, those identified from trypsin digestion of the three lighter Mr isoforms are in white font on grey background. The peptides found after trypsin digestion of all isoforms are on black background.
Mentions: We detected two putative isoforms (spots 12 and 13) of the 21.5 kDa proteolytic subunit ClpP. Spot 11 was also identified as ClpP, although its molecular mass (33 kDa) and isoelectric point did not correspond to the theoretically predicted ones. This particular form of ClpP was previously observed by Cohen et al. [15] in L. plantarum WCFS1 at stationary-phase, suggesting that this might be due to a complex with an unidentified small molecule. Three isoforms (spots 21–23) with a Mr of 95.6 kDa were identified as the ATP-binding subunit ClpB, varying slightly in their isoelectric points (Figure 2). Seven well-defined spots (14–20) were identified as being isoforms of the ATP-binding subunit ClpE. These peptides were resolved in the gel into two main groups (four, spots 17–20, and three, spots 14–16) with a Mr difference of 5.8 kDa (Figure 2). Recently, Elsholz et al. [7] demonstrated that the heat stress regulation of CtsR was regarded to be dependent on an intrinsic heat-sensing property of CtsR in all low GC, Gram-positive bacteria. Therefore, de-repression is considered as a physiological process, which is independent from CtsR degradation. It was reported that ClpE plays a role in the control of CtsR stability, but the mechanisms of how this occurs has not yet been clarified [7,22–24]. Two ClpE forms, differing by 6 kDa in their Mr, as revealed by western blot analysis, were previously reported in extracts from Lactococcus lactis strains subjected to either high temperature [22] or oxidative stress [24]. Based on these previous studies and on our data, we can therefore suggest that a variation of the relative concentration of these forms may be associated to a cellular heat stress condition. However, in contrast to what was observed by Elsholz et al. [24], and taking into account our proteomic analyses, it is the small subunit that seems to characterize physiological conditions in the wild type, whereas the heavier Mr isoforms appear only in response to heat shock. Accordingly, in the mutant strain, which constitutively exhibits a proteomic pattern that is typical of a stress condition, all ClpE forms are evident in extracts from both unstressed and heat stressed cells. The peptide mass fingerprint of the four heavier isoforms revealed the occurrence of a 15 amino acid fragment corresponding to the N-terminal zinc finger region (Figure 3). However, this peptide was never detected in the digestion pattern of the three small ClpE isoforms; instead, a different 13 amino acids N-terminus proximal fragment, starting from position 66, was found. Such a fragment was never observed after trypsin digestion of ClpE spots with heavier Mr (Figure 3).

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