In recent years, several studies used the two-dimensional gel electrophoresis approach in combination with mass spectrometry to study listerial gene expression upon changes in growth condition including salt stress [101], acid [102], high pressure and freezing [103], carbon starvation [104], or transition to the stationary phase [105] - conditions also encountered by the bacteria during food processing and storage. Of particular interest is the finding by Weeks et al. [105] that the bacteria perceived stress and began preparations for stationary phase much earlier (already in mid-exponential phase) than predicted by growth characteristics alone, and that the expression levels of more than 50% of all proteins observed changed significantly during the transition into stationary phase. Transition into stationary phase was also analyzed by Folio et al. [106] in a study primarily aiming to establish a two-dimensional electrophoresis database of L. monocytogenes EGD (see:, as also done before by Ramnath et al. [107].

Multiple classes of putative cell-wall-anchored surface proteins which are believed to be important for interaction with the host cell were identified in the L. monocytogenes genome [2, 65]. Thus, the listerial cell wall subproteome is of special interest and was analyzed in detail in two recent studies [108, 109] using either standard two-dimensional protein separation [108] or a two-dimensional nanoliquid chromatography approach designed to specifically identify proteins linked to the peptidoglycan network [109]. For the standard approach [108], the proteins were extracted from the listerial surface by serial treatment with different salts at high concentration, and a total of 55 proteins were identified by N-termi-nal sequencing and mass spectrometry. Remarkably, besides lipoproteins, transporters, and other proteins (of often unknown function), a relatively high number of proteins with a function in the cytoplasmic compartment were identified in this surface proteome, which had neither predicted or detectable signal peptides, nor could any modification be observed. Among these unexpected proteins are enolase (Lmo2455), DnaK (Lmo1473), elongation factor Tu (Lmo2653), and glycer-aldehyde-3-phosphate dehydrogenase (Lmo2459). In contrast, the extraction method used by Calvo et al. [109] resulted in the identification of primarily LPXTG motif-harboring proteins, which were most likely covalently linked to the peptido-glycan. Among them were InlA, InlG, InlH, and seven further LPXTG proteins (putative internalins) of unknown function.

Two-dimensional gel electrophoresis also represents a powerful approach to characterizing regulons once a regulatory mutant is available. Thus, sigma B-con-trolled and DegU-dependent genes were identified in L. monocytogenes [103, 110]. Nine proteins were identified to accumulate in the wild-type strain but not in the DsigB strain [103]. These proteins included Pfk, GalE, ClpP, and Lmo1580, all of which are typical proteins involved in acid adaptation. However, the gad operon, coding for the glutamate decarboxylase (GAD) acid resistance system, a specific mechanism for acid adaptation in L. monocytogenes aimed at maintenance of the internal pH and shown to be SigB-dependent by reverse transcription polymerase chain reaction (RT-PCR), was not identified in the proteomic approach pointing to limitations of this otherwise powerful technique. The analysis of the supernatant subproteome of L. monocytogenes WT and a DdegU mutant resulted in the identification of nine proteins, encoded in three operons coding for the flagellar apparatus, the expression of which is DegU-dependent [110]. Similar experiments can now be performed by comparison of WT bacteria with a wide array of L. monocytogenes in-frame deletion mutants in known or putative regulatory genes.

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