Acidic environments encountered in food products and during gastrointestinal tract passage affect the survival of bacteria that are marketed as probiotics. In this study, the global proteome responses of the probiotic lactic acid bacterium Lactobacillus rhamnosus GG to two physiologically relevant pH conditions (pH 4.8 and pH 5.8) were studied by 2-D DIGE. The proteomics data were complemented with transcriptome analyses by whole-genome DNA microarrays. The cells were cultured in industrial-type whey medium under a strictly defined bioreactor conditions. In total, 2-D DIGE revealed the pH-dependent formation of 92 protein spots. In response to lower pH conditions, the strongest up-regulation of all proteins was detected for a predicted surface antigen, LGG_02016. In addition, the acid pH was found to up-regulate the expression of F(0)F(1)-ATP synthase genes whereas the abundance of proteins participating in nucleotide biosynthesis and protein synthesis was significantly diminished. Moreover, the results suggest that L. rhamnosus GG modulates its pyruvate metabolism depending on the growth pH. Furthermore, a growth pH-dependent protein phosphorylation phenomenon was detected in several L. rhamnosus GG proteins with ProQ Diamond 2-DE gel staining. Proteins participating in central cellular pathways were shown to be phosphorylated, and the phosphorylation of glycolytic enzymes was found to be especially extensive.

In Staphylococcus aureus, ClpP proteases were previously shown to be essential for virulence and stress tolerance in strains derived from NCTC8325. Because these strains exhibit a severely reduced activity of the alternative sigma factor, SigB, we here re-assessed the role of ClpP in SigB-proficient clinical strains. To this end, clpP was deleted in strains COL, Newman, and SA564, and the strains were characterized phenotypically. The proteomic changes accomplished by the clpP deletion in the different strains were analyzed using the 2-D DIGE technique. The proteomic analyses revealed mostly conserved changes in the protein profiles of the ClpP-deficient strains. Among the strain-specific changes were the up-regulation of prophage proteins that coincided with an increased spontaneous release of prophages and the relatively poorer growth of the clpP mutants in some strain backgrounds. Interestingly, the effect of ClpP on the expression of selected virulence genes was strain-dependent despite the fact that the expression of the global virulence regulators RNAIII, mgrA, sarZ, sarR, arlRS was similarly changed in all clpP mutants. ClpP affected the expression of sarS in a strain-dependent manner, and we propose that the differential expression of sarS is central to the strain-dependent effect of ClpP on the expression of virulence genes.

The growth phase during which probiotic bacteria are harvested and consumed can strongly influence their performance as health-promoting agents. In this study, global transcriptomic and proteomic changes were studied in the widely used probiotic Lactobacillus rhamnosus GG during growth in industrial-type whey medium under strictly defined bioreactor conditions. The expression of 636 genes (P ≤ 0.01) and 116 proteins (P < 0.05) changed significantly over time. Of the significantly differentially produced proteins, 61 were associated with alterations at the transcript level. The most remarkable growth phase-dependent changes occurred during the transition from the exponential to the stationary growth phase and were associated with the shift from glucose fermentation to galactose utilization and the transition from homolactic to mixed acid fermentation. Furthermore, several genes encoding proteins proposed to promote the survival and persistence of L. rhamnosus GG in the host and proteins that directly contribute to human health showed temporal changes in expression. Our results suggest that L. rhamnosus GG has a highly flexible and adaptable metabolism and that the growth stage during which bacterial cells are harvested and consumed should be taken into consideration to gain the maximal benefit from probiotic bacteria.

The present study reports an in-depth proteome analysis of two Lactobacillus rhamnosus strains, the well-known probiotic strain GG and the dairy strain Lc705. We used GeLC-MS/MS, in which proteins are separated using 1-DE and identified using nanoLC-MS/MS, to generate high-quality protein catalogs. To maximize the number of identifications, all data sets were searched against the target databases using two search engines, Mascot and Paragon. As a result, over 1600 high-confidence protein identifications, covering nearly 60% of the predicted proteomes, were obtained from each strain. This approach enabled identification of more than 40% of all predicted surfome proteins, including a high number of lipoproteins, integral membrane proteins, peptidoglycan associated proteins, and proteins predicted to be released into the extracellular environment. A comparison of both data sets revealed the expression of more than 90 proteins in GG and 150 in Lc705, which lack evolutionary counterparts in the other strain. Differences were noted in proteins with a likely role in biofilm formation, phage-related functions, reshaping the bacterial cell wall, and immunomodulation. The present study provides the most comprehensive catalog of the Lactobacillus proteins to date and holds great promise for the discovery of novel probiotic effector molecules.

Lactobacillus rhamnosus GG (LGG) is one of the most extensively studied and widely used probiotic bacteria. While the benefits of LGG treatment in gastrointestinal disorders and immunomodulation are well-documented, functional genomics research of this bacterium has only recently been initiated. In the present study, a 2-D DIGE approach was used for the quantitative analysis of growth media-dependent changes in LGG protein abundance. Proteins were isolated from cells grown in industrial-type whey-based medium or in rich laboratory medium for subsequent 2-D DIGE. The analysis revealed patterns of protein abundance unique to each growth condition. In total, 196 quantitatively altered protein spots (at least 1.5-fold change in relative abundance, p < 0.05) representing approximately 13% of all protein spots in the gel were detected. From these protein spots, 157 were identified by mass spectrometry and were found to represent 100 distinct gene products. Collectively, these data show that growth of LGG in whey medium increased the relative abundance of proteins involved in purine biosynthesis, galactose metabolism, and fatty acid biosynthesis. In comparison, growth of LGG in laboratory medium resulted in an increase in the amount of proteins involved in translation and the general stress response, as well as pyrimidine and exopolysaccharide biosynthesis. Moreover, several enzymes of the proteolytic system of LGG demonstrated growth medium-dependent production. The present study demonstrates the fundamental effects of culture conditions on the proteome of LGG, which are likely to affect the functionality and characteristics of its use as a probiotic.

Summary Nodularia spumigena is a filamentous nitrogen-fixing cyanobacterium that forms toxic blooms in brackish water bodies worldwide. Spumigins are serine protease inhibitors reported from a single strain of N. spumigena isolated from the Baltic Sea. These linear tetrapeptides contain non-proteinogenic amino acids including a C-terminal alcohol derivative of arginine. However, very little is known about these compounds despite the ecological importance of N. spumigena. We show that spumigins are assembled by two non-ribosomal peptide synthetases encoded in a 21 kb biosynthetic gene cluster. The compact non-ribosomal peptide synthetase features a reductive loading and release mechanism. Our analyses demonstrate that the bulk of spumigins produced by N. spumigena are released as peptide aldehydes in contrast to earlier findings. The main spumigin E variant contains an argininal residue and is a potent trypsin inhibitor. Spumigins were present in all of the N. spumigena strains isolated from the Baltic Sea and comprised up to 1% of the dry weight of the cyanobacterium. Our results demonstrate that bloom-forming N. spumigena strains produce a cocktail of enzyme inhibitors, which may explain in part the ecological success of this cyanobacterium in brackish water bodies worldwide.

In this article we summarize the current knowledge of Baltic Sea cyanobacteria, focusing on diversity, toxicity, and nitrogen fixation in the filamentous heterocystous taxa. We also review the recent results of our microbial diversity studies in planktonic and benthic habitats in the Baltic Sea. Based on molecular analyses, we have improved the understanding of cyanobacterial population structure by assessing genetic diversity within species that are morphologically inseparable. Moreover, we have studied microbial functions such as toxin production and nitrogen fixation in situ under different environmental conditions. Phosphorus limitation of bloom-forming, nitrogen-fixing cyanobacteria was clearly verified, emphasizing the importance of continuous efforts to reduce this element in the Baltic Sea. We have designed a rapid and reliable detection method for the toxic cyanobacterium Nodularia spumigena, which can be used to study bloom formation of this important toxin producer.

A specific quantitative real-time PCR (qPCR) method was developed for quantification of hepatotoxin nodularin-producing Nodularia, one of the main bloom-forming cyanobacterium in the Baltic Sea. Specific PCR primers were designed for subunit F of nodularin synthetase gene (ndaF), which encodes the NdaF subunit of the nodularin synthetase gene complex needed for nodularin production. The qPCR method was applied to water samples (a total of 120 samples) collected from the Baltic Sea in July 2004. As few as 30 ndaF gene copies ml(-1) of sea water could be detected, and thus the method was very sensitive. The ndaF gene copy numbers and nodularin concentrations were shown to correlate in the Baltic Sea water indicating constant production of nodularin by Nodularia. This qPCR method for the ndaF gene can be used for detailed studies about Nodularia blooms and their formation. ndaF gene copies and nodularin were detected mostly in the surface water but also in deeper water layers (down to 30 m). Toxic Nodularia blooms are not only horizontally but also vertically widely distributed, and thus the Baltic fauna is extensively exposed to nodularin.