KiBeom Lee

The effect of low pH on protein expression by the probiotic bacterium Lactobacillus reuteri

The ability of a lactic acid bacterium to survive passage through the gastrointestinal tract is a key point in its function as a probiotic. In this study, protein synthesis by the probiotic bacterium, Lactobacillus reuteri, was analyzed under transiently decreased pH conditions. L. reuteri cells grown to the midexponential growth phase at 37°C were exposed to transient (1 h) low‐pH stresses from pH 6.8 to pH 5.0, 4.5, or 4.0. 2‐DE allowed us to identify 40 common proteins that were consistently and significantly altered under all three low‐pH conditions. PMF was used to identify these 40 proteins, and functional annotation allowed them to be distributed to six major classes: (i) transport and binding proteins; (ii) transcription–translation; (iii) nucleotide metabolism and amino acid biosynthesis; (iv) carbon energy metabolism; (v) pH homeostasis and stress; and (vi) unassigned. These findings provide new insight into the inducible mechanisms underlying the capacity of gastrointestinal L. reuteri to tolerate acid stress.

Proteomic analysis of the effect of bile salts on the intestinal and probiotic bacterium Lactobacillus reuteri

Lactobacillus reuteri is a resident of the human and animal intestinal tracts. The ability of L. reuteri to survive passage through the intestinal tract is a key point in its function as a probiotic. In order to examine the nature of bile salt tolerance by L. reuteri, its protein synthesis was analyzed in liquid cultures containing two different bile salt conditions. Significant cell growth inhibition was observed in the presence of 1.2g/L (higher concentration) bile salts. Two-dimensional gel electrophoresis allowed us to identify 28 proteins spots that were consistently and significantly altered in the presence of bile in the growth medium. Peptide mass fingerprinting was used to identify these 28 proteins, and functional annotation revealed their involvement in carbohydrate metabolism, transcription-translation, nucleotide metabolism, amino acid biosynthesis, pH homeostasis and stress responses, oxidation-reduction reactions, and unknown functions. These findings, which suggest that bile salts induce complex physiological responses in L. reuteri may provide early new insights into the inducible mechanisms underlying the capacity of intestinal L. reuteri to tolerate bile stress.

Proteome response of Escherichia coli fed-batch culture to temperature downshift

During fed-batch cultivation of Escherichia coli K-12, the proteomic response to a temperature downshift from 37 to 20°C was quantitatively monitored and analyzed by using two-dimensional electrophoresis. When the temperature of exponentially growing E. coli K-12 culture was downshifted to 20°C, the synthesis level of 57 intracellular proteins showed significant changes for a prolonged period of time, compared to the fed-batch culture controlled at 37°C. Thus, these proteins are regarded as important stress proteins responsive to cold shock, which were analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and identified using the E. coli SWISS-2DPAGE database. Most of the identified proteins were shown to be involved in energy metabolism, several cellular molecule biosynthetic pathways and catabolism, cell processes, flagellar biosynthesis and motility, and protein translation and folding. The systematic approach to the monitoring of proteomic responses and the detailed analysis results reported in this article would be useful in understanding the metabolic adaptation to lowered culture temperature and designing efficient fermentation strategies for the production of recombinant proteins and metabolites using E. coli strains.

Electroenzymatic oxidation of veratryl alcohol by lignin peroxidase

This paper reports the formation of veratraldehyde by electroenzymatic oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) hybridizing both electrochemical and enzymatic reactions and using lignin peroxidase. The novel electroenzymatic method was found to be effective for replacement of hydrogen peroxide by an electrochemical reactor, which is essential for enzyme activity of lignin peroxidase. The effects of operating parameters such as enzyme dosage, pH, and electric potential were investigated. Further, the kinetics of veratryl alcohol oxidation in an electrochemical reactor were compared to oxidation when hydrogen peroxide was supplied externally.

Effect of transient acid stress on the proteome of intestinal probiotic Lactobacillus reuteri

We report the acid tolerance response and changes in the level of protein expression of probiotic Lactobacillus reuteri subjected to transient (1.5 h) acid stress at pH 3.0. Sixteen acid-responsive proteins were identified by peptide mass fingerprinting including members of five broad functional categories: metabolism, transcription/translation, DNA replication/repair, transport and binding proteins, and pH homeostasis and stress responses. This work can provide some new and relevant information on the inducible mechanisms underlying the capacity of probiotic L. reuteri to tolerate acid stress.

Proteomic analysis of protein expression in Lactobacillus plantarum in response to alkaline stress

Lactobacillus plantarum, a probiotic organism that plays an important role in the microbial fermentation of alkaline materials in fermenting foods, faces alkaline stress during the fermentation process. Here, we report the patterns of protein expression in L. plantarum subjected to transient (1h) alkaline stress at pH 7.7, 8.7 or 9.7. Thirty-three alkaline-responsive proteins were identified by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Identification of proteins showing differential expression in response to alkaline stress revealed that the alkaline stress response of L. plantarum is a complex process. Some proteins appear to be induced, others repressed. These proteins could be clustered into nine groups based on their probable functions: energy metabolism, transport system, purine/pyrimidine metabolism, amino acid metabolism, proteolytic activity, transcription-translation, stress-related, general function, and unknown functions. These proteomic analyses are expected to prove useful in understanding the adaptive response of L. plantarum strains to alkaline stress and may facilitate future investigations into the genetic and physiological aspects of this response.

Unique Properties of Four Lactobacilli in Amino Acid Production and Symbiotic Mixed Culture for Lactic Acid Biosynthesis

With four Lactobacilli—L. delbrueckii subsp. lactis (ATCC 12315), L. casei (NRRL-B1445), L. delbrueckii (NRRL-B445), and L. heveticus (NRRL-B1937)—the characteristics of cell growth and production of lactate and amino acids were investigated. Especially, the time-course variation in concentration of amino acids (classified into alanine, serine, aspartate, glutamate, aromatic amino acid, and histidine families) was estimated in detail, and the results were systematically compared. It was elucidated that L. delbrueckii (NRRL-B445) and L. helveticus (NRRL-B1937) had quite different characteristics in growth, lactic acid synthesis, and amino acid production. L. helveticus (NRRL-B1937) was superior in the production of amino acids as well as in cell growth, but showed very poor ability in lactic acid production. However, L. delbrueckii (NRRL-B445) showed higher yield of lactic acid despite repressed cell growth, but suffered from severe amino acid deficiency in culture. By modulating the initial concentration of each strain in the mixed culture containing both L. delbrueckii (NRRL-B445) and L. helveticus (NRRL-B1937), the lactic acid production (i.e., the amount of lactic acid produced and lactic acid yield to glucose consumed) was significantly improved, presumably via symbiotic interaction between the two strains.

Growth Kinetics of Lactococcus lactis ssp. diacetylactis Harboring Different Plasmid Content

The effect of plasmid content on growth of Lactococcus lactis ssp. diacetylactis harboring different plasmids and on plasmid stability was studied. Strain DRC-2C is a plasmid Lac+- and Prt+-free strain. Strain DRC-2 utilizes lactose as carbohydrate and has proteinase activity. The plasmid-free strain DRC-2C exhibited none of these features. Plasmid-encoded properties were clearly identified. Results showed that plasmid content decreased bacterial growth in terms of the specific growth rate determined. Slightly lower specific growth rate and lactic acid production were observed in the strain of higher plasmid content owing to the plasmid presence, causing metabolic burden to the host cell. The plasmid profile results showed that the number of bands in the two strains before and after fermentation were the same. This indicated that the plasmids were stably maintained and unchanged during the fermentation.

Electrophoretic Prefractionation for Comprehensive Analysis of Proteomes

Publisher Summary Two-dimensional polyacrylamide gel electrophoresis (2DE) has dominated protein profile analysis for more than 25 years, and it is still the method of choice in many laboratories for quantitatively comparing changes of proteins for proteome analysis experiments. Due to the limited capacities of both gel based and nongel based protein profiling methods, it has become apparent that more powerful and reliable methods are needed for prefractionation of complex proteomes prior to 2D gels and alternative LC–MS analysis. Although preparative IEF prefractionation methods are not orthogonal to 2D gels, they show the most promise due to the very high resolution that can be obtained. Alternative lower resolution prefractionation methods severely compromise the ability to perform comprehensive quantitative comparisons due to variable cross-contamination between adjacent fractions and greater fraction complexity. A number of preparative solution-based IEF methods have been productively integrated into quantitative protein profiling strategies including Rotofor, FFE, IsoPrime, the MCE and μsol-IEF. However, some of these methods require large sample amounts and result in large dilute fractions that are not compatible with direct analysis using downstream protein profiling methods.

Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis.

A problem in proteomic analysis of lung cancer tissue is the presence of complex components of different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma, and adenocarcinoma). The efficient solubilization of protein components before two-dimensional electrophoresis (2-DE) is a very critical. Poor solubilization has been associated with a failure to detect proteins and diffuse, streaked and/or trailing protein spots. Here, we have optimized the solubilization of human lung cancer tissue to increase protein resolution. Isoelectric focusing (IEF) rehydration buffer containing a thiourea–urea mixture provided superior resolution, whereas a buffer without thiourea yielded consistently poor results. In addition, IEF rehydration buffers containing CHAPS and DTT gave superior resolution, whereas buffers containing Nonidet P-40 (NP-40) and/or Triton X-100 did not. A tributylphosphine-containing buffer gave consistently poor results. Using optimized conditions, we used 2-D gel analysis of human lung cancer tissue to identify 11 differentially-expressed protein spots by MALDI-mass spectrometry. This study provides a methodological tool to study the complex mammalian proteomes.