Identification of the DNA-binding sites for two response regulators involved in control of bacteriocin synthesis in Lactobacillus plantarum C11

In Lactobacillus plantarum C11, bacteriocin production has previously been shown to be an inducible process, in which a secreted peptide, produced by the host itself, is involved. The inducing factor, designated plantaricin A (PlnA), is a bacteriocin-like peptide encoded by a gene (plnA) located on the same operon as the genes for a two-component regulatory system (plnBCD). This system consists of a histidine kinase (PlnB) and two response regulators (PlnC,D), and belongs to a recently defined subfamily of two-component regulatory systems, which are activated by secreted peptide pheromones through a quorum-sensing mechanism. We show here that the two response regulators PlnC and PlnD bind specifically to imperfect direct repeats found within the adjacent promoter of the plnABCD operon, and to similar sequences found within the promoter regions of two nearby operons containing bacteriocin structural genes (plnEFI and plnJKLR). Binding of PlnC and PlnD was increased two to three fold in the presence of acetyl phosphate. The results suggest that bacteriocin synthesis in L. plantarum C11 is regulated by the DNA-binding activity of the two response regulators PlnC and PlnD.

     

The activity of the extracellular signal-regulated kinase 2 is regulated by differential phosphorylation in the activation loop

The mitogen-activated protein kinases (MAP kinases) play a central role in signaling pathways initiated by extracellular stimuli such as growth factors, cytokines, and various forms of environmental stress. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Interestingly, down-regulation of MAP kinase activity can be initiated by multiple Ser/Thr phosphatases, Tyr-specific phosphatases, and dual-specificity phosphatases. This would inevitable lead to the formation of monophosphorylated MAP kinases. However, in much of the literature investigating MAP kinase signaling, there has been the implicit assumption that the monophosphorylated forms are inactive. Thus, the significance for the need of multiple phosphatases in regulating MAP kinase activity is not clear, and the biological functions of these monophosphorylated MAP kinases are currently unknown. We have prepared extracellular signal-regulated protein kinase 2 (ERK2) in all phosphorylated forms and kinetically characterized them using two proteins (the myelin basic protein and Elk-1) and ATP as substrates. Our results revealed that a single phosphorylation in the activation loop of ERK2 produces an intermediate activity state. Thus, the catalytic efficiencies of the monophosphorylated ERK2/pY and ERK2/pT (ERK2 phosphorylated on Tyr-185 and Thr-183, respectively) are approximately 2-3 orders of magnitude higher than that of the unphosphorylated ERK2 and are only 1-2 orders of magnitude lower than that of the fully active bisphosphorylated ERK2/pTpY. This raises the possibility that the monophosphorylated ERK2s may have distinct biological roles in vivo. Different phosphorylation states in the activation loop could be linked to graded effects on a single ERK2 function. Alternatively, they could be linked to distinct ERK2 functions. Although less active than the bisphosphorylated species, the monophosphorylated ERK2s may differentially phosphorylate pathway components.     

The calcium-binding activity of a vacuole-associated,dehydrin-like protein is regulated by phosphorylation

A vacuole membrane-associated calcium-binding protein with an apparent mass of 45 kD was purified from celery (Apium graveolens). This protein, VCaB45, is enriched in highly vacuolate tissues and is located within the lumen of vacuoles. Antigenically related proteins are present in many dicotyledonous plants. VCaB45 contains significant amino acid identity with the dehydrin family signature motif, is antigenically related to dehydrins, and has a variety of biochemical properties similar to dehydrins. VCaB45 migrates anomalously in sodium dodecyl sulfate-polyacrylamide gel electrophoresis having an apparent molecular mass of 45 kD. The true mass as determined by matrix-assisted laser-desorption ionization time of flight was 16.45 kD. VCaB45 has two characteristic dissociation constants for calcium of 0.22 +/- 0.142 mM and 0.64 +/- 0.08 mM, and has an estimated 24.7 +/- 11.7 calcium-binding sites per protein. The calcium-binding properties of VCaB45 are modulated by phosphorylation; the phosphorylated protein binds up to 100-fold more calcium than the dephosphorylated protein. VCaB45 is an "in vitro" substrate of casein kinase II (a ubiquitous eukaryotic kinase), the phosphorylation resulting in a partial activation of calcium-binding activity. The vacuole localization, calcium binding, and phosphorylation of VCaB45 suggest potential functions.     

Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus

A total of 52 strains of Lactobacillus acidophilus were examined for production of bacteriocins. A majority (63%) demonstrated inhibitory activity against all members of a four-species grouping of Lactobacillus leichmannii, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus lactis. Four L. acidophilus strains with this activity also inhibited Streptococcus faecalis and Lactobacillus fermentum, suggesting a second system of antagonism. Under conditions eliminating the effects of organic acids and hydrogen peroxide, no inhibition of other gram-positive or -negative genera was demonstrated by L. acidophilus. The agent produced by L. acidophilus N2 and responsible for inhibition of L. leichmannii, L. bulgaricus, L. helveticus, and L. lactis was investigated. Ultrafiltration studies indicated a molecular weight of approximately 100,000 for the crude inhibitor. The agent was sensitive to proteolytic enzymes and retained full activity after 60 min at 100 degrees C (pH 5). Activity against sensitive cells was bactericidal but not bacteriolytic. These characteristics identified the inhibitory agent as a bacteriocin, designated lactacin B. Examination of strains of L. acidophilus within the six homology groupings of Johnson et al. (Int. J. Syst. Bacteriol. 30:53-68, 1980) demonstrated that production of the bacteriocin lactacin B could not be used in classification of neotype L. acidophilus strains. However, the usefulness of employing sensitivity to lactacin B in classification of dairy lactobacilli is suggested.     

PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP,the cognate phospho-Ser/Thr phosphatase,in Mycobacterium tuberculosis

Bacterial genomics revealed the widespread presence of eukaryotic-like protein kinases and phosphatases in prokaryotes, but little is known on their biochemical properties, regulation mechanisms and physiological roles. Here we focus on the catalytic domains of two trans-membrane enzymes, the Ser/Thr protein kinase PknB and the protein phosphatase PstP from Mycobacterium tuberculosis. PstP was found to specifically dephosphorylate model phospho-Ser/Thr substrates in a Mn2+-dependent manner. Autophosphorylated PknB was shown to be a substrate for Pstp and its kinase activity was affected by PstP-mediated dephosphorylation. Two threonine residues in the PknB activation loop, found to be mostly disordered in the crystal structure of this kinase, namely Thr171 and Thr173, were identified as the target for PknB autophosphorylation and PstP dephosphorylation. Replacement of these threonine residues by alanine significantly decreased the kinase activity, confirming their direct regulatory role. These results indicate that, as for eukaryotic homologues, phosphorylation of the activation loop provides a regulation mechanism of mycobacterial kinases and strongly suggest that PknB and PstP could work as a functional pair in vivo to control mycobacterial cell growth.     

Modelling the growth and bacteriocin production by Lactobacillus amylovorus DCE 471 in batch cultivation

A model was set up to describe the production of amylovorin L471 by Lactobacillus amylovorus DCE 471, on a laboratory scale, in which the cells are grown in MRS (deMau-Rogosa-Sharpe) broth. The main features of the dynamic model are : (i) increase of the biomass according to a logistic equation ; (ii) non-growth-associated consumption of substrate (maintenance metabolism) ; and (iii) primary metabolite kinetics for the bacteriocin production. The main purpose was to set up a simple empirical model to examine growth and bacteriocin production in different conditions. Parameters estimated from a fermentation with 20 g l⁻¹ glucose (w/v) could be used to predict the evolution of cell dry mass, glucose and lactic acid concentration of fermentations, performed with 5, 30, 40 and 60 g l⁻¹ initial glucose. The influence of the operating temperature (30, 37 and 45 °C) on the model parameters was also investigated. The proposed model was able to describe growth and bacteriocin production in all cases. The specific bacteriocin production rate was found to vary strongly with temperature, with 30 °C as the best value. Variation of the operating temperature from 37 to 30 °C appeared to significantly enhance the specific bacteriocin production.     

Purification and partial amino acid sequence of plantaricin S, a bacteriocin produced by Lactobacillus plantarum LPCO10, the activity of which depends on the complementary action of two peptides.

Plantaricin S, one of the two bacteriocins produced by Lactobacillus plantarum LPCO10, which was isolated from a green-olive fermentation (R. Jiménez-Díaz, R.M. Ríos-Sánchez, M. Desmazeaud, J.L.Ruiz-Barba, and J.-C. Piard, Appl. Environ. Microbiol. 59:1416-1424, 1993), has been purified to homogeneity by ammonium sulfate precipitation, by binding to SP-Sepharose fast-flow, phenyl-Sepharose CL-4B, and C2/C18 reverse-phase chromatographies. The purification resulted in a final yield of 91.6% and a 352,617-fold increase in the specific activity. The bacteriocin activity was associated with two distinct peptides, termed alpha and beta, which were separated by C2/C18 reverse-phase chromatography. Although beta alone appeared to retain a trace of inhibitory activity, the complementary action of both the alpha and beta peptides was required for full bacteriocin activity, as judged by both the agar well diffusion and the microtiter plate assays. From the N-terminal end, 26 and 24 amino acids residues of alpha and beta, respectively, were sequenced. Further attempts at sequencing revealed no additional amino acids residues, suggesting that either modifications in the next amino acid residue blocked the sequencing region or that the C-terminal end had been reached. The amino acid sequences of alpha and beta show no apparent homology to each or to other bacteriocins purified from lactic acid bacteria.     

PHAX, a mediator of U snRNA nuclear export whose activity is regulated by phosphorylation

In metazoa, assembly of spliceosomal U snRNPs requires nuclear export of U snRNA precursors. Export depends upon the RNA cap structure, nuclear cap-binding complex (CBC), the export receptor CRM1/Xpo1, and RanGTP. These components are however insufficient to support U snRNA export. We identify PHAX (phosphorylated adaptor for RNA export) as the additional factor required for U snRNA export complex assembly in vitro. In vivo, PHAX is required for U snRNA export but not for CRM1-mediated export in general. PHAX is phosphorylated in the nucleus and then exported with RNA to the cytoplasm, where it is dephosphorylated. PHAX phosphorylation is essential for export complex assembly while its dephosphorylation causes export complex disassembly. The compartmentalized PHAX phosphorylation cycle can contribute to the directionality of export.     

Catalase activity is regulated by c-Abl and Arg in the oxidative stress response

The Abl family of mammalian non-receptor tyrosine kinases includes c-Abl and Arg. Recent studies have demonstrated that c-Abl and Arg are activated in the response of cells to oxidative stress. This work demonstrates that catalase, a major effector of the cellular defense against H2O2, interacts with c-Abl and Arg. The results show that H2O2 induced binding of c-Abl and Arg to catalase. The SH3 domains of c-Abl and Arg bound directly to catalase at a P293FNP site. c-Abl and Arg phosphorylated catalase at Tyr231 and Tyr386 in vitro and in the response of cells to H2O2. The functional significance of the interaction is supported by the demonstration that cells deficient in both c-Abl and Arg exhibit substantial increases in H2O2 levels. In addition, c-abl-/- arg-/- cells exhibited a marked increase in H2O2-induced apoptosis compared with that found in the absence of either kinase. These findings indicate that c-Abl and Arg regulate catalase and that this signaling pathway is of importance to apoptosis in the oxidative stress response.     

CcpA affects expression of the groESL and dnaK operons in Lactobacillus plantarum

Background  

Lactic acid bacteria (LAB) are widely used in food industry and their growth performance is important for the quality of the fermented product. During industrial processes changes in temperature may represent an environmental stress to be overcome by starters and non-starters LAB. Studies on adaptation to heat shock have shown the involvement of the chaperon system-proteins in various Gram-positive bacteria. The corresponding operons, namely the dnaK and groESL operons, are controlled by a negative mechanism involving the HrcA repressor protein binding to the cis acting element CIRCE.     

Inducible expression of Bcl-XL inhibits sodium butyrate-induced apoptosis in hybridoma, resulting in enhanced antibody production

Sodium butyrate (NaBu) can increase the specific Mab production rate of hybridomas by enhancing histone hyperacetylation and influencing the cell cycle, but it can also inhibit cell growth and induce apoptosis. Thus, the beneficial effect of NaBu on Mab secretion is compromised by its cytotoxic effect. In the present study, expression of the anti-apoptotic protein human Bcl-XL was made inducible in hybridoma H18 to overcome the cytotoxic effect of NaBu, circumventing the detrimental effects of constitutive high-level expression. We constructed an expression vector in which the promoter of a mammalian metallothionein (MT) gene drove the expression of bcl-XL in response to metal exposure. The vector was then used to exogenously control the expression of bcl-XL in H18 hybridoma cells. Our data showed that stably transfected H18.D4 cells expressed high levels of Bcl-X(L), which was induced within 24 h of addition of ZnSO4. NaBu (0.4 mM) increased antibody production by more than 3-fold in H18.D4. This effect resulted from the suppression of NaBu-induced apoptosis, allowing the H18.D4 cells to grow at higher viability and extending culture longevity by >3 days.     

ERK7 expression and kinase activity is regulated by the ubiquitin-proteosome pathway

ERK7 is a unique member of the extracellular signal-regulated kinase (ERK) subfamily of MAP kinases. Although ERK7 shares a TEY motif in the activation loop of the kinase, it displays constitutive activation, nuclear localization, and growth inhibitory properties that are regulated by its C-terminal domain. Because ERK7 is expressed at low levels compared with ERK2 and its activity is dependent upon its expression level, we investigated the mechanism by which ERK7 expression is regulated. We now show that ERK7 expression is regulated by ubiquitination and rapid proteosomal turnover. Furthermore, both the kinase domain and the C-terminal tail are independently degraded at a rate comparable with that of the intact protein. Analysis of a series of chimeras between ERK2 and ERK7 reveal that the N-terminal 20 amino acids of the kinase domain are a primary determinant of ERK7 degradation. Fusion of the N-terminal 20 amino acids is both necessary and sufficient to cause proteolytic degradation of both ERK2 and green fluorescent protein. Finally, ERK7 is stabilized by an N-terminal mutant of Cullin-1 suggesting that ERK7 is ubiquitinated by the Skip1-Cullin-F box complex. These results indicate that ERK7 is a highly regulated enzyme whose cellular expression and kinase activation level is tightly controlled by the ubiquitin-proteosome pathway.