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PubMed1993

The Mode of Replication Is a Major Factor in Segregational Plasmid Instability in Lactococcus lactis.

The effects of the rolling-circle and theta modes of replication on the maintenance of recombinant plasmids in Lactococcus lactis were studied. Heterologous Escherichia coli or bacteriophage lambda DNA fragments of various sizes were inserted into vectors based on either the rolling-circle-type plasmid pWV01 or the theta-type plasmid pAMbeta1. All pAMbeta1 derivatives were stably maintained. pWV01 derivatives, however, showed size-dependent segregational instability, in particular when large DNA fragments were inserted. All recombinant pWV01 derivatives generated high-molecular-weight plasmid multimers (HMW) in amounts that were positively correlated with plasmid size and inversely correlated with the copy numbers of the monomeric plasmid forms. Formation of HMW or reductions in copy numbers were not observed with pAMbeta1 derivatives. The results indicate that HMW formation and/or reduction in plasmid copy numbers is an important factor in the maintenance of pWV01 derivatives. It is concluded that theta-type plasmids are superior to rolling-circle-type plasmids for cloning in lactococci.

Journal Article↗
PubMed2000

Heterologous coproduction of enterocin A and pediocin PA-1 by Lactococcus lactis: detection by specific peptide-directed antibodies.

Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH. Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4. In contrast, anti-PH5-KLH antibodies only reacted with enterocin A because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable. Enterocin A and/or pediocin PA-1 structural and immunity genes were introduced in Lactococcus lactis IL1403 to achieve (co)production of the bacteriocins. The level of production of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacteriocin translocation machinery of IL1403. Despite the low production levels, both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the anti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (J. M. Martínez, M. I. Martínez, A. M. Suárez, C. Herranz, P. Casaus, L. M. Cintas, J. M. Rodríguez, and P. E. Hernández, Appl. Environ. Microbiol. 64:4536-4545, 1998). In this work, the availability of antibodies for the specific detection and quantification of enterocin A and pediocin PA-1 was crucial to demonstrate coproduction of both bacteriocins by L. lactis IL1403(pJM04), because indicator strains that are selectively inhibited by each bacteriocin are not available.

Animals↗
PubMed1997

Double-glycine-type leader peptides direct secretion of bacteriocins by ABC transporters: colicin V secretion in Lactococcus lactis.

Many non-lantibiotic bacteriocins of lactic acid bacteria are produced as precursors which have N-terminal leader peptides that share similarities in amino acid sequence and contain a conserved processing site of two glycine residues in positions -1 and -2. A dedicated ATP-binding cassette (ABC) transporter is responsible for the proteolytic cleavage of the leader peptides and subsequent translocation of the bacteriocins across the cytoplasmic membrane. To investigate the role that these leader peptides play in the recognition of the precursor by the ABC transporters, the leader peptides of leucocin A, lactococcin A or colicin V were fused to divergicin A, a bacteriocin from Carnobacterium divergens that is secreted via the cell's general secretion pathway. Production of divergicin was monitored when these fusion constructs were introduced into Leuconostoc gelidum, Lactococcus lactis and Escherichia coli, which carry the secretion apparatus for leucocin A, lactococcins A and B, and colicin V, respectively. The different leader peptides directed the production of divergicin in the homologous hosts. In some cases production of divergicin was also observed when the leader peptides were used in heterologous hosts. For ABC-transporter-dependent secretion in E. coli the outer membrane protein TolC was required. Using this strategy, colicin V was produced in L. lactis by fusing this bacteriocin behind the leader peptide of leucocin A.

ATP-Binding Cassette Transporters↗
PubMed1998

Binding properties of Streptococcus gordonii SspA and SspB (antigen I/II family) polypeptides expressed on the cell surface of Lactococcus lactis MG1363.

The oral bacterium Streptococcus gordonii expresses two cell wall-associated polypeptides, designated SspA (1,542 amino acid residues) and SspB (1,462 amino acid residues), that have 70% sequence identity. These polypeptides are members of the antigen I/II family of oral streptococcal adhesins and mediate the binding of streptococci to salivary glycoproteins, collagen, and other oral microorganisms such as Actinomyces naeslundii. To determine if SspA and SspB have differential binding properties, the coding sequences of the sspA and sspB genes were cloned into expression plasmid vector pTREX1-usp45LS to generate pTREX1-sspA and pTREX1-sspB, respectively, and the Ssp polypeptides were displayed on the cell surface of Lactococcus lactis MG1363. Lactococcal cells expressing similar levels of surface SspA or SspB polypeptide were then compared for their abilities to adhere to a range of antigen I/II polypeptide substrates. More than twice as many L. lactis cells expressing SspA bound to immobilized salivary agglutinin glycoprotein (SAG) as did L. lactis cells expressing SspB. In contrast, lactococci expressing SspB adhered twice as well as lactococci producing SspA to collagen type I and to Candida albicans. The binding of A. naeslundii to lactococci was only weakly enhanced by surface expression of Ssp polypeptides. L. lactis(pTREX1-sspB) cells bound in greater numbers to SAG than did Enterococcus faecalis JH2-2 cells expressing SspB from pAM401EB-5. The results suggest that SspA and SspB have markedly different binding affinities for their oral substrates and thus may function to promote site diversity in colonization by S. gordonii.

Adhesins, Bacterial↗
PubMed2000

Heterologous inducible expression of Enterococcus faecalis pCF10 aggregation substance asc10 in Lactococcus lactis and Streptococcus gordonii contributes to cell hydrophobicity and adhesion to fibrin.

Aggregation substance proteins encoded by the sex pheromone plasmid family of Enterococcus faecalis have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further, prgB, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin-inducible nisA promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of E. faecalis and the heterologous bacteria Lactococcus lactis and Streptococcus gordonii was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces of E. faecalis and L. lactis but not on that of S. gordonii. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of E. faecalis and L. lactis revealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on L. lactis also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the nisA promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor.

Bacterial Adhesion↗
PubMed1999

A general method for selection of alpha-acetolactate decarboxylase-deficient Lactococcus lactis mutants to improve diacetyl formation.

The enzyme acetolactate decarboxylase (Ald) plays a key role in the regulation of the alpha-acetolactate pool in both pyruvate catabolism and the biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine (ILV). This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of Lactococcus lactis subsp. lactis biovar diacetylactis. Such mutants can be selected as leucine-resistant mutants in ILV- or IV-prototrophic strains. Most dairy lactococcus strains are auxotrophic for the three amino acids. Therefore, the plasmid pMC004 containing the ilv genes (encoding the enzymes involved in the biosynthesis of IV) of L. lactis NCDO2118 was constructed. Introduction of pMC004 into ILV-auxotrophic dairy strains resulted in an isoleucine-prototrophic phenotype. By plating the strains on a chemically defined medium supplemented with leucine but not valine and isoleucine, spontaneous leucine-resistant mutants were obtained. These mutants were screened by Western blotting with Ald-specific antibodies for the presence of Ald. Selected mutants lacking Ald were subsequently cured of pMC004. Except for a defect in the expression of Ald, the resulting strain, MC010, was identical to the wild-type strain, as shown by Southern blotting and DNA fingerprinting. The mutation resulting in the lack of Ald in MC010 occurred spontaneously, and the strain does not contain foreign DNA; thus, it can be regarded as food grade. Nevertheless, its application in dairy products depends on the regulation of genetically modified organisms. These results establish a strategy to select spontaneous Ald-deficient mutants from transformable L. lactis strains.

Amino Acids, Branched-Chain↗
PubMed2005

Expression of SARS-coronavirus nucleocapsid protein in Escherichia coli and Lactococcus lactis for serodiagnosis and mucosal vaccination.

The nucleocapsid (N) protein of the severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) is an important antigen for the early diagnosis of SARS and the development of vaccines. It was expressed in Escherichia coli as a fusion with human glutathione S-transferase (hGST) and was confirmed by Western blotting analysis. This recombinant N protein (hGST-N) was purified and used to measure the SARS-CoV N-specific antibody in the sera of eight SARS patients by enzyme-linked immunosorbent assay. Specific antibody response to this purified recombinant N protein was 100% positive in the SARS patients' sera, while none of the control sera from 30 healthy people gave a positive reaction in the same assay. The SARS-CoV N protein was also expressed in Lactococcus lactis in the cytoplasm or secreted into the medium. The N-producing strain MG1363/pSECN and the purified hGST-N protein were respectively administered to mice, either orally or intranasally. Results indicated that orally delivered MG1363/pSECN induced significant N-specific IgG in the sera. In conclusion, our work provides a novel strategy to produce the SARS-CoV N protein for serodiagnosis and for L. lactis-based mucosal vaccines.

Administration, Intranasal↗
PubMed2005

On-line monitoring of CO2 production in Lactococcus lactis during physiological pH decrease using membrane inlet mass spectrometry with dynamic pH calibration.

Monitoring CO2 production in systems, where pH is changing with time is hampered by the chemical behavior and pH-dependent volatility of this compound. In this article, we present the first method where the concentration and production rate of dissolved CO2 can be monitored directly, continuously, and quantitatively under conditions where pH changes rapidly ( approximately 2 units in 15 min). The method corrects membrane inlet mass spectrometry (MIMS) measurements of CO2 for pH dependency using on-line pH analysis and an experimentally established calibration model. It is valid within the pH range of 3.5 to 7, despite pH-dependent calibration constants that vary in a non-linear fashion with more than a factor of 3 in this interval. The method made it possible to determine the carbon dioxide production during Lactococcus lactis fermentations, where pH drops up to 3 units during the fermentation. The accuracy was approximately 5%. We used the method to investigate the effect of initial extracellular pH on carbon dioxide production during anarobic glucose fermentation by non-growing Lactocoocus lactis and demonstrated that the carbon dioxide production rate increases considerably, when the initial pH was increased from 6 to 6.8.

Calibration↗
PubMed2003

Active lipoprotein precursors in the Gram-positive eubacterium Lactococcus lactis.

Lipid-modified proteins play important roles at the interface between eubacterial cells and their environment. The importance of lipoprotein processing by signal peptidase II (SPase II) is underscored by the fact that this enzyme is essential for viability of the Gram-negative eubacterium Escherichia coli. In contrast, SPase II is not essential for growth and viability of the Gram-positive eubacterium Bacillus subtilis. This could be due to alternative amino-terminal lipoprotein processing, which was shown previously to occur in SPase II mutants of B. subtilis. Alternatively, uncleaved lipoprotein precursors might be functional. To explore further the importance of lipoprotein processing in Gram-positive eubacteria, an SPase II mutant strain of Lactococcus lactis was constructed. Although some of the 39 (predicted) lactococcal lipoproteins, such as PrtM and OppA, are essential for growth in milk, the growth of SPase II mutant L. lactis cells in this medium was not affected. Furthermore, the activity of the strictly PrtM-dependent extracellular protease PrtP, which is required for casein degradation, was not impaired in the absence of SPase II. Importantly, no alternative processing of pre-PrtM and pre-OppA was observed in cells lacking SPase II. Taken together, these findings show for the first time that authentic lipoprotein precursors retain biological activity.

Aspartic Acid Endopeptidases↗
PubMed1998

Nucleotide sequence and analysis of the new chromosomal abortive infection gene abiN of Lactococcus lactis subsp. cremoris S114.

A 7.275-kb DNA fragment which encodes resistance by abortive infection (Abi+) to bacteriophage was cloned from Lactococcus lactis subsp. cremoris S114. The genetic determinant for abortive infection was subcloned from this fragment. This gene was found to confer a reduction in efficiency of plating and plaque size for prolate-headed bacteriophage phi 53 (group I homology) and for small isometric-headed bacteriophage phi 59 (group III homology). This new gene, termed abiN, is predicted to encode a polypeptide of 178 amino acid residues with a deduced molecular mass of 20,461 Da and an isoelectric point of 4.63. No homology with any previously described genes was found. A probe was used to determine the presence of this gene only in S114 from 31 strains tested.

Bacteriophages↗
PubMed2006

Specific effects of denaturation, hydrolysis and exposure to Lactococcus lactis on bovine beta-lactoglobulin transepithelial transport, antigenicity and allergenicity.

BACKGROUND: Food allergy in developed countries represents a growing concern as reflected by epidemiological studies, indicating that up to 4% of the overall population is affected. Reduction of symptoms takes place following eviction or processing of some allergens. However, it cannot be predicted which structural changes will be associated with significant effects on the allergenicity. OBJECTIVE: To determine how various treatments of bovine beta-lactoglobulin (BLG) used as a model antigen alters its immunoreactivity and transepithelial transport, and whether this correlates with reduced allergenicity using an in vitro basophil activation assay. METHODS: BLG was subjected to reduction/alkylation, trypsin digestion or exposed to Lactococcus lactis. The remaining immunoreactivity toward IgG raised against native BLG was assessed by ELISA. Transepithelial transport of BLG and derivatives was examined using polarized Caco-2 cell monolayers mimicking the intestinal epithelium. Selective passage of tryptic peptides was determined using colchicine and cytochalasin D. Basophil activation was measured following stimulation with BLG and derivatives. RESULTS: Reduction/alkylation, trypsin digestion or incubation with L. lactis was associated with decreased BLG recognition by IgG antibodies raised against the native protein. All treatments also resulted in a more efficient transepithelial transport of BLG. BLG crossed the Caco-2 monolayer through passage across the cell, whereas tryptic peptides followed both the para- and transcellular routes. With the exception of denaturation by reduction/alkylation, cross-linking of IgE antibodies by BLG derivatives led to lower basophil degranulation. CONCLUSION: In vitro dissection of antigenicity and allergenicity may be a valid and convenient alternative to evaluate the effects of biotechnological processing on dietary proteins. In addition, it can help to define the molecular and cellular mechanisms that will provide improved means of diagnosis and possibly therapy of food-allergic disorders.

Allergens↗
PubMed2003

Cooperation between Lactococcus lactis and nonstarter lactobacilli in the formation of cheese aroma from amino acids.

In Gouda and Cheddar type cheeses the amino acid conversion to aroma compounds, which is a major process for aroma formation, is essentially due to lactic acid bacteria (LAB). In order to evaluate the respective role of starter and nonstarter LAB and their interactions in cheese flavor formation, we compared the catabolism of phenylalanine, leucine, and methionine by single strains and strain mixtures of Lactococcus lactis subsp. cremoris NCDO763 and three mesophilic lactobacilli. Amino acid catabolism was studied in vitro at pH 5.5, by using radiolabeled amino acids as tracers. In the presence of alpha-ketoglutarate, which is essential for amino acid transamination, the lactobacillus strains degraded less amino acids than L. lactis subsp. cremoris NCDO763, and produced mainly nonaromatic metabolites. L. lactis subsp. cremoris NCDO763 produced mainly the carboxylic acids, which are important compounds for cheese aroma. However, in the reaction mixture containing glutamate, only two lactobacillus strains degraded amino acids significantly. This was due to their glutamate dehydrogenase (GDH) activity, which produced alpha-ketoglutarate from glutamate. The combination of each of the GDH-positive lactobacilli with L. lactis subsp. cremoris NCDO763 had a beneficial effect on the aroma formation. Lactobacilli initiated the conversion of amino acids by transforming them mainly to keto and hydroxy acids, which subsequently were converted to carboxylic acids by the Lactococcus strain. Therefore, we think that such cooperation between starter L. lactis and GDH-positive lactobacilli can stimulate flavor development in cheese.

Amino Acids↗
PubMed1996

Beta-casomorphins: analysis in cheese and susceptibility to proteolytic enzymes from Lactococcus lactis ssp. cremoris.

Commercial cheese products were surveyed for beta-casomorphin peptides. Two extraction methods were compared: 1) water and 2) chloroform and methanol. Peptide profiles were determined using reverse-phase HPLC and multiple wavelength detection. beta-Casomorphin standards were used for comparison with cheese peptide profiles. Results indicated that peptides were present in cheeses with HPLC elution times that were similar to those for beta-casomorphins. However, comparison of absorbancies of the peaks at multiple wavelengths did not indicate peptides similar to beta-casomorphins. Therefore, beta-casomorphins were absent, or concentrations were below the HPLC detection threshold for beta-casomorphin of 2 micrograms/ml of cheese extract. The susceptibility of beta-casomorphins to the proteolytic system of a commercial strain of Lactococcus lactis ssp. cremoris was investigated. beta-Casomorphin standards were incubated at 4 degrees C with bacterial cell lysate at pH 5.0, 5.2, 5.4, and 5.7. Salt concentrations varied among 0, 1.5, and 5%. The concentration of added beta-casomorphins and the degradation products were monitored over 15 wk using HPLC. Enzymatic degradation of beta-casomorphins was influenced by the combination of pH and salt concentrations at cheese ripening temperatures. Therefore, if formed in cheese, beta-casomorphins may be degraded under conditions common for Cheddar cheese.

Amino Acid Sequence↗
PubMed1991

Study of the Citrate Metabolism of Lactococcus lactis subsp. lactis Biovar Diacetylactis by Means of C Nuclear Magnetic Resonance.

The metabolic fate of citrate and pyruvate in four strains of Lactococcus lactis subsp. lactis biovar diacetylactis has been studied by means of C nuclear magnetic resonance, using as a substrate either [3-C]pyruvic acid or custom-synthesized citric acid that is C labeled either at carbons 2 and 4 or at carbon 3. The fermentations were carried out batchwise in modified M17 broth. For the actual conversions of the C-labeled substrates, cells at the end of their logarithmic growth phase were used to minimize the conversion to lactic acid. A mass balance of the main citric acid metabolites was obtained; the four strains produced from 50 to 70% (on a molar basis) lactic acid from either citrate or pyruvate. The remaining 50 to 30% was converted mainly to either alpha-acetolactic acid (for one strain) or acetoin (for the other three strains). One of the strains produced an exceptionally high concentration of the diacetyl precursor alpha-acetolactic acid. Another strain (SDC6) also produced alpha-acetolactic acid, but this was decarboxylated to acetoin at a high rate. The C nuclear magnetic resonance method confirmed that the biosynthesis of alpha-acetolactic acid occurs via condensation of pyruvate and "active" acetaldehyde. Diacetyl was not found as a direct metabolite of citrate or pyruvate metabolism.

Journal Article↗
PubMed1993

Effect of X-Prolyl Dipeptidyl Aminopeptidase Deficiency on Lactococcus lactis.

The genetic determinant (pepXP) of an X-prolyl dipeptidyl aminopeptidase (PepXP) has recently been cloned and sequenced from both Lactococcus lactis subsp. cremoris (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and L. lactis subsp. lactis (M. Nardi, M.-C. Chopin, A. Chopin, M.-M. Cals, and J.-C. Gripon, Appl. Environ. Microbiol. 57:45-50, 1991). To examine the possible role of the enzyme in the breakdown of caseins required for lactococci to grow in milk, integration vectors have been constructed and used to specifically inactivate the pepXP gene. After inactivation of the gene in L. lactis subsp. lactis MG1363, which is Lac and Prt, the Lac Prt determinants were transferred by conjugation by using L. lactis subsp. lactis 712 as the donor. Since growth of the transconjugants relative to the PepXP strains was not retarded in milk, it was concluded that PepXP is not essential for growth in that medium. It was also demonstrated that the open reading frame ORF1, upstream of pepXP, was not required for PepXP activity in L. lactis. A marked difference between metenkephalin degradation patterns was observed after incubation of this pentapeptide with cell extracts obtained from wild-type lactococci and pepXP mutants. Therefore, altered expression of the pepXP-encoded general dipeptidyl aminopeptidase activity may change the peptide composition of fermented milk products.

Journal Article↗
PubMed2005

Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications.

BACKGROUND: The nisin-controlled gene expression system NICE of Lactococcus lactis is one of the most widely used expression systems in Gram-positive bacteria. Despite its widespread use, no optimization of the culture conditions and nisin induction has been carried out to obtain maximum yields. As a model system induced production of lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) produced by S. simulans biovar. Staphylolyticus, was used. Three main areas need optimization for maximum yields: cell density, nisin-controlled induction and protein production, and parameters specific for the target-protein. RESULTS: In a series of pH-controlled fermentations the following parameters were optimized: pH of the culture, use of NaOH or NH4OH as neutralizing agent, the addition of zinc and phosphate, the fermentation temperature, the time point of induction (cell density of the culture), the amount of nisin added for induction and the amount of three basic medium components, i.e. yeast extract, peptone and lactose. For each culture growth and lysostaphin production was followed. Lysostaphin production yields depended on all parameters that were varied. In the course of the optimization a three-fold increase in lysostaphin yield was achieved from 100 mg/l to 300 mg/l. CONCLUSION: Protein production with the NICE gene expression system in L. lactis strongly depends on the medium composition, the fermentation parameters and the amount of nisin added for induction. Careful optimization of key parameters lead to a significant increase in the yield of the target protein.

Journal Article↗
PubMed1996

Lactococcus lactis and stress.

It is now generally recognized that cell growth conditions in nature are often suboptimal compared to controlled conditions provided in the laboratory. Natural stresses like starvation and acidity are generated by cell growth itself. Other stresses like temperature or osmotic shock, or oxygen, are imposed by the environment. It is now clear that defense mechanisms to withstand different stresses must be present in all organisms. The exploration of stress responses in lactic acid bacteria has just begun. Several stress response genes have been revealed through homologies with known genes in other organisms. While stress response genes appear to be highly conserved, however, their regulation may not be. Thus, search of the regulation of stress response in lactic acid bacteria may reveal new regulatory circuits. The first part of this report addresses the available information on stress response in Lactococcus lactis. Acid stress response may be particularly important in lactic acid bacteria, whose growth and transition to stationary phase is accompanied by the production of lactic acid, which results in acidification of the media, arrest of cell multiplication, and possible cell death. The second part of this report will focus on progress made in acid stress response, particularly in L. lactis and on factors which may affect its regulation. Acid tolerance is presently under study in L. lactis. Our results with strain MG1363 show that it survives a lethal challenge at pH 4.0 if adapted briefly (5 to 15 minutes) at a pH between 4.5 and 6.5. Adaptation requires protein synthesis, indicating that acid conditions induce expression of newly synthesized genes. These results show that L. lactis possesses an inducible response to acid stress in exponential phase. To identify possible regulatory genes involved in acid stress response, we determined low pH conditions in which MG1363 is unable to grow, and selected at 37 degrees C for transposition insertional mutants which were able to survive. About thirty mutants resistant to low pH conditions were characterized. The interrupted genes were identified by sequence homology with known genes. One insertion interrupts ahrC, the putative regulator of arginine metabolism; possibly, increased arginine catabolism in the mutant produces metabolites which increase the pH. Several other mutations putatively map at some step in the pathway of (p)ppGpp synthesis. Our results suggest that the stringent response pathway, which is involved in starvation and stationary phase survival, may also be implicated in acid pH tolerance.

Adaptation, Physiological↗
PubMed2001

Location effects of a reporter gene on expression levels and on native protein synthesis in Lactococcus lactis and Saccharomyces cerevisiae.

The engineering of industrially important genetically modified organisms by the integration of heterologous genes into the chromosome is often the method of choice for several reasons concerned with long-term stability, homogeneous population distribution, and the enabling of selection without the addition of antibiotics. However, integration may disrupt endogenous gene expression, giving rise to increased levels of toxic metabolic byproducts or activating otherwise silent genes. The position of integration of a foreign gene in the chromosome can also influence its expression levels, and this effect will be of relevance in terms of optimizing protein production parameters. In this study, we determine how the random integration of a foreign reporter gene might affect expression levels and assess the use of proteome analysis to investigate possible effects on synthesis of endogenous proteins in two important food-relevant microorganisms, Saccharomyces cerevisiae and Lactococcus lactis. Eleven L. lactis integrants carrying the gusA gene were analyzed, and expression levels were found to vary by a factor of threefold in contrast to expression levels of lacZ in 18 S. cerevisiae integrants, which showed a 14-fold variation. Of relevance to industry is whether any changes in expression levels might occur as a consequence of storage of the modified strains. Here it is also shown that the above differences in expression levels were not significantly affected by storage of frozen cultures over a period of several months. Analysis of the protein composition of the yeast and lactococcal integrant strains by separation on one-dimensional (1D) and 2D gels showed no significant variations in position beyond those observed in control samples.

Bacterial Proteins↗