Citrate permease gene expression in Lactococcus lactis subsp. lactis strains IL1403 and MG1363

Abstract Citrate permease gene expression in the plasmid-free Lactococcus lactis strains IL1403 and MG1363 was studied. The ability to transport citrate results in diacetyl and acetoin production in IL1403 but not in MG1363. Citrate lyase, α-acetolactate decarboxylase, diacetyl and acetoin reductase were detected in IL1403. These data show that L. lactis ssp. lactis strain IL1403 is a citrate permease mutant of the biovar. diacetylactis . Immunological analysis revealed the α-and β-subunits of citrate lyase not only in IL1403 but also in MG1363 where no citrate lyase activity was found.     

Mannoproteins from the cell wall of Kluyveromyces lactis

Abstract Wall mannoproteins from Kluyveromyces lactis have been solubilised by treatment of cell walls with sodium dodecyl sulphate (SDS) or zymolyase. While the former reagent liberates a large number of molecular species, zymolyase preferentially releases a high-molecular-weight material that is sensitive to endo- β - N -acetylglucosaminidase H, and a 29-kDa molecule that reacts with the antiserum raised against a similar species from walls of Saccharomyces cerevisiae . In contrast with observations on isolated walls of S. cerevisiae , dithiothreitol pretreatment of K. lactis walls does not enhance the effect of zymolyase upon mannoprotein release. However, the action of thiol agents is still necessary to obtain protoplasts by zymolyase digestion from K. lactis whole cells.     

Ubiquity and diversity of multidrug resistance genes in Lactococcus lactis strains isolated between 1936 and 1995

The presence and the nucleotide sequence of four multidrug resistance genes, lmrA, lmrP, lmrC, and lmrD, were investigated in 13 strains of Lactococcus lactis ssp. lactis, four strains of Lactococcus lactis ssp. cremoris, two strains of Lactococcus plantarum, and two strains of Lactococcus raffinolactis. Multidrug resistance genes were present in all L. lactis isolates tested. However, none of them could be detected in the strains belonging to the species L. raffinolactis and L. plantarum, suggesting a different set of multidrug resistance genes in these species. The analysis of the four deduced amino acid sequences established two different variants depending on the subspecies of L. lactis. Either lmrA, or lmrP, or both were found naturally disrupted in five strains, while full-length lmrD was present in all strains.     

The apyrase KlYnd1p of Kluyveromyces lactis affects glycosylation, secretion, and cell wall properties

The Kluyveromyces lactis ORF r_klactIV3,463 on chromosome IV, hereafter named KlYND1, encodes an endoapyrase that has nucleoside phosphatase activity with a lumenal orientation. The enzyme showed equally high activity towards GDP/UDP and ADP, and also showed activity, although to a lesser extent, towards GTP. No activity was detected with the other triphosphates and all monophosphates. The overexpression of KlYND1 in Klgda1Delta cells of K. lactis, devoid of the encoded GDPase/UDPase activity, suppressed the loss of O-glycosylation and cell wall-related defects described in such mutants, and suggests a partial overlap of function between the two genes, and therefore some redundancy. The overexpression of KlYND1 in wild-type cells enhanced the secretion of the recombinant human serum albumin and glucoamylase employed as reporters.     

Lactose-induced cell death of beta-galactosidase mutants in Kluyveromyces lactis

The Kluyveromyces lactis lac4 mutants, lacking the beta-galactosidase gene, cannot assimilate lactose, but grow normally on many other carbon sources. However, when these carbon sources and lactose were simultaneously present in the growth media, the mutants were unable to grow. The effect of lactose was cytotoxic since the addition of lactose to an exponentially-growing culture resulted in 90% loss of viability of the lac4 cells. An osmotic stabilizing agent prevented cells killing, supporting the hypothesis that the lactose toxicity could be mainly due to intracellular osmotic pressure. Deletion of the lactose permease gene, LAC12, abolished the inhibitory effect of lactose and allowed the cell to assimilate other carbon substrates. The lac4 strains gave rise, with unusually high frequency, to spontaneous mutants tolerant to lactose (lar1 mutation: lactose resistant). These mutants were unable to take up lactose. Indeed, lar1 mutation turned out to be allelic to LAC12. The high mutability of the LAC12 locus may be an advantage for survival of K. lactis whose main habitat is lactose-containing niches.     

Pore selectivity analysis of an aquaglyceroporin by stopped-flow spectrophotometry on bacterial cell suspensions

Background information. Transport of water and small neutral solutes across plasma membranes is facilitated by AQP (aquaporin) and aquaglyceroporin channels, which belong to the MIP (major intrinsic protein) family. So far, more than 800 MIP proteins have been identified on the basis of sequence homology, but only less than 10% of them have been functionally characterized. In most studies, the channel properties of MIP proteins have been determined by using Xenopus oocyte swelling assays or stopped-flow spectrophotometry on proteoliposomes. As both methods sometimes present disadvantages, we developed an alternative method for analysing MIP function.Results. The kinetics of plasmolysis or deplasmolysis of Escherichia coli cells in suspension, in response to osmotic challenges, was analysed by stopped-flow spectrophotometry. Cytoplasmic volume variations were monitored either by GFP (green fluorescent protein) fluorescence quenching or by 90 degrees scattered light. The single exponential response to up-shocks in the impermeant solute mannitol was strongly accelerated when the cells expressed the native E. coli AQP AqpZ (rate constant 37.24 versus 3.05 s(-1) for control cells). The responses to hyperosmotic shocks realized with glycerol were biphasic. First, a light-scattering increase corresponded to cell plasmolysis. Secondly, deplasmolysis occurred when glycerol entered into the cell. Both phases were accelerated when the aquaglyceroporin GlpF was present in cell membranes. We concluded that the behaviour of MIP-expressing bacteria in the stopped-flow system was qualitatively identical with that reported for MIP-expressing oocytes or MIP-containing proteoliposomes. We then used this system to analyse the effects of mutations in the pore constriction of Gla(Llac), the aquaglyceroporin from Lactococcus lactis. In the present study, we show that Gla(Llac) loses its ability to transport glycerol but retains its ability to transport water when Val(223) was replaced by a histidine, the residue at the equivalent position in strict AQPs.Conclusions. These results show that stopped-flow spectrophotometry performed on E. coli cell suspensions is a useful experimental system to analyse the selectivity of wild-type or mutant MIP proteins and that a bifunctional aquaglyceroporin switches to an AQP by a single amino acid mutation in the pore constriction.     

α-Ketoglutarate biosynthesis in wild and industrial strains of Lactococcus lactis

Aims:  This study was carried out to explore the ability of wild and industrial strains of Lactococcus lactis to produce α-ketoglutarate (α-KG), which is essential during the conversion of amino acids to flavour compounds.
Methods and Results:  Two pathways in α-KG biosynthesis were explored in strains of L. lactis isolated from dairy products, vegetables and commercial dairy starter cultures. Half of the strains efficiently converted glutamine to glutamate (Glu) and grew in Glu-free medium. Strains did not present isocitrate dehydrogenase and aconitase activities. However, half of the strains presented glutamate dehydrogenase (GDH) activity.
Conclusions:  The ability of L. lactis to synthesize either α-KG or Glu via GDH was confirmed. However, L. lactis strains were not able to biosynthesize α-KG by the citrate–isocitrate pathway. NADP-GDH activity was mainly found in strains isolated from vegetables, whereas NAD-GDH activity was mainly found in strains isolated from dairy products.
Significance and Importance of the Study:  The origin of isolation highly influenced NAD or NADP-GDH activities. These enzymatic activities may be correlated to the flavour production capacity of the different strains.     

Alteration of cell wall composition leads to amphotericin B resistance in Aspergillus flavus

An amphotericin B (AmB)-resistant mutant was isolated from a wild-type AmB-susceptible strain of Aspergillus flavus by serial transfer of conidia on agar plates containing stepwise increased concentrations of AmB up to 100 microg ml-1. The acquired resistance of mycelia was specific for polyene-antibiotics AmB, nystatin and trichomycin. Spheroplasts derived from the resistant mycelia were as susceptible to AmB as the wild-type. Chemical analysis of the cell wall revealed that levels of alkali-soluble and -insoluble glucans were significantly higher in the resistant mycelia as compared to those in the wild-type. When resistant mycelia were treated with SDS, they adsorbed as much AmB as wild-type mycelia. These results suggest that alterations in the cell wall components of mycelia, especially 1,3-alpha-glucan and protein complex in the outermost wall layer, lead to AmB resistance in A. flavus.     

Optimized production and quantification of the tryptophan-deficient sweet-tasting protein brazzein in Kluyveromyces lactis

Abstract

The sweet-tasting protein brazzein is a candidate sugar substitute owing to its sweet, sugar-like taste and good stability. To commercialize brazzein as a sweetener, optimization of fermentation and purification procedure is necessary. Here, we report the expression conditions of brazzein in the yeast Kluyveromices lactis and purification method for maximum yield. Transformed K. lactis was cultured in YPGlu (pH 7.0) at 25?°C and induced by adding glucose:galactose at a weight ratio of 1:2 (%/%) during the stationary phase, which increased brazzein expression 2.5 fold compared to the previous conditions. Cultures were subjected to heat treatment at 80?°C for 1?h, and brazzein containing supernatant was purified using carboxymethyl-sepharose cation exchange chromatography using 50?mM NaCl in 50?mM sodium acetate buffer (pH 4.0) as a wash buffer and 400?mM NaCl (pH 7.0) for elution. The yield of purified brazzein under these conditions was 2.0-fold higher than that from previous purification methods. We also determined that the NanoOrange assay was a suitable method for quantifying tryptophan-deficient brazzein. Thus, it is possible to obtain pure recombinant brazzein with high yield in K. lactis using our optimized expression, purification, and quantification protocols, which has potential applications in the food industry.     

Gene cloning, functional expression and secretion of the S-layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a in Lactococcus lactis

The ~93-kDa surface layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a forms a regular crystalline array providing a nanopatterned matrix for the future display of biologically relevant molecules. Lactococcus lactis NZ9000 was established as a safe expression host for the controlled targeted production of SgsE based on the broad host-range plasmid pNZ124Sph, into which the nisA promoter was introduced. SgsE devoid of its signal peptide-encoding sequence was cloned into the new vector and purified from the cytoplasm at a yield of 220 mg l- of expression culture. Secretion constructs were based on the signal peptide of the Lactobacillus brevis SlpA protein or the L. lactis Usp45 protein, allowing isolation of 95 mg of secreted rSgsE l-1. N-terminal sequencing confirmed correct processing of SgsE in L. lactis NZ9000. The ability of rSgsE to self-assemble in suspension and to recrystallize on solid supports was demonstrated by electron and atomic force microscopy.     

Proteome analysis of the purine stimulon from Lactococcus lactis

A comparative expression proteome analysis was carried out by analyzing differential expression patterns of pulse-labelled proteins on two-dimensional gels under standard conditions and during purine nucleotide starvation, followed by mass spectrometric identification of regulated proteins. Based upon the expression patterns, three stimulons could be identified in Lactococcus lactis subsp. cremoris. The Psu proteins (purine starvation up-regulated) had increased synthesis during purine depletion in a purine auxotroph. Among these proteins were enzymes of the purine biosynthesis pathways (PurE, PurS, PurM, PurL), and enzymes involved in the generation of C1 units (GlyA, Fhs). C1 units are primarily required for purine biosynthesis. Upon analysis of the nucleotide sequence preceding the structural genes for these proteins in the L. lactis IL1403 genome sequence showed that all contained PurBox-Pribnov box structures resembling the PurR activated promoters for the purDEK and purCSQLF operons. Most, and possibly all members of the Psu stimulon are thus members of the PurR regulon. Five Psu proteins could not be identified. The second stimulon, the Psd stimulon (purine starvation decreased), whose members are down-regulated during purine depletion, contained proteins related to protein synthesis (PpsB, EF-TS, trigger factor), or to GTPases (FtsZ, EF-TS); or are involved in energy metabolism (GapB, CcpA). No common regulatory elements could be found for members of this stimulon. Two Psd proteins escaped identification. The last, Dcu (decoynine up-regulated), stimulon contained proteins whose synthesis escaped the severe general depression during inhibition of the GMP synthetase by decoynine. This regulon was comprised of mostly glycolytic enzymes (fructose bisphosphate aldolase, enolase, pyruvate kinase) and translation elongation factors (GTPases: EF-TU, EF-G). Two Dcu proteins could not be identified. Out of 28 proteins subjected to mass spectrometry, 19 could be readily identified despite the fact that only the genome sequence of a strain of L. lactis subsp. lactis was available. The two subspecies share about 85% sequence identity, comparable to the genetic distance between Escherichia coli and Salmonella typhimurium. A success rate of 68% indicates that it may be feasible to perform proteomics based upon genomic sequences of relatives outside the genus.     

表达猪传染性胃肠炎病毒S蛋白重组乳酸乳球菌在模拟动物胃肠道内的稳定性检测

为确定本实验室研究构建的表达猪传染性胃肠炎病毒S蛋白重组乳酸乳球菌pNZ8112-Sa/NZ9000在模拟动物肠道内稳定性,对重组菌株的培养条件、蛋白表达和质粒携带以及在模拟胃肠道环境中的稳定性进行了检测。实验结果表明能够保持其蛋白表达的稳定性及重组质粒的稳定性;模拟胃肠道环境实验结果表明重组菌能够耐受胰蛋白酶溶液、0.1%的胆汁及在含有胃蛋白酶pH 1.5的盐酸存活1 h和在pH 2.5的盐酸耐受性良好。     

乳酸乳球菌食品级诱导表达系统的构建及异源蛋白的表达

以α-aga基因为食品级选择标记构建了乳酸乳球菌食品级高效诱导细胞内和细胞壁锚定表达系统,并用这一表达系统表达了铜绿假单胞菌融合外膜蛋白基因OprF/H。首先以pRAF800和pNZ8048构建了含有α-aga、PnisA-MCS-TpepN和θ复制子的乳酸乳球菌食品级细胞内诱导表达载体pRNA48,再以pRNA48和pVE5524为出发载体构建了含有α-aga、PnisA-SPUsp45-nucA-CWAM6-t1t2和θ复制子的乳酸乳球菌细胞壁锚定诱导表达载体pRNV48。然后以食品级载体pRNA48和pRNV48为基础,构建了不含抗生素抗性选择标记的铜绿假单胞菌融合外膜蛋白基因的表达质粒pRNA48-OprF/H和pRNV48-OprF/H。利用nisin进行重组乳酸乳球菌菌株的诱导表达,通过SDS-PAGE和Western blot分析,检测到表达蛋白分别占细胞内可溶蛋白的9.6%和细胞壁锚定蛋白的9.8%,表达产物具有免疫原性,可与含OprF/H的乳球菌以及铜绿假单胞菌发生特异性的凝集反应。     

Isoamyl alcohol-induced morphological change in Saccharomyces cerevisiae involves increases in mitochondria and cell wall chitin content

Isoamyl alcohol reduced growth and induced filament formation in Saccharomyces cerevisiae. Isoamyl alcohol-induced filamentation was accompanied by an almost threefold greater increase in the specific activity of succinate dehydrogenase than in untreated cells, which suggested that isoamyl alcohol treatment caused the cells to produce more mitochondria than in normal yeast form proliferation. This was supported by measuring the dry weight of purified, isolated mitochondria. Filaments have an increased chitin content which is distributed over the majority of their surface, and is not confined to bud scars and the chitin ring between mother and daughter cells as in yeast-form cells.     

Comparative studies on cell stimulatory, permeabilizing and toxic effects induced in sensitive and multidrug resistant fungal strains by amphotericin B (AMB) and N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME)

N-Methyl-N-D-fructosyl-amphotericin B methyl ester (MFAME) is a new derivative of amphotericin B, which is characterised by low toxicity to mammalian cells and good solubility in water of its salts. The antifungal activity and effects of MFAME towards Candida albicans and Saccharomyces cerevisiae multidrug resistant MDR(+) and sensitive MDR(-) strains was compared with those of parent compound. The results obtained indicate that MDR(+) S. cerevisiae was sensitive to MFAME as well as to AMB. MFAME exhibited the same effects on fungal cells studied as parent antibiotic. The two antibiotics, depending on the dose applied induced cell stimulation, K+ efflux, and/or had a toxic effect.     

Variation of the sterols from Kluyveromyces lactis and from a resistant mutant by culture in the presence of sublethal doses of amphotericin B

A mutant strain of Kluyveromyces lactis resistant to amphotericin B and weakly to nystatin has been isolated from subcultures of the wild strain grown in the presence of sublethal doses of amphotericin B. The mutant and the wild strain were equally sensitive to pimaricin, filipin, and candicidin. The efficacy of fungizone was very low. In comparison with the wild strain the level of sterols was two times lower in the resistant strain but the composition of these sterols was about the same in the two strains. The action of sublethal doses of amphotericin B on the composition of the sterols was the same in these two yeasts and brought a 40% decrease of the total sterol level and a modification in their distribution. This variation cannot fully explain the resistance of the yeast but it may be associated to other changes of the membranes.     

Plasmacytoid dendritic cell dysfunction caused by heat stress is improved by administration of Lactococcus lactis strain plasma in mice

ABSTRACT

Plasmacytoid dendritic cells (pDCs) are crucial in anti-viral immunity, acting as regulators in both adaptive and innate immunity. In this study, brief heat stress caused a decrease in splenic pDC activity in mice. Administration of Lactococcus lactis strain Plasma (LC-Plasma) significantly suppressed the decrease in pDC activity and IFN-α production.

Abbreviations: LC-Plasma: Lactococcus lactis strain Plasma; LAB: lactic acid bacteria; pDC: plasmacytoid dendritic cell; IFN: interferons; mDC: myeloid dendritic cells