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T Abee

Publications and source records attributed to T Abee.

At least 37 records · Page 2Linked to original sources
PubMed2000

Characteristics of the biologically active 35-kDa metalloprotease virulence factor from Listeria monocytogenes.

Listeria monocytogenes, a facultative intracellular pathogen, synthesizes an extracellular protease which is responsible for the maturation of phosphatidylcholine phospholipase C (lecithinase), a virulence factor involved in cell-to-cell spread. This work describes the environmental parameters necessary for increased production of mature, 35-kDa active protease in strains of L. monocytogenes, and its detection using polyclonal antibodies raised against Bacillus subtilis neutral protease. High performance liquid affinity chromatography was exploited to isolate the biologically active form of the mature protease, which was then subjected to biochemical characterization using casein as a substrate. The protease is a zinc-dependent metalloprotease which degrades casein over a wide range of temperatures and pH values. It can also degrade actin, the most abundant protein in many eukaryotic cells. The Listeria protease was shown to exhibit a high thermal stability and a relatively narrow substrate specificity. A three-dimensional model built on the basis of the homology with thermolysin was used to understand the structural basis of these characteristics.

Actins↗
PubMed2000

Rapid fluorescence assessment of intracellular pH as a viability indicator of Clavibacter michiganensis subsp. michiganensis.

The viability of Clavibacter michiganensis subsp. michiganensis (Cmm) was determined by measuring the intracellular pH (pHin) as a viability parameter. This was based on the observation that growth of Cmm was inhibited at pH 5.5 and below. Therefore, viable cells should maintain their pHin above this pH value. The pHin of Cmm was determined using the fluorescent probe 5(and 6-)-carboxyfluorescein succinimidyl ester (cFSE). The pHin of Cmm cells exposed to acid treatments was determined using fluorescence spectrofluorometry, and for cells exposed to elevated temperatures, the pHin was determined using fluorescence spectrofluorometry and flow cytometry (FCM). A good correlation was found between the presence of a pH gradient and the number of colony-forming units (cfu) observed in plate counts. However, with the spectrofluorometry technique, the analysis is based on the whole cell population and the detection sensitivity of this technique is rather low, i.e., cell numbers of at least 107 cfu ml-1 are needed for the analysis. Using FCM, heat-treated and non-treated Cmm cells could be distinguished based on the absence and presence of a pH gradient, respectively. The major advantage of FCM is its high sensitivity, allowing analysis of microbial populations even at low numbers, i.e., 102-103 cfu ml-1.

Actinomycetales↗
PubMed2000

Characterization of a novel beta-galactosidase from Bifidobacterium adolescentis DSM 20083 active towards transgalactooligosaccharides.

This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel beta-galactosidase (beta-Gal II). In cells grown on TOS, in addition to the lactose-degrading beta-Gal (beta-Gal I), another beta-Gal (beta-Gal II) was detected and it showed activity towards TOS but not towards lactose. beta-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. Beta-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. Beta-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35 degrees C. The enzyme showed highest V(max) values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. Beta-Gal II was active towards beta-galactosyl residues that were 1-->4, 1-->6, 1-->3, and 1 <--> 1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.

Bifidobacterium↗
PubMed2000

Changes in glycolytic activity of Lactococcus lactis induced by low temperature.

The effects of low-temperature stress on the glycolytic activity of the lactic acid bacterium Lactococcus lactis were studied. The maximal glycolytic activity measured at 30 degrees C increased approximately 2.5-fold following a shift from 30 to 10 degrees C for 4 h in a process that required protein synthesis. Analysis of cold adaptation of strains with genes involved in sugar metabolism disrupted showed that both the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) subunit HPr and catabolite control protein A (CcpA) are involved in the increased acidification at low temperatures. In contrast, a strain with the PTS subunit enzyme I disrupted showed increased acidification similar to that in the wild-type strain. This indicates that the PTS is not involved in this response whereas the regulatory function of 46-seryl phosphorylated HPr [HPr(Ser-P)] probably is involved. Protein analysis showed that the production of both HPr and CcpA was induced severalfold (up to two- to threefold) upon exposure to low temperatures. The las operon, which is subject to catabolite activation by the CcpA-HPr(Ser-P) complex, was not induced upon cold shock, and no increased lactate dehydrogenase (LDH) activity was observed. Similarly, the rate-limiting enzyme of the glycolytic pathway under starvation conditions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not induced upon cold shock. This indicates that a factor other than LDH or GAPDH is rate determining for the increased glycolytic activity upon exposure to low temperatures. Based on their cold induction and involvement in cold adaptation of glycolysis, it is proposed that the CcpA-HPr(Ser-P) control circuit regulates this factor(s) and hence couples catabolite repression and cold shock response in a functional and mechanistic way.

Bacterial Proteins↗
PubMed2000

Physiological and regulatory effects of controlled overproduction of five cold shock proteins of Lactococcus lactis MG1363.

The physiological and regulatory effects of overproduction of five cold shock proteins (CSPs) of Lactococcus lactis were studied. CspB, CspD, and CspE could be overproduced at high levels (up to 19% of the total protein), whereas for CspA and CspC limited overproduction (0.3 to 0.5% of the total protein) was obtained. Northern blot analysis revealed low abundance of the cspC transcript, indicating that the stability of cspC mRNA is low. The limited overproduction of CspA is likely to be caused by low stability of CspA since when there was an Arg-Pro mutation at position 58, the level of CspA production increased. Using two-dimensional gel electrophoresis, it was found that upon overproduction of the CSPs several proteins, including a number of cold-induced proteins of L. lactis, were induced. Strikingly, upon overproduction of CspC induction of CspB, putative CspF, and putative CspG was also observed. Overproduction of CspB and overproduction of CspE result in increased survival when L. lactis is frozen (maximum increases, 10- and 5-fold, respectively, after 4 freeze-thaw cycles). It is concluded that in L. lactis CSPs play a regulatory role in the cascade of events that are initiated by cold shock treatment and that they either have a direct protective effect during freezing (e.g., RNA stabilization) or induce other factors involved in the freeze-adaptive response or both.

Adaptation, Physiological↗
PubMed1999

Microbial stress response in minimal processing.

"Bacteria have evolved adaptive networks to face the challenges of changing environments and to survive under conditions of stress. Therefore, the efficiencies of inactivation and preservation methods need to be assessed, especially with regard to the enormous potential of food pathogens to adapt to a wide variety of stress conditions. All adaptive responses, whether to changing nutrients or to various stresses encountered in minimal processing, involve a series of genetic switches that control the metabolic changes taking place. A common regulatory mechanism involves the modification of sigma (sigma) factors whose primary role is to bind to core RNA polymerase conferring promoter specificity directing expression of specialty regulons involved in heat-shock response, the chemotactic response, sporulation, and general stress response. Examples of the latter regulon in Gram-positive bacteria (the sigmaB regulon) and in Gram-negative bacteria (the RpoS regulon) will be discussed in more detail. Cellular adaptive mechanisms to starvation, cold shock, heat shock, (weak) acids, high osmolarity and high hydrostatic pressure will be described and their significance in food preservation and safety will be discussed."

Adaptation, Physiological↗
PubMed1999

The cellular target of histatin 5 on Candida albicans is the energized mitochondrion.

Histatin 5 is a human basic salivary peptide with strong fungicidal properties in vitro. To elucidate the mechanism of action, the effect of histatin 5 on the viability of Candida albicans cells was studied in relation to its membrane perturbing properties. It was found that both the killing activity and the membrane perturbing activity, studied by the influx of a DNA-specific marker propidium iodide, were inhibited by high salt conditions and by metabolic inhibitors, like sodium azide. In addition, exposure to histatin 5 resulted in a loss of the mitochondrial transmembrane potential in situ, measured by the release of the potential-dependent distributional probe rhodamine 123. Localization studies using tetramethylrhodamine isothiocyanate-labeled histatin 5 or fluorescein isothiocyanate-labeled histatin 5 showed a granular intracellular distribution of the peptide, which co-localized with mitotracker orange, a permeant mitochondria-specific probe. Like the biological effects, uptake of labeled histatin 5 was inhibited by mitochondrial inhibitors and high salt conditions. Our data indicate that histatin 5 is internalized, and targets to the energized mitochondrion.

Amino Acid Sequence↗
PubMed1999

Cold shock proteins and low-temperature response of Streptococcus thermophilus CNRZ302.

Low-temperature adaptation and cryoprotection were studied in the thermophilic lactic acid bacterium Streptococcus thermophilus CNRZ302. S. thermophilus actively adapts to freezing during a pretreatment at 20 degrees C, resulting in an approximately 1, 000-fold increased survival after four freeze-thaw cycles compared to mid-exponential-phase cells grown at an optimal temperature of 42 degrees C. No adaptation is observed when cells are exposed to a temperature (10 degrees C) below the minimal growth temperature of the strain (just below 15 degrees C). By two-dimensional gel electrophoresis several 7-kDa cold-induced proteins were identified, which are the major induced proteins after a shift to 20 degrees C. These cold shock proteins were maximally expressed at 20 degrees C, while the induction level was low after cold shock to 10 degrees C. To confirm the presence of csp genes in S. thermophilus, a PCR strategy was used which yielded products of different sizes. Sequence analysis revealed csp-like sequences that were up to 95% identical to those of csp genes of S. thermophilus ST1-1, Streptococcus dysgalactiae, Streptococcus pyogenes, and Lactococcus lactis. Northern blot analysis revealed a seven- to ninefold induction of csp mRNA after a temperature shift to 20 degrees C, showing that this thermophilic bacterium indeed contains at least one cold-inducible csp gene and that its regulation takes place at the transcriptional level.

Adaptation, Physiological↗
PubMed1999

Identification and disruption of BetL, a secondary glycine betaine transport system linked to the salt tolerance of Listeria monocytogenes LO28.

The trimethylammonium compound glycine betaine (N,N, N-trimethylglycine) can be accumulated to high intracellular concentrations, conferring enhanced osmo- and cryotolerance upon Listeria monocytogenes. We report the identification of betL, a gene encoding a glycine betaine uptake system in L. monocytogenes, isolated by functional complementation of the betaine uptake mutant Escherichia coli MKH13. The betL gene is preceded by a consensus sigmaB-dependent promoter and is predicted to encode a 55-kDa protein (507 amino acid residues) with 12 transmembrane regions. BetL exhibits significant sequence homologies to other glycine betaine transporters, including OpuD from Bacillus subtilis (57% identity) and BetP from Corynebacterium glutamicum (41% identity). These high-affinity secondary transporters form a subset of the trimethylammonium transporter family specific for glycine betaine, whose substrates possess a fully methylated quaternary ammonium group. The observed Km value of 7.9 microM for glycine betaine uptake after heterologous expression of betL in E. coli MKH13 is consistent with values obtained for L. monocytogenes in other studies. In addition, a betL knockout mutant which is significantly affected in its ability to accumulate glycine betaine in the presence or absence of NaCl has been constructed in L. monocytogenes. This mutant is also unable to withstand concentrations of salt as high as can the BetL+ parent, signifying the role of the transporter in Listeria osmotolerance.

Amino Acid Sequence↗
PubMed1999

Rapid fluorescence assessment of the viability of stressed Lactococcus lactis.

The aim of this study was to establish the use of the fluorescent probes carboxyfluorescein (cF) and propidium iodide (PI) for rapid assessment of viability, using Lactococcus lactis subsp. lactis ML3 exposed to different stress treatments. The cF labeling indicated the reproductive capacity of mixtures of nontreated cells and cells killed at 70 degrees C very well. However, after treatment up to 60 degrees C the fraction of cF-labeled cells remained high, whereas the survival decreased for cells treated at above 50 degrees C and was completely lost for those treated at 60 degrees C. In an extended series of experiments, cell suspensions were exposed to heating, freezing, low pH, or bile salts, after which the colony counts, acidification capacity, glycolytic activity, PI exclusion, cF labeling, and cF efflux were measured and compared. The acidification capacity corresponded with the number of CFU. The glycolytic activity, which is an indicator of vitality, was more sensitive to the stress conditions than the reproduction, acidification, and fluorescence parameters. The cF labeling depended on membrane integrity, as was confirmed by PI exclusion. The fraction of cF-labeled cells was not a general indicator of reproduction or acidification, nor was PI exclusion or cF labeling capacity (the internal cF concentration). When the cells were labeled by cF, a subsequent lactose-energized efflux assay was needed for decisive viability assessment. This novel assay proved to be a good and rapid indicator of the reproduction and acidification capacities of stressed L. lactis and has potential for physiological research and dairy applications related to lactic acid bacteria.

Bile Acids and Salts↗
PubMed1998

A possible role of ProP, ProU and CaiT in osmoprotection of Escherichia coli by carnitine.

Exogenously provided carnitine (beta-hydroxy-L-tau-N-trimethyl aminobutyrate) was found to stimulate aerobic growth of enterohaemorrhagic Escherichia coli O157:H7 in a medium of inhibitory osmotic strength. Its osmoprotective ability is comparable with that of betaine. As carnitine is an important compound in mammalian tissues, it is suggested that it might play a role in the growth of the pathogen on low water activity (aw) meat products. Using specific uptake mutants of E. coli K-12, it was established that, under osmotic stress, carnitine accumulates in the cytoplasm following import through the ProP and ProU transport systems. Betaine and carnitine also protect E. coli cells while growing anaerobically at inhibitory osmolarity. Under these conditions, an E. coli K-12 strain with lesions in both proP and proU accumulates low levels of L-carnitine but fails to accumulate betaine when these compounds are supplied in the external medium. This is probably a result of uptake of L-carnitine by the secondary transporter CaiT. The caiT gene forms part of the caiTABCDE operon which encodes the carnitine pathway, and is transcribed during anaerobic growth in the presence of carnitine. However, further experiments revealed that the carnitine pathway, including CaiT, does not play a significant role in osmoregulation of E. coli during anaerobiosis. Together, the results indicate that ProP and ProU are the sole transport systems involved in carnitine influx, both in aerobically and anaerobically osmotically stressed E. coli cells.

Aerobiosis↗
PubMed1998

Physiological activity of Campylobacter jejuni far below the minimal growth temperature.

The behavior of Campylobacter jejuni at environmental temperatures was examined by determining the physiological activities of this human pathogen. The minimal growth temperatures were found to be 32 and 31 degrees C for strains 104 and ATCC 33560, respectively. Both strains exhibited a sudden decrease in growth rate from the maximum to zero within a few degrees not only near the maximal growth temperature but also near the minimal growth temperature. This could be an indication that a temperature-dependent transition in the structure of a key enzyme(s) or regulatory compound(s) determines the minimal growth temperature. Oxygen consumption, catalase activity, ATP generation, and protein synthesis were observed at temperatures as low as 4 degrees C, indicating that vital cellular processes were still functioning. PCR analysis showed that cold shock protein genes, which play a role in low-temperature adaptation in many bacteria, are not present in C. jejuni. The fact that chemotaxis and aerotaxis could be observed at all temperatures shows that the pathogen is able to move to favorable places at environmental temperatures, which may have significant implications for the survival of C. jejuni in the environment.

Adenosine Triphosphate↗
PubMed1998

Lacticin 3147, a broad-spectrum bacteriocin which selectively dissipates the membrane potential.

Lacticin 3147 is a broad-spectrum bacteriocin produced by Lactococcus lactis subsp. lactis DPC3147 (M. P. Ryan, M. C. Rea, C. Hill, and R. P. Ross, Appl. Environ. Microbiol. 62:612-619, 1996). Partial purification of the bacteriocin by hydrophobic interaction chromatography and reverse-phase fast protein liquid chromatography revealed that two components are required for full activity. Lacticin 3147 is bactericidal against L. lactis, Listeria monocytogenes, and Bacillus subtilis; at low concentrations of the bacteriocin, bactericidal activity is enhanced when target cells are energized. This finding suggests that the presence of a proton motive force promotes the interaction of the bacteriocin with the cytoplasmic membrane, leading to the formation of pores at these low lacticin 3147 concentrations. These pores were shown to be selective for K+ ions and inorganic phosphate. The loss of these ions resulted in immediate dissipation of the membrane potential and hydrolysis of internal ATP, leading to an eventual collapse of the pH gradient at the membrane and ultimately to cell death. Our results suggest that lacticin 3147 is a pore-forming bacteriocin which acts on a broad range of gram-positive bacteria.

Adenosine Triphosphate↗
PubMed1998

Utilization of oligopeptides by Listeria monocytogenes Scott A.

For effective utilization of peptides, Listeria monocytogenes possesses two different peptide transport systems. The first one is the previously described proton motive force (PMF)-driven di- and tripeptide transport system (A. Verheul, A. Hagting, M.-R. Amezaga, I. R. Booth, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol, 61:226-233, 1995). The present results reveal that L. monocytogenes possesses an oligopeptide transport system, presumably requiring ATP rather than the PMF as the driving force for translocation. Experiments to determine growth in a defined medium containing peptides of various lengths suggested that the oligopeptide permease transports peptides of up to 8 amino acid residues. Peptidase activities towards several oligopeptides were demonstrated in cell extract from L. monocytogenes, which indicates that upon internalization, the oligopeptides are hydrolyzed to serve as sources of amino acids for growth. The peptide transporters of the nonproteolytic L. monocytogenes might play an important role in foods that harbor indigenous proteinases and/or proteolytic microorganisms, since Pseudomonas fragi as well as Bacillus cereus was found to enhance the growth of L. monocytogenes to a large extent in a medium in which the milk protein casein was the sole source of nitrogen. In addition, growth stimulation was elicited in this medium when casein was hydrolyzed by using purified protease from Bacillus licheniformis. The possible contribution of the oligopeptide transport system in the establishment of high numbers of L. monocytogenes cells in fermented milk products is discussed.

Biological Transport↗
PubMed1997

Nonanoic Acid, a Fungal Self-Inhibitor, Prevents Germination of Rhizopus oligosporus Sporangiospores by Dissipation of the pH Gradient.

Germination of Rhizopus oligosporus sporangiospores is characterized by swelling of the spores and subsequent emergence of germ tubes. Changes in spore morphology and alterations in intracellular pH (pH(infin)) of the sporangiospores were assessed during the germination process by flow cytometry. Sporangiospores were stained with carboxyfluorescein by incubation with carboxyfluorescein diacetate. The nonfluorescent carboxyfluorescein diacetate is passively transported into intact cells and subsequently cleaved by esterases, which results in intracellular accumulation of the fluorescent carboxyfluorescein. Given that the fluorescence of carboxyfluorescein is pH dependent, the pH(infin) of the individual spores could be assessed simultaneously with spore size. For R. oligosporus, swelling of the sporangiospores was accompanied by an increase of pH(infin). In the presence of nonanoic acid, a self-inhibitor produced by various fungi, increase of the pH(infin) was prevented and swelling was inhibited at concentrations of less than 1 mM. Octanoic acid and decanoic acid were equally effective. Acetic acid also inhibited germination but at much higher concentrations (>8 mM). The mechanism of action of these fatty acids is most likely dissipation of the pH gradient. A model is proposed in which the pH(infin) plays a crucial role in the germination of R. oligosporus sporangiospores.

Journal Article↗
PubMed1997

Protamine-induced permeabilization of cell envelopes of gram-positive and gram-negative bacteria.

The inhibitory effect of the cationic peptide protamine on Listeria monocytogenes, Escherichia coli, and Shewanella putrefaciens has been studied in detail. The addition of protamine (10 to 1,000 micrograms/ml) resulted in inhibition of oxygen consumption after less than 1 min and loss of intracellular carboxyfluorescein and ATP after 2 to 5 min. Maximum antibacterial activity was reached at alkaline pH and in the absence of divalent cations. The efficient permeabilization of cell envelopes of both gram-positive and gram-negative bacteria suggests that protamine causes a general disruption of the cell envelope, leading to a rapid and nonspecific efflux of low- and high-molecular-weight compounds.

Adenosine Triphosphate↗
PubMed1997

Modifications of membrane phospholipid composition in nisin-resistant Listeria monocytogenes Scott A.

A nisin-resistant (NISr) variant of Listeria monocytogenes Scott A was isolated by stepwise exposure to increasing concentrations of nisin in brain heart infusion (BHI) broth. The NISr strain was about 12 times more resistant to nisin than was the wild-type (WT) strain. Accordingly, higher nisin concentrations were required to dissipate both components of the proton motive force in the NISr strain than in the WT strain. Comparison of the membrane fatty acyl composition of the sensitive strain with that of its NISr derivative revealed no significant differences. From phospholipid head group composition analysis and phospholipid biosynthesis measurements during growth in the absence and presence of nisin, it could be inferred that the NISr strain produces relatively more phosphatidylglycerol (PG) and less diphosphatidylglycerol (DPG) than the parent strain does. Monolayer studies with pure lipid extracts from both strains showed that nisin interacted more efficiently with lipids derived from the WT strain than with those derived from the NISr strain, reflecting qualitative differences in nisin sensitivity. Involvement of the cell wall in acquisition of nisin resistance was excluded, since the WT and NISr strains showed a comparable sensitivity to lysozyme. Recently, it has been demonstrated that nisin penetrates more deeply into lipid monolayers of DPG than those of other lipids including PG, phosphatidylcholine, phosphatidylethanolamine, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol (R.A. Demel, T. Peelen, R.J. Siezen, B. de Kruijff, and O.P. Kuipers, Eur. J.Biochem. 235:267-274, 1996). Collectively, the mechanism of nisin resistance in this L. monocytogenes NISr strain is attributed to a reduction in the DPG content of the cytoplasmic membrane.

Drug Resistance, Microbial↗