Acid resistance contributes to the high-pressure carbon dioxide resistance of Escherichia coli K-12.

Effect of deletion of acid resistant genes of E. coli on the high-pressure carbon dioxide (HPC) resistance was investigated. Genes coding amino acid decarboxylases, such as lysine, arginine, and glutamate decarboxylase, were found to contribute to HPC resistance. Protonophore-treated cells showed hypersensitivity to HPC, confirming that HPC induced cytoplasm acidification and exerted severe damage on cells by intrusion of gaseous carbon dioxide into cytoplasm.

Mixed-species biofilm formation by direct cell-cell contact between brewing yeasts and lactic acid bacteria.

Mixed-species biofilm was remarkably formed in a static co-culture of Lactobacillus plantarum ML11-11 and Saccharomyces cerevisiae Y11-43 isolated from brewing samples of Fukuyama pot vinegar. Mixed-species biofilm is probably formed by direct cell-cell contact between ML11-11 and S. cerevisiae including Y11-43 and laboratory yeast strains. Scanning electron microscopic observation suggested that the mixed-species biofilm had a thick, bi-layer structure.

Microbiological and biochemical survey on the transition of fermentative processes in Fukuyama pot vinegar brewing.

Traditional brewing of Fukuyama pot vinegar is a process that has been continued in Fukuyama, Kagoshima, Japan, for almost 200 years. The entire process proceeds from raw materials, including steamed rice, rice koji (steamed rice grown with a fungus, Aspergillus oryzae) and water, to produce vinegar in roughly capped large pots laid in the open air. No special fermentative manipulation is required, except for scattering dried rice koji (called furi-koji) on the surface of the mash to form a cap-like mat on the surface at the start of brewing. As the biochemical mechanism of the natural transition of the fermentative processes during brewing has not been fully explained, we conducted a microbiological and biochemical study on the transition. First, a distinct biochemical change was observed in the brewing of spring preparation; that is, a sharp decline in pH from 6.5 to 3.5 within the first 5 days of brewing was observed due to lactic acid fermentation. Alcoholic fermentation also proceeded with a sharp increase to 4.5% ethanol within the first 5 days under the acidic conditions, suggesting that saccharification and both fermentations proceed in parallel. Acidic conditions and ethanol accumulation restricted the growth of most microorganisms in the mash, and in turn provided a favorable growth condition for acetic acid bacteria which are acid resistant and "ethanol-philic." Acetic acid was detected from day 16 and gradually increased in concentration, reaching a maximum of 7% at day 70 that was maintained thereafter. Empirically furi-koji naturally sinks into the mash after around day 40 by an unknown mechanism, allowing acetic acid bacteria to easily form pellicles on the mash surface and promoting efficient acetic acid fermentation. Dominant microbial species involved in the three fermentations were identified by denaturing gradient gel electrophoresis analysis using PCR-amplified defined-regions of small rDNA from microorganisms in the brewing mash or colony direct PCR applied to isolated microorganisms from the mash.

Cell-cycle independent chromosome condensation in Schizosaccharomyces pombe induced by high hydrostatic pressure treatment.

We exposed Schizosaccharomyces pombe to high hydrostatic pressure treatment (HPT) of 75 MPa at 28 degrees Celsius for 30 min and then observed that the DAPI-stained chromosomal DNA had shrunk compactly. We termed this phenomenon HPT-induced chromosome condensation (HPT-CC). HPT did not significantly decrease viability. The condensed state was released when HPT cells were cultured at 28 degrees Celsius for 30 min. The condensation was not caused by shrinking of the nuclear envelope, which was visualized by YFP-tagged importin alpha. HPT-CC was cell cycle independent, because it was observed in almost all randomly cultured cells. The condensin complex (Cut3, Cut14, and three other proteins) is responsible for cell cycle dependent CC. Studies with Cut3-YFP and ts mutants of Cut3 and Cut14 confirmed that HPT-CC was independent of condensin molecules. HPT-CC was also observed in Saccharomyces cerevisiae. HPT-CC appears likely to be a temporal stress response to high hydrostatic pressure found at least in yeasts.

Biofilm formation by Escherichia coli in hypertonic sucrose media.

High osmotic environments produced by NaCl or sucrose have been used as reliable and traditional methods of food preservation. We tested, Escherichia coli as an indicator of food-contaminating bacterium, to determine if it can form biofilm in a hyperosmotic environment. E. coli K-12 IAM1264 did not form biofilm in LB broth that contained 1 M NaCl. However, the bacterium formed biofilm in LB broth that contained 1 M sucrose, although the planktonic growth was greatly suppressed. The biofilm, formed on solid surfaces, such as titer-plate well walls and glass slides, solely around the air-liquid interface. Both biofilm forming cells and planktonic cells in the hypertonic medium adopted a characteristic, fat and filamentous morphology with no FtsZ rings, which are a prerequisite for septum formation. Biofilm forming cells were found to be alive based on propidium iodide staining. The presence of 1 M sucrose in the food environment is not sufficient to prevent biofilm formation by E. coli.

Effect of skimmed milk and its fractions on the inactivation of Escherichia coli K12 by high hydrostatic pressure treatment.

We investigated the effects of skimmed milk and its protein fractions (casein, whey, globulin, and albumin) on the injury and inactivation of Escherichia coli K-12 by high hydrostatic pressure (HHP) treatment. The protective effect of skimmed milk on HHP-mediated inactivation and injury of E. coli increased with increases in the skimmed milk concentration. However, protein fractions derived from skimmed milk did not exhibit this protective effect. Microscopy analysis by DAPI/PI staining indicated that some cells were localized in the solid portion of skimmed milk, and some of these cells were alive. The coagulated fraction derived from the autoclaved whey fraction also showed a significant protective effect. We speculate that the solid portion in skimmed milk could provide the protective effect to bacterial cells.

Cessation of cytokinesis in Schizosaccharomyces pombe during growth after release from high hydrostatic pressure treatment.

On the basis of our previous study concerning the effect of high hydrostatic pressure treatment (HPT) on Escherichia coli FtsZ ring (bacterial cytoskeleton) formation, we aimed to determine the effect of HPT on the growth properties of a representative eukaryotic microbe, Schizosaccharomyces pombe, in relation to the behavior of genuine cytoskeletons. Microtubules were visualized with GFP-linked alpha-tubulin. Actin-related cytoskeletons were fluorescently stained with rhodamine-phalloidin. We observed growth retardation of about 10 h in post growth after HPT (75 MPa, 30 min, 28 degrees C), which caused only a little loss of viable cells. In accordance with the period of growth retardation, cessation of cytokinesis and disappearance of the contractile ring (composed of actin, myosin II, and other proteins), directly participates in cytokinesis, continued for 18 h after HPT. On the other hand, the microtubules disappeared only for 6 h after HPT. Based on these observations, the contractile ring was the site most sensitive to HPT resulting in the cessation of cytokinesis.

Cytoplasmic acidification may occur in high-pressure carbon dioxide-treated Escherichia coli K12.

While studying the mechanism by which high-pressure carbon dioxide treatment (HCT) inactivates bacteria, we found that the efficiency of DNA recovery via phenol extraction was extraordinarily low from E. coli K12 cells that had been subjected to HCT. DAPI staining of the treated cells, however, revealed that nuclear DNA was present. Most DNA from the cells subjected to HCT was probably caught in the denatured protein layer during phenol extraction. The efficiency of DNA recovery from proteinase-treated crude extracts from cells subjected to HCT was high. Crude extracts of E. coli K12 cells that had not undergone HCT were intentionally acidified with acetic acid to pH 5.2 to cause acidic coagulation of cytoplasmic proteins. The efficiency of DNA recovery from the acidified extracts was low. These results suggest that in cells subjected to HCT, cytoplasmic pH is reduced to around pH 5.2, and that nuclear DNA becomes entangled in coagulated cytoplasmic proteins. Acidification of the cytoplasm might be the primary mechanism by which HCT inactivates bacteria.

Mixed-species biofilm formation by lactic acid bacteria and rice wine yeasts.

We found that species combinations such as Lactobacillus casei subsp. rhamnosus IFO3831 and Saccharomyces cerevisiae Kyokai-10 can form a mixed-species biofilm in coculture. Moreover, the Kyokai-10 yeast strain can form a biofilm in monoculture in the presence of conditioned medium (CM) from L. casei IFO3831. The active substance(s) in bacterial CM is heat sensitive and has a molecular mass of between 3 and 5 kDa. In biofilms from cocultures or CM monocultures, yeast cells had a distinct morphology, with many hill-like protrusions on the cell surface.

High-hydrostatic-pressure treatment impairs actin cables and budding in Saccharomyces cerevisiae.

We previously reported that the postgrowth of Escherichia coli K-12 after high-hydrostatic-pressure treatment (HPT) as moderate as 75 MPa for 30 min at 37 degrees C induced the formation of elongated cells due to an HPT-induced disorder in FtsZ ring formation, which is essential for cell division. Because an FtsZ ring is known as a bacterial cytoskeleton, we examined the effect of HPT on a eukaryotic cytoskeleton, such as actin cables (long bundles of actin filaments), of Saccharomyces cerevisiae. We found that actin cables disappeared after HPT (100 MPa) and were not reorganized until 3.5 h of growth after HPT. As long as actin cables disappeared, budding did not start. We also demonstrated that the in vitro polymerization of actin monomers was highly sensitive to HPT.

Effect of high pressure carbon dioxide on the clumping of the bacterial spores.

The formation of spore clumps of Bacillus coagulans and Bacillus licheniformis during high-pressure carbon dioxide treatment (HCT) was investigated. As the treatment time increased, the number of spore clumps increased. After 120 min, single spore decreased to 20-35% of the population. Addition of a surfactant decreased the hydrophobicity of spore surface and increased both the number of single spores and the rate of inactivation ratio of B. coagulans and B. licheniformis spores.

Estimation of the biofilm formation of Escherichia coli K-12 by the cell number.

We developed a method of estimating the biofilm formation of Escherichia coli K-12 strains in microtiter-plate wells by the cell number. Regression lines between the cell number and absorbance of crystal violet that stained the E. coli biofilm consisted of high and low slope lines, respectively.

Vacuolar H(+)-ATPase and plasma membrane H(+)-ATPase contribute to the tolerance against high-pressure carbon dioxide treatment in Saccharomyces cerevisiae.

As a non-thermal sterilization process, high-pressure carbon dioxide treatment (HPCT) is considered to be promising. The main sterilizing effect of HPCT is thought to be acidification in cytoplasm of microorganisms. We investigated the tolerance mechanism of Saccharomyces cerevisiae to HPCT with special reference to vacuolar and plasma membrane H(+)-ATPases. HPCT was imposed at 35 degrees C, 4 to 10 MPa, for 10 min. slp1 mutant defective in vacuole morphogenesis was more sensitive to HPCT than its isogenic parent. Concanamycin A, a specific inhibitor of vacuolar H(+)-ATPase (V-ATPase), at 10 microM rendered the parent more HPCT-sensitive to the level of slp1. To confirm further the contribution of V-ATPase to the tolerance against HPCT in S. cerevisiae, we compared vma1 mutant of V-ATPase with its isogenic parent for their HPCT sensitivity. vma1 mutant was more sensitive to HPCT than its parent. Addition of 10 microM vanadate, an inhibitor of plasma membrane H(+)-ATPase (P-ATPase), to the wild type strains also increased the inactivation ratio. These results clearly show that V- and P-ATPases contribute to the tolerance against HPCT in S. cerevisiae by accumulating excess H(+) from cytoplasm to vacuole and excluding H(+) outside of the cell, respectively.

Formation of the spore clumps during heat treatment increases the heat resistance of bacterial spores.

Effects of the clumping of bacterial spores on their heat resistance as a result of heat treatment were investigated. Spore suspensions of Bacillus cereus, Bacillus coagulans and Bacillus licheniformis were heated at 85 degrees C. Survivor curves of the three strains showed tailing in all treatments after 30 min. As the treatment time increased, the formation of spore clumps increased in all strains after 20 min. Relative hydrophobicity of the spore surface increased as a result of heat treatment. The effect of spore concentration on the inactivation of the B. licheniformis spores was investigated, and surviving curves showed no tailing below a concentration of 4.9 log CFU/ml.

High hydrostatic pressure treatment impairs AcrAB-TolC pump resulting in differential loss of deoxycholate tolerance in Escherichia coli.

We reported previously that high hydrostatic pressure-injured stationary phase cells of Escherichia coli K-12 lost their intrinsic deoxycholate tolerance. The AcrAB-TolC multi-drug resistance pump driven by proton motive force has been argued to be responsible for the tolerance to deoxycholate. In this report, we tested the sensitivity of the AcrAB-TolC (three components) pump to high hydrostatic pressure treatment (HPT). E. coli K-12 treated with HPT became sensitive to AcrAB-TolC-specific drugs such as ethidium bromide, but not to tetracycline which is pumped out by a one-component transporter, Tet. Only E. coli K-12 overproducing both AcrAB and TolC exhibited restored tolerance to deoxycholate after HPT but not E. coli overproducing either TolC or AcrAB. These observations strongly suggest that three-component pumps such as AcrAB-TolC are more susceptible to HPT than one-component pumps such as Tet, resulting in the differential loss of deoxycholate tolerance in high hydrostatic pressure-injured E. coli cells.

Inactivation of Geobacillus stearothermophilus spores by high-pressure carbon dioxide treatment.

High-pressure CO2 treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO2 treatment at 30 MPa and 35 degrees C, to high-hydrostatic-pressure treatment at 200 MPa and 65 degrees C, or to heat treatment at 0.1 MPa and 85 degrees C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO2 treatment. We also investigated the influence of temperature on CO2 inactivation of G. stearothermophilus. Treatment with CO2 and 30 MPa of pressure at 95 degrees C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95 degrees C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95 degrees C for 120 min had little effect. The activation energy required for CO2 treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2 treatment of G. stearothermophilus spores, CO2 treatment at 95 degrees C was more effective than treatment at 95 degrees C alone.

Fosmidomycin resistance in adenylate cyclase deficient (cya) mutants of Escherichia coli.

Adenylate cyclase deficient (cya) mutants of E. coli K-12 were found to be resistant to fosmidomycin, a specific inhibitor of the non-mevalonate pathway, just like to fosfomycin. E. coli glpT mutants were resistant to fosfomycin and also to fosmidomycin. This fact shows that fosmidomycin was transported inside via the glycerol-3-phosphate transporter, GlpT. DNA micro-array analysis showed that the transcription of glpT and other genes concerning glycerol utilization were highly dependent on the presence of cAMP.

Efficient export of alkaline phosphatase overexpressed from a multicopy plasmid requires degP, a gene encoding a periplasmic protease of Escherichia coli.

Escherichia coli DegP is an inducible serine protease which is involved in the breakdown of abberant proteins arising in the periplasmic compartment. Overexpression of alkaline phosphatase (PhoA) increased transcription of degP by twofold. To examine the significance of its induction, we overexpressed PhoA in a mutant strain deficient in the degP gene. Upon PhoA overexpression, the degP mutant produced a smaller amount of active PhoA, about one half of the enzymatic activity of its isogenic wild-type strain, and accumulated a larger amount of its precursor, indicating that degP is required for efficient export of overexpressed PhoA. Pulse-chase experiment showed that PhoA overexpression in the absence of degP causes a severe defect in the export of several proteins tested. Examination of the synthesis and the accumulation of the phoA gene products revealed that a part of them, synthesized in the wild-type strain, undergoes relatively rapid proteolysis and that degP is necessary for such a process. From these results, we discuss a possible role of DegP in facilitating protein export under stress conditions.

A glutamine synthetase mutant of Saccharomyces cerevisiae shows defect in cell wall.

To study the organization and biosynthesis of the yeast cell wall, hypo-osmolarity-sensitive mutants of Saccharomyces cerevisiae were analyzed. Cells of JS4 were irregular in shape and fragile. Calcofluor staining and quantitative analysis indicated that the chitin content was reduced. By DNA cloning and genetic analysis, the mutation hpo1-1 was found to be allelic to GLN1 which encodes glutamine synthetase. The glutamine content was significantly low in JS4, and the mutant was recovered from the cell wall defect by supplying glutamine in the medium. Partial inhibition of glutamine synthetase by phosphinothricin also induced defects in the cell wall. These results indicate that the shortage of glutamine affects cell wall integrity prior to other cellular functions.