Cellular Envelopes and Tolerance to Acid pH in Mesorhizobium loti

Changes in the cell envelopes in response to acidity were studied in two strains of Mesorhizobium loti differing in their tolerance to pH. When the less acid-tolerant strain LL22 was grown at pH 5.5, membrane phosphatidylglycerol decreased and phosphatidylcholine increased, compared with cells grown at pH 7.0. On the other hand, when the more acid-tolerant strain LL56 was grown at pH 5.5, phosphatidylglycerol, phosphatidylethanolamine, and lysophospholipid decreased 25%, 39%, and 51% respectively, while phosphatidyl-N-methylethanolamine and cardiolipin increased 26% and 65% respectively compared with cells grown at pH 7.0. The longest-chain fatty acids (19:0 cy and 20:0) increased in both strains at pH 5.5, while in LL56 these fatty acids increased still further at pH 4.0. Variations in other wall and membrane properties such as cell hydrophobicity, lypopolysaccharides, and protein composition of the outer membrane in relation to acid pH are also discussed. Received: 22 December 1998 / Accepted: 2 February 1999     

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyrhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH. 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acidadapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.     

Mechanism of acid tolerance in a rhizobium strain isolated from Pueraria lobata (Willd.) Ohwi

The Rhizobium sp. strain PR389 was isolated from the root nodules of Pueraria lobata (Willd.) Ohwi, which grows in acidic (pH 4.6) yellow soil of the Jinyun Mountains of Beibei, Chongqing, China. While rhizobia generally have a pH range of 6.5-7.5 for optimum growth, strain PR389 grew in a liquid yeast extract - mannitol agar medium at pH 4.6, as well as in a pH 4.1 soil suspension, suggesting acid tolerance in this specific strain of rhizobium . However, at pH 4.6, the lag phase before vigorous growth was 40 h compared with 4 h under neutral conditions (pH 7.0). For PR389, the generation time after the lag phase remained the same at different pH levels despite the different durations of the lag phase. Except in the pH 4.4 treatment, the pH of the culturing media increased from 4.6, 4.8, 5.0, and 5.5 to neutral and slightly alkaline after 70 h of culture. Chloramphenicol was added to determine if protein production was involved in the increasing pH process. Chloramphenicol significantly inhibited PR389 growth under acid stress but had little effect under neutral conditions. Proton flux measured during a short acid shock (pH 3.8) revealed that this strain has an intrinsic ability to prevent H(+) from entering cells when compared with acid-sensitive rhizobia. We propose that the mechanism for acid tolerance in PR389 involves both intracellular and extracellular processes. When the extracellular pH is lower than pH 4.4, the cell membrane blocks hydrogen from entering the cell. When the pH exceeds 4.4, the rhizobium strain has the ability to raise the extracellular pH, thereby, potentially decreasing the toxicity of aluminum in acid soil.     

一株耐酸野生葛藤(Pueraria lobata)根瘤菌的筛选与耐酸机理初步研究

从重庆市北碚区缙云山酸性黄壤(pH4.6)上生长的葛藤根瘤中分离到一株耐酸葛藤根瘤菌PR389,能在pH4.3的YMA培养基上正常生长,而一般根瘤菌最适生长pH值为6.5~7.5,说明PR389为一株耐酸葛藤根瘤菌。通过质子通量试验发现,与不耐酸的菌株相比,PR389的细胞膜能阻止过量的H 进入细胞,表明PR389具有某种能力使之在低酸性环境下不受伤害。在耐酸性试验中,PR389在加氯霉素的强酸性(pH3.8)YMA培养液中表现出来的耐酸性被氯霉素抑制,推测胞内特异蛋白质的诱导合成是PR389具有耐酸性的原因。     

Effects of temperature stress on bean-nodulating Rhizobium strains

High soil temperatures in tropical areas limit nodulation and dinitrogen fixation by strains of Rhizobium. Several heat-tolerant bean-nodulating Rhizobium strains have been isolated previously. However, the basis of their resistance to heat remains unknown. In this study, we compared the effects of heat on symbiotic nitrogen fixation, cell survival, amino acid uptake, and protein synthesis in a heat-tolerant (CIAT899) and a heat-sensitive (CNPAF512) bean-nodulating Rhizobium strain. Acetylene reduction activity of nodulated roots excised from unstressed plants was strongly diminished at 35 or 40 degrees C when plants were nodulated either by CIAT899 or by CNPAF512. When these strains were tested under free-living conditions, survival at 40 degrees C as well as the kinetics of l-[S]methionine uptake and protein synthesis at 35 and 40 degrees C indicated the higher tolerance of CIAT899 than of CNPAF512 to thermal stress. The synthesis of heat shock proteins was detected in both strains, although at different temperatures. Increased synthesis of 14 heat shock proteins in CNPAF512 and of 6 heat shock proteins in CIAT899 was observed at 40 and 45 degrees C, respectively. A heat shock protein of approximately 21 kDa, of which the synthesis was strongest in both Rhizobium strains upon a temperature shift up, was also conserved in several other bean-nodulating rhizobia. Acquired thermotolerance in CIAT899 was shown to depend on protein synthesis.     

百脉根根瘤菌群体感应相关基因的克隆与表达

在实验室的纯培养条件下,检测不到百脉根根瘤菌自体诱导物的产生,但是通过基因序列同源性比对分析发现,百脉根根瘤菌基因组中至少含有4种自体诱导物合成酶基因;将其中一个自体诱导物合成酶基因ml4543克隆到大肠杆菌表达载体pET30a,构建得到能异源表达该基因的大肠杆菌重组菌株;对该大肠杆菌重组菌株进行自体诱导物检测发现,该合成酶基因在大肠杆菌中能合成至少3种自体诱导物因子。     

THE OPTIMUM PH OF FOUR STRAINS OF ACIDOPHILIC SNOW ALGAE IN THE GENUS CHLOROMONAS (CHLOROPHYTA) AND POSSIBLE EFFECTS OF ACID PRECIPITATION1

Four axenic strains of snow algae were examined for optimum pH under laboratory conditions using M-1 growth medium. Growth was measured using cell counts, cell measurements and absorbance readings at 440 nm. Strains C204 and C479A of Chloromonas sp. from the Adirondack Mountains, New York, grew optimally at pH 4.0 to 5.0. Strains C381F and C381G, Chloromonas polyptera (Fritsch) Hoh., Mull. & Roem. from the White Mountains, Arizona, grew optimally at pH 4.5 to 5.0. Growth was significantly higher at pH 4.0 in the northeastern species (Chloromonas sp.), but no significant difference was observed in final growth at pH 4.5, 5.0 and 5.5 between species. It is postulated that the more acidic precipitation in the northeastern United States may be selecting for strains of snow algae with greater tolerance to acidity than in strains from the southwestern United States or that the different pH optima reported are simply species differences. New York strain C204 was also grown in heavily buffered AM medium where it had an optimum pH of 5.0, but cells became irregularly shaped and tended to clump at pH 6.0 to 7.0. Growth of C204 in AM medium was significantly lower than in M-1 medium for snow algae. These findings justify the use of M-1 medium for this type of experimentation.     

Evaluation and improvement of the energy efficiency of nitrogen fixation in Lotus corniculatus nodules induced by Rhizobium loti strains indigenous to Uruguay

In order to evaluate energy efficiency of nitrogen fixation by the Lotus corniculatus/Rhizobium loti symbiosis, Uruguayan R. loti strains were tested for hydrogen-uptake (Hup) status. Nodules induced in L. corniculatus by all eight R. loti strains tested evolved high amounts of hydrogen (2.0–8.7 mol H2/h.g nodule fresh weight). This production of hydrogen corresponds to 38–69% of total nitrogenase activity estimated as acetylene reduction, suggesting that hydrogen is not recycled within these nodules. This was confirmed by the lack of hydrogenase activity in bacteroid suspensions. Additionally, no hybridization signals were observed in total DNA restriction digests from these strains when a DNA fragment containing part of hydrogenase structural genes from Rhizobium leguminosarum bv. viciae was used as probe. Cosmid pHU52, containing the complete gene cluster required for hydrogen oxidation in Bradyrhizobium japonicum, was introduced into two R. loti strains. Transconjugants from only one of the strains were able to express hydrogenase activity in vegetative cells incubated under the derepression conditions described for B. japonicum. Bacteroids induced by both transconjugant strains in L. corniculatus and Lotus tenuis expressed hydrogenase activity in nodules. The level of hydrogenase activity induced in L. tenuis nodules was two-fold higher than those induced in L. corniculatus. This implies the existence of a strong host effect on hydrogenase expression in this symbiotic system.     

Effect of ammonia on the growth of Bacillus species and some other bacteria

The tolerance of 26 Bacillus species isolated from alkaline fermented foods, five other bacilli and nine non spore-forming bacteria to alkaline pH and ammonia was determined. All grew at pH 7, 8 and 9 in the presence of 930 mmol l-1 NH4 + at pH 7.0, and in the presence of NH3 concentrations up to 5 mmol l-1 at pH 7.0 and 8.0. At higher NH3 concentrations, growth of some of the bacteria was inhibited and at 500 mmol l-1 only B. pasteurii and B. pumilus grew. Bacteria from alkaline food fermentations included strains relatively sensitive to NH3 (inhibited by 50 mmol l-1) and relatively tolerant strains (grew in the presence of 300 mmol l-1) and there was no evidence that they were more tolerant to NH3 than bacteria not associated with these fermentations.     

Acid resistance of Helicobacter pylori depends on the UreI membrane protein and an inner membrane proton barrier

ureI encodes an inner membrane protein of Helicobacter pylori. The role of the bacterial inner membrane and UreI in acid protection and regulation of cytoplasmic urease activity in the gastric microorganism was studied. The irreversible inhibition of urease when the organism was exposed to a protonophore (3,3',4', 5-tetrachlorsalicylanide; TCS) at acidic pH showed that the inner membrane protected urease from acid. Isogenic ureI knockout mutants of several H. pylori strains were constructed by replacing the ureI gene of the urease gene cluster with a promoterless kanamycin resistance marker gene (kanR). Mutants carrying the modified ureAB-kanR-EFGH operon all showed wild-type levels of urease activity at neutral pH in vitro. The mutants resisted media of pH > 4.0 but not of pH < 4.0. Whereas wild-type bacteria showed high levels of urease activity below pH 4.0, this ability was not retained in the ureI mutants, resulting in inhibition of metabolism and cell death. Gene complementation experiments with plasmid-derived H. pylori ureI restored wild-type properties. The activation of urease activity found in structurally intact but permeabilized bacteria treated with 0.01% detergent (polyoxy-ethylene-8-laurylether; C12E8), suggested a membrane-limited access of urea to internal urease at neutral pH. Measurement of 14C-urea uptake into Xenopus oocytes injected with ureI cRNA showed acid activation of uptake only in injected oocytes. Acceleration of urea uptake by UreI therefore mediates the increase of intracellular urease activity seen under acidic conditions. This increase of urea permeability is essential for H. pylori survival in environments below pH 4.0. ureI-independent urease activity may be sufficient for maintenance of bacterial viability above pH 4.0.     

Occurrence of choline and glycine betaine uptake and metabolism in the family rhizobiaceae and their roles in osmoprotection

The role of glycine betaine and choline in osmoprotection of various Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, and Bradyrhizobium reference strains which display a large variation in salt tolerance was investigated. When externally provided, both compounds enhanced the growth of Rhizobium tropici, Sinorhizobium meliloti, Sinorhizobium fredii, Rhizobium galegae, Agrobacterium tumefaciens, Mesorhizobium loti, and Mesorhizobium huakuii, demonstrating their utilization as osmoprotectants. However, both compounds were inefficient for the most salt-sensitive strains, such as Rhizobium leguminosarum (all biovars), Agrobacterium rhizogenes, Rhizobium etli, and Bradyrhizobium japonicum. Except for B. japonicum, all strains exhibit transport activity for glycine betaine and choline. When the medium osmolarity was raised, choline uptake activity was inhibited, whereas glycine betaine uptake was either increased in R. leguminosarum and S. meliloti or, more surprisingly, reduced in R. tropici, S. fredii, and M. loti. The transport of glycine betaine was increased by growing the cells in the presence of the substrate. With the exception of B. japonicum, all strains were able to use glycine betaine and choline as sole carbon and nitrogen sources. This catabolic function, reported for only a few soil bacteria, could increase competitiveness of rhizobial species in the rhizosphere. Choline dehydrogenase and betaine-aldehyde dehydrogenase activities were present in the cells of all strains with the exception of M. huakuii and B. japonicum. The main physiological role of glycine betaine in the family Rhizobiaceae seems to be as an energy source, while its contribution to osmoprotection is restricted to certain strains.     

Identification of Rhizobium phaseoli Strains That Are Tolerant or Sensitive to Soil Acidity

A study was conducted to determine whether the survival of Rhizobium phaseoli in acid soils could be predicted on the basis of the tolerance of the organism to acidity in culture. Of 16 strains tested, all grew in culture at pH 4.6, but only those that grew at pH 3.8 survived in soils having pH values of 4.1 to 4.6. Strains that tolerated the lowest pH values in culture were tolerant of the highest aluminum concentrations. In one acid soil, an acid-tolerant strain was unable to survive in numbers greater than 100/g, but the poor survival was not related to the level of extractable aluminum or manganese in the soil. Reproduction of an acid-tolerant strain of R. phaseoli was enhanced in the rhizosphere of Phaseolus vulgaris in both acid and limed soils, but stimulation of an acid-sensitive strain by the plant occurred only in the limed soil. These results indicate that cultural tests can be used to predict the ability of R. phaseoli to survive in acid soil.     

Zygosaccharomyces lentus: a significant new osmophilic, preservative-resistant spoilage yeast, capable of growth at low temperature

Zygosaccharomyces lentus is a yeast species recently identified from its physiology and 18S ribosomal sequencing (Steels et al. 1999).The physiological characteristics of five strains of this new yeast so far isolated were investigated, particularly those of technical significance for a spoilage yeast, namely temperature range, pH range, osmotolerance, sugar fermentation, resistance to food preservatives such as sorbic acid, benzoic acid and dimethyldicarbonate (DMDC; Velcorin). Adaptation to benzoic acid, and growth in shaking and static culture were also investigated. Zygosaccharomyces lentus strains grew over a wide range of temperature (4-25 degrees C) and pH 2.2-7.0. Growth at 4 degrees C was significant. Zygosaccharomyces lentus strains grew at 25-26 degrees C in static culture but were unable to grow in aerobic culture close to their temperature maximum. All Z. lentus strains grew in 60% w/v sugar and consequently, are osmotolerant. Zygosaccharomyces lentus strains could utilize sucrose, glucose or fructose as a source of fermentable sugar, but not galactose. Zygosaccharomyces lentus strains were resistant to food preservatives, growing in sorbic acid up to 400 mg l-1 and benzoic acid to 900 mg l-1 at pH 4.0. Adaptation to higher preservative concentrations was demonstrated with benzoic acid. Resistance to DMDC was shown to be greater than that of Z. bailii and Saccharomyces cerevisiae. This study confirms that Z. lentus is an important food spoilage organism potentially capable of growth in a wide range of food products, particularly low pH, high sugar foods and drinks. It is likely to be more significant than Z. bailii in the spoilage of chilled products.     

On the resistance of effectiveness ofRhizobium trifolii to a low pH

Summary ThreeRhizobium trifolii strains were subcultured repeatedly on agar medium of pH values ranging from 3.5 to 7.0. Growth was retarded at pH 4.5 and very poor at pH 4.0 and 3.5, although the cells were not killed at the lowest pH. After 12 subcultures during a period of 300 days on these media, the symbiotic effectiveness was estimated by inoculating aseptic seedlings of white clover in test tubes containing Jensen's agar medium at pH 6.5.The number of nodules formed on the plants and the effectiveness of some strains seemed to be affected by growing the rhizobium for a long period at a low pH. All test plants produced nodules with red pigment.     

Deionized distilled water as a medium for aluminium toxicity studies of Rhizobium

A method has been developed to study aluminium (A1) toxicity towards Rhizobium. This involves growth in broth followed by washing and measurement of cell viability in deionized distilled water plus A1. The results illustrate the high degree of sensitivity and rapid response of Rhizobium leguminosarum biovar trifolii and R. loti to A1 under acid conditions but confirm earlier results on the relative tolerance of these two species.     

Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer.

Induction of acid resistance (habituation) in Escherichia coli at pH 5.0 took ca 5 min in broth at 37 degrees C and 30-60 min in minimal medium. Induction occurred at a range of pH values from 4.0 to 6.0; it was dependent on continuing protein and RNA synthesis but substantial acid resistance appeared in the presence of nalidixic acid. Acid resistance was long-lasting; organisms grown at pH 5.0 retained most of their resistance after 2 h growth at pH 7.0. Organisms grown at pH 5.0 showed increased synthesis of a number of cytoplasmic proteins compared with the level in cells grown at pH 7.0. DNA repair-deficient strains carrying recA, uvrA or polA1 mutations were more acid-sensitive than the repair-proficient parents but were able to habituate at pH 5.0. Organisms grown at pH 5.0 transferred the ColV plasmid much more effectively at acid pH than did those grown at pH 7.0 and habituated recipients appeared better able to repair incoming acid-damaged plasmid DNA than did those that were non-habituated. Induction of acid resistance at pH 5.0 may be significant for the survival of organisms exposed to periodic discharges of acid effluent in the aquatic environment and habituation may also allow plasmid transfer and repair of acid-damaged plasmid DNA during or after such exposure.     

Electrophoretic profile of hybrids between cryotolerant and non-cryotolerant Saccharomyces strains

Escherichia coli O157 : H7 (O157) has unusual acid tolerance. The influence of heat shock on acid tolerance of O157 was studied. Seven strains of O157 and E. coli K-12 were tested for their ability to survive in minimum glucose medium (pH 2·5) at 37 °C. The survival of heat-shocked (10 min at 48 °C) cells was about 10–100 times greater compared with untreated cells depending on the strain. No significant difference ( P > 0·05) for O157 strain 932 was observed between heat shock-induced and acid adaptation-induced (pH 5·0) acid tolerance. Chloramphenicol prevented heat shock-induced acid tolerance, indicating the requirement of newly synthesized protein(s). Two outer membrane proteins (OMP) (22 and 15 kDa) were synthesized within 10 min of heat shock and were expressed for at least 6 h by cells held at 37 °C. N-terminal amino acid sequence analysis suggested that the 22 kDa OMP is a component of an alkyl hydroperoxide reductase. This protein contains a redox active disulphide, which is probably involved in H⁺ transport. Results indicate that sublethal heat treatment of O157 cells substantially increases their tolerance to acidic conditions. This could have practical implications for foods that receive a mild heat treatment and rely on acid as a preservative.     

Stability of short and long O-chain lipopolysaccharide types in Rhizobium galegae and their correlation with symbiotic properties and growth conditions, tolerance of low pH, aluminum and salt in the growth medium

We recently showed that Rhizobium galegae strains had two different lipopolysaccharide types, short and long O-chain. In the present study we observed that the lipopolysaccharide type was a stable feature of the strain. Both types persisted in cells at all phases of the growth cycle, during differentiation of bacteria into bacteroids and when the cells were grown under environmental stress. When R. galegae strains were grown at low pH or in medium containing aluminum or salt, simulating conditions in acid soils of temperate regions and osmotic stress, respectively, the tolerance of low pH was associated with the long O-chain lipopolysaccharide and abundant acidic polysaccharide production.     

pH effects on 10 Streptomyces spp. growth and sporulation depend on nutrients

AIMS: Streptomycetes are regarded to prefer neutral to alkaline environmental pH, although they commonly occur at remarkably variable pH and nutritional conditions. Therefore, the dependence of 10 Streptomyces spp. pH tolerance on nutrients was determined. METHODS AND RESULTS: Ten environmental Streptomyces spp. were grown and sporulated between pH 4.0 and 11.5, at the interval of 1.5, on starch-casein-KNO(3), tryptone-yeast extract-glucose, glycerol-arginine and tryptone-soy agars, and three their modifications. On media with starch and casein; glucose, tryptone and yeast extract; tryptone and soy peptone; and glycerol-arginine and yeast extract strains grew over a broad pH range between 4.0-5.5 and 10.0-11.5. On glycerol-arginine and on medium with Na-propionate, NH(4)NO(3) and yeast extract, streptomycetes grew optimally at pH 7.0 and above. The high organic load enabled the growth over a wide pH range. The sporulation pH ranges followed those for growth. CONCLUSIONS: The high organic load enabled the growth over a wide pH range. The strain-specific differences in sporulation were greater than those caused by pH. The best medium for sporulation contained glucose and tryptone with minerals of glycerol-arginine agar at pH 5.5. SIGNIFICANCE AND IMPACT OF THE STUDY: The growth pH ranges, pH ranges for the optimal growth, and sporulation were strongly dependent on nutrients.