Interrelation between synthesis and uptake of ectoine for the growth of the halotolerant Brevibacterium species JCM 6894 at high osmolarity

The growth of a halotolerant Brevibacterium sp. JCM 6894 was examined in the presence of compatible solutes such as glycine betaine, ectoine (2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine) and ectoine derivatives. The effect of competition between their uptake and synthesis in the cells was subjected to osmotic shift towards the higher salinity. Among each solute examined the supplement of ectoine or hydroxyectoine exhibited a remarkable stimulation on the growth of strain JCM 6894, regardless of the range of osmotic shifts, where the largest was 0-->2 M NaCl, the intermediate was 1-->2 M NaCl, and no shift was 2-->2 M NaCl. The growth rates of this strain were dependent on the amount of ectoine taken up, which was conspicuous for the largest osmotic shift and during the first few hours of incubation after transfer. The cells subjected to 1-->2 M NaCl and 2-->2 M NaCl transfers took up less ectoine and this resulted in lower growth rates than those of cells with the largest osmotic shift (0-->2 M NaCl). The role of other compatible solutes which accumulated is discussed in relation to growth stimulation of strain JCM 6894.     

Efficient cyclic system to yield ectoine using Brevibacterium sp. JCM 6894 subjected to osmotic downshock

Brevibacterium sp. JCM 6894 cells grown in the presence of 1.5-2.5 M NaCl for 24 h at 30 degrees C were subjected to the osmotic downshock. Downshocked cells after ectoine release were grown for further 24 h in the fresh medium with same salinity as before shock. When this cyclic system was applied to the strain JCM 6894, the amount of ectoine in the cells increased with an increase of incubation time, which indicates that the cells manipulated by the present conditions were enough active to survive and synthesize ectoine after several times of osmotic downshock. In the presence of 2 M NaCl, the highest yield of ectoine released was achieved in this cyclic system, more than 2.4 g/L during 7 days of incubation. (1)H and (13)C-NMR analyses of solutes released from the cells by the osmotic downshock showed the presence of only ectoine with high purity. Release of ectoine from the cells was carried out within 5 min and its rates were increased by the dilution in the downshock treatment. For the convenience of operations, non-sterilized medium containing 2 M NaCl was examined for the cell growth in the present system, in which almost same level of ectoine yield, release rates, and cell viability were observed as those of sterilized medium.     

Acidophilic haloarchaeal strains are isolated from various solar salts

Haloarchaeal strains require high concentrations of NaCl for their growth, with optimum concentrations of 10–30%. They display a wide variety of morphology and physiology including pH range for growth. Many strains grow at neutral to slightly alkaline pH, and some only at alkaline pH. However, no strain has been reported to grow only in acidic pH conditions within the family Halobacteriaceae. In this study, we isolated many halophiles capable of growth in a 20% NaCl medium adjusted to pH 4.5 from 28 commercially available salts. They showed growth at pH 4.0 to 6.5, depending slightly on the magnesium content. The most acidophilic strain MH1-52-1 isolated from an imported solar salt (pH of saturated solution was 9.0) was non-pigmented and extremely halophilic. It was only capable of growing at pH 4.2–4.8 with an optimum at pH 4.4 in a medium with 0.1% magnesium chloride, and at pH 4.0–6.0 (optimum at pH 4.0) in a medium with 5.0% magnesium. The 16S rRNA and DNA-dependent RNA polymerase subunit B' gene sequences demonstrated clearly that the strain MH1-52-1 represents a new genus in the family Halobacteriaceae.     

Effects of sodium ions on the electrical and pH gradients across the membrane of streptococcus lactis cells

Energized cells of Streptococcus lactis conserve and transduce energy at the plasma membrane in the form of an electrochemical gradient of hydrogen ions (Δp). An increase in energy-consuming processes, such as cation transport, would be expected to result in a change in the steady state Δp. We determined the electrical gradient (ΔΨ) from the fluorescence of a membrane potential-sensitive cyanine dye, and the chemical H⁺ gradient (ΔpH) from the distribution of a weak acid. In glycolyzing cells incubated at pH 5 the addition of NaCl to 200 mM partially dissipated the Δp by decreasing ΔΨ, while the ΔpH was constant. The Δp was also determined independently from the accumulation levels of thiomethyl-β-galactoside. The Δp values decreased in cell fermenting glucose at pH 5 or pH 7 when NaCl was added, while the ΔpH values were unaffected; cells fermenting arginine at pH 7 showed similar effects. Thus, these nongrowing cells cannot fully compensate for the energy demand of cation transport.     

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.     

Intracellular Changes in Ions and Organic Solutes in Halotolerant Brevibacterium sp. Strain JCM 6894 after Exposure to Hyperosmotic Shock

In the present study we aimed to observe the intracellular responses when there was a hyperosmotic shock with a large shift in ionic strength in nutrient-rich and nutrient-poor external environments in order to clarify the availability of substrates. To do this, we used the halotolerant organism Brevibacterium sp. strain JCM 6894, which is able to grow in the presence of a wide range of salt concentrations. Hyperosmotic shock was induced by transferring cells in the late exponential phase of growth in a complex medium containing 0.5 M NaCl into either old or fresh culture medium containing 2 M NaCl. Changes in the growth rate, in the pH of the medium, and in the internal cation or organic solute concentrations in the cytosol after an upshock were analyzed as a function of incubation time. The cells exhibited very different responses to upshocks in fresh culture medium and in old culture medium; in fresh culture medium, growth was stimulated and the medium became more acidic, whereas the old culture medium repressed growth and the medium became more alkaline. The intracellular free Na⁺ concentrations remained low (80 nmol mg of protein⁻¹) after an upshock in fresh culture medium, although they quickly increased twofold in the old culture medium. In contrast, K⁺ ions immediately accumulated in the cells in fresh culture medium, whereas K⁺ ions were taken up quite slowly in old culture medium. Furthermore, the cells placed in fresh culture medium transiently accumulated alanine and glutamine in response to the upshock, but the cells placed in old culture medium did not. Growth of the Brevibacterium strain at higher levels of salinity was supported by ectoine synthesis but was not observed after the shift to high-osmolarity conditions in the old culture. In the fresh culture, however, ectoine was vigorously synthesized in cells for more than 5 h after the upshock; the concentration of ectoine in cells was more than 3,500 nmol mg of protein⁻¹ at 10 h, which corresponded to a ninefold increase compared to the concentration before the shock. These findings are consistent with the results of an analysis of the extracellular medium composition before and after the upshock.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO COPPER AND MERCURY SALTS

The susceptibility of Botrytis cinerea to copper sulphate in liquid media increased when the volume, and therefore the depth, of the medium in culture bottles exceeded certain values; when the volume was 40 ml. the maximum concentration allowing growth was 300 p.p.m.
By growing mycelium in media containing progressively higher concentrations of copper sulphate a strain was produced which grew at a concentration of 750 p.p.m.
In high concentrations of copper sulphate growth always started at the edge of the liquid, and inocula grew only if they were placed in this position.
In germination tests spores from the resistant strain were more resistant to copper sulphate than were spores of the parent strain.
The resistance of mycelium, and to a lesser extent of spores, was retained after growth of the resistant strain for six months in fungicide-free media.
Spore and mycelial inocula grew in much higher concentrations of copper sulphate when nutrient media were solidified with agar.
The strain resistant in liquid media was no more resistant than the parent strain on agar media.
The resistance of the fungus was not increased after growth for long periods on agar containing high concentrations of copper sulphate. The resistance of the strain resistant in liquid media was not lost after growth on agar media for 3 months.
Attempts to produce strains more resistant than the parent to mercuric chloride were unsuccessful.
The results obtained with phenyl-mercuric acetate were essentially similar to those obtained with copper sulphate, but relatively much more resistant strains were produced.     

Characteristics and Living Patterns of Marine Myxobacterial Isolates

The growth, morphology, and life cycle of two marine myxobacterial isolates, halotolerant Myxococcus fulvus strain HW-1 and halophilic Haliangium ochraceum strain SMP-2, were studied as models to determine the living patterns of myxobacteria in the ocean. The growth, morphology, and development of halotolerant strain HW-1 shifted in response to salinity. The optimal seawater concentration for growth of HW-1 was 0 to 80% (salinity, 0.1 to 2.9%), and the strain grew poorly in media with a salinity of more than 4%. The cells became shorter as the seawater concentration increased. The fruiting body structure was complete only on agar prepared with low concentrations of seawater or salts (less than 60% seawater; salinity, 2.1%), and rudimentary structures or even simple cell mounds appeared as the seawater concentration increased. In contrast, the halophilic strain SMP-2 was unable to grow without NaCl. The cell length and the morphology of the fruiting body-like structure did not change in response to salts. In seawater liquid medium, the cells of both strains were confirmed to be able to form myxospores directly from vegetative cells, but they could not do so in medium containing a low seawater concentration (10% or less). HW-1 cells from medium containing a high concentration of seawater grew independent of cell density, while cells from medium containing a low concentration of seawater (10% or less) showed density-dependent growth. SMP-2 cells showed density-dependent growth under all salinity conditions. The results suggest that the halotolerant myxobacteria are the result of degenerative adaptation of soil myxobacteria to the marine environment, while the halophilic myxobacteria form a different evolutionary group that is indigenous to the ocean.     

Characteristics and living patterns of marine myxobacterial isolates

The growth, morphology, and life cycle of two marine myxobacterial isolates, halotolerant Myxococcus fulvus strain HW-1 and halophilic Haliangium ochraceum strain SMP-2, were studied as models to determine the living patterns of myxobacteria in the ocean. The growth, morphology, and development of halotolerant strain HW-1 shifted in response to salinity. The optimal seawater concentration for growth of HW-1 was 0 to 80% (salinity, 0.1 to 2.9%), and the strain grew poorly in media with a salinity of more than 4%. The cells became shorter as the seawater concentration increased. The fruiting body structure was complete only on agar prepared with low concentrations of seawater or salts (less than 60% seawater; salinity, 2.1%), and rudimentary structures or even simple cell mounds appeared as the seawater concentration increased. In contrast, the halophilic strain SMP-2 was unable to grow without NaCl. The cell length and the morphology of the fruiting body-like structure did not change in response to salts. In seawater liquid medium, the cells of both strains were confirmed to be able to form myxospores directly from vegetative cells, but they could not do so in medium containing a low seawater concentration (10% or less). HW-1 cells from medium containing a high concentration of seawater grew independent of cell density, while cells from medium containing a low concentration of seawater (10% or less) showed density-dependent growth. SMP-2 cells showed density-dependent growth under all salinity conditions. The results suggest that the halotolerant myxobacteria are the result of degenerative adaptation of soil myxobacteria to the marine environment, while the halophilic myxobacteria form a different evolutionary group that is indigenous to the ocean.     

In vitro symplasmata formation in the rice diazotrophic endophyte Pantoea agglomerans YS19

Pantoea (formerly Enterobacter) agglomerans YS19 is an endophytic diazotrophic bacterium isolated from rice (Oryza sativa cv. Yuefu) grown in temperate climatic regions in west Beijing (China). The bacterium forms aggregate structures called `symplasmata'. A symplasmatum is a multicellular aggregate structure in which several (at least two) to hundreds of individual cells tightly bind together. The studies on the symplasmata formation of YS19 showed that there were two growth stages for YS19, including the single cell stage existing before exponential growth phase and the symplasmata forming stage starting at the early stationary growth phase in liquid GY (glucose yeast extract) medium or at the end of the exponential growth phase in liquid LB (Luria-Bertani) medium. There was a correlation between symplasmata formation and bacterial growth phase. When the medium was acidified, the cell growth rate was affected by the low pH of the medium, but the time required for symplasmata formation was not influenced by it. YS19 also formed symplasmata on agar medium, where more symplasmata were formed than in liquid medium. The volume of individual constitutional cells of symplasmata was sharply decreased by more than a half in comparison with that of the single cells existing before symplasmata formation. On all the media tested, YS19 formed symplasmata in most of the cell growth phases. The genome DNA/DNA homology between P. agglomerans YS19 and type strain P. agglomerans JCM1236^T (ATCC27155^T) was determined as 90.1%, confirming its membership of P. agglomerans. In order to investigate the phylogenetic relationships of YS19 at the intraspecific, intrageneric and super-generic level, the 16S rDNA similarities between strain YS19 and 17 other strains of Pantoea and 4 representatives of the closely related genera were analyzed. All the strains of Pantoea were clustered into 5 groups, and YS19 was clustered in a unique branch. The 16S rDNA similarity between YS19 and type strain JCM1236^T was 93.9%, much lower than the generally accepted value (=97%) for members of the same species, indicating that the 16S rDNA of YS19 has a distinct molecular characteristic.     

Osmotic stress leads to decreased intracellular pH of Listeria monocytogenes as determined by fluorescence ratio-imaging microscopy

Intracellular pH (pH(i)) of Listeria monocytogenes was determined after exposure to NaCl or sorbitol in liquid and solid media (agar). Both compounds decreased pH(i), and recovery on solid medium was impaired compared to that in liquid medium. N,N'-dicyclohexylcarbodiimide abolished pH(i) recovery, and lowering a(w) with glycerol showed no effect on pH(i).     

Expression of outer membrane proteins in Escherichia coli growing at acid pH

It is generally accepted for Escherichia coli that (i) the level of OmpC increases with increased osmolarity when cells are growing in neutral and alkaline media, whereas the level of OmpF decreases at high osmolarity, and that (ii) the two-component system composed of OmpR (regulator) and EnvZ (sensor) regulates porin expression. In this study, we found that OmpC was expressed at low osmolarity in medium of pH below 6 and that the expression was repressed when medium osmolarity was increased. In contrast, the expression of ompF at acidic pH was essentially the same as that at alkaline pH. Neither OmpC nor OmpF was detectable in an ompR mutant at both acid and alkaline pH values. However, OmpC and OmpF were well expressed at acid pH in a mutant envZ strain, and their expression was regulated by medium osmolarity. Thus, it appears that E. coli has a different mechanism for porin expression at acid pH. A mutant deficient in ompR grew slower than its parent strain in low-osmolarity medium at acid pH (below 5.5). The same growth diminution was observed when ompC and ompF were deleted, suggesting that both OmpF and OmpC are required for optimal growth under hypoosmosis at acid pH.     

Recovery of a marker strain of Escherichia coli from ozonated water by membrane filtration.

Selective and nonselective growth media were evaluated at two incubation temperatures, 35 and 44.5 degrees C, for the recovery of a nalidixic acid-resistant marker strain of Escherichia coli ATCC 11775 by membrane filtration from ozonated 0.05 M phosphate buffer (pH 6.9). There were significantly fewer bacteria recovered with the standard m-FC agar when compared with the same growth medium prepared without bile salts and rosolic acid. This effect was particularly noticeable at the elevated incubation temperature of 44.5 degrees C. These findings are contrary to previous work which concluded that the standard American Public Health Association membrane filtration procedure is suitable for recovery of fecal coliform indicator bacteria from ozonated wastewater.     

Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum.

Competition experiments between wild-type Azotobacter vinelandii and a mutant lacking Mo-independent nitrogenase 3 indicate that nitrogenase 3 provides an advantage during diazotrophic growth on agar media containing 100 to 500 nM Na2MoO4 but not in liquid media under the same conditions. Expression of nitrogenase 3 in wild-type cells growing on agar surfaces was verified with an anfH-lacZ fusion and by detection of nitrogenase 3 subunits. These results show that nitrogenase 3 is important for diazotrophic growth on agar medium at molybdenum concentrations that are not limiting for Mo-dependent diazotrophic growth in liquid medium.     

Minimal Medium Recovery of Thermally Injured Salmonella senftenberg 4969

Exposure of Salmonella senftenberg 4969 to sublethal heating in phosphate buffer, pH 7·0, at 52· produced thermally injured cells characterized by their relative inability to form colonies on trypticase soy yeast extract agar compared to minimal medium (M9) agar. During subsequent incubation at 37· in liquid media, more injured cells were capable of repair in M9 than in nutrient media used for pre-enrichment purposes. M9 was superior to lactose broth as a liquid holding medium to restore the ability of injured cells to grow on both rich and selective agar media. The addition of food products produced a more favourable environment for the repair of thermally injured cells in M9 rather than lactose broth. Pre-enrichment in M9 was 100 times more effective than using lactose broth as the preliminary step in the detection of S. senftenberg in laboratory pasteurized liquid egg albumen.     

Characterization of Campylobacter jejuni RacRS reveals roles in the heat shock response, motility, and maintenance of cell length homogeneity

Campylobacter jejuni commensally colonizes the cecum of birds. The RacR (reduced ability to colonize) response regulator was previously shown to be important in avian colonization. To explore the means by which RacR and its cognate sensor kinase RacS may modulate C. jejuni physiology and colonization, ΔracR and ΔracS mutations were constructed in the invasive, virulent strain 81-176, and extensive phenotypic analyses were undertaken. Both the ΔracR and ΔracS mutants exhibited a ~100-fold defect in chick colonization despite no (ΔracS) or minimal (ΔracR) growth defects at 42 °C, the avian body temperature. Each mutant was defective for colony formation at 44°C and in the presence of 0.8% NaCl, both of which are stresses associated with the heat shock response. Promoter-reporter and real-time quantitative PCR (RT-qPCR) analyses revealed that RacR activates racRS and represses dnaJ. Although disregulation of several other heat shock genes was not observed at 38°C, the ΔracR and ΔracS mutants exhibited diminished upregulation of these genes upon a rapid temperature upshift. Furthermore, the ΔracR and ΔracS mutants displayed increased length heterogeneity during exponential growth, with a high proportion of filamented bacteria. Filamented bacteria had reduced swimming speed and were defective for invasion of Caco-2 epithelial cells. Soft-agar studies also revealed that the loss of racR or racS resulted in whole-population motility defects in viscous medium. These findings reveal new roles for RacRS in C. jejuni physiology, each of which is likely important during colonization of the avian host.     

Induction of Cell Differentiation of Isolated Cells in Dictyostelium discoideum by Low Extracellular pH

In Dictyostelium discoideum , the formation of multicellular masses is necessary for cell differentiation. However, the present study shows that amoebae of strain V12M2 efficiently differentiate to prespore or stalk cells under submerged incubation in a simple medium containing cAMP and salts without cell contact, only if the pH of the medium is maintained at acidic values; differentiation scarcely occurs in the neutral pH range. The optimum pH values for prespore and stalk cell differentiation are 5.1 and 4.5, respectively. In addition to the extracellular pH, Mg ions and the concentration of cAMP also affect the choice of the differentiation pathway. The time courses of differentiation of both cell types under optimum conditions are also presented.     

Stimulation of Mycelial Growth of Endothia parasitica by Heavy Metals

Of 16 metal cations tested on agar medium, only copper and iron stimulated mycelial growth of Endothia parasitica in relatively high concentrations. Similarly enhanced growth was produced in high (32%) glucose concentrations and also when the fungus was grown on cellophane placed over the agar surface. E. parasitica secreted large amounts of oxalate that precipitated primarily as calcium oxalate at the periphery of the fungal colony, causing an opaque halo in the medium. Mycelial growth was retarded greatly when calcium oxalate accumulated, but retardation was reversed by copper and iron salts that prevented accumulation of the calcium oxalate crystals. E. parasitica grew well on media containing copper oxalate and copper-calcium oxalate but grew poorly with calcium oxalate as the carbon source and was inhibited by sodium oxalate in the medium. The specificity by which only copper and iron salts stimulated mycelial growth suggested that the metal and oxalate ions interact to form specific oxalate complexes that reverse the inhibition of simple oxalate salts. This probably accounts for enhanced growth in the presence of otherwise toxic levels of metals and oxalate. The stimulation did not occur in liquid cultures.     

Sublethal heat stress of Vibrio parahaemolyticus.

When Vibrio parahaemolyticsu ATCC 17802 was heated at 41 degrees C for 30 min in 100 mM phosphate-3% NaCl buffer (pH 7.0), the plate counts obtained when using Trypticase soy agar containing 0.25% added NaCl (0.25 TSAS) were nearly 99.9% higher than plate counts using Trypticase soy agar containing 5.5% added NaCl (5.5 TSAS). A similar result was obtained when cells of V. parahaemolyticus were grown in a glucose salts medium (GSM) and heated at 45 degrees C. The injured cells recovered salt tolerance within 3 h when placed in either 2.5 TSBS or GSM at 30 degrees C. The addition of chloramphenicol, actinomycin D, or nalidixic acid to 2.5 TSBS during recovery of cells grown in 2.5 TSBS indicated that recovery was dependent upon protein, ribonucleic acid (RNA, and deoxyribonucleic acid (DNA) synthesis. Penicillin did not inhibit the recovery process. Heat-injured, GSM-grown cells required RNA synthesis but not DNA synthesis during recovery in GSM. Chemical analyses showed that total cellular RNA decreased and total cellular DNA remained constant during heat injury. The addition of [6-3H]uracil, L-[U-14C]leucine, and [methyl-3H]thymidine to the recovery media confirmed the results of the antibiotic experiments.     

Expression of Outer Membrane Proteins in Escherichia coli Growing at Acid pH

It is generally accepted for Escherichia coli that (i) the level of OmpC increases with increased osmolarity when cells are growing in neutral and alkaline media, whereas the level of OmpF decreases at high osmolarity, and that (ii) the two-component system composed of OmpR (regulator) and EnvZ (sensor) regulates porin expression. In this study, we found that OmpC was expressed at low osmolarity in medium of pH below 6 and that the expression was repressed when medium osmolarity was increased. In contrast, the expression of ompF at acidic pH was essentially the same as that at alkaline pH. Neither OmpC nor OmpF was detectable in an ompR mutant at both acid and alkaline pH values. However, OmpC and OmpF were well expressed at acid pH in a mutant envZ strain, and their expression was regulated by medium osmolarity. Thus, it appears that E. coli has a different mechanism for porin expression at acid pH. A mutant deficient in ompR grew slower than its parent strain in low-osmolarity medium at acid pH (below 5.5). The same growth diminution was observed when ompC and ompF were deleted, suggesting that both OmpF and OmpC are required for optimal growth under hypoosmosis at acid pH.