Physiology and ecology of bacteriophages of the marine bacterium Beneckea natriegens: salinity.

The effects of variation in ionic levels on the stability and replication of two bacteriophages (nt-1 and nt-6) host specific for the marine bacterium Beneckea natriegens were examined. Monovalent cations influenced the adsorption of the nt-1 but not the nt-6 phage; however, one-step growth studies showed that NaCl was required for replication of both phage. The NaCl optimum for nt-1 production was 0.25 M NaCl, the same as the growth optimum for B. natriegens. However, the optimum for nt-6 production was 0.16 M NaCl. These NaCl optima for host and phage are at estuarine rather than oceanic levels. The nt-1 phage was better suited to replicate at NaCl levels typical of higher salinity areas (18-35%) and the nt-6 phage was better suited to replicate at lower salinities (5-18%). The nt phage were more resistant to low NaCl levels than their host bacterium and appeared limited to marine waters by the lower survival salinity of B. natriegens coupled with phage inactivation processes occurring in natural estuarine waters.     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

Lipolytic activity from Halobacteria: screening and hydrolase production

Strains of Halobacteria from an Algerian culture collection were screened for their lipolytic activity against p-nitrophenyl butyrate (PNPB) and p-nitrophenyl palmitate (PNPP). Most strains were active on both esters and 12% hydrolyzed olive oil. A strain identified as Natronococcus sp. was further studied. It grew optimally at 3.5 M NaCl, pH 8 and 40 degrees C. An increase in temperature shifted the optimum salt concentration range for growth from a wider range of 2-4 M, obtained at 25-30 degrees C, to a narrower range of 3.5-4 M, obtained at 35-40 degrees C. At 45 degrees C the optimum salt concentration was 2 M. These results show a clear correlation between salt and temperature requirement. The optimum conditions for the production of hydrolytic activity during growth were: 3.5 M NaCl and pH 8 for PNPB hydrolytic activity and 4 M NaCl and pH 7.5 for PNPP hydrolytic activity; both at 40 degrees C. The clear supernatant of cells grown at 4 M NaCl showed olive oil hydrolysis activity (in presence of 4 M NaCl) demonstrating the occurrence of a lipase activity in this strain. To our knowledge, this is the first report of a lipase activity at such high salt concentration.     

Interactive effects of salt concentration and temperature on growth and lipid composition in the moderately halophilic bacterium Vibrio costicola.

The interactive effects of NaCl concentration and growth temperature on the growth and lipid composition of the moderately halophilic eubacterium Vibrio costicola have been investigated. Vibrio costicola was shown to be capable of growth over the temperature range 4-37 degrees C. Maximum growth yields were obtained at 30 degrees C when the optimum NaCl concentration was 1.0 M NaCl. In contrast with some previous studies, at higher or lower growth temperatures both the optimum and lower limit of NaCl concentration were higher, but there was no change in the upper limit of NaCl concentration for growth. There were no differences between the lipid compositions of cultures grown in 1 M NaCl at 30 or 37 degrees C, but as the growth temperature was lowered from 30 to 10 or 4 degrees C, the ratio of phosphatidylethanolamine to phosphatidylglycerol increased significantly as a result of the conversion of phosphatidylglycerol to diphosphatidylglycerol; in addition, at the lower growth temperatures the phospholipid fatty acyl composition became more unsaturated and the mean acyl chain length was shorter. It is suggested that the altered salt dependence of V. costicola at temperatures below the optimum for growth is due to a modification in membrane lipid phase behavior and stability brought about by changes in lipid composition, whereas a different mechanism operates above the growth temperature optimum.     

The role of osmotic effects in haloadaptation of Vibrio costicola

Growth rates of Vibrio costicola showed a broad optimum between 0.8 and 1.5 M-NaCl, and there was no growth above 3.3 M-NaCl in a peptone-based medium. The minimum requirement of 0.5 M-NaCl for growth in NaCl alone was reduced to 0.3 M-NaCl when the total solute concentration was raised to 0.5 to 1.0 M equivalent with sucrose or glycerol. Compared with equivalent NaCl concentrations, higher concentrations of sucrose were more inhibitory to growth, whereas glycerol had less effect. Increasing the medium NaCl concentration suddenly by 2- or 3-fold with either a constant starting, or final, salt concentration showed that, after the shift-up, the lag in growth, the rate of growth, and the inhibition of phospholipid synthesis depended both on the final NaCl concentration and the magnitude of the shift in salinity. The time-courses of phospholipid synthesis following a 2- or 3-fold shift-up in NaCl or sucrose media were very similar and exhibited a relative increase in phosphatidylglycerol synthesis over that of phosphatidylethanolamine. This 'switch-over' was not seen following shift-up in glycerol media when there was also a stimulation, rather than inhibition, of phospholipid synthesis. It is concluded that during phenotypic haloadaptation of V. costicola, osmotic effects play a significant part in the sensing of and response to raised external salinity.     

Amphibacillus fermentum sp. nov., Amphibacillus tropicus sp. nov.--new alkaliphilic, facultatively anaerobic, saccharolytic Bacilli from Lake Magadi]

New alkaliphilic, saccharolytic, rod-shaped, gram-positive bacteria resistant to heating and drying and phylogenetically affiliated to the Bacillus lineage were isolated under strictly anaerobic conditions from sediments of the alkaline and highly mineralized Lake Magadi. Strain Z-7792 forms endospores; in strain Z-7984, endospore formation was not revealed. The strains are capable of both anaerobic growth (at the expense of fermentation of glucose and certain mono- and disaccharides with the formation of formate, ethanol, and acetate) and aerobic growth. Among polysaccharides, the strains hydrolyze starch, glycogen, and xylan. Yeast extract or methionine are required for growth. The strains are strict alkaliphiles exhibiting obligate requirement for Na+ and carbonate ions but not for Cl- ion. Growth occurs at a total mineralization as high as 3.3-3.6 M Na+, with an optimum at 1-1.7 M Na+. Strain Z-7792 is an obligate alkaliphile with a pH growth range of 8.5-11.5 and an optimum of 9.5-9.7. Strain Z-7984 grows in a pH range of 7.0-10.5 with an optimum at 8.0-9.5. Both strains are mesophiles having a growth optimum at 37-38 degrees C. They belong to bacilli with a low G + C content. The G + C contents of the DNA of strains Z-7792 and Z-7984 are 39.2 and 41.5 mol%, respectively. These isolates of facultatively anaerobic, strictly alkaliphilic, Na(+)-dependent bacilli can be considered representatives of the ecological group adapted to the life at drying-up shoars of soda lakes. Because of their independence of NaCl and lack of obligate dependence on sodium carbonates, the isolates are to be assigned to athalassophilic organisms. According to their physiological and phylogenetic characteristics, they taxonomically belong to group 1 of the species of bacilli, occupying a position intermediate between the genera Amphibacillus and Gracilibacillus. The isolates are described as new species of Amphibacillus: A. fermentum (type strain, Z-7984T) and A. tropicus (type strain, Z-7792T).     

Characterization of a new marine methylotroph

Abstract A methanol-oxidizing bacterium from a marine environment has been isolated and characterized. The bacterium was a Gram-negative rod, capable of growth on methanol and methylamine, but not on multicarbon compounds. It showed a temperature optimum of 30°C, a salt optimum of 0.4% (w/v) and the mol % G + C of its DNA was 46%. Carbon was assimilated via the ribulose monophosphate pathway for formaldehyde fixation during growth on methanol. This bacterium superficially resembled other obligate methylotrophs requiring NaCl reported previously which were designated Methylomonas thalassica . It also appeared similar to many strains of obligate freshwater methylotrophs, except for its NaCl requirement and its lower mol % G + C.     

Nutrition and growth of the moderately halophilic bacteria Micrococcus morrhuae K-17 and Micrococcus luteus K-15

Chemically defined media have been developed for the growth of two moderately halophilic bacteria, Micrococcus morrhuae K-17 and Micrococcus luteus K-15. M. morrhuae K-17 grows well in a synthetic medium (SM-1) which contains a number of salts, 0.21 M KCl, 2 M NaCl, D-mannose, five vitamins and ten amino acids. The synthetic medium (SM-2) for M. luteus K-15 contains a number of salts, 0.21 M KCl, 1 M NaCl, D-fructose, nine vitamins and nine amino acids. Nutritional studies show that M. morrhuae K-17 can utilize a large number of organic compounds as carbon and energy source while the ability of M. luteus K-15 in utilizing the organic compounds is rather limited. The minimum salt requirement is 0.5 M NaCl for both strains when growth at the optimum temperature of 30 degrees C. However, this requirement can be lowered to 0.2 M in M. luteus K-15 when grown at a lower temperature of 25 degrees C. It is concluded that the ability to grow in a wider range of salt concentrations in response to temperature is species specific in moderate halophiles. The salt range for growth to occur can be extended when cells of both strains are grown in complex medium which might provide the amino acids and growth factors that cannot be synthesized by these strains at high salt concentrations.     

嗜盐菌Haloferax mediterranei R4胞外淀粉酶的研究

HaloferaxmediterraneiR4是从地中海分离得到的一株极端嗜盐古生菌,属于富盐菌属(Haloferax)。该菌在适当条件下可以产胞外淀粉酶,此酶需要高浓度NaCl维持其活性及稳定性,在NaCl浓度为2～5mol·L-1时维持较高活性。在NaCl浓度为3mol·L-1时该酶的最适反应pH为6.0～7.0,最适反应温度为50℃,且最适反应温度与NaCl浓度有一定的相关性。Ca2+对酶反应活性有影响。用KCl代替NaCl,该酶可以保留约50%活性,而用Na2SO4代替NaCl则完全失活。     

Isolation of sodium chloride tolerant cell lines and plants in finger millet

Sodium chloride tolerant cell lines of finger millet were isolated from embryogenic cultures growing on MS medium supplemented with picloram (2 mg I^-1), kinetin (0.1 mg l^-1) and sodium chloride (1 %) at the end of 6 passages. The sodium chloride tolerant cell lines showed better growth in comparison with control at all concentrations of sodium chloride tested, with optimum growth at 0.25 % NaCl. When the tolerant lines were grown for 3 passages in absence of NaCl, the growth was lower than that of the tolerant lines tested immediately at the end of 6 passages of selection. NaCl tolerant calli had more Na¹ in comparison with control and they regenerated plants in presence of 1 % NaCl, while the control lines failed to differentiate. When screened in a hydroponics system with 1 % NaCl, the tolerant plants grew to maturity while the control plants failed to grow.     

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.     

Auxin is a positive regulator for ethylene-mediated response in the growth of Arabidopsis roots

The requirement of auxin for the ethylene-mediated growth response in the root of Arabidopsis thaliana seedlings was investigated using two ethylene-resistant mutants, aux1-7 and eir1-1, whose roots have been shown to have a defect in the auxin influx and efflux carriers, respectively. A 50% inhibition of growth (I(50)) was achieved with 0.84 microl liter(-1) ethylene in wild-type roots, but 71.3 microl liter( -1) ethylene was required to induce I(50) in eir1-1 roots. In aux1-7 roots, I(50) was not obtained even at 1,000 microl liter(-1) ethylene. By contrast, in the presence of 10 nM 1-naphthaleneacetic acid (NAA), the concentrations of ethylene required to induce I(50) in eir1-1 and aux1-7 roots were greatly reduced nearly to the level required in wild-type roots. Since the action of NAA to restore the ethylene response in aux1-7 roots was not replaced by IAA, an increase in the intracellular level of auxin is likely to be the cause for the restoration of ethylene response. NAA at 10 nM did not inhibit root growth when applied solely, but it was the optimum concentration to recover the ethylene response in the mutant roots. These results suggest that auxin is a positive regulator for ethylene-induced inhibition in root elongation.     

The effect of incubation temperature and sodium chloride concentration on the growth kinetics of Vibrio anguillarum and Vibrio anguillarum-related organisms

The effect of temperature and NaCl concentration on the growth kinetics of Vibrio anguillarum and V. anguillarum -related (VAR) strains was studied.
For one wild VAR strain, NaCl concentration interfered with growth temperature parameters, in particular, with the maximum growth temperature but also with the optimum temperature (defined as the temperature at which μ_max equals its maximal value μ_opt), and with μ_opt itself. For the same strain, optimal growth required the adding of NaCl to the medium to a final concentration of 1·5%. These results were not confirmed by tests on a V. anguillarum collection strain.
When the NaCl concentration in the culture media was 1.5%, the optmum temperature for the nine strains studied ranged from 29.7°C to 34°C whereas the maximum temperature ranged between 35.3°C and 38.5°C.
Hence, antbiotic susceptibility testing as well as biochemical identification might be carried out at 30°C in the presence of 1.5% NaCl, which corresponded to a suboptimal growth.     

Physiological Characteristics of Mycosphaerella ascophylli, a Fungal Endophyte of the Marine Brown Alga Ascophyllum nodosum

Mycosphaerella ascophylli Cotton, a fungal endosymbiont in the thallus of the marine brown alga, Ascophyllum nodosum (L.) Le Jol., was brought into axenic culture. Most of its nutritional requirements proved to be similar to those characteristic of marine saprophytic fungi: growth optimum at pH 7 to 8, good growth only with a high concentration of NaCl, ability to utilize nitrate as a nitrogen source and glucose, galactose and mannose as carbon sources. The fungus was more specifically characterized by a low temperature optimum (20°C, or lower), a requirement for both thiamin and biotin, and inability to metabolize xylose and fructose.     

A thermophilic amyloglucosidase fromHalobacterium sodomense,a halophilic bacterium from the Dead Sea

Halobacterium sodomense, a halophilic bacterium from the Dead Sea, degraded starch to glucose by means of an extracellular amyloglucosidase with a temperature optimum of around 65°C in the presence of 1.4 M NaCl, and around 75°C in the presence of 3.9 M NaCl. The enzyme required salt concentrations higher than 1 M for optimal activity, NaCl, KCl, and MgCl₂ being equally suitable as activators. The optimum pH was 7.5.H. sodomense culture supernatants showed only a very low maltose degrading activity. H. sodomense excreted amyloglucosidase constitutively, and relatively high activities were found in cultures grown in the absence of starch; when glucose was added to the growth medium, the amount of enzyme excreted into the medium decreased.     

Expression of luminescence in Photobacterium phosphoreum: Na+ regulation of in vivo luminescence appearance

In Photobacterium phosphoreum strain 496, growth and luminescence in a complex medium are optimal with 3% NaCl. However, in the same medium with 1% NaCl growth is similar, but the development of bioluminescence does not occur. In cells grown to mid or late-log phase in 1% NaCl, light emission can be triggered by the addition of NaCl, but the time required for its appearance is quite long, at least 30–45 min. The synthesis of m-RNA and protein are required for the development of luminescence, but the long time interval suggests that some intermediate steps are required. The time required is not less in conditioned 3% NaCl medium.     

Optimization of culture conditions for the production of haloalkaliphilic thermostable protease from an extremely halophilic archaeon Halogeometricum sp. TSS101

AIMS: Isolation and screening of extreme halophilic archaeon producing extracellular haloalkaliphilic protease and optimization of culture conditions for its maximum production. METHODS AND RESULTS: Halogeometricum sp. TSS101 was isolated from salt samples and screened for the secretion of protease on gelatin and casein plates containing 20% NaCl. The archaeon was grown aerobically in a 250 ml flask containing 50 ml of (w/v) NaCl 20%; MgCl(2) 1%; KCl 0.5%; trisodium citrate 0.3%; and peptone 1%; pH 7.2 at 40 degrees C on rotary shaker. The production of enzyme was investigated at various pH, temperatures, NaCl concentrations, metal ions and different carbon and nitrogen sources. The partially purified protease had activity in a broad pH range (7.0-10.0) with optimum activity at pH 10.0 and a temperature (60 degrees C). The enzyme was thermostable and retained 70% initial activity at 80 degrees C. Maximum protease production occurred at 40 degrees C in a medium containing 20% NaCl (w/v) and 1% skim milk powder after 84 h in shaking culture. Enzyme secretion was observed at a broad pH range of 7.0-10.0. Addition of CaCl(2) (200 mmol) to the culture medium enhanced the production of protease. Protein rich flours proved to be cheap and good alternative source for enzyme production. Different osmolytes were tested for the growth and production of haloalkaliphilc protease and found that betaine and glycerol enhanced growth without secretion of the protease. Immobilization studies showed that whole cells immobilized in 2% alginate beads were stable up to 10 batches and able to secrete the protease, which attained maximum production within 60 h under shaking conditions. CONCLUSIONS: Halogeometricum sp. TSS101 secreted an extracellular haloalkaliphilic and thermostable protease. The optimum conditions required for maximum production are 20% NaCl, 1% skim milk powder and temperature at 40 degrees C. Addition of CaCl(2) (200 mmol) enhanced the enzyme production. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of haloalkaliphilic protease. SIGNIFICANCE AND IMPACT OF THE STudy: The low cost protein rich flours were used as an alternative carbon and nitrogen sources for enzyme production. Immobilization of halophilic cells in alginate beads can be used in continuous production of halophilic enzyme. The halophilic and thermostable protease from Halogeometricum sp. TSS101 is good source for industrial applications and can be a suitable source for preparation of fish sauce.     

Effect of growth conditions on the motility of<Emphasis Type="Italic"> Photorhabdus temperata</Emphasis>

Photorhabdus temperata is a bioluminescent bacterium that lives in mutualistic association with entomopathogenic nematodes of the genus Heterorhabditis. The bacterium exists in two morphologically distinguishable phases (primary and secondary). The swimming behavior of P. temperata was investigated. Both the primary and secondary variants were able to swim in liquid or semisolid media under appropriate conditions. Variation in the oxygen levels had little affect on the chemotaxis and motility of the primary form, but greatly influenced the behavior of the secondary form. Under oxic conditions the secondary form was nonmotile, but motility was induced under anoxic conditions. Several phenotypic traits of the primary form were not expressed under anoxic conditions. The constituents of the growth media affected the motility of both variants. P. temperata required additional NaCl or KCl for optimum motility and chemotaxis. Optimal chemotactic behavior required the presence of bacto-peptone and yeast extract in the swim-migration medium. A mutant that was isolated from the secondary form was able to swim under oxic conditions and possessed an altered salt requirement for motility.     

Optimum bacteriocin production by Lactobacillus plantarum 17.2b requires absence of NaCl and apparently follows a mixed metabolite kinetics

Bacteriocins from lactic acid bacteria are ribosomally synthesized anti-microbial compounds that may find applications from food preservation to healthcare. Food preservation, using in situ bacteriocin production is the most obvious and simple. Frequently, the best conditions for bacteriocin production are those prevailing during food fermentation but a better understanding of the relationship between growth and bacteriocin production is required. In this work, we evaluate the effects of some environmental factors on bacteriocin production by Lactobacillus plantarum 17.2b. A first screening design showed that NaCl, temperature, pH and the type and concentration of carbon and nitrogen sources were most influents. A moderate stimulatory effect of ethanol and oleuropein was also registered. Two consecutive central composite designs were used to examine the effect of the selected variables and to compute its optimum. The evolution of changes produced by the alterations in environmental factors was further examined trough perturbation plots. Bacteriocin production by L. plantarum 17.2b was very sensitive to environmental conditions and uncoupled from growth. Maximum production required suboptimal growth temperatures, pH values above growth's optimum and no NaCl. A preliminary approach to kinetics showed that bacteriocin production by this strain apparently follows mixed metabolite kinetics.     

Attachment and growth of mammalian cells on microcarriers with different ion exchange capacities

In the design of microcarriers for animal cell growth, the exchange capacity has been considered a critical factor. However, charge densities of microcarriers under culture conditions are not the same as the exchange capacities. Furthermore, the charge density requirement for optimum attachment is not necessarily the same as that required for optimum growth. We demonstrate that charge is not the sole factor affecting the attachment and growth of animal cells on microcarriers. We also show that supplemental serum in the growth medium has a negative effect on cell attachment to microcarriers.