Mitigation of Hyper KCl Stress at 42ºC with Externally Existing Sodium Glutamate to a Halotolerant Brevibacterium sp. JCM 6894.

Halotolerant Brevibacterium sp. JCM 6894 grew at 37ºC in the presence of 2.3 M KCl, while the growth was repressed with the same concentration of NaCl. When resting cells, 107.4 ± 0.1 (CFU·mL-1), prepared from cells grown in the absence of salts at 30ºC, were exposed to 3.3 M NaCl for 36 h at 42ºC, reduction of the number of resting cells was maintained within a 1-log cycle in the presence of proline, betaine, or ectoine (50 mM). In the presence of 3.3 M KCl, the most functional osmoprotectant was sodium glutamate (50 mM), and the value was 107.2 ± 0.1 (CFU·mL-1) when exposed for 72 h at 42ºC. In the absence of osmoprotectants, the value was reduced to four orders of magnitude in each experimental condition. The number of resting cells, 106.8 ± 0.1 (CFU·mL-1), prepared from grown cells pre-adapted to 2.3 M KCl at 37ºC, was hardly reduced when exposed to 3.3 M KCl in the presence of sodium glutamate more than 50 mM for 72 h at 42ºC. Those results indicate that the isolate can sense the difference in hyper KCl stress as opposed to hyper NaCl stress, and different kinds of osmoadaptation systems can function to cope with each hyper salt stress.

Changes in the Survivability of Marine Vibrio sp. under Hyper KCl Stress in the Presence of Betaine as Well as with Exposure to 37ºC.

Survivability at hyper KCl stress was examined at 30ºC and 37ºC in the presence and absence of an osmoprotectant by using resting cells prepared from marine Vibrio sp. grown at early stationary phase. Survivability was decided by counting colonies. The number of initial cells, 107.1 ± 0.2 (CFU·mL-1), was reduced to 105.1 ± 0.5 and < 101.0 (CFU·mL-1) at 30ºC and 37ºC, respectively, by the exposure of resting cells, that were prepared from cells grown for 8 h at 0.5 M NaCl at 30ºC, to 1.2 M KCl and 50 mM NaCl for 3 h. Betaine externally existed as a final concentration of 50 mM mitigated hyper KCl stress to the resting cells at 37ºC. The number of surviving cells was maintained 104.9 ± 0.3 (CFU·mL-1) when resting cells, 106.5 ± 0.1 (CFU·mL-1), that were prepared from pre-adapted cells to relatively high concentration of KCl in the growth for 10 h at 0.8 M KCl and 50 mM NaCl at 37ºC, were exposed to 1.2 M KCl, 50 mM NaCl, and 50 mM betaine at 37ºC for 3 h. The results indicate that osmoadaptation system(s) in resting cells is temperature sensitive and betaine functions to mitigate hyper KCl stress to the resting cells at 37ºC.

Environmental Impact of Tributyltin-Resistant Marine Bacteria in the Indigenous Microbial Population of Tributyltin-Polluted Surface Sediments.

　We compared the TBT-resistant ability of resting cells prepared from isolates that formed colonies on nutrient agar plates containing 100 µM tributyltin (TBT) chloride, such as Photobacterium sp. TKY1, Halomonas sp. TKY2, and Photobacterium sp. NGY1, with those from taxonomically similar type strains. Photobacterium sp. TKY1 showed the highest ability among those three isolates. The number of surviving Photobacterium sp. TKY1 cells was hardly decreased after 1 h of exposure to 100 µM TBTCl, regardless of the number of resting cells in the range from 109.4 to 104.2 CFU mL-1. In such an experimental condition, the maximum number of TBT molecules available to associate with a single cell was estimated to be approximately 6.0 x 1011.8. Resting cells prepared from type strains Photobacterium ganghwense JCM 12487T and P. halotolerans LMG 22194T, which have 16S rDNA sequences highly homologous with those of Photobacterium sp. TKY1, showed sensitivity to TBT, indicating that TBT-resistant marine bacterial species are not closely related in spite of their taxonomic similarity. We also estimated the impact of TBT-resistant bacterial species to indigenous microbial populations of TBT-polluted surface sediments. The number of surviving TBT-sensitive Vibrio natriegens ATCC 14048T cells, 106.2±0.3 CFU mL-1, was reduced to 104.4±0.4 CFU mL-1 when TBT-resistant Photobacterium sp. TKY1 cells, 109.1±0.2 CFU mL-1, coexisted with 109.4±0.2 CFU mL-1 of V. natriegens ATCC 14048T cells in the presence of 100 µM TBTCl. These results indicate that the toxicity of TBT to TBT-sensitive marine bacterial populations might be enhanced when a TBT-resistant marine bacterial species inhabits TBT-polluted surface sediments.

Growth enhancement of the halotolerant Brevibacterium sp. JCM 6894 by methionine externally added to a chemically defined medium.

We examined amino acid requirements for the growth of the halotolerant Brevibacterium sp. JCM 6894 in the absence and presence of 1.2 M NaCl in a chemically defined medium. The experiment was also carried out in the presence of 1.2 M KCl. As a result, growth was highly enhanced by methionine in the absence and presence of KCl as well as NaCl up to 1.2 M. However, growth in the presence of 150 mM methionine was repressed by leucine (up to 100 mM）and valine (up to 100 mM). Concentration-dependent growth inhibition was observed in the presence of isoleucine (up to 150 mM) and threonine (up to 300 mM). When the cells were incubated in the absence of externally added K+, growth was strongly repressed, even in the presence of 150 mM methionine. The growth, however, recovered drastically by the addition of 1 mM KCl, regardless of the presence and absence of 1.2 M NaCl. These results indicate that methionine, which seems to be symported into cytoplasm with K+, plays an important role in the growth of the strain under salt stress.

Adsorption of tributyltin by tributyltin resistant marine Pseudoalteromonas sp. cells.

The isolate, Pesudoalteromonas sp. TBT1, could grow to overcome the toxicity of tributyltin chloride (TBTCl) up to 30 microM in the absence of Cl(-) in the medium until the cells reached an exponential phase of growth. The viability, however, was reduced after the cells reached a stationary phase. The degradation products, such as dibutyltin (DBT) and monobutyltin (MBT), were not detected in the growth medium, indicating that the isolate has no ability to degrade TBT into less toxic DBT and MBT. Up to about 10(7.5) TBT molecules were adsorbed by a single cell. The observation of morphological changes with an electron microscope showed that the cell surface became wrinkled after exposure to the lethal concentration of 10 mM TBTCl. These results indicate that the resistance of the isolate toward the toxicity of TBTCl is not related to the unique cell surface, which seems to play an important role in preventing the diffusion of TBTCl into the cytoplasm.

Accelerator analysis of tributyltin adsorbed onto the surface of a tributyltin resistant marine Pseudoalteromonas sp. cell.

Tributyltin (TBT) released into seawater from ship hulls is a stable marine pollutant and obviously remains in marine environments. We isolated a TBT resistant marine Pseudoalteromonas sp. TBT1 from sediment of a ship's ballast water. The isolate (10(9.3 +/- 0.2) colony-forming units mL(-1)) adsorbed TBT in proportion to the concentrations of TBTCl externally added up to 3 mM, where the number of TBT adsorbed by a single cell was estimated to be 10(8.2). The value was reduced to about one-fifth when the lysozyme-treated cells were used. The surface of ethanol treated cells became rough, but the capacity of TBT adsorption was the same as that for native cells. These results indicate that the function of the cell surface, rather than that structure, plays an important role to the adsorption of TBT. The adsorption state of TBT seems to be multi-layer when the number of more than 10(6.8) TBT molecules is adsorbed by a single cell.

Lethality of shock pressures to a marine Vibrio sp. isolated from a ship's ballast water.

The lethal effects of shock pressure treatment on suspended Vibrio sp. cells were examined. Lethality of shock pressures to the Vibrio sp. cells increased with the increase in the values of maximum shock pressures generated in the cell suspension. When the value was around 114 MPa, the total number of colony-forming cells was reduced from 10(8.5+/-0.1) colony-forming units (CFU) to 10(3.3) - 10(3.4) CFU/ml, and complete loss of colony-forming ability was seen at the maximum value of 282 MPa. Almost all the cells could survive after the exposure to shock pressures including the maximum value of around 189 MPa in the presence of 2% sodium ascorbate (VitC-Na), whereas the total number of colony-forming cells was reduced to 10(1.6)-10(2.1) CFU/ml in the absence of VitC-Na. The surviving cells, however, showed sensitivity to 0.8% sodium cholate, a strong detergent. About 11% of cell-associated proteins had leaked out when the cells were exposed to lethal shock pressures including the maximum value of around 290 MPa in the absence of VitC-Na. These results indicate that the radicals generated in the cell suspension may be closely related to the loss of colony-forming ability of the Vibrio sp. cells. Damage to the outer membrane structure also seems to have occurred by the exposure to shock pressures.

Changes of microbial populations in a ship's ballast water and sediments on a voyage from Japan to Qatar.

Colony-forming eutrophic marine microorganisms in ballast water were counted in samples taken on board in 2002 and 2003. In the ballast water in Japan, viable cell numbers were highly variable but not by more than 10(5.1) colony-forming units (CFU)ml(-1) regardless of season. Even when ballast water was discharged offshore, values varied but not by more than 10(5.0) CFUml(-1). The effectiveness of the ballast water exchange was unconfirmed, except for the February 2003 voyage. No microbial colonies were counted in the reloaded ballast water in the high seas on that voyage, which contributed to the reduction of the total number of viable cells sampled in the discharged ballast water at the Ras Laffan port in Qatar. In sediment samples, the values of 10(5.2) - 10(6.0) CFUml(-1) were estimated for all seasons in which voyages took place. The maximum of the marine Vibrio species, 110 CFUml(-1), was observed in the ballast water sample taken in July 2003. The estimated total viable cell numbers in sediments were higher than those counted in the ballast water throughout the experiments, indicating the importance of sediment management as well as ballast water management on vessels traveling from Japan.