Extracellular polysaccharides of Rhodococcus rhodochrous S-2 stimulate the degradation of aromatic components in crude oil by indigenous marine bacteria

Rhodococcus rhodochrous S-2 produces extracellular polysaccharides (S-2 EPS) containing D-glucose, D-galactose, D-mannose, D-glucuronic acid, and lipids, which is important to the tolerance of this strain to an aromatic fraction of (AF) Arabian light crude oil (N. Iwabuchi, N. Sunairi, H. Anzai, M. Nakajima, and S. Harayama, Appl. Environ. Microbiol. 66:5073-5077, 2000). In the present study, we examined the effects of S-2 EPS on the growth of indigenous marine bacteria on AF. Indigenous bacteria did not grow significantly in seawater containing AF even when nitrogen, phosphorus, and iron nutrients were supplemented. The addition of S-2 EPS to seawater containing nutrients and AF resulted in the emulsification of AF, promotion of the growth of indigenous bacteria, and enhancement of the degradation of AF by the bacteria. PCR-denaturing gradient gel electrophoresis analyses show that addition of S-2 EPS to the seawater containing nutrients and AF changed the composition of the bacterial populations in the seawater and that bacteria closely related to the genus Cycloclasticus became the major population. These results suggest that Cycloclasticus was responsible for the degradation of hydrocarbons in AF. The effects of 15 synthetic surfactants on the degradation of AF by indigenous marine bacteria were also examined, but enhancement of the degradation of AF was not significant. S-2 EPS was hence the most effective of the surfactants tested in promoting the biodegradation of AF and may thus be an attractive agent to use in the bioremediation of oil-contaminated marine environments.     

The systematic substitutions around the conserved charged residues of the cytoplasmic loop of Na+-driven flagellar motor component PomA

PomA, a homolog of MotA in the H+-driven flagellar motor, is an essential component for torque generation in the Na+-driven flagellar motor. Previous studies suggested that two charged residues, R90 and E98, which are in the single cytoplasmic loop of MotA, are directly involved in this process. These residues are conserved in PomA of Vibrio alginolyticus as R88 and E96, respectively. To explore the role of these charged residues in the Na+-driven motor, we replaced them with other amino acids. However, unlike in the H+-driven motor, both of the single and the double PomA mutants were functional. Several other positively and negatively charged residues near R88 and E96, namely K89, E97 and E99, were neutralized. Motility was retained in a strain producing the R88A/K89A/E96Q/E97Q/E99Q (AAQQQ) PomA protein. The swimming speed of the AAQQQ strain was as fast as that of the wild-type PomA strain, but the direction of motor rotation was abnormally counterclockwise-biased. We could, however, isolate non-motile or poorly motile mutants when certain charged residues in PomA were reversed or neutralized. The charged residues at positions 88-99 of PomA may not be essential for torque generation in the Na+-driven motor and might play a role in motor function different from that of the equivalent residues of the H+-driven motor.     

Prevention by polyhydroxysteroids and saponins of Asterina pectinifera of the desquamation of stratum corneum cells

Two polyhydroxysteroids and three steroidal saponins (pectiniosides A, B and C) were isolated as bioactive substances from Asterina pectinifera. These compounds inhibited the release of guinea-pig skin stratum corneum cells by 48-67% (control, 0%) at 2 mg/ml. It is suggested that the bioactivity of these compounds is connected with the remedial and preventive effects of an aqueous extract of A. pectinifera on rough human skin.     

Determination of the absolute configuration of (+)-2,7(14),10-bisabolatrien-1-ol-4-one from Japanese cedar,Cryptomeria japonica

The absolute configuration of (+)-2,7(14),10-bisabolatrien-1-ol-4-one, a peculiar sesquiterpenol in the Japanese cedar, Cryptomeria japonica, was determined as (1S,6R)-2,7(14),10-bisabolatrien-1-ol-4-one by comparing the specific rotation values of cryptomeriones respectively converted from (+)-2,7(14),10-bisabolatrien-1-ol-4-one and synthesized from (R)-(-)-carvone.     

1H, 15N and 13C resonance assignments of yeast Saccharomyces cerevisiae calmodulin in the Ca2+-free state

Calmodulin (CaM) is a small Ca²⁺-binding protein, which has been found in all of eucaryotic cells examined. CaMs isolated from various species have highly conserved amino acid sequence (more than 90% identical), and show the same biological functions. CaM isolated from the baker's yeast (Saccharomyces cerevisiae) (yCaM), however, shares only 60% identity in the amino acid sequence with CaM from vertebrate, and shows quite distinct conformational and biochemical properties compared with those of CaM from other species. The conformational details of yCaM, however, have not been revealed yet. We achieved the chemical shift assignments of yCaM (146 amino acids) in the apo-state using uniformly ¹⁵N- and ¹³C-labeled protein. Consequently, the resonances of 95% atoms in the backbone amides were successfully assigned.     

Involvement of surface polysaccharides in the organic acid resistance of Shiga Toxin-producing Escherichia coli O157:H7

In general, wild Escherichia coli strains can grow effectively under moderately acidic organic acid-rich conditions. We found that the Shiga Toxin-producing E. coli (STEC) O157:H7 NGY9 grows more quickly than a K-12 strain in Luria-Bertani (LB)-2-morpholinoethanesulphonic acid (MES) broth supplemented with acetic acid (pH 5.4). Hypothesizing that the resistance of STEC O157:H7 to acetic acid is as a result of a mechanism(s) other than those known, we screened for STEC mutants sensitive to acetic acid. NGY9 was subjected to mini-Tn5 mutagenesis and, from 50,000 colonies, five mutants that showed a clear acetic acid-sensitive phenotype were isolated. The insertion of mini-Tn5 in three mutants occurred at the fcl, wecA (rfe) and wecB (rffE) genes and caused loss of surface O-polysaccharide, loss of both O-polysaccharide and enterobacterial common antigen (ECA) and loss of ECA respectively. The other two mutants showed inactivation of the waaG (rfaG) gene but at different positions that caused a deep rough mutant with loss of the outer core oligosaccharide of lipopolysaccharide (LPS) as well as phenotypic loss of O-polysaccharide and ECA. With the introduction of plasmids carrying the fcl, wecA, wecB and waaG genes, respectively, all mutants were complemented in their production of O-polysaccharide and ECA, and normal growth was restored in organic acid-rich culture conditions. We also found that the growth of Salmonella LPS mutants Ra, Rb1, Rc, Rd1, Rd2 and Re was suppressed in the presence of acetic acid compared with that of the parents. These results suggest that the full expression of LPS (including O-polysaccharide) and ECA is indispensable to the resistance against acetic acid and other short chain fatty acids in STEC O157:H7 and Salmonella. To the best of our knowledge, this is a newly identified physiological role for O-polysaccharide and ECA as well as an acid resistance mechanism.     

MotX and MotY,specific components of the sodium-driven flagellar motor,colocalize to the outer membrane in Vibrio alginolyticus

Rotation of the sodium-driven polar flagella of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX and MotY. MotX and MotY, which are unique components of the sodium-driven motor of Vibrio, have been believed to be localized in the inner (cytoplasmic) membrane via their N-terminal hydrophobic segments. Here we show that MotX and MotY colocalize to the outer membrane. Both proteins, when expressed together, were detected in the outer membrane fraction separated by sucrose density gradient centrifugation. As mature MotX and MotY proteins do not have N-terminal hydrophobic segments, the N-termini of the primary translation products must have signal sequences that are removed upon translocation across the inner membrane. Moreover, MotX and MotY require each other for efficient localization to the outer membrane. Based on these lines of evidence, we propose that MotX and MotY form a complex in the outer membrane. This is the first case that describes motor proteins function in the outer membrane for flagellar rotation.     

Quantitative determination of glufosinate in biological samples by liquid chromatography with ultraviolet detection after p-nitrobenzoyl derivatization

We have established a new HPLC method for derivatizing and quantifying glufosinate (GLUF) in human serum and urine using p-nitrobenzoyl chloride (PNBC). The p-nitrobenzoyl derivative of GLUF (PNB-GLUF) was produced quantitatively over 10 min at room temperature. PNB-GLUF possesses the property of ultraviolet (UV) light absorption with a lambda(max) of 272.8 nm, and was isolated from biological specimens by reversed-phase chromatography using Inertsil Ph-3. In experiments at a UV wavelength of 273 nm, GLUF has a quantitative detection limit of 0.005 microg/ml, and when it was added to both serum and urine to yield concentrations of 0.1-1000 microg/ml, its recovery rate was quite satisfactory: at least 93.8% in all cases. Further, the measured amounts of GLUF in 23 serum samples from patients intoxicated by ingestion of GLUF compared favorably with those obtained by fluorescence derivatization-HPLC using 9-fluorenylmethyl chloroformate (R=0.998). This technique of analysis is, in addition, applicable for Glyphosat, which possesses a chemical structure resembling that of GLUF, and it will be of great use in the determination of these two compounds.