Biphenyl degradation by recombinant photosynthetic cyanobacterium Synechocystis sp. PCC6803 in an oligotrophic environment using unphysiological electron transfer.

Cyanobacteria are potentially useful photosynthetic microorganisms for bioremediation under oligotrophic environments. Here, the biphenyl degradation pathway genes of ß-proteobacterium Acidovorax sp. strain KKS102 were co-expressed in cyanobacterium Synechocystis sp. PCC6803 cells under control of the photo-inducible psbE promoter. In the KKS102 cells, biphenyl is dioxygenated by bphA1 and bphA2 gene products complex using electrons supplied from NADH via bphA4 and bphA3 gene products (BphA4 and BphA3, respectively), and converted to benzoic acid by bphB, bphC and bphD gene products. Unexpectedly, biphenyl was effectively hydroxylated in oligotrophic BG11 medium by co-expressing the bphA3, bphA1 and bphA2 genes without the bphA4 gene, suggesting that endogenous cyanobacteria-derived protein(s) can supply electrons to reduce BphA3 at the start of the biphenyl degradation pathway. Furthermore, biphenyl was converted to benzoic acid by cyanobacterial cells co-expressing bphA3, bphA1, bphA2, bphB, bphC and bphD. Structural gene-screening using recombinant Escherichia coli cells co-expressing bphA3, bphA1, bphA2, bphB and bphC suggested that petH, which encodes long- and short-type NADP-ferredoxin oxidoreductase isomers (FNRL and FNRS, respectively), and slr0600, which is annotated as an NADPH-thioredoxin reductase gene in CyanoBase, were BphA3-reducible proteins. Purified FNRL and FNRS, and the slr0600 gene product showed BphA3 reductase activity dependent on NADPH and the reduced form of glutathione, respectively, potentially shedding light on the physiological roles of the slr0600 gene product in cyanobacterial cells. Collectively, our results demonstrate the utility of Synechocystis sp. PCC6803 cells as a host for bioremediation of biphenyl compounds under oligotrophic environments without an organic carbon source.

Design of UCST polymers for chilling capture of proteins.

Ureido-derivatized polymers, such as poly(allylurea) (PU) and poly(L-citrulline) derivatives, exhibited upper critical solution temperature (UCST) behavior under physiological buffer conditions as we previously reported. The PU derivatives having amino groups (PU-Am) also showed UCST behavior. In this study, we modified the amino groups of the polymer with succinyl anhydride (PU-Su) or acetyl anhydride (PU-Ac) to determine the effects of these ionic groups on the UCST behavior and to control interactions between the PU derivatives and biocomponents such as proteins and cells. Succinylation of PU-Am resulted in a significant decrease in phase separation temperature (Tp), whereas acetylation of PU-Am resulted in an increase in Tp. As expected, the Tp of PU-Am and PU-Su changed when the pH of the solution was changed. The Tp of PU-Am increased at higher pH, whereas that of PU-Su increased at lower pH, indicating that ionic charge decreases Tp of PU derivatives by increasing osmotic pressure and by increasing hydrophilicity of the polymer chains. Interestingly, these groups did not significantly change UCST when these groups were nonionic. We then examined capture and separation of particular proteins from a protein mixture by cooling-induced phase separation. Selective and rapid capture of particular proteins from protein mixture by PU derivatives was shown, indicating that the ureido-derivatized polymers are potential media for bioseparation under biofriendly conditions.

Formation of rectangular packing and one-dimensional lines of C60 on 11-phenoxyundecanethiol self-assembled monolayers on Au(111).

The behavior of C(60) molecules deposited onto 11-phenoxyundecanethiol (phenoxy) self-assembled monolayers (SAMs) is studied using ultrahigh vacuum scanning tunneling microscopy (UHV-STM) and spectroscopy. We observe that after thermally annealing between 350 and 400 K in vacuum a combination of hexagonally close-packed islands, rectangularly packed islands, and isolated single lines of C(60) is observed when the C(60) is initially deposited on an unannealed phenoxy SAM. However, only rectangularly packed islands are found when they are deposited on a preannealed phenoxy SAM. We determine the rectangular packing to have a (2√3 × 4) rectangular unit cell with respect to the underlying Au(111) substrate. This type of C(60) structure has not been observed previously for multicomponent self-assemblies on a surface. We discuss the possible causes for the formation of this structure as well as the differences between starting on an unannealed SAM and an annealed one. This study demonstrates the capability of functionalized alkanethiol SAMs to control the growth and structure of C(60) islands during annealing depending on the structural changes of the SAM itself; by preannealing the SAM, the motion of the C(60) can be confined and unique structures resulting from interactions between the SAM molecules and C(60) can be produced.

Ureido-derivatized polymers based on both poly(allylurea) and poly(L-citrulline) exhibit UCST-type phase transition behavior under physiologically relevant conditions.

There are few examples of polymers that exhibit upper critical solution temperature (UCST) behavior under physiological conditions of temperature, pH, and ionic strength. In this study, we demonstrated that polymers with ureido groups undergo UCST-type phase transitions under physiologically relevant conditions. Poly(allylurea) copolymers showed UCST behavior at pH 7.5 in 150 mM NaCl even at the low polymer concentration of 0.13 mg/mL. Their phase separation temperatures (T(p)) could be controlled up to 65 °C. Similar thermosensitivity was observed with copolypeptides consisting of L-citrulline having an ureido group. This is the first demonstration of a non-vinyl polymer that shows UCST behavior under physiologically relevant conditions. We suggest that the ureido modification will be useful for production of polymer materials with UCST behavior in aqueous media.

Ribosome-binding site interference caused by Shine-Dalgarno-like nucleotide sequences in Escherichia coli cells.

Two-cistronic expression plasmids are useful for high-level expression of heterologous genes in Escherichia coli cells by preventing the inhibition of translational initiation. In the process of constructing a two-cistronic expression plasmid pCbSTCR-4 containing the fragments of the porcine cytochrome b(5) (Psb5) and NADPH-cytochrome P450 reductase (PsCPR) genes as the first and second cistrons, respectively, the presence of a specific region in the first cistron that lowered the accumulation level of the PsCPR was suggested [Kimura, S., et al. (2005) J. Biochem. 137, 523-533]. In this study, a disturbing nucleotide sequence similar to a Shine-Dalgarno (SD) sequence (SD-like sequence), AGGAG, was identified at the 5'-upstream region near the SD sequence for the second cistron. Silent mutations in the SD-like sequence that lowered the similarity to a typical SD sequence increased the accumulation level of PsCPR. SD-like sequences introduced into mono-cistronic expression plasmids for the Psb5 and PsCPR genes also decreased the accumulation level of these proteins. The SD-like sequence also decreased the accumulation level of the insoluble PsCPR protein. This type of ribosome-binding site interference is useful not only for precise control of protein accumulation but also for increasing the soluble form of recombinant proteins in E. coli cells.

Characterization of adducts formed in the reaction of 2-chloro-4-methylthiobutanoic acid with 2'-deoxyguanosine.

2-chloro-4-methylthiobutanoic acid (CMBA) is a direct-acting mutagen found in salt-nitrite-treated Sanma fish or similarly treated methionine solution. In this study, CMBA was reacted with 2'-deoxyguanosine (dG) in phosphate buffer (pH 7.4) at 37 degrees C. The HPLC-UV analysis showed that two products were mainly formed during the reaction. These were isolated, purified by semipreparative HPLC, and characterized as N7-guanine adducts: N7-(3-carboxy-3-methylthiopropyl)guanine (A1) and N7-(1-carboxy-3-methylthiopropyl)guanine (A2). Furthermore, liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) analysis was employed to investigate the possible formation of minor products during the time-course of the reaction of CMBA with dG. It was found that N7-dG adducts, the precursors of A1 and A2, were formed early in the reaction and that subsequently the spontaneous depurination occurred to yield stable N7-guanine adducts A1 and A2. Stability studies in phosphate buffer (pH 7.4) at 37 degrees C showed that the amount of each N7-dG adduct decreased rapidly with a half-life of 6 h and 4 h to yield A1/A2, respectively. A regioisomer of N7-dG adducts was also observed in the LC/ESI-MS/MS analysis, but it was not characterized in detail because it was present only in trace amounts. On the basis of structural features, A1 and A2 seemed to be formed from the reaction of dG with 1-methyl-2-thietaniumcarboxylic acid, an intermediate resulting from the cyclization of CMBA. However, A2 might also have formed from the direct reaction of dG and CMBA. N7-Alkylation of the guanine residue and subsequent depurination are known to produce apurinic sites in DNA that induce point mutations and may be responsible for the observed CMBA-induced mutagenesis.