Functional analysis of arabinofuranosidases and a xylanase of <Emphasis Type="Italic">Corynebacterium alkanolyticum</Emphasis> for arabinoxylan utilization in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Xylooligosaccharides (XOSs) and arabinoxylooligosaccharides (AXOSs) are major oligosaccharides derived from arabinoxylan. In our previous report, Corynebacterium glutamicum was engineered to utilize XOSs by introducing Corynebacterium alkanolyticum xyloside transporter and β-xylosidase. However, this strain was unable to consume AXOSs due to the absence of α-l-arabinofuranosidase activity. In this study, to confer AXOS utilization ability on C. glutamicum, two putative arabinofuranosidase genes (abf51A and abf51B) were isolated from C. alkanolyticum by the combination of degenerate PCR and genome walking methods. Recombinant Abf51A and Abf51B heterologously expressed in Escherichia coli showed arabinofuranosidase activities toward 4-nitrophenyl-α-l-arabinofuranoside with k _cat values of 150 and 63, respectively, with optimum at pH 6.0 to 6.5. However, Abf51A showed only a slight activity toward AXOSs and was more susceptible to product inhibition by arabinose and xylose than Abf51B. Introduction of abf51B gene into the C. glutamicum XOS-utilizing strain enabled it to utilize AXOSs as well as XOSs. The xylI gene encoding a putative xylanase was found upstream of the C. alkanolyticum xyloside transporter genes. A signal peptide was predicted at the N-terminus of the xylI-encoding polypeptide, which indicated XylI was a secreted protein. Recombinant mature XylI protein heterologously expressed in E. coli showed a xylanase activity toward xylans from various plant sources with optimum at pH 6.5, and C. glutamicum recombinant strain expressing native XylI released xylose, xylobiose, xylotriose, and arabino-xylobiose from arabinoxylan. Finally, introduction of the xylI gene into the C. glutamicum AXOS-utilizing strain enabled it to directly utilize arabinoxylan.     

Genome 3'-end repair in dengue virus type 2

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3′-end repair in dengue virus-2 in mammalian cells, a series of 3′-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7–10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3′ end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3′-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3′ stem–loop (3′ SL) structure was abrogated by mutations, viruses eventually restored the 3′ SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3′-end sequences based on the best-fit RNA structure that can support viral replication.     

Effects of detergents on the West Nile virus protease activity

Detergents such as Triton X-100 are often used in drug discovery research to weed out small molecule promiscuous and non-specific inhibitors which act by aggregation in solution and undesirable precipitation in aqueous assay buffers. We evaluated the effects of commonly used detergents, Triton X-100, Tween-20, Nonidet-40 (NP-40), Brij-35, and CHAPS, on the enzymatic activity of West Nile virus (WNV) protease. Unexpectedly, Triton X-100, Tween-20, and NP-40 showed an enhancement of in vitro WNV protease activity from 2 to 2.5-fold depending on the detergent and its concentration. On the other hand, Brij-35, at ?0.001% enhanced the protease activity by 1.5-fold and CHAPS had the least enhancing effect. The kinetic analysis showed that the increase in protease activity by Triton X-100 was dose-dependent. Furthermore, at Triton X-100 and Tween-20 concentrations higher than 0.001%, the inhibition of compound B, one of the lead compounds against WNV protease identified in a high throughput screen (IC₅₀ value of 5.7 ± 2.5 μM), was reversed. However, in the presence of CHAPS, compound B still showed good inhibition of WNV protease. Our results, taken together, indicate that nonionic detergents, Triton X-100, Tween, and NP-40 are unsuitable for the purpose of discrimination of true versus promiscuous inhibitors of WNV protease in high throughput assays.     

Structural basis for the broad range substrate specificity of a novel mouse cytosolic sulfotransferase—mSULT1D1

The mouse cytosolic sulfotransferase, mSULT1D1, catalyzes the sulfonation of a wide range of phenolic molecules including p-nitrophenol (pNP), α-naphthol (αNT), serotonin, as well as dopamine and its metabolites. To gain insight into the structural basis for its broad range substrate specificity, we solved two distinct ternary crystal structures of mSULT1D1, complexed with 3′-phosphoadenosine-5′-phosphate (PAP) plus pNP or PAP plus αNT. The structures revealed that the mSULT1D1 contains an L-shaped accepter-binding site which comprises 20 amino acid residues and four conserved water molecules. The shape of the accepter-binding site can be adjusted by conformational changes of two residues, Ile148 and Glu247, upon binding with respective substrates.     

On the similar spatial arrangement of active site residues in PAPS-dependent and phenolic sulfate-utilizing sulfotransferases

Mammalian sulfotransferases (STs) utilize exclusively the sulfuryl group donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to catalyze the sulfurylation reactions based on a sequential transfer mechanism. In contrast, the commensal intestinal bacterial arylsulfate sulfotransferases (ASSTs) do not use PAPS as the sulfuryl group donor, but instead catalyze sulfuryl transfer from phenolic sulfate to a phenol via a Ping-Pong mechanism. Interestingly, structural comparison revealed a similar spatial arrangement of the active site residues as well as the cognate substrates in mouse ST (mSULT1D1) and Escherichia coli CFT073 ASST, despite that their overall structures bear no discernible relationship. These observations suggest that the active sites of PAPS-dependent SULT1D1 and phenolic sulfate-utilizing ASST represent an example of convergent evolution.     

A case of maxillary sarcoma in a chimpanzee (Pan troglodytes)

Oral malignancy is rare in chimpanzees. A 34‐year‐old female chimpanzee (Pan troglodytes) at Kumamoto Sanctuary, Japan, had developed it. Treatment is technically difficult for chimpanzees while malignant neoplasm is seemingly rising in captive populations. Widespread expert discussion, guidelines for treatment, especially for great apes in terminal stages is urgently needed.     

Real-time vibrational dynamics in chlorophyll a studied with a few-cycle pulse laser

We use a 6.8-fs laser as the light source for broad-band femtosecond pump-probe real-time vibrational spectroscopy to investigate both electronic relaxation and vibrational dynamics of the Q_y-band of Chl-a at 293 K. More than 25 vibrational modes coupled to the Q_y transition are observed. Eleven of them have been clarified predominantly due to the excited state, and six of them are concluded to be nearly exclusively resulting from the ground-state wave-packet motion. Moreover, thanks to the broad-band detection over 5000 cm⁻¹, the modulated signals due to the excited state vibrational coherence are observed on both sides of the 0-0 transition with equal separation. The corresponding nonlinear process has been studied using a three-level model, from which the probe wavelength dependence of the phase of the periodic modulation can be calculated. The probe wavelength dependence of the vibrational amplitude is interpreted in terms of the interaction between the “pump” or “laser,” Stokes, and anti-Stokes field intermediated by the molecular vibrations. In addition, an excited state absorption peak at ∼709 nm has been observed. To the best of our knowledge, this is the first study of broad-band real-time vibrational spectroscopy in Chl-a.     

Genome-wide identification of in vivo binding sites of GlxR, a cyclic AMP receptor protein-type regulator in Corynebacterium glutamicum

Corynebacterium glutamicum GlxR is a cyclic AMP (cAMP) receptor protein-type regulator. Although over 200 GlxR-binding sites in the C. glutamicum genome are predicted in silico, studies on the physiological function of GlxR have been hindered by the severe growth defects of a glxR mutant. This study identified the GlxR regulon by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analyses. In total, 209 regions were detected as in vivo GlxR-binding sites. In vitro binding assays and promoter-reporter assays demonstrated that GlxR directly activates expression of genes for aerobic respiration, ATP synthesis, and glycolysis and that it is required for expression of genes for cell separation and mechanosensitive channels. GlxR also directly represses a citrate uptake gene in the presence of citrate. Moreover, ChIP-chip analyses showed that GlxR was still able to interact with its target sites in a mutant with a deletion of cyaB, the sole adenylate cyclase gene in the genome, even though binding affinity was markedly decreased. Thus, GlxR is physiologically functional at the relatively low cAMP levels in the cyaB mutant, allowing the cyaB mutant to grow much better than the glxR mutant.     

Differential regulation of TRPM channels governs electrolyte homeostasis in the C. elegans intestine

The transient receptor potential (TRP) channels are implicated in various cellular processes, including sensory signal transduction and electrolyte homeostasis. We show here that the GTL-1 and GON-2 TRPM channels regulate electrolyte homeostasis in the C. elegans intestine. GON-2 is responsible for a large outwardly rectifying current of intestinal cells, and its activity is tightly regulated by intracellular Mg(2+) levels, while GTL-1 mainly contributes to appropriate Mg(2+) responsiveness of the outwardly rectifying current. We also used nickel cytotoxicity to study the function of these channels. Both GON-2 and GTL-1 are necessary for intestinal uptake of nickel, but GTL-1 is continuously active while GON-2 is inactivated at higher Mg(2+) levels. This type of differential regulation of intestinal electrolyte absorption ensures a constant supply of electrolytes through GTL-1, while occasional bursts of GON-2 activity allow rapid return to normal electrolyte concentrations following physiological perturbations.