Indigo is an insoluble blue dye historically used for dyeing textiles. A traditional approach for indigo dyeing involves microbial reduction of polygonum indigo to solubilize it under alkaline ...conditions; however, the mechanism by which microorganisms reduce indigo remains poorly understood. Here, we aimed to identify an enzyme that catalyzes indigo reduction; for this purpose, from alkaline liquor that performed microbial reduction of polygonum indigo, we isolated indigo carmine-reducing microorganisms. All isolates were facultative anaerobic and alkali-tolerant
Bacillus
spp. An isolate termed AO1 was found to be an alkaliphile that preferentially grows at pH 9.0–11.0 and at 30–35 °C. We focused on flavin-dependent azoreductase as a possible enzyme for indigo carmine reduction and identified its gene (
azoA
) in
Bacillus
sp. AO1 using homology-based strategies.
azoA
was monocistronic but clustered with ABC transporter genes. Primary sequence identities were < 50% between the
azoA
product (AzoA) and previously characterized flavin-dependent azoreductases. AzoA was heterologously produced as a flavoprotein tolerant to alkaline and organic solvents. The enzyme efficiently reduced indigo carmine in an NADH-dependent manner and showed strict specificity for electron acceptors. Notably, AzoA oxidized NADH in the presence, but not the absence, of indigo. The reaction rate was enhanced by adding organic solvents to solubilize indigo. Absorption spectrum analysis showed that indigo absorption decreased during the reaction. These observations suggest that AzoA can reduce indigo in vitro and potentially in
Bacillus
sp. AO1. This is the first study that identified an indigo reductase, providing a new insight into a traditional approach for indigo dyeing.
Thermostable enzymes are commonly produced in mesophilic hosts for research and bioengineering purposes. However, these hosts do not overexpress the active forms of some biologically functional ...thermoenzymes. Therefore, an efficient thermophilic expression system is needed. Thermus thermophilus contains an easily manipulable genome and is therefore among the best candidate microbes for a "hot" expression system. We previously identified a strong and inducible promoter that was active in T. thermophilus under supersaturated silica conditions. Here, we report a new heterologous gene expression system based on a silica-inducible promoter in T. thermophilus.
A Thermus sp. A4 gene encoding thermostable β-galactosidase was cloned as a reporter gene into the expression vector pSix1, which contains a selection marker that confers thermostable resistance to hygromycin and a 600 bp DNA region containing a putative silica-inducible promoter. β-galactosidase activity was 11-fold higher in the presence than in the absence of 10 mM silicic acid. SDS-PAGE revealed a prominent band corresponding to 73 kDa of β-galactosidase, and this enzyme was expressed as an active and soluble protein (yield: 27 mg/L) in Thermus but as an inclusion body in Escherichia coli. Truncation of the putative silica-inducible promoter region in Thermus expression vector improved the yield of the target protein, possibly by avoiding plasmid instability due to homologous recombination. Finally, we developed an expression vector containing the pSix1 backbone and a 100 bp DNA region corresponding to the silica-inducible promoter. We used this vector to successfully express the active form of glutamate dehydrogenase from Pyrobaculum islandicum (PisGDH) without additional treatment (yield: 9.5 mg/L), whereas the expression of active PisGDH in E. coli required heat treatment.
We successfully expressed the thermostable β-galactosidase and PisGDH in T. thermophilus as active and soluble forms and achieved with our system the highest known protein expression levels in this species. These thermoenzymes were expressed in active and soluble forms. Our results validate the use of our silica-inducible expression system as a novel strategy for the intracellular overexpression of thermostable proteins.
Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified ...from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The Km and Vmax values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min−1·mg−1, respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min−1·mg−1, respectively. Hydroxylamine and other inhibitors of pyridoxal 5′-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5′-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.
Excitable cochlear hair cells convert the mechanical energy of sounds into the electrical signals necessary for neurotransmission. The key process is cellular depolarization via K+ entry from ...K+‐enriched endolymph through hair cells’ mechanosensitive channels. Positive 80 mV potential in endolymph accelerates the K+ entry, thereby sensitizing hearing. This potential represents positive extracellular potential within the epithelial‐like stria vascularis; the latter potential stems from K+ equilibrium potential (EK) across the strial membrane. Extra‐ and intracellular K+ determining EK are likely maintained by continuous unidirectional circulation of K+ through a putative K+ transport pathway containing hair cells and stria. Whether and how the non‐excitable tissue stria vascularis responds to acoustic stimuli remains unclear. Therefore, we analysed a cochlear portion for the best frequency, 1 kHz, by theoretical and experimental approaches. We have previously developed a computational model that integrates ion channels and transporters in the stria and hair cells into a circuit and described a circulation current composed of K+. Here, in this model, mimicking of hair cells’ K+ flow induced by a 1 kHz sound modulated the circulation current and affected the strial ion transport mechanisms; the latter effect resulted in monotonically decreasing potential and increasing K+ in the extracellular strial compartment. Similar results were obtained when the stria in acoustically stimulated animals was examined using microelectrodes detecting the potential and K+. Measured potential dynamics mirrored the EK change. Collectively, because stria vascularis is electrically coupled to hair cells by the circulation current in vivo too, the strial electrochemical properties respond to sounds.
Key points
A highly positive potential of +80 mV in K+‐enriched endolymph in the mammalian cochlea accelerates sound‐induced K+ entry into excitable sensory hair cells, a process that triggers hearing.
This unique endolymphatic potential represents an EK‐based battery for a non‐excitable epithelial‐like tissue, the stria vascularis.
To examine whether and how the stria vascularis responds to sounds, we used our computational model, in which strial channels and transporters are serially connected to those hair cells in a closed‐loop circuit, and found that mimicking hair cell excitation by acoustic stimuli resulted in increased extracellular K+ and decreased the battery's potential within the stria.
This observation was overall verified by electrophysiological experiments using live guinea pigs.
The sensitivity of electrochemical properties of the stria to sounds indicates that this tissue is electrically coupled to hair cells by a radial ionic flow called a circulation current.
figure legend Evidence for functional linkage between a double‐layered epithelial‐like tissue, stria vascularis, and sensory hair cells in the cochlea of the mammalian inner ear. The endolymph, which is highlighted by blue in the cochlear cross section, exhibits a highly positive potential of +80 mV. This potential is essential for the excitation of hair cells and is dependent upon the K+ homeostasis in the extracellular space in the stria. The K+ balance is likely to be maintained by ‘circulation current’, a radial ionic flow in the cochlea. Not only computational simulation but also in vivo electrophysiological experiments using guinea pigs showed that stimulation of the hair cells by sounds changed the strial K+ property. This result indicates that the circulation current electrically couples the stria vascularis to the hair cells.
To assess the prevalence of vestibular schwannoma (VS) in patients with sudden sensorineural hearing loss (SSNHL).
This is a retrospective chart review of 861 patients who were diagnosed with or ...treated for SSHNL between January 2008 and February 2017 at our department in a tertiary academic center. We retrospectively analyzed the medical charts and MRI findings of 499 patients who had undergone MRI.
Fifteen (3.0%) of the 499 patients exhibited tumors at the cerebellopontine angle on the same side affected by SSNHL. In one patient, a tumor was incidentally detected in the contralateral ear. The 15 VS lesions were graded using the Koos acoustic neuroma grading system as follows: grade I (intracanalicular tumor), n=8; grade II (up to 2cm), n=6; and grade III (up to 3cm), n=1. Koos grade IV tumors, which are large tumors that displace the trunk or cranial nerves, were not found.
The prevalence of VS in patients with SSNHL was 3.0% in the present study. Considering this high prevalence, clinicians should consider detailed examinations in addition to audiometry for patients with SSNHL.
Coronavirus disease 2019 (COVID-19) occasionally causes acute laryngitis, requiring emergency treatment. Understanding the characteristic laryngeal findings can help diagnose COVID-19 earlier, ...prevent worsening infection, and properly manage airway obstruction. Herein, we report the case of a 44-year-old male with acute epiglottitis likely caused by COVID-19. On presentation, chest computed tomography (CT) showed no signs of pneumonia. However, the larynx had extensive necrotic-like erosive lesions resembling those of tuberculous laryngitis. COVID-19 was diagnosed by reverse-transcription polymerase chain reaction, and secondary bacterial superinfections were suspected after blood testing. The symptoms improved after administration of antibiotics (sulbactam sodium/ampicillin sodium), steroids (dexamethasone), and favipiravir. The patient developed a high fever on the sixth day of hospitalization, and pneumonia was identified on CT. Various culture tests, including tuberculosis, were negative. Thus, remdesivir was administered for COVID-19-induced pneumonia. The patient gradually recovered, was transferred to another hospital, and was discharged on the 35th day of hospitalization. Six previous case reports of COVID-19-induced acute epiglottitis suggested that acute epiglottitis preceded the onset of pneumonia. The laryngeal findings from this report may be useful for diagnosing COVID-19 that does not cause pneumonia and for bringing attention to pneumonia after a COVID-19 diagnosis.
An endocochlear potential (EP) of +80 mV is essential for audition. Although the regulation of K⁺ concentration (K⁺) in various compartments of the cochlear stria vascularis seems crucial for the ...formation of the EP, the mechanism remains uncertain. We have used multibarreled electrodes to measure the potential, K⁺, and input resistance in each compartment of the stria vascularis. The stria faces two fluids, perilymph and endolymph, and contains an extracelluar compartment, the intrastrial space (IS), surrounded by two epithelial layers, the marginal cell (MC) layer and that composed of intermediate and basal cells. Fluid in the IS exhibits a low K⁺ and a positive potential, called the intrastrial potential (ISP). We found that the input resistance of the IS was high, indicating this space is electrically isolated from the neighboring extracellular fluids. This arrangement is indispensable for maintaining positive ISP. Inhibiting the K⁺ transporters of the stria by anoxia, ouabain, or bumetanide caused the K⁺ of the IS to increase and the intracellular K⁺ of MCs to decrease, reducing both the ISP and the EP. Calculations indicate that the ISP represents the K⁺ diffusion potential across the apical membranes of intermediate cells through Ba²⁺-sensitive K⁺ channels. The K⁺ diffusion potential across the apical membranes of MCs also contributes to the EP. Because the EP depends on two K⁺ diffusion potentials and an electrical barrier in the stria vascularis, interference with any of these elements can interrupt hearing.
Study Design: A retrospective case-control propensity score-matching study.Purpose: This study aimed to longitudinally evaluate whether preoperative ligamentous stenosis at the spondylolisthetic ...segments could affect the incidence of symptomatic adjacent canal stenosis following one-segment fusion surgery.Overview of Literature: Several risk factors for symptomatic adjacent canal stenosis following fusion surgery have been assessed. Patients with lumbar canal stenosis mainly due to ligamentum flavum (LF) hypertrophy (ligamentous stenosis) also have LF hypertrophy in other segments.Methods: In total, 76 patients participated in this case-control study (neurologically symptomatic adjacent canal stenosis, n=33; neurologically asymptomatic cases at follow-up, n=43). Their risk factors during surgery and magnetic resonance (MR) images before the surgery and at follow-up were evaluated. Data from the two groups (n=25 each) were matched using propensity scores for age, sex, time to MR imaging at follow-up, surgical procedure, and LF hypertrophy in adjacent segments before the surgery and analyzed.Results: Compared with the asymptomatic group, the symptomatic adjacent canal stenosis group had a significantly larger LF area/spinal canal area in the spondylolisthetic segments before the surgery. During the follow-up periods (in months), they had a larger LF area/ spinal canal area in the adjacent segments: the two values were significantly correlated. The sensitivity, specificity, and positive and negative predictive values for determining symptomatic adjacent canal stenosis were high compared with on the cutoff value for the LF area/spinal canal area at the spondylolisthetic segments before the surgery. These results were the same after matching.Conclusions: Symptomatic adjacent canal stenosis is mainly caused by LF hypertrophy. Ligamentous stenosis at the spondylolisthetic segments before fusion surgery might be strongly associated with symptomatic adjacent canal stenosis at follow-up.
Thermostability is an important property of enzymes utilized for practical applications because it allows long-term storage and use as catalysts. In this study, we constructed an error-prone strain ...of the thermophile Geobacillus kaustophilus HTA426 and investigated thermoadaptation-directed enzyme evolution using the strain. A mutation frequency assay using the antibiotics rifampin and streptomycin revealed that G. kaustophilus had substantially higher mutability than Escherichia coli and Bacillus subtilis. The predominant mutations in G. kaustophilus were A · T→G · C and C · G→T · A transitions, implying that the high mutability of G. kaustophilus was attributable in part to high-temperature-associated DNA damage during growth. Among the genes that may be involved in DNA repair in G. kaustophilus, deletions of the mutSL, mutY, ung, and mfd genes markedly enhanced mutability. These genes were subsequently deleted to construct an error-prone thermophile that showed much higher (700- to 9,000-fold) mutability than the parent strain. The error-prone strain was auxotrophic for uracil owing to the fact that the strain was deficient in the intrinsic pyrF gene. Although the strain harboring Bacillus subtilis pyrF was also essentially auxotrophic, cells became prototrophic after 2 days of culture under uracil starvation, generating B. subtilis PyrF variants with an enhanced half-denaturation temperature of >10°C. These data suggest that this error-prone strain is a promising host for thermoadaptation-directed evolution to generate thermostable variants from thermolabile enzymes.
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