Isolation of nabscessin C from nocardia abscessus IFM 10029T and A study on biosynthetic pathway for nabscessins

Article abstract of DOI:10.1248/cpb.c18-00430

A new aminocyclitol derivative, designated nabscessin C (1), was isolated from Nocardia abscessus IFM 10029^T. Nabcessin C is an isomer of nabscessins A (2) and B (3) with different positioning of the acyl group. Absolute configuration of nabscessin A was determined by conversion into the 2-deoxy-scyllo-inosamine pentaacetyl derivative (4) by hydrolysis and acetylation of 2. The biosynthetic pathway of nabscessins is proposed based on gene expression analysis.

Full text of DOI:10.1248/cpb.c18-00430

976
Chem. Pharm. Bull. 66, 976–982 (2018)
Vol. 66, No. 10
Regular Article
Isolation of Nabscessin C from Nocardia abscessus IFM 10029T and a
Study on Biosynthetic Pathway for Nabscessins
Shoko Hara,a Yasumasa Hara,a Midori A. Arai,a Yoko Kusuya,b Hiroki Takahashi,b
,a
Takashi Yaguchi,b and Masami Ishibashi*
a Graduate School of Pharmaceutical Sciences, Chiba University; 1–8–1 Inohana, Chuo-ku, Chiba 260–8675,
b
Japan: and Medical Mycology Research Center, Chiba University; 1–8–1 Inohana, Chuo-ku, Chiba 260–8673,
Japan.
Received June 6, 2018; accepted July 18, 2018
A new aminocyclitol derivative, designated nabscessin C (1), was isolated from Nocardia abscessus IFM
10029T. Nabcessin C is an isomer of nabscessins A (2) and B (3) with different positioning of the acyl group.
Absolute configuration of nabscessin A was determined by conversion into the 2-deoxy-scyllo-inosamine
pentaacetyl derivative (4) by hydrolysis and acetylation of 2. The biosynthetic pathway of nabscessins is pro-
posed based on gene expression analysis.
Key words actinomycete; Nocardia; aminocyclitol; biosynthetic pathway
Actinomycetes of the genus Nocardia are Gram-positive clusters, thirteen strains (Nocardia abscessus IFM 10029T,
bacteria widely present in nature. Many species in this genus Nocardia africana IFM 10147T, Nocardia anaemiae IFM
possess weak virulence. In humans, Nocardia infect the 0323T, Nocardia arthritidis IFM 10035T, Nocardia asiatica
lungs and disseminate hematogenously, causing visceral no- IFM 0245T, Nocardia exalbida IFM 0803T, Nocardia inoha-
cardiosis, as well as skin nocardiosis (in the dermis). Many nensis IFM 0092T, Nocardia kruczakiae IFM 10565T, No-
cases of infection occur in humans with reduced immunity. cardia sienata IFM 10088T, Nocardia terpenica IFM 0706T,
Some species of the genus Nocardia are known to infect other Nocardia transvalensis IFM 0333T, Nocardia vinacea IFM
animals and plants. Several secondary metabolites have been 10175T, and Nocardia yamanashiensis IFM 0265T) were se-
isolated from the genus Nocardia. Nocardicin A, a monocyclic lected for growth as small-scale cultures. Metabolite produc-
β-lactam, was isolated from Nocardia uniformis ssp. Tsuyama- tion in the thirteen selected strains was examined using four
nensis ATCC 21806,1) and the nargenicins were isolated from different media [mCD, nutrient broth (NB),9) Waksman,10) and
Nocardia argentinensis HUANG ATCC 31306.2) Although these Yeast-Malt-Glucose (YMG)11)] and incubating the cultures at
metabolites have been isolated, research on the genus Nocar- 28°C in ambient air with rotary shaking at 160rpm in 50-mL
dia has not progressed as enough as that on the genus Strep- Erlenmeyer flasks.
tomyces. The genome sequences of several Nocardia strains
have been reported, but members of this genus have not yet under various conditions led us to focus our attention on the
been the subject of detailed analyses of gene function.3) extract of Nocardia abscessus IFM 10029T cultured in the
The LC-MS analysis of the culture broth extracts obtained
In our previous report,4) new aminocyclitol derivatives, mCD medium. In the corresponding extract, three character-
nabscessins A and B, were isolated from Nocardia abscessus istic peaks were observed; the UV absorption spectra and the
IFM 10029T grown in modified Czapek-Dox (mCD)5) medium. MS spectra of the 3 peaks largely coincided with each other,
In the present study, we describe the isolation and structural even though the compounds represented by the 3 peaks exhib-
elucidation of a new compound (1) obtained from Nocardia ited distinct retention times. A large-scale culture (2.0L) of
abscessus IFM 10029T grown in mCD medium. We extend Nocardia abscessus IFM 10029T was performed by growing
that work by determining the absolute configurations of the the strain in mCD medium for 1 week at 28°C in ambient air
nabscessins, and additionally use molecular genetic analyses as a shaking culture (160rpm) in five 1-L Erlenmeyer flasks.
to propose the pathway for the biosynthesis of this class of After centrifugation of the culture, the supernatant and a
compounds.
methanol extract of the mycelia were combined and subjected
to partitioning between ethyl acetate (EtOAc) and water. The
EtOAc-soluble fraction was subjected to fractionation using
Results and Discussion
Strains for this study were initially selected from among 76 silica gel column chromatography (the CHCl3–MeOH system),
strains belonging to the genus Nocardia; all of these strains followed by reverse-phase HPLC separation (40% MeOH) to
were obtained from the Medical Mycology Research Cen- obtain a new compound (1), designated nabscessin C (Fig. 1),
ter, Chiba University, Japan. A phylogenetic tree for these along with the previously identified nabscessins A (2) and B
strains was constructed based on 16S ribosomal RNA (rRNA) (3).
sequence analysis using two analytical software programs,
High resolution (HR) electrospray ionization (ESI)-
Clustal X6) and MEGA.7) This tree classified the strains into MS revealed that the molecular formula of compound 1
nine clades. Biosynthetic gene clusters were examined in is C22H24N2O9 (obsd. m/z 483.1426 [M+Na]+, Calcd for
a gene analysis of the genus Nocardia using antiSMASH.8) C22H24N2O9Na, 483.1380). The H-NMR spectrum of 1 mea-
1
Based on these clades and the number of biosynthetic gene sured in acetone-d6 showed seven aromatic hydrogens (δH
*To whom correspondence should be addressed. e-mail: mish@chiba-u.ac.jp
© 2018 The Pharmaceutical Society of Japan

Vol. 66, No. 10 (2018)
Chem. Pharm. Bull.
Table 1. 1H- and 13C-NMR Chemical Shifts for Nabscessin C (1)
977
Position
δH
δC
1
2
3
4
5
6
4.20 (m)
50.9
73.8
79.6
74.1
73.0
33.9
3.99 (dd, 9.6, 9.6)
5.34 (dd, 9.6, 9.6)
3.81 (dd, 9.6, 9.6)
4.82 (ddd, 12.0, 9.6, 4.6)
1.71 (ddd, 12.0, 12.0, 12.0)
2.42 (ddd, 12.0, 4.6, 4.6)
7.72 (d, 8.3)
1-NH
1′
2′
3′
4′
5′
6′
7'
137.0
115.3
158.4
119.0
130.0
119.0
167.8
115.3
162.0
115.9
134.3
123.2
141.6
171.3
23.2
7.44 (dd, 2.7, 1.4)
6.95 (ddd, 7.8, 2.7, 0.9)
7.23 (dd, 7.8, 7.8)
Fig. 1. Structure of 1
7.34 (ddd, 7.8, 1.4, 0.9)
7.44–6.75), five sp3 methines (δH 5.34–3.81), one methylene (δH
2.42 and 1.71), one methyl (δH 2.56), and one broad signal [δH
7.72 (1H)]. The 13C-NMR spectrum of 1 exhibited 21 peaks
(Table 1), which were assignable to seven aromatic methines,
seven quaternary carbons, five sp3 methines, one methylene,
1″
2″
3″
4″
5″
6″
7″
8″
6.77 (d, 8.7)
7.29 (dd, 8.7, 7.8)
6.75 (d, 7.8)
1
and one methyl, based on analysis of the H-detected het-
eronuclear multiple quantum coherence (HMQC) spectrum.
1
The cross-peaks observed in the H–1H correlation spectros-
2.56 (s)
n.d.a)
copy (COSY) data of 1 suggested the presence of three partial
structures A, B, and C as indicated in Fig. 2. Based on COSY
and heteronuclear multiple bond correlation (HMBC) correla-
tions (Fig. 2) and comparison to the 13C-NMR spectra for 2
and 3, partial structures A, B, and C were assigned to 3-hy-
droxybenzoic acid (3-HBA), 6-methylsalicylic acid (6-MSA),
and a cyclohexane ring moiety, respectively; all three cor-
respond to structures that are shared with those of 2 and 3.
The difference in structure of 1 from those of 2 and 3 was
revealed to be the position of the 6-MSA group. The HMBC
correlation observed from H-3 (δH 5.34) in partial structure C
to the C-7″ carbonyl (δC 171.3) in the 6-MSA moiety suggested
that the 6-MSA ester group is located on C-3. Although the
spectral data elucidating connection between partial structures
A and C as well as the connection between partial structure
C and the carbamoyl group (position 1″) were not obtained,
the positions of partial structure A and the carbamoyl groups
were deduced as the same as those of nabscessins A (2) and B
1‴-NH2
5.88 (brs)
a)
Measured in acetone-d6, 600MHz (1H), 150MHz (13C) δ in ppm. J in Hz.; n.d.:
not determined.
Fig. 2. Partial Structures A–C for 1
1
(3) based on the comparison of the H-NMR chemical shifts
(Fig. S1, supplementary materials) as well as the observa-
tion of interconversion of compound 1 into 2 and 3 (Fig. S2, cessin B (3) recently reported by total synthesis.13)
supplementary materials). The structure of nabscessin C thus
The 2-deoxy-scyllo-inosamine moiety contained in the
was revealed to be 1, as shown in Fig. 1.
structure of nabscessins A–C is known to be a biosynthetic
To elucidate the absolute stereochemistry of nabscessins intermediate of aminoglycoside antibacterial drugs such as
A–C, nabscessin A (2) was converted to a 2-deoxy-scyllo- kanamycin,14) and 2-deoxy-scyllo-inosose synthase has been
inosamine pentaacetyl derivative (4), since the specific rota- reported as a biosynthetic enzyme.15) The gene encoding this
tions of both enantiomers of 4 have been reported in the enzyme therefore served as the “bait” in a search for the bio-
literature.12) Compound 4 was obtained by hydrolysis and synthetic gene cluster for nabscessins A–C. MiGAP analysis16)
acetylation of 2, and the spectral data of the resulting penta- and BLASTP search17) of the draft genome of Nocardia ab-
acetyl compound 4 derived from 2 was identical with those scessus identified contig 00601 as a region harboring a gene
of literature data,12) including the sign of the specific rotation with sequence similarity to the gene encoding 2-deoxy-scyllo-
(levorotatory), which established the absolute configuration of inosose synthase. Analysis of the sequences of contig 00601
2 as shown in Fig. 3. Since nabscessins A–C were obtained (GeneBank accession no. BAFP01000039.1, BAFP01000256.1,
from the same Nocardia strain, the absolute configuration of BAFP01000258.1) surrounding the synthase-encoding gene
nabscessins A–C were presumed to be the same; this infer- suggested the presence of 6 open reading frames (ORFs) that
ence was consistent with the absolute configuration of nabs- were possibly involved in the biosynthesis of the nabscessins

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Chem. Pharm. Bull.
Vol. 66, No. 10 (2018)
Fig. 3. The Determination of Absolute Configuration of 2
Table 2. (a) A Schematic of the Putative Cluster and (b) Putative Nabcessin Biosythetic Genes Selectively Expressed in mCD (Production) Medium
a
b
Closest protein homolog
ORF
A
Size (aa)
331
Proposed protein function
3-Hydroxybenzoate synthase
Protein identity (%)
(source organism)
3-Hydroxybenzoate synthase
(Streptomyces hygroscopius)
51
58
B
450
2-Deoxy-scyllo-inosose aminotransferase
L-Glutamine:2-deoxy-scyllo-inosose/3-
amino-2,3-dideoxy-scyllo-inosose
aminotransferase
(Saccharopolyspora antimicrobica)
C
D
E
F
347
658
2-Deoxy-scyllo-inosose synthase
Carbamoyltransferase
2-Deoxy-scyllo-inosose synthase
(Streptomyces tenjimariensis)
62
42
56
47
Carbamoyltransferase
(Streptomyces niveiscabiei)
225
AMP ligase
2,3-Dihydroxybenzoate-AMP ligase
(Nocardia terpenica)
1611
6-Methylsalicylic acid synthase
6-Methylsalicylic acid synthase
(Streptomyces gandocaensis)
ORF : open reading frame; AMP : adenosine monophosphate.
(Table 2). Based on the structure of this putative gene cluster, A–C were not produced in Waksman medium, while these
the pathway for biosynthesis of the nabscessins is proposed compounds were produced in mCD medium (Fig. 5). Analysis
as shown in the Fig. 4. The partial structures of the nabsces- of the RNA-seq data revealed that the expression of the genes
sin moieties are presumed to be constructed by three separate coding for the six putative nabscessin biosynthetic enzymes
sub-pathways, as follows: (1) 3-HBA is derived from choris- (A–F), was 30-fold or more higher in mCD medium than in
mate by 3-hydroxybenzoate synthase18); (2) 2-Deoxy-scyllo- Waksman medium (Fig. 6). Enhancement of the expression of
inosamine is obtained from glucose-6-phosphate via 2-deoxy- these genes in production medium is consistent with the use
scyllo-inosose by 2-deoxy-scyllo-inosose aminotransferase and of the corresponding proteins in the proposed biosynthetic
2-deoxy-scyllo-inosose synthase15); and (3) 6-MSA is generat- pathway (Fig. 4).
ed from acetyl CoA and malonyl CoA by 6-MSA synthase.19)
Production conditions for nabscessins A–C were further
These three moieties of the nabscessins then are connected examined, as shown in Fig. 7. Figure 7c, which corresponds
by the activities of an AMP ligase20) and a carbamoyltransfer- to Fig. 5a, shows the HPLC profile for the extract of Nocardia
ase,21) yielding nabscessins A–C (1–3).
abcessus obtained when the strain was precultured in BHI+
The expression levels of these putative biosynthesis genes in medium5) before shifting to mCD medium (mCD with BHI+).
cells grown in mCD medium and in Waksman medium then Figure 7b shows the HPLC profile for the extract of Nocardia
were compared by the RNA-seq method. Notably, nabscessins abcessus obtained when the BHI+ medium used in precultur-

Vol. 66, No. 10 (2018)
Chem. Pharm. Bull.
979
Fig. 4. The Proposed Biosynthetic Pathway for Nabscessins A–C
sion into a tetraacetyl derivative (4) through hydrolysis and
acetylation. Gene analysis suggested the involvement of six
enzymes (A–F) in a proposed pathway for the biosynthesis
of nabscessins (Fig. 4); RNA-seq identified a corresponding
gene cluster that is selectively expressed in mCD, the growth
medium in which biosynthesis of the nabscessins is observed.
Experimental
General Experimental Procedures The following in-
struments were used in the present study: a P-2200 polarim-
eter (JASCO) for optical rotations; an ECZ-600 spectrometer
(JEOL) for NMR spectroscopy (solvent chemical shifts were
used as the internal standard); and a JMS-T100LP (JEOL)
for HR-ESI-MS. A HPLC system consisting of a PU1580
(JASCO) pump, along with UV 970 (JASCO) UV and RI
Fig. 5. Comparison of Extracts of Culture Broth Following Growth in
mCD or Waksman Medium
a) mCD medium, b) Waksman medium.
ing was removed from the strain (by centrifugation and wash- 1530 detectors, was used; chromatographic data were col-
ing with mCD medium before shifting to mCD medium; mCD lected using a chart recorder (ROSS). A Shimadzu LCMS
without BHI+). Notably, nabscessins A–C were produced in system (Shimadzu, Japan) consisting of LC-20AD pumps,
mCD with BHI+ (Fig. 7c), while these compounds were not DGU-12A3 online degasser, CTO-20A column oven, SIL-20A
produced in mCD without BHI+ (Fig. 7b). Thus, the pres- autosampler, SPD-M20A PDA detector, FCV-20AH2 valve
ence of the BHI+ medium used for preculture was essential unit, LCMS 2020 for ESIMS, and N2 Supplier Model 24F
for the production of nabscessins A–C in mCD medium. On for the N2 generator was used; chromatographic data were
the other hand, when Nocardia abcessus was co-cultured with collected and processed using LabSolution software (version
the mouse macrophage-like cell line J774.122) in mCD medium 5.42 SP4, Shimadzu). The conditions of the LCMS analysis
without BHI+, nabscessins A–C were produced (Fig. 7d); a were as follows : 0−100% MeOH in 0.1% HCOOH, 0−30min,
culture of the J774.1 cell line alone (single culture) did not linear gradient, and 100% MeOH in 0.1% HCOOH, isocratic
yield nabscessins A–C (Fig. 7a). These observations demon- 30−60min; flow rate: 0.2mL/min; UV detection: photodiode
strate that nabscessins A–C were produced by Nocardia ab- array (190−600nm); MS detection: ESI (positive and nega-
scessus only when the bacteria were cultured in mCD medium tive) (m/z 100−2000); guard column: Develosil ODS-HG-S
with BHI+ medium or J774.1.
(ϕ1.5×10mm, Nomura chemical, Japan); column: COSMOSIL
In the present study, a new molecule, nabscessin C (1), 5C18-AR-II (ϕ2.0×150mm, Nacalai Tesque, Japan). The fol-
was isolated from Nocardia abscessus IFM 10029T, and the lowing adsorbents were used for purification: Silica gel 60F254
absolute structure of nabscessin A was determined by conver- (0.25mm, Merck, Germany) and Silica gel 60 RP-8F254
S

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Chem. Pharm. Bull.
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Fig. 6. (a) A Schematic of the Putative Cluster and (b) Expression of Putative Nabscessin Biosynthetic Genes (A–F) in mCD Medium (Production)
Compared to That in Waksman Medium (Non-production) Based on Fold Change of RPKM (Reads per Kilobase of Exon per Million Mapped Se-
quence Read) Values
and Company), glucose (1g/100mL, Wako, Japan), and glyc-
erol (1mL/100mL, Nacalai Tesque) in a 10-mL Erlenmeyer
flask at 28°C for 5d with shaking (160rpm). Five milliliters
of the culture broth were added to 25mL of modified Cza-
pek-Dox medium consisting of sucrose (3g/100mL, Wako),
NaNO3 (0.3g/100mL, Wako), KH2PO4 (0.1g/100mL, Nacalai
Tesque), KCl (0.05g/100mL, Nacalai Tesque), MgSO4·7H2O
(0.05g/100mL, Wako), and FeSO4·7H2O (0.001g/100mL,
Nacalai Tesque), in a 50-mL Erlenmeyer flask. The strains
were cultured at 28°C for 1 week on mCD medium. The cul-
ture broth was centrifuged at 3000rpm, 1674×g for 20min
to give the supernatant and mycelial cake; the mycelial cake
was extracted with MeOH (12.5mL×2). The MeOH extract
was combined with the supernatant obtained above, and the
Fig. 7. Comparison of Extracts of Culture and Co-culture Broth in
mCD Medium
combined materials were partitioned with EtOAc (25mL×3)
and water. The EtOAc extracts then were analyzed by LC-MS.
a) Single culture, J774.1, b) single culture, Nocardia abscessus in mCD without
Fermentation for mCD Medium and Isolation An
BHI+, c) single culture, Nocardia abscessus in mCD with BHI+, d) co-culture,
aliquot (100µL) of the stock of strain Nocardia abscessus
Nocardia abscessus and J774.1 in mCD without BHI+.
IFM 10029T was added to 5mL of BHI+ liquid medium in
(0.25mm, Merck) for analytical TLC; Chromatrex ODS a 10-mL Erlenmeyer flask and cultured at 160rpm for 5d at
(Fuji Silysia Chemical, Japan) for column chromatography; 28°C. Aliquots (500µL each) of the resulting culture broth
COSMOSIL 5C18-AR-II (ϕ10.0×250mm, Nacalai Tesque) for were added to each of two 25-mL volumes of BHI+ liquid
preparative HPLC. The following instruments were used for medium in 200-mL Erlenmeyer flasks, and the resulting
cultivation and extraction: GeneQuant pro (GE) for OD600 cultures were incubated with shaking at 28°C for 5d. Por-
spectroscopy; FMC 1000 (EYELA), SOFT INCUBATOR tions (15mL each) of the resulting culture broth in BHI+
SLI-450ND (EYELA), and LTI-1200E (EYELA) for incuba- liquid medium were added to each of five 500-mL volumes
tion; DOUBLE SHAKER NR-30 (TAITEC) and Bio-Shaker of mCD medium in 1-L Erlenmeyer flasks, and the resulting
BR-30L (TAITEC) for shaking incubation; BIOLABO BL-171 cultures were incubated with shaking at 28°C for 1 week. The
(Juji Field, Japan) for CO2 incubation; and M150-IVD (Sa- resulting culture broth (2.0L) was centrifuged at 3000rpm,
kuma, Japan), HIMAC CENTRIFUGE (Hitachi, Japan), 1674×g for 20min to give the supernatant and mycelial cake;
KS-5000 (Kubota, Japan), and Avanti centrifuge HP-26XP the mycelial cake then was extracted with MeOH (1.0L×2).
(Beckman Coulter) for centrifugation.
The MeOH extract was combined with the 2-L supernatant
Microbial Strain Strains of the genus Nocardia were obtained above, and the combined materials were partitioned
stored in a freeze-dried state at the Medical Mycology Re- with EtOAc (2.0L×3) and water. The EtOAc layer (202.9mg)
search Center, Chiba University, Japan. The GenBank ac- was subjected to ODS column chromatography (ϕ14×320mm,
cession number of Nocardia abscessus IFM 10029T was MeOH–H2O system) to give fractions 1A–1O. Fraction 1F
BAFP00000000.1.
(MeOH:H2O=3:2, 6.7mg) was subjected to reverse-phase
Culture of Nocardia sp. in Modified Czapek-Dox Me- HPLC [COSMOSIL 5C18-AR-II (ϕ10.0×250mm); eluent: 40%
dium for LC-MS Analysis Each strain of Nocardia sp. MeOH; flow rate: 1.5mL/min; UV detection: photodiode array
was cultured in 5mL of BHI+ liquid medium consisting of (190−400nm)] to give compound 1 (0.5mg, tR 28.0min) and 3
Bacto™ brain heart infusion (3.7g/100mL, Becton, Dickinson (0.8mg, tR 40.0min). Compound 2 was obtained in fraction 1E

Vol. 66, No. 10 (2018)
Chem. Pharm. Bull.
981
(MeOH:H2O=7:3, 9.5mg).
A suspension of Nocardia abscessus was added to the flasks
Nabscessin C (1) Colorless amorphous solid. HR-ESI-MS until the cell number ratio was reached (J774.1: Nocardia ab-
m/z: 483.1426 [M+Na]+ (Calcd for C22H24N2O9Na: 483.1380). scessus.=1:10). Flasks were incubated at 28°C for 1 weeks.
1H- and 13C-NMR data are provided in Table 1. As shown in
(1R,2S,3S,4R,5S)-5-Acetamidocyclohexane-1,2,3,4-
Fig. S2 (Supplementary materials), compound 1 was unstable tetrayl Tetraacetate (4) Six mole aq. HCl (1.5mL) was
and other physical data of 1 were not determined. added to a solution of nabscessin A (2, 7.0mg, 0.015mmol),
Transcriptional Analysis RNA was extracted from the which was then stirred at 100°C for approximately 5d. After
strain Nocardia abscessus IFM 10029T cultured under nab- addition of DOWEX 2×8, the reaction mixture was evapo-
scessin-producing and non-producing conditions. mCD me- rated and EtOAc was added to the residue. The EtOAc solu-
dium was used for the nabscessin-producing conditions, while tion was extracted 3 times with H2O. After removal of H2O by
Waksman medium was used for the non-producing conditions. evaporation, pyridine (1.0mL) and acetic anhydride (500µL)
Specifically, the strain Nocardia abscessus IFM 10029T, pre- were added to the residue (5.6mg), and the mixture was then
cultivated on BHI+ medium at 28°C for 3d, was inoculated stirred at room temperature for 18h. After the solvent was
into nabscessin-producing and non-producing media. After in- removed, H2O was added and then the H2O solution was
cubation at 28°C for 7d, the cells of Nocardia abscessus IFM extracted 3 times with EtOAc. The organic layer was evapo-
10029T cultured in either of the two media were collected and rated in vacuo and the residue was separated by reverse-phase
resuspended in RNeasy Mini Kit (Qiagen, U.S.A.) Buffer RLT HPLC [COSMOSIL 5C18 AR-II (ϕ10.0×250mm); eluent: 40%
with β-mercaptoethanol. The resuspended cells were homog- MeOH; flow rate: 1.5mL/min; UV detection: photodiode array
enized using a multi-bead beater (Yasui Kikai) at 2500rpm for (190−400nm)] to give 4 (1.0mg, 0.0026mmol, 18% yield, tR
60s at 4°C, and RNA was extracted according to the RNeasy 18.0 min).
Mini Kit protocol. Following the deoxyribonuclease (DNase)
1H-NMR (600MHz, CDCl3) δ: 5.62 (1H, d, J=8.3Hz),
treatment, total RNA was treated with a Ribo-Zero Gram-pos- 5.17 (1H, t, J=9.6Hz), 5.10 (1H, t, J=9.6Hz), 4.98 (1H, ddd,
itive bacteria Kit (Illumina) to remove rRNA. For construction J=12.4, 9.6, and 4.8Hz), 4.89 (1H, dd, J=11.0 and 9.6Hz), 4.15
of the RNA-seq libraries, each of the mRNA samples obtained (1H, m), 2.42 (1H, dt, J=12.4 and 4.8Hz), 2.03 (3H, s), 1.99
were treated with a KAPA Stranded RNA-seq Kit (Kapa (3H, s), 1.90 (9H, s), 1.47 (1H, q, J=12.4Hz). [α]D26−10.6 (c 0.5,
Biosystems, U.S.A.) according to the manufacturer’s protocol. CHCl3). ESI-MS m/z: 374 [M+H]+.
The constructed libraries were sequenced using the HiSeq
system (Illumina). The resulting data were mapped against the
sequenced genome data of Nocardia abscessus IFM 10029T KAKENHI Grant Number 17H03992 and the Strategic Prior-
using CLC GenomicsWorkbench 6.5.1 (CLC bio). ity Research Promotion Program of Chiba University, titled
Acknowledgments This study was supported by JSPS
Seed Culture of a Mouse Macrophage-Like Cell Line “Phytochemical Plant Molecular Sciences.”
(J774.1) in a 75-cm2 Cell Culture Flask A mouse macro-
phage-like cell line (J774.1) was incubated at 37°C in a 5%
CO2 atmosphere in 15mL of Dulbecco’s modified Eagle’s interest.
medium (DMEM) (Wako) with 10% fetal bovine serum (FBS,
Conflict of Interest The authors declare no conflict of
Bio West) in 75-cm2 cell culture flasks (Violamo), which
Supplementary Materials The online version of this ar-
reached approximately 80–90% confluency after 5–7d. Old ticle contains supplementary materials.
culture medium was then removed and 5mL of DMEM with
10% FBS medium was added to the flask. Cells were scraped
and collected from the bottom of the flask with a cell scraper.
Cell medium was transferred to a 50-mL tube, which was
centrifuged at 2000rpm at 20°C for 5min. After the removal
of medium, 5mL of DMEM with 10% FBS medium was
added to the tube. The residue was stirred by pipetting and
transferred to a hemocytometer. The number of viable cells in
the hemocytometer was counted using trypan blue. Cell me-
dium was added to each new cell culture flask with 25mL of
DMEM with 10% FBS medium. Cells were cultured for 24h.
Seed Culture of Nocardia abscessus for a Co-culture in
mCD Medium Nocardia abscessus was cultivated in 5mL
of BHI+ liquid medium in a 10-mL Erlenmeyer flask at 28°C
for 5d with shaking (160rpm). The culture broth was added to
a 50-mL tube. The supernatant was removed after centrifuga-
tion at 3000rpm at 20°C for 2min. 2mL of mCD medium was
added to the tube. The supernatant was removed after cen-
trifugation at 3000rpm at 20°C for 2min. A strain suspension
was prepared by the addition of 11mL of mCD medium to the
tube. OD600 was measured for counting the number of strain.
Co-culture in mCD Medium in a Cell Culture Flask
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