Supplementary Information
Supplementary Materials and Methods
N-acetylglucosamine Identification
N-acetylglucosamine was identified according to He et al. [1] with some minor modifications as
follows. The sample was heated at 105 °C with 6 M HCl for 8 h. After cooling, the hydrolysate was
centrifuged, the supernatant was mixed with myoinositol as an internal standard, and the solution was
then dried by a stream of N2 gas. Sample derivatization was performed as follows: methoxyamine
hydrochloride dissolved in pyridine (20 mg/ml) was added and incubated at 37 °C for 2 h, then
N,O-bis(trimethylsily)trifluoroacetamide + trimethychlorosilane (Supelco, USA) was added and
incubated for another 30 min. The trimethylsilyl methoxime derivatives were run on the GC/MS
instrument (6890N-5975B, Agilent Technologies, USA) with helium as the carrier gas on a J&W
DB-5MS column (30 m × 0.25 mm × 0.25 m, Agilent Technologies, USA). The molecular mass was
confirmed by mass spectrometry in electron ionization mode. The derivative groups on
N-acetylglucosamine were lost during fragmentation, which produced the characteristic base peaks at
217.1 (m/z) in Supplementary information, Figure S1.
Southern Blot Hybridization
Southern blot hybridization was performed using DIG High Prime DNA labeling and detection
starter kit I (Cat# 11745832910), and the primers of the probe to detect the Synechococcus 7002 cesA
mutant and wild type (Supplementary information, Figure S5) were:
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7002 cesA-1 5’-TCTGTAAACCTTCCGTTACATCTTCAGAGG-3’
7002 cesA-2 5’-GCTCGCCAAGAAGTTAAATTAAAGAAAAATAAATTCG-3’
Primers for the probes to detect the genes (Supplementary information, Figure S2) from G. xylinus were:
cmc-1 5-TGCCCGACAAGAACAATGCCACAG-3
cmc-2 5-GGAATACTGCCCACCTTCGACACCA-3
ccp-1 5-AGGCACTTCGATCAGGCGGAGCA-3
ccp-2 5-CATGAACACTTCGGCCATTGTGGG-3
acsA-1 5-ATGCCAGAGGTTCGGTCGTCAACGCAG-3
acsA-2 5-GGTCGCATCCCAGAAATCGTTGCC-3
acsB-1 5-CGAGCAGTATATCGGCACGTTGCGTC-3
acsB-2 5-GCCACGTATAAAGATCGGGCGGAATG-3
acsC-1 5-CAATCGTGATGCCGCCCTTGATACG-3
acsC-2 5-GACGCCAGTGCGCCGCATTCTTG-3
acsD-1 5-CCCTGTTTCTTCAGACCCTGTCATGG-3
acsD-2 5-GGAACTGCGCACGCGCATGTACATGAT-3
bgl-1 5-GTTGCTGTCTGCCGTGGCGTTTG-3
bgl-2 5-GCCGGGATATTTCCAGTCCTGCTTC-3
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Supplementary References
1.
He HB, Xie HT, Zhang XD, Wang YH, Wu YY. A gas chromatographic/mass spectrometric
method for tracing the microbial conversion of glucose into amino sugars in soil. Rapid Commun
Mass Sp 2005; 19:1993-1998.
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Supplementary Figures S1 to S6
Figure S1 Analysis of components of the PG layer by GC-MS. The isolated PG was hydrolyzed in 6 M
HCl before analysis by gas chromatography (GC) and mass spectroscopy (MS). Panels A through D show
GC analyses of an N-acetylglucosamine standard (A), isolated peptidoglycan from Synechococcus 7002
WT (B), isolated peptidoglycan from Synechococcus 7002 WT after treatment with cellulase (C), and
isolated peptidoglycan from the Synechococcus 7002 cesA mutant (D), respectively. The fractions
containing N-acetylglucosamine were subjected to further analysis by mass spectroscopy using electron
impact mode, and the results are shown in panels E through H, respectively. The base peak at m/z = 217.1
is characteristic of N-acetylglucosamine from which four trimethylsilyl groups have been removed by
fragmentation during the mass spectroscopic analysis.
Figure S2 (A) Physical map of the gene organization in the cellulose synthesis module (CSM) from G.
xylinus. (B) Analysis of individual genes of the CSM in strain CM12 by Southern blot hybridization.
Figure S3 Low-temperature (77 K) fluorescence emission spectra of cells of strain CM 12 grown in
high-salinity medium A+ over a 12-day time course of cellulose production. The excitation wavelength
was 440 nm to excite chlorophyll a selectively. The emission at 685 to 695 reflects emission from
Photosystem II and that at ~720 nm emission from Photosystem I. Note that the amplitude on day 12 is
approximately half that on day 1, which agrees well with the images shown in Figure 6B.
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Figure S4 Low-temperature (77 K) fluorescence emission spectra of cells of strain CM 12 grown in
low-salinity medium BG-11 over a 12-day time course of cellulose production. The excitation wavelength
was 440 nm to excite chlorophyll a selectively. The emission at 685 to 695 reflects emission from
Photosystem II and that at ~720 nm emission from Photosystem I. Note the dramatic decrease in
fluorescence amplitude between day 5 and day 10, which agrees well with the microscopic images shown
in Figure 6B. Also note that cellulose production ceases at about day 11 (see Figure 6A), when there is no
longer any fluorescence from chlorophyll a.
Figure S5 (A) Analysis of the cesA insertion mutant strain of Synechococcus 7002 by Southern blot
hybridization. Total DNA was isolated from the wild type and the mutant strains and subjected to BamHI
and HincII digestion. The DNA fragments were transferred to a nylon membrane after agarose gel
electrophoresis. Fragments hybridizing to the probe described in Materials and Methods were detected by
enhanced chemiluminescence. The molecular size markers are shown on the left side of the gel. (B)
Schematic drawing of the construction of cesA mutant by insertion of an aphA-II cartridge, which confers
resistance to kanamycin (KmR), into the cesA gene of Synechococcus 7002.
Figure S6 Diagram showing the physical map for plasmid pAQ1EX::PcpcBA::cesA-yfp.
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