DSSG11 : A bacterial dipeptide as potential high pressure

DSSG11 : A bacterial dipeptide as potential high pressure osmolyte
Adaptation in Deep Sea Bacteria The deep sea still presents many mysteries, with details of how
microorganisms are capable of growth under such extreme pressures being very scarce. With regard to growth
under elevated pressures, organisms can be divided into 3 classes: 1) those that grow optimally under
pressures between >0.1 MPa and <60 MPa are piezophiles, 2) Organisms that are capable of growth under high
pressure, but not optimally, are piezotolerant, 3) and those which have reduced growth at highpressures are
piezosensitive1.
Bacterial adaptation to high pressure that has been studied to date shows either change in membrane and/or
change in enzyme composition. The outer membrane prevents the diffusion of molecules, such as detergents
(SDS and DOC), antibiotics (rifampicin and actinomycin), and dyes (eosine and methylene blue). This change in
membrane composition affects sensitivity of the bacteria to certain stressors2. It is likely that in order to
maintain normal growth that primary and secondary metabolism is affected, but very little has been published
in this regard. We propose that small molecule compatible solutes (osmolytes) are produced in response to
elevated hydrostatic pressures and act as high pressure osmolytes (piezolytes).
Secondary metabolites from Deep Sea Microorganism Micromonospora is a genus of bacteria belonging to the
Phylum Actinobacteria and is known as a prolific producer of novel bioactive natural products with interesting
chemical structures3. Many secondary metabolites have been found since then such as thiocoraline from
Micromonospora sp. L-13-ACM2-0924, micromonospolides5, micromonomycin6, indolocarbazole derivatives
staurosporines7, lomaiviticin A from Micromonospora lomaivitiensis8, and diazepinomicin from
Micromonospora DPJ129.
Figure 1. N-acetylglutaminyl glutamine amide
from Mariana Trench Micromonospora strain MT25
Preliminary data obtained on the piezotolerant Micromonospora strain MT2510 isolated from sediment
obtained from Challenger deep in the Mariana trench (ca 11,000 m) shows that it produces a dipeptide Nacetylglutaminyl glutamine amide (NAGGN) which is a known osmolyte11 (Wael Abdel-Mageed PhD Thesis
2010, University of Aberdeen).
The aim of this project is to investigate the correlation between concentrations of NAGGN in Micromonospora
MT25 at different high pressures.
Table 1. Procurement of NAGGN in different media composition.
Media
Culture Growth
NAGGN Product
ISP2
+++
-
ISP2 + CaCO3
+++
-
ISP2 + Monosodium Glutamic Acid
++
+
ISP2 + Monosodium Glutamic Acid
+ (NH4)2SO4 in pH 9
+
-
ISP2 + L-Glutamine
-
-
GYE
+++
-
Marine Broth
+++
-
Experimental Results Micromonospora strain MT25 was grown at atmospheric pressure in different medium
and we used standard isolation and characterisation procedures available in the Marine Biodiscovery Centre to
obtain NAGGN. The result was shown in table 1.
Using HPLC-MS based quantification procedure to allow us to identify and quantify the amount of NAGGN
produced under elevated pressures. The LC-MS chromatogram showed that NAGGN occurred at early
retention time (Figure 2). Due to the small amount of NAGGN that being produce by Micromonospora MT25,
two other methods to obtain NAGGN have been conducted. First method is producing NAGGN synthetically
using precursor compounds and the second method is biosynthesis of NAGGN with Sinorhizobhium meliloti.
D:\LCMS-Data\20140116\HSP217
1/17/2014 11:24:16 AM
B:30
NL:
4.83E4
Total Scan
P DA HSP 217
0.47
uAU
40000
20000
0.67 1.83
2.70
0
Relative Abundance
0
2
Relative Absorbance
4
13.40
8.23 9.47 10.57 11.47 12.40
7.33
6
8
10
12
14
16
Time (min)
25.70
21.80 22.67
18.63
15.93
25.87
26.20
18
20
22
24
27.67 28.57
26
28
30
NL: 1.33E6
Base Peak F:
FTMS + p ESI Full
ms
[100.00-2000.00]
MS HSP217
25.95
100
0.58
50
2.83
3.11
3.84
4.14
5.99 6.86 7.46 8.23
9.98 10.55 12.14
16.26
18.98 19.34 19.92 20.59
14.23 14.83
22.71
22.87
26.50 27.36 29.03
25.32
0
0
Relative Abundance
4.63 4.90 5.10
2
4
6
8
10
12
14
16
Time (min)
18
20
22
24
26
28
30
100
220.0000
249.0000
50
0
200
378.0000
210
220
240
250
260
270
280
290
NH2
527.1582
338.1435
100
354.1175
476.1380
190.0125
314.0849
200
300
H2N
585.3188
381.0795
400
500
600
700
HN
H
N
O
320
330
340
350
360
370
380
851.2651
800
900
390.0000
390
400
O
NH2
O
O
C hem ica l Form ula: C 12 H 21 N 5 N aO 5 +
E xact M ass : 338 .14 35
689.2112
705.1854
300
310
wavelength (nm)
Na
O
50
0
100
230
1013.3149 1114.3969
1000
1100
1875.1384
1234.4668 1311.7848
1200
1300
1463.6653 1556.4650
1400
1500
1600
1734.6232
1700
1800
1900
2000
m/z
Figure 2. Presence of NAGGN [M+Na]+ in media ISP2+MSG
Synthesis of NAGGN using Rapid Repetitive Solution Phase Peptide Synthesis (RRSPS)12 is undergoing and
showed positive result in its half steps. In the same time, the production of NAGGN using biosynthesis from
Sinorhizobium meliloti13 is being carried out at early step of cultivation and upscaling the culture.
The Micromonospora MT25 culture in the high-pressure vessel is being carried out at constant temperature
(28oC) in ISP2+MSG in sealed bags containing a small amount of Fluorinert FC72 saturated with oxygen to
provide oxygen during cultivation.(Wael Abdel-Mageed PhD Thesis 2010, University of Aberdeen).
At the moment, the experiment results have not shown that NAGGN works as an piezolytes just yet, since
obtaining pure NAGGN as calibration standard was challenging. But the results had shown that for
Micromonospora MT25, NAGGN did not show any activity as an osmolytes. Hence, the possibilities of the use
of NAGGN in Micromonospora MT25 still remain undiscovered.
References.
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