Osmotically induced synthesis of the dipeptide N-acetylglutaminylglutamine amide is mediated by a new pathway conserved among bacteria

Article abstract of DOI:10.1073/pnas.1003063107

The dipeptide N-acetylglutaminylglutamine amide (NAGGN) was discovered in the bacterium Sinorhizobium meliloti grown at high osmolarity, and subsequently shown to be synthesized and accumulated by a few osmotically challenged bacteria. However, its biosyn

Full text of DOI:10.1073/pnas.1003063107

Osmotically induced synthesis of the dipeptide
N-acetylglutaminylglutamine amide is mediated
by a new pathway conserved among bacteria
Brice Sagota, Marc Gaysinskib, Mohamed Mehiric, Jean-Marie Guigonisd, Daniel Le Ruduliera, and Geneviève Alloinga,1
aLaboratoire Interactions Biotiques et Santé Végétale Unité Mixte de Recherche, Centre National de la Recherche Scientifique 6243, Institut National de la
Recherche Agronomique 301, Université de Nice-Sophia Antipolis, 06903 Sophia Antipolis, France; bPlateforme Technologique de Chimie, Institut de Chimie
de Nice, 06108 Nice, France; cLaboratoire de Chimie des Molécules Bioactives et des Arômes Unité Mixte de Recherche, Centre National de la Recherche
Scientifique 6001, 06108 Nice, France; and dPlateforme Bernard Rossi Protéome et Métabolome, Institut Fédératif de Recherche 50, 06107 Nice, France
Edited by Sharon R. Long, Stanford University, Stanford, CA, and approved May 14, 2010 (received for review March 11, 2010)
The dipeptide N-acetylglutaminylglutamine amide (NAGGN) was
discovered in the bacterium Sinorhizobium meliloti grown at high
osmolarity, and subsequently shown to be synthesized and accu-
mulated by a few osmotically challenged bacteria. However, its
biosynthetic pathway remained unknown. Recently, two genes,
which putatively encode a glutamine amidotransferase and an ace-
tyltransferase and are up-regulated by osmotic stress, were identi-
fied in Pseudomonas aeruginosa. In this work, a locus carrying the
orthologous genes in S. meliloti, asnO and ngg, was identified, and
the genetic and molecular characterization of the NAGGN biosyn-
thetic pathway is reported. By using NMR experiments, it was
found that strains inactivated in asnO and ngg were unable to pro-
duce the dipeptide. Such inability has a deleterious effect on S.
meliloti growth at high osmolarity, demonstrating the key role of
NAGGN biosynthesis in cell osmoprotection. β-Glucuronidase activ-
ity from transcriptional fusion revealed strong induction of asnO
expression in cells grown in increased NaCl concentration, in good
agreement with the NAGGN accumulation. The asnO–ngg cluster
encodes a unique enzymatic machinery mediating nonribosomal
peptide synthesis. This pathway first involves Ngg, a bifunctional
enzyme that catalyzes the formation of the intermediate N-
acetylglutaminylglutamine, and second AsnO, required for subs-
equent addition of an amide group and the conversion of N-
acetylglutaminylglutamine into NAGGN. Interestingly, a strong
conservation of the asnO–ngg cluster is observed in a large num-
ber of bacteria with different lifestyles, such as marine, symbi-
otic, and pathogenic bacteria, highlighting the ecological impor-
tance of NAGGN synthesis capability in osmoprotection and also
potentially in bacteria host–cell interactions.
volved in uptake or synthesis of compatible solutes are often reg-
ulated in an osmotically fashion at the transcriptional level, and
this process contributes to the finely tuned osmolyte buildup (3).
Among endogenous synthesized osmolytes, the dipeptide
NAGGN has been originally identified in osmotically stressed
cultures of the alfalfa root-symbiont Sinorhizobium meliloti (4).
Ever since, its presence has only been reported in a very limited
number of other soil bacteria from the Pseudomonas genus, and in
few marine bacteria (5, 6). Through physiological and biochemi-
cal approaches, some characteristics of NAGGN accumulation
have been analyzed in S. meliloti (4) and in the human pathogen
Pseudomonas aeruginosa (7). In both bacteria, the endogenous
pool of NAGGN increases with osmolarity and, in the absence of
exogenous osmoprotective compound, the dipeptide is the pre-
dominant solute at high salt concentration. Interestingly, when
GB is added to the growth medium the amount of endogenously
synthesized NAGGN is severely reduced (7, 8). In a pioneering
study, Smith and collaborators have showed that the dipeptide
synthesis does not occur ribosomally, and using an in vitro ap-
proach with crude extracts of S. meliloti cells, they have proposed
that an intermediate, the N-acetylglutaminylglutamine (NAGG),
can be produced from N-acetylglutamine and glutamine. In ad-
dition, they have suggested that the osmotically stimulated accu-
mulation of the dipeptide requires a genetic induction. Although
several mutants were isolated, none was completely devoid of
NAGGN (9), and the molecular characterization of the dipeptide
biosynthetic pathway has not been conducted so far. Recently,
a microarray analysis of P. aeruginosa PAO1 transcriptional re-
sponse to osmotic stress has revealed the up-regulation of two
contiguous genes that putatively encode a glutamine amidotran-
sferase and an acetyltransferase (10). Mutant strains in these two
genes were impaired for growth in the presence of high NaCl
concentration, and the authors made the presumption that these
two genes might be implicated in the synthesis of NAGGN.
In this study, we report a genetic and molecular analysis of
genes involved in the biosynthesis of NAGGN, the osmotically
induced genes asnO and ngg of S. meliloti. We present evidence
that Ngg triggers the formation of the dipeptide bond, producing
the intermediate NAGG that is subsequently converted into
NAGGN by AsnO. Remarkably, Ngg represents a unique enzy-
matic machinery mediating nonribosomal peptide synthesis.
Furthermore, a comparison with all available bacteria sequences
revealed that ngg and asnO are highly conserved in a large range of
osmotic stress rhizobium peptide synthesis GCN5-related
|
|
|
N-acetyltransferase glutamine amidotransferase
|
acteria that thrive in environments of elevated osmolarity
Bpossess specific mechanisms to adjust their internal osmotic
status, and one of the widely used is the ability to accumulate low
molecular weight organic osmolytes, the so-called compatible
solutes (1), which do not interfere with cellular functions. The
spectrum of compatible solutes identified is limited and com-
prises sugars, polyols, amino acids and derivatives, quaternary
amines and their sulfonium analogues, sulfate esters, and the di-
peptide N-acetylglutaminylglutamine amide (NAGGN). Usually,
a given bacterium employs a selection of several osmolytes, ac-
cumulated from external source or from de novo synthesis, and
the composition of intracellular compatible solutes largely de-
pends on the growth conditions (2). The genetic processes in-
volved in the accumulation of compatible solutes have been in-
vestigated in many bacteria, and various genes encoding uptake
systems have been cloned and functionally characterized. In
contrast, only few biosynthetic pathways of osmolytes have been
completely elucidated at the molecular level, such as the most
widely used ectoine, glycine betaine (GB), or trehalose. Genes in-
Author contributions: B.S., M.M., and G.A. designed research; B.S., M.G., M.M., J.-M.G.,
and G.A. performed research; B.S., M.G., M.M., J.-M.G., D.L.R., and G.A. analyzed data;
and D.L.R. and G.A. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: alloing@unice.fr.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1003063107/-/DCSupplemental.
12652–12657
|
PNAS
|
July 13, 2010
|
vol. 107
|
no. 28
www.pnas.org/cgi/doi/10.1073/pnas.1003063107

bacteria with different lifestyles, including halophiles, mesophiles,
and both plant and human pathogens. Thus, contrary to the pre-
sumption that NAGGN biosynthesis is limited to a few bacterial
species, this study provides a broader insight into the possible role
of NAGGN in enhancing the ability of bacteria to adapt flexibly to
varying conditions in their habitats and to colonize different
ecological niches.
The product of SMb20481 has been previously named AsnO
in view of its similarity with the AsnO protein of Bacillus subtilis
(11). The sequence of both proteins shows residues conserved
in members of the PurF family of glutamine-dependent amido-
transferases, which use glutamine as nitrogen source for the bio-
synthesis of various compounds (12). Within this superfamily,
proteins that belong to the classB asparagine synthetase family,
like Escherichia coli AsnB or B. subtilis AsnO, catalyze specifically
the transfer of the amide nitrogen of glutamine to aspartic acid,
and produce asparagine (13, 14). Such specificity could not be
ascribed to the S. meliloti AsnO, suggesting that another acceptor
molecule is aminated by the enzyme (11). This work also reported
that the product of SMb20482 encodes a protein with two distinct
regions, an N-terminal domain conserved in acetyltransferases
and a C-terminal domain conserved in cyanophycin synthetases.
The refined sequence analysis conducted here reveals that this
protein might be a bifunctional enzyme with N-acetylating and
peptide bond–forming activities. For the reasons developed here
and described later, the SMb20482 product was called Ngg for
N-acetylglutaminylglutamine synthetase. The N-terminal domain
is typical of N-acetyltransferases from the GCN5-related N-
acetyltranferase (GNAT) superfamily. These enzymes are involved
in the N-acylation of aminoglycosides, peptides, polyamines, pro-
teins, and other molecules (15). They acylate their substrate by
using an acylCoA and share a structurally conserved fold, the
GNAT fold, involved in binding of the acyl donor (16). Alignment
between a part of the Ngg N-terminal sequence and three GNAT
members showed the presence of the motifs involved in this fold
in the S. meliloti sequence (Fig. 1B), suggesting that Ngg catalyzes
an N-acylation reaction. On the contrary to the N-terminal domain,
the C-terminal domain of Ngg groups within the ATP-grasp family
of ATP-dependent ligases. These enzymes, which catalyze the
ATP-dependent ligation of a carboxylate containing molecule to an
Results
S. meliloti Genome Contains a Locus Putatively Involved in NAGGN
Synthesis. A BLAST search for proteins homologous to the prod-
ucts of PA3459 and PA3460 from P. aeruginosa PAO1 (10) was
performed against the S. meliloti genome database (http://iant.
toulouse.inra.fr/bacteria/annotation/cgi/rhime.cgi). Two proteins
encoded by the contiguous ORFs SMb20481 and SMb20482 of
the pSymb replicon were identified. The SMb20481 product is
homologous to the putative glutamine amidotranferase encoded
by PA3459 (55% identical amino acids) and the SMb20482 pro-
duct shares 53% identical amino acids with the putative acetyl-
transferase encoded by PA3460. These high identities extend over
the entire length of the proteins, which have similar size (591 res-
idues vs. 589 for the products of SMb20481 and PA3459, respec-
tively, and 595 vs. 585 for the products of SMb20482 and PA3460,
respectively). DNA sequence analysis suggested that the two coding
sequences belong to the same transcriptional unit, as they are sep-
arated by only 1 bp. A cluster of five genes potentially encoding
the four components of a dipeptide ABC transporter (SMb20476
to SMb20479) and a transcriptional regulator (SMb20480) is lo-
cated 122 bp upstream of SMb20481. At the 3′ extremity of the
locus, another putative transcriptional regulator is encoded by
a gene (SMb20483) divergently transcribed compared with SMb-
20482 (Fig. 1A).
Fig. 1. The NAGGN encoding region of S. meliloti. (A) Genetic organization of the asnO–ngg region on the pSymb replicon. The location of the Tn5 insertions
(arrowheads) and the position of the restriction sites and primers used for ngg cloning in pSM37 and for ngg inactivation by pSM34 integration are indicated.
(B, BamHI, E, EcoRI, H, HindIII, S, SalI, X, XhoI.) (B) Homology of Ngg with members of the GNAT and ATP-grasp superfamilies. (Upper) Alignment of the
N-terminal region of Ngg with three GNAT proteins. [AAC(6′)-Ii, aminoglycoside 6′-N-acetyltransferase type Ii from Enterococcus faecium (accession no.
1N71D); YpeA, N-acetyltransferase from E. coli (accession no. A1ADU8); YsnE, acetyltransferase from B. subtilis (accession no. 1YX0A).] The boxes correspond
to the regions designated motifs C, D, A, and B by Neuwald and Landsman (16). (Lower) Alignment of the C-terminal region of Ngg with the conserved
regions in three ATP-grasp proteins. [CphA, cyanophycin synthetase from Synechocystis sp. strain PCC6308 (accession no. AAF43647); GshAB, glutathione
biosynthesis bifunctional protein from Streptococcus agalactiae (accession no. Q8DXM9); DlbB, D-alanine:D-alanine ligase from E. coli (accession no.
BAB96660).] *DlbB residues interacting with ATP; °DlbB residues interacting with Mg2+ (17).
Sagot et al.
PNAS
|
July 13, 2010
|
vol. 107
|
no. 28
|
12653

amino or thiol group–containing molecule, present an ATP-grasp
fold with conserved amino acid residues in the regions of the
phosphate-binding loop and the Mg2+ binding site (Fig. 1B) (17).
They participate to the synthesis of molecules containing peptide
bonds such as cyanophycin, peptidoglycan, or glutathione (18, 19).
In view of the fact that NAGGN is a modified dipeptide formed
by an N-acetylated glutamine and a glutamine carrying an extra
amide group, the potential catalytic properties of AsnO and Ngg
are consistent with their involvement in NAGGN biosynthesis, as
we propose in Fig. 2. In a first step, Ngg catalyzes both the N-
acetylation of one glutamine and the formation of a peptide bond
with a second glutamine, producing the intermediate NAGG. In
a second step, the protein AsnO transfers the amide nitrogen of
another free glutamine to the second glutamine of NAGG, giving
rise to NAGGN.
A
Rm2011 (WT)
AsnO°
UNA442 (ngg)
glutamate
NAGGN
185
180
ppm
55
55
55
50
45
40
40
40
35
30
ppm
asnO or ngg Inactivation Stops NAGGN Synthesis. To assess the role
of the asnO–ngg cluster in NAGGN synthesis by S. meliloti, we
compared the capacity of the WT strain Rm2011, an asnO mutant,
isolated after transposon mutagenesis (20) and called here AsnO°,
and an ngg mutant (strain UNA442) to accumulate NAGGN. Cul-
tures were grown to late exponential phase in MCAA growth me-
dium containing 0.3 M NaCl, and the cellular content was analyzed
by 13C NMR. NAGGN and glutamate were accumulated in the
WT strain as previously observed (4), whereas only glutamate could
be detected in both mutant strains (Fig. 3A), demonstrating that
the integrity of asnO–ngg cluster is required for NAGGN synthesis.
B
AsnO1/2
* *
*
*
*
* *
*
*
AsnO° pSM37
185
180
ppm
50
45
35
30
ppm
synthetic NAGG
C
purified NAGG
Ngg Is Involved in the Synthesis of NAGG. According the pathway
proposed earlier (Fig. 2), a strain deficient in AsnO should ac-
cumulate a NAGGN intermediate generated by Ngg. To explain
why this intermediate was not characterized from the AsnO°
extracts, we postulated that the Tn5 insertion in asnO gene had
a polar effect on the expression of the downstream ngg gene
because the asnO–ngg cluster is an operon. Consequently, an
asnO mutant created by a potentially leakier insertion and called
here AsnO1/2 was analyzed as described before. In this strain, the
transposon was inserted 1,103 bp downstream the beginning of
asnO coding sequence instead of 164 bp in AsnO° (Fig. 1 and
Table S1; also see http://www.cebitec.uni-bielefeld.de/CeBiTec/
rhizogate). A 13C NMR spectrum of an extract from salt-stressed
cells of AsnO1/2 showed the presence of fair unidentified signals
in addition to the already observed glutamate signals (Fig. 3B).
To confirm the correlation between these signals and a functional
ngg, this gene was expressed from a stable plasmid (pSM37) in the
185
180
ppm
50
45
35
30
ppm
Fig. 3. The asnO–ngg cluster is involved in NAGGN synthesis pathway in
S. meliloti. The solutes accumulated by strains (A) Rm2011 (WT), AsnO°,
UNA442; and (B) Asno1/2, AsnO° pSM37, grown in MCAA medium containing
0.3 M NaCl were extracted and analyzed by 13C NMR. Spectra of chemically
synthesized glutamate and NAGGN are also shown. Asterisks indicate the
fair signals detected in addition to glutamate signals in AsnO1/2 spectrum.
(C) Carbon 13 NMR spectra of synthetic NAGG and of purified NAGG from
AsnO° pSM37. Only regions that contain osmolyte signals (ranges of 185–175
and 60–24 ppm) are shown.
AsnO° mutant. Carbon 13 NMR analysis of osmolytes accumu-
lated in such salt-stressed cells revealed the signals detected in the
cell extracts of AsnO1/2, and thus corroborated their Ngg origin
(Fig. 3B). To obtain structural information on the product syn-
thesized by Ngg, a combination of one-dimensional and 2D NMR
analyses was performed on the extracts from the strain AsnO°
pSM37. These analyses offer evidence for the formation of
a peptide compound structurally similar to NAGG (SI Materials
and Methods, Table S2, and Fig. S1). Additional support for this
structural assignment was obtained by comparison of NMR data
for the purified compound with those for a synthetically prepared
NAGG (Fig. 3C and SI Materials and Methods). The slight var-
iations in chemical shift positions between the NAGG in cellu-
lar extracts and after purification (compare Fig. 3B with Fig. 3C)
are attributed to differences in the osmolyte environment. Fur-
thermore, the purified NAGG exhibited the expected molecular
mass (SI Materials and Methods and Fig. S2). Altogether, these
results demonstrated that the ngg gene mediated the NAGG
synthesis and confirmed the proposed pathway.
NH2-CH-COOH
(CH2)2
NH2-CH-COOH
(CH2)2
CO
NH2
CO
NH2
+
Gln
AcCoA
Ngg
CoA
CH3-CO-NH-CH-CO-NH-CH-COOH
(CH2)2
CO
(CH2)2
CO
NH2
NH2
NAGG
Gln
Glu
AsnO
Incapacity to Synthesize NAGGN Is Deleterious for Growth of S. meliloti
at High Osmolarity and Fairly Compensated by Exogenous GB. NAGGN
is accumulated only at high osmolarity and thus, has been con-
sidered as a compatible solute. To get direct evidence, various
growth experiments using, in parallel, the WT strain Rm2011 and
the AsnO° mutant strain were performed. Clearly, growth of the
asnO mutant was more affected than growth of the WT strain in
CH3-CO-NH-CH-CO-NH-CH-CO-NH2
(CH2)2
CO
(CH2)2
CO
NH2
NH2
NAGGN
Fig. 2. Pathway for the biosynthesis of NAGGN in S. meliloti.
12654
|
www.pnas.org/cgi/doi/10.1073/pnas.1003063107
Sagot et al.

NAGGN Biosynthesis Pathway Is Conserved. Until now, accumula-
tion of NAGGN has been demonstrated only in few bacteria such
as various Pseudomonas species (22). To investigate the possible
presence of NAGGN synthesis pathway in other organisms,
a search for proteins homologous to AsnO and Ngg has been
conducted. Such proteins have been detected only in the bacterial
databases; 60 species have a gene encoding a protein closely re-
lated to AsnO, and among them 39 have an ngg homologue. The
asnO and ngg genes are always contiguous and form a potential
operon in which asnO is the first gene transcribed. The identities
between AsnO and Ngg from S. meliloti and their respective
homologues are particularly high, varying in the case of AsnO
from a maximum of 94% identical residues (Sinorhizobium med-
icae) to a minimum of 51% (Mycobacterium sp. MCS), and in the
case of Ngg from 91% (S. medicae) to 49% (Hahella chejuensis;
Table S3).
The asnO–ngg arrangement is present in the genome of bac-
teria from Proteobacteria (α, β, γ classes), Actinobacteria (acti-
nobacteria class), and chlorobi (chlorobia class) phyla. It is
scattered within a few families and can occur in certain species of
a given genus and not in others in which the complete sequence
is available, e.g., an ansO–ngg type cluster is present in Methyl-
obacterium nodulans and sp. 4-46 but not in Methylobacterium
radiotolerans, Methylobacterium populi, or Methylobacterium
extorquens, or in several Mycobacterium species, but not in My-
cobacterium leprae, Mycobacterium tuberculosis, Mycobacterium
marinum, or Mycobacterium ulcerans. This type of sporadic dis-
tribution in taxonomically distant bacteria is often associated to
operon genes encoding secondary metabolites and inherited by
horizontal gene transfer (23). Phylogenetic relationships among
homologues of AsnO and Ngg are consistent with an acquisition
via horizontal gene transfer, as the corresponding phylogenetic
trees revealed clustering patterns that are not congruent with the
taxonomic status of the strains based on the topology of 16S
rRNA gene tree (Fig. S3).
Several marine bacteria possess an asnO–ngg cluster, i.e., the
three chlorobia, several proteobacteria (Aurantimonas sp. SI85-
9A1, Fulvimarina pelagi, α-proteobacterium, H. chejuensis, Stappia
aggregata, Labrenzia alexandrii, Brevundimonas sp. BAL3), and
actinobacteria (Salinospora arenicola and Salinospora tropica).
Among them, the chlorobia Prosthecochloris aestuarii and
Chlorobaculum parvum and the two members of the Salinospora
genus are seawater-requiring bacteria undoubtedly adapted to
permanent high salinity. The cluster also occurs in strains coping
with frequent salinity fluctuations such as Magnetococcus sp.
MC-1 strain isolated in Pettaquamscutt Estuary (Rhode Island,
United States) and Mycobacterium vanbaaleni strain isolated in
the watershed of Redfish Bay (Texas, United States). Soil bac-
teria, also exposed to large modulation of osmolarity, living in
a free-living form (Azotobacter vinelandii, M. sp. 4-46, Mycobac-
terium gilvum, Mycobacterium smegmatis, Kineococcus ratiotolerans)
or frequently associated to plant or human (Aromatoleum aroma-
ticum sp. EbN1, M. nodulans, Sinorhizobium and Pseudomonas
species, Mycobacterium avium, Ochrobactrum anthropi) also con-
tain an asnO–ngg. Taken together, these observations suggested
that this region has been widespread between bacteria living in dif-
ferent ecosystems and, although detailed studies are required to
verify and characterize the effective production of NAGGN in such
bacteria, underscore the physiological relevance of the dipeptide in
organism adaptation to fluctuations imposed by the environment.
MCAA medium containing 0.4 or 0.6 M NaCl, whereas the two
strains had a similar growth rate in MCAA medium without NaCl
(Fig. 4A). Inhibition was drastic upon the addition of 0.6 M NaCl,
a growth condition in which NAGGN has been shown to be the
major osmolyte in WT strain (8). Growth of the two strains was
also compared in the presence of GB, a very efficient osmopro-
tectant in S. meliloti (6). The addition of 1 mM GB in MCAA
medium containing 0.4 or 0.6 M NaCl improved the growth rate of
the asnO mutant (Fig. 4B), which suggested that the accumulated
GB counteracted the defect in NAGGN. Taken together, these
results demonstrated that NAGGN synthesis and accumulation are
important for bacterial growth and division at high NaCl concen-
trations in the absence of exogenous osmoprotective compound.
Osmotic Induction of asnO and Reverse Effect of GB. The accumu-
lation of NAGGN has been shown to increase as a function of salt
concentration in the growth medium, and more largely to depend
on medium osmolarity; such accumulation is limited by the addi-
tion of GB (8, 21). To examine if these factors control the ex-
pression of NAGGN biosynthesis genes, β-glucuronidase activity
was monitored in the AsnO° strain, which contains an asnO–gus
transcriptional fusion. First, gene expression was analyzed in cul-
tures grown for 24 h in media of various osmolarities (Fig. 4C). The
level of asnO–gus expression increased with NaCl concentration
and reached a maximum value at 0.5 M NaCl, showing that the
induction level of NAGGN synthesis genes largely reflected the
amount of NAGGN previously quantified. Similarly, high osmo-
larity resulting from the addition of KCl or sucrose induced asnO–
gus expression (Fig. 4C). Second, the effect of exogenous GB on
asnO expression was investigated by comparing β-glucuronidase
activity in AsnO° cultures grown in MCAA medium containing 0.4
M or 0.6 M NaCl, with or without 1 mM GB. After 24 h growth in
the presence of GB, the NaCl stimulation of the β-glucuronidase
activity was reduced by approximately twofold, suggesting that
the accumulated GB modulated the osmotic induction of asnO
expression (Fig. 4D). In cells grown in the absence of NaCl, ex-
ogenous GB had no effect on asnO expression. Thus, the accu-
mulation of NAGGN results from a tight control of the expression
of genes involved in the dipeptide synthesis relative to external
osmolarity and GB availability.
1
1
0,1
A
B
0,1
0,01
0,01
0
10 20 30 40 50
time (h)
0
10 20 30 40 50
time (h)
C
D 800
800
600
400
200
0
600
400
200
0
0.6
0
0
+
0.4 0.4 0.6
+
0
0.1 0.2 0.3 0.4 0.5 0.6 0.4 0.6
+
Osmolyte concentration (M)
NaCl concentration (M)
Fig. 4. Role of NAGGN in S. meliloti and regulation of NAGGN synthesis.
(Upper) Growth curves of strains Rm2011 (white symbols) and AsnO° (black
symbols) in MCAA medium containing (A) 0 NaCl (◇, ◆), 0.4 M NaCl (△, ▲), or
0.6 M NaCl (○, ●). (B) GB (1 mM) and 0.4 M NaCl (△, ▲) or 0.6 M NaCl (○, ●).
(Lower) Effects of osmolytes on asnO expression. β-Glucuronidase activity of
AsnO° (asnO–gus fusion) cells grown in MCAA medium containing
(C) increasing NaCl concentrations (white bars), KCl (dashed bars), or sucrose
(dotted bars), and (D) 0, 0.4, or 0.6 M NaCl with (+) or without 1 mM GB. Re-
sults are means of three independent cultures, and SDs are shown.
Discussion
In this study, we presented a functional characterization of genes
involved in NAGGN synthesis, a dipeptide previously identified
as an osmolyte in only few bacteria, and we showed that these
genes and their organization are conserved among many di-
vergent bacterial species. We demonstrated that NAGGN syn-
thesis in S. meliloti required the integrity of asnO–ngg cluster,
Sagot et al.
PNAS
|
July 13, 2010
|
vol. 107
|
no. 28
|
12655

Ngg triggering first the synthesis of the intermediate NAGG and
AsnO its subsequent conversion into NAGGN. These two pro-
is also obvious that NAGGN is produced within a taxonomically
and physiologically diverse set of bacterial species. In particular,
teins represent a unique enzymatic machinery mediating non- the presence of asnO–ngg cluster on the genome of several ma-
ribosomal peptide synthesis in bacteria, which differs from the rine bacteria and in bacteria subjected to osmolarity fluctuations
nonribosomal peptide synthetases, the poly-γ-glutamate, cyano- in their environment indicate that NAGGN could participate in
phycin, and glutathione synthetases, the D-alanine:D-alanine li- the responsiveness to seawater salinity and osmolarity variation.
gase, the L-amino acid α-ligase, and others (24).
Our results (Fig. 4A) show that the growth of an asnO mutant
that does not produce any NAGGN is much more affected than
the growth of the WT strain in a medium of elevated osmolarity,
and thus offer definitive evidence of the efficiency of the di-
peptide for osmoprotection of the cells. Interestingly, the asnO–
ngg cluster of S. meliloti is located within a region of the pSymb
replicon that has been shown recently to be important for osmo-
protection, and hence for the survival of the free-living form in
the rhizosphere (31). Our analysis with an asnO transcriptional
fusion revealed that, in the absence of exogenous osmoprotective
compound, an increasing NaCl concentration triggers an in-
crease in the expression of the asnO–ngg operon (Fig. 4C). It
is also noteworthy that the presence of exogenous compatible
solute, such as GB, has a reverse effect on this induction (Fig.
4D). Thus, it is obvious that the cells sensitively adjust the ex-
pression of the asnO and ngg genes with a direct consequence on
the NAGGN content. It has already been assumed that the
biosynthesis of organic compatible solute is energetically more
costly than the uptake of osmolytes from the environment when
available (32).
All the genomes carrying the asnO–ngg cluster encode a con-
served putative peptidase, with the exception of Mesorhizobium
sp., M. avium, M. vanbaalenii, M. gilvum, M. smegmatis, and M.
sp. MCS. Two different organizations are observed: (i) the gene
encoding the peptidase is located immediately downstream ngg
for 26 of the 40 asnO–ngg clusters, such as in P. aeruginosa (ORF
PA3461), or (ii) this gene is found elsewhere on the genome in
eight cases, like in S. meliloti (ORF SMb20466). This peptidase
belongs to the M42 peptidase family that contains aminopepti-
dases hydrolyzing acylated N-terminal residues (33). Despites the
fact that its role still remains to be established, one can suggest that
such peptidase could play a role in balancing NAGGN pool during
adaptation to osmotic fluctuations. In addition, particularly during
an osmotic down-shock, a direct release of the dipeptide cannot be
excluded. The presence of genes encoding a putative dipeptide
ABC transporter located directly upstream of asnO might suggest
such possibility or an efflux of hydrolysates.
Another attractive and intriguing feature about the asnO–ngg
cluster comes from the work of Berges and collaborators (11).
The authors have shown that a mutation in the asnO gene im-
paired the activity of FixT, an antikinase that inhibits the FixL–
FixJ two-component system that controls the expression of ni-
trogen fixation genes in bacteroid, the symbiotic form of S. meli-
loti. It is thus tempting to argue that the NAGGN biosynthetic
pathway could play an important role inside the nitrogen-fixing
nodules. These data, together with the presence of an asnO–ngg
cluster on the genome of pathogenic or symbiotic bacteria, offer
insights into the relationships between osmoadaptation and host
interactions. In view of the variety of stresses encountered by
pathogenic bacteria during the course of infection, a number of
osmoprotective compounds have already been linked to the vir-
ulence potential of certain pathogens (34). Therefore, our study
raises questions about the effectiveness of NAGGN as a latent
virulence factor in pathogenic bacteria. AsnO and Ngg proteins,
which are conserved in animal and human pathogens (M. avium,
P. aeruginosa, Pseudomonas mendocina, and O. anthropi), but
absent in eukaryotes, could be consequently attractive targets for
antibacterial drugs.
The dipeptide formation is mediated by Ngg, which appears to
be a bifunctional protein also catalyzing an acetylation reaction.
Peptide synthetases with modular organization have been de-
scribed previously. However, conversely to Ngg and excepting
some nonribosomal peptides (25), they catalyze only peptide-
forming reactions. For example, CyaH incorporates an Asp or
Arg residue into a cyanophycin primer using two distinct ATP-
dependent ligase activities (19), and the synthesis of the tripeptide
glutathione (γ-glutamyl-cysteinylglycine) in S. agalactiae is medi-
ated by a bifunctional enzyme, GshAB, that contains the γ-glu-
tamylcysteine and glutathione synthetase activities usually found
on two distinct proteins (26). By its N-terminal domain, the Ngg
protein belongs to the enormous family of GCN5-related N-ace-
tyltransferases. In bacteria, these enzymes participate in impor-
tant metabolic pathways such as the synthesis of spermidine and
mycothiol, or in the extension mechanism of pepdidoglycan in
Gram-positive bacteria (15, 27). An example of a bifunctional
enzyme containing a GNAT domain is GlmU, which catalyzes the
acetylation of glucosamine-1-phophate and its subsequent uridy-
lylation to give UDP-N-acetylglucosamine (28). Multiple repre-
sentatives of this family are present on any sequenced genome,
but most of them have not been functionally characterized and
their substrates are still unknown. Within the S. meliloti genome,
22 of the 38 ORFs annotated as encoding acetyltransferases be-
long to the GNAT family, two have been ascribed as acetylating
ribosomal proteins, and one as a GlmU homologue. The dem-
onstration that Ngg is involved in the biosynthesis of NAGG is
a unique functional characterization of a GNAT protein in this
symbiotic bacterium. Our results give a molecular support to the
previous in vitro experiments that have proposed the formation of
NAGGN with NAGG as a potential intermediate (9). However,
conversely to these preliminary results, the bioinformatic analysis
of the Ngg sequence indicated that NAGG formation can occur
directly from two glutamines and not from one N-acetylglutamine
and one glutamine. A possible explanation for this discrepancy is
the absence of AcCoA in the in vitro reaction mixture, and thus
only the Ngg activity making a peptide bond between N-acetyl-
glutamine and glutamine could be seen. Nevertheless, additional
biochemical work is still needed to establish (i) the sequence of
the two reactions catalyzed in vivo by Ngg and (ii) if N-acetyl-
glutamine can directly be used in vivo as a substrate.
The asnO gene is required for the final step of NAGGN syn-
thesis, i.e., the addition of a NH2 group to the NAGG moiety thus
creating a C-terminal amide group. As a member of the PurF
family, AsnO most likely catalyzes the removal of the NH2 group
from glutamine via its N-terminal domain, then transfers it to
NAGG within its C-terminal domain. This is an example of an
enzyme similar to the E. coli AsnB that is not involved in aspar-
agine synthesis (29, 30). Sequence comparisons indicate that the
proteins closely related to the S. meliloti AsnO group into the
amidotransferase AsnB family, but that they are distinct from
previously identified members of this family (Fig. S4). Such dis-
tribution supports the idea that AsnO-like proteins have their own
catalytic properties. Consequently, the presence of AsnO homo-
logues in bacteria that do not encode a Ngg homologue, such as in
Frankia alni or in Methylobacterium extorquens, remains in-
triguing, and its biological significance is still far from clear.
The high degree of sequence identity among the AsnO and
Ngg proteins and the preservation of the asnO–ngg organization
in various bacteria showed that the NAGGN biosynthetic path-
way is evolutionally well conserved in the bacterial kingdom. It
Finally, the characterization of the asnO–ngg cluster offers a set
of biosynthetic genes for metabolic engineering technology, to in-
stall the production of NAGGN in bacteria or agriculturally im-
portant crop plants to improve their tolerance to osmotic stress.
12656
|
www.pnas.org/cgi/doi/10.1073/pnas.1003063107
Sagot et al.

and the reactions were stopped with 250 μL 2M Na2CO3. After 1 min cen-
trifugation at room temperature, the absorbance of the supernatant was
read at 405 nm. β−Glucuronidase specific activity was calculated as (1,000 ×
OD405) / (time × mg protein).
The introduction of novel pathways in various organisms increases
the demand for precursors, and impacts metabolic fluxes, pool
sizes, and gene expression (35). Because glutamate and glutamine
are abundant compounds in most organisms, engineering NAGGN
production might overcome the problem of precursor limitation.
Furthermore, in vitro biotechnological applications would benefit
of the asnO–ngg cluster for an efficient manufacturing production
of the dipeptide.
NMR Spectral Determination of Intracellular Osmolytes. Ethanolic extraction
of cellular osmolytes and sample preparation were performed as previously
(21). Details on NAGG purification and NMR spectral analyses are given in
SI Materials and Methods.
Materials and Methods
MS Analyses. Purified and synthetic NAGG were analyzed by electrospray
ionization (ESI) MS. Low-resolution MS (i.e., ESIMS) were obtained with
a Bruker Esquire 3000 Plus spectrometer in the positive and negative mode.
High-resolution ESIMS (HRESIMS) were conducted on an LTQ Orbitrap mass
spectrometer (Thermo Fisher Scientific). Details are given in SI Materials
and Methods.
Bacterial Strains and Growth Conditions. Strains and plasmids used in this
study are listed in Table S1. S. meliloti strains were maintained at 30 °C in LB
medium supplemented with 2.5 mM MgSO4 and 2.5 mM CaCl2, then grown
in MCAA semisynthetic medium (4). The medium osmolarity was increased
by the addition of NaCl as indicated, or by the addition of 0.4 KCl or 0.6 M
mannitol, which are osmotically equivalent to 0.4 M NaCl.
Enzyme Assay. β-Glucuronidase was measured from 50 to 200 μL culture with
Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 1 mM
BSA) containing 0.01% SDS, 0.8 mg/mL p-nitrophenyl β-D-glucuronide, and
40 mM β-mercaptoethanol. Reaction mixtures (1 mL) were incubated at 30 °C
ACKNOWLEDGMENTS. We thank Aurélie Kiers for technical assistance and
Renaud Brouquisse and Pierre Vierling for helpful discussions. This work was sup-
ported by the Centre National de la Recherche Scientifique. B.S. received a
doctoral fellowship from Institut National de la Recherche Agronomique–Région.
1. Csonka LN, Hanson AD (1991) Prokaryotic osmoregulation: Genetics and physiology.
Annu Rev Microbiol 45:569–606.
2. Ventosa A, Nieto JJ, Oren A (1998) Biology of moderately halophilic aerobic bacteria.
Microbiol Mol Biol Rev 62:504–544.
3. Bremer E, Krämer R (2000) Bacterial Stress Responses, eds Stortz GT, Hengge Aronis R
(ASM, Washington, DC), pp 79–97.
19. Berg H, et al. (2000) Biosynthesis of the cyanobacterial reserve polymer multi-L-
arginyl-poly-L-aspartic acid (cyanophycin): Mechanism of the cyanophycin synthetase
reaction studied with synthetic primers. Eur J Biochem 267:5561–5570.
20. Pobigaylo N, Szymczak S, Nattkemper TW, Becker A (2008) Identification of genes
relevant to symbiosis and competitiveness in Sinorhizobium meliloti using signature-
tagged mutants. Mol Plant Microbe Interact 21:219–231.
4. Smith LT, Smith GM (1989) An osmoregulated dipeptide in stressed Rhizobium
meliloti. J Bacteriol 171:4714–4717.
5. Imhoff JF (2001) True marine and halophilic anoxygenic phototrophic bacteria. Arch
Microbiol 176:243–254.
6. Le Rudulier D, Mandon K, Dupont L, Trinchant JC (2002) Encyclopedia of Environmental
Microbiology (Wiley, Hoboken, NJ), pp 2774–2789.
7. D’Souza-Ault MR, Smith LT, Smith GM (1993) Roles of N-acetylglutaminylglutamine
amide and glycine betaine in adaptation of Pseudomonas aeruginosa to osmotic
stress. Appl Environ Microbiol 59:473–478.
8. Smith LT, et al. (1994) Osmoregulation in Rhizobium meliloti: Mechanism and control
by other environmental signals. J Exp Zool 268:162–165.
9. Smith LT, Allaith AM, Smith GM (1994) Mechanism of osmotically regulated N-
acetylglutaminylglutamine amide production in Rhizobium meliloti. Plant Soil 161:
103–108.
10. Aspedon A, Palmer K, Whiteley M (2006) Microarray analysis of the osmotic stress
response in Pseudomonas aeruginosa. J Bacteriol 188:2721–2725.
11. Bergès H, et al. (2001) A glutamine-amidotransferase-like protein modulates FixT
anti-kinase activity in Sinorhizobium meliloti. BMC Microbiol 1:6.
12. Boehlein SK, Richards NG, Schuster SM (1994) Glutamine-dependent nitrogen transfer
in Escherichia coli asparagine synthetase B. Searching for the catalytic triad. J Biol
Chem 269:7450–7457.
13. Larsen TM, et al. (1999) Three-dimensional structure of Escherichia coli asparagine
synthetase B: A short journey from substrate to product. Biochemistry 38:16146–16157.
14. Yoshida K, Fujita Y, Ehrlich SD (1999) Three asparagine synthetase genes of Bacillus
subtilis. J Bacteriol 181:6081–6091.
21. Talibart R, et al. (1997) Transient accumulation of glycine betaine and dynamics of
endogenous osmolytes in salt-stressed cultures of Sinorhizobium meliloti. Appl Environ
Microbiol 63:4657–4663.
22. Pocard JA, Smith LT, Smith GM, Le Rudulier
D (1994) A prominent role for
glucosylglycerol in the adaptation of Pseudomonas mendocina SKB70 to osmotic
stress. J Bacteriol 176:6877–6884.
23. Fischbach MA, Walsh CT, Clardy J (2008) The evolution of gene collectives: How
natural selection drives chemical innovation. Proc Natl Acad Sci USA 105:4601–4608.
24. Yagasaki M, Hashimoto S (2008) Synthesis and application of dipeptides; Current
status and perspectives. Appl Microbiol Biotechnol 81:13–22.
25. Sieber SA, Marahiel MA (2005) Molecular mechanisms underlying nonribosomal
peptide synthesis: Approaches to new antibiotics. Chem Rev 105:715–738.
26. Janowiak BE, Griffith OW (2005) Glutathione synthesis in Streptococcus agalactiae.
One protein accounts for gamma-glutamylcysteine synthetase and glutathione
synthetase activities. J Biol Chem 280:11829–11839.
27. Rajan LA, et al. (2008) Characterization and phylogenetic analysis of ectoine
biosynthesis genes from Bacillus halodurans. Arch Microbiol 190:481–487.
28. Olsen LR, Roderick SL (2001) Structure of the Escherichia coli GlmU pyrophosphorylase
and acetyltransferase active sites. Biochemistry 40:1913–1921.
29. Ren H, Liu J (2006) AsnB is involved in natural resistance of Mycobacterium smegmatis
to multiple drugs. Antimicrob Agents Chemother 50:250–255.
30. Miller MT, Bachmann BO, Townsend CA, Rosenzweig AC (2001) Structure of beta-
lactam synthetase reveals how to synthesize antibiotics instead of asparagine. Nat
Struct Biol 8:684–689.
31. Domínguez-Ferreras A, et al. (2006) Transcriptome profiling reveals the importance of
15. Vetting MW, et al. (2005) Structure and functions of the GNAT superfamily of
acetyltransferases. Arch Biochem Biophys 433:212–226.
16. Neuwald AF, Landsman D (1997) GCN5-related histone N-acetyltransferases belong to
a diverse superfamily that includes the yeast SPT10 protein. Trends Biochem Sci 22:
154–155.
17. Galperin MY, Koonin EV (1997) A diverse superfamily of enzymes with ATP-dependent
carboxylate-amine/thiol ligase activity. Protein Sci 6:2639–2643.
18. Fan C, Moews PC, Shi Y, Walsh CT, Knox JR (1995) A common fold for peptide
synthetases cleaving ATP to ADP: Glutathione synthetase and D-alanine:d-alanine
ligase of Escherichia coli. Proc Natl Acad Sci USA 92:1172–1176.
plasmid pSymB for osmoadaptation of Sinorhizobium meliloti.
7617–7625.
J Bacteriol 188:
32. Oren A (1999) Bioenergetic aspects of halophilism. Microbiol Mol Biol Rev 63:334–348.
33. Ando S, et al. (1999) Thermostable aminopeptidase from Pyrococcus horikoshii. FEBS
Lett 447:25–28.
34. Sleator RD, Hill C (2002) Bacterial osmoadaptation: The role of osmolytes in bacterial
stress and virulence. FEMS Microbiol Rev 26:49–71.
35. Rontein D, Basset G, Hanson AD (2002) Metabolic engineering of osmoprotectant
accumulation in plants. Metab Eng 4:49–56.
Sagot et al.
PNAS
|
July 13, 2010
|
vol. 107
|
no. 28
|
12657