Adaptation to osmotic stress can be achieved by the accumulation of compatible solutes that aid in turgor maintenance and macromolecule stabilization. The genetic regulation of solute accumulation is poorly understood, and has been described well at the molecular level only in enterobacteria. In this study, we show the importance of the alternative sigma factor RpoE2 in Sinorhizobium meliloti osmoadaptation. Construction and characterization of an S. meliloti rpoE2 mutant revealed compromised growth in hyperosmotic media. This defect was due to the lack of trehalose, a minor carbohydrate osmolyte normally produced in the initial stages of growth and in stationary phase. We demonstrate here that all three trehalose synthesis pathways are RpoE2 dependent, but only the OtsA pathway is important for osmoinducible trehalose synthesis. Furthermore, we confirm that the absence of RpoE2-dependent induction of otsA is the cause of the osmotic phenotype of the rpoE2 mutant. In conclusion, we have highlighted that, despite its low level, trehalose is a crucial compatible solute in S. meliloti, and the OtsA pathway induced by RpoE2 is needed for its accumulation under hyperosmotic conditions.
BeckerA.,
SchmidtM.,
JagerW.,
PuhlerA.1995; New gentamicin-resistance and lacZ promoter–probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions. Gene 162:37–39
BourotS.,
SireO.,
TrautwetterA.,
TouzéT.,
WuL. W.,
BlancoC.,
BernardT.2000; Glycine betaine-assisted protein folding in a lysA mutant of Escherichia coli. J Biol Chem 275:1050–1056
CardosoF. S.,
CastroR. F.,
BorgesN.,
SantosH.2007; Biochemical and genetic characterization of the pathways for trehalose metabolism in Propionibacterium freudenreichii, and their role in stress response. Microbiology 153:270–280
CarpinelliJ.,
KramerR.,
AgosinE.2006; Metabolic engineering of Corynebacterium glutamicum for trehalose overproduction: role of the TreYZ trehalose biosynthetic pathway. Appl Environ Microbiol 72:1949–1955
CowieA.,
ChengJ.,
SibleyC. D.,
FongY.,
ZaheerR.,
PattenC. L.,
MortonR. M.,
GoldingG. B.,
FinanT. M.2006; An integrated approach to functional genomics: construction of a novel reporter gene fusion library for Sinorhizobium meliloti. Appl Environ Microbiol 72:7156–7167
da CostaM. S.,
SantosH.,
GalinskiE. A.1998; An overview of the role and diversity of compatible solutes in Bacteria and Archaea. Adv Biochem Eng Biotechnol 61:117–153
De SmetK. A.,
WestonA.,
BrownI. N.,
YoungD. B.,
RobertsonB. D.2000; Three pathways for trehalose biosynthesis in mycobacteria. Microbiology 146:199–208
DiamantS.,
EliahuN.,
RosenthalD.,
GoloubinoffP.2001; Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses. J Biol Chem 276:39586–39591
EncarnaciónS.,
del Carmen VargasM.,
DunnM. F.,
DávalosA.,
MendozaG.,
MoraY.,
MoraJ.2002; AniA regulates reserve polymer accumulation and global protein expression in Rhizobium etli. J Bacteriol 184:2287–2295
FellayR.,
FreyJ.,
KrischH.1987; Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram-negative bacteria. Gene 52:147–154
FinanT. M.,
HirschA. M.,
LeighJ. A.,
JohansenE.,
KuldauG. A.,
DeeganS.,
WalkerG. C.,
SignerE. R.1985; Symbiotic mutants of Rhizobium meliloti that uncouple plant from bacterial differentiation. Cell 40:869–877
FlechardM.,
FontenelleC.,
TrautwetterA.,
ErmelG.,
BlancoC.2009; Sinorhizobium meliloti rpoE2 is necessary for H2O2 stress resistance during the stationary growth phase. FEMS Microbiol Lett 290:25–31
GalibertF.,
FinanT. M.,
LongS. R.,
PuhlerA.,
AbolaP.,
AmpeF.,
Barloy-HublerF.,
BarnettM. J.,
BeckerA.other authors2001; The composite genome of the legume symbiont Sinorhizobium meliloti. Science 293:668–672
GouffiK.,
PicaN.,
PichereauV.,
BlancoC.1999; Disaccharides as a new class of nonaccumulated osmoprotectants for Sinorhizobium meliloti. Appl Environ Microbiol 65:1491–1500
Hengge-AronisR.2002; Signal transduction and regulatory mechanisms involved in control of the σS (RpoS) subunit of RNA polymerase. Microbiol Mol Biol Rev 66:373–395
Hengge-AronisR.,
KleinW.,
LangeR.,
RimmeleM.,
BoosW.1991; Trehalose synthesis genes are controlled by the putative sigma factor encoded by rpoS and are involved in stationary-phase thermotolerance in Escherichia coli. J Bacteriol 173:7918–7924
HumannJ. L.,
ZiemkiewiczH. T.,
YurgelS. N.,
KahnM. L.2009; Regulatory and DNA repair genes contribute to the desiccation resistance of Sinorhizobium meliloti Rm1021. Appl Environ Microbiol 75:446–453
JebbarM.,
Sohn-BosserL.,
BremerE.,
BernardT.,
BlancoC.2005; Ectoine-induced proteins in Sinorhizobium meliloti include an ectoine ABC-type transporter involved in osmoprotection and ectoine catabolism. J Bacteriol 187:1293–1304
KaasenI.,
FalkenbergP.,
StyrvoldO. B.,
StromA. R.1992; Molecular cloning and physical mapping of the otsBA genes, which encode the osmoregulatory trehalose pathway of Escherichia coli: evidence that transcription is activated by KatF (AppR. J Bacteriol 174:889–898
KovachM. E.,
ElzerP. H.,
HillD. S.,
RobertsonG. T.,
FarrisM. A.,
RoopR. M.II,
PetersonK. M.1995; Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176
McIntyreH. J.,
DaviesH.,
HoreT. A.,
MillerS. H.,
DufourJ. P.,
RonsonC. W.2007; Trehalose biosynthesis in Rhizobium leguminosarum bv. trifolii and its role in desiccation tolerance. Appl Environ Microbiol 73:3984–3992
McLeodS. M.,
XuJ.,
JohnsonR. C.2000; Coactivation of the RpoS-dependent proP P2 promoter by Fis and cyclic AMP receptor protein. J Bacteriol 182:4180–4187
MeadeH. M.,
LongS. R.,
RuvkunG. B.,
BrownS. E.,
AusubelF. M.1982; Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn 5 mutagenesis. J Bacteriol 149:114–122
MillerJ. H.1972Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
MonteroM.,
EydallinG.,
VialeA. M.,
AlmagroG.,
MunozF. J.,
RahimpourM.,
SesmaM. T.,
Baroja-FernandezE.,
Pozueta-RomeroJ.2009; Escherichia coli glycogen metabolism is controlled by the PhoP–PhoQ regulatory system at submillimolar environmental Mg2+ concentrations, and is highly interconnected with a wide variety of cellular processes. Biochem J 424:129–141
PichereauV.,
PocardJ.-A.,
HamelinJ.,
BlancoC.,
BernardT.1998; Differential effects of dimethylsulfoniopropionate, dimethylsulfonioacetate, and other S-methylated compounds on the growth of Sinorhizobium meliloti at low and high osmolarities. Appl Environ Microbiol 64:1420–1429
PinedoC. A.,
BringhurstR. M.,
GageD. J.2008; Sinorhizobium meliloti mutants lacking phosphotransferase system enzyme HPr or EIIA are altered in diverse processes, including carbon metabolism, cobalt requirements, and succinoglycan production. J Bacteriol 190:2947–2956
SambrookJ.,
FritschE. F.,
ManiatisT.1989Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
SauviacL.,
PhilippeH.,
PhokK.,
BruandC.2007; An extracytoplasmic function sigma factor acts as a general stress response regulator in Sinorhizobium meliloti. J Bacteriol 189:4204–4216
SchaferA.,
TauchA.,
JagerW.,
KalinowskiJ.,
ThierbachG.,
PuhlerA.1994; Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73
StreeterJ. G.,
GomezM. L.2006; Three enzymes for trehalose synthesis in Bradyrhizobium cultured bacteria and in bacteroids from soybean nodules. Appl Environ Microbiol 72:4250–4255
TalibartR.,
JebbarM.,
GouffiK.,
PichereauV.,
GouesbetG.,
BlancoC.,
BernardT.,
PocardJ.1997; Transient accumulation of glycine betaine and dynamics of endogenous osmolytes in salt-stressed cultures of Sinorhizobium meliloti. Appl Environ Microbiol 63:4657–4663
TavernierP.,
BessonI. I.,
PortaisJ. C.,
CourtoisJ.,
CourtoisB.,
BarbotinJ. N.1998; In vivo 13C-NMR studies of polymer synthesis in Rhizobium meliloti M5N1 strain. Biotechnol Bioeng 58:250–253
VriezenJ. A.,
de BruijnF. J.,
NussleinK.2007; Responses of rhizobia to desiccation in relation to osmotic stress, oxygen, and temperature. Appl Environ Microbiol 73:3451–3459
WolfA.,
KrämerR.,
MorbachS.2003; Three pathways for trehalose metabolism in Corynebacterium glutamicum ATCC13032 and their significance in response to osmotic stress. Mol Microbiol 49:1119–1134
YanceyP. H.2005; Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J Exp Biol 208:2819–2830