nitrogen metabolism and regulation in Grampositive bacteria

18 Pages·2010·0.46 MB·English

REVIEW ARTICLE
Commonpatterns^uniquefeatures:nitrogenmetabolismand
regulationinGram-positivebacteria
JohannesAmon1,FritzTitgemeyer2&AndreasBurkovski1
1Lehrstuhlfu¨rMikrobiologie,Friedrich-Alexander-Universita¨tErlangen-Nu¨rnberg,Erlangen,Germany;and2DepartmentOecotrophologie,Universityof
AppliedSciencesMu¨nster,Mu¨nster,Germany
D
o
w
n
Correspondence:AndreasBurkovski, Abstract lo
a
Lehrstuhlfu¨rMikrobiologie,Friedrich- Gram-positivebacteriahavedevelopedelaboratemechanismstocontrolammonium ded
AStlaeuxadntsdter.r-5U,n9i1ve0r5s8ita¨EtrlEarnlagnegne,nG-eNrum¨rnabney.rgTe,l.: assimilation,atthelevelsofbothtranscriptionandenzymeactivity.Inthisreview,the from
14991318528086;fax:[phone] commonandspecificmechanismsofnitrogenassimilationandregulationinGram- h
8082;e-mail:[email protected] positivebacteriaaresummarizedandcomparedforthegeneraBacillus,Clostridium, ttp
s
erlangen.de Streptomyces,MycobacteriumandCorynebacterium,withemphasisonthehighG1C ://a
c
genera. Furthermore, the importance of nitrogen metabolism and control for the a
d
Received17December2009;revised1 pathogeniclifestyleandvirulenceisdiscussed.Insummary,theregulationofnitrogen em
February2010;accepted3February2010. ic
metabolisminprokaryotesshowsanimpressivediversity.Virtuallyeveryphylumof .o
Finalversionpublishedonline11March2010. u
bacteria evolved its own strategy to react to the changing conditions of nitrogen p
.c
supply.Notonlydothetranscriptionfactorsdifferbetweenthephylaandsometimes o
DOI:10.1111/j.1574-6976.2010.00216.x m
even between families, but the genetic targets of a given regulon can also differ /fe
m
Editor:KeithChater betweencloselyrelatedspecies. sre
/a
Keywords rtic
le
Streptomyces;Mycobacterium; -a
b
Corynebacterium;Bacillus;AmtR; s
S GlnR. tra
W ct/3
4
/4
E s54-dependent promoters in response to nitrogen limitation /5
I Introduction 88
V (Weissetal.,2002).Phosphorylationoftheresponseregulator /5
4
E Regulationofnitrogenmetabolisminenterobacteriawasthe NtrCbytheNtrBkinaseiscontrolledbythePII-typeprotein 14
2
R paradigmfornitrogencontrolinprokaryotesformanyyears. depending on the cellular glutamine concentration (via uri- 8
b
Forexample,themetabolicpathwaysusedbyEscherichiacoli dylylation/deuridylylationofP byUTase)andonthecellular y
Y II g
toassimilateammoniumdependontheconcentrationofthis 2-oxoglutarateconcentration(Jiang&Ninfa,2009b,c).High u
e
G s
nitrogensourceinthemedium,andareunderstrictcontrolat 2-oxoglutarateconcentrationspreventbindingofPIItoNtrB, t o
O theleveloftranscriptionandactivityregulation(forareview, leadingtoadecreaseofNtrBphosphataseandstimulationof n 1
1
L see Merrick & Edwards, 1995; Leigh & Dodsworth, 2007). NtrB kinase activity. As a consequence, NtrC is phosphory- A
p
O Duringgrowthinanammonium-richmedium,thisnitrogen latedandNtrBC-regulatedgenesareexpressed. ril 2
source is primarily assimilated by glutamate dehydrogenase In enteric bacteria, the activity of GS is regulated by 0
BI (GDH),whileatammoniumconcentrationsbelow1mM,the adenylylation/deadenylylation,dependingonnitrogenavail- 19
O affinityofGDHforammoniumistoolow.Inthissituation, ability. This modification is catalyzed by the bifunctional
the glutamine synthetase/glutamate synthase (GS/GOGAT) enzyme adenylyltransferase (ATase/GlnE), whose activity is
R
systemtakesover.ThefluxthroughGSissubjecttoregulation controlled by the signal transduction protein P (Jiang &
C II
atboththeleveloftranscriptionoftheGS-encodingglnAgene Ninfa,2009a,c).P occupiesapivotalpositioninnitrogen-
II
I
M andenzymeactivity. regulatory networks – not only in enterobacteria – as a
TheexpressionofglnAandseveralothergenesinvolvedin sensory protein and signal transducer (for recent reviews,
nitrogen metabolism in enterobacteria is controlled by the see Forchhammer, 2007, 2008). The nitrogen status of the
NtrBC nitrogen regulation system. This two-component cell,as sensedbyuridylyltransferase (GlnD),issignalledto
signal transduction system activates the transcription of P by adjusting the degree of uridylylation of the latter.
II
(cid:2)c2010FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolRev34(2010)588–605
PublishedbyBlackwellPublishingLtd.Allrightsreserved
NitrogencontrolinGram-positivebacteria 589
Native P indicates a nitrogen-rich status, whereas The low G1C Firmicutes are divided into the classes
II
P (cid:3)UMPflagsanitrogen-poorstatusofthecell.Fromthe BacilliandClostridia.TheBacilliarefurthersubdividedinto
II
P protein,thesignalistransferredtoATase,whichregulates twoorders,namelytheBacillalesandtheLactobacillales.The
II
GSactivity.TwoP -typeproteinsarepresentinE.coli:the phylumofthehighG1CActinobacteriacomprisesonlythe
II
products of glnB (Bueno et al., 1985; van Heeswijk et al., class of Actinobacteria, which is subdivided into various
2009) and glnK (van Heeswijk et al., 1995, 1996). P and subclasses,ofwhichtheordersBifidobacterialesandActino-
II
GlnKformheterotrimersthatareproposedtobeimportant mycetales contain the largest numbers of species, and
for fine tuning the nitrogen regulatory cascade (van Hees- include such important families as the Streptomycetaceae,
wijk et al., 2000). The complete E. coli signal transduction MycobacteriaceaeandCorynebacteriaceae.
cascade was investigated in a reconstituted system (Jiang This work presents a summary of the nitrogen-control
et al., 1998a–c; Jiang & Ninfa, 2009a–c). Subsequent work mechanisms in Gram-positive bacteria, with emphasis on
D
andcomparativegenomicanalyseswithagrowingnumber the genera Bacillus, Clostridium, Streptomyces, Mycobacter- o
w
n
of whole-genome sequences showed a rather uniform reg- iumandCorynebacterium.Wefocusonnitrogen-dependent lo
a
ulation of nitrogen metabolism in the Gram-negative Pro- transcription control of genes encoding systems of core d
e
teobacteriasimilartothesystemdescribedindetailabovefor nitrogen metabolism (ammonium, glutamine) and the d fro
E.coli(Leigh&Dodsworth,2007). related post-translational control mechanisms. Further- m
Outside this group, the situation is more complex and more,theconnectionofnitrogenmetabolism,apathogenic http
diverse.Inthelastdecade,nitrogencontrolwasanalyzedin lifestyleandvirulenceisdiscussedforseveralspecies. s
severalGram-negativeandespeciallyGram-positivebacter- ://ac
a
ia,anditsoonbecameclearthatforvirtuallyeveryphylum d
Nitrogencontrol in lowG1CDNA e
m
of the bacteria, there are different strategies for transcrip- Gram-positive bacteria ic
tional regulation, while the assimilatory enzymes (e.g. .o
u
p
glutamine synthetase and glutamate synthase) as well as .c
Regulation of nitrogen metabolism in o
signal transduction proteins (adenylyltransferase, uridylyl- m
transferase, PII) are widely conserved. For example, the Bacillussubtilis /fem
Gram-negative cyanobacteria evolved the Crp–Fnr family The transcriptional regulation of nitrogen metabolism has sre
2re0g0u5l)atfoorrNthtecAtra(nHscerrirpetriooneatlaclo.,nt2r0o0l1o;fMamurmo-oPnaisutmoraestsiaml.-, bFeisehnert,ho1r9o9u9g;hSlyoninevnesshtiegiant,ed20fo0r7)B.assubtthileism(foorstaprervoimewin,esneet /article
ilation, whereas the post-translational signal transduction modelorganismforlowG1CGram-positivebacteria.Two -ab
s
viathePIIsystem–whilesportingsomeuniquefeatures–is very similar regulatory proteins of the MerR-type, namely tra
c
highly conserved (Forchhammer, 2004; Osanai & Tanaka, GlnR and TnrA (Schreier et al., 1989; Wray et al., 1996, t/3
2007). When looking into the Gram-positive phyla of 1998),andtoacertaindegreetheglobalregulatoryprotein 4/4
Firmicutes and Actinobacteria, the situation becomes even CodY (Slack et al., 1995) cooperate to ensure the optimal /5
8
8
morecomplex,withvariousdifferentstrategiesoftranscrip- growthofB.subtiliswithrespecttonitrogensupply(foran /5
4
tionalcontroldowntotheleveloffamilies. overview,seeFig.1).While theoccurrenceofhomologsof 1
4
2
8
b
y
g
u
e
s
t o
n
1
glnR glnA tnrA amtB glnK 1 A
p
ril 2
0
Fig.1. Regulatorynetworkofnitrogen 1
9
metabolisminBacillussubtilis.Arrowsindicatethe
GGllnnRR GGSS TTnnrrAA AAmmttBB GGllnnKK
relativelengthandorganizationofgenesonthe
chromosome.Dottedblackarrowsillustratethe
regulatoryinteractionsoftherespectiveregulator
A B ureC
ofnitrogenmetabolism(orange)includingits
functioninactivating(plus)orrepressing(minus)
gltB gltA gltC
thetargetgene(s).Graylineswithcirclesindicate
nasC nasB
post-translationalinteractions(blue,glutamine
synthetase;yellow,ammoniumtransporterAmtB
andPIIproteinGlnK;gray,furthergenesinvolvedin nasF E nasD GltC
nitrogenassimilation;forfurtherdetails,seetext).
FEMSMicrobiolRev34(2010)588–605 (cid:2)c2010FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
590 J.Amonetal.
Table1. DistributionofkeyplayersofammoniumassimilationandnitrogencontrolintheavailablegenomesofGram-positiveorganisms
Firmicutes Actinobacteria Proteobacteria
Bacilli Clostridia
Protein Function Bacillales Lactobacillales ClostridialesStreptomycetes MycobacteriaCorynebacteria
GS(typeI) Glutaminesynthesis, a a a b b b b
GlnA2-A4 ammoniumassimilation No No No Yes Yes Some No
GS(typeII) No No No Yes No No Some
GS(typeIII) No No Some No Some No Some
GOGAT Glutamatesynthesis, Yes Yes Yes Yes Yes Yes Yes
ammoniumassimilation
GlnE Adenylyl-transferase, No No No Yes Yes Yes Yes D
o
regulationofGSactivity w
n
AmtB Ammoniumpermease Yes Yes Yes Yes Yes Yes Yes lo
a
GlnK PII-typesignaltransduction Yes Yes Yes Yes Yes Yes Yes de
d
protein fro
GlnD Adenylyl-/uridylyl- No No No Yes Yes Yes Yes m
transferase,regulation h
ofGlnK ttps
Transcriptional GlnR/TnrA GlnR NitR GlnR/GlnRII GlnR AmtR NtrC ://a
c
regulator a
d
e
m
Notethatforclostridia,thedistributionofgenesencodingglutaminesynthetase-likeproteinsisdiverse,andtheregulatorNitRisonlyfoundinsome
ic
Clostridiumspecies.IntheorderBacillales,theoccurrenceofTnrAisspecificforthefamilyBacillaceae.aandbdesignatetherespectivesubgroupsof .o
u
thetypeIGSenzyme(Brownetal.,1994;Eisenbergetal.,2000).TheGOGATusuallycomprisesgenesencodingalargeandsmallsubunit,designated p
.c
gltABforFirmicutesandgltBDforActinobacteriaandProteobacteria,inabicistronicoperon.Gram-negativeProteobacteriaaregivenasreference.For o
m
detailsaboutdistribution,seecorrespondingsections. /fe
m
s
re
TexncreAptisoenemosf ttohebeclospseelcyifircelfaoterdthBeacBilalucisllacceeraeeu,s,wBitahciltlhues erengcuoldaitnogr(gFeinge.s1)a.reReapdrdeistsioionnalblyyuGnltdCeriscodnirtercotllyodfetpheenGdeltnCt /article
anthracis and Bacillus thuringiensis according to currently ontheintracellularconcentrationof2-oxoglutarate(Picossi -ab
s
available genome sequencing data (fordetails, see Dorosh- etal.,2007). tra
c
chuketal.,2006),theregulationofnitrogenmetabolismin The metabolite indirectly sensed by GlnR and TnrA is t/3
othermembersoftheclassBacillisuchastheStaphylococca- glutamine, which is also the amino acid preferred as the 4/4
ceae,ListeriaeandLactobacillales(includingenterococciand nitrogensourcefortheoptimalgrowthofB.subtilis(Sonen- /5
8
8
streptococci)isexclusivelydependentonhomologsofGlnR shein, 2007). Recent studies showed that the modulation /5
4
(Table 1; Schreier et al., 2000; Varmanen et al., 2000; of the activity of the two regulators depends on direct 1
4
2
Doroshchuk et al., 2006; Kloosterman et al., 2006; Larsen interactionswithglutaminesynthetase,whichbelongstothe 8
b
etal.,2006;Hendriksenetal.,2008b). a-subgroupofGSI-typeenzymes(Brownetal.,1994;Eisen- y
g
GlnR acts as a specific regulatorof nitrogen metabolism berg et al., 2000). This group is distinguished from the u
e
s
and represses the glnRA operon (Brown & Sonenshein, b-subgroupbyhavingregulatoryproperties(fordetailsabout t o
n
1996), the urease operon ureABC (Wray et al., 1997; thephylogenyanddistribution,seeTable1andFig.2).GlnRis 1
1
Brandenburgetal.,2002)andalsothetnrAgene(Zalieckas directly regulated by repressing its own transcription. A A
p
etal.,2006).TnrAactivatesthetranscriptionoftheammo- GlnR–DNA complex forms upstream of the gnlRA operon, ril 2
niumtransporter-encodinggeneamtB(formerlynrgA),the with feedback-inhibited GS acting as a chaperone (Fisher & 0
1
P proteinencodingglnK(formerlynrgB)(Wrayetal.,1996, Wray,2008,2009).Ontheotherhand,TnrAisinactivatedby 9
II
1998), the urease operon (Wray et al., 1997; Brandenburg adirectinteractionofitscarboxy-terminalregionwithfeed-
et al., 2002) and the nitrate and nitrite reductase gene back-inhibited GS, effectively blocking the DNA-binding
clusters nasBC and nasDEF (Nakano et al., 1998), among ability of TnrA (Wray & Fisher, 2008). As a result, GlnR is
other putative targets (Yoshida et al., 2003; Doroshchuk only active in cells growing with excess nitrogen and TnrA
etal.,2006).Furthermore,TnrAispositivelyautoregulated, isonlyactiveinnitrogen-limitedcells.
whileatthesametimeexhibitingnegativecross-regulation Recently,Kayumovetal.(2008)showedthatTnrAisalso
for glnRA (Zalieckas et al., 2006) and repressing the gltAB subjecttoproteolysis,dependingonthenitrogensupplyof
operon encoding glutamate synthase (Belitsky et al., 2000; the cell and its interaction with a complex of ammonium
Belitsky & Sonenshein, 2004). The glutamate synthase- transporter AmtB and the P protein GlnK (Detsch &
II
(cid:2)c2010FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolRev34(2010)588–605
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NitrogencontrolinGram-positivebacteria 591
Mycobacterium smegmatis MC2 155
Mycobacterium tuberculosis H37rv
β
S I- Streptomyces coelicolor A3(2) Gram-positive, high G+C
G
Corynebacterium glutamicum ATCC 13032
Escherichia coli K12 Gram-negative
Lactobacillus acidophilus NCFM
Clostridium botulinum E3
Clostridium beijerinckii NCIMB 8052
D
Clostridium butyricum 5521 ow
αS I- Lactococcus lactis ssp. lactis IL1403 Gram-positive, low G+C nload
G Streptococcus pneumoniae R4 ed
Enterococcus faecalis V583 from
h
Bacillus subtilis 168 ttp
s
Staphylococcus aureus NCTC 8325 ://a
c
a
Listeria monocytogenes F2365 d
e
0.1 m
ic
Fig.2. PhylogenetictreeofhomologsofthetypeIglutaminesynthetase.AnunrootedphylogenetictreewascalculatedusingtheCLUSTALWsoftwareusing .ou
theimplementedneighbor-joiningmethodwiththefunctionforevolutionarydistancecorrection.Evolutionarydistancesareproportionaltothebranch p.c
length.Fifteenproteinsequenceswereselectedasindicatedinthefigure.Scalebarindicatesthenumberofsubstitutionsperaminoacidposition. om
/fe
m
s
re
Sutnu¨dlkere,t2h0e0d3i;rHecetintrraicnhscertipatli.o,n2a0l0r6e)g,ubloattihonofowfhTinchrAa.rHeaolwso- pboattuhloinguenms,anthdeCmloostsrtidwiuemll-ktentaonwin, wohnicehs pbreoindguceCleoxsttrreidmiuemly /article
ever, the physiological benefit of the rapid proteolysis of potent toxins. Because of this medical importance, research -a
b
s
TnrAinresponsetonitrogenstarvationandthecorrespond- overthepastfewyearshasfocusedespeciallyonthevirulence tra
c
ingproteaseremainsunknown. factorsandtoxinsofthesebacteria,andnotmuchisknown t/3
While GlnR and TnrA are supposedly specific for the about their central metabolism and regulation. Clostridia 4/4
subdivisionBacilli,homologs oftheglobal regulator CodY share homologs of some global regulators of the Firmicutes, /5
8
8
that directly senses GTP and branched-chain amino acids suchasCcpA(Tangneyetal.,2003;Vargaetal.,2004,2008) /5
4
can be found in all available genomes of Firmicutes. CodY and CodY (Dineen et al., 2007), whereas homologs of the 1
4
2
playsacentralroleattheintersectionofnitrogenandcarbon B.subtilisnitrogen-regulatoryproteinsGlnRorTnrAhavenot 8
b
metabolism, mostly by direct repression of the urease- been found in the available clostridial genome sequences, y
g
encoding operon and control of genes involved in the indicating different regulatory networks for at least central u
e
s
transport, catabolism and biosynthesis of amino acids nitrogenmetabolismanditskeyplayers(Table1). t o
n
(Sonenshein,2007).Recentstudieshavefocusedontherole ForClostridiumsaccharobutylicumNCP262,theregulation 1
1
ofCodYinvirulenceandtoxingeneexpressioninpathogens of the assimilatory nitrogen gene cluster (Fig. 3) is well A
p
suchasStaphylococcusaureus(forareview,seeSomerville& characterized.ItcomprisesaspecificRNA-bindingregulatory ril 2
Proctor, 2009), B. anthracis (van Schaik et al., 2009), protein of the ANTAR family (Shu & Zhulin, 2002), desig- 0
1
Streptococcus pneumoniae (Hendriksen et al., 2008a) and nated NitR, which is characterized by an RNA-binding 9
Clostridium difficile (Dineen et al., 2007) (for details, see domain exerting transcription antitermination; furthermore,
Nitrogenmetabolismandvirulence). post-transcriptional regulation of the key enzymes via anti-
sense RNAwas shown (Stutz et al., 2007). Under nitrogen-
limiting conditions, genes encoding the GS/GOGATsystem
Regulation of nitrogen metabolism inClostridia
are induced by an as yet unknown signal transducer that
The genus Clostridium, endospore-forming obligate anae- activatestheantiterminationfunctionofNitR,whichinturn
robes, includes a number of biotechnologically important activatesthebicistronicoperonsglnA–nitRandgltAB.Simul-
speciessuchasClostridiumacetobutylicum,whichisableto taneously,anantisenseRNAthatisencodedintheglnA–nitR
produceacetoneandbutanolfromstarch,aswellashuman operon can bind to the ribosome-binding site and the start
FEMSMicrobiolRev34(2010)588–605 (cid:2)c2010FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
592 J.Amonetal.
?
NitR GlnK AmtB
Fig.3. Regulatorynetworkofnitrogenmetabo-
lisminClostridiumsaccharobutylicumNCP262.
glnA nitR glnK amtB
Arrowsindicatetherelativegenelengthand
organizationonthechromosomewiththe
?
respectivegenenameasdescribedinFig.1.
Questionmarksindicateputativeinteractions.
Thecoiledarrow(red)representstheantisense D
o
gltA gltB RNA(forexplanationsofthesymbolsandcolor w
n
code,seeinFig.1;forfurtherdetails,seetext). lo
a
d
e
d
fro
m
amtB glnK glnA nitR gltA gltB h
ttp
s
Fig.4. Genesencodingammoniumuptake,assimilationandsignaltransductionproteinsinClostridiumbutyricum5521.ThegenomeofC.butyricum ://a
featuresanunusuallyconservednitrogenuptakeandassimilationclustercomprisinggenesencodinganammoniumtransporterandPII-typeprotein ca
(yellow),atypeIglutaminesynthetase(blue),theputativeregulatoryproteinNitR(orange)andthelargeandsmallsubunitsoftheglutamatesynthase d
e
(gray).Notethatthisgenomicarrangementishighlyconservedinotherclostridia(colorcodeasinFig.1;forfurtherdetails,seetext). m
ic
.o
u
p
.c
o
codon of the respective operons, effectively downregulating homologsofthisGlnNenzymeinadditiontotheregulartype m
thelevelsofactiveGSandGOGATenzymeinthecellunder IglutaminesynthetaseGlnA(Reid&Stutz,2005). /fem
nitrogen-richconditions(Fig.4). sre
Thissystemseemstobeconservedforatleastsomeother /a
Clostridium species, including Clostridium saccharolyticum, Npoitsriotigveenbcaocntetrroial in highG1CDNAGram- rticle
Clostridiumbutyricum,ClostridiumbeijerinckiiandC.botuli- -a
b
s
ncoummp.rIinsetsergeesntiensgleyn,cfoodrinCg.thbeutaymricmumon,iuthmetgreannespcolrutsetrerAmaltsBo SRtergeupltaotmioynceosfcnoiterloicgoelonrmetabolism in tract/3
and the PII protein GlnK, which seem to form a bicistronic 4/4
operon and might be regulated in a manner similar to that Transcriptional regulation of the S. coelicolor glutamine /5
8
8
describedaboveforC.saccharobutylicum.Toourknowledge, synthetase was shown to be under the control of GlnR /5
4
thisisthefirstexampleofabacterialgeneclusterthatencodes (Wray et al., 1991; Wray & Fisher, 1993). Despite sharing 1
4
not only the complete GS/GOGAT system but also the the same name with the nitrogen regulatory protein of the 28
b
ammonium transporter and GlnK protein together with genusBacillus,whichisamemberoftheMerR-typefamily y
g
thecorrespondingregulatoryprotein(seeFig.4). oftranscriptioncontrolproteins,homologsofGlnRbelong u
e
s
Thedistributionofvarioustypesofglutaminesynthetases to the OmpR-type family of transcription factors with a t o
showsasurprisingdiversityamongthegenus.Accordingto C-terminalhelix-turn-helixDNA-bindingdomain(Kenney, n 1
1
thegenomesequencesofC.tetaniandClostridiumperfrin- 2002). Furthermore, streptomycetes have another OmpR- A
p
gsuenasll,ythlaersgeetwpuotastpievceiegsluptoasmseisnseasnynOthReFtaesenc(oadpipnrgoxainmuanteuly- teytpael.,re2g0u0l2a)to,rwthhiachtirsesciomginlaizretsomGolsntRo,fntahmeeslaymGelnpRroIIm(oFtienrks ril 20
1
70kDa in comparison with a typical glnA type I gene as GlnR, including the promoter of the Streptomyces- 9
encodinga50kDaGlnAtypeImonomer)thathasnotbeen (andFrankia-)specifictypeIIglutaminesynthetase-encod-
further characterized. No homologs of this enzyme have ing gene, glnII (Behrmann et al., 1990; Weisschuh et al.,
been found outside of the genus Clostridium. Clostridium 2000;forareview,seeReuther&Wohlleben,2007).Because
acetobutylicumATCC824 seemstobequite uniqueinthat OmpR proteins are typical response regulators of bacterial
its genome features exclusively a GSIII enzyme (GlnN) two-component systems, it can be assumed that GlnR is
possibly of deltaproteobacterial origin, but unfortunately, subject to phosphorylation by a yet unknown histidine
nothing is known about its function or regulation. Other kinase.AnoverviewofnitrogenregulationinS.coelicoloris
clostridia such as C. butyricum, C. beijerinckii, Clostridium given in Fig. 5. GlnR apparently binds DNA using the
thermocellumandvariousThermoanaerobacterspeciespossess ‘galloping model’ proposed for the E. coli OmpR protein
(cid:2)c2010FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolRev34(2010)588–605
PublishedbyBlackwellPublishingLtd.Allrightsreserved
NitrogencontrolinGram-positivebacteria 593
AMP
? ?
GlnK GlnK AmtB
?
GlnD
P
? ?
GlnRII ? GlnR
Fig.5. Regulatorynetworkofnitrogen
metabolisminStreptomycescoelicolorA3(2).
Arrowsindicatetherelativegenelengthand ≥ 7 genes ? ureA ureB ureC ureF ureG
organizationonthechromosome;dottedblack
arrowsillustratetheregulatoryinteractionsofthe
nirB nirD
GlnRandGlnRIItranscriptionfactor(orange)
includingitsfunctioninactivating(plus)or D
sco1863 o
repressing(minus)thetargetgene(s).Pleasenote w
n
thatGlnRIIalsoregulatessomeoftheGlnRtarget AMP lo
genes(forexplanationsofthesymbolsandcolor GS II GS GlnE GS ade
code,seeinFig.1;forfurtherdetails,seetext). d
fro
m
h
ttp
(Yoshidaetal.,2006).Inthismodel,theconsensussequence Besides the type I glutamine synthetase of the b-group s
in the promoter regions of the respective target genes is (Eisenbergetal.,2000;Brownetal.,1994;seeFig.2fordetails) ://ac
a
broken down into binding boxes that feature sites with a termedGlnA1andtheeukaryotic-typeGlnII,streptomycetes d
e
m
relativelylow (a-site) and a higher binding affinity (b-site) possess additional genes encoding GSI-homologs, namely ic
(Tiffert et al., 2008). This results in multiple binding of GlnA2, GlnA3 and GlnA4 (Table 1); however, not much is .o
u
p
GlnR,whichinturncouldallowafine-tunedmodulationof known about their physiological relevance or regulation, .c
o
the corresponding promoter in response to the nitrogen although they seem not to be subject to GlnE-mediated m
statusofthecell.Reflectingtheirpost-translationalinterac- adenylylation(Rexeretal.,2006).Incontrasttothesituation /fem
tions, GlnK and GlnD are encoded in a highly conserved found for Firmicutes, the post-translational regulation of sre
gtreannespcolurtseterrAtomgteBthe(rTawbilteh 1a).hoTmhoislogapopfartehnetatmrimcisotnroiunmic nsyistrteomgenomf eentatebroiclisbmacattefirirast(ssiegehtInsetreomdsutcotiroense).mTbolegetthheeNrtwrBitCh /article
operonisregulatedbyGlnR,whichactsasatranscriptional the planctomycetes (Fuerst, 1995; Wagner & Horn, 2006), -ab
s
activator under conditions of nitrogen starvation (Fink actinomycetesaretheonlyprokaryotesoutsideoftheProteo- tra
c
et al., 2002). Further genes that show GlnR-controlled bacteriathatpossesshomologsoftheadenylyltransferaseGlnE t/3
transcription are the type I glutamine synthetase-encoding andtheuridylyltransferaseGlnD,withthespecialfeaturethat 4/4
geneglnA1andtheeukaryotic-typeglnII(Finketal.,2002) GlnD actually adenylylates or deadenylylates the P protein /5
II 8
8
as well as the nirBD operon (Tiffertet al., 2008),encoding GlnKinresponsetonitrogenavailability(Heskethetal.,2002; /5
4
subunitsofthenitritereductase.Recently,agenecodingfor Stro¨sser etal., 2004; Forchhammer, 2008).Incontrast to the 1
4
2
aproteinsimilartoperiplasmicnitratereductaseprecursors, proteobacterial Ntr system, the GlnD-PII pathway is not 8 b
termednasA,wasshowntobeunderthepositivecontrolof required for GlnE-dependent GSI regulation (Hesketh et al., y
g
GlnR (Wang & Zhao, 2009), which extends the role of the 2002). Also, S. coelicolor GlnK is subject to a nonreversible u
e
s
regulator also to nitrate-dependent growth. Interestingly, modificationby removalofthefirstthreeN-terminalamino t o
n
GlnRcanalsoactasaninhibitoroftranscription,repressing acidsinresponsetoanammoniumshock.Thephysiological 1
1
the transcription of the gdhA, ureA and various other, relevance of this modification and the responsible cleaving A
p
not further characterized ORFs of putative or unknown enzyme(s)areunknown(Heskethetal.,2002). ril 2
function(Tiffertetal.,2008).TherepressionoftheGDHby Justrecently,adirectnegativecontrolonthetranscription 0
1
GlnR seems to make sense under nitrogen-limited condi- ofglnRanditsmostprominenttargetsglnA,glnIIandamtB 9
tions, as the GDHis thought to be ineffective at lowNH1 by the phosphate response regulator PhoP was shown
4
concentrations compared with the GS/GOGAT system. (Rodr´ıguez-Garc´ıaetal.,2007,2009):PhoPproteinsseemto
However,theinhibitoryeffectonthetranscriptionofureA, compete with GlnR in binding to overlapping sites on the
which is part of an operon encoding the various subunits DNAaswellasposingaroadblockoftranscription,depending
of the urease holoenzyme, seems puzzling under nitrogen- onthepromoterstructureoftherespectivegene(Rodr´ıguez-
limiting conditions because urease represents a major Garc´ıa et al., 2009). Paradoxically, for wild-type cells, tran-
pathway for the release and utilization of ammonia from scriptomestudiesshowednoresponseoftheGlnRregulonto
urea, either as an external source or as an intermediate of phosphatelimitation(Rodr´ıguez-Garc´ıaetal.,2007),andso
purinedegradation. the physiological relevance of this observation, besides the
FEMSMicrobiolRev34(2010)588–605 (cid:2)c2010FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
594 J.Amonetal.
deducedtranscriptionalrelationbetweenthecontrolofphos- regulated genes also yielded cis-elements that are highly
phateandnitrogenmetabolism,isstilldebatable. similartotheconsensussequencesfoundforGlnR,andthus
Homologs of GlnR have so far been experimentally suggestsasimilarmodeofDNAinteraction(seethesection
verifiedtoregulatethetranscriptionofatleasttheglutamine aboutstreptomycetes).
synthetase in the antibiotic-producing pseudonocardia ForM.smegmatis,thehighnumberofgenesdirectlyand
Amycolatopsis mediterranei (Yu et al., 2006, 2007) and indirectly related to primary nitrogen metabolism is note-
members of the mycobacteria, for example Mycobacterium worthy.Twogenesencodingadditionalputativeammonium
smegmatis and Mycobacterium tuberculosis. Bioinformatic transporters, amtA and amt1, were reported (Amon et al.,
database screenings and homology analyses with available 2009).Theamt1geneisorganizedinacluster,togetherwith
genome sequences reveal homologs of GlnR in virtually genes that apparently encode proteins with the functional
everyactinobacterialgenomepublishedthusfar,exceptfor domains of a class III glutamine synthetase, a class II
D
the Corynebacteriaceae, where AmtR is the regulator of glutamine amidotransferase and domains of a glutamate o
w
n
nitrogenmetabolism.ItissupposedthatGlnR(andpossibly synthase,whilenoapparentoperonwasobservedforamtA. lo
a
GlnRII)areorphanregulatorsofabacterialtwo-component Thedomainpredictionstronglysuggeststheinvolvementof d
e
system, but the putative sensor histidine kinase or the this operon in general ammonium assimilation (Amon d fro
molecule that serves as an indicator of the nitrogen status et al., 2009). Interestingly, a gdhA gene coding for an m
have not been determined as yet. Furthermore, the exact assimilatory NADPH-dependent GDH is observed only in http
physiological roleofthe Streptomyces-specific GlnRII regu- thegenomeofM.smegmatis.Theabsenceofthisgenefrom s
lator and its putative function in secondary metabolism the other four mycobacterial species suggests that ammo- ://ac
a
needtobeelucidatedinfuturestudies. nium assimilation depends on the GS/GOGAT pathway in d
e
m
these bacteria. The genes glnA1 and glnA2 are found in all ic
mycobacterialgenomesinaconservedregiontogetherwith .o
u
Regulation of nitrogen metabolism p
glnE, encoding an adenylyltransferase that regulates gluta- .c
in mycobacteria o
minesynthetaseactivity(Carrolletal.,2008).Homologsof m
ThegenusMycobacteriumincludessaprophyticsoilbacteria theGlnA3andGlnA4proteinswerefoundinallmycobac- /fem
such as M. smegmatis as well as major human pathogens terialgenomes,exceptforM.leprae,whileforGlnA4,atleast sre
sucGheansetMic.tuscbreerecnuilonsgisaannddMsyucbobseaqcuteerniutmmleuptraane.t analyses tMhr.esemheogmmaotloisg.oBuessipdreostehionmswoleorgesidoefngtlinfiAed1-i4n,tMhe.gsemneogmmeatoisf /article
revealedthattranscriptionofM.smegmatisamt1,theamtB– possesses various additional genes that seem to encode -ab
s
glnK–glnDoperonandglnAispositivelycontrolledbyGlnR, homologsofglutaminesynthetasesand,basedonhomology tra
c
aGlnR-likeprotein(Amonetal.,2008,2009).Additionally, studies,mayhavebeenacquiredviahorizontalgenetransfer t/3
Malm et al. (2009) demonstrated a GlnR-dependent upre- from different bacteria including other actinomycetes, rhi- 4/4
gulationoftheM.tuberculosisH37rvgenesnirBD,encoding zobiaandDeltaproteobacteria(Amonetal.,2009).Whileit /5
8
8
subunits of the nitrite reductase, in response to nitrogen- was already shown for M. tuberculosis that only GlnA1 is /5
4
limiting conditions (see Fig. 6). Further screening of the abundantlyexpressedandessentialforbacterialhomeostasis 1
4
2
upstreamregionsofthecorrespondingmycobacterialGlnR- (Harth et al., 2005), the physiological role and function of 8
b
y
g
u
e
s
AAMMPP ? ? t on
GlnK GlnK AmtB 1
? glnR 1
GlnD A
p
ril 2
0
? P 19
? GlnR glnD glnK amtB
Fig.6. Regulatorynetworkofnitrogenmetabo-
nirB nirD lisminMycobacteriumsmegmatisMC2155.
glnA Pleasenotethattheregulationofnitrite
reductase-encodinggenes(nirBD)isdeduced
AMP
GlnE fromMycobacteriumtuberculosisH37rv
GGSS GGSS (forexplanationsofthesymbolsandcolorcode,
seeinFig.1;forfurtherdetails,seetext).
(cid:2)c2010FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolRev34(2010)588–605
PublishedbyBlackwellPublishingLtd.Allrightsreserved
NitrogencontrolinGram-positivebacteria 595
the various other glutamine synthetase-encoding genes, mycobacteria. Importantly, the sensory protein for the
especially in the genome of M. smegmatis, remain to be nitrogenstatusofthecellthatputativelycontrolstheGlnR
verified. In all mycobacterial genomes, a highly conserved responseregulatoraswellasthesignalsensedbythisprotein
apparent operon encoding the large and small subunit of is still unknown, as is the function of the P -type signal
II
glutamate synthase (GOGAT; gltB and gltD) was observed. transduction proteins. Also, not much is known about the
In addition, the genome of M. smegmatis features several role of AmtR in the genomes of M. smegmatis and closely
additionalcopiesofgltBandgltDthatarenotfoundinother relatedactinomycetessuchasNocardiafarcinica,Rhodococ-
mycobacteria. Furthermore, two putative urease-encoding cus sp., Clavibacter michiganensis or Kineococcus radiotoler-
operons were found, of which onlyone exhibits homology ans, all of which also have homologs of GlnR. First
totheuregeneclustersinM.tuberculosisandMycobacterium experiments point to a regulatory function of AmtR in
bovis. The otheroperon exhibits striking similarities to the uptake and assimilation of amino acids (J. Amon & Y. Lu,
D
genes encoding urease subunits in Deltaproteobacteria. No unpublisheddata).Furtherexperimentsarenecessarytonot o
w
n
ureaseoperonorurease-relatedgeneswerefoundinMyco- only expand the mycobacterial GlnR regulon but also lo
a
bacterium avium and M. leprae. Also, M. smegmatis is the elucidate the exact function of AmtR, which has been d
e
only mycobacterial species to feature a distinct operon studiedinfarmoredetailincorynebacteria. d fro
encoding the subunits of a putative urea ABC transporter, m
emphasizing – together with the presence of the different Regulation of nitrogen metabolism http
urease-encoding operons – the importance of urea as a s
nitrogensourceforthisspecies. in corynebacteria ://ac
a
Amongmycobacteria,proteinsinvolvedinsignallingand AmtRactsastheglobalregulatorofnitrogenmetabolismin d
e
m
post-translational modification are best characterized in C.glutamicumandothercorynebacteria(Jakobyetal.,2000; ic
M.tuberculosis(Parish&Stoker,2000;Table1).Mutagenesis Walter et al., 2007). By a combination of bioinformatics, .o
u
p
experiments point to the fact that glnE is essential in this transcriptome and proteome analyses and DNA-binding .c
o
organism(Parish&Stoker,2000),anobservationthatisin studies, it was shown that 35genes are directly regulated by m
agreementwiththecrucialfunctionofadenylyltransferasein AmtR (Beckers et al., 2005; Buchinger et al., 2009). These /fem
the regulation of GS activity (Carroll et al., 2008). Because includegenesencoding proteinsinvolvedinthetransportof sre
gimluptaomrtiannetdsyrnutghteatragseeti(nHMar.thtuebtearlc.,u1lo9s9is4,is20e0ss5e;nTtuiallli,uisteitsaaln., namitrmogoenniusmou(racmestA(c,ranmT,tBu)rtaAsBwCelDlaEs,tgrlaunAspBoCrDte)r,saonfdalteenrznyamtivees /article
2003;Nordqvistetal.,2008).Workconcentratedfirstonthe for ammonium assimilation (dapD, gdh, glnA, gltBD) and -ab
s
glnE gene product, adenylyltransferase. The M. tuberculosis utilization of alternative nitrogen sources such as creatinine tra
c
glnE promoter is upregulated in ammonia- or glutamine- (codA)andurea(ureABCEFGD).Furthermore,theexpression t/3
containing media, at least in the heterologous host of proteins that are part of the signal transduction cascade 4/4
M.smegmatis(Pashleyetal.,2006),whilethetranscriptional (GlnKandGlnD)isAmtR-regulated(Fig.7). /5
8
8
organizationoftheglnA1–glnE–glnA2geneclusterremains The characterization of the AmtR regulon was a prere- /5
4
to be elucidated further (Hotter et al., 2008). Compared quisiteforamoredetailedinvestigationofAmtR-mediated 1
4
2
with GlnE, GlnD seems to have no crucial function in the repression.Forinstance,asdescribedforNtrBC-controlled 8
b
regulation of ammonium assimilation in M. tuberculosis transcriptionoftargetgenesinE.coli(Atkinsonetal.,2002), y
g
(Read et al., 2007). This is in agreement with the observa- nitrogen-dependent gene expression in C. glutamicum is u
e
s
tionsmadeinS.coelicolorandCorynebacteriumglutamicum alsomarkedbygradualexpressionofgenesbelongingtothe t o
(Heskethetal.,2002;Stro¨sseretal.,2004).Incontrasttothe AmtRregulon.WhiletheamtAandamtBgenescodingfor n 1
1
signaltransferviaGlnD,GlnKandGlnEtoGSasshownin ammonium uptake systems are not transcribed under A
p
E. coli, enzyme activity measurements in glnK deletion nitrogen surplus, glnA is expressed at a low level in the ril 2
strains showed that the activity of ATase GlnE is not presence of ammonium. This differential regulation of 0
1
regulatedbyGlnKinS.coelicolor(Heskethetal.,2002)and transcription fulfills the physiological need for glutamine 9
C.glutamicum(Stro¨sseretal.,2004).FortheP proteinof synthesisbyGSwithoutturningonthenitrogenstarvation
II
M. tuberculosis H37rv, biochemical studies revealed a good response. Upstream of strongly repressed genes such as
conformitytootherP orthologswithrespecttobindingof amtAandamtB,canonicalAmtR-bindingsiteswereidenti-
II
ATPand 2-oxoglutarate, and emphasize the importance of fied(Jakobyetal.,2000),whereastheAmtR-bindingmotif
the T-loop for the flexibility of interactions with other upstream of glnA differs significantly from the deduced
proteinsandmodifications(Bandyopadhyayetal.,2009). consensus motif (Nolden et al., 2001a). In addition, the
Therearestillanumberofotheropenquestionsregard- AmtR-binding sites upstream of strictly regulated genes
ingthepost-translationalinteractionsbetweenandmodifi- such as amtA and the gltBD operon are located near the
cations of the key players of nitrogen control in transcriptionstart,sothattranscriptionmightbeblockedin
FEMSMicrobiolRev34(2010)588–605 (cid:2)c2010FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
596 J.Amonetal.
AMP
amtR AmtR GlnK GlnK AmtB
GGllnnDD
gglnD gglnK aammttBB
≥ 25 genes
A B ureC E F G D
gltB gltD
D
o
ggllnnAA ggddhh w
n
lo
Fig.7. Regulatorynetworkofnitrogen a
d
metabolisminCorynebacteriumglutamicum e
d
GGllnnEE AMP ATCC13032.Forexplanationsofthesymbols fro
GS GS andcolorcode,seeFig.1;forfurtherdetails, m
seetext. http
s
://a
c
a
the presence of AmtR. The AmtR-binding site of glnA is Adenylylation and deadenylyation of GlnK are not only d
e
m
locatedupstreamofthe (cid:4)10region,whichmightdiminish crucialforitsinteractionwiththetranscriptionalregulator ic
theblockingeffect. AmtR but also for its localization. During nitrogen starva- .o
u
p
The characterization of mutant AmtR proteins by gel tion, GlnK is adenylylated and exclusively found in the .c
o
retardation assays and surface plasmon resonance and cytoplasm (Stro¨sser et al., 2004) and is demodified and m
molecular modelling revealed amino acid residues that are sequestered to the cytoplasmic membrane by binding to /fem
directly involved in DNA binding. Furthermore, it was AmtB in response to improving nitrogen supply (Stro¨sser sre
schruocwianlftohratretphreessspioancibnygAomf ttRhe(Mbiunhdlinegtaml.,o2t0if09h)a.lf sites is emtoaslt.,o2f0t0h4e)G.lOnnKciesldoecgarlaizdeedd.aLtetshsethcaynto5p%lasomfiGclnmKemsubrrvaivnees, /article
AmtR is a member of the TetR family of transcriptional the first 30min after an ammonium pulse. The residue is -ab
s
regulators(forareview,seeRamosetal.,2005).Typically,these protectedfromproteolysisbyanunknownmechanism. tra
c
repressors interact with small molecules, as shown for TetR, Perceptionofthecellularnitrogenstatusisaprerequisite t/3
whichbindstetracyclines,andQacR,whichbindsanumberof foranadequateresponseofC.glutamicumtochangesinthe 4/4
drugs and dyes such as rhodamine G, crystal violet and nitrogen supply. Various nitrogen sources such as ammo- /5
8
8
ethidium bromide. In contrast to these TetR-type repressors, nium, different amino acids, creatinine and urea can be /5
4
the interaction of C. glutamicum AmtR with its target DNA metabolized by C. glutamicum (Burkovski, 2005, 2007; 1
4
2
does not depend on the presence or the absence of small Ha¨nßler & Burkovski, 2008) and contribute to nitrogen 8
b
ligands.Instead,bindingtopromotersofnitrogen-dependent supply,butitisunlikelythateverysinglenitrogensourceis y
g
genesisregulatedbyaspecificprotein–proteininteraction:at sensed. More likely, a single solute or only a very limited u
e
s
decreasingammoniumconcentrations,AmtRisreleasedfrom numberofkeynitrogenmetabolites aresensed. Pioneering t o
n
theDNAby interactionwiththePII-typesignaltransduction workinSalmonellaentericaserovarTyphimurium,Klebsiella 1
1
proteinGlnK(Beckersetal.,2005).Toelucidatetheinteraction pneumoniae (Ikeda et al., 1996; Schmitz, 2000), B. subtilis A
p
domains, which are still unclear, crystallization of AmtR (Hu et al., 1999a) and cyanobacteria (Forchhammer, 2004, ril 2
(Hasselt et al., 2009) and GlnK is in progress. However, it is 2007) suggested that 2-oxoglutarate and L-glutamine were 0
1
knownthatasaprerequisiteforthisinteraction,thetrimeric likelycandidatesforthisfunction. 9
GlnK complex has to be adenylylated at tyrosyl residue 51 A clue for a possible signal molecule of low nitrogen
located in the T-loop of the protein. This is catalyzed in supplycamefromanalysesofGDH-deficientC.glutamicum
responsetonitrogenstarvationbyGlnD.Besidesadenylylation, strains. RNA hybridization revealed that deletion of gdh
GlnD is also responsible for the demodification of GlnK leads to the deregulation of nitrogen control, including
depending on the nitrogen status of the cell. Studies with deregulated expression of the gltBD operon (Mu¨ller et al.,
mutants expressing a truncated GlnD protein indicated that 2006).Metaboliteanalysesindicatedthatahigh2-oxogluta-
themodifyingactivityofthisbifunctionalenzymeislocatedin rate concentration found in the gdh mutant under any
theN-terminalpart,whereastheC-terminalpartoftheprotein growth conditions and in the wild type under nitrogen
iscrucialforitsdemodificationactivity(Stro¨sseretal.,2004). starvationmightbeperceivedasmarkerofapoornitrogen
(cid:2)c2010FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolRev34(2010)588–605
PublishedbyBlackwellPublishingLtd.Allrightsreserved
NitrogencontrolinGram-positivebacteria 597
supply. The most likely sensor protein of 2-oxoglutarate is andtheimportanceofGlnDinnitrogencontrolwasverified
signal transduction protein GlnK (for a review, see Arcon- by the characterization of a mutant strain (Nolden et al.,
deguyetal.,2001),althoughdirectbiochemicalevidencefor 2002). Modification of Corynebacterium efficiens GlnK in
2-oxoglutaratebindingbyC.glutamicumGlnKandalinkto response to nitrogen starvationwas shown by biochemical
regulationismissing. analyses (Walter et al., 2007). Further genome sequencing
IncontrasttothesituationinenterobacteriaandB.subtilis, has revealed AmtR homologs in virtually every corynebac-
L-glutamine seems notto be a markerofnitrogen supply in terialspecies,exceptCorynebacteriumjeikeium(Tauchetal.,
C.glutamicum.TheL-glutamineconcentrationisveryhighin 2005),Corynebacteriumkroppenstedtii(Tauchetal.,2008a)
C. glutamicum compared with other bacteria (Nolden et al., andCorynebacteriumurealyticum(Tauchetal.,2008b).
2001b; Niebisch et al., 2006; Rehm et al., 2010), and more
strikingly,theL-glutaminepoolreactsonlyslowlytochanges Influence ofa pathogenic lifestyle on nitrogen D
in the nitrogen supply (Nolden et al., 2001b; Mu¨ller et al., o
w
metabolismand control n
2006). Most likely, this amino acid (like L-glutamate, which lo
a
also has an extremely high concentration in the cytoplasm) Acommonpropertyofthegenomesofpathogenicbacteria d
e
aacstswaesllasatsornaigterocgoemnpomuentadbaonlidsmbu,ffrearthfoerrctahrabnonaasndnietrnoegregny itsioanclteearrmteednd‘ernedcyucttoivwearedvoreludtuioctni’on(Goof´mgeenzo-Vmailcerionfoetrmaal.-, d from
marker. This is in accordance with previous findings that 2007).Asthepathogenbecomesmoreandmoreadaptedto http
C.glutamicumGlnDlacksthetypicalglutamine-bindingsite –andthusdependenton–itshost,itbeginstolosegenetic s
found in enterobacterial proteins and is consequently most information that is superfluous for nutrient acquisition ://ac
a
likelynotabletosenseL-glutamine(Tønderviketal.,2006). (Titgemeyeretal.,2007).Incontrasttothesituationoftheir de
m
ForC.glutamicum,observationsfromseveralstudiesstrongly free-living relatives, carbohydrates, fatty acids or amino ic
indicatethatammoniummightfunctionasapositivemarker acidsareabundantlyavailabletopathogens,andarereadily .o
u
p
fornitrogensupply(Noldenetal.,2001b;Stro¨sseretal.,2004; assimilated. Thus, the need for a primary metabolism is .c
o
Mu¨lleretal.,2006). alleviated to the point where gene sets for complete path- m
Glutamine synthetase is regulated at the level of tran- ways disappear without any effect on the fitness or the /fem
scription in an AmtR-dependent manner (Nolden et al., survivaloftheorganism. sre
2ch0a0n1ag)e.siAndndiittrioogneanllsyu,pfpolry,atheraapcitdiviatydaopfttahteioennzytomesuisdadlesno higAhmesotnngummbyceorboafcgteernieas,,Men.asbmlienggmtahteisuispetaqkueipapneddawssiitmhitlhae- /article
regulated via adenylylation/deadenylylation (Jakoby et al., tionofvariousnitrogensources.Mycobacteriumtuberculosis, -ab
s
1999) by GlnE. A deletion of the glnE gene led to a M. bovis BCG and M. avium ssp. paratuberculosis show a tra
c
deregulation of GS activity in response to the nitrogen reduced set of nitrogen metabolism-related genes, while t/3
availability (Nolden et al., 2001a). Nucleotide sequence M.lepraerevealsthestrongestreduction(Amonetal.,2009). 4/4
comparisons revealed a typical adenylylation site, a tyrosyl Asimilarsituationwasfoundforcorynebacteria.Inthis /5
8
8
residue at amino acid position 405 (Jakoby et al., 1997), genus, the soil-dwelling species C. efficiens and C. glutami- /5
4
whichwasverifiedbymutantanalyses(Jakobyetal.,1999). cumhaveabroadsetofgenesencodinguptakesystemsfor 1
4
2
Interestingly, in a C. glutamicum glnE deletion strain, nitrogensources,assimilatoryenzymesandregulatorypro- 8
b
nitrogen control is partially deregulated (Rehm et al., teins,whileacleartendencytowardgenedecaywasobserved y
g
2010).Studiestoinvestigatewhetherthisisduetoindirect for the pathogens C. diphtheriae and C. jeikeium. This u
e
s
metaboliteeffectsoradirectregulatoryfunctionofGlnEare includesammoniumandureauptakesystems,ammonium t o
n
in progress. Characterization of a glnK deletion strain assimilation pathways, urea-cleaving enzymes and regula- 1
1
showed that ATase works independently of GlnK in toryproteins(Walteretal.,2007). A
p
C.glutamicum(Stro¨sseretal.,2004)asinS.coelicolor(Hesketh The most prominent examples of genome reduction are ril 2
et al., 2002). In both C. glutamicum and S. coelicolor, it is found for the Mollicutes, i.e. the genera Mycoplasma and 0
1
completelyunclearhowGlnEperceivesthenitrogenstatus. Ureaplasma. These bacteria have some of the smallest 9
Nitrogen control mechanisms shown for C. glutamicum genomes among self-replicating organisms, ranging in size
seemtobeconservedinothercorynebacteria(seeTable1): from 0.6 to 1.35Mbp, with just around 500–800 reading
for Corynebacterium diphtheriae, the function of AmtR in frames actually coding for proteins (Fadiel et al., 2007).
nitrogen control was verified by mutant generation and Genescodingforglutaminesynthetase,glutamatesynthase
RNA hybridization analyses (Nolden et al., 2002), and the or GDH are absent from all the published sequences,
AmtR DNA-binding motif seems to be conserved (Walter indicating a complete loss of ammonium assimilation in
et al., 2007). As described above, C. glutamicum AmtR is these organisms. Conserved genes encoding ammonium
controlled by a signal cascade including GlnK and GlnD. transporterswerespottedonlyinthegenomesofUreaplas-
The corresponding genes are conserved in C. diphtheriae maparvum and Ureaplasma urealyticum, where theyoccur
FEMSMicrobiolRev34(2010)588–605 (cid:2)c2010FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved