DCW-2008-Cell growth and cell division in the rod-shaped actinomycete Corynebacterium glutamicum

ORIGINAL PAPERCell growth and cell division in the rod-shaped actinomycete Corynebacterium glutamicumMichal Letek ÆMarı´a Fiuza ÆEfre ´n Ordo ´n ˜ez ÆAlmudena F.Villadangos ÆAstrid Ramos ÆLuı´s M.Mateos ÆJose ´A.Gil Received:3December 2007/Accepted:29January 2008/Published online:19February 2008ÓSpringer Science+Business Media B.V.2008Abstract Bacterial cell growth and cell division are highly complicated and diversified biological pro-cesses.In most rod-shaped bacteria,actin-like MreB homologues produce helicoidal structures along the cell that support elongation of the lateral cell wall.An exception to this rule is peptidoglycan synthesis in the rod-shaped actinomycete Corynebacterium glutamicum ,which is MreB-independent.Instead,during cell elongation this bacterium synthesizes new cell-wall material at the cell poles whereas the lateral wall remains inert.Thus,the strategy employed by C.glutamicum to acquire a rod-shaped morphology is completely different from that of Escherichia coli or Bacillus subtilis .Cell division in C.glutamicum also differs profoundly by the apparent absence in its genome of homologues of spatial or temporal regu-lators of cell division,and its cell division apparatus seems to be simpler than those of other bacteria.Here we review recent advances in our knowledge of the C.glutamicum cell cycle in order to furtherunderstand this very different model of rod-shape acquisition.Keywords Corynebacterium ÁCell division ÁCell growth ÁFtsZ ÁDivIVA ÁFtsI ÁHMW-PBP ÁCell wallIntroductionCorynebacterium glutamicum is a soil-borne actino-mycete that is widely used in the industrial production of amino acids,such as L -lysine,an essential amino acid used in animal nutrition,or the taste enhancer L -glutamic acid (Hermann 2003).The biotechnological production of each of these amino acids by corynebacteria is estimated to be more than 1million tons per year (Hermann 2003).Interest in corynebacteria stems from the late 1950s,when it was discovered that the bacterium is a natural producer of amino acids (Kinoshita et al.1957).Since that time and continuing into the present,C.glutamicum has been extensively studied,leading to the development of efficient transformation systems (Santamaria et al.1985)and cloning vectors (Santamaria et al.1984,1987;Cadenas et al.1991,1996),the cloning of genes involved in amino-acid production (Mateos et al.1987a,b ;Martin et al.1987),genome sequencing by three independent laboratories (Kalinowski et al.2003;Ikeda and Nakagawa 2003;Yukawa et al.2007),proteomicElectronic supplementary material The online version of this article (doi:10.1007/s10482-008-9224-4)containssupplementary material,which is available to authorized users.M.Letek ÁM.Fiuza ÁE.Ordo´n ˜ez ÁA.F.Villadangos ÁA.Ramos ÁL.M.Mateos ÁJ.A.Gil (&)Departamento de Biologı´a Molecular.A ´rea de Microbiologı´a.Facultad de Biologı´a,Universidad de Leo´n,Leon 24071,Spain e-mail:jagils@unileon.esAntonie van Leeuwenhoek (2008)94:99–109DOI 10.1007/s10482-008-9224-4and phosphoproteomic maps(Hermann et al.2001; Bendt et al.2003),DNA microarrays(Polen and Wendisch2004),and,finally,the use of all of these technologies in‘‘genome breeding’’to improve amino-acid production(Ohnishi et al.2002,2003).The genus Corynebacterium also comprises other relevant species.In fact,nearly coincident with the first report of the genome sequence of C.glutam-icum,the sequence of the very closely related Corynebacterium efficiens(Nishio et al.2003)as well as that from the well-known human pathogen Corynebacterium diphtheriae(Cerdeno-Tarraga et al.2003)were released.The genus also includes emergent pathogens,such as Corynebacterium jeikeium(Tauch et al.2005),Corynebacterium urealyticum(Fernandez-Natal et al.2001),and Corynebacterium amycolatum(Letek et al.2006a), which are frequently multi-drug resistant and cause nosocomial infections in immunosuppressed patients.The study of bacterial cell division and cell-shape acquisition could be applied to the rational design of new compounds tofight bacterial infections,since most of the genes involved in these processes are essential and thus may serve as targets of new antimicrobial agents or new antibiotics(Vicente et al.2006).The rate at which new antibiotics are being discovered by traditional approaches is much slower than the rate at which bacteria are becoming resistant to currently available antibiotics(Courvalin and Davies2003).In addition,manipulation of the cellular growth rate or the properties of the bacterial cell envelope could be used to improve the biotechnological production of amino acids in C.glutamicum(Hirasawa et al.2000;Radmacher et al.2005).Snapping division and club shape Corynebacteria are gram-positive,non-sporulating, pleomorphic bacilli that belong to the group of mycolata,which also includes the genera Mycobac-terium and Nocardia.The pleomorphism of C.glutamicum is influenced by the culture conditions and in general the cell poles of cultured C.glutam-icum are frequently engrossed,which confers a ‘‘club’’shape to the cells(Cure and Keddie1973). The mycolata are characterized by a lipid-rich cell envelope surrounding the cell wall,which has been described as an outer membrane of gram-positive bacteria and is thought to act as a permeability barrier (Minnikin1982;Dover et al.2004).In Corynebac-terium,this lipid domain is composed of corynemycolic acids,which contain30–36carbon atoms and a non-reduced b-keto group(Collins et al. 1982;Puech et al.2001).In addition,several species of corynebacteria possess an all-embracing and well-documented S-layer(Peyret et al.1993;Chami et al. 1997;Hansmeier et al.2006).After the bacteria undergo‘‘snapping’’cell division(Video S1),the daughter cells remain joined together,forming a characteristic V-shape(Collins and Cummins1986) and often lie in clusters resembling Chinese letters or palisades.Transmission electron microscopy of C.glutamicum cells showed that newly formed cells remain together,linked by a yet-unknown compound present on the surface of C.glutamicum(Fig.1)that could be the extensive S-layer present in this microorganism.Most of the studies on corynebacterial morphology have focused on non-pathogenic species.However, there are also reports in which the human pathogen C.diphtheriae was studied(Sall et al.1958;UmedaFig.1Transmission electron micrograph of two Corynebac-terium glutamicum V-shaped cells,the products of snapping division.The unknown compound present on the surface of C.glutamicum(arrow)could maintain the link between the two daughter cells.The experimental procedure used was described previously in Flardh,K.(2003a)and Amako1983).Electron microscopic examination of C.diphtheriae stained with antiserum against total wall material suggested that cell division occurred at the middle of the cell,with the existing cell-surface divided between the two daughter cells.Cell elonga-tion was found to resemble the growth pattern of fungal or Streptomyces hyphae and was therefore called apical growth(Umeda and Amako1983; Flardh2003b).Similar patterns of cell division and elongation were recently recognized in C.glutamicum by staining of newly synthesized peptidoglycan(PG)(see below and Daniel and Errington2003).Genes involved in cell division and cell growthin corynebacteriaThe entire process of cell division in bacteria requires the products of several fts genes.These genes are located in the conserved dcw(division cell wall) cluster,the organization of which has been analyzed in several species of bacteria(Tamames et al.2001). These phylogenetic analyses led to the conclusion that the different arrangements of the dcw clusters clearly separate the rod-shaped actinomycetes M.tuberculosis and C.diphtheriae from other bacteria.The genetics of cell growth and cell division of the non-pathogenic C.glutamicum were studied even before complete genome sequences were available. The earliest studies were focused on the sequencing and characterization of several corynebacterial genes present in the dcw cluster,such as ftsZ(Kobayashi et al.1997;Honrubia et al.1998),ftsI(Wijayarathna et al.2001;Valbuena et al.2006),murE (Wijayarathna et al.2001),murD,murC,and ftsQ (Wachi et al.1999;Honrubia et al.2001;Ramos et al. 2004),and divIVA(Ramos et al.2003).From these studies,a transcription profile of the dcw cluster from C.glutamicum(Fig.2)was obtained as well as an early indication that cell division of this bacterium must differ given the lack of cell-division genes essential to the process in other bacteria,like ftsA or ftsL(see below).However,recent experimental data showed that the overexpression or alteration of several fts-encoded proteins in E.coli can make up for the absence of other essential members of the cell-division apparatus,or divisome(Geissler and Margolin2005;Bernard et al.2007).Thus,C.glutamicum may have a reduced version of the more sophisticated divisome present in other bacteria, since some proteins in the latter have functional redundancies.For a detailed description of the proteins involved in bacterial cytokinesis,see the review by Errington et al.2003.The sequencing of the genomes of different Corynebacterium species and comparison of the sequences to those of other well-known model bacteria(Table1),together with the application of new molecular biology and microscopy techniques, have provided a major impulse to our understanding of the cell-cycle in these bacteria.Analysis of the genes present in the genome of several corynebac-teria species revealed the absence of any MreB actin-like homologues,which are essential for cell elongation of E.coli(Kruse et al.2005),Bacillus subtilis(Jones et al.2001),and Caulobacter cres-centus(Figge et al.2004),among others. Furthermore,this analysis confirmed that the con-served dcw cluster of C.glutamicum is a simplified version of the homologous clusters found in most bacteria(Fig.2)and that the well-known positive or negative regulators of cell division are missing (Ramos et al.2003;Letek et al.2007).Similar to other Actinobacteria,such as M.tuberculosis or S.coelicolor(Table1),there are no homologues to ftsA(an actin-homologue)in the C.glutamicum genome,neither homologous genes been found to positive regulators involved in FtsZ polymerization e.g.,zipA or zapA,or to negative regulators,e.g., ezrA,noc,slmA,sulA,and minCD(Letek et al.2007). Moreover,essential cell-division genes(those encod-ing FtsN and FtsL)are also not present in C.glutamicum.An exception is the poorly understood ftsEX cluster,which is present in C.glutamicum (cg0914and cg0915)and in M.tuberculosis(Mir et al.2006).Recently,it was suggested that,in E.coli, FtsEX proteins form an ABC transporter that imports certain substrates during cell division in order to maintain osmotic pressure in the cell during formation of the cell envelope(Schmidt et al.2004;Reddy 2007).The absence of any positive regulators could be compensated by FtsW,as is the case in M.tuberculosis where the protein is involved in the stabilization of Z-ring polymerization(Datta et al.2006).Although in C.glutamicum FtsW interacts directly with FtsZ (Valbuena et al.2007),other proteins could participateF i g .2G e n e t i c o r g a n i z a t i o n o f t h e d c w c l u s t e r i n d i f f e r e n t m i c r o o r g a n i s m s c l u s t e r e d b y t h e i r c e l l m o r p h o l o g y :b a c i l l a r (r e d s q u a r e ),h y p h a l (g r e e n s q u a r e )o r c o c c o i d (b l u e s q u a r e ).G e n e s a r e r e p r e s e n t e d b y t h i c k a r r o w s i n d i c a t i n g d i r e c t i o n o f t r a n s c r i p t i o n a n d h o m o l o g o u s g e n e s a r e s h o w n i n t h e s a m e c o l o r ;g e n e s n o t r e l a t e d t o c e l l d i v i s i o n o r p e p t i d o g l y c a n b i o s y n t h e s i s a r e c o l o r l e s s .T h i n a r r o w s r e p r e s e n t t r a n s c r i p t s d e t e c t e d i n C .g l u t a m i c u m ,a s d e s c r i b e d i nH o n r u b i a e t a l .(2001),R a m o s e t a l .(2003),V a l b u e n a e t a l .(2006)a n d L e t e k e t a l .(2007)in the Z-ring stabilization,such as the product of cg2363(Letek et al.2007),a sepF homologue that has been described as a positive cell-division regulator in B.subtilis(Hamoen et al.2006).However,the cg2363 gene(previously named OFR6by our laboratory)is not essential for either the growth or the viability of C.glutamicum(Honrubia et al.2001).The lack of any nucleoid occlusion effectors in C.glutamicum,such as noc(Wu and Errington2004)and slmA(Bernhardt and de Boer2005),was further confirmed as Z-rings can assemble even before nucle-oids are completely segregated(Ramos et al.2005). The spatial regulation of minCD requires the topolog-ical effectors MinE in E.coli(de Boer et al.1989), which is also absent in the C.glutamicum genome,and divIVA in B.subtilis(Cha and Stewart1997),which although present in Actinobacteria has a completely different function(see below and Letek et al.2008).Table1Genes related to cell division and cell growth present in different bacterial genomesGene name EscherichiacoliBacillussubtilisCorynebacteriumglutamicumMycobacteriumtuberculosisStreptomycescoelicoloramiC b2817BSU35620cg3424Rv3915SCO2345 divIVA–BSU15420cg2361Rv2145c SCO2077ezrA–BSU29610–––ftsA b0094BSU15280–––ftsB b2748–cg1112Rv1024SCO3095(divIC)ftsE b3463BSU35260cg0914Rv3102c SCO2969ftsI b0084BSU15170cg2375Rv2163c SCO2090ftsK b0890BSU29800(ytpT)cg2158Rv2748c SCO3934BSU16800(spoIIIE)SCO4508SCO5750ftsL b0083BSU15150–––ftsN b3933––––ftsQ b0093–cg2367Rv2151c SCO2083ftsW b0089BSU14850(ftsW)cg2370Rv2154c SCO2085(ftsW) BSU15210(spoVE)SCO2607(sfr) ftsX b3462BSU35250cg0915Rv3101c SCO2968ftsZ b0095BSU15290cg2366Rv2150c SCO2082 minC b1176BSU28000–––minD b1175BSU27990–––minE b1174––––mreB b3251BSU14470(mreBH)––SCO2611BSU28030(mreB)BSU36410(mbl)mreC b3250BSU28020––SCO2610 mreD b3249BSU28010––SCO2609noc–BSU40990––-rodA b0634BSU38120cg0061Rv0017c SCO3846sepF–BSU15390cg2363RV2147c SCO2079smlA b3641––––sulA b0958––––zapA b2910––––zipA b2412––––The functions of the codified proteins were recently reviewed(Errington et al.2003;Goehring and Beckwith2005)Cell elongation at the cell polesThe year2003can be considered as a milestone in the study of rod-shape acquisition by Corynebacterium. In that year Daniel and Errington(2003)determined the pattern of PG synthesis in several strains of gram-positive bacteria by staining withfluorescently labeled vancomycin.These authors found that cells from two different Actinobacteria,Corynebacterium and Streptomyces,elongate by PG synthesis at the poles,confirming the model of growth previously described for C.diphtheriae(Umeda and Amako 1983).In that model,Actinobacteria were proposed to employ a strategy for cell elongation different from the one used by representative gram-positive bacteria such as B.subtilis and Staphylococcus aureus(Daniel and Errington2003).In the same year,two different reports provided evidence that divIVA,a gene located downstream from the dcw cluster of many gram-positive bacteria (Massidda et al.1998),was involved in cell-wall synthesis in Corynebacterium and Streptomyces (Ramos et al.2003;Flardh2003a).DivIVA has a conserved,short N-terminal domain and at least two coiled-coil regions by which the protein oligomerizes to producefilaments(Stahlberg et al.2004).Among the different species of Corynebacterium,the divIVA genes show poor DNA-sequence conservation(Letek et al.2006a)and their protein products exhibit a broad diversity of functions in gram-positive bacteria. For example, B.subtilis DivIVA sequesters the MinCD complex to the poles,thus participating in the spatial regulation of cell division and in chromo-some segregation during sporulation(Cha and Stewart1997;Thomaides et al.2001).DivIVA is also present in a variety of gram-positive cocci that lack the MinCD system,such as Enterococcus faecalis,Streptococcus pneumoniae,and Staphylo-coccus aureus.In E.faecalis and S.pneumoniae, DivIVA is required for nucleoid segregation,cell division,and cell growth(Ramirez-Arcos et al.2005; Fadda et al.2007),whereas in S.aureus the protein is not essential for growth,viability,or nucleoid segregation but localizes at the septum(Pinho and Errington2004).In C.glutamicum,DivIVA is essential for cell elongation(Ramos et al.2003;Letek et al.2008),as in other actinomycetes including Streptomyces and Mycobacterium(Flardh2003a;Nguyen et al.2007). In Corynebacterium,DivIVA localizes at both cell poles and the septum,where the cell wall is being synthesized(Fig.3).When the protein is overexpres-sed,the cells become larger and take on an‘‘ice-cream-cone’’shape(Ramos et al.2003;Letek et al. 2006b),as the accumulated excess DivIVA localizes mainly at one cell pole,which in turn becomes a very active site of PG synthesis(Letek et al.2008).Low-level expression of DivIVA in C.glutamicum results in a loss of the rod-shaped cellular morphology, yielding coccoid cells(Letek et al.2008).Moreover, DivIVA interacts with PBP1a,a high-molecular-weight penicillin-binding protein(HMW-PBP) homologous to PBP1c from E.coli,involved in polar cell-wall synthesis in C.glutamicum(Valbuena et al. 2007).Therefore,it has been postulated that DivIVA oligomerizes creating an internal scaffold for the PG synthesis machinery at the cell poles(Fig.4)(Ramos et al.2003;Flardh2003a;Letek et al.2008).InFig.3Vancomycin-FL staining of C.glutamicum.The wild-type strain synthesizes peptidoglycan at the cell poles(cell elongation,left photograph);when the correct size is reached the cells begin to synthesize peptidoglycan at the septum(cell division,right photograph).The experimental procedure used was described previously in Daniel and Errington(2003)addition,DivIVA also localizes at the septum;thus,it may have additional functions related to a final stage in cell division or cell-pole maturation,as sug-gested for S.pneumoniae DivIVA (Fadda et al.2007),or in DNA segregation,as demonstrated in other gram-positive bacteria (Ramirez-Arcos et al.2005;Thomaides et al.2001).Penicillin-binding proteinsThe penicillin-binding proteins (PBPs)are involved in the last steps of cell-wall biosynthesis inC.glutamicum and some of them have been recently characterized (Valbuena et al.2007).PBPs are often the targets of b -lactam antibiotics and in many cases are essential for PG synthesis (Scheffers and Pinho 2005).However,these proteins are functionally redundant in E.coli and B.subtilis (Goffin and Ghuysen 1998;Scheffers et al.2004),and several of them could be depleted from the cell without affecting cell shape or viability.From the nine proteins identified as putative penicillin-binding proteins in C.glutamicum ,five of them are HMW-PBPs (Valbuena et al.2007)and involved in PG synthesis.Of these five,two are class A HMW-PBPs (PBP1a and PBP1b)with transglycosylase and transpeptidase domains,and three are class B HMW-PBPs (FtsI,PBP2a and PBP2b)with only the transpeptidase domain char-acteristic of this family of proteins.Except for FtsI,which is found only at the septum (Valbuena et al.2006),all HMW-PBPs are present at both cell poles and at the septum.However,when the transpepti-dase domain of the PBPs is specifically blocked by b -lactam treatment,these proteins lose their local-ization in the cell (Valbuena et al.2007),indicating that HMW-PBP localization is dependent upon substrate recognition.C.glutamicum cells lose their rod shape only when they are deprived of both class A HMW-PBPs,indicating that PBP1a and PBP1b are essential for cell-wall synthesis at the poles (Valbuena et al.2007).Class B HMW-PBPs are closely associated with septal PG synthesis during cell division,with FtsI being the only essential HMW-PBP as no other protein of this type can fulfill its function (Valbuena et al.2006).In support of this hypothesis,class B HMW-PBPs interact more prominently with cell-division proteins,such as FtsZ or FtsW,whereas class A HMW-PBPs are associated with DivIVA and RodA (Valbuena et al.2007).The latter protein is required for the control of rod shape in E.coli and B.subtilis (Matsuzawa et al.1989;Henriques et al.1998)and is also essential for cell elongation in C.glutamicum (Maria Fiuza,unpublished observa-tions).All of the HMW-PBPs show a certain level of interaction between them,suggesting that they are part of the same machinery of PG synthesis;depending on the stage of the cell cycle,they direct PG synthesis at the septum (cell division)or at the cell poles (cellelongation).Fig.4Comparison of the division and elongation machineries in C.glutamicum .The key component of the divisome (cell-division machinery)is FtsZ.This protein polymerizes to form a Z-ring that acts as a scaffold of the multi-protein complex comprising the divisome.The hierarchy for assembly of the remaining proteins (FtsEX,FtsK,FtsQ,FtsW,FtsI,Pbp2a,and PBP2b)is still unclear,but FtsZ is known to interact with FtsW,and FtsZ/FtsW with FtsI and PBP2a/2b (Valbuena et al.2007).The essential component of the cell-elongation machin-ery is DivIVA,which polymerizes at the cell poles and permits the cell-wall synthesis carried out by RodA,PPB1a,and PBP1b (Valbuena et al.2007)Towards a new model of bacterial cell division and elongationMost rod-shaped cells require MreB,which assem-bles into wire-like structures that run between the poles of the cell and distributes various components of PG metabolism along the cell’s length.When the cell has acquired the appropriate length,it enters into the cell-division process.The divisome is responsible for the formation of two symmetrical daughter cells after constriction of the Z-ring and synthesis of the cell envelope.C.glutamicum lacks MreB and thus represents a model of cell elongation/division distinct from that of typical rod-shaped bacteria such as E.coli or B.subtilis,which express MreB.During cell elonga-tion,the polar PG-synthesis protein complex is mainly formed by DivIVA,RodA,and class A HMW-PBPs(PBP1a and PBP1b)(Fig.4),whereas the core of the cell-division machinery(divisome) comprises FtsZ,FtsEX,FtsK,FtsQ,FtsB,FtsW,and three class B HMW-PBPs(FtsI,PBP2a,and PBP2b) (Fig.4).It seems that Actinobacteria,like Corynebacterium or Mycobacterium,are able to produce rod-shaped cells in the absence of MreB homologues(Letek et al. 2008;Nguyen et al.2007).However,S.coelicolor contains a cluster of mreBCD genes(Table1), although the mreB gene seems to be involved only in sporulation(Mazza et al.2006);this was supported by the following observations:(i)a null mreB-mutant shows normal vegetative growth but defective spores; (ii)the mreB gene is only expressed during sporulation; (iii)mreB is only detected in genera of actinomycetes that sporulate and form an aerial mycelium.Thus,actin homologues may long ago have lost their roles in the cytoskeleton of actinomycetes,with the function of these proteins now limited to sporulation in some genera.How the cell‘‘decides’’between synthesizing PG at mid-cell vs.at the cell poles is still unknown,but these two pathways might be regulated by phosphor-ylation,as in the case of M.tuberculosis,in which the protein kinases PknA and PknB regulate cell growth (Kang et al.2005)and cell division(Dasgupta et al. 2006).Therefore,future efforts should focus on the characterization and identification of these cell-cycle regulators as well as other proteins involved in cell-shape acquisition by C.glutamicum.All these proteins are known to orchestrate the localization of cell-wall synthetic complexes,resulting in co-ordi-nated and efficient PG synthetic activity at the septum (cell division)or at the cell poles(cell elongation). However,to date,the details of these processes remain poorly understood in Actinobacteria. Acknowledgments M.Letek and M.Fiuza were beneficiaries of fellowships from the Ministerio de Educacio´n y Ciencia (Spain);E.Ordo´n˜ez and A.Villadangos from the Junta de Castilla y Leo´n,and A.Ramos from the ALFA project II-0313-FA-FCB.This work was funded by grants from the Junta de Castilla y Leo´n(Ref.LE040A07),University of Leo´n (ULE2001-08B),and Ministerio de Ciencia y Tecnologı´a (BIO2002-03223and BIO2005-02723).We thank Dr.Ramon Santamarı´a(Universidad de Salamanca,Spain)for the TEM image of C.glutamicum.ReferencesBendt AK,Burkovski A,Schaffer S,Bott M,Farwick M, HermannT(2003)Towards a phosphoproteome map of Corynebacterium glutamicum.Proteomics3:1637–1646 Bernard CS,Sadasivam M,Shiomi D,Margolin W(2007)An altered FtsA can compensate for the loss of essential cell division protein FtsN in Escherichia coli.Mol Microbiol 64:1289–1305Bernhardt TG,de Boer PA(2005)SlmA,a nucleoid-associ-ated,FtsZ binding protein required for blocking septal ring assembly over chromosomes in 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