A Vitamin B12-Based System for Conditional Expression Reveals...

JOURNAL OF BACTERIOLOGY, May 2009, p. 3108–3119 Vol. 191, No. 90021-9193/09/$08.00�0 doi:10.1128/JB.01737-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A Vitamin B12-Based System for Conditional Expression Reveals dksATo Be an Essential Gene in Myxococcus xanthus�

Diana García-Moreno,1 María Carmen Polanco,1 Gloria Navarro-Aviles,1† Francisco J. Murillo,1S. Padmanabhan,2 and Montserrat Elías-Arnanz1*

Departamento de Genetica y Microbiología, Area de Genetica (Unidad Asociada al IQFR-CSIC), Facultad de Biología,Universidad de Murcia, Murcia 30100, Spain,1 and Instituto de Química Física Rocasolano, Consejo Superior de

Investigaciones Científicas, Serrano 119, 28006 Madrid, Spain2

Received 11 December 2008/Accepted 17 February 2009

Myxococcus xanthus is a prokaryotic model system for the study of multicellular development and theresponse to blue light. The previous analyses of these processes and the characterization of new genes wouldbenefit from a robust system for controlled gene expression, which has been elusive so far for this bacterium.Here, we describe a system for conditional expression of genes in M. xanthus based on our recent finding thatvitamin B12 and CarH, a MerR-type transcriptional repressor, together downregulate a photoinducible pro-moter. Using this system, we confirmed that M. xanthus rpoN, encoding �54, is an essential gene, as reportedearlier. We then tested it with ftsZ and dksA. In most bacteria, ftsZ is vital due to its role in cell division, whereasnull mutants of dksA, whose product regulates the stringent response via transcriptional control of rRNA andamino acid biosynthesis promoters, are viable but cause pleiotropic effects. As with rpoN, it was impossible todelete endogenous ftsZ or dksA in M. xanthus except in a merodiploid background carrying another functionalcopy, which indicates that these are essential genes. B12-based conditional expression of ftsZ was insufficientto provide the high intracellular FtsZ levels required. With dksA, as with rpoN, cells were viable underpermissive but not restrictive conditions, and depletion of DksA or �54 produced filamentous, aberrantlydividing cells. dksA thus joins rpoN in a growing list of genes dispensable in many bacteria but essential in M.xanthus.

The gram-negative Myxococcus xanthus and other myxobac-teria are striking in the prokaryotic world for their ability toundergo a complex developmental program that culminates inthe production of multicellular fruiting bodies. This process,triggered by starvation, has served as a prokaryotic model forthe study of the molecular mechanisms underlying coordinatedcell movements and intercellular interactions, as well as for thecellular differentiation process that occurs during multicellulardevelopment (22, 23, 59). M. xanthus is also undoubtedly animportant model system for the genetic-molecular analysis ofthe reception and transduction of the blue light signal and ofthe consequent transcriptional regulation of structural genesinvolved in carotenoid biosynthesis (11). Genetic and molecu-lar biological tools such as transposon insertions of transcrip-tional reporters and nonpolar in-frame gene deletions haveenabled a functional analysis and characterization of the genesinvolved in these processes. However, many studies in M. xan-thus have been hindered by the lack of autonomously replicat-ing plasmids (one has only recently been reported [58]) and ofa tightly regulated gene expression system that allows func-tional analysis of essential genes. One system that employslight activation of the carQRS promoter, which depends on the

extracytoplasmic function � factor CarQ, has been used in thecontrolled expression of genes such as csgA and socE, impli-cated in developmental cell-cell interactions, or relA, requiredfor (p)ppGpp synthesis in M. xanthus (7, 17, 27, 28, 50). Theapplicability of this system to the study of essential genes in M.xanthus, however, remains to be demonstrated. A second sys-tem designed for conditional gene expression in M. xanthusmakes use of the PpilA-lacO-RBS promoter (where RBS is ribo-some binding site), an isopropyl-�-D-thiogalactopyranoside(IPTG)-inducible version of the native pilA promoter, whosetranscription initiation depends on the alternative sigma factor�54 (19). In this system, the target gene is cloned in a plasmidunder the control of PpilA-lacO-RBS, which integrates by homol-ogous recombination at the pilA chromosomal locus. The geneencoding the LacI repressor is supplied in a separate plasmidthat integrates in the chromosome by recombination at theMx8 attB site. A limitation of this system for characterizingessential genes by studying the conditional mutant phenotypeproduced on turning off their expression is the relatively highlevel of basal expression observed from PpilA-lacO-RBS (19).Moreover, the system as designed requires two plasmids anduses up both of the available selectable markers typically usedin M. xanthus (kanamycin or tetracycline resistance), which isan impediment in the study of essential genes.

An alternative design for conditional gene expression in M.xanthus is reported here. It is based on our recent finding thatvitamin B12 and a MerR-type transcriptional repressor, CarH,collaborate to downregulate the light-inducible M. xanthus PB

promoter in the dark, whereas light derepresses it (Fig. 1) (45).PB, a �A-dependent promoter, drives the expression of the

* Corresponding author. Mailing address: Departamento de Geneticay Microbiología, Area de Genetica (Unidad Asociada al IQFR-CSIC), Facultad de Biología, Universidad de Murcia, Murcia 30100,Spain. Phone: 34 968 367134. Fax: 34 968 363963. E-mail: [email protected].

† Present address: Department of Environmental Protection, Esta-cion Experimental del Zaidín, Consejo Superior de InvestigacionesCientíficas, 18008 Granada, Spain.

� Published ahead of print on 27 February 2009.

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carB-carA gene cluster, which groups together all but one ofthe structural genes encoding the enzymes involved in carote-noid synthesis (reviewed in reference 11). In addition to re-pression by B12 and CarH, expression of PB is also repressed inthe dark by CarA, another MerR-type transcriptional factorremarkably similar to CarH in domain organization but whoseaction does not depend on B12. CarH (in the presence of B12)and CarA bind to the same bipartite operator to impede pro-moter access to RNA polymerase (RNAP) and thereby repressPB (29, 30, 44, 45). RNAP holoenzyme containing the extra-cytoplasmic function � factor CarQ, together with a host oftranscriptional factors (11), drives expression of the carQRSoperon to produce CarS, an antirepressor that physically bindsto CarA and CarH to derepress PB in the light. Mutuallyexclusive binding of CarA or CarH to operator DNA and toCarS is the molecular basis for this mechanism of repressionand antirepression (38, 45).

We have exploited the repression of PB by CarH and B12 todesign a system for the regulatable expression of genes in M.xanthus. In this single-plasmid-based system, the gene of inter-est is placed under the control of PB and CarH so as to tightlyrepress its expression in the dark when B12 is present. Weemployed the system to confirm a previous finding that rpoN,which encodes the alternative sigma factor �54, is an essentialgene in M. xanthus, unlike in other bacteria, where it appearsto be required for growth only under specific nutrient-limitingconditions (25). We then tested our system with other candi-date genes. One of these was the M. xanthus dksA gene. InEscherichia coli, DksA has been shown to regulate the strin-gent response via transcriptional control of rRNA and aminoacid biosynthesis promoters (18, 40, 41, 43, 51). Gene disrup-tion of dksA indicated that it is not essential in E. coli, but astrain lacking DksA grew poorly under nutrient-limiting con-ditions (24). Moreover, DksA has been implicated in the reg-ulation of �54-dependent transcription in vivo, thus establish-ing a functional link between rpoN and dksA (3, 52). TheB12-based system for conditional expression described in thisstudy shows that dksA, like rpoN, is an essential gene in M.xanthus.

MATERIALS AND METHODS

Bacterial strains and growth conditions. Table 1 lists the strains and plasmidsused in this study. M. xanthus was grown at 33°C in rich Casitone-Tris ([CTT](�1 nM B12, according to the manufacturer estimates) medium (4) supple-mented, when indicated (see below and see the figure legends), with antibiotics(kanamycin at 40 �g/ml or tetracycline at 10 �g/ml) and 0.75 �M B12. Theconcentration of B12 in the medium was based on the observation that PB

repression levels are maintained for a 1 nM to 1 �M concentration range of B12

(cyanocobalamin) or derivatives such as methylcobalamin, adenosylcobalamin,or hydroxycobalamin (45). E. coli strain DH5� was used for plasmid construc-tions, and BL21(DE3) was used for overexpressing His-tagged proteins. E. coligrowth was carried out at 37°C in Luria broth supplied with the appropriateantibiotic as needed, while protein overexpression was induced overnight at25°C.

Plasmid and strain constructions. Standard protocols and commercial kitswere used in the preparation and manipulation of chromosomal and plasmidDNA. All constructs were verified by DNA sequencing. For protein overexpres-sion, the M. xanthus dksA gene was PCR amplified from genomic DNA andcloned into the NdeI site of the pET15b vector (Novagen) to generate pMR3042.Plasmids pMR2915 and pMR2979, described next, were used in the constructionof pMR2991. Plasmid pMR2915 was generated by replacing the Kmr selectablemarker in pMR2700, a plasmid that contains a 1.38-kb fragment of M. xanthusDNA for chromosomal integration by homologous recombination (44), by a Tcr

selectable marker. For this, pMR2700 was digested with MluI to release a 1.5-kbDNA fragment containing the Kmr marker and then ligated to a 1.3-kb PCR-amplified DNA fragment containing the Tcr marker from pACYC184 (47).Plasmid pMR2979 was generated by ligating into PstI/KpnI-digested pMR2915 a121-bp PstI/XbaI-digested DNA fragment containing the PB promoter/operatorregion and an XbaI/KpnI-digested 3.26-kb DNA fragment containing the lacZgene with its ribosomal binding site. To construct pMR2991, the �3.4-kb PstI/KpnI fragment from pMR2979 containing the PB-lacZ fusion and a �0.9-kbKpnI/EcoRI fragment corresponding to carH were first ligated into PstI/EcoRI-digested pMR2915 to generate pMR2981. This plasmid was then cut with KpnIin order to insert a 92-bp DNA fragment containing Pc, a �A-dependent pro-moter that has been previously used in M. xanthus for constitutive expression ofgenes at a heterologous site (44). The resulting plasmid, pMR2991, which inte-grates into the M. xanthus chromosome by homologous recombination via the1.38-kb DNA fragment, therefore carries carH under the control of a constitutivepromoter and a reporter lacZ gene under PB control. Plasmids pMR3120,pMR3168, and pMR3085 are derivatives of pMR2991 that were obtained bycloning the M. xanthus rpoN, ftsZ, or dksA (each with its own RBS) genes,respectively, into the XbaI site of pMR2991. Plasmids pMR3121, pMR3165, andpMR3254 are pBJ113/pBJ114 derivatives (21) that were generated to createcomplete in-frame deletions of the M. xanthus rpoN, ftsZ, or dksA genes, respec-tively, as described next. All three plasmids contain a Kmr gene for positiveselection and a galK gene that confers sensitivity to galactose (Gals) for negativeselection. To construct pMR3121, two chromosomal fragments were PCR am-plified and ligated into pBJ114 to replace the complete rpoN gene by an XbaIsite: (i) a 1.01-kb fragment corresponding to the DNA immediately upstream ofrpoN, with a HindIII site added to its 5� end and an XbaI site added to its 3� end;(ii) a 0.99-kb fragment corresponding to the DNA immediately downstream ofrpoN, with an XbaI site added to its 5� end and a KpnI site added to its 3� end.Plasmid pMR3165, with a replacement of the ftsZ gene by an EcoRI site, wasconstructed by ligating into pBJ113 a 0.99-kb fragment of chromosomal DNAupstream of ftsZ (flanked by KpnI and EcoRI sites) and a 1.02-kb fragment ofchromosomal DNA downstream of ftsZ (flanked by EcoRI and HindIII sites).Plasmid pMR3254 was constructed by PCR amplification of 0.85 kb of DNAupstream of dksA (flanked by SalI and XbaI sites) and 1.02 kb of DNA down-stream of dksA (flanked by XbaI and EcoRI sites). After the two DNA fragmentswere ligated into pBJ114, an XbaI site replaced the dksA gene. PlasmidpMR3202 was constructed by ligating into PstI/KpnI-digested pMR2915 a1.54-kb PstI/XbaI DNA fragment including ftsZ and 330 bp of upstream DNAand an XbaI/KpnI 3.26-kb DNA fragment containing the lacZ gene with itsribosomal binding site.

Plasmids were introduced into M. xanthus cells by electroporation, and inte-gration of the plasmids by homologous recombination was selected for on CTTplates containing the appropriate antibiotic. Plasmid integration at the correctchromosomal site in M. xanthus was verified by PCR in all cases. Integration ofplasmids pMR3121, pMR3165, and pMR3254 into the chromosome yielded Kmr

merodiploids containing both the wild-type and deletion alleles of rpoN, ftsZ, anddksA, respectively. Independent merodiploids were grown for several genera-tions in CTT medium in the absence of kanamycin and then plated on CTT

FIG. 1. Schematic of the M. xanthus PB promoter region and itsregulation. Promoter PB drives expression of the gene clusters repre-sented by the squat arrows (constituent genes are labeled); the arrow-head indicates the direction of transcription. Ovals depict proteinfactors involved in PB regulation, as labeled. Arrows indicate positiveregulation, and blunt-ended lines indicate negative regulation. Otherregulatory proteins or light-dependent genes or operons implicated incarotenogenesis are omitted for clarity (see reference 11 for details).

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plates supplemented with 10 mg/ml galactose to select for the loss of the vectorGals marker. This evicts the vector DNA together with either the wild-type or thedeletion allele by intramolecular recombination to yield haploid Gal� Kms cells.The presence of the wild-type or the deletion allele was diagnosed by PCR.

Purification and characterization of DksA. E. coli BL21(DE3) freshly trans-formed with pMR3042 was grown in 10 ml of Luria broth-ampicillin medium toan A600 of 0.6 to 1.0 at 37°C and then inoculated into 1 liter of fresh medium.After growth to an A600 of 0.6 to 1.0 at 37°C, 0.5 mM IPTG was added to inducedksA expression overnight at 25°C. Analysis of cell extracts from 1 ml of cultureshowed that His6-DksA is overexpressed as a soluble protein. The protein waspurified using Talon metal affinity resin (Clontech) and the accompanying nativeprotein purification protocol. The imidazole was eliminated by extensive dialysisin buffer A (50 mM phosphate buffer, pH 7.5, 5 mM �-mercaptoethanol) plus150 mM NaCl, and the sample was further purified by passage through a phos-phocellulose column equilibrated with the same buffer or, when required, treatedwith thrombin (GE Health Sciences), passed through phosphocellulose, anddialyzed extensively against buffer A plus 150 mM NaCl. Analysis of DksA forbound zinc was carried out using the colorimetric 4-(2-pyridylazo)resorcinol(PAR) assay (reference 42 and references therein). Briefly, DksA without theHis6 tag was preincubated with p-chloromercuribenzoate to modify sulfhydryl(-SH) groups and release Zn. Freshly prepared PAR (100 mM) was then addedto complex the freed Zn, and the absorbance at 500 nm was used to estimate Zn.

In the pull-down assay to confirm interaction between DksA and M. xanthusRNA polymerase (obtained as described previously [30]), 200 �l of Talon in two2-ml SigmaPrep (Sigma-Aldrich) columns was washed five times with 600 �l oflow-salt buffer (buffer A plus 50 mM NaCl). A total of 400 �l of His6-DksA (�25nmol) was applied to one column but not to the other, which served as a control.After incubation for 1 h, the resin in each column was washed with 30 volumesof low-salt buffer. To each column 20 pmol of RNAP (200 �l) was added andincubated for 1 to 2 h with gentle rotary agitation. Flowthrough from eachcolumn was collected separately. The resin was then extensively washed withlow-salt buffer and subsequently eluted in five 200-�l fractions with high-saltbuffer (buffer A plus 500 mM NaCl). These and the low-salt flowthrough (afterRNAP incubation) were analyzed using 10% sodium dodecyl sulfate-polyacryl-amide gel electrophoresis with silver staining.

�-Galactosidase activity assays. �-Galactosidase activity was qualitatively as-sessed on CTT plates with 40 mg ml1 of X-Gal (5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside) and measured (in nmol of o-nitrophenyl �-D-galactosidehydrolyzed min1 mg1 of protein) as described elsewhere for vegetativelygrowing liquid cultures of M. xanthus (1). Comparison of reporter lacZ expres-sion from pMR2991 in the dark and in the light and in the presence or absenceof B12 was carried out as follows. A starter culture of the corresponding M.xanthus strain was grown in the dark in 10 ml of CTT medium supplemented withthe required antibiotic. The culture (0.25 ml) was added to each of two separate

TABLE 1. Bacterial strains and plasmids used in this work

Strain or plasmid Relevant genotype or description Source or reference

StrainsM. xanthus

DK1050 Wild type 48MR1461 carA carH PB::lacZ Pc::carH This workMR1716 carA carH 45MR1859 carA carH rpoN/rpoN This workMR1860 carA carH ftsZ/ftsZ This workMR1862 carA carH ftsZ/ftsZ PB::ftsZ-lacZ Pc::carH This workMR1866 carA carH ftsZ/ftsZ P330::ftsZ-lacZ This workMR1867 carA carH rpoN/rpoN PB::rpoN-lacZ Pc::carH This workMR1870 carA carH rpoN PB::rpoN-lacZ Pc::carH This workMR1871 carA carH rpoN PB::rpoN-lacZ Pc::carH This workMR1925 carA carH dksA PB::dksA-lacZ Pc::carH This workMR1926 carA carH dksA PB::dksA-lacZ Pc::carH This workMR1929 carA carH dksA/dksA This workMR1930 carA carH dksA/dksA PB::dksA-lacZ Pc::carH This workMR1933 dksA/dksA This work

E. coliBL21(DE3) F lon ompT rB

mB �DE3 Novagen

DH5� F supE44 lacU169 (�80d lacZM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Gibco-BRL

PlasmidspET15b Vector for protein overexpression; Ampr NovagenpACYC184 Cloning vector; Tcr Cmr 47pBJ113 Cloning vector; Gals Kmr 21pBJ114 Cloning vector; Gals Kmr; same as pBJ113, but with polylinker in opposite orientation 21pMR2700 Contains 1.38-kb M. xanthus DNA fragment for chromosomal integration; Kmr 44pMR2915 Tcr derivative of pMR2700 This workpMR2979 pMR2915 plus PstI/XbaI fragment containing PB and XbaI/KpnI fragment containing lacZ This workpMR2981 pMR2915 plus PstI/KpnI fragment from pMR2979 containing PB::lacZ and KpnI/EcoRI

fragment containing carHThis work

pMR2991 pMR2981 plus KpnI/KpnI promoter fragment This workpMR3042 Vector for protein overexpression of dksA; Ampr This workpMR3085 pMR2991 plus XbaI/XbaI fragment containing dksA This workpMR3120 pMR2991 plus XbaI/XbaI fragment containing rpoN This workpMR3121 pBJ114 plus 1.01-kb HindIII/XbaI fragment upstream of rpoN and 0.99-kb XbaI/KpnI

fragment downstream of rpoNThis work

pMR3165 pBJ113 plus 0.99-kb KpnI/EcoRI fragment of chromosomal DNA upstream of ftsZ and1.02-kb EcoRI/HindIII fragment of chromosomal DNA downstream of ftsZ

This work

pMR3168 pMR2991 plus XbaI/XbaI fragment containing ftsZ This workpMR3202 pMR2915 plus PstI/XbaI fragment containing P330::ftsZ and XbaI/KpnI fragment

containing lacZThis work

pMR3254 pBJ114 plus 0.85-kb SalI/XbaI fragment of chromosomal DNA upstream of dksA and 1-kbXbaI/EcoRI fragment of chromosomal DNA downstream of dksA

This work

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culture flasks containing 12 ml of fresh CTT medium and antibiotic, with 0.75�M B12 included in one of the two flasks. After 16 h of growth in the dark at33°C, each 12-ml culture was split into two; one of these remained in the dark,and the other was exposed to light while growth continued for another 8 h.Samples were then harvested for quantitative analysis of �-galactosidase activity.

Cell growth and microscopy. Cells were grown at 33°C and 300 rpm underpermissive conditions (in the light) to an optical density at 550 nm (OD550) of 0.5. Ten-microliter aliquots (�106 cells) were transferred to each of threeseparate culture flasks containing 10 ml of fresh CTT medium. One of thecultures was incubated in the light (permissive conditions), a second was incu-bated in the dark, and a third also was incubated in the dark but after theaddition of B12 to 0.75 �� (restrictive conditions). All three cultures were grownat 33°C and 300 rpm, and the OD550 was monitored periodically. Samples of 100�l of each culture were taken at different time points during the incubation andmixed with the fluorescent dye 4�-6-diamino-2-phenylindole (DAPI; excitationmaximum, 350 nm; emission maximum, 461 nm) to a final concentration of 1ng/�l. A 4-�l drop of this cell suspension was placed on a glass plate coated with3-aminopropyl-triethoxysilane to facilitate cell immobilization and then exam-ined using a Nikon Eclipse 80i microscope equipped with a Nikon Plan Apo VC100� oil immersion objective (numerical aperture, 1.40) and a HamamatsuORCA-AG charge-coupled-device camera. Images were processed with Meta-morph, version 4.5 (Universal Imaging Group).

RESULTS

Design of a vitamin B12-based system for regulatable ex-pression in M. xanthus. In the dark, PB is repressed by CarA orCarH-B12 binding to a bipartite operator that consists of ahigh-affinity site pI (a perfect interrupted palindrome locatedupstream of PB) and a low-affinity site pII (a pI-like pseudo-palindrome that straddles the 35 region of PB) (30). CarS,produced on illumination, relieves repression of PB by physi-cally interacting with CarA or CarH to dismantle repressor-operator DNA complexes (Fig. 1) (29, 30, 44, 45). To designthe vitamin B12-based system for conditional gene expressionin M. xanthus, we first constructed a plasmid (pMR2991) witha reporter lacZ probe under the transcriptional control of a121-bp DNA fragment that includes PB and its bipartite oper-ator, a Tcr marker for selection, and a 1.38-kb DNA fragmentfor plasmid integration into the M. xanthus chromosome byhomologous recombination. In addition, pMR2991 bears acassette for constitutive expression of the CarH repressor (Fig.2A) (see Materials and Methods for details); in its naturalcontext, expression of carA and carH appears to be driven by alight-independent �54-dependent promoter as well as by tran-scriptional readthrough from PB in the light (11, 49). PlasmidpMR2991 was then introduced by electroporation into the M.xanthus MR1716 strain, which lacks both carA (whose productrepresses PB in a B12-independent manner) as well as theadjacent carH, to generate strain MR1461. The levels of re-porter lacZ expression for MR1461 cells grown in the dark andin the light in the absence or presence of B12 were then as-sessed (Fig. 2B) (see also Materials and Methods). In the darkand with B12 present, �-galactosidase activity was very low,consistent with the role of B12 in mediating PB repression byCarH. Accordingly, �-galactosidase activity in the dark wasabout sixfold higher when B12 was absent. An additional 1.4-fold increase in PB expression was observed for samples grownin the light, which induces production of the antirepressorCarS. The maximum level of reporter lacZ expression from PB

was observed in the light with B12 also present. This stimula-tory effect of B12 on the light activation of PB occurs even instrains lacking CarA and CarH (45). Although the reasons forthis effect of B12 in the light remain to be deciphered, it pro-

vides an added level of modulating gene expression in thisB12-regulated system. In sum, the results demonstrate the fol-lowing for a gene (e.g., lacZ) under PB control: (i) that there isessentially no expression for growth in the dark with B12

present; (ii) that the expression is significantly greater forgrowth in the dark with no B12 added; and (iii) that expressionlevels can be further enhanced by light and even more so byincluding B12. The system thus provides a graduated ability tofine-tune expression of a candidate gene.

Use of the B12-based system for conditional expression ofthe essential rpoN gene in M. xanthus. Keseler and Kaiser (25)showed that rpoN, which encodes �54, is an essential gene in M.xanthus based on the impossibility of obtaining rpoN null mu-tants using various strategies for gene knockout (direct disrup-tion by fragment insertions, resolution of tandem gene dupli-cations, or transduction of the null allele). The finding was alsonoteworthy in that no other member of the �54 family had beenreported to be essential for growth until then. Moreover, it hasbeen reported that many developmentally regulated genes inM. xanthus are expressed from �54-dependent promoters (20).Since conditional expression of rpoN has not been investigated,we reasoned that this would be a suitable test case for theapplicability of the B12-based system that we have designed.

To confirm that rpoN is essential for cell viability in M.xanthus, strain MR1716 was first electroporated with plasmidpMR3121, which bears a complete in-frame deletion of rpoN

FIG. 2. Regulated gene expression mediated by CarH and B12.(A) Schematic of plasmid pMR2991. The plasmid is introduced intothe carA carH double-deleted M. xanthus strain MR1716, where itintegrates into the M. xanthus chromosome by homologous recombi-nation via the 1.38-kb DNA fragment to yield strain MR1461. (B) Ex-pression of the reporter lacZ in MR1461 in the dark or in the light withor without 0.75 �M B12. Samples were obtained as described in Ma-terials and Methods and then harvested for quantitative analysis of�-galactosidase activity (in nmol of o-nitrophenyl �-D-galactoside hy-drolyzed/min/mg of protein). Mean values and errors are from two ormore independent measurements.



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(rpoN allele) flanked by about 1 kb of the genomic DNA oneither side of the gene for homologous recombination. Plasmidintegration at the correct chromosomal locus to produce amerodiploid (strain MR1859) with the wild type as well as therpoN allele was verified by PCR. The resulting Gals Kmr

merodiploid was then electroporated with pMR3120 (Fig. 3), aplasmid that was constructed by cloning the rpoN gene and itsRBS immediately downstream of the PB promoter inpMR2991, to generate strain MR1867 (with pMR3120 inte-grated by homologous recombination via the 1.38-kb DNAfragment, as verified by PCR). Haploids were generated bygrowing MR1867 for several generations in the absence ofkanamycin and, in order to optimize rpoN expression from PB,in the light. Haploid Galr Kms cells arising from recombinationbetween the two rpoN alleles were selected on plates contain-ing galactose and grown in the light. A total of 133 recombi-nants were picked, and the presence of the wild-type or therpoN allele at the native chromosomal locus was diagnosed byPCR. Out of these, 24 recombinants had the rpoN allele whilethe rest retained the wild-type allele. One of each type was

then selected for comparing the ability to grow on CTT platesin the light or in the dark and in the absence or the presenceof B12. As shown in Fig. 4A, the recombinant bearing thewild-type rpoN allele (strain MR1871) grew equally well underthe four conditions tested, and the red colony color in the lightwas the result of light-induced carotenogenesis. In markedcontrast, the recombinant with the rpoN allele and an rpoNcopy under PB control (strain MR1870) grew well in the lightwith or without B12, less so in the absence of B12 in the dark,and hardly at all when B12 was also present. Thus, if suppliedwith rpoN under the control of PB, a strain with the rpoNallele is viable when grown in the light but not when grown inthe dark with B12 present.

Cultures of MR1870 and MR1871 were grown under per-missive conditions (light) and then shifted for subsequentgrowth in the light, in the dark with no B12, and in the darkwith B12. The growth curve for MR1870 in the light (Fig. 4B)resembled curves obtained for MR1871 under the threegrowth conditions (data not shown). Consistent with the ap-parent lower growth rate of MR1870 on plates in the dark with

FIG. 3. Schematic showing construction of a M. xanthus strain conditionally expressing rpoN. Plasmid pMR3120, which expresses the rpoN geneunder the control of the PB promoter, is introduced into the merodiploid strain MR1859 (containing wild-type and deleted copies of rpoN in thecarA carH double-deleted genetic background). Rectangles in gray and black indicate genomic regions upstream and downstream of the deletedgene, respectively. Integration of pMR3120 into the chromosome by homologous recombination via the 1.38-kb DNA fragment yields strainMR1867. From this, subsequent loss of the rpoN allele at the native site yields strain MR1871 (with two copies of rpoN, of which one is expressedfrom PB at an unlinked site), while loss of the wild-type rpoN allele at the native site yields strain MR1870 (with the rpoN allele at the native siteand rpoN expressed from PB at the unlinked site).



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no B12 (Fig. 4A), liquid cultures were also observed to growmore slowly in the dark than in the light (Fig. 4B). The pres-ence of B12 not only caused MR1870 to grow even more slowlyin the dark but also arrested growth at a significantly lowerOD550 relative to that in the light or in the dark without B12

(Fig. 4B). We examined the cellular morphology of MR1870under each of these conditions by differential interference con-trast (DIC) and fluorescence microscopy of cells stained withDAPI to visualize the nucleoid and compared it to that ofMR1871. As can be seen in Fig. 4C, both strains exhibited

normal cell lengths (�4 to 8 �m) under permissive conditions(light), with cells containing one or two nucleoids at the mid-cell section. A similar, normal cellular morphology character-ized MR1870 grown in the dark with no B12 present (data notshown), despite the slower-growth phenotype observed. Bycontrast, on shifting to restrictive conditions (dark with B12),MR1870 produced cells that were �10 �m (about 48% of thetotal examined), with some cells reaching lengths of up to �35�m (Fig. 4C, inset). These abnormally long, filamentous cellsappeared to contain several DAPI-stained foci distributed

FIG. 4. PB-dependent expression of rpoN confirms it to be an essential gene. (A) Growth under the conditions indicated for M. xanthus strainswith the wild-type rpoN allele (MR1871) or the rpoN allele (MR1870) at the native site and with an rpoN copy under PB control at an unlinkedsite. Cells grown on CTT plates under permissive conditions (light) were streaked on CTT plates with or without B12 and incubated at 33°C underthe indicated conditions for 2 days. The cells are red in the light due to carotenoid synthesis. In the dark, cells are yellow when B12 is present, butyellow gradually turns orange when B12 is absent (due to expression of the carB operon). Note that there is essentially no growth in the dark whenB12 is present for strain MR1870. (B) Growth curves for MR1870 in the light (filled circles) and in the dark with no B12 (open circles) or with B12(filled triangles), obtained as described in Materials and Methods. The abscissa indicates time (in hours) after the shift in growth conditions.(C) Cellular morphology of M. xanthus strains MR1870 and MR1871, under the conditions indicated (permissive, light; restrictive, dark and B12).Cells were examined at an OD550 of �0.8 by DIC and fluorescence microscopy after DAPI staining to visualize the nucleoid, as described inMaterials and Methods. Note the aberrant cell division phenotype of MR1870 on shifting to restrictive conditions. Bar, 5 �m.



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along the cell that were less distinct and well defined thanthose observed for MR1870 under permissive conditions or forMR1871 under restrictive conditions. This aberrant cell divi-sion phenotype that we observe on limiting rpoN expression inM. xanthus is reminiscent of that reported in Caulobacter cres-centus when rpoN (not essential in this bacterium) is disrupted(5). In this latter case, it appears to be a consequence of therpoN disruption mutant’s being impaired in a global regulatorymechanism that links �54-dependent flagellar biogenesis to celldivision. In sum, our results show that the B12-based systemengineered here is suitable for conditionally expressing rpoN,whose limited expression is detrimental to M. xanthus growth,as would be expected for an essential gene. This prompted usto test the applicability of this system for some other M. xan-thus genes, as discussed next.

Analysis of the M. xanthus ftsZ gene. The highly conservedcell division protein FtsZ is vital in most bacteria including E.coli, Bacillus subtilis, and C. crescentus (2, 8, 56), with Strepto-myces coelicolor being one known exception (36). We tested ifM. xanthus ftsZ is also essential and whether it could be con-ditionally expressed using our B12-based system. For this, weused the same strategy as described above for rpoN and de-picted in Fig. 3. Plasmid pMR3165, with a complete in-framedeletion of ftsZ (the ftsZ allele), was used to generate strainMR1860, a Gals Kmr merodiploid resulting from integration ofthe plasmid at the correct chromosomal locus in MR1716, asverified by PCR. MR1860 was then electroporated withpMR3168, which expresses ftsZ under the control of PB (Fig.5A). Plasmid integration at the heterologous site (via recom-bination at the 1.38-kb M. xanthus DNA fragment) generated

FIG. 5. Schematic showing construction of a M. xanthus strain with ftsZ expressed from promoters PB or P330. (A) Plasmid pMR3168, with theftsZ gene under the control of PB, is electroporated into the merodiploid strain MR1860 (containing wild-type and deleted copies of ftsZ in thecarA carH genetic background). Rectangles in gray and black indicate genomic regions upstream and downstream of the deleted gene,respectively. Integration of pMR3168 into the chromosome at a heterologous site via recombination through the 1.38-kb DNA fragment generatesstrain MR1862, which was grown in the light and in the absence of kanamycin to generate Galr Kms recombinants. These latter were analyzed forthe presence of the wild-type or deleted ftsZ allele. (B) Plasmid pMR3202 is similar to pMR3168 in panel A except that P330 (see text) replacesPB, and the carH expression cassette is absent. As in panel A, it was electroporated into strain MR1860 and the Tcr transformants were grown inthe absence of kanamycin to generate Galr Kms recombinants, which were then analyzed for the presence of the wild-type or deleted ftsZ allele.



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strain MR1862. After MR1862 was grown for several genera-tions in the absence of kanamycin under permissive conditions,200 Galr Kms recombinants obtained were picked. All of thesegrew well under restrictive conditions, and PCR analysis of 48of them did not reveal any recombinant that carried the ftsZallele. In bacteria where it has been studied, FtsZ is typicallypresent in high amounts in vivo, and such elevated intracellularlevels are presumably required for viability of actively dividingcells (10, 31, 32, 35). The inability to obtain recombinants withthe ftsZ allele in M. xanthus could therefore be a consequenceof the levels of FtsZ produced, when PB drives its expression,being lower than those required for viability. This possibilitywas supported by the following experiment. We first con-structed pMR3202 (Fig. 5B), which contains ftsZ together with0.33 kb of intergenic DNA (P330) immediately preceding ftsZin the genome. The latter has promoter activity, as inferredfrom expression of the reporter lacZ gene in pMR3202, whichwas about fivefold higher than the maximum levels observedwhen expressed from PB (data not shown). Strain MR1860 wasthen electroporated with pMR3202, whose chromosomal inte-gration at a heterologous site (via recombination at the 1.38-kbM. xanthus DNA fragment) generated strain MR1866. Thelatter was grown for several generations in the absence ofkanamycin, and the resulting Galr Kms recombinants wereanalyzed by PCR for the presence of the wild-type or ftsZallele at the natural site. In this case, out of the 15 haploidcolonies that we checked by PCR, 11 were found to contain theftsZ allele. Thus, the natural ftsZ allele can be deleted in amerodiploid background in which a second copy of ftsZ isexpressed from P330 but not PB, consistent with the fivefoldhigher expression from P330. Thus, we found that the B12-basedsystem for conditional expression of ftsZ is likely insufficient toprovide the higher intracellular levels of FtsZ required andthat, as with its counterparts in most other bacteria, ftsZ ap-pears to be an essential gene in M. xanthus.

The M. xanthus dksA gene. The finding that rpoN is requiredfor growth only under specific nutrient-limiting conditions inmost bacteria but is essential in M. xanthus and the implicationof DksA in the regulation of �54-dependent transcription invivo prompted us to examine dksA, whose counterparts inother bacteria have a role in response to starvation conditions.As noted before, E. coli dksA is not an essential gene, but astrain lacking it grows poorly on nutrient limitation (24). In E.coli, DksA regulates the stringent response by enhancing theinhibitory effects of (p)ppGpp (GDP 3�-diphosphate and itspentaphosphate derivative) on rRNA transcription and by ac-tivating promoters for amino acid biosynthesis and transport(18, 40, 41, 43). Accumulation of (p)ppGpp in response toamino acid starvation has been shown to initiate the early geneexpression leading to fruiting body formation in M. xanthus(17). The role of DksA in the M. xanthus life cycle, however,remains to be examined.

A protein BLAST (http://www.ncbi.nlm.nih.gov/BLAST) searchof the M. xanthus genome (13) with E. coli DksA as query revealsfour proteins with predicted zinc-binding motifs (made up of Cys)of the type found in the DksA/TraR family. These four proteins(annotated as MXAN_3200, MXAN_5718, MXAN_3006, andMXAN_7086 with NCBI accession codes YP_631401,YP_633856, YP_631215, and YP_635199, respectively) are 120,130, 130, and 101 residues long, respectively, compared to the

151-residue E. coli DksA (Fig. 6A). We assigned the 120-residueprotein as DksA based on the observations that its sequenceshares the maximum identity/similarity (37%/58%) to E. coliDksA and that it conserves all four Cys and the two Asp residuesimplicated in function (Fig. 6A) (40, 43). A putative promoterwith a consensus 35 TTGACA hexamer and a less-conservedTGAAGC at the 10 position can be identified 60 bp upstreamof the start codon of dksA, which appears to be expressed as anindependent unit with both genes flanking it transcribed in theopposite sense. The one immediately upstream of dksA encodesa putative uncharacterized protein while the gene downstreamexpresses a protein with a forkhead-associated domain (a phos-phoprotein recognition module with diverse roles in cellular phys-iology) (13, 20, 39). Further downstream of dksA are genes en-coding a serine/threonine protein kinase and, more notably giventhe link between DksA function and the stringent response, apppGpp synthetase.

DksA is an acidic protein (for example, the theoretical pI is4.9 for E. coli DksA and 5.04 for Pseudomonas aeruginosaDksA) that has been shown to contain zinc and to interactdirectly with native RNAP in vitro (40, 46). The protein that weassigned as DksA is also acidic (theoretical pI of 5.22). More-over, after removal of the His6 tag, purified DksA was con-firmed to contain zinc using the colorimetric PAR assay, asdescribed in Materials and Methods (data not shown). RNAPretention at low salt and elution at high salt by His6-DksAimmobilized on a metal affinity resin has been employed todemonstrate direct binding of E. coli DksA to RNAP in vitro(40). Using this pull-down assay, we also observed that His6-DksA retained RNAP (Fig. 6B). Thus, the protein that weassigned as DksA binds to zinc and RNAP, as has been re-ported for its counterparts in other bacteria.

dksA is an essential gene in M. xanthus. To assess the im-portance of dksA function in M. xanthus, we set out to generatea strain with a complete deletion of the gene. For this, wegenerated plasmid pMR3254, which bears a complete in-framedeletion of the dksA gene (dksA allele) flanked by genomicDNA on either side of the gene for homologous recombination(0.85 kb and 1 kb of DNA upstream and downstream of dksA,respectively). Plasmid pMR3254 was introduced by electropo-ration into the wild-type strain DK1050. A Gals Kmr merodip-loid, strain MR1933, resulting from integration of the plasmidat the correct chromosomal locus (as verified by PCR) waspicked. Strain MR1933 was then grown for several generationsin the absence of kanamycin, and haploid cells arising fromrecombination between the DNA segments flanking the wild-type and dksA alleles were selected on plates containinggalactose. Out of 86 recombinants diagnosed by PCR, nonewas found to have the dksA allele, suggesting that dksA is anessential gene in M. xanthus.

Conditional expression using the B12-dependent system de-scribed here was used to further confirm if dksA is indeed anessential gene. Strain MR1716 was first electroporated withplasmid pMR3254 to generate a merodiploid with the wild-type and dksA alleles in the carA carH genetic back-ground. Such a merodiploid was then electroporated with plas-mid pMR3085, a derivative of pMR2991 (Fig. 2) that wasconstructed by cloning the dksA gene and its RBS immediatelydownstream of the PB promoter. Haploids were generated bygrowing the resulting strain for several generations in the ab-



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sence of kanamycin and in the light for optimal expression ofdksA from PB. Haploid cells arising from recombination be-tween the two dksA alleles were selected on plates containinggalactose and grown in the light. A total of 87 recombinantswere picked, and the presence of the wild-type or the dksAallele at the native chromosomal locus was diagnosed by PCR.Out of these, 17 had the dksA allele while the rest retained

the wild-type allele. One of each type was then selected, andthe ability to grow on CTT plates in the light or in the dark,with and without B12, was compared. As shown in Fig. 6C, therecombinant bearing the wild-type dksA allele (strainMR1925) grew equally well under all four growth conditionstested. In marked contrast, the recombinant with the dksAallele (strain MR1926) grew well in the light with or without

FIG. 6. Analysis of M. xanthus DksA. (A) Sequence alignment of E. coli DksA (NCBI accession code NP_414687) and its sequence homologsin M. xanthus (MXAN_3200, NCBI accession code YP_631401; MXAN_5718, NCBI accession code YP_633856; MXAN_3006, NCBI accessioncode YP_631215; MXAN_7086, NCBI accession code YP_635199). In the alignment, identical residues are shaded black (with an asterisk in theconsensus line), and similar residues are in gray. Black arrows indicate the four conserved zinc-binding Cys residues and the unfilled arrows arethe two Asp residues implicated in function. (B) Analysis of RNAP retention by His6-DksA immobilized on TALON metal affinity resin. Shownis a silver-stained 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel of low-salt (50 mM) and high-salt (500 mM) eluates onpassing M. xanthus core RNAP through a column of Talon resin alone (Control) or that bound to His6-DksA. The �, �, and �� RNAP subunitsare indicated. (C) Growth on CTT plates, under the conditions indicated, for M. xanthus strains with the wild-type dksA allele (MR1925) or thedksA allele (MR1926) at the native site and a dksA copy under PB control at an unlinked site. Cells grown on CTT plates under permissiveconditions (light) were streaked on CTT plates with or without B12 and incubated at 33°C under the indicated conditions for 2 days. Both strainsappear red in the light due to carotenoid synthesis. In the dark, MR1925 is yellow if B12 is present, but yellow gradually turns to orange when B12is absent (due to expression of the carB operon). MR1926 in the dark appears to have lost the yellow, suggesting a link between DksA depletionand impaired yellow pigment synthesis. Note that there is virtually no growth in the dark with B12 present for strain MR1926.



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B12, less in the absence of B12 in the dark, and very poorly, ifat all, in the dark with B12 present. Thus, if provided with dksAunder PB control, a strain with the dksA allele is viable whengrown in the light but not when grown in the dark with B12

present. Moreover, these data also indicate a lack of functionalredundancy between the gene that we have assigned to be dksAand any of the other three that code for the similar proteinsshown in Fig. 6A.

Examination of cellular morphology by DIC and fluores-cence microscopy after DAPI staining of the cells showed thatboth MR1925 and MR1926 grew normally under permissiveconditions to produce similar, rod-shaped cells of normallengths (�4 to 8 �m), with one or two nucleoids discernible atthe mid-cell section (Fig. 7). However, on shifting to restrictiveconditions, MR1926 displayed an aberrantly dividing cellularmorphology. In this case, about 56% of the cells examined hadcell lengths of �10 �m, with about half of these being �15 �m.Moreover, a skewed distribution of the DAPI-stained foci toone portion of the cell was frequently observed in these longercells. Interestingly, a filamentous phenotype has also been re-ported with a dksA E. coli strain (34) even though dksA is notessential in this bacterium, as noted earlier (24). Thus, consis-tent with the finding that dksA is indispensable in M. xanthus,limiting its expression is deleterious to M. xanthus growth, andthis directly or indirectly affects cell division.

DISCUSSION

B12-dependent conditional expression in M. xanthus. Theability to change the expression levels of a given gene using atightly regulated promoter cassette allows the study of a con-

ditional mutant phenotype for essential genes simply by turn-ing off their expression. Controlled gene expression systemsthat have been developed for use in bacteria include thosebased on effectors such as short aliphatic amides like acet-amide (10) or drugs like the antibiotic anhydrotetracycline(33), but among the regulatory switches most widely used arepromoters that control expression of genes and operons in-volved in the utilization of carbon sources. In these, promoteractivity is modulated by repressors or activators in response tothe availability of sugars such as lactose, arabinose, or xylose;the IPTG-inducible promoter for the lac operon is possibly thebest known such system for regulating the expression of a geneof interest (9, 15, 26, 33, 37). There is a dearth of conditionalexpression systems available for use in the myxobacteria, andthe one lac-based system described for M. xanthus has thedrawback of a relatively high level of basal expression undernoninduced conditions, thereby limiting its use for the study ofessential genes (19). This report describes a system for condi-tional gene expression in M. xanthus whose modulation de-pends on a novel effector: vitamin B12. In this system, thecandidate gene is placed under the control of the PB promoterwhich is repressed in the dark by the CarH repressor whenvitamin B12 is present. Incremental levels of gene expressioncan be achieved by the absence of B12 in the dark, by illumi-nation with no B12 present, or by exposure to light in thepresence of B12, in that order.

An important prerequisite for studying essential genes andtheir cellular roles is that their expression can be completelyturned off, as required, by the tightly regulated promoter underwhose control they are placed. In this respect, the B12-basedsystem does repress the expression of a candidate gene to

FIG. 7. Cellular morphology of M. xanthus strains MR1925 and MR1926 under permissive (light) and restrictive (dark with B12) conditions.Cells were examined at an OD550 of �0.8 by DIC and fluorescence microscopy after DAPI staining to visualize the nucleoid, as described inMaterials and Methods. Arrows point to regions depleted in DAPI fluorescence in abnormally long MR1926 cells observed on shifting to restrictivegrowth conditions (dark and B12). Scale bar, 5 �m.



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marginal levels under restrictive conditions. Thus, the systemallowed us to confirm that M. xanthus rpoN is essential, as waspreviously reported based on different experimental criteria(25). Additionally, our system has revealed that the gene weassigned as dksA in M. xanthus is also essential for viability.This system has also revealed that a gene coding for a proteinof unknown function, whose details will be reported elsewhere,is an essential gene in M. xanthus (García-Moreno et al., un-published data). A fourth gene in M. xanthus that we examined,ftsZ, was concluded to be essential based on the observationthat the natural ftsZ allele could be disrupted only in a mero-diploid background carrying another functional ftsZ copy.However, expression of ftsZ from PB in the B12-based system,in the absence of the gene at its native site, did not allow cellsurvival. Our data suggest that this may be because PB-drivenexpression of ftsZ produces lower than the normal amountsrequired for viability of M. xanthus. Thus, for the B12-basedsystem to be broadly applicable, it will be necessary to engineerPB promoter strength for greater expression levels of targetgenes on activation without compromising low expression un-der conditions of repression. This remains an objective forfuture studies and would certainly benefit from our ongoingwork aimed at understanding the molecular basis of how B12

and CarH act jointly in repressing PB.The applicability of the B12-based system to conditionally

express genes involved in M. xanthus development remains tobe examined. With this system, achieving maximal levels ofexpression requires illumination, but light has been reported toinhibit sporulation though not fruiting body formation (28).Since a candidate gene can also be expressed in the dark withno B12 present, the need for light could be dispensed with,especially once a PB of optimal promoter strength is engi-neered. PB and the promoter from which CarH is expressed inthe B12-based system are both �A dependent. Since �A levelshave been shown to persist at a high level until 12 h intodevelopment (16), our B12-based system could be applicable atleast for the genes expressed early in development.

Why might dksA be essential in M. xanthus? The choice ofdksA as a candidate gene for study was motivated by its func-tional link to �54-dependent transcription and the involvementof both in the response to nutrient-limiting conditions that isobserved in bacteria such as E. coli. In contrast to other bac-teria, rpoN coding for �54 is a vital gene in M. xanthus (25), andit is not entirely surprising that we found dksA also to beessential, given the functional link between the two genes. Athird example of a gene known to be dispensable in many otherbacteria but not in M. xanthus is lonV, which encodes an ATP-dependent protease (53). This is striking, given that a secondgene, lonD, that encodes a very similar product also exists in M.xanthus (12, 54). Both lonV and lonD are expressed duringvegetative growth and development, but while lonV is essentialunder all conditions of growth, lonD is required only for mul-ticellular development (12, 53, 54). The functions underpin-ning the essential nature of rpoN and lonV in M. xanthusremain to be elucidated, and an in-depth experimental analysisof this for dksA is also outside the scope of the present study.A common feature of rpoN, lonV, and dksA is that they areimplicated in many functions, and their absence causes pro-nounced pleiotropic phenotypes (5, 6, 14, 18, 24, 51, 57). More-over, even in bacteria where these genes can be deleted with-

out impairing cell viability, growth in rich medium is required,and they are indispensable under specific, nutrient-limitingconditions.

Given that dksA, like rpoN, is implicated in regulation oftranscription, the essential nature of dksA and rpoN may stemfrom controlling expression of a gene essential for M. xanthusviability. For instance, �54-dependent promoters require, inaddition to �54-RNAP holoenzyme, a specialized transcriptionfactor called an enhancer-binding activator protein (EBP) thatusually binds to regulatory DNA sequences upstream of thepromoter (6). Analysis of the M. xanthus genome indicates thatit has an unusually high number of EBP-encoding genes (52such putative EBP genes have been identified [20]). A numberof these EBPs have been implicated in development, heatshock response, and motility, but the functions of many othersremain to be discovered. The high abundance of these EBPs(possibly the largest number found so far in bacteria) suggestsa mode of regulation that is very likely critical in the life styleof M. xanthus. It also implies that �54 is at the nexus of a largeregulatory network, of which at least some parts may be es-sential for viability. Likewise, dksA may be a vital part of achain of transcriptional control. Studies in numerous otherbacteria have also implicated DksA in chaperone activity,DNA replication, recombination and repair, cell division, quo-rum sensing, responses to envelope stress, and a number ofother critical cellular processes in concert with or independentof (p)ppGpp (18, 34, 51, 55), and our data indicate that theprogressive depletion of DksA (or �54) in M. xanthus results inaberrant cell division. Thus, the direct or indirect role in any ofthe essential processes attributed to dksA may underlie its vitalnature in M. xanthus.

ACKNOWLEDGMENTS

We thank Rick Gourse (University of Wisconsin-Madison) for use-ful discussions on DksA and for a short stay in his laboratory by one ofus (G.N.A.). We also thank J. A. Madrid for technical assistance andC. Flores for DNA sequencing.

This work was supported by MEC (Spain) grants BFU2006-14524(to M.E.A.) and BFU2005-01040 (to S.P.) and FPI fellowships(D.G.M. and G.N.A.).

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