JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010

Jan. 2000, p. 546–550 Vol. 182, No. 2

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Disruption of aldA Influences the Developmental Process inMyxococcus xanthus

MANDY J. WARD, HELEN LEW, AND DAVID R. ZUSMAN*

Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720

Received 5 August 1999/Accepted 26 October 1999

Previously, we identified a gene (aldA) from Myxococcus xanthus, which we suggested encoded the enzymealanine dehydrogenase on the basis of similarity to known Ald protein sequences (M. J. Ward, H. Lew, A.Treuner-Lange, and D. R. Zusman, J. Bacteriol. 180:5668–5675, 1998). In this study, we have confirmed thataldA does encode a functional alanine dehydrogenase, since it catalyzes the reversible conversion of alanine topyruvate and ammonia. Whereas an aldA gene disruption mutation did not significantly influence the rate ofgrowth or spreading on a rich medium, AldA was required for growth on a minimal medium containingL-alanine as the major source of carbon. Under developmental conditions, the aldA mutation caused delayedaggregation in both wild-type (DZ2) and FB (DZF1) strains. Poorly formed aggregates and reduced levels ofspores were apparent in the DZ2 aldA mutant, even after prolonged development.

Myxococcus xanthus is a gram-negative bacterium that un-dergoes a starvation-induced developmental cycle. The devel-opmental program results in the morphogenesis of vegetativerod-shaped cells into spherical myxospores within multicellularaggregates termed fruiting bodies. Starvation for certain aminoacids, carbon, energy, or inorganic phosphate is sufficient toinitiate the process. The first obvious signs of developmentoccur after approximately 6 h of starvation, at which time thecells start streaming into aggregation centers. Mounds developas more and more cells enter the aggregates, which thendarken into fruiting bodies as the cells differentiate into spores.

Amino acids, rather than sugars, are of particular nutritionalimportance to M. xanthus, since this species hydrolyzes proteinas both an energy and a carbon source. Dworkin (5) showedthat M. xanthus can, in fact, grow on a mixture of amino acids,which are present as the only organic constituent of the growthmedium. Certain amino acids (methionine, leucine, isoleucine,and valine) are specifically required for growth. The remainderare degraded to acetate, pyruvate, and other tricarboxylic acidcycle intermediates to act as sources of energy, carbon, andnitrogen (1). Conversely, during vegetative growth, glucose isnot converted into pyruvate for use as an energy source, andcarbon from glucose is not incorporated into the cell (7, 18).

Many of the nutritional conditions and events which initiatedevelopment in M. xanthus also initiate development in otherbacteria, including Bacillus subtilis. For example, after starva-tion for amino acids, both B. subtilis and M. xanthus transientlydecrease the cellular GTP pool, while increasing (p)ppGpp(guanosine 39-di[tri]phosphate-59-diphosphate) levels (10, 11).The enzyme alanine dehydrogenase, which catalyzes the re-versible conversion of alanine to pyruvate and ammonia, hasalso been shown to be required for normal sporulation in B.subtilis (14). During sporulation, this enzyme is thought to beinvolved in the generation of pyruvate from alanine for theproduction of energy by metabolism through the tricarboxylicacid cycle. Alanine dehydrogenase activity has previously beenreported in M. xanthus (9), and while studying a region of DNAinvolved in directed motility, we identified an open reading

frame which could encode this enzyme (16). In this study, weconfirm that this open reading frame, renamed aldA, doesindeed encode a functional alanine dehydrogenase and showthat cells with a mutation in this gene have developmentaldefects. These defects seem unlikely to be due to a lack ofpyruvate in developing cells but may be the result of an in-creased cellular pool of L-alanine.

Identification of a potential transcriptional start site for thealdA gene. Further analysis of the aldA gene was performed todetermine whether the gene, because of its chromosomal lo-cation near the frz genes (Fig. 1), might be functionally asso-ciated with the Frz signal transduction system and therebyinvolved with regulating directed motility behavior. The M.xanthus aldA gene was suggested to be 1,125 bp long (Gen-EMBL accession no. AF049107) based on preferential codonusage, which is a good indicator of translational potential inGC-rich genomes (13). The proposed GTG start codon lies 39bp (13 codons) downstream of an in-frame TAG stop codon,and no alternative start codons are present in the interveningsequence. A potential ribosome binding site (GGAGG) waslocated 6 bp upstream of the proposed start codon (16).

In this study, we have used primer extension analysis toidentify potential transcriptional start sites for aldA. Severalpotential start sites were identified approximately 100 bp up-stream of the GTG translational start codon, and the oneshowing the strongest signal was designated 11 as the mostfrequently used start site (Fig. 2). Most of the potential startsites were seen to be present in both vegetative (V) and de-velopmental (D; 6 h of starvation) RNA transcripts preparedfrom both DZ2 (wild type) (4) and DZF1 (FB) (3) strains (Fig.2). While several additional faint start sites were present in thevegetative extracts (Fig. 2), most of the transcripts appeared tobe present at similar levels in both vegetative and developmen-tal samples, suggesting that the aldA gene is transcribed simi-larly during vegetative growth and early development. How-ever, we were unable to identify a potential promoter upstreamof this group of proposed transcriptional start sites. The primerextension analysis also identified four additional transcrip-tional start sites (not shown) upstream of those indicated inFig. 2. These results suggest that there may be multiple pro-moters upstream of aldA. A potential transcriptional termina-tor structure (CGGATG-4 bp-CATCCG) was identified down-

* Corresponding author. Mailing address: Department of Molecularand Cell Biology, 401 Barker Hall, University of California at Berke-ley, Berkeley, CA 94720-3204. Phone: (510) 642-2293. Fax: (510) 643-6334. E-mail: zusman@uclink4.berkeley.edu.

546

on March 3, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

stream of the aldA gene, in the aldA-rpoE1 intergenic region(16).

Construction of aldA mutants. The aldA gene encodes aprotein containing 374 amino acids with strong homology(58% identity) to the enzyme alanine dehydrogenase from B.subtilis (14). Since alanine dehydrogenase activity has previ-ously been identified in crude extracts of M. xanthus (9), in-sertion mutations were constructed in the aldA gene in orderto analyze both wild-type and mutant cell extracts for enzymeactivity. M. xanthus DZ2 (wild type) and DZF1 (pilQ1) wereused for the construction of aldA mutants DZ24280 (DZ2aldA) and DZF4281 (DZF1 pilQ1 aldA). Mutants were con-structed by cloning a 750-bp internal fragment of the aldA geneinto the 3.3-kb kanamycin resistance-encoding vector, pZErO-2(Invitrogen), to create the plasmid pZALD. The internal frag-ment of the gene was prepared by PCR, using Taq polymerase(Promega). The following primer pair combination was usedfor the amplification: forward primer, 59-CGGACGAGGTCTGGAAGCGC (bp 182 to 201 from the GTG start codon);reverse primer, 59-AGGTGGACGTCTGCGGCACG (bp 915to 934 from the GTG start codon). The plasmid pZALD waselectroporated into M. xanthus strains. Growth on CYE agar(3) containing 25 mg of kanamycin per ml was used to select formutants. Since the vector is unable to replicate in M. xanthus,kanamycin resistance could be maintained after integration ofthe plasmid into the host chromosome only by homologousrecombination. Chromosomal DNA was prepared from bothmutant and parent strains, and insertions within the aldA genewere confirmed by Southern blotting. The hybridization probewas constructed by PCR amplification of the internal 750-bpfragment of aldA, incorporating the hapten digoxigenin-11-dUTP (Boehringer Mannheim), and detection was performedby enzyme immunoassay and an enzyme-catalyzed color reac-tion. The aldA gene is present on a 3.3-kb SphI fragment in thewild-type strains. This fragment was missing and replaced bytwo SphI fragments of approximately 3.7 kb each in the mutantstrains (not shown). The positioning of the aldA gene upstreamof three genes (rpoE1, orf5, and frzS) in the same transcrip-tional orientation might suggest that all four genes are part ofan operon and therefore that mutations in aldA could havepolar effects on downstream gene expression. However, wehave previously identified a strong transcriptional start siteupstream of the rpoE1 gene (16). This transcriptional start sitewas present in both vegetative and developmental extracts andwas shown to be positioned downstream of a potential sA-dependent promoter. Therefore, it seems likely that the genesdownstream of aldA can be transcribed independently of aldA.

The aldA gene encodes a functional alanine dehydrogenase.To determine whether the aldA gene encodes a functionalalanine dehydrogenase, we prepared crude cell extracts fromvegetatively growing cultures and assayed them for enzymeactivity by the protocol of Kottel et al. (9). This assay measures

the reaction in which pyruvate and ammonia are converted toalanine and requires NADH, which is oxidized to NAD1.Alanine dehydrogenase activity was measured by monitoringthe loss in absorbancy at 340 nm in a cuvette containing thefollowing: 5 mM potassium phosphate buffer (pH 7.5), 52 mMNH4Cl, 0.1 mM reduced NAD, 100 mg of cell extract, and 5.2mM sodium pyruvate. While both the wild-type DZ2 andDZF1 extracts showed rapid oxidation of NADH, as indicatedby the loss of absorbance at 340 nm (20.8 absorbance [Abs]/min/mg of crude protein for DZ2 and 20.73 Abs/min/mg ofcrude protein for DZF1), the aldA mutant extracts showedonly low level residual activity (,20.08 Abs/min/mg of crudeprotein). These results show that the aldA gene does, indeed,encode a functional alanine dehydrogenase.

Effects of aldA mutations on vegetative growth and spread-ing. Since alanine dehydrogenase is required for the conver-sion of alanine to pyruvate, which can then be utilized as anenergy and carbon source, we measured the ability of thewild-type DZ2 and the DZ2 aldA mutant to grow on a modi-fied version of A1 minimal medium (1) in which the normalcarbon sources, pyruvate and aspartate, were replaced by L-alanine. Figure 3 shows that, after 35 days of incubation, singlecolonies of the wild-type DZ2 were growing on this medium,whereas no growth was visible on the plates inoculated with theDZ2 aldA mutant. This confirms that the aldA gene is requiredfor growth when L-alanine is the major carbon source.

Under nutrient-rich conditions, the aldA mutation was de-termined to have a minimal effect on growth rate, since bothparent and mutant strains showed similar doubling times ofjust over 4 h in CYE liquid medium. Liquid cultures (50 ml)were grown in 500-ml Erlenmeyer flasks, with a side arm, anddoubling times were calculated between Klett units of 50 to 100(approximately 2 3 108 to 4 3 108 CFU/ml). In a representa-tive experiment, DZ2 had a doubling time of 4 h 3 min, whilethe DZ2 aldA mutant doubled in 4 h 12 min. Similarly, theDZF1 strain had a doubling time of 4 h 6 min, while the DZF1aldA mutant had a doubling time of 4 h 9 min.

The aldA mutation was also shown to cause only slight dif-ferences in cell spreading behavior on CYE agar. Cells wereconcentrated to 4 3 109 CFU/ml, prior to spotting (5-mm-diameter spots) on plates. Both DZ2 and DZ2 aldA cellsspread identically on plates containing 1.5% agar (spread di-ameter of 9 mm after 18 h of incubation), whereas a smalldifference in spreading diameter was apparent on plates con-taining 0.3% agar (DZ2 spreading diameter, 12 mm; DZ2 aldAspreading diameter, 10 mm). Spreading movement on 1.5%agar was slightly reduced in the DZF1 aldA mutant. After 6days of growth, the DZF1 colony had a diameter of 12 mm,while that of the DZF1 aldA mutant was only 9 mm (notshown). Spreading in the DZF1 background was not analyzedon low-percentage agars, since the DZF1 strain contains aleaky social motility defect (pilQ1) which results in poor move-

FIG. 1. Map of the region surrounding aldA, showing frz genes (black) both upstream and downstream of the aldA gene (grey). Proposed sA-dependent promotersites (P) are shown. Arrows denote the direction of gene transcription.

VOL. 182, 2000 NOTES 547

on March 3, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

ment on soft agar (12, 15). The slightly reduced spreadingphenotypes of the aldA mutants could be due to the marginallyreduced growth rates on nutrient-rich CYE medium but seemunlikely to be associated with the Frz signal transduction sys-tem and directed motility behavior, since mutations in the frzgenes result in significantly reduced and disorganized spread-ing (17).

Developmental effects of aldA mutations. The most strikingeffect of the aldA mutation was seen under developmentalconditions. In the DZ2 background, the aldA mutation delayedaggregation by more than 24 h (Fig. 4). The DZ2 aldA mutantdid not form truly wild-type fruiting bodies. The aggregatesremained poorly formed and did not darken even after pro-longed (7-days) development, and a reduced number of sporeswas present in the fruiting bodies (approximately 10% of thewild-type number as evaluated microscopically). These sporeswere also less viable on germination than those of the DZ2parent (germination was reduced approximately 10-fold whenequivalent numbers of spores were plated on CYE agar). Thisphenotype is distinct from that of cells with mutations in therpoE1 gene which show developmental defects only whenplated at high cell density (16), confirming that the aldA mu-tant phenotype is unlikely to be due to polar effects on down-stream gene expression. In the DZF1 background, aggregationwas delayed 8 to 10 h with respect to the parent (not shown).However, after 4 days of development, the DZF1 aldA mutanthad formed normal fruiting bodies containing wild-type levelsof spores. These spores were shown to germinate at a fre-quency similar to that of DZF1 spores (data not shown). Cur-rently, it is not known why a mutation in the pilQ gene shouldpartially suppress the developmental defects of the aldA mu-tant. Mutations in the frz genes also result in both develop-mental aggregation and strain-dependent sporulation defects.However, since the frz phenotype is highly distinctive and dis-similar to that shown for the aldA mutants, we conclude that,although the aldA and frz genes are positioned close togetheron the chromosome, these genes are unlikely to be functionallyassociated. However, it is worth noting that the pilQ1 mutationalso partially suppresses frz sporulation deficiencies (8).

FIG. 2. Primer extension analysis of the region upstream of the aldA trans-lational start codon, highlighting the strongest of the proposed transcriptionalstart sites (11). The translational start codon (GTG) is shown next to thesequencing ladder (TCGA), with the potential Shine-Dalgarno (SD) site(GGAGG) shown upstream. RNA from vegetative (V) and developmental (D)M. xanthus cells was prepared with the RNeasy kit (QIAGEN). RNA was dilutedto 1 mg/ml in RNase-free water for primer extension analysis. Primer extensionswere performed as described previously (16), using 6 mg of RNA for cDNAsynthesis. The oligonucleotide 59-GGTTTTGATCTCCTTGGG (17 pmol) wasused as the primer for this reaction. Sequencing reactions for primer extensionanalysis were performed, using the same primer, by the dideoxynucleotide chaintermination method, using Sequenase (U.S. Biochemical Corp.) and[a-35S]dATP (410 Ci mmol21; Amersham) on double-stranded DNA. Sequenc-ing and primer extension reactions were run for 2 h on a 6% polyacrylamide gelprepared with National Diagnostics Sequagel reagents.

FIG. 3. Growth of DZ2 cells streaked on A1 minimal medium containingL-alanine as the major carbon source. Minimal A1 medium was prepared asdescribed by Bretscher and Kaiser (1) with the following modification: the majorcarbon sources, sodium pyruvate and potassium aspartate, were replaced by 10mg of L-alanine per ml. Media were solidified, using 0.8% ultrapure agarose(Gibco BRL). Cells were incubated at 34°C for 35 days and photographed witha dissecting scope at a magnification of 312.

548 NOTES J. BACTERIOL.

on March 3, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

That mutations in ald genes result in developmental defectsin both M. xanthus and B. subtilis suggests that alanine dehy-drogenase might play a similar role during development inboth species. However, in B. subtilis, it has been suggested thatthe ald mutation results in reduced levels of pyruvate, therebydepleting energy levels in the cell. This explanation seemsunlikely in the case of M. xanthus, as the cells are provided withsignificant amounts of pyruvate (9.1 mM sodium pyruvate) inCF fruiting medium (6). Pyruvate is included in CF medium

because cells are considered unlikely to suddenly encounterabsolute starvation conditions in the natural environment. Onthe other hand, exclusion of pyruvate from CF medium did notsignificantly alter the timing of developmental aggregation ineither the wild-type or aldA mutant strains (data not shown).This suggests that it may be the increased pool of L-alanine inthe cell which results in the delayed aggregation and otherdevelopmental defects seen in M. xanthus aldA mutants. Weare currently unaware of the physiological significance of in-

FIG. 4. Developmental aggregation of DZ2 and DZ2 aldA cells on CF fruiting medium. Cells for developmental analyses were concentrated to 4 3 109 CFU/ml,and then 5-ml volumes were spotted onto CF agar. Aggregation patterns were photographed at various time points. Spore counts were performed after removing thecells from CF agar and resuspending them in water. Spore clumps were dispersed by sonication, and appropriate dilutions were placed in a Petroff-Hausser chamberfor counting under magnification. Germination frequencies were determined by plating known spore numbers (from spore counts) onto CYE agar and incubating theplates for 4 or 5 days at 34°C.

VOL. 182, 2000 NOTES 549

on March 3, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

creased levels of a single amino acid on the cell, and alternativeexplanations for the aldA mutant phenotype are certainly pos-sible. However, in E. coli, alanine, as well as leucine, has beenshown to have a strong effector role by stimulating Lrp, theleucine response regulator (2, 19). The possibility that M. xan-thus could also utilize a global regulator, similar to Lrp, duringdevelopment would be an interesting area for future research.

Research in our laboratory is supported by Public Health Servicegrant GM20509 from the National Institutes of Health.

REFERENCES

1. Bretscher, A. P., and D. Kaiser. 1978. Nutrition of Myxococcus xanthus, afruiting bacterium. J. Bacteriol. 133:763–768.

2. Calvo, J. M., and R. G. Matthews. 1994. The leucine-responsive regulatoryprotein, a global regulator of metabolism in Escherichia coli. Microbiol. Rev.58:466–490.

3. Campos, J. M., J. Geisselsoder, and D. R. Zusman. 1978. Isolation of bac-teriophage Mx4, a generalized transducing phage for Myxococcus xanthus. J.Mol. Biol. 119:167–178.

4. Campos, J. M., and D. R. Zusman. 1975. Regulation of development inMyxococcus xanthus: effect of 39:59 cyclic AMP, ADP and nutrition. Proc.Natl. Acad. Sci. USA 72:518–522.

5. Dworkin, M. 1962. Nutritional requirements for vegetative growth of Myxo-coccus xanthus. J. Bacteriol. 84:250–257.

6. Hagen, D. C., A. P. Bretscher, and D. Kaiser. 1978. Synergism betweenmorphogenic mutants of Myxococcus xanthus. Dev. Biol. 64:284–296.

7. Hemphill, H. E., and S. A. Zahler. 1968. Nutritional induction and suppres-sion of fruiting in Myxococcus xanthus FBa. J. Bacteriol. 95:1018–1023.

8. Kashefi, K., and P. L. Hartzell. 1995. Genetic suppression and phenotypicmasking of a Myxococcus xanthus frzF defect. Mol. Microbiol. 15:483–494.

9. Kottel, R. H., M. Orlowski, D. White, and J. Grutsch. 1974. Presence ofamino acid dehydrogenases and transaminases in Myxococcus xanthus duringvegetative growth and myxospore formation. J. Bacteriol. 119:650–651.

10. Lopez, J. M., A. Dromerick, and E. Freese. 1981. Response of guanosine59-triphosphate concentration to nutritional changes and its significance forBacillus subtilis sporulation. J. Bacteriol. 146:605–613.

11. Manoil, C., and D. Kaiser. 1980. Accumulation of guanosine tetraphosphateand guanosine pentaphosphate in Myxococcus xanthus during starvation andmyxospore formation. J. Bacteriol. 141:297–304.

12. Shi, W., and D. R. Zusman. 1993. The two motility systems of Myxococcusxanthus show different selective advantages on various surfaces. Proc. Natl.Acad. Sci. USA 90:3378–3382.

13. Shimkets, L. J. 1993. The myxobacterial genome, p. 85–107. In M. Dworkinand D. Kaiser (ed.), Myxobacteria II. American Society for Microbiology,Washington, D.C.

14. Siranosian, K. J., K. Ireton, and A. D. Grossman. 1993. Alanine dehydro-genase (ald) is required for normal sporulation in Bacillus subtilis. J. Bacte-riol. 175:6789–6796.

15. Wall, D., P. E. Kelenbrander, and D. Kaiser. 1999. The Myxococcus xanthuspilQ (sglA) gene encodes a secretin homolog required for the type IV pilusbiogenesis, social motility, and development. J. Bacteriol. 181:24–33.

16. Ward, M. J., H. Lew, A. Treuner-Lange, and D. R. Zusman. 1998. Regulationof motility behavior in Myxococcus xanthus may require an extracytoplasmic-function sigma factor. J. Bacteriol. 180:5668–5675.

17. Ward, M. J., and D. R. Zusman. 1997. Regulation of directed motility inMyxococcus xanthus. Mol. Microbiol. 24:885–893.

18. Watson, B. F., and M. Dworkin. 1968. Comparative intermediary metabolismof vegetative cells and microcysts of Myxococcus xanthus. J. Bacteriol. 96:1465–1473.

19. Zhi, J., E. Mathew, and M. Freundlich. 1999. Lrp binds to two regions in thedadAX promoter region of Escherichia coli to repress and activate transcrip-tion directly. Mol. Microbiol. 32:29–40.

550 NOTES J. BACTERIOL.

on March 3, 2020 by guest

http://jb.asm.org/

Dow

nloaded from