APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2002, p. 2943–2949 Vol. 68, No. 60099-2240/02/$04.00�0 DOI: 10.1128/AEM.68.6.2943–2949.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification and Isolation of Genes Involved inPoly(�-Hydroxybutyrate) Biosynthesis in Azospirillum brasilense

and Characterization of a phbC MutantDaniel Kadouri, Saul Burdman, Edouard Jurkevitch, and Yaacov Okon*

Department of Plant Pathology and Microbiology and The Otto Warburg Center for Agricultural Biotechnology,Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Received 27 November 2001/Accepted 29 March 2002

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly(�-hydroxy-butyrate) (PHB) under suboptimal growth conditions. Utilization of PHB by bacteria under stress has beenproposed as a mechanism that favors their compatible establishment in competitive environments, thusshowing great potential for the improvement of bacterial inoculants for plants and soils. The three genes thatare considered to be essential in the PHB biosynthetic pathway, phbA (�-ketothiolase), phbB (acetoacetylcoenzyme A reductase), and phbC (PHB synthase), were identified in Azospirillum brasilense strain Sp7, cloned,and sequenced. The phbA, -B, and -C genes were found to be linked together and located on the chromosome.An A. brasilense phbC mutant was obtained by insertion of a kanamycin resistance cassette within the phbCgene. No PHB production was detected in this mutant. The capability of the wild-type strain to endurestarvation conditions was higher than that of the mutant strain. However, motility, cell aggregation, rootadhesion, and exopolysaccharide (EPS) and capsular polysaccharide (CPS) production were higher in thephbC mutant strain than in the wild type.

A wide variety of microorganisms are known to produceintracellular energy and carbon storage compounds knownas poly(�-hydroxybutyrate) (PHB) or polyhydroxyalkanoates(PHA) (44). Knowledge of the structure and organization ofthe PHA biosynthetic genes from a wide range of bacteria hasincreased as a result of intensive research for industrial appli-cations. Various pathways have been found among differentbacteria (16).

PHA biosynthetic genes and other related genes in the PHAmetabolism are often clustered together within bacterial ge-nomes, as was revealed by nucleotide sequence mapping anal-yses (26, 27, 28, 34).

The gram-negative nitrogen-fixing rhizobacterium Azospiril-lum brasilense lives in close association with plant roots, whereit exerts beneficial effects on plant growth and yield of manycrops of agronomic importance (22, 23). Enzymes involved inthe synthesis, accumulation, and degradation of PHAs in A.brasilense have been examined in detail (36, 37, 38). It wasshown that in contrast to other bacterial species, A. brasilensedoes not produce copolymers of hydroxyalkanoates but ratheronly homopolymers of PHB (14).

It has been suggested for diverse ecological systems that theaccumulation, degradation, and utilization of PHAs by severalbacteria under stress is a mechanism that favors their estab-lishment, proliferation, survival, and competition, especially incompetitive environments where carbon and energy sourcesare limiting, such as those encountered in the soil and rhizo-sphere (24). Understanding the role that PHAs play as internal

storage polymers is of fundamental importance in microbialecology.

The role played by PHB in the survival, proliferation, andplant root colonization of Azospirillum spp. is not yet fullyknown due to the lack of mutants impaired in their ability tosynthesize PHB. To gain insight into the possible influences ofPHB in the free-living state and in the rhizosphere, we isolatedand sequenced the phbA, -B, and -C genes from A. brasilensestrain Sp7. We also report on the construction and character-ization of a phbC mutant strain of A. brasilense unable tosynthesize PHB.

MATERIALS AND METHODS

Bacterial strains, plasmid, and growth conditions. The bacterial strains andplasmids used in this work are listed in Table 1. For Escherichia coli growth,Luria Bertani (LB) (19) media was used. A medium with a high carbon-to-nitrogen (C:N) ratio (4, 25) was used to induce the accumulation of PHB in A.brasilense. Azospirillum transconjugants were selected on minimal medium for A.brasilense (MMAB) (42).

DNA manipulations, sequencing, and analysis. Subcloning, transformation,and DNA extractions were carried out according to standard methods (31). DNAsequencing was carried out with an ABI Prism 377 DNA sequencer (AppliedBiosystem Inc., Foster City, Calif.). Sequence data were analyzed with the Uni-versity of Wisconsin Genetic Computer Group software. Homology searcheswere performed using the Blast network service (1). Sequence alignments weredone with the Clustal W program (40) and edited using GeneDoc (K. B. Nicholasand J. B. Nicholas, Jr., GeneDoc [http://www.cris.com/�Ketchup/genedoc.shtml]).

Synthesis of oligonucleotides and PCR. Oligonucleotide primers were synthe-sized (General Biotechnology, Rehovot, Israel) by the phosphoramidate method,using a Pharmacia 4-Primer Gene Assembler, according to the codon frequencyfor A. brasilense extracted from the Codon Usage Database compiled from thecodon usage tabulated from GenBank (21). Based on known phbC sequences,two degenerate primers were designed to amplify the putative phbC fragmentfrom total DNA of A. brasilense Sp7: phbC-R, 5�-ATC-AAC-AAG-TTC-TAY-ATC-3�, and phbC-L, 5�-GTT-CCA-GTA-SAG-SAG-GTC-GAA-3� (S � G orC; Y � C or T). The primers anneal with phbC of Rhodospirillum rubrum,Azorhizobium caulinodans, and Sinorhizobium meliloti in positions 780 to 1308,744 to 1269, and 831 to 1356, respectively. PCR was performed with an auto-

* Corresponding author. Mailing address: Department of Plant Pa-thology and Microbiology, Faculty of Agricultural, Food and Environ-mental Quality Sciences, The Hebrew University of Jerusalem, P.O.Box 12, Rehovot 76100, Israel. Phone: 972 8 9489216. Fax: 972 89466794. E-mail: okon@agri.huji.ac.il.

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mated PCR thermoblock (Mastercycler gradient; Eppendorf, Netheler, Ham-burg). The PCR product was labeled with digoxigenin (DIG)-dUTP using thePCR-DIG probe synthesis kit (Roche Diagnostics Corp.).

Southern blot analysis and hybridization. Total DNA from A. brasilense,Azospirillum lipoferum, Azospirillum amazonense, Azospirillum halopraeferens,Azospirillum irakense, and Azospirillum doebereinerae was isolated, digested withrestriction enzymes, electrophoresed, and blotted onto MSI nylon transfer mem-branes (Roche Diagnostics Corp.) by standard methods (31). Prehybridizationand hybridization were carried out at 68°C and detection was performed with theDIG luminescent detection kit (Roche Diagnostics Corp.)

Construction of A. brasilense Sp7 phbC::Km mutant. The Tn5-derived 1.4-kbHincII kanamycin (Km) resistance cassette of pUCA800 was inserted in theunique Sma� restriction site of pPEB1.2 to yield pPEBKm2.6. The 2.6-kbphbC::Km EcoRI fragment of pPEBKm2.6 was subsequently cloned into theEcoRI site of the suicide vector pSUP202 to yield pSPEAKm. E. coli S17.1 wastransformed with pSPEAKm, which was further mobilized to A. brasilense Sp7through biparental mating. Azospirillum transconjugants were selected onMMAB supplemented with Km. Km-resistant clones were isolated. Southernblot hybridization, DNA sequencing, and PCR screening with specific oligonu-cleotides against either the 1.2-kb phbC fragment of pPEB1.2 or the 1.4-kbHincII Km resistance cassette of pUCA800, showed the correct genetic config-uration. One of these A. brasilense Sp7 phbC::Km mutants was used for furtheranalysis.

PHB production. PHB conccntration was assayed by gas chromatography (2),using PHB from Alcaligenes (Sigma) as a standard and wild-type Sp7 as a control.

Transmission electron microscopy. Bacteria were embedded in a low-viscosityepoxy resin as described by Burdman et al. (3). Thin sections were cut with adiamond knife on a 8800 ultramicrotome (LKB, Stockholm, Sweden) and exam-ined on a JEM 100-CX transmission electron microscope (JEOL, Tokyo, Japan).

Starvation experiments. Overnight cultures of A. brasilense (2-ml aliquots)were used to inoculate flasks containing 25 ml of high-C:N-ratio medium. After24 h of growth, bacteria were collected and washed twice by centrifugation at4,000 � g for 10 min in 0.06 M potassium phosphate buffer (pH 6.8). Bacteria

were then resuspended in the same buffer and incubated on a shaker at 170 rpmfor 12 days under starvation conditions (38). Bacterial density (CFU/milliliter)was further determined by dilution plating.

Adhesion assay of bacteria to intact roots. Seeds of sweet corn (cv. Jubilee)and wheat (cv. Atir) were surface-sterilized for 2 min in 95% ethyl alcohol,followed by 1 min in 1% sodium hypochlorite, and then washed five times withsterile distilled water. An A. brasilense overnight culture (1 ml) was used toinoculate flasks containing 0.4 or 0.2 g of freshly harvested 1-week-old roots ofsweet corn and wheat seedlings, respectively, in 5 ml of 0.06 M potassiumphosphate buffer (pH 6.8). After 2 h of incubation on a rotary shaker (170 rpm)at 30°C, the roots were washed three times by immersion in 0.06 M potassiumphosphate buffer for 1 min without agitation. Buffer (6 ml) was added and theroots were washed intensively for 2 min by vortex. The supernatant was taken todetermine the amount of bacteria attached to the roots. The percentage ofadhesion was estimated as follows: % adhesion � [(CFUt � CFUs) � 100%]/CFUt; with t and s being total and supernatant, respectively.

Extraction of extracellular polysaccharides. Polysaccharides were extractedfrom a 48-h culture (3, 7). The amounts of sugar in the polysaccharide fractionswere evaluated by the anthrone method, using glucose as the standard (9).

Bacterial dry weight. Cell dry weight per milliliter of culture broth was deter-mined by weighing dry cells with a microbalance (Mettler, Zurich, Switzerland)as described by Wang and Lee (43).

Nucleotide sequence accession number. The sequences of A. brasilense corre-sponding to �-ketothiolase, acetoacetyl-CoA reductase, PHB synthase, andacyl-CoA dehydrogenase genes were submitted to GenBank (accession no.AF353206, AY046923, AF353205, and AY046924, respectively).

RESULTS

Cloning and sequence analysis of A. brasilense Sp7 phbABC.Two degenerate primers were designed based on the phbCsequences of R. rubrum, A. caulinodans, and S. meliloti. Using

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Relevant characteristic(s)a Reference or source

StrainsAzospirillum spp.A. brasilense Sp7 (ATCC 29145) Wild-type strain 39A. brasilense 7030 Smr nitroso-guanidine-induced mutant of Sp7, lacking 115-MDa megaplasmid 12A. lipoferum (ATCC 29708) Wild-type strain 39A. amazonense (ATCC 35119) Wild-type strain 17A. halopraeferens (ATCC 43709) Wild-type strain 30A. irakense KBC1 Wild-type strain 15A. doebereinerae GSF-71T Wild-type strain 10A. brasilense phbC mutant Sp7 phbC::Km mutant This studyEscherichia coli

DH5 hsdR17 endA1 thi-1 gyrA96 relA1 recA1 supE44 lacU169(�80lacZM15) 31HB101 F� hsdS20 (r�

B m�B) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 supE44 31

S17.1 pro thi endA recA hsdR with RP4-2-Tc::Mu-Km::Tn7 integrated inchromosome, Smr

32

PlasmidsPUC18/19 Apr, ColE1 replicon, lacZ, cloning vector 45PLAFR3 Tcr, pLAFR1 derivative containing HaeII fragment of pUC8 33pSUP202 Apr, Tcr, Cmr, ColE1 replicon, mobilizable plasmid, suicide vector for A.

brasilense32

PRK2073 Smr, ColE1 replicon, Tra�, Mob�, helper plasmid for triparental conjugations 11PUCA800 pUC8 with Kmr GenBlock from Puc-4K 20P90 Tcr, pLAFR1 containing A. brasilense Sp7 p90 cosmid library 6pP2EP5 Apr, pUC19, containing 5-kb EcoRI-PstI A. brasilense Sp7 phbC fragment This studypP13S2 Apr, pUC18, containing 2-kb SalI A. brasilense Sp7 phbA fragment This studypP13S8 Apr, pUC18, containing 8-kb SalI A. brasilense Sp7 phbB fragment This studypPEB1.2 Apr, pUC18, containing 1.2-kb EcoRI-BamHI A. brasilense Sp7 phbC fragment This studypPEBKm2.6 Apr, Kmr, pUC18, containing 2.6-kb EcoRI-AatII A. brasilense Sp7 phbC

fragment with Kmr cassette inserted in SmaI siteThis study

PSPEAKm Apr, Kmr pSUP202, containing 2.6-kb EcoRI-AatII A. brasilense Sp7 phbCfragment with Kmr cassette inserted in SmaI site

This study

a Abbreviations: Tc, tetracycline; Ap, ampicillin; Km, kanamycin; Cm, cycloheximide, Sm, streptomycin.

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total DNA of A. brasilense Sp7 as the template in PCR, a500-bp product, homologous to known phbC genes, was ob-tained. Following sequencing, specific oligonucleotide primerswere synthesized and used for PCR screening of an E. coliHB101 cosmid library containing partially EcoRI-restricted to-tal DNA of A. brasilense strain Sp7 in pLAFR3. A clone con-taining a 20-kb A. brasilense DNA fragment including the re-gion encoding PhbC was isolated. This DNA fragment wassubcloned in pUC19 and pUC18, resulting in clones pP2EP5,pP13S2, and pP13S8.

The above subclones were sequenced by primer walking, andfour reliable open reading frames (ORFs) were detected. Thededuced amino acid sequence of the first ORF exhibited highsimilarity with an internal part of the PhbC proteins of R.rubrum (GenBank accession no. CAB65395; 69% identity,83% similarity), A. caulinodans (GenBank accession no.CAA06928; 55% identity, 71% similarity), Mesorhizobium loti(GenBank accession no. BAB48418; 53% identity, 71% simi-larity), S. meliloti (GenBank accession no. AAC61899; 52%identity, 70% similarity), and R. eutropha (GenBank accessionno. AAB24910; 44% identity, 69% similarity). Multiple align-ment of the deduced amino acid sequence of the phbC from A.

brasilense strain Sp7 with homologous phbC genes from otherbacteria is shown in Fig. 1. The second ORF showed highsimilarity with an internal part of the PhbA proteins of Pseudo-monas aeruginosa (GenBank accession no. AAG06328; 64%identity, 78% similarity), Bacillus halodurans (GenBank acces-sion no. BAB07206; 49% identity, 64% similarity), and S. me-liloti (GenBank accession no. CAC49901; 46% identity, 58%similarity). The third ORF showed similarity with an internalpart of PhbB of Bacillus subtilis (GenBank accession no.CAB15273; 39% identity, 55% similarity) and Staphylococcusaureus (GenBank accession no. AAK51157; 30% identity, 48%similarity). Finally, the fourth ORF showed high similarity withan internal part of the acyl coenzyme (acyl-CoA) dehydroge-nase proteins of P. aeruginosa (GenBank accession no.AAG03896; 57% identity, 70% similarity) and M. loti (Gen-Bank accession no. BAB52037; 54% identity, 71% similarity).This protein is involved in fatty acid metabolism. The A.brasilense ORFs were therefore designated phbA, -B, and -Cand fadE.

A physical map of the phbABC and fadE cluster was estab-lished (Fig. 2). The phbC ORF of A. brasilense is putatively1,854-bp long and is transcribed in a direction opposite to the

FIG. 1. Multiple alignment of the deduced amino acid sequence of phbC of A. brasilense (accession no. AF353205) with correspondingsequences of phbC from R. rubrum (accession no. CAB65395), A. caulinodans (accession no. CAA06928), S. meliloti (accession no. AAC61899),M. loti (accession no. BAB48418), and R. eutrophus (accession no. AAB24910). Three levels of similarity are shown according to the default settingsof GeneDoc.

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other three ORFs. The putative phbA gene is 1,161-bp long,followed by the putative 2,332-bp-long phbB and the putative1,788-bp fadE genes. Purine-rich tracts were found to precedethe putative start codons of the four ORFs, as characterizedfor prokaryotes. Typical Shine-Dalgarno sequences were found10 nucleotides upstream from the putative start codon of fadEand phbB and 12 nucleotides upstream of that of phbA (notshown).

To establish whether phbABC is located in the chromosomeor in one of the A. brasilense megaplasmids (either the 115-MDa or the 90-MDa plasmid), total DNA was extracted fromA. brasilense Sp7, A. brasilense 7030 (a strain lacking the 115-MDa megaplasmid), and a pooled Sp7-p90 cosmid library.PCRs were performed, using a specific oligonucleotide for thephbABC cluster. Positive reactions were observed only withDNA from A. brasilense Sp7 and A. brasilense 7030 (data notshown), suggesting that phbABC is located in the chromosomeand not in one of the megaplasmids.

Southern blot hybridization. The original 500-bp PCR prod-uct corresponding to an internal region of phbC was DIG-labeled and used in Southern blot hybridization against totalDNA from other Azospirillum species digested with EcoRI(Table 1). DNA from A. brasilense Sp7 and from E. coli DH5were used as positive and negative controls, respectively. Allthe Azospirillum DNA tested yielded a positive reaction, hy-bridizing with the phbC probe (Fig. 3), suggesting the presence

of the phbC gene in all of them.Construction and characterization of A. brasilense Sp7

phbC::Km mutant. An A. brasilense phbC mutant was isolatedfollowing the cloning of a Tn 5-derived Km resistance cassetteinto the unique Sma� restriction site of pPEB1.2 (Fig. 2) anddouble homologous recombination into the A. brasilense Sp7phbC locus.

No substantial differences were found between Sp7 and thephbC mutant strain in their growth curves and generation timein LB media. A. brasilense Sp7 showed a generation time of2.1 h, whereas for the phbC mutant, the generation time was2.2 h (Fig. 4). A few phenotypic differences between the wildtype and the mutant were detected: the motility exhibited bythe mutant was considerably higher than that of the wild-typestrain, as observed by contrast microscopy, and the mutantstrain exhibited higher aggregation after 24 h of growth inhigh-C:N-ratio media (Fig. 5).

No apparent PHB was detected in the phbC mutant grownfor 24 h on high-C:N-ratio medium (not shown), indicatingthat the phbC locus is required for synthesis of PHB. Thisresult was confirmed by transmission electron microscopy,which showed large PHB granules in the wild type (Fig. 6A)but none in the phbC mutant (Fig. 6B).

Starvation experiments. Upon starvation in a phosphatebuffer suspension, without exogenous carbon or energy sourcesin the medium, A. brasilense Sp7 survived longer than the phbCmutant strain (Fig. 7). The density of viable cells of the phbCmutant decreased from the onset of the starvation period. Incontrast, strain Sp7 showed a density increase at the beginningof the starvation period, followed by a moderate decreaseduring the time course of the experiment.

FIG. 2. Physical map of A. brasilense Sp7 phbABC and fadE region. Open arrows indicate the locations and directions of transcription of phbC,phbA, phbB, and fadE. The triangle represents the location of the insertion of the kanamycin resistance cassette in the phbC mutant strain. E,EcoRI; B, BamHI; S, SmaI.

FIG. 3. Southern blot hybridization using a DIG-labeled 500-bplong PCR-generated probe representing an internal region of the A.brasilense Sp7 phbC gene. Total DNA from A. doebereinerae (lane 1),A. brasilense Sp7 (lane 2), A. lipoferum (lane 3), A. irakense (lane 4), A.amazonense (lane 5), A. halopraeferens (lane 6), and E. coli (lane 7).The DNA was digested with EcoRI. Arrows indicate the estimatedposition of the putative phbC bands.

FIG. 4. Growth curves of A. brasilense Sp7 (Œ) and of the phbCmutant (F) in LB media. Bacteria were grown in a shaker (170 rpm at30°C). Each value represents the mean of three replicates from onerepresentative experiment. Each experiment was carried out threetimes, each yielding similar results.

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Adhesion assay of bacteria to intact roots. Bacteria weregrown for 24 h in high-C:N-ratio medium and used in theadhesion assay. The percentage of adhering phbC mutant cellsto roots of sweet corn and wheat was five to six times higherthan that of A. brasilense Sp7 (Table 2).

Extraction of extracellular polysaccharides. The amount ofexopolysaccharide (EPS) and capsular polysaccharide (CPS)

extracted after 48 h of growth in medium with a high C:N ratiowas considerably higher in the phbC mutant than in strain Sp7(Table 3).

DISCUSSION

In this study, we cloned and sequenced three genes from A.brasilense that are traditionally considered to be necessary inthe PHB biosynthetic pathway of a wide variety of microor-ganisms. A mutant strain impaired in PHB synthesis was alsoconstructed and characterized. The PHB biosynthetic pathwaydescribed for R. eutrophus is probably present in most bacteriaable to accumulate PHB. The pathway begins with the con-densation of two acetyl-CoA molecules catalyzed by �-keto-thiolase. An NADPH-dependent acetoacyl-CoA reductasecatalyzes the conversion of acetoacyl-CoA to �-hydroxybu-tyryl–CoA. Finally, the resulting �-hydroxybutyryl–CoA ispolymerized by a PHB synthase (35).

The PHB synthase gene of A. brasilense (phbC) was identi-fied by means of PCR, using two degenerate primers designedon the basis of the PhbC sequences of other bacteria. Analysisof the deduced amino acid sequence of the A. brasilense phbCgene showed a high degree of similarity with other PHB syn-thases. Disruption of this gene suppressed PHB accumulation,as evidenced by gas chromatography and electron microscopy.

Additional sequencing from the phbC region and homologyanalysis of the identified putative ORFs suggested the pres-

FIG. 5. Aggregation of A. brasilense Sp7 (A) and of the phbC mu-tant (B) after 24 h of growth in medium with a high C:N ratio. Bacteriawere transferred to Petri dishes before the photograph was taken.

FIG. 6. Electron micrographs of thin sections of A. brasilense(A) and of the phbC mutant (B), grown for 24 h in medium with a highC:N ratio. The arrow indicates PHB granules. Bars, 1 �m. At least 10sections of each strain were examined.

FIG. 7. The effect of PHB on survival capability of starved bacteria.Cells of A. brasilense Sp7 (Œ) and phbC mutant (F) were grown onmedium with a high C:N ratio for 24 h and transferred to phosphatebuffer, where they were incubated for 12 days. Bacterial density wasdetermined using dilution plating. Each value represents the mean ofthree replicates from one representative experiment. Each experimentwas carried out three times, each yielding similar results.

TABLE 2. Adhesion of A. brasilense Sp7 and phbC mutantto roots of sweet corn and wheata

StrainAdhesion (%)

Sweet corn Wheat

Sp7 4.5 (1.2)a 4.5 (2.5)a

phbC mutant 30.5 (2.3)b 26.75 (6.0)b

a Percentage adhesion was measured according to the described methodology.This experiment was performed three times yielding similar results. Valuesrepresent the mean and standard error (in parentheses) of three replicates pertreatment. Different letters within columns indicate significant differences at a Pvalue of 0.05 according to t-test analysis.

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ence of other PHA biosynthetic genes, phaA and phaB, codingfor �-ketothiolase and NADPH-dependent acetoacetyl-CoAreductase, respectively. An additional ORF was detecteddownstream of the phbB gene. Sequence homology analysissuggested that this ORF encodes acyl-CoA dehydrogenase(FadE), a protein that is known to play a role in fatty acidmetabolism (18).

Therefore, our findings indicate that the phbABC genes in A.brasilense are linked in a cluster similar to the ones found inAcinetobacter sp., Alcaligenes latus, Pseudomonas acidophila,and R. eutrophus and most recently in Azotobacter sp. (16, 28,44). Southern blot hybridization suggested that all known spe-cies of Azospirillum contain at least one phbC gene similar tothat of A. brasilense. In A. doebereinerae, two bands hybridizedto the probe (Fig. 3). This bacterium was recently character-ized and proposed to belong to the Azospirillum genus (10).Although it exhibits general characteristics of Azospirillum, A.doebereinerae differs from its nearest relatives in some physio-logical properties (10). The fact that two bands were observedfor A. doebereinerae could be due to a nonspecific reaction orto the presence of an EcoR� site in the reacting fragment.However, we cannot discard the possibility that this bacteriumcarries a second copy of the gene, as reported for some otherbacteria (41).

Starvation experiments showed a clear decrease in the sur-vival ability of the phbC mutant compared to the wild-typestrain Sp7. This is in accordance with previous results by Taland Okon (38), in which bacteria containing high levels of PHBsurvived and proliferated to a higher extent than bacteria con-taining low levels of PHB. These findings demonstrate the roleof PHB in A. brasilense as an intracellular energy and carbonstorage compound, which can enhance survival when thesesources are limited.

In A. brasilense, PHB oxidation involves a specific NADH-dependent dehydrogenase (36), which competes for tricarbox-ylic acid (TCA) cycle intermediates (for example, NADH,NADPH, ATP, and acetyl-CoA) in the electron transport sys-tem (37). When PHB accumulation is disrupted, more re-sources are accessible to the TCA cycle. The presence of in-tracellular PHB in the cells has been previously shown toprevent chemotaxis to any chemoattractant (13). Therefore,with no PHB being produced by the mutant, the bacteria maybe in a constant chemotactic “spree,” responding to chemoat-tractants present in the medium. This could explain the in-creased motility in the phbC mutant compared to that of thewild type, as seen by contrast microscopy.

The phbC mutant also showed more aggregation than thewild-type strain in a medium containing a relatively high C:Nratio. Azospirilla are known for their capacity to aggregate andflocculate under diverse stress conditions. Previous studieshave suggested the involvement of extracellular polysaccha-rides in cell aggregation (5). The concentrations of EPS pro-duced by four different strains of A. brasilense differing in theirability to aggregate increased with the extent of aggregation(3). Similarly, in our experiments, there was an associationbetween the quantities of EPS and CPS and the extent ofaggregation.

In a root adhesion assay, the phbC mutant strain exhibitedincreased ability to adhere to roots relative to the wild-typeSp7 (Table 2). As for cell aggregation, the differences in EPSand CPS contents between wild-type and mutant strains mightexplain the difference observed in root adhesion properties (8).In A. brasilense, chemotaxis and motility are probably involvedin promoting root colonization (29). Therefore, in addition todifferences in polysaccharides, the enhanced motility observedfor the mutant strain could also contribute to stronger rootadhesion than that of the wild type.

In conclusion, a cluster of genes involved in the biosynthesisof PHB was isolated from A. brasilense and a PhbC-defectivemutant was shown to have decreased in survival while exhib-iting increased motility, production of extracellular polysaccha-rides, and adhesion to cereal roots. With the commercial ap-plication of field inoculations using A. brasilense, there isincreasing interest in the development of efficient and reliableinoculants. We are also testing the effects of PHB synthesis onthe ability of A. brasilense to endure stress factors involved ininoculum efficiency and viability.

ACKNOWLEDGMENTS

This research was supported by “The Israel Science Foundation”founded by “The Academy of Sciences and Humanities,” and by theEuropean Union-5th Framework contract QLK3-CT-2000-31759-ECO-SAFE.

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TABLE 3. Extraction of extracellular polysaccharides fromA. brasilense Sp7 and phbC mutant after 48 h of growth

in high C:N-ratio mediuma

Strain Dry wtb EPSc CPSc

Sp7 0.178 (0.014) 61.4 (4.3) 1.0 (0.2)phbC mutant 0.160 (0.005) 106.2 (5.2) 1.7 (0.1)

a Each value represents the mean and standard error (in parentheses) of threereplicates from one representative experiment. Each experiment was carried outthree times, each yielding similar results. EPS, exopolysaccharide; CPS, capsularpolysaccharide.

b Values are expressed as grams of dried bacteria (10 ml of growth medium)�1.c Values are expressed as milligrams of sugar (grams of dried bacteria)�1.

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