Changes in Azospirillum brasilense motility and the effect of wheat seedling exudates

ARTICLE IN PRESS Microbiological Research 164 (2009) 578—587

www.elsevier.de/micres

Changes in Azospirillum brasilense motility and the effect of wheat seedling exudates Igor V. Borisov, Andrei V. Schelud’ko, Lilia P. Petrova, Elena I. Katsy Laboratory of Microbial Genetics, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospekt Entuziastov, Saratov 410049, Russian Federation Received 22 January 2007; received in revised form 5 July 2007; accepted 16 July 2007

KEYWORDS Azospirillum brasilense; Wheat seedling exudates; Motility; Mutation

Summary The rhizobacterium Azospirillum brasilense Sp245 swims, swarms (Swa+ phenotype) or, very rarely, migrates with the formation of granular macrocolonies (Gri+ phenotype). Our aims were (i) to identify Sp245 mutants that swarm faster than the parent strain or differ from it in the mode of spreading and (ii) to compare the mutants’ responses to wheat seedling exudates. In isotropic liquid media, the swimming speeds of all motile A. brasilense strains were not influenced by the exudates. However, the exudates significantly stimulated the swarming of Sp245. In several Sp245 mutants, the superswarming phenotype was insensitive to local colonial density and to the presence of wheat seedling exudates. An A. brasilense polar-flagellum-defective Gri+ mutant BK759.G gave rise to stable Swa++ derivatives with restored flagellum production. This transition was concurrent with plasmid rearrangements and was stimulated in the presence of wheat seedling exudates. The swarming rate of the Swa++ derivatives of BK759.G was affected by the local density of their colonies but not by the presence of the exudates. & 2007 Elsevier GmbH. All rights reserved.

Introduction Plant-growth-promoting rhizobacteria of the genus Azospirillum have been widely used as models for studying the mechanisms of the associative plant–bacterial interactions (Steenhoudt and Vanderleyden, 2000). All Azospirillum species Corresponding author. Tel.: +7 8452 970403;

fax: +7 8452 970383. E-mail address: [email protected] (E.I. Katsy).

are motile because of the presence of a single polar flagellum (Fla), and several of them have a mixed type of flagellation. For example, in A. brasilense and A. lipoferum, Fla is produced in a liquid environment, and numerous lateral flagella (Laf) are induced in addition to Fla on viscous and solid media (Tarrand et al., 1978). Fla is responsible for swimming (Mot+ phenotype), whereas Laf are used for swarming (Swa+ phenotype) (Tarrand et al., 1978; Hall and Krieg, 1983). After being stabinoculated into media containing 0.2–0.5% Bacto

0944-5013/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2007.07.003

ARTICLE IN PRESS Changes in A. brasilense motility and the effect of wheat seedling exudates agar, wild-type A. brasilense spreads with the formation of ring structures (Moens et al., 1995; Scheludko et al., 1998). As in the closely related a-purple photosynthetic bacterium Rhodospirillum centenum (Jiang et al., 1998), not only Laf but also a functional polar flagellum seems to be required for A. brasilense swarming (Scheludko et al., 1998). Recently, spreading with the formation of microcolonies (Swa Gri+ phenotype) was revealed in A. brasilense strain Sp245. Under standard laboratory conditions, the culture of Sp245 consisted of Swa+ Gri (95–91% of the clones), Swa Gri (4.7–8% of the clones) and Swa Gri+ (0.3–1% of the clones) subpopulations (Shelud’ko and Katsy, 2001). As compared with the aerated cultures, precultivation of Sp245 in liquid media under nonaerated, anoxic conditions before sowing them into semisolid media slightly increased the frequencies of occurrence of the Swa Gri and Swa Gri+ clones. The Swa Gri and Swa Gri+ clones of Sp245 gave rise to Swa+ Gri descendants. In the non-swarming Omegon-Km mutants of Sp245, the quantity of clones with a stable Swa Gri+ phenotype approximated to 70–90%, and the rest were Swa Gri (Shelud’ko and Katsy, 2001). In all the mutants with the predominant Swa Gri+ phenotype, the polar flagellum was not produced or was paralysed. The diameter of the granular dispersal zones did not depend on the number of lateral flagella on the A. brasilense cells. For instance, the Fla , Fla Laf and Fla leaky Laf mutants of Sp245 formed Gri+ colonies of approximately the same diameter. No effects of the nutrients on the dispersal of A. brasilense cells in semiliquid media were revealed. For example, irrespective of the presence of NH4Cl, malate, leucine, proline, tryptophan, arabinose and rhamnose in semisolid media (0.2–0.6% agar), the nonswarming Omegon-Km mutants formed colonies with either the Swa Gri+ or the Swa Gri phenotype (Shelud’ko and Katsy, 2001). It was demonstrated that adsorption of the vital sulphonated azodye Congo Red confers on wildtype A. brasilense the ability to consistently spread in semiliquid agar with the formation of microcolonies. Rapidly swarming spontaneous derivatives of A. brasilense Sp245 and derivatives that swarmed in the presence of Congo Red were identified (Shelud’ko et al., 2006). Seed and root exudates are a major source of nutrients and signals for rhizobacteria (Lynch and Whipps, 1990; Zhu et al., 1997; Fan et al., 2001; Bacilio-Jime´nes et al., 2003). Tactic responses of Azospirillum and other plant-associated bacteria towards plant exudates are important for successful bacterial establishment on plant roots (Vande

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Broek et al., 1998; Bacilio-Jime´nes et al., 2003). On and inside the roots, the appearance of non-motile variants of A. lipoferum was much more frequent (40% of the clones) than it was outside the rhizosphere and in a laboratory culture (5% of the clones) (Alexandre et al., 1996). When colonizing plant roots, azospirilla frequently form microcolonies; however, the underlying mechanisms are not well understood (Burdman et al., 2000). The relationship between the Gri+ Azospirillum spreading under laboratory conditions and the rhizosphere microcolonies has not been investigated. The aim of this work was to identify A. brasilense Sp245 mutants that swarm faster than the parent strain or differ from it in the mode of spreading. We also intended to analyse whether the complex of substances exuded by the seedlings of wheat, an associative partner of A. brasilense, could affect bacterial swimming and swarming speeds and the phenotypic differences in bacterial behaviour in soft media (transitions between Swa+ Gri , Swa Gri and Swa Gri+ phenotypes).

Materials and methods Bacterial strains, plasmids and growth conditions The bacteria and plasmids used are listed in Table 1. The mutants of Sp245 were obtained by Katzy et al. (1998) by mating Sp245 with Escherichia coli S17-1, harbouring a vector for OmegonKm mutagenesis, pJFF350 (Fellay et al., 1989). Azospirilla were routinely grown in malate–salt medium (Do ¨bereiner and Day, 1976) (MSM; pH 7.0) at 30 1C. Semisolid media contained Bacto agar at 4 or 5 g l 1. Adsorption of Congo Red by the A. brasilense colonies was determined on a solid medium containing 37.5 mg ml 1 of the dye (Bastarrachea et al., 1988). Calcofluor white (Fluorescent Brightener 28, Aldrich) was added to tryptic soya agar medium (TSA, Serva) to a final concentration of 1 mg ml 1. When appropriate, kanamycin (Km) and NH4Cl were added to the media to final concentrations of 30 mg ml 1 and 0.5 g l 1, respectively. To examine the effect of wheat seedling exudates (SE) on bacterial spreading, we added 10 ml of SE (pH 5.9), preheated to 50 1C, to an equal volume of melted MSM containing 10 g l 1 of Bacto agar (SE+MSM; pH after mixing, 6.3). In control experiments, SE were replaced with plant medium (PM) (PM+MSM; pH after mixing, 6.2). Overnight liquid cultures of azospirilla (at equal OD540 0.1) were serially diluted with sterile MSM.

ARTICLE IN PRESS 580 Table 1.

I.V. Borisov et al. A. brasilense strains and plasmids used in the study

Strain or plasmid

Relevant characteristics

Source

Strain Sp245

Wild-type strain isolated from surface-sterilized wheat roots in Brazil

Baldani et al. (1983) Katzy et al. (1998) Katzy et al. (1998) This work

KM018 KM252 BK468 BK571 BK759.G BK759.P1–BK759.P5 Plasmid pJFF350 pOmegon-Kmlps348X pOmegon-Kmfla048X pEK051X

Mot Swa LpsII Cal mutant of Sp245 (p120::Omegon-Km) with paralysed Fla and Laf, KmR LpsI Cal mutant of Sp245 (p120::Omegon-Km) with wild-type flagellation, KmR Swa++ mutant of Sp245 with wild-type flagellation, KmR, chosen from our collection of the Omegon-Km mutants of Sp245 Katzy et al. (1998) Swa++ mutant of Sp245 with wild-type flagellation, KmR, chosen from our collection of the Omegon-Km mutants of Sp245 Katzy et al. (1998) Gri+ Fla mutant of Sp245 with inducible lateral flagella, KmR, chosen from our collection of the Omegon-Km mutants of Sp245 Katzy et al. (1998) Five independent Swa++ derivatives of BK759.G with wild-type flagellation, KmR Omegon-Km (3.8 kb) with ori from pBR322, Mob+, KmR, 5.3 kb XhoI fragment of p120, carrying Omegon-Km (3.8 kb) and the lps/cal loci from A. brasilense KM348 (LpsI mutant of Sp245), 18.8 kb, KmR. A 15-kb BamHI fragment of this plasmid was used in DNA hybridizations XhoI fragment of p120, carrying Omegon-Km and the fla/swa locus from A. brasilense SK048 (Fla Swa mutant of Sp245), 12 kb, KmR. A 8.3-kb BamHI fragment of this plasmid was used in DNA hybridizations XhoI fragment of the cointegrate p85::pJFF350 from A. brasilense SK051 (Fla Laf mutant of Sp245), 23 kb, KmR. A 2.4-kb EcoRI fragment of this cointegrate was used in DNA hybridizations

One millilitre of the dilutions was mixed with 20 ml of melted (50 1C) SE+MSM or PM+MSM, and the mixture was poured into Petri dishes (90 mm in diameter). The media used for testing A. brasilense growth and speeds of swimming were liquid SE+MSM, PM+MSM and MSM (with or without added NH4Cl). Bacterial motility was evaluated by using phasecontrast microscopy of liquid cultures and by measuring the diameter of spreading zones formed after inoculation of bacteria into the semisolid media. The motility of 50–100 individual bacteria in a drop of liquid was observed with a Jenaval phasecontrast microscope (Carl Zeiss, Jena, Germany) and was video-recorded with a Sony DCR-TRV900E digital camera (Sony, Tokyo, Japan). Bacterial swimming speeds were determined by analysing the video-recorded data with a computer program designed by V.A. Krestinenko (this institute), as described in detail earlier (Shelud’ko et al., 2006). All experiments were done at least five times, each run in triplicate. The confidence intervals were determined for a 95% significance level. Statistical analyses were done with the program Microsoft Office Excel 2003 (11.6355.6360) SP1.

This work This work This work

Fellay et al. (1989) Katzy et al. (1998) Katzy and Shelud’ko (1999) Katzy et al. (2001)

Transmission electron microscopy Transmission electron microscopy was used to study bacterial flagellation. Aliquots (20 ml) taken from an 18-h-old liquid bacterial culture or from a suspension of 48-h-old cells washed off the solid MSM were applied onto a Formvar-coated grid. The specimens thus prepared were kept for 20 min, dried on a sheet of filter paper (Whatman no. 1), washed with distilled water and dried again. The specimens were then contrasted with a 2% solution of uranyl acetate for 1–5 min and were examined under a Tesla BS-500 electron microscope (Czech Republic) at an accelerating voltage of 50 kV.

DNA techniques Plasmids were visualized by the method of Eckhardt (1978). Extraction and purification of plasmid and total bacterial DNA, restriction endonuclease digestion and electroelution of DNA fragments from agarose gels were performed as described in Sambrook et al. (1989). Labelling of DNA fragments with peroxidase and Southern

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hybridization using an enhanced chemiluminescence (ECL) gene detection system were performed as recommended by the manufacturer (Amersham).

Sterilization, germination and cultivation of wheat seeds Soft red spring wheat (Triticum aestivum L. var. lutescens) seeds, cv. Saratovskaya 29, were obtained from the Agricultural Research Institute for the South-East Region, Russian Academy of Agricultural Sciences, Saratov, Russia. Seeds were washed in 0.5% SDS, rinsed with tap water and surface sterilized by soaking for 3 min in 70% ethanol, rinsing with sterile distilled water, and immersing for 3 min in a solution of ethanol mercuric chloride (330 mg l 1) and cetylpyridinium chloride (660 mg l 1). The seeds were then thoroughly rinsed several times with sterile distilled water and were germinated in the dark on nutrientagar plates at room temperature. After 3 days, the sterile seedlings were transferred individually to 25  195 mm glass tubes standing in a support of stainless steel and containing 10 ml of liquid sterile PM per seedling. Each seed was placed on a 200-ml plastic pipette tip floating in the medium. The growth medium was collected after 7 days from the tubes without microbial contamination and was used as SE. PM contained the following (in g l 1): KH2PO4
3H2O, 4; MgSO4
7H2O, 0.09; CaCl2
H2O, 0.02; ZnSO4
7H2O, 0.015; KJ, 0.008; Na2MoO4
2H2O, 0.003; H3BO3, 0.002; and CuSO4
5H2O, 0.0003. Fifteen millilitres of a stock solution containing FeSO4
7H20 (2 g l 1) and nitrilotriacetic acid (NTA; 5.6 g l 1) was added to 1 l (final volume) of PM. The pH of the medium was adjusted to 6.0 with 2 N NaOH.

Results Motility behaviour and flagellation of A. brasilense Sp245 mutants After the matings between E. coli S17-1 (pJFF350) (Fellay et al., 1989) and A. brasilense Sp245, we obtained a collection of 879 KmR exconjugants of Sp245 (Katzy et al., 1998). Several non-swarming Omegon-Km Sp245 mutants with various defects in Fla and/or Laf production or rotation were identified and characterized (Scheludko et al., 1998; Katzy et al., 1998). In this study, in order to identify A. brasilense Sp245 mutants that swarm faster than the parent

Figure 1. Swa+ phenotype of A. brasilense Sp245 (a), Gri+ phenotype of A. brasilense BK759.G and the appearance of its superswarming derivative (b) after stab inoculation and 72-h incubation on malate–salt medium containing 0.4% Bacto agar. Bars represent 1 cm.

strain or differ from it in the mode of spreading, KmR mutants of this strain were taken at random and were inoculated into semisolid media. Three mutants of Sp245 were found to swarm faster than the parent strain, and one mutant formed granular spreading zones in semisolid media. During the first 48 h of incubation after stab inoculation of the bacteria into the soft media, one of the mutants, BK759.G, formed granular (Gri+) macrocolonies. Within the next 24 h or so, one or two swarming zones (‘‘protuberances’’) usually appeared in the granular spreading zones formed from the compact microcolonies of BK759.G (Figure 1). All our attempts to obtain a culture of BK759.G with a uniform spreading phenotype were unsuccessful: on prolonged incubation (X3 days) in the semisolid media, BK759.G produced macrocolonies with a mixed phenotype. Preincubation of BK579.G cells on the solid and in the liquid media before their transfer to soft agar did not change the behavioural phenotype of this strain. Stab inoculation of several hundred colonies from solid to soft media and mixing of serial dilutions of the liquid cultures with aliquots of the soft medium before plating gave the above-described picture of the time-dependent transition of BK759.G from microcolonial spreading to swarming. The more prolonged the bacterial incubation on soft agar, the more the BK579.G colonies switched from Gri+ to swarming phenotype. The swarming behaviour of five randomly chosen derivatives of BK759.G (designated BK759.P1–BK759.P5) was stable during subsequent subculturing and testing. We never observed reversion of BK759.P1–BK759.P5 to microcolonial spreading. On the semisolid media, the rates of expansion of A. brasilense BK468, BK571, BK759.P1, BK759.P2, BK759.P3, BK759.P4 and BK759.P5 were much higher (Swa++ phenotype) than that of Sp245 (Swa+ phenotype) (Table 2). However, the

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Table 2. Swarming of A. brasilense Sp245 and its mutants on semisolid (0.5% Bacto agar) media with (SE+MSM) or without (PM+MSM) wheat seedling exudates A. brasilense strain Diameter of the swarms (mm)a formed in 72 h on

Sp245 BK468 BK571 BK759.G BK759.P1 KM252

PM+MSM

SE+MSM

3.770.4 28.470.5 28.770.5 4.070.1 24.771.8 6.270.4

8.870.5 30.070.4 30.070.5 4.370.1 26.871.5 7.870.2

PM+MSM, plant medium+malate–salt medium (1:1, vol/vol); SE+MSM, wheat seedling exudates+malate–salt medium (1:1, vol/vol). a Statistical results of four to seven independent experiments; confidence intervals at the 95% significance level.

swimming speeds of the Sp245 Swa++ derivatives grown in liquid PM+MSM and in liquid MSM (32.972.6 and 31.972.3 mm s 1 in BK468; 32.67 2.7 and 29.272.5 mm s 1 in BK571; and 29.672.1 and 34.372.3 mm s 1 in BK759.P1–P5) did not differ significantly from those of the wild-type strain (29.572.0 and 31.972.3 mm s 1, respectively). Tracing individual tracks of the free-swimming Sp245 and mutant cells did not reveal evident differences in the frequencies of the reversals in their swimming directions. Transmission electron microscopy of the A. brasilense cells revealed that BK759.P1, BK468 and BK571 retained wild-type flagellation and possessed a single Fla in the liquid media and Fla and numerous Laf of normal morphology on the solid media. Cells of BK759.G, immotile in the liquid media, were devoid of Fla but produced Laf of normal morphology on the solid media (Figure 2). (The flagellation of BK759.P2–P5 was not analysed.) To analyse whether alterations in cell-surface polysaccharides affect motility of A. brasilense and its response to wheat SE, we also used in this study two previously characterized mutants, KM018 and KM252 (Katzy et al., 1998). The behaviour of A. brasilense KM252 (an lpsI cal mutant with wildtype flagellation) in the liquid media did not differ significantly from that of Sp245. On the soft media, this mutant spread significantly faster than did Sp245 but significantly slower than did BK468, BK571 and BK759.P1 (Table 2). The lpsII cal mot swa mutant KM018 retained wild-type flagellation; however, it was immotile in liquid media and did not spread on the soft media. Less than 1% of the KM018 cells retained swimming and swarming motility. A molecular reason for the pleiotropic

Figure 2. Cell morphology of A. brasilense (a, b) Sp245, (c, d) BK571, (e, f) BK759.G and (g, h) BK759.P1. Bacteria were grown in liquid malate–salt medium (MSM) for 18 h (a, c, e, g) or on solid MSM for 42 h (b, d, f, h). The arrows point at the polar flagellum. The specimens for transmission electron microscopy were prepared as described in the Materials and methods section. Bars represent 1 mm.

lpsII cal mot swa mutation in KM018 was not characterized (Katzy et al., 1998). It was accidentally noticed that in A. brasilense Sp245 and in BK759.P1, a superswarming derivative of BK759.G (the other derivatives were not studied), the diameter of the spreading zones formed on soft MSM (data not shown) and on MSM+PM (Table 3) increased with decreasing number of bacterial colonies per Petri dish. Yet, at the bacterial concentrations used, the diameters of

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Table 3. Effect of wheat seedling exudates on the motility phenotypes of A. brasilense Sp245 and its mutant derivatives incubated on soft (0.5% agar) media during 72 h A. brasilense strain CFU per plate, motility variants (% of the total CFU number) and mean diameter (mm, in parentheses) of the swarmsa formed on PM+MSM

SE+MSM

CFU

Phenotype

CFU

Phenotype

Sp245

150 12

100% Swa+ (3) 100% Swa+ (4)

180 48

100% Swa+ (6) 100% Swa+ (8)

BK468

100 12

100% Swa++ (28) 100% Swa++ (28)

78 20

100% Swa++ (29) 100% Swa++ (30)

BK571

110 30

100% Swa++ (28) 100% Swa++ (29)

100 34

100% Swa++(28) 100% Swa++ (30)

BK759.G

85 10

75% Swa+ (4); 25% Gri+ 80% Swa+ (4); 20% Gri+

86 7

89% Swa+ (4); 11% Gri+ 85% Swa+ (6); 15% Gri+

BK759.P1

160 33

100% Swa++ (12) 100% Swa++ (29)

180 36

100% Swa++ (12) 100% Swa++ (29)

KM018

108 14

1% Swa+ (2); 99% Swa 100% Swa

112 18

2.7% Swa+ (2); 97.3% Swa 100% Swa

KM252

100 10

100% Swa+ (5) 100% Swa+ (6)

110 9

100% Swa+ (7) 100% Swa+ (8)

CFU, colony-forming units per 90-mm Petri dish; PM+MSM, plant medium+malate–salt medium (1:1, vol/vol); SE+MSM, wheat seedling exudates+malate–salt medium (1:1, vol/vol). a Representative results of one experiment.

macrocolonies formed by the mutants with LpsI Cal , Gri+ and Swa++ phenotypes seemed independent of microbial density (Table 3).

Cell-surface characteristics of A. brasilense Sp245 mutants The cell-surface polymers of A. brasilense are capable of adsorbing the vital dye Congo Red (Bastarrachea et al., 1988). Unlike A. brasilense Sp245, BK468 and BK571, which formed red colonies on solid MSM containing Congo Red, the colonies of BK759.G, BK759.P1, BK759.P2, BK759.P3, BK759.P4, BK759.P5, KM018 and KM252 were pale pink. Azospirillum produces some uncharacterized surface polysaccharides detectable by binding a specific fluorescent dye, Calcofluor white (Cal+ phenotype) (Del Gallo et al., 1989). On TSA with Calcofluor white, the colonies of Sp245 and its superswarming mutants had a Cal+ phenotype; KM018 and KM252 were Cal . As determined by our colleagues L.Yu. Matora and G.L. Burygin (this institute), BK468, BK571, BK759.G and BK759.P1 retained the wild-type LPS antigenic structure (personal communication).

Comparative analysis of plasmid and total DNAs of mutants of A. brasilense Sp245 using pJFF350 DNA as a probe In A. brasilense Sp245, plasmids with molecular weights of 85 (p85), 120 (p120) and more than 300 MDa could be revealed by the Eckhardt (1978) procedure (Figure 3). In mutants KM018 and KM252, which have the same plasmid profile as Sp245, single Omegon-Km insertions were localized in different loci of p120 (Katzy et al., 1998). Plasmid profiles of Sp245 and mutants BK468, BK571 and BK759.G were found to be similar (Figure 3). An 36-MDa plasmid was revealed instead of p85 in the superswarming derivatives of BK759.G (e.g., Figure 3). To localize pJFF350 in BK468, BK571 and BK759.G, their plasmids were separated by the method of Eckhardt (1978), Southern blotted and ECL hybridized to peroxidase-labelled pJFF350 DNA. In BK571 and BK759.G, hybridization signals were detected at the migration distance of p85 (data not shown). To determine whether insertion sites were identical or different in the mutants, we also compared the numbers and sizes of the restriction

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Figure 3. Plasmid patterns of the A. brasilense strains Sp245 (1), BK468 (2), BK571 (3), BK759.G (4) and BK759.P1 (5). The molecular weights of plasmid DNA are indicated on the right.

I.V. Borisov et al. insertions, the mutants of Sp245 should reveal only a 3.8-kb BamHI or a XhoI hybridizing band. In BK468, BK571, BK759.G and BK759.P1, the unique XhoI DNA fragments of different sizes hybridized to pJFF350 (Figure 4A). However, in the BamHI digests of the BK468, BK571, BK759.G and BK759.P1 DNAs, several positive fragments were revealed (Figure 4B). We conclude that single positive XhoI fragments of the mutant DNAs harboured not a single copy of Omegon-Km but an integrated vector molecule pJFF350. In the case of BK759.G, a cointegrate of p85 and pJFF350 seemed to be unstable and subjected to further rearrangements during prolonged incubation of the bacteria in semisolid media.

Using the 2.4-kb fragment of p85 for physical analysis of DNAs of A. brasilense Sp245, BK468, BK571, BK759.G and BK759.P1

Figure 4. Identification of the XhoI (panel A) and BamHI (panel B) restriction fragments of the total A. brasilense DNAs that hybridize with pJFF350. Lanes: (1) pJFF350; (2) Sp245; (3) BK468; (4) BK571; (5) BK759.P1; (6) BK759.G. l EcoRI restriction fragments (21.2, 7.4, 5.8, 5.6, 4.9 and 3.5 kb) (lane 7) and l HindIII restriction fragments (23.1, 9.4, 6.7, 4.4, 2.3 and 2.0 kb) (lane 8) were used as molecular size markers. DNA restriction fragments were subjected to electrophoresis on 0.7% agarose gel, Southern blotted to Hybond-N+ membrane and ECL hybridized with pJFF350.

fragments of the total DNAs of BK468, BK571, BK759.G and BK759.P1 hybridizing to the peroxidase-labelled DNAs of pJFF350 (Figure 4). In pJFF350, there are two BamHI sites at the ends of Omegon-Km (3.8 kb) and no XhoI sites (Fellay et al., 1989). Therefore, in the case of single Omegon-Km

To further characterize alterations of p85 in the mutants, hybridization of the A. brasilense total DNAs was also carried out with the 2.4-kb EcoRI fragment of p85 (Table 1) (Figure 5C). A. brasilense Sp245 total DNA was shown to contain two homologous EcoRI fragments (2.7 and 2.4 kb) that hybridized to this probe (Katsy et al., 2002). In this work, we found a single positive 15-kb BamHI fragment in the total DNAs of Sp245, BK468, BK571 and BK759.G and an additional positive 25-kb BamHI fragment in the DNA of BK759.G. In BK759.P1, the size of the single positive BamHI DNA fragment hybridizing to the 2.4-kb fragment of p85 slightly exceeded 15 kb (data not shown). In the XhoI digests of the total DNAs of Sp245, BK468 and BK571, two fragments of equal lengths gave positive signals in the hybridizations with the 2.4kb EcoRI fragment of p85 (Figure 5C). The DNAs of A. brasilense BK759.G and BK759.P1 demonstrated other patterns of hybridization with this fragment (Figure 5C).

Use of two p120 fragments to study possible DNA rearrangements in the A. brasilense mutants BK468, BK571, BK759.G and BK759.P1 In an attempt to reveal whether differences in the spreading characteristics of A. brasilense Sp245 and the mutant strains could also be attributed to the rearrangements in p120, we used two peroxidase-labelled fragments (15 and 8.3 kb) of p120 (Table 1) in Southern hybridizations (Figure 5A and B). As shown in Figure 5A and B, Sp245 and all the mutants except BK759.P1 had identical DNA hybri-

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Figure 5. Identification of the XhoI restriction fragments of the total A. brasilense DNAs hybridizing with the p120 fragments from pOmegon-Km-lps348X (panel A) and pOmegon-Km-fla048X (panel B) and with the 2.4-kb EcoRI fragment of p85 from pEK051X (panel C). Lanes: (1) Sp245; (2) BK468; (3) BK571; (4) BK759.P1; (5) BK759.G; (6) marker l HindIII restriction fragments (23.1, 9.4, 6.7 and 4.4 kb).

dization patterns with both probes. In BK759.P1, the size of the single positive XhoI fragment was slightly greater than that in the other strains (Figure 5A and B).

Effects of wheat seedling exudates on the behaviour of A. brasilense Sp245 and its mutants We asked whether differences in the behaviour of A. brasilense Sp245 and its derivatives could be affected by the complex of substances exuded by the seedlings of wheat, a well-known associative partner of azospirilla. After 48 h of incubation, we detected 81% Swa+, 18.3% Swa and 0.7% Gri+ Sp245 colonies in PM+MSM and 100% Swa+ colonies in SE+MSM. During prolonged incubation (X72 h), these differences disappeared and 100% colonies of Sp245 swarmed in both media (Table 3). The mean diameter of the swarming macrocolonies of Sp245 in SE+MSM was twice as large as that in PM+MSM. The increase in the swarming rate of KM252 was less than that observed for Sp245 (Tables 2 and 3). The number of the swarming colonies of A. brasilense KM018 and BK759.G increased in the presence of wheat SE. The initially granular colonies of BK759.G frequently became Swa+. This switch was more evident when the incubation was prolonged (X72 h) and microbial density per Petri dish approximated to 100 colony-forming units than when microbial density was 10-fold smaller. The mean diameter of the macrocolonies formed by A. brasilense BK468, BK571, BK759.G and

BK759.P1, and the reverse dependence of the diameter of the BK759.P1 and (to some extent) Sp245 swarms on the number of their colonies per Petri dish were unaffected by the exudates. The mutants and Sp245 grew at the same rate as did the wild type in shake cultures in liquid MSM with NH4Cl (0.5 g l 1) (data not shown). In liquid PM+MSM and SE+MSM, under conditions of nitrogen (with NH4Cl omitted) and oxygen (without mixing) limitation similar to those in the soft media, A. brasilense Sp245 and the mutants grew poorly (OD540 0.1 after 18 h of incubation). After overnight growth, PM+MSM was equally alkalinized by all the strains (the pH changed from 6.2 to 6.6); the pH of SE+MSM shifted from 6.3 to 6.4 in Sp245 and to 6.5 in the other cultures. The consequences (if any) of the slight differences in pH between the two media and between wild-type and mutant cultures in the same medium remain unclear. At least the swimming speeds of Sp245 and its superswarming derivatives did not differ significantly and were unaffected by the presence of wheat SE (data not shown).

Discussion The purposes of this work were to identify A. brasilense Sp245 mutants that swarm faster than the parent strain or differ from it in the mode of spreading and to analyse whether substances exuded by the seedlings of wheat, an associative partner of A. brasilense, could affect bacterial behaviour in soft media.

ARTICLE IN PRESS 586 Three mutants of A. brasilense Sp245 – BK468, BK571 and KM252 – were found to swarm faster than the parent strain. Those mutants retained the flagellation of the wild type and did not differ from Sp245 in the swimming rates. The vector plasmid pJFF350, used for Omegon-Km mutagenesis, integrated in the DNA of BK468 and BK571. In BK571, pJFF350 formed a cointegrate with the resident 85-MDa plasmid. In KM252, the single insertion of Omegon-Km was previously localized in the 120-MDa plasmid (Katzy et al., 1998). Another A. brasilense Sp245 mutant, the Fla Gri+ strain BK759.G, spread in semisolid media with the formation of microcolonies and gave rise to stable Fla+ derivatives (BK759.P) with a Swa++ phenotype, which was affected by the local density of the colonies. We believe that the Fla Gri+-Fla+ Swa++ transition has now been described for the first time, at least for Azospirillum. In this mutant, pJFF350 also integrated in p85. During prolonged incubation of BK759.G in semisolid media, the p85–pJFF350 cointegrate molecule was subject to further rearrangement causing a large deletion in p85. Interestingly, the slight increases in the lengths of single XhoI fragments of the total DNAs hybridizing to two p120-specific probes were also revealed in BK759.P1. Those probes carried p120 loci involved in the production of the polar flagellum, lipopolysaccharides and calcofluor-binding polysaccharides in A. brasilense Sp245. Coordinated bacterial spreading depends not only on the function of the motility apparatus but also on the intercellular interactions. It is known that alterations in bacterial motility can be caused by changes in the cell-surface polymers (Harshey, 2003). In this study, we have not found a strict correlation between the intensity of adsorption of the specific dyes (Calcofluor white and Congo Red) by A. brasilense grown on the solid media and the bacterial motility phenotypes. On the soft media, A. brasilense wild-type strain Sp245 displayed a strong tendency towards swarming, fully realized on prolonged incubation. In the presence of wheat SE, the swarming rate of Sp245 was significantly higher than in the control medium without the exudates. The potential for further motility stimulation seemed exhausted in the superswarming A. brasilense mutants BK468, BK571 and BK759.P. The causes for the more or less notable increase in the number of swarming colonies of BK759.G and KM018 growing in the presence of wheat SE are not clear. For BK759.G, it can be supposed, for example, that in the soft media (especially in the presence of the exudates) bacterial metabolism is changed in such a way as to promote a genetic

I.V. Borisov et al. rearrangement of the mutant DNA, leading to the restoration of polar-flagellum production and the swarming potential. It has not yet been established which components of wheat SE influence the spreading phenotypes of A. brasilense Sp245 on soft media. Some plants are shown to be capable of producing substances that mimic bacterial extracellular regulators (Teplitski et al., 2000). Hypothetical regulators of bacterial social behaviour could be present in wheat SE as well and could stimulate swarming in A. brasilense Sp245 through an unknown mechanism. Yet another possibility is that the cell-surface polymers of Sp245 were modified in soft SE+MSM in a way beneficial for bacterial swarming. For example, under certain conditions in the presence of plant exudates, the A. brasilense exopolysaccharides (EPS) and LPS could be altered (Fischer et al., 2003). Since bacterial EPS and LPS are important for swarming, probably acting as wetting agents (Toguchi et al., 2000; Harshey, 2003), their modification by mutation (as in KM252) or by environmental factors (e.g., wheat seedling exudation) could be the cause for the accelerated swarming rate of A. brasilense. Whatever the mechanism for the observed positive effect of wheat SE on the transition of A. brasilense to (accelerated) swarming, this point deserves further investigation. An understanding of the genetic and biochemical causes for the different motility responses to colonial density and to wheat SE shown by A. brasilense wild-type strain Sp245 and by its superswarming mutants BK759.P and BK468/BK571 would help to gain an insight into the intercellular and plant–bacterial communication networks.

Acknowledgements This work was supported by the President of the Russian Federation (Grants NSh-6177.2006.4, MK235.2003.04 and MK-948.2005.4) and by the Russian Foundation for Basic Research (Grant 06-04-48204a). We thank Drs. L.Yu. Matora and G.L. Burygin for the comparative immunochemical analyses of LPS in the A. brasilense wild-type and mutant strains and Mr. D.N. Tychinin for correcting our English.

References Alexandre G, Jacoud C, Faure D, Bally R. Population dynamics of a motile and non-motile Azospirillum lipoferum strain during rice colonization and motility variation in the rhizosphere. FEMS Microbiol Ecol 1996;19:271–8.

ARTICLE IN PRESS Changes in A. brasilense motility and the effect of wheat seedling exudates Bacilio-Jime´nes M, Aguilar-Flores S, Ventura-Zapata E, Pe ´rez-Campos E, Bouquelet S, Zenteno E. Chemical characterization of root exudates from rice (Oryza sativa) and their effects on the chemotactic response of endophytic bacteria. Plant Soil 2003;249:271–7. Baldani VLD, Baldani JI, Do ¨bereiner J. Effects of Azospirillum inoculation on root infection and nitrogen incorporation in wheat. Can J Microbiol 1983; 29:924–9. Bastarrachea F, Zamudio M, Rivas R. Non-encapsulated mutants of Azospirillum brasilense and Azospirillum lipoferum. Can J Microbiol 1988;34:24–9. Burdman S, Okon Y, Yurkevitch E. Surface characteristics of Azospirillum brasilense in relation to cell aggregation and attachment to plant roots. Crit Rev Microbiol 2000;26:91–110. Del Gallo M, Negi M, Neyra CA. Calcofluor and lectinbinding exocellular polysaccharides of Azospirillum brasilense and Azospirillum lipoferum. J Bacteriol 1989;171:3504–10. Do ¨bereiner J, Day JM. Associative symbiosis in tropical grasses: characterization of microorganisms and dinitrogen fixing sites. In: Newton WE, Nijmans CJ, editors. Symposium on nitrogen fixation, vol. 2. Pullman, WA: Washington State University Press; 1976. p. 518–38. Eckhardt T. A rapid method for the identification of plasmid deoxyribonucleic acid in bacteria. Plasmid 1978;1:584–8. Fan TWM, Lane AN, Shenker M, Bartley JP, Crowley D, Higashi RM. Comprehensive chemical profiling of gramineous plant root exudates using high-resolution NMR and MS. Phytochemistry 2001;57:209–21. Fellay R, Krisch HM, Prentki P, Frey J. Omegon-Km: a transposable element designed for in vivo insertional mutagenesis and cloning of genes in Gram-negative bacteria. Gene 1989;76:215–26. Fischer SE, Miguel MJ, Mori GB. Effect of root exudates on the exopolysaccharide composition and the lipopolysaccharide profile of Azospirillum brasilense Cd under saline stress. FEMS Microbiol Lett 2003;219:53–62. Hall PG, Krieg NR. Swarming of Azospirillum brasilense on solid media. Can J Microbiol 1983;29:1592–4. Harshey RM. Bacterial motility on a surface: many ways to a common goal. Annu Rev Microbiol 2003;57: 249–73. Jiang Z-Y, Rushing BG, Bai Y, Bauer C. Isolation of Rhodospirillum centenum mutants defective in phototactic colony motility by transposon mutagenesis. J Bacteriol 1998;180:1248–55. Katzy EI, Shelud’ko AV. Mapping of the fla locus in a plasmid with molecular weight of 120 MDa in Azospirillum brasilense Sp245. Russ J Genet 1999;35: 1177–82. Katzy EI, Matora LYu, Serebrennikova OB, Scheludko AV. Involvement of a 120-MDa plasmid of Azospirillum brasilense Sp245 in the production of lipopolysaccharides. Plasmid 1998;40:73–83.

587

Katzy EI, Borisov IV, Scheludko AV. Effect of the integration of vector pJFF350 into plasmid 85-MDa of Azospirillum brasilense Sp245 on bacterial flagellation and motility. Russ J Genet 2001;37:129–34. Katsy EI, Borisov IV, Petrova LP, Matora LYu. The use of fragments of the 85- and 120-MDa plasmids of Azospirillum brasilense Sp245 to study the plasmid rearrangement in this bacterium and to search for homologous sequences in plasmids of Azospirillum brasilense Sp7. Russ J Genet 2002;38:124–31. Lynch JM, Whipps JM. Substrate flow in the rhizosphere. Plant Soil 1990;129:1–10. Moens S, Michiels K, Keijers V, van Leuven F, Vanderleyden J. Cloning, sequencing, and phenotypic analysis of laf1, encoding the flagellin of the lateral flagella of Azospirillum brasilense Sp7. J Bacteriol 1995;177: 5419–26. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989. Shelud’ko AV, Katsy EI. Formation of polar bundles of pili and the behavior of Azospirillum brasilense cells in a semiliquid agar. Microbiology 2001;70:570–5. Scheludko AV, Katsy EI, Ostudin NA, Gringaus OK, Panasenko VI. Novel classes of Azospirillum brasilense mutants with defects in the assembly and functioning of polar and lateral flagella. Mol Genet Mikrobiol Virusol 1998;4:33–7. Shelud’ko AV, Borisov IV, Krestinenko VA, Panasenko VI, Katsy EI. Effect of Congo red on the motility of the bacterium Azospirillum brasilense. Microbiology 2006;75:48–54. Steenhoudt O, Vanderleyden J. Azospirillum, a freeliving nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiol Rev 2000;24:487–506. Tarrand JX, Krieg NE, Do ¨bereiner J. A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov. and two species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Can J Microbiol 1978; 24:967–80. Teplitski M, Robinson JB, Bauer WD. Plants secrete substances that mimic bacterial N-acyl homoserine lactone signal activities and affect population densitydependent behaviors in associated bacteria. Mol Plant Microbe Interact 2000;13:637–48. Toguchi A, Siano M, Burkart M, Harshey RM. Genetics of swarming motility in Salmonella enterica serovar Typhimurium: critical role for lipopolysaccharide. J Bacteriol 2000;182:6308–21. Vande Broek A, Lambrecht M, Vanderleyden J. Bacterial chemotactic motility is important for the initiation of wheat root colonization by Azospirillum brasilense. Microbiology 1998;144:2599–606. Zhu Y, Pierson LS, Hawes MC. Induction of microbial genes for pathogenesis and symbiosis by chemicals from root border cells. Plant Physiol 1997;115:1691–8.