Vol. 56, No. 4

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1990, p. 990-996

0099-2240/90/040990-07$02.00/0 Copyright © 1990, American Society for Microbiology

Isolation of Behavioral Mutants of Azospirillum brasilense by Using Tn5 lacZ PIETERNEL VAN RHIJN, MARC VANSTOCKEM, JOS VANDERLEYDEN,* AND RENE DE MOT

F. A. Janssens Memorial Laboratory of Genetics, University of Leuven, B-3030 Heverlee, Belgium Received 14 June 1989/Accepted 18 December 1989

TnS lacZ mutants were generated with Azospirillum brasilense 7030 by mating that strain with Escherichia coli strains carrying suicide plasmid pCIB100 or pCIB110. Kanamycin-resistant Azospirillum colonies were obtained with a maximum frequency of 10-6 per recipient cell. The potential of Tn5 lacZ for random transposon mutagenesis coupled to transcription analysis in A. brasilense 7030 was demonstrated. Sixty percent of all Kmr A. brasilense 7030 mutants expressed ,8-galactosidase activity. Mutants affected in motility (Fla-) and general chemotaxis (Che-) were identified. Chromosomal insertions of TnS lacZ are involved, except for two Che- mutants. The latter che loci reside on a 90-megadalton plasmid. Expression of an acidic protein (Mr, 110,000) was abolished in these mutants.

coli donor and helper cells were grown overnight at 37°C, diluted 100-fold, and grown for another 5 h. Samples of donor, helper, and acceptor cells (1:1:2 ratio) were pooled, washed, and suspended in 10 mM MgSO4. Mating mixtures were spread on YEP agar (supplemented with 10 mM MgSO4) and incubated overnight at 30°C in a humid atmosphere. They were scraped off with a sterile loop, washed twice in 10 mM MgSO4, and spread on selective plates in an appropriate dilution. DNA isolation and manipulation. Single colonies of E. coli and A. brasilense were screened for the presence of plasmid DNA by the rapid detection method of Kado and Liu (26). Azospirillum total DNA was prepared by a modification of the method of Marmur (33) by using 2-ml cultures. Plasmid DNA from E. coli was prepared with the cleared-lysate procedure (10) and purified by centrifugation in a CsClethidium bromide gradient. Restriction endonucleases (Boehringer Mannheim Biochemicals) were used as recommended by the manufacturer. DNA hybridizations. DNA hybridizations were conducted overnight on nylon membranes (Hybond-N; Amersham Corp.) as described by Silhavy et al. (48). [a-32P]dCTPlabeled probes (specific activity, >5 x 107 cpm/,ug of DNA) were obtained by using the nick translation kit of Amersham. Blots were autoradiographed at -80°C by using Fuji RX films and intensifying screens (Kyokko Special). Detection of transcriptional fusions. Transcription of lacZ in TnS lacZ-induced mutants of A. brasilense 7030 was detected by transferring Kmr exconjugants to MMAB plates supplemented with kanamycin and 5-bromo-4-chloro-3-indolyl-3-D-galactopyranoside (40 Fg/ml). The quantitative assay for 3-galactosidase activity was performed as described by Miller (36), with cells grown to an optical density of 0.5 (600 nm). The microdilution plate assay for ,3-galactosidase activity has been described by Olson et al. (38). Identification of mutants affected in motility or chemotaxis. The Kmr exconjugants were screened for mutants impaired in motility and chemotaxis toward L-malate by scoring MMAB swarm plates (0.4% agar) incubated for 48 h at 30°C. Motility was examined by light microscopy. The presence of flagella was verified by transmission electron microscopy

The behavioral process of chemotaxis allows bacteria to move to higher concentrations of attractants or down gradients of repellants and has a considerable impact on microbial ecology (14). Major progress toward elucidation of the phenomenon at the level of molecular biology and genetics has been made by focusing on a few species, namely, Escherichia coli, Salmonella typhimurium, and Bacillus subtilis (30, 39). The involvement of chemotaxis in the interaction of bacteria with plants has been investigated mainly for legume-nodulating rhizobia (9, 12, 13, 16, 25, 42), tumorinducing Agrobacterium tumefaciens (4, 29, 41), fluorescent pseudomonads (46, 47), and Azospirillum species (5, 6, 22, 32, 37, 45). Azospirilla are diazotrophs closely associated with roots of various grasses, where they can exert a beneficial effect on plant growth under certain environmental conditions (18). There are indications that chemotaxis to root exudates is involved in the attraction of Azospirillum species to the rhizosphere, mediating a certain degree of host specificity (22, 45). Tn5 has previously been used successfully to mutagenize A. brasilense (1, 54). Here we report on the use of Tn5 lacZ for isolation and characterization of A. brasilense mutants with impaired motility and chemotaxis. MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. Media and growth conditions. Escherichia coli strains were maintained on LB agar and grown in LB broth supplemented with the appropriate antibiotics. The concentrations of antibiotics used for E. coli were 100 ,ug of ampicillin per ml and 25 ,ug of kanamycin per ml. Azospirillum strains were maintained on nutrient agar and grown in YEP broth or minimal medium (MMAB) at 30°C (54). Tn5 lacZ-induced mutants were selected on MMAB agar supplemented with 25 ,ug of kanamycin per ml. Bacterial matings. Azospirillum strains were grown overnight at 30°C in YEP broth to a density of 108 CFU/ml. E. *

Corresponding author. 990

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TABLE 1. Bacterial strains and plasmids used Strain or plasmid

Strains E. coli HB101 A. brasilense Sp7 A. brasilense 7030

Plasmids pCIB100 pCIB110 ColEl: :TnS ColE1::Tn5 lac pSUP2021 pSUP5011 pRK2013

Relevant properties

F- hsd2O (rB- mB-) recAJ3 ara-14 proA2 lacYl galK2 rpsl-20 (Smr) xyl-5 mtl-L supE44 XApr Apr Smr; chemical mutant of Sp7; lacks 115-MDa plasmid

ColEl replicon; Kmr; TnS lacZ Mob donor pCIB100; unique Sall site removed ColEl ColEl ColEl ColEl

replicon; replicon; replicon; replicon;

Kmr; Tn5 lacZ donor Apr Cmr Tcr Kmr; Tn5 donor; Tra- Mob' Apr Cmr Kmr Tn5 Mob donor; Tra- Mob' Kmr Tra+; helper plasmid

of negatively stained cells (31). Chemotactic responses to a wide range of organic acids, carbohydrates, and amino acids were evaluated as described by Adler (2). Genetic complementation. A cosmid library of the 90megadalton (MDa) plasmid from A. brasilense 7030 in pLAFR3 contained in E. coli HB101 was used in triparental matings with HB1O1(pRK2013) as a helper strain. Tcr exconjugants were screened for restoration of spreading ability on MMAB (0.4% agar). EcoRI-digested plasmid DNAs of the corrected clones were analyzed electrophoretically. Two-dimensional protein analysis. Two-dimensional protein patterns were determined as described elsewhere (16a).

RESULTS Isolation of A. brasilense Sp7 and 7030 clones with Tn.5 lacZ insertions. In matings between E. coli HB1O1(pCIB100) or HB101 (pCIB110) and A. brasilense Sp7 or 7030, Azospirillum exconjugants were isolated on MMAB medium containing 25 ,ug of kanamycin per ml at frequencies ranging from 10-6 to 10-' (Table 2). With all of the TnS constructs tested, the higher number of Kmr colonies was always obtained with A. brasilense 7030. This is consistent with earlier observations from experiments in which several A. brasilense strains were compared for random TnS mutagenesis (55). For this reason, strain 7030 was used in subsequent experiments. Strain 7030 is a chemically induced mutant of strain Sp7. Compared with strain Sp7, the mutant is streptomycin resistant and forms pronounced red-pink colonies on plates (20). Moreover, the mutant lacks one of the indigenous TABLE 2. Frequencies of transposition of various Tn5 constructs in matings between E. coli and A. brasilense frequency of Acceptor strai pMean Suie Krtasojgns Acceptor strain Suicide plasmid Kmr transconjugantSa

A. brasilense Sp7

A. brasilense 7030

10-7

pSUP2021 pSUP5011 ColE1::Tn5 lac pCIB100

2.9 2.6 6.9 1.2

x x

pSUP2021 pSUP5011

4.4 4.8 3.9 4.7

x 10-5 x 10-5 x 10-6 x 10-6

ColE1::Tn5 lac pCIB100

10-7

x 10-9 x 10-8

a Transconjugants were selected for Kmr on MMAB. Transfer frequencies with respect to the final number of recipient cells are shown; these represent the mean values of three independent matings.

Source or reference

11

ATCC 29145 20 S. T. Lam CIBA-GEIGY Our laboratory 27 50 49 19

plasmids of strain Sp7, namely, the 115-MDa plasmid (56). A number of differences in the respective two-dimensional protein patterns have also been demonstrated recently (16a). Five hundred exconjugants of A. brasilense 7030 were assayed for B-galactosidase activity by inoculation on MMAB agar containing 5-bromo-4-chloro-3-indolyl-i-D-galactopyranoside. Sixty percent of the exconjugants became blue, covering a broad range of color intensities. Strain 7030 does not develop blue colonies on 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside plates, even after prolonged incubation for 72 h. In the quantitative assay, the clones that became blue on 5-bromo-4-chloro-3-indolyl-4-D-galactopyranoside plates revealed 3-galactosidase activities of 2 to 99 Miller units. With the parental strain, ,B-galactosidase activity never exceeded 1 Miller unit. Physical analysis of A. brasilense 7030 clones with TnS lacZ insertions. Sall-restricted total DNAs of eight randomly selected exconjugants, of which three were obtained from matings between A. brasilense 7030 and E. coli HB101 (pCIB100) and five were from matings of A. brasilense 7030 with E. coli HB1O1(pCIB110), were Southern blotted and hybridized to 32P-labeled ColEl DNA, the vector DNA of pCIB100 and pCIB110. The lack of hybridization indicated the absence of vector DNA (data not shown). The same Southern blot was hybridized with 32P-labeled ColEl::TnS lacZ DNA (Fig. 1). The TnS lacZ construct of pCIB100 contains one Sall restriction site, which is removed in the corresponding construct of pCIB110. Exconjugants obtained with pCIB100 should therefore reveal two hybridizing bands (Fig. 1, lanes 1 to 3), whereas exconjugants obtained with pCIB110 should reveal only one hybridizing band (Fig. 1, lanes 5 to 9). The positions of the bands revealed that TnS lacZ apparently integrates at random in the genome of A. brasilense, generating a single copy in each exconjugant, as does TnS. Supporting evidence for random integration of TnS lacZ in the A. brasilense genome comes from the large variation in P-galactosidase levels among the different exconjugants (see above). Identification and characterization of TnS lacZ-induced mutants of A. brasilense 7030. Of 500 TnS lacZ-induced mutants of A. brasilense 7030 obtained with the pCIB100 construct (see above), 6 that were unable to spread in semisolid MMAB medium were isolated. Light microscopic observations showed that three of them were nonmotile (NM304, NM309, and NM310). Since transmission electron microscopy revealed a lack of flagella (data not shown), these mutants are Fla-. When assayed for P-galactosidase,

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1

2

3

4

5

6

7

8

9

TABLE 3. Chemotaxis of A. brasilense 7030 and three Chemutants toward various carbon sources Extent of swarmingb of strain: Substratea

Malate Succinate Citrate Fumarate Oxalate Sucrose Glucose Arabinose Fructose Glutamate Glutamine FIG. 1. Physical analysis of eight randomly chosen TnS lacZinduced insertion mutants of A. brasilense 7030. Safl-restricted total DNA was transferred to a nylon membrane and hybridized with 32P-labeled ColE1::TnS DNA. Lanes: 1 to 3, mutants obtained with pCIB100; 5 to 9, mutants obtained with pCIB110; 4, A. brasilense 7030.

activity levels of 2, 3, and 4.5 U were obtained with NM310, NM304, and NM309, respectively. SalI-restricted total DNAs of these mutants were hybridized with 32P-labeled ColEl::TnS lacZ DNA (Fig. 2). A size estimation of the hybridizing bands (two for each mutant) indicated that among the Fla- mutants, TnS lacZ had inserted itself into at least two different Sall restriction fragments. Possibly, NM309 and NM310 have the TnS derivative in the same Sall fragment. Since motility was not abolished in the other three mutants, these were likely to be affected in their chemotactic response. In the initial screening, malate was used as an attractant, because it constitutes the major low-molecularweight compound in root exudates of Digitaria. sp. Strain Sp7 was isolated from the rhizosphere of Digitaria decumbens (53). These mutants (NM306, NM313, and NM315) were further characterized with regard to their chemotactic

FIG. 2. Physical analysis of TnS lacZ-induced Fla- (NM304, NM309, and NM310) and Che- (NM306 and NM313) mutants of A. brasilense 7030. Sall-restricted total DNAs of the mutants were transferred to a nylon membrane and hybridized with 32P-labeled ColE1::TnS DNA.

Aspartate Asparagine Serine Methionine Lysine Threonine Tryptophan Leucine Isoleucine Valine Alanine Arginine Proline Glycine Tyrosine Histidine Phenylalanine

NM315

7030

NM306

NM313

++ +++ ++ +++ + + + ++ ++ + + +

-

-

+ + +

+ +

-

+ ++

-

-

-

-

-

-

-

-

-

-

-

-

+

-

-

-

+ -

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+ + + + +

-

-

-

+ +

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

a All were used at 1 mM. b The extent of swarming from the inoculation point into the 0.2% agar medium is indicated as follows: -, 1.5 cm.

behavior toward several organic acids, carbohydrates, and amino acids (Table 3). Since they failed to be attracted to a wide range of tested compounds, these mutants are considered generally nonchemotactic (Che-). Compared with the Fla- mutants, considerably more variation in the expressed ,-galactosidase activity was observed, namely, 7 U for NM313, 15 U for NM306, and as high as 65 U for NM315. The latter value suggests a transcriptional fusion to a strong promotor. Physical analysis of NM306 and NM315 revealed that TnS lacZ had inserted itself into different SalI fragments (Fig. 2). A further characterization of these chemotaxis mutants was performed by two-dimensional electrophoresis of total proteins (Fig. 3). Mutants NM306 and NM313 both lack an acidic protein with an Mr of about 110,000 (Fig. 3A, arrowhead). This observation was not made with NM315. It was previously shown that the very same protein is absent in the protein pattern of an exoC mutant of A. brasilense Sp7 (16a, 35). The appearance of a truncated form (Mr, 87,000) which was demonstrated with the latter Sp7 mutant was not observed in this study with the chemotaxis mutants of strain 7030. The additional small acidic protein (Mr, 28,000; circled in Fig. 3B) represents TnS-encoded neomycin phosphotransferase (16a). In A. brasilense 7030, a plasmid of 90 MDa and two megaplasmids of over 300 MDa can be detected (20). To determine the locations of the TnS lacZ insertions, cell lysates of the Fla- and Che- mutants were electrophoresed to separate plasmid and chromosomal DNAs, blotted, and hybridized to 32P-labeled ColEl::TnS (Fig. 4). In both

TnS lacZ MUTAGENESIS IN A. BRASILENSE

VOL. 56, 1990

A

(DO)OC OOO

C

993

LO

Z--O-

ZZ ZZZ

sZ

4WA* -.

CHR

FIG. 4. Location of Tn5 lacZ insertions in A. brasilense Fla- and Che- mutants by Southern blot hybridization of plasmid patterns with 32P-labeled ColE1::TnS as the probe. Strain 7030 was the negative control. The positions of the 90-MDa plasmid (p90) and the chromosomal DNA (CHR) are indicated.

X0

ff

,

;lS

FIG. 3. Two-dimensional protein patterns of A. brasilense 7030 (A) and its Che- mutant, NM313 (B). The pH gradient extends from the left (high) to the right (low). The Mr markers (103) are as follows (from the top): 97.4, 66.2, 42.7, 31.0, 21.5, and 14.4. The marked proteins are discussed in the text.

NM306 and NM313, a hybridization signal was detected at the migration distance of the 90-MDa plasmid, whereas for the other mutants homology with the chromosomal DNA was demonstrated. A cosmid library of the 90-MDa plasmid (p90) DNA was conjugated en masse to the three chemotaxis mutants (NM306, NM313, and NM315). Complementation of the chemotaxis phenotype was found for NM306 and NM313 but not for NM315 as expected, since the latter mutant is the result of a chromosomal TnS-lacZ insertion. Recombinant cosmids were isolated from two complemented transconjugants of NM306 (p90-001 and p90-002) and two complemented transconjugants of NM313 (p90-003 and p90-004). Upon EcoRI restriction analysis of the isolated DNAs, it was found that clones p90-001 and p90-004 are identical. Clones p90-001 and p90-002 have a 16-kilobase (kb) EcoRI fragment in common, whereas clones p90-001 and p90-003 have a 3.5-kb EcoRI fragment in common. On the basis of this, we can conclude that in mutant NM306, TnS lacZ inserted itself into a 16-kb EcoRI fragment, whereas in mutant NM313, TnS lacZ inserted itself into the 3.5-kb EcoRI fragment. The two EcoRI fragments must be closely linked on the 90-MDa plasmid, since they occur in a particular recombinant cosmid (p90-001). Previously, we have characterized two recombinant cosmids of the 90-MDa

plasmid DNA that are able to complement the exoB and exoC mutations, respectively, in Rhizobium meliloti 1021 (34). Given a similar change in the protein profile of the constructed exoC mutant of A. brasilense and the abovementioned che mutants (truncated form or lack of the 110-kilodalton protein, respectively) it was tempting to determine the relationship between the exo-complementing cosmid clones (pCall02 for exoB and pCalll2 for exoC) and the che-complementing clones (Fig. 5). Interestingly, the pCalll2 clone contains two EcoRI fragments also present in p90-003, whereas clone pCallO2 contains the above-mentioned 16-kb EcoRI fragment. These data encourage determination of the physical map of the 90-MDa plasmid and conduction of a more detailed genetic analysis of the loci already located on the 90-MDa plasmid. Preliminary data indicate that the exoC mutant of A. brasilense, with the truncated form of the 110-kilodalton protein, displays wildtype chemotactic behavior. The precise function of this protein in exopolysaccharide synthesis (34) and chemotaxis of A. brasilense remains to be determined. DISCUSSION Previously, we and others have shown that TnS can be used as a mutagen for A. brasilense (1, 54). The results presented here demonstrate that TnS lacZ is suitable for random transposon mutagenesis of A. brasilense and subsequent detection of transcriptional activity originating from sequences upstream of the inserted ,B-galactosidase gene. In heterospecific matings with E. coli cells carrying suicide plasmid pCIB100 or pCIB110, TnS lacZ insertion mutants of A. brasilense 7030 were obtained at a frequency of 10-6 per recipient cell, implying a 10-fold reduction compared with TnS. The same observation was made with Myxococcus xanthus and E. coli and was ascribed to the increased distance between the ends of the transposon upon introduction of the trp-lacZ fusion gene (27). The oriT sequence of plasmid RP4 was introduced into TnS lacZ to facilitate its transfer into Pseudomonas sp. and other gram-negative bacteria (S. T. Lam, personal communication). For A. brasilense, we observed no difference in transposition frequency between ColEl::TnS lacZ and a Tn5 lacZ Mob (pCIB100) construct. Sixty percent of the TnS lacZ-induced mutants examined

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branch of a dual chemotaxis pathway that is specialized in the detection of nutrients but contain an intact second branch that is responsible for responding to plant signals (9). In two Che- mutants of A. brasilense 7030, Tn5 lacZ inserted itself into the 90-MDa plasmid. Both mutants lacked an acidic protein that was previously reported to be absent in an A. brasilense Sp7 exoC mutant as well. The exo genes from A. brasilense can correct R. meliloti mutations for exopolysaccharide synthesis and are also located on the 90-MDa plasmid (34, 35). We obtained evidence that the che and exoC loci are linked but not identical. Thus, an operonlike structure on the 90-MDa plasmid covering these loci may be postulated. However, the possibility cannot be ruled out that the Che- phenotype of both mutants arose from a pleiotrophic effect due to alterations in the cell envelope similar to the one reported for some Exo- mutants of R. meliloti that are affected not only in exopolysaccharide synthesis but also in resistance to bacteriophages and nodule invasion (28). Finally, the isolation of chemotaxis-deficient mutants of A. brasilense with a mutation in the 90-MDa plasmid DNA stresses the relevance of the information carried by this particular plasmid, as suggested previously

1 23

. --

16

_= _n

_

_

_

_3.5

_ _

_ _,

_

1-

(34). ACKNOWLEDGMENTS

FIG. 5. EcoRl restriction patterns of pCalll2, pCallO2, and Che--correcting clones p90-001 to p90-004. Lanes: 1, HindIIIEcoRl-digested lambda DNA; 2, p90-001; 3, p90-002; 4, pCal1l2; 5, p90-003; 6, p90-004; 7, pCallO2; 8, HindIll-digested lambda DNA. The molecular sizes of common EcoRI restriction fragments are given in kilobase pairs on the right.

expressed ,B-galactosidase above the background level. This activity results from transcriptional fusions with the galactosidase gene, since a stop codon in all three reading frames in the first 80 base pairs of the trp-lacZ fusion segment prevents the formation of translational fusions. Physical analysis of randomly selected TnS lacZ-induced mutants provided proof of the random insertion of the TnS lacZ construct and its presence as a single copy in the Azospirillum genome. The closely linked phenomena of flagellum-driven movement and chemotactic attraction are thought to play a significant role in the establishment of several types of plant-bacterium interactions, namely, symbiosis (3, 9, 16, 25, 42), pathogenesis (4, 15, 21, 29, 40, 41, 43, 44), and associative colonization (5-8, 17, 22, 24, 32, 37, 45-47). When pCIB100 was used, mutants affected in motility (Fla-) and general chemotaxis (Che-) were isolated from A. brasilense 7030. Such mutants will be very useful for further elucidation of the involvement of motility and chemotaxis in the process of root colonization. Furthermore, by virtue of the promoter probe transposon used, it will be possible to monitor plant-inducible gene expression in this process. Previously, this approach proved quite successful for analysis of nodulation and virulence genes in Rhizobium sp. (23, 51) and Agrobacterium sp. (52), respectively. The Chemutants characterized here are generally nonchemotactic and can be used to examine whether a dual chemotaxis pathway exists in Azospirillum sp. Generally, nonchemotactic mutants of R. meliloti having lost responses to all amino acid and sugar attractants tested but retaining chemotaxis toward unidentified compounds from alfalfa root extracts have been isolated. It is believed that they are affected in one I-

R.D.M. is a Research Associate and M.V. is a Research Assistant of the National Fonds voor Wetenschappelijk Onderzoek. This work was supported by grants from the FGWO (3.0093.89) and the Onderzoeksraad, Katholieke Universiteit Leuven (OT/88/20). We gratefully acknowledge the assistance of B. Van Der Schueren (CME, Katholiek Universiteit Leuven) with transmission electron microscopy. We thank S. T. Lam (CIBA-GEIGY Corp., Summit, N.C.) for providing plasmids pCIB100 and pCIB110 and Els Van Bastelaere and Mark Eyers for constructing the cosmid library of the 90-MDa plasmid. LITERATURE CITED 1. Abdal-Salam, M. S., and W. Klingmuller. 1987. Transposon TnS mutagenesis in Azospirillum lipoferum: isolation of indole acetic acid mutants. Mol. Gen. Genet. 210:165-170. 2. Adler, J. 1966. Chemotaxis of bacteria. Science 153:709-716. 3. Ames, P., and K. Bergman. 1981. Competitive advantage provided by bacterial motility in the formation of nodules by Rhizobium meliloii. J. Bacteriol. 148:728-729. 4. Ashby, A. M., M. D. Watson, G. J. Loake, and C. H. Shaw. 1988. Ti plasmid-specified chemotaxis of Agrobacterium tume-

faciens C58C1 toward vir-inducing phenolic compounds and soluble factors from monocotyledonous and dicotyledonous plants. J. Bacteriol. 170:4181-4187. 5. Barak, R., I. Nur, and Y. Okon. 1983. Detection of chemotaxis in Azospirillum brasilense. J. Appl. Bacteriol. 54:399-403. 6. Barak, R., I. Nur, Y. Okon, and Y. Henis. 1982. Aerotactic response of Azospirillum brasilense. J. Bacteriol. 152:643-649. 7. Bashan, Y. 1986. Migration of the rhizosphere bacteria Azospirillum brasilense and Pseudomonas fluorescens towards wheat roots in the soil. J. Gen. Microbiol. 132:3407-3414. 8. Bashan, Y., and H. Levanony. 1987. Horizontal and vertical movement of Azospirillum brasilense Cd in the soil and along the rhizophere of wheat and weeds in controlled and field environments. J. Gen. Microbiol. 133:3473-3480. 9. Bergman, K., M. Gulash-Hoffee, R. E. Hovestadt, R. C. Larosiliere, P. G. Ronco II, and L. Su. 1988. Physiology of behavioral

mutants of Rhizobium meliloti: evidence for a dual chemotaxis pathway. J. Bacteriol. 170:3249-3254. 10. Blair, D. G., D. J. Sherrat, D. B. Clewell, and D. R. Helinski. 1972. Isolation of supercoiled colicinogenic factor E, DNA sensitive to ribonuclease and alkali. Proc. Natl. Acad. Sci. USA 69:2518-2522. 11. Boyer, H. W., and D. Roulland-Dussoix. 1969. A complementation analysis of the restriction and modification of DNA in

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Isolation of behavioral mutants of Azospirillum brasilense by using Tn5 lacZ.

Tn5 lacZ mutants were generated with Azospirillum brasilense 7030 by mating that strain with Escherichia coli strains carrying suicide plasmid pCIB100...
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