1 Dip. Biologia Animale e Genetica, Università di Firenze, 50125 Firenze, Italy
2 Dip. Agrobiologia e Agrochimica, Università della Tuscia, 01100 Viterbo, Italy
3 Dip. Scienze Biochimiche, Università La Sapienza, 00185 Roma, Italy
4 Dip. Biologia Vegetale, Università di Firenze, 50100 Firenze, Italy
5 Dip. Biologia di Base e Applicata, Università dell'Aquila, Via Vetoio, Coppito, 67100 L'Aquila, Italy
Correspondence
Maddalena del Gallo
delgallo{at}aquila.infn.it/maddalena.delgallo@libero.it
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the A. brasilense mreB-like gene sequence reported in this paper is AF438483.
Present address: Bauer Center for Genomics Research, Harvard University, 7 Divinity Ave, 02138, Cambridge, MA, USA.
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INTRODUCTION |
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Members of the genus Azospirillum are capable of nitrogen fixation under microaerophilic conditions in association with the roots of several grasses (Döbereiner, 1991). The vibroid form of Azospirillum brasilense has a polar flagellum and is highly motile (Döbereiner & Day, 1976
; Tarrand et al., 1978
). However, under certain environmental conditions, particularly when inside plant tissue, Azospirillum cells become round and non-motile, and are referred to as encapsulated or C-forms (Becking, 1985
; Berg et al., 1979
; Döbereiner & Day, 1976
), or as cysts (Sadasivan & Neyra, 1985
, 1987
). The cellular envelopes of these forms are thicker than those of vegetative cells (Murray & Moyles, 1987
). Polymorphic forms of Azospirillum appear in response to different factors, such as medium composition (C/N ratio), pH, age of the culture, polysaccharide production and plant colonization (Bashan et al., 1991
; Becking, 1985
; Berg et al., 1980
; Burdman et al., 2000
, 2001
; Sadasivan & Neyra, 1985
, 1987
; Tal & Okon, 1985
). Azospirillum appears to form several different types of cyst-like cell: pleomorphic cyst-like forms associated with cultured sugarcane-callus tissue and with root colonization (Bashan et al., 1991
; Berg et al., 1979
, 1980
; Whallon et al., 1985
). Unlike true cysts, these forms are not resting and are able to divide and to fix nitrogen.
Capsular and exocellular polysaccharides (PS) are responsible for the binding of the fluorescent dye Calcofluor White (CFW) to Azospirillum cells (Del Gallo et al., 1989; Michiels et al., 1991
). These PS and other capsular material are also involved in the interaction between Azospirillum and roots (Michiels et al., 1990
; Burdman et al., 2000
). This process involves the adsorption of bacteria to the root surface and the colonization of the root. A number of bacterial cell-surface proteins and carbohydrates are involved in the attachment to plant surfaces and may be involved in the Azospirillumroot interaction. Burdman et al. (2001)
, in particular, isolated a major outer-membrane protein involved in root adhesion and adsorption.
We used Tn5 mutagenesis to create A. brasilense mutants with altered CFW binding. We then used these mutants to study the role of exocellular PS in cell behaviour and in morphological differentiation. These mutants had a round morphotype and altered production of capsular components. Cloning and sequencing revealed that the gene responsible for this phenotype was homologous to mreB, which is involved in the maintenance of bacterial shape.
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METHODS |
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DNA manipulations.
Azospirillum DNA was extracted by adding SDS and proteinase K, as previously described (Giovannetti et al., 1990). Southern blotting was performed by standard protocols (Sambrook et al., 1989
), using as probe the 4·6 kb NotI fragment of Tn5 and the A. brasilense mreB fragments, labelled with digoxigenin according to the supplier's instructions (Roche).
The 18 kb EcoRI fragment of strain SPFE6, containing Tn5, was cloned into pUC18, and kanamycin-resistant (40 µg ml1) transformants were selected. The resulting construct was named pAFE61. The two A. brasilense DNA fragments surrounding Tn5 were subcloned from pAFE61 that had been digested with SalI. The construct containing the 3' side of Tn5 together with A. brasilense DNA (4800 bp) was named pAFE62, and the construct containing the 5' side of Tn5 together with A. brasilense DNA (4800 bp) was named pAFE63. These plasmids were then used to transform competent E. coli DH5. We then sequenced pAFE62 and pAFE63 by use of the M13 forward and reverse primers and four more primers, designed to anneal to the new sequence. The two sequences were assembled. We ensured that they corresponded to the Azospirillum genome sequence by amplifying DNA from strain SPF94 with primers pmreBfor (5'-CGAAGGGGCATTCGTCTAT-3') and pmreBrev (5'-ATCAGTAGGCGCTGACCAAG-3'), which spanned the entire gene, and sequencing the amplified fragment. The sequence of the mreB gene was analysed with the MacVector 4.0 software (Life Science Products). Homology analysis was performed using the BLAST database (Altschul et al., 1990
, 1997
) from the National Center for Biotechnology Information (NCBI). Sequence alignments were obtained with MultAlin (Corpet, 1988
), available from the INRA Toulouse website.
To confirm the phenotype of the mreB mutant, a second independent mreB mutant was constructed by inserting a kanamycin-resistance cassette. The first 500 bp of the mreB gene were amplified using pmreB(a)for (5'-GAGAGCATGCTGCTTTCCAAACTCCTC-3') and pmreB(a)rev (5'-GAGACTGCAGCCGATGTCCACGACC-3'). The second half was amplified using pmreB(b)for (5'-GAGACTGCAGGGCATCGTCTATTCCC-3') and pmreB(b)rev (5'-GAGAGGATCCCGTTTCGCAGGTCTTC-3'). These two fragments were restricted with appropriate enzymes and ligated outside of the kanamycin-resistance cassette (GenBlock; Pharmacia). The whole construction was cloned into pUC18 and used to transform competent E. coli DH5 cells. Ampicillin- (100 g ml1) and kanamycin- (40 µg ml1) resistant colonies were selected, and the plasmid named pUCmreB5. The mreB mutagenesis cassette was then transferred into the conjugative plasmid, pSUP202 (Simon et al., 1983
), yielding pSUPmreB6 (Cmr, Ampr, Kanr, Tets). pSUPmreB6 was used to transform streptomycin-resistant E. coli S17.1 cells (Simon et al., 1983
). Finally, pSUPmreB6 was conjugated to A. brasilense SPF94 (ampicillin and chloramphenicol sensitive), as described by Singh & Klingmüller (1986)
. Double crossing-over events generated kanamycin-resistant, tetracycline-sensitive mutants. We used Southern blotting and PCR to check that the kanamycin cassette was correctly inserted within the mreB gene.
Cell-surface characterization
Cell aggregation.
Cells were grown in LB to stationary phase (OD600=1·6), and then observed under a light microscope. The number of microaggregates (more than eight cells) present in 10 microscopical fields (400x magnification) was counted 5 and 10 minutes after laying a drop of the culture on the microscope slide.
Cell dispersion.
Cells were grown in NB to mid-log (OD600=0·8) or to stationary phase (OD600=1·6), and then harvested by centrifugation. We measured the vortexing time necessary to resuspend the resulting pellet in PBS buffer (Del Gallo et al., 1989), as described by Arunakumari et al. (1992)
. Dispersion was monitored by measuring OD600 in a spectrophotometer.
Cohesiveness and autoagglutination.
Cells were grown in NB to late-log (OD600=1·2) or to stationary phase (OD600=1·6). The cells were harvested and resuspended in PBS. The sedimentation rate of the cells was measured by monitoring changes in OD600 (Arunakumari et al., 1992). The net electrical charge of cells was determined by electrophoresis, as described by Sakai (1986)
. The effect of differences in cell-surface electrical charges was assayed by measuring the sedimentation rate of stationary-phase cells that had been resuspended in 0·1 M phosphate buffer at pH 4 and at pH 10 (Sakai, 1986
).
Hydrophobicity.
Hydrophobicity was determined by measuring the adhesion of a cell pellet to tetradecane (Rosemberg, 1984).
Staining.
Cells were stained with Alcian Blue and with Congo Red, as previously described (Del Gallo et al., 1989; Forni et al., 1992
). Both stains are known to bind exocellular PS: Alcian Blue stains acidic PS, Congo Red all neutral PS.
Epifluorescence microscopy.
Cells were grown for 24 and 48 h on MM-fructose or MM-succinate (both 5 g l1). After staining with CFW, they were observed under an epifluorescence microscope (Leitz Dialux 20, equipped with an excitation filter A2, 270380 nm).
Electron microscopy.
Transmission electron microscopy (TEM) samples were prepared as follows: bacterial cells were grown up to mid-exponential phase (OD600=0·8) in LB, collected by centrifugation, embedded in 4 % agar, fixed for 1 h with 4 % glutaraldehyde in 0·2 M phosphate buffer (pH 7·2), washed with the same buffer, and post-fixed for 2 h in 2 % OsO4. The samples were then dehydrated in ethanol and embedded in Epon-Araldite (Fluka), according to standard procedures. Sections were made with an LKB IV ultramicrotome. The sections were stained with uranyl acetate and lead citrate, and observed with a Philips 201-C electron microscope working at 80 kV.
Scanning electron microscopy (SEM) samples were prepared as follows: a drop of cell culture grown in LB up to mid-exponential phase (OD600=0·8) was spread on polycarbonate membranes (0·6 µm pore size; Millipore), fixed for 1 h with 4 % glutaraldehyde in 0·2 M phosphate buffer (pH 7·2), washed with the same buffer and dehydrated through an alcohol series, finishing with absolute alcohol. The samples were then critical-point-dried with a Blazer CPD 030, mounted on stubs, coated with carbon and gold, and examined with a Philips 515 scanning electron microscope.
Capsule extraction and analysis.
Cells were grown for 48 h in MM, then washed and resuspended in one-tenth the volume of PBS and stored at 4 °C for 3 days until the capsule had dissolved (Del Gallo & Haegi, 1990). The cells were centrifuged at 8000 g for 10 min. The supernatant, which contained the dissolved capsular material, was collected, and total PS content was determined by the Anthrone method, using glucose as a standard (Dische, 1962
). The protein concentration was determined either by the method of Lowry et al. (1951)
or by a derived protein assay (DC Protein Assay; Bio-Rad). Protease inhibitors, 3 mM Pefabloc (Roche) and 1·3 mM EDTA, were added to the capsular material.
Capsular material was dialysed against HPLC-grade water and analysed by HPLC after PS hydrolysis, as described previously (Del Gallo & Haegi, 1990). Quantitative analyses were carried out by use of the two-point absolute titration curve described in the Shimatzu Chromatopac C-3A manual, with Bio-Rad Aminex HPX87C and Aminex HPX87H columns.
SDS-PAGE was carried out as described by Laemmli (1970) with a 4 % stacking gel and a 10 % resolving gel. The separated proteins were either stained with Coomassie Brilliant Blue R250 or electroblotted onto PVDF membranes (Immobilon; Millipore) by use of a semi-dry electroblotting apparatus (Hoefler; Pharmacia). Molecular mass markers were purchased from Sigma.
The blots were stained with Coomassie Brilliant Blue R250. Proteins of interest were excised and subjected to sequence analysis by automated Edman degradation, using a Perkin Elmer AB476 gas-phase sequencer.
Glycoproteins were detected either on Alcian Blue-stained (1 % ethanol : water) SDS-PAGE gels or on blots stained with specific DIG-labelled lectins (DIG Glycan Detection Kit; Roche).
Effect of lysozyme and temperature
Lysozyme.
Bacteria were grown to the end of the exponential phase (OD600=1·2) in MM supplemented with 10 mM NH4Cl. They were then washed in 50 mM Tris/HCl, pH 7·5, and incubated in the same buffer plus 0·5 % Triton X-100 for 30 min. Cells were centrifuged and resuspended in SMM, pH 5·5, with 1 µg lysozyme ml1 and 4 % EDTA. Aliquots were taken at different time points. The aliquots were diluted in SMM (isotonic) or water (hypotonic) and plated out on PY agar plates. Viable cells were counted after incubation for 2 days at 33 °C.
Temperature.
Cells grown in MM or NB were treated up to 60 °C, following the procedure described by Matsuzawa et al. (1972).
Nitrogen metabolism.
Nitrogenase activity was assayed by the acetylene reduction method, and nitrate utilization was assayed using cells grown anaerobically in the presence of 25 mM KNO3,, as previously described (Bani et al., 1980).
Adsorption of A. brasilense strains to plant surfaces.
Bacteria were adsorbed to wheat roots, as described by Michiels et al. (1990), with the following modifications: cultures of A. brasilense SPF94, A. brasilense SPFE6 and E. coli HB101 (as negative control) were labelled by inoculating 4 ml LB medium supplemented with 8 µCi AA3H (Tritiated Amino Acid Mixture; Amersham Pharmacia) with a single colony. This culture was grown to OD600=1·2 (about 5x108 c.f.u. ml1). The cells were then washed twice in saline solution (0·85 % NaCl) and resuspended in 4 ml MM medium to a final concentration of 107 bacteria ml1. Ten-day-old sterile wheat-seedling roots (Triticum aestivum cv. Aurelio) were cut into 2 cm pieces. Five pieces were added to each tube of MM. The negative control consisted of tubes containing the same amount of heat-killed SPF94 (15 min at 80 °C). After 1 h at 33 °C in a rotary shaker at 65 r.p.m., the roots were washed three times in 10 ml saline solution, and the associated radioactivity was measured in a liquid scintillation counter. The percentage of the total added radioactivity remaining on the roots was calculated.
Potato cells isolated from callus were cultured on LS medium (Linsmaier & Skoog, 1965) to a density of 1x105 cells ml1, and filtered through a nylon filter (200 µm pore size) every ten days. The bacteria were grown on LB at 30 °C up to 5x108 c.f.u. ml1. The bacteria were then washed with saline solution and added to the potato-cell suspension at a final concentration of 3x105 ml1. The mixture was incubated for 1 h at 30 °C with gentle shaking, filtered through a nylon filter (10 µm pore size), washed with two volumes of saline solution and ground in a mortar. Disrupted cells were diluted in saline solution, plated on LB plates and potato-cell-bound bacteria were counted.
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RESULTS |
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We used Southern blotting with a Tn5 probe to ensure that the mutant contained Tn5. When total SPFE6 DNA was digested with EcoRI (which does not cut the transposon sequence), a single 18 kb band was observed (not shown). Double digests with EcoRI and SalI (which cuts Tn5 once) produced two bands of approximately 7 and 11 kb. Thus, SPFE6 only contains one copy of the transposon within a 13 kb EcoRI fragment.
Sequence editing
Sequence analysis showed that the 2012 bp nucleotide sequence surrounding Tn5 contained an ORF (from nt 697, ATG start codon, to nt 1819, TGA stop codon). The 374-amino-acid sequence was highly homologous to the MreB-like proteins of Magnetospirillum magnetotacticum (identity 90 %, similarity 93 %, accession no. ZP00055538), Caulobacter crescentus (identity 72 %, similarity 84 %, accession no. NC002696) and Rickettsia prowazekii (identity 71 %, similarity 82 %, accession no. NC000963). The sequence of the A. brasilense mreB-like gene was submitted to the NCBI database (accession no. AF438483). No recognizable promoter sequences were found upstream of the ATG start codon. The conserved domain BLAST analysis program revealed two domains typical of MreB proteins: the HSP70-like domain corresponding to the first 200 amino acids and the FtsA-like domain corresponding to amino acids 140 to 320.
The phenotype of SPFE6 was highly pleiotropic; thus, to exclude the possibility that this pleiotropy was due to secondary mutations that accidentally occurred during Tn5 mutagenesis, we constructed two new mreB mutants using the Kan cassette. All of the characteristics described below were identical in the new mutants and in SPFE6.
Cell-surface properties
We compared the cell-surface properties of SPFE6 with those of the parental strain. The aggregation assay demonstrated that the mutant strain was four to five times more likely to form microaggregates than the parental strain. Accordingly, SPFE6 cells in stationary phase dispersed more slowly than the parental cells (data not shown). The hydrophobicity test and the net-electrical-charge assay did not show any significant differences between the mutant and parental strains, both of which were negatively charged (data not shown). The cohesiveness assay suggested that there may be differences in surface electrical charge at different pHs. The mutant strain agglutinated after long incubation at low pHs; in particular, after 7 h at pH 4·0 the OD600 of the mutant suspension was 0·25±0·1, while that of the parental strain was 1·4±0·2.
Staining also revealed some differences between the parental and mutant strains. Both strains bound comparable amounts of Congo Red, but the parental strain bound much more Alcian Blue than the mutant (data not shown), indicating that the mutant produces a larger amount of acidic PS.
Capsule composition
Quantitative analysis showed some clear differences in the composition of the capsule in the mutant and parental strains. There was up to tenfold more capsule protein in the mutant than in the parental strain, whereas both strains contained similar amounts of PS (Table 1). HPLC analysis (Table 1
) showed that the mutant contained twofold more fucose than the parental strain, whereas both strains contained similar amounts of the other monosaccharides.
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The N-terminal sequence of the 40 kDa protein, which is specific to SPF94, was TTSSGINGQT. This sequence was compared with the SWISS-PROT protein databases by use of a BLAST search and found, in the absence of the signal peptide, to be 70 % identical to the N-terminal sequence (FTSSGTNGKV; Labigne-Reussel et al., 1985) of the afimbrial adhesin AFA-1 of uropathogenic E. coli.
Adsorption of A. brasilense strains to plant surfaces
The lack of an adhesin-like protein suggested that the mutant could be impaired in the capability to attach to plant surfaces. Thus, we compared the ability of the parental and mutant strains to adsorb to wheat roots and to a suspension of cultured potato cells. The adsorption capabilities of the two strains did not differ significantly (Table 2). Between six and nine times fewer E. coli cells adsorbed. This is comparable to the data obtained with the heat-killed A. brasilense cells. This suggests that a specific and active process is involved, and that this process does not involve the 40 kDa surface adhesin-like protein lacking in the mutant.
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The round shape of the mutant could also be due to the impaired synthesis of cell-wall components, giving rise to a kind of spheroplast. To test whether the cell wall of the mutant was altered, we tested the sensitivity of the peptidoglycan layer to lysozyme. Our results (Fig. 5) clearly demonstrated that mutant cells were more resistant to lysozyme and to osmotic shock than the parental strain. Therefore, the rounded shape is not due to an impairment in the cell wall. In fact, the higher level of resistance could be related to the thicker envelope structure, as revealed by electron microscopy. This thick envelope may somehow prevent lysozyme from reaching the mutant cell wall.
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DISCUSSION |
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Although the shape of the mutant cells was similar to that of E. coli mreB mutants (Labigne-Reussel et al., 1985; Matsuzawa et al., 1972
), the A. brasilense phenotype seemed to be more complex. The phenotype of SPFE6 cells was in many ways reminiscent of the differentiated cystic forms that occur in cultures of A. brasilense. After particular environmental changes, Azospirillum species undergo a series of physiological modifications, including changes in the capsular composition (Sadasivan & Neyra, 1987
). This in turn affects the ability to bind to CFW (De Troch et al., 1992
) and leads to changes in poly-
-hydroxybutyrate accumulation (Tal & Okon, 1985
), cell shape and motility, while other metabolic functions become less active, tending to dormancy (Bastarrachea et al., 1988
; Becking, 1985
; Sadasivan & Neyra, 1987
). All of these changes eventually result in the formation of cyst-like forms that are larger, round and more resistant to high temperature and desiccation (Sadasivan & Neyra, 1985
). Interestingly, the cells of many strains of Azospirillum form cyst-like structures when they colonize roots or other plant tissues (Bashan et al., 1991
; Berg et al., 1979
, 1980
; Madi et al., 1989
; Michiels et al., 1989
; M. Grilli Caiola and others, unpublished results.). However, this process has been described in a rather fragmented manner, with phenotypes indiscriminately described as cysts, cyst-like forms, coccoid forms' (C-forms), etc., some related to stress resistance, others associated with the colonization of plant tissues.
Genetic studies have improved our knowledge of the encystment of Azospirillum cells. Some mutants with altered encystment have been isolated and characterized (De Troch et al., 1992; Michiels et al., 1989
, 1990
). However, the relationships between these mutations and encystment have not been assessed, with the possible exception of the enc mutants (Bastarrachea et al., 1988
). Examination of the Sp7-S mutant described by Katupitiya et al. (1995)
and Castellanos et al. (1997)
revealed a relationship between the surface properties of the bacterium, its shape, and its ability to colonize plants. This mutant stained weakly with Congo Red, did not flocculate in the presence of fructose or nitrate, lacked the thick exopolysaccharide layer, had the same general nutritional properties and growth rate as the wild-type and had the same vibroid morphology and motility as the wild-type. Like Sp7-S, SPEF6 displayed a highly pleiotropic phenotype with respect to capsular protein production, Calcofluor staining abilities, aggregation in liquid cultures, resistance to lysozyme and resistance to osmotic shock.
Many of the cell-surface properties of the mutant strain SPEF6 differed from those of the parental strain: the mutant aggregated more readily than the parental strain, and its surface components bound much more CFW and less Alcian Blue. The study of A. brasilense 7030 mutants (Michiels et al., 1989, 1990
, 1991
) also indicates a relationship between surface properties and aggregation. These mutants were Calcofluor dark and had lost the ability to form flocs in liquid cultures, and their exopolysaccharides were more acidic than those of the parental strain. These features were similar, though different, to those of SPFE6. Mutants of A. brasilense strain Sp7, recently described by Burdman et al. (2000)
, showed instead a positive correlation between exopolysaccharide composition and aggregation. The monosaccharide composition of the SPF94 capsules was virtually identical to that of SPFE6 (Table 1
), with the exception of fucose content, although the significance of this to the mutant phenotype is unclear. The protein composition of the capsular extracts, on the other hand, was quantitatively and qualitatively different in the two strains.
The larger amount of protein in the mutant extract may be related to an altered membrane permeability, resulting in the export of cytoplasmic proteins. As a further pleiotropic effect, the mutant lacked a specific glycosylated 40 kDa protein that is normally present in other related species. A 40 kDa protein has in fact been found in the capsular protein preparation of Azospirillum lipoferum strains grown under different conditions (E. G. Biondi and others, unpublished results), and also in A. brasilense Sp7 (Schloter et al., 1994). Whether this protein was absent in the SPF94 mutant because it is not synthesized or because it is rapidly digested by proteases, we cannot decide on the basis of present data. The N-terminal sequence of the 40 kDa protein showed homology to the E. coli afimbrial adhesin AFA-1. Adhesins are extracellular proteins responsible for the attachment of bacteria to different surfaces, including animal (Hultgren et al., 1996
) and plant (Romantschuk, 1992
) tissues. However, we cannot reach any conclusions on the role of this protein, since two adsorption assays both demonstrated that the parental and mutant strains did not differ greatly in this characteristic.
MreB is a structural protein, governing the shape of B. subtilis cells. The phenotype of the Azospirillum mreB-like mutant suggests that the inactivation of this gene also affects other functions: MreB may be involved in the definition of surface properties, and more generally in the control of cellular differentiation. The mutant constitutively showed some of the features of differentiated Azospirillum cells: it was round, produced large amounts of exocellular material, formed large aggregates and had multiple encapsulated cells containing abundant poly--hydroxybutyrate granules even during exponential growth. The mutant cells (metabolically active, actively dividing and able to fix nitrogen) resembled some of the forms observed in association with plants, instead of the resistant cyst-like forms. Thus, mreB may always be active during the vegetative growth of Azospirillum cells, and may be repressed when environmental conditions signal that it is time to start to differentiate. Though the absence of ORFs immedately downstream of mreB suggests that it is a single gene, we cannot exclude that the insertion affected the expression of other genes associated in the same putative operon of mreB, resulting in a sort of polar effect. Whatever its mode of action, the mutation in the mreB-like gene is responsible for a number of important phenotypic effects in Azospirillum, particularly in functions related to plant interaction, and should be further characterized.
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ACKNOWLEDGEMENTS |
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Received 12 November 2003;
revised 18 December 2003;
accepted 16 April 2004.
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