Construction of a recA mutant of Azospirillum lipoferum and involvement of recA in phase variation

The plant-growth promoting rhizobacterium Azospirillum lipoferum strain 4B generates in vitro a stable phase variant designated 4VI at frequencies of 10(-4) to 10(-3) per cell per generation. Variant 4VI displays pleitropic modifications, such as the loss of swimming motility and the inability to assimilate certain sugars compared to the wild type. The mechanism underlying phase variation is unknown. To determine whether RecA-mediated processes are involved in phase variation, the recA gene of A. lipoferum 4B was cloned and sequenced and a recA mutant (termed 4BrecA) was constructed by allelic exchange. Strain 4BrecA showed increased sensitivity to UV and MMS compared with 4B and impaired recombinase activity. The ability to generate variants in vitro was not altered; the variants from 4BrecA exhibited all morphological and biochemical features characteristic of the variant generated by strain 4B. However, the frequency of variants generated by 4BrecA was increased by up to 10-fold. So, in contrast with many studies showing the abolition or a large reduction of the frequency of phase variation in recA mutants, this study describes an enhancement of phase variation in the absence of a functional recA.     

Emergence of a laccase-positive variant of Azospirillum lipoferum occurs via a two-step phenotypic switching process

Two variants have been isolated from the wild-type Azospirillum lipoferum strain 4B. The first variant, 4V(I), spontaneously emerged from the wild-type at frequencies in the order of 10(-4) to 10(-3) per cell generation. Compared to the wild-type, the 4V(I) variant gained (production of a carotenoid-like pigment, assimilation of certain carbohydrates) and lost (swimming motility, reduction of triphenyl tetrazolium chloride, acid production from certain sugars) apparently unrelated phenotypic characteristics. Only from the 4V(I) variant, a second atypical stable form, variant 4V(II), which acquired laccase activity and ability to produce melanin, appeared under very specific conditions, namely growth at extremely low oxygen concentrations. Neither of the variants was able to revert to the parental phenotype. The results suggest that atypical non-motile laccase-positive isolates of A. lipoferum that are found in the rice rhizosphere originate from wild-type (motile, laccase-negative) cells via a two-step phenotypic switching event, a non-motile laccase-negative variant being an intermediate phase.     

Population dynamics of a motile and a non-motile Azospirillum lipoferum strain during rice root colonization and motility variation in the rhizosphere

Abstract: Azospirillum lipoferum 4B and non-motile A. lipoferum 4T have been simultaneously isolated from rice rhizosphere at the same frequency. A. lipoferum 4T showed stable morphological and metabolic traits which are atypical for A. lipoferum species such as lack of motility, carbohydrate metabolism and laccase activity. Inoculation experiments showed that A. lipoferum 4T, but not A. lipoferum 4B, needed rice roots to stabilize in sterile soil. Both strains were able to colonize efficiently rice roots (10⁸ cfu g⁻¹ fresh roots) but motile form 4B remained dominant. In spite of their phenotypical differences, A. lipoferum 4B and 4T co-existed without exclusion in sterile soil (planted or not) and rice rhizosphere. Inoculation of rice roots with A. lipoferum 4B showed that rice rhizosphere enhanced the frequency of appearance of stable non-motile forms (40%). This percentage was weaker in plantlet growth medium (4%). However, these non-motile bacteria kept the same biochemical traits than the motile parental strain 4B (carbohydrates metabolism, laccase activity).     

N-acyl-homoserine lactone-mediated quorum-sensing in Azospirillum: an exception rather than a rule

Forty Azospirillum strains were tested for their ability to synthesize N-acyl-homoserine lactones (AHLs). AHL production was detected for four strains belonging to the lipoferum species and isolated from a rice rhizosphere. AHL molecules were structurally identified for two strains: Azospirillum lipoferum TVV3 produces 3O,C(8)-HSL (N-3-oxo-octanoyl-homoserine-lactone), C(8)-HSL (N-3-octanoyl-homoserine-lactone), 3O,C(10)-HSL (N-3-oxo-decanoyl-homoserine-lactone), 3OH,C(10)-HSL (N-3-hydroxy-decanoyl-homoserine-lactone) and C(10)-HSL (N-3-decanoyl-homoserine-lactone), whereas A. lipoferum B518 produced 3O,C(6)-HSL (N-3-oxo-hexanoyl-homoserine-lactone), C(6)-HSL (N-3-hexanoyl-homoserine-lactone), 3O,C(8)-HSL, 3OH,C(8)-HSL and C(8)-HSL. Genes involved in AHL production were characterized for A. lipoferum TVV3 by generating a genomic library and complementing an AHL-deficient strain with sensor capabilities. Those genes, designated alpI and alpR, were found to belong to the luxI and luxR families, respectively. When cloned in a suitable heterologous host, alpI and alpR could direct the synthesis of the five cognate AHLs present in A. lipoferum TVV3. These two adjacent genes were found to be located on a 85 kb plasmid. Southern hybridization experiments with probes alpI/R indicated that genes involved in AHL production in the three other AHL-producing strains were not closely related to alpI and alpR. This study demonstrates that AHL-based quorum-sensing is not widespread among the genus Azospirillum and could be found only in some A. lipoferum strains.     

Loss of Cytochrome c Oxidase Activity and Acquisition of Resistance to Quinone Analogs in a Laccase-Positive Variant of Azospirillum lipoferum

Laccase, a p-diphenol oxidase typical of plants and fungi, has been found recently in a proteobacterium, Azospirillum lipoferum. Laccase activity was detected in both a natural isolate and an in vitro-obtained phase variant that originated from the laccase-negative wild type. In this study, the electron transport systems of the laccase-positive variant and its parental laccase-negative forms were compared. During exponential (but not stationary) growth under fully aerobic (but not under microaerobic) conditions, the laccase-positive variant lost a respiratory branch that is terminated in a cytochrome c oxidase of the aa(3) type; this was most likely due to a defect in the biosynthesis of a heme component essential for the oxidase. The laccase-positive variant was significantly less sensitive to the inhibitory action of quinone analogs and fully resistant to inhibitors of the bc(1) complex, apparently due to the rearrangements of its respiratory system. We propose that the loss of the cytochrome c oxidase-containing branch in the variant is an adaptive strategy to the presence of intracellular oxidized quinones, the products of laccase activity.     

Isolation and characterization of nitrogen-fixing bacteria of the genus Azospirillum from the soil of a Sphagnum peat bog

The presence of nitrogen-fixing bacteria of the genus Azospirillum in the soils of acidic raised Sphagnum bogs is revealed for the first time. Three Azospirillum strains, B2, B21, and B22, were isolated as a component of methane-oxidizing enrichment cultures, whereas attempts to isolate them directly from peat samples have failed. The results of comparative analysis of the nucleotide sequences of 16S rRNA genes, DNA-DNA hybridization, and the analysis of the sequences of the functional genes encoding nitrogenase and ribulose-1, 5-bisphosphate carboxylase reveal that all the newly obtained strains can be classified as Azospirillum lipoferum. Yet, unlike A. lipoferum. the isolates do not require biotin and utilize sucrose, inositol, and glycerol for growth. The cell morphology of strain B2 differs from that of the type strain and strains B21 and B22. The results obtained indicate the variability of morphological, physiological, and biochemical properties in closely related Azospirillum strains and suggest the existence of metabolic relationships between methanotrophic bacteria and the representatives of the genus Azospirillum under peat bog conditions.     

Heterologous hybridization of Azospirillum DNA to Rhizobium nod and fix genes

Abstract Probes containing the nod and hsn regions of Rhizobium meliloti and the fixABC genes of Rhizobium japonicum were used to perform hybridization experiments with endonuclease-restricted DNA from Azospirillum brasilense strains and 2 Azospirillum lipoferum strains. Homology to nod, hsn and fixA was found in the 4 Azospirillum strains.     

Mobilization and transfer of Azospirillum lipoferum plasmid by the Tn5-Mob transposon into a plasmid-free Agrobacterium tumefaciens strain

Azospirillum lipoferum 4B harbors five cryptic plasmids. Several suicide plasmids were used to transfer Tn5-Mob to A. lipoferum 4B. Tn5-Mob insertion mutations of this strain could be obtained at frequencies of 10(-8)-10(-7) per recipient cell. One hundred Tn5-Mob A. lipoferum 4B mutants were used in bacterial matings with a plasmid-free Agrobacterium tumefaciens recipient strain. This is the first report of mobilization, transfer, and replication of an Azospirillum plasmid in Agrobacterium tumefaciens. One transconjugant was found which had lost an indigenous plasmid.     

A phase variant of Azospirillum lipoferum lacks a polar flagellum and constitutively expresses mechanosensing lateral flagella.

Flagellation of a nonswimming variant of the mixed flagellated bacterium Azospirillum lipoferum 4B was characterized by electron microscopy, and polyclonal antibodies were raised against polar and lateral flagellins. The variant cells lacked a polar flagellum due to a defect in flagellin synthesis and constitutively expressed lateral flagella. The variant cells were able to respond to conditions that restricted the rotation of lateral flagella by producing more lateral flagella, suggesting that the lateral flagella, as well as the polar flagellum, are mechanosensing.     

Physical map and properties of a 90-MDa plasmid of Azospirillum brasilense Sp7

Homology was previously detected between the DNA restriction fragments containing Rhizobium meliloti nodulation genes and the 90-MDa plasmid, p90, of Azospirillum brasilense Sp7. Two DNA loci from Sp7 genome that complement mutations in the exopolysaccharide synthesis genes, exoB and exoC, of R. meliloti were also shown to be present on the plasmid. A more detailed characterization of the plasmid was undertaken to establish its physical map and to localize the nod homologies and other specific regions. Six loci were mapped, the region homologous to the nodulation genes, nodPQ, of R. meliloti, the exoB and exoC mutation-correcting loci, a locus for Ap resistance, a bla homology region different from the Ap resistance locus, and a region necessary for the maintenance of p90 as an independent replicon. Mobilization into Agrobacterium tumefaciens of p90-Tn5-Mob was obtained at a frequency of 10(-4), with the plasmid helper pJB3JI. Self-transfer of p90 was not demonstrated. Fragments of p90 hybridized with a plasmid of 90 MDa present in most A. brasilense and some A. lipoferum strains, suggesting a plasmid family in Azospirillum.     

Wheat root colonization and nitrogenase activity by Azospirillum isolates from crop plants in Korea

Nitrogen-fixing bacteria were isolated from the rhizosphere of different crops of Korea. A total of 16 isolates were selected and characterized. Thirteen of the isolates produced characteristics similar to those of the reference strains of Azospirillum, and the remaining 3 isolates were found to be Enterobacter spp. The isolates could be categorized into 3 groups based on their ARDRA patterns, and the first 2 groups comprised Azospirillum brasilense and Azospirillum lipoferum. The acetylene reduction activity (ARA) of these isolates was determined for free cultures and in association with wheat roots. There was no correlation between pure culture and plant-associated nitrogenase activity of the different strains. The isolates that showed higher nitrogenase activities in association with wheat roots in each group were selected and sequenced. Isolates of Azospirillum brasilense CW301, Azospirillum brasilense CW903, and Azospirillum lipoferum CW1503 were selected to study colonization in association with wheat roots. We observed higher expression of beta-galactosidase activity in A. brasilense strains than in A. lipoferum strains, which could be attributed to their higher population in association with wheat roots. All strains tested colonized and exhibited the strongest beta-galactosidase activity at the sites of lateral roots emergence.     

Use of random amplified polymorphic DNA markers for the detection of Azospirillum strains in soil microcosms

Probes for the detection of Azospirillum strains were obtained from DNA fragments generated by random amplification of polymorphic DNA (RAPD) and tested to assess their specificity towards DNA extracted from pure cultures. The most specific probe, referred to as α4, produced a hybridization signal only with amplified DNA of A. lipoferum ATCC29731. This strain was inoculated, together with two other Azospirillum strains, in soil microcosms of different complexity and its presence tested with the probe α4. This probe confirmed its high specificity with amplified DNA extracted from the soil microcosm and in the presence of other A. lipoferum strains, indicating that the strategy for bacterial detection, based on RAPD markers, is useful for monitoring the presence of a particular strain under environment-like conditions. Other RAPD-derived probes, when tested on soil samples, did not show the same level of specificity as that shown on DNA from pure cultures. This result suggests that some precautions are necessary in the choice of a really specific RAPD marker. In a further development of this strategy, the α4 probe was sequenced and two pairs of “nested” primers were designed, which enabled a diagnostic polymerase chain reaction from soil samples that was specific for the A. lipoferum species. Received: 7 July 1997 / Accepted: 14 October 1997     

Applicability of the 16S–23S rDNA internal spacer for PCR detection of the phytostimulatory PGPR inoculant Azospirillum lipoferum CRT1 in field soil

Aims:  To assess the applicability of the 16S–23S rDNA internal spacer regions (ISR) as targets for PCR detection of Azospirillum ssp. and the phytostimulatory plant growth-promoting rhizobacteria seed inoculant Azospirillum lipoferum CRT1 in soil.
Methods and Results:  Primer sets were designed after sequence analysis of the ISR of A. lipoferum CRT1 and Azospirillum brasilense Sp245. The primers fAZO/rAZO targeting the Azospirillum genus successfully yielded PCR amplicons (400–550 bp) from Azospirillum strains but also from certain non- Azospirillum strains in vitro , therefore they were not appropriate to monitor indigenous Azospirillum soil populations. The primers fCRT1/rCRT1 targeting A. lipoferum CRT1 generated a single 249-bp PCR product but could also amplify other strains from the same species. However, with DNA extracts from the rhizosphere of field-grown maize, both fAZO/rAZO and fCRT1/rCRT1 primer sets could be used to evidence strain CRT1 in inoculated plants by nested PCR, after a first ISR amplification with universal ribosomal primers. In soil, a 7-log dynamic range of detection (10²–10⁸ CFU g⁻¹ soil) was obtained.
Conclusions:  The PCR primers targeting 16S–23S rDNA ISR sequences enabled detection of the inoculant A. lipoferum CRT1 in field soil.
Significance and Impact of the Study:  Convenient methods to monitor Azospirillum phytostimulators in the soil are lacking. The PCR protocols designed based on ISR sequences will be useful for detection of the crop inoculant A. lipoferum CRT1 under field conditions.     

Search for conjugative plasmids in Azospirillum brasilense and Azospirillum lipoferum associated with plants

The conjugative plasmids in Azospirillum brasilense strains S17. Sp107, Sp245, SpBr14, JM6B2, JM82Al, UQ1794, UQI796 and in Azospirillum lipoferum strain RG20 were prove to exist for the first time in connection with their potency to mobilize a non-conjugative IncQ-plasmid pVZ361 from IncQ-group (ori RSF1010, KmR, SuR. 11.4 kb) for conjugated transfer to aplasmid strains Agrobacterium tumefaciens and Pseudomonas putida at high frequencies.     

Influence of amino acids on nitrogen fixation ability and growth of Azospirillum spp

The utilization of amino acids for growth and their effects on nitrogen fixation differ greatly among the several strains of each species of Azospirillum spp. that were examined. A. brasiliense grew poorly or not at all on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources. Nitrogen fixation by most A. brasiliense strains was inhibited only slightly even by 10 mM concentrations of these amino acids. In contrast, A. lipoferum and A. amazonense grew very well on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources; nitrogen fixation, which was measured in the presence of malate or sucrose, was severely inhibited by these amino acids. It was concluded that growth on histidine as the sole source of nitrogen, carbon, and energy may be used for the taxonomic characterization of Azospirillum spp. and for the selective isolation of A. lipoferum. The different utilization of various amino acids by Azospirillum spp. may be important for their establishment in the rhizosphere and for their associative nitrogen fixation with plants. The physiological basis for the different utilization of glutamate by Azospirillum spp. was investigated further. A. brasiliense and A. lipoferum exhibited a high affinity for glutamate uptake (Km values for uptake were 8 and 40 microM, respectively); the Vmax was 6 times higher in A. lipoferum than in A. brasiliense. At high substrate concentrations (10 mM), the nonsaturable component of glutamate uptake was most active in A. lipoferum and A. amazonense.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of amino acids on nitrogen fixation ability and growth of Azospirillum spp.

The utilization of amino acids for growth and their effects on nitrogen fixation differ greatly among the several strains of each species of Azospirillum spp. that were examined. A. brasiliense grew poorly or not at all on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources. Nitrogen fixation by most A. brasiliense strains was inhibited only slightly even by 10 mM concentrations of these amino acids. In contrast, A. lipoferum and A. amazonense grew very well on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources; nitrogen fixation, which was measured in the presence of malate or sucrose, was severely inhibited by these amino acids. It was concluded that growth on histidine as the sole source of nitrogen, carbon, and energy may be used for the taxonomic characterization of Azospirillum spp. and for the selective isolation of A. lipoferum. The different utilization of various amino acids by Azospirillum spp. may be important for their establishment in the rhizosphere and for their associative nitrogen fixation with plants. The physiological basis for the different utilization of glutamate by Azospirillum spp. was investigated further. A. brasiliense and A. lipoferum exhibited a high affinity for glutamate uptake (Km values for uptake were 8 and 40 microM, respectively); the Vmax was 6 times higher in A. lipoferum than in A. brasiliense. At high substrate concentrations (10 mM), the nonsaturable component of glutamate uptake was most active in A. lipoferum and A. amazonense.(ABSTRACT TRUNCATED AT 250 WORDS)     

Restriction endonucleases in Azospirillum

Azospirillum brasilense, A. amazonense, and A. lipoferum strains were screened for restriction endonucleases using phage lambda DNA. The extract of A. brasilense 29711 cleaved lambda DNA into specific fragments. It was concluded that this strain possesses a class II restriction endonuclease which was named AbrI. AbrI has a single recognition site on lambda DNA at position of approx. 33 500 bp. AbrI was characterized as an isoschizomer of XhoI, which cuts lambda DNA at 33 498 bp and cleaves double-stranded DNA at the sequence 5'-C TCGAG-3'. From other Azospirilla strains only A. amazonense QRZ42 extracts (AamI activity) cleaved DNA into specific fragments under certain conditions.     

Isolation and cloning of a Azospirillum lipoferum locus that complements Escherichia coli proU mutant

Glycine betaine relieved sodium chloride-mediated inhibition of growth in Azospirillum lipoferum ATCC 29708. ³⁵S-methionine labelling of proteins after salinity up-shock revealed strong induction of a 30 kDa protein which cross-reacted with the anti-glycine betaine binding protein antibody from Escherichia coli. This suggested that A. lipoferum had a salinity-induced ProU-like high-affinity glycine betaine transport system. A genomic library of A. lipoferum ATCC 29708 was screened for the proU-like gene by complementation of a proU mutant of E. coli. Four recombinant cosmids, capable of restoring growth of the proU mutant on plates containing 600 mM NaCl and 1 mM glycine betaine were selected. Selected recombinant cosmids hybridized with a proU gene probe from E. coli. Complementation of E. coli proU mutant with the A. lipoferum genomic DNA was evident by the ability of proU mutant (containing selected recombinant cosmids) to grow on minimal medium supplemented with 600 mM NaCl and 1 mM glycine betaine.     

The effect of combined and separate inoculation of alfalfa plants with Azospirillum lipoferum and Sinorhizobium meliloti on denitrification and nitrogen-fixing activities

The effects of associative nitrogen fixer Azospirillum lipoferum strain 137 and root nodule bacteria Sinorhizobium meliloti after combined and separate inoculation of alfalfa seedlings on the background of mineral nitrogen applied at various times were studied. It was demonstrated that exudates of the alfalfa seedlings with the first pair of cotyledonary leaves already provide a high activity of these bacteria in the rhizosphere. To 74.6% of the introduced nitrate was transformed into N2O when the binary preparation of these bacteria was used. In an extended experiment (30 days), an active reduction of nitrates to N2O (11 micromol N2O/pot x 24 h) with inhibition of nitrogen fixation was observed in all of the experimental variants during the formation of legume-rhizobial and associative symbioses and simultaneous introduction of nitrates and bacteria. The most active enzyme fixation was observed in the case of a late (after 14 days) application of nitrates in the variants with both separate inoculations and inoculation with the binary preparation of A. lipoferum and S. meliloti. Separation in time of the application of bacterial preparations and mineral nitrogen assisted its preservation in all of the experimental variants. The variant of alfalfa inoculation with the binary preparation of A. lipoferum and S. meliloti and application of nitrates 2 weeks after inoculation was optimal for active nitrogen fixation (224.7 C2H4 nmol/flask x 24 h) and low denitrification activity (1.8 x micromol N2O/flask x 24 h). These results are useful in applied developments aimed at the use of bacterial and mineral fertilizers for leguminous plants.