BACKGROUND
many of the negative ecological impacts of agriculture originate from the high input of fertilizers.
the increase of crop production in the future raises concerns about how to establish sustainable agriculture; that is, agricultural practices that are less adverse to the surrounding environment  <cit> .
the use of microorganisms capable of increasing harvests is an ecologically compatible strategy as it could reduce the utilization of industrial fertilizers and, therefore, their pollutant outcomes  <cit> .
azospirillum is a well-known genus that includes bacterial species that can promote plant growth.
this remarkable characteristic is attributed to a combination of mechanisms, including the biosynthesis of phytohormones and the fixation of nitrogen, the most intensively studied abilities of these bacteria  <cit> .
the species azospirillum amazonense was isolated from forage grasses and plants belonging to the palmaceae family in brazil by magalhães et al.
<cit> , and subsequent works demonstrated its association with rice, sorghum, maize, sugarcane, and brachiaria, mainly in tropical countries  <cit> .
when compared with azospirillum brasilense, the most frequently studied species of the genus, a. amazonense has prominent characteristics such as its ability to fix nitrogen when in the presence of nitrogen  <cit>  and its better adaptations to acidic soil, the predominant soil type in brazil  <cit> .
moreover, rodrigues et al.
<cit>  reported that the plant growth promotion effect of a. amazonense on rice plants grown under greenhouse conditions is mainly due to its biological nitrogen fixation contribution, in contrast to the hormonal effect observed in the other azospirillum species studied.
despite the potential use of a. amazonense as an agricultural inoculant, there is scarce knowledge of its genetics and, consequently, its physiology.
currently, the genome of a. amazonense is being analyzed by our group and its completion will be forthcoming; therefore, the development of specific genetic tools is crucial for taking full benefit of the data.
that will be generated.
hence, in this work we describe methods for the genetic manipulation of a. amazonense: dna transfer methodologies , reporter vectors, and site-directed mutagenesis.
in order to demonstrate the applicability of the optimized techniques, we show the results obtained in the study of the pii signaling proteins of a. amazonense, starting from their gene isolation.
RESULTS
isolation of glnb and glnk genes from a. amazonense
the pii proteins are pivotal regulators of the nitrogen metabolism, controlling the activities of transporters, enzymes and transcriptional factors implicated in this process  <cit> .
these proteins are highly conserved and are widely distributed throughout prokaryotes  <cit> .
in proteobacteria in particular, there are two main types of pii proteins, glnb and glnk.
in this work, two pii protein encoding genes from a. amazonense were isolated.
southern blot analysis utilizing a pcr-generated glnb fragment as the probe revealed two distinct signals in the genomic dna of a. amazonense digested with sali: the strongest at the ~ <dig> kb dna fragments and the weakest at the ~ <dig> kb dna fragments .
based on these results, a genomic library enriched with 2- <dig> kb sali fragments was constructed.
the library was partially sequenced and a pii protein homolog was identified.
the deduced amino acid sequence of this gene was found to be highly similar to that of the glnz proteins  from a. brasilense and azospirillum sp.
b <dig> , and rhodospirillum.
centenum .
arcondéguy et al.
<cit>  suggested that the glnz genes should be termed glnk, since their deduced proteins are highly similar to the glnk proteins.
furthermore, there is a functional correspondence between these proteins, as both regulate the uptake of ammonium through the amtb transporters  <cit> .
therefore, we adopted the glnk designation for this a. amazonense homolog, mainly because this nomenclature could facilitate comparisons between other bacterial systems.
the glnk gene from a. amazonense is flanked by the aat gene in the downstream region, which codes a putative aspartate aminotransferase and the ubih gene in the upstream region, which codes an enzyme implicated in ubiquinone biosynthesis .
this genetic organization resembles that found in other species from the rhodospirillales order, namely a. brasilense, azospirillum sp.
b <dig> and r. centenum.
since the glnb gene was not found in the genomic library, the inverse pcr methodology was carried out to isolate this gene.
a ~ <dig> kb amplicon that contained the glnb gene was obtained .
it was found that the protein of this gene displays 92% identity and 98% similarity to the glnb proteins from azospirillum sp.
b <dig> and a. brasilense, and 96% identity and 98% similarity to the glnb protein of r. centenum.
the glnb gene is located upstream of the glna gene , the same genetic context observed in these bacteria .
in a. brasilense, glnb has a key role in nitrogen fixation because its protein product regulates the activity of nifa, the transcriptional factor of nitrogen fixation  <cit> .
furthermore, both of the glnz  and glnb proteins are also implicated in the drat/drag system, which regulates dinitrogenase reductase activity by covalent modifications  <cit> .
however, fu et al.
<cit>  verified that a. amazonense does not have the drat/drag system.
hence, in the near future, the interaction targets of the pii protein in a. amazonense should be determined to better understand their roles in the nitrogen metabolism of this microorganism.
antibiotic minimum inhibitory concentration
most dna manipulation is dependent on the use of vectors containing resistance markers to antibiotics  <cit> .
in a previous work using antibiotic susceptibility test discs, magalhães et al.
<cit>  showed that a. amazonense is sensitive to kanamycin and gentamicin, tolerant to tetracycline, and resistant to penicillin.
in this work, we determined the minimum inhibitory concentrations of a. amazonense to antibiotics that are normally used to provide a selective pressure for vectors.
the susceptibility of a. amazonense to kanamycin and gentamicin was confirmed, since no growth was observed in concentrations of these antibiotics of  <dig>  μg/ml; therefore, vectors that contain selection markers for these compounds are appropriate for use.
high concentrations of ampicillin  were required for complete growth inhibition, showing that a. amazonense is also resistant to this beta-lactam antibiotic.
it is worth noting that the growth of a. amazonense was absent in a relatively high concentration of tetracycline , indicating that this species is, in fact, resistant to this antibiotic, instead of tolerant, as pointed out by magalhães et al.
<cit> .
these findings about the latter two antibiotics are relevant because they could be used in counter-selection procedures in conjugation experiments, as there is a variety of e. coli strains that are susceptible to them.
conjugation
conjugation mediated by e. coli is the standard dna transfer technique of the azospirillum genus  <cit> .
therefore, in this work the conjugation ability of a. amazonense was evaluated.
unlike a. brasilense, a. amazonense cannot grow in lb medium.
furthermore, e. coli cannot grow in m <dig> medium; therefore, the first concern was to establish a medium that provided appropriate growth conditions for the donor and recipient strains.
hence, different medium compositions, containing distinct ratios of m <dig> and lb media , were prepared.
the medium mixture of m79:lb at a proportion of 8: <dig> was the most suitable for culturing both bacteria and it was designated as mlb medium.
another requisite for the conjugation procedure is to select vectors that contain proper selection markers that are mobilizable and able to replicate inside the receptor cell  <cit> .
therefore, the phrgfpgus  and the ppzplaceyfp  plasmids were tested by tri-parental conjugation.
these plasmids are mobilizable broad-host vectors harboring kanamycin resistance markers and fluorescent protein coding genes, which could promptly report achievement of the dna transfer.
the transconjugants exhibited kanamycin resistance and fluorescence.
the conjugation frequencies were  <dig>  × 10- <dig> per recipient cell for the phrgfpgus vector and  <dig>  × 10- <dig> for the ppzplaceyfp vector.
different ratios of recipient to donor and helper strains  were also tested.
the best efficiencies were obtained with the ratios 10:1: <dig> and 5:1:1; however, no obvious differences between these latter ratios were observed .
in conclusion, conjugation is an appropriate method for dna transfer to a. amazonense.
although only tri-parental mating was tested in this work, it is important to mention that bi-parental conjugation could be an alternative test, due to the possibility of increasing the conjugation efficiencies.
electrotransformation
since suitable vectors for a. amazonense were defined and since conjugation is a time-consuming procedure, the transformation of a. amazonense via electroporation was tested.
the eletrocompetence of the cells is greatly influenced by the growth phase  <cit> .
therefore, a. amazonense cells were harvested at different growth phases to evaluate their effect on electroporation efficiency.
cells from the late-log phase  and the stationary phase  were not electrocompetent.
electroporation utilizing cells from the early-log growth phase  generated a significant number of transformants.
therefore, all subsequent tests were performed utilizing cells cultivated at this growth phase.
in the electrocompetent cell preparation, the cells were harvested and washed continuously until the solution had a low-ionic strength.
the mgcl <dig> hepes-sucrose buffer was found to be the most suitable solution for the preparation of a. amazonense electrocompetent cells.
although 10% glycerol solution is commonly used for electrocompetent cell preparation in a diverse number of species , it was not appropriate for a. amazonense, as no transformants were obtained when this solution was used.
different electroporation parameters were tested.
the increase in electrical field strength had a positive effect on electroporation efficiency .
the highest electrical field strength tested was  <dig>  kv/cm, and this condition was found to be the most efficient, generating about  <dig> transformants/μg of phrgfpgus .
the effect of pulse length on electroporation efficiency was also investigated .
a pulse length of  <dig>  ms  was the most efficient.
the pulse lengths of  <dig>  ms  and  <dig>  ms  had a dramatic negative effect on transformation efficiency, where only few transformants were obtained .
these conditions are in agreement with the general parameters of bacterial electroporation  <cit> .
in conclusion, the transfer of dna to a. amazonense by means of electroporation was demonstrated.
although the efficiency of electrotransformation was far from desirable, this result is supported by previous works showing that bacteria closely related to a. amazonense, such as a. brasilense  <cit> , r. rubrum  <cit>  and magnetospirillum gryphiswaldense  <cit> , are recalcitrant to electrotransformation.
nonetheless, this technique is an easy and a rapid method of dna transfer to the cells of a. amazonense.
site-directed mutagenesis
site-directed mutagenesis is a fundamental tool for correlating cellular functions with specific regions of the dna.
therefore, once dna transfer techniques were established for a. amazonense, the next step was to determine a site-directed mutagenesis protocol for this species.
most of the a. brasilense mutants have been generated by the disruptive insertion of an antibiotic resistance cassette into the target gene  <cit> .
this approach is not recommended when the target gene composes an operon, since the resistance cassette could introduce a polar effect on the expression of the surrounding genes and, consequently, make it difficult to assign a mutant phenotype to the disrupted gene  <cit> .
therefore, in this work, a site-directed mutagenesis methodology that generates in-frame mutants without the disruptive insertion of a resistance cassette was evaluated.
the glnk gene was selected for this methodology because subsequent studies of our laboratory will aim to determine the role of the pii proteins in a. amazonense metabolism.
the mutagenesis methodology is depicted in figure 3a.
firstly, an amplicon containing an in-frame deletion of the glnk gene was generated through crossover pcr, and it was subsequently cloned in the suicide replacement vector pk19mobsacb, generating the pkΔk plasmid.
this vector contains a kanamycin resistance gene  that allows the selection of bacteria that would have integrated the plasmid into the chromosome.
this vector was delivered to a. amazonense by means of conjugation  and one colony resistant to kanamycin was obtained, suggesting that the integration of the plasmid was successfully accomplished.
the sacb gene  of the vector is lethal in the presence of sucrose; therefore, the merodiploid strain  was unable to grow in m <dig> .
subsequently, expecting that a recombination event could replace the wild-type allele, the merodiploid strain was cultured for many generations in m <dig> containing maltose instead of sucrose.
finally, this culture was plated in m <dig> containing sucrose to eliminate the bacteria that did not accomplish the second recombination event.
seven sucrose-resistant/kanamycin-sensitive colonies were chosen for pcr evaluation of the substitution of the mutant allele for the wild-type gene.
four colonies presented a band of  <dig> bp, indicating that the wild-type glnk was successfully substituted, whereas three colonies presented the  <dig> bp band, corresponding to the wild-type allele .
furthermore, an additional pcr with primers flanking the recombination sites was performed, and it also demonstrated a reduction of the amplicon sizes originated from the glnk mutants in relation to the wild type strain .
this latter result demonstrates that recombination occurred in the target site.
altogether, these results show that an in-frame glnk gene mutant strain of a. amazonense was successfully generated by this mutagenesis system.
reporter gene system
the study of promoters is fundamental to elucidation of the genetic regulatory mechanisms of bacterial species.
up until now, there has been neither a report of heterologous gene expression in a. amazonense, nor a reporter system designed for this species.
in this work, a reporter system based on expression of the enhanced yellow fluorescent protein  was developed to analyze the regulatory regions of a. amazonense genes in vivo.
in silico analysis using a sinorhizobium meliloti sigma  <dig> promoter weight matrix revealed that the genes aat, glnk, and glnb of a. amazonense have putative promoter sequences in their upstream regions .
in e. coli, sigma  <dig> is considered to be the vegetative sigma factor, as it is responsible for the expression of the majority of genes  <cit> .
therefore, one could expect that these putative a. amazonense sigma  <dig> promoters could act under standard laboratory growth conditions .
consequently, different vectors were constructed to determine the activity of the upstream regulatory sequences of a. amazonense genes in the expression of eyfp.
the lac promoter was utilized as a positive control since there is a report showing that this promoter has high activity in a. brasilense  <cit> .
two different vectors were constructed with the lac promoter, one derived from ppzplaceyfp  and the other derived from phrgfpgus .
the upstream regions of the genes glnb, glnk, and aat were cloned into the phrgfpgus derivative.
the lac promoter had the best score in the in silico analysis from among the promoters detected, and, as expected, the highest fluorescence levels were observed in the lac constructions .
the difference in the fluorescence levels between the phrlaceyfp and ppzplaceyfp transformants could be a product of the difference in the copy number between these vectors.
although the in silico analysis revealed that the glnk promoter had a higher score than the aat and glnb promoters, its in vivo activity under the conditions tested did not differ significantly from the negative controls  .
one of the possible reasons for this is that this gene was repressed under these conditions.
the reporter gene analysis also demonstrated that the aat and glnb promoters were active under the conditions tested, although the aat promoter showed a higher activity than the glnb promoter.
these observations show that a reporter system based on eyfp can be used for in vivo promoter analyses in a. amazonense.
CONCLUSIONS
genetic manipulation is fundamental for taking full advantage of the information generated by dna sequences  <cit> .
thus, in the present work, we described a series of tools that could assist genetic studies of the diazotrophic bacteria a. amazonense, a microorganism presenting potential for use as an agricultural inoculant.

 many of the negative ecological impacts of agriculture originate from the high input of fertilizers. the increase of crop production in the future raises concerns about how to establish sustainable agriculture; that is, agricultural practices that are less adverse to the surrounding environment. the use of microorganisms capable of increasing harvests is an ecologically compatible strategy as it could reduce the utilization of industrial fertilizers and, therefore, their pollutant outcomes. azospirillum is a well-known genus that includes bacterial species that can promote plant growth. this remarkable characteristic is attributed to a combination of mechanisms, including the biosynthesis of phytohormones and the fixation of nitrogen, the most intensively studied abilities of these bacteria. the species azospirillum amazonense was isolated from forage grasses and plants belonging to the palmaceae family in brazil by magalhães et al., and subsequent works demonstrated its association with rice, sorghum, maize, sugarcane, and brachiaria, mainly in tropical countries. when compared with azospirillum brasilense, the most frequently studied species of the genus, a. amazonense has prominent characteristics such as its ability to fix nitrogen when in the presence of nitrogen and its better adaptations to acidic soil, the predominant soil type in brazil. moreover, rodrigues et al. reported that the plant growth promotion effect of a. amazonense on rice plants grown under greenhouse conditions is mainly due to its biological nitrogen fixation contribution, in contrast to the hormonal effect observed in the other azospirillum species studied. despite the potential use of a. amazonense as an agricultural inoculant, there is scarce knowledge of its genetics and, consequently, its physiology. currently, the genome of a. amazonense is being analyzed by our group and its completion will be forthcoming; therefore, the development of specific genetic tools is crucial for taking full benefit of the data. that will be generated. hence, in this work we describe methods for the genetic manipulation of a. amazonense: dna transfer methodologies, reporter vectors, and site-directed mutagenesis. in order to demonstrate the applicability of the optimized techniques, we show the results obtained in the study of the pii signaling proteins of a. amazonense, starting from their gene isolation.  isolation of glnb and glnk genes from a. amazonense the pii proteins are pivotal regulators of the nitrogen metabolism, controlling the activities of transporters, enzymes and transcriptional factors implicated in this process. these proteins are highly conserved and are widely distributed throughout prokaryotes. in proteobacteria in particular, there are two main types of pii proteins, glnb and glnk. in this work, two pii protein encoding genes from a. amazonense were isolated. southern blot analysis utilizing a pcr-generated glnb fragment as the probe revealed two distinct signals in the genomic dna of a. amazonense digested with sali: the strongest at the ~ kb dna fragments and the weakest at the ~ kb dna fragments. based on these results, a genomic library enriched with 2- kb sali fragments was constructed. the library was partially sequenced and a pii protein homolog was identified. the deduced amino acid sequence of this gene was found to be highly similar to that of the glnz proteins from a. brasilense and azospirillum sp. b, and rhodospirillum. centenum. arcondéguy et al. suggested that the glnz genes should be termed glnk, since their deduced proteins are highly similar to the glnk proteins. furthermore, there is a functional correspondence between these proteins, as both regulate the uptake of ammonium through the amtb transporters. therefore, we adopted the glnk designation for this a. amazonense homolog, mainly because this nomenclature could facilitate comparisons between other bacterial systems. the glnk gene from a. amazonense is flanked by the aat gene in the downstream region, which codes a putative aspartate aminotransferase and the ubih gene in the upstream region, which codes an enzyme implicated in ubiquinone biosynthesis. this genetic organization resembles that found in other species from the rhodospirillales order, namely a. brasilense, azospirillum sp. b and r. centenum. since the glnb gene was not found in the genomic library, the inverse pcr methodology was carried out to isolate this gene. a ~ kb amplicon that contained the glnb gene was obtained. it was found that the protein of this gene displays 92% identity and 98% similarity to the glnb proteins from azospirillum sp. b and a. brasilense, and 96% identity and 98% similarity to the glnb protein of r. centenum. the glnb gene is located upstream of the glna gene, the same genetic context observed in these bacteria. in a. brasilense, glnb has a key role in nitrogen fixation because its protein product regulates the activity of nifa, the transcriptional factor of nitrogen fixation. furthermore, both of the glnz and glnb proteins are also implicated in the drat/drag system, which regulates dinitrogenase reductase activity by covalent modifications. however, fu et al. verified that a. amazonense does not have the drat/drag system. hence, in the near future, the interaction targets of the pii protein in a. amazonense should be determined to better understand their roles in the nitrogen metabolism of this microorganism. antibiotic minimum inhibitory concentration most dna manipulation is dependent on the use of vectors containing resistance markers to antibiotics. in a previous work using antibiotic susceptibility test discs, magalhães et al. showed that a. amazonense is sensitive to kanamycin and gentamicin, tolerant to tetracycline, and resistant to penicillin. in this work, we determined the minimum inhibitory concentrations of a. amazonense to antibiotics that are normally used to provide a selective pressure for vectors. the susceptibility of a. amazonense to kanamycin and gentamicin was confirmed, since no growth was observed in concentrations of these antibiotics of μg/ml; therefore, vectors that contain selection markers for these compounds are appropriate for use. high concentrations of ampicillin were required for complete growth inhibition, showing that a. amazonense is also resistant to this beta-lactam antibiotic. it is worth noting that the growth of a. amazonense was absent in a relatively high concentration of tetracycline, indicating that this species is, in fact, resistant to this antibiotic, instead of tolerant, as pointed out by magalhães et al.. these findings about the latter two antibiotics are relevant because they could be used in counter-selection procedures in conjugation experiments, as there is a variety of e. coli strains that are susceptible to them. conjugation conjugation mediated by e. coli is the standard dna transfer technique of the azospirillum genus. therefore, in this work the conjugation ability of a. amazonense was evaluated. unlike a. brasilense, a. amazonense cannot grow in lb medium. furthermore, e. coli cannot grow in m medium; therefore, the first concern was to establish a medium that provided appropriate growth conditions for the donor and recipient strains. hence, different medium compositions, containing distinct ratios of m and lb media, were prepared. the medium mixture of m79:lb at a proportion of 8: was the most suitable for culturing both bacteria and it was designated as mlb medium. another requisite for the conjugation procedure is to select vectors that contain proper selection markers that are mobilizable and able to replicate inside the receptor cell. therefore, the phrgfpgus and the ppzplaceyfp plasmids were tested by tri-parental conjugation. these plasmids are mobilizable broad-host vectors harboring kanamycin resistance markers and fluorescent protein coding genes, which could promptly report achievement of the dna transfer. the transconjugants exhibited kanamycin resistance and fluorescence. the conjugation frequencies were × 10- per recipient cell for the phrgfpgus vector and × 10- for the ppzplaceyfp vector. different ratios of recipient to donor and helper strains were also tested. the best efficiencies were obtained with the ratios 10:1: and 5:1:1; however, no obvious differences between these latter ratios were observed. in conclusion, conjugation is an appropriate method for dna transfer to a. amazonense. although only tri-parental mating was tested in this work, it is important to mention that bi-parental conjugation could be an alternative test, due to the possibility of increasing the conjugation efficiencies. electrotransformation since suitable vectors for a. amazonense were defined and since conjugation is a time-consuming procedure, the transformation of a. amazonense via electroporation was tested. the eletrocompetence of the cells is greatly influenced by the growth phase. therefore, a. amazonense cells were harvested at different growth phases to evaluate their effect on electroporation efficiency. cells from the late-log phase and the stationary phase were not electrocompetent. electroporation utilizing cells from the early-log growth phase generated a significant number of transformants. therefore, all subsequent tests were performed utilizing cells cultivated at this growth phase. in the electrocompetent cell preparation, the cells were harvested and washed continuously until the solution had a low-ionic strength. the mgcl hepes-sucrose buffer was found to be the most suitable solution for the preparation of a. amazonense electrocompetent cells. although 10% glycerol solution is commonly used for electrocompetent cell preparation in a diverse number of species, it was not appropriate for a. amazonense, as no transformants were obtained when this solution was used. different electroporation parameters were tested. the increase in electrical field strength had a positive effect on electroporation efficiency. the highest electrical field strength tested was kv/cm, and this condition was found to be the most efficient, generating about transformants/μg of phrgfpgus. the effect of pulse length on electroporation efficiency was also investigated. a pulse length of ms was the most efficient. the pulse lengths of ms and ms had a dramatic negative effect on transformation efficiency, where only few transformants were obtained. these conditions are in agreement with the general parameters of bacterial electroporation. in conclusion, the transfer of dna to a. amazonense by means of electroporation was demonstrated. although the efficiency of electrotransformation was far from desirable, this result is supported by previous works showing that bacteria closely related to a. amazonense, such as a. brasilense, r. rubrum and magnetospirillum gryphiswaldense, are recalcitrant to electrotransformation. nonetheless, this technique is an easy and a rapid method of dna transfer to the cells of a. amazonense. site-directed mutagenesis site-directed mutagenesis is a fundamental tool for correlating cellular functions with specific regions of the dna. therefore, once dna transfer techniques were established for a. amazonense, the next step was to determine a site-directed mutagenesis protocol for this species. most of the a. brasilense mutants have been generated by the disruptive insertion of an antibiotic resistance cassette into the target gene. this approach is not recommended when the target gene composes an operon, since the resistance cassette could introduce a polar effect on the expression of the surrounding genes and, consequently, make it difficult to assign a mutant phenotype to the disrupted gene. therefore, in this work, a site-directed mutagenesis methodology that generates in-frame mutants without the disruptive insertion of a resistance cassette was evaluated. the glnk gene was selected for this methodology because subsequent studies of our laboratory will aim to determine the role of the pii proteins in a. amazonense metabolism. the mutagenesis methodology is depicted in figure 3a. firstly, an amplicon containing an in-frame deletion of the glnk gene was generated through crossover pcr, and it was subsequently cloned in the suicide replacement vector pk19mobsacb, generating the pkΔk plasmid. this vector contains a kanamycin resistance gene that allows the selection of bacteria that would have integrated the plasmid into the chromosome. this vector was delivered to a. amazonense by means of conjugation and one colony resistant to kanamycin was obtained, suggesting that the integration of the plasmid was successfully accomplished. the sacb gene of the vector is lethal in the presence of sucrose; therefore, the merodiploid strain was unable to grow in m. subsequently, expecting that a recombination event could replace the wild-type allele, the merodiploid strain was cultured for many generations in m containing maltose instead of sucrose. finally, this culture was plated in m containing sucrose to eliminate the bacteria that did not accomplish the second recombination event. seven sucrose-resistant/kanamycin-sensitive colonies were chosen for pcr evaluation of the substitution of the mutant allele for the wild-type gene. four colonies presented a band of bp, indicating that the wild-type glnk was successfully substituted, whereas three colonies presented the bp band, corresponding to the wild-type allele. furthermore, an additional pcr with primers flanking the recombination sites was performed, and it also demonstrated a reduction of the amplicon sizes originated from the glnk mutants in relation to the wild type strain. this latter result demonstrates that recombination occurred in the target site. altogether, these results show that an in-frame glnk gene mutant strain of a. amazonense was successfully generated by this mutagenesis system. reporter gene system the study of promoters is fundamental to elucidation of the genetic regulatory mechanisms of bacterial species. up until now, there has been neither a report of heterologous gene expression in a. amazonense, nor a reporter system designed for this species. in this work, a reporter system based on expression of the enhanced yellow fluorescent protein was developed to analyze the regulatory regions of a. amazonense genes in vivo. in silico analysis using a sinorhizobium meliloti sigma promoter weight matrix revealed that the genes aat, glnk, and glnb of a. amazonense have putative promoter sequences in their upstream regions. in e. coli, sigma is considered to be the vegetative sigma factor, as it is responsible for the expression of the majority of genes. therefore, one could expect that these putative a. amazonense sigma promoters could act under standard laboratory growth conditions. consequently, different vectors were constructed to determine the activity of the upstream regulatory sequences of a. amazonense genes in the expression of eyfp. the lac promoter was utilized as a positive control since there is a report showing that this promoter has high activity in a. brasilense. two different vectors were constructed with the lac promoter, one derived from ppzplaceyfp and the other derived from phrgfpgus. the upstream regions of the genes glnb, glnk, and aat were cloned into the phrgfpgus derivative. the lac promoter had the best score in the in silico analysis from among the promoters detected, and, as expected, the highest fluorescence levels were observed in the lac constructions. the difference in the fluorescence levels between the phrlaceyfp and ppzplaceyfp transformants could be a product of the difference in the copy number between these vectors. although the in silico analysis revealed that the glnk promoter had a higher score than the aat and glnb promoters, its in vivo activity under the conditions tested did not differ significantly from the negative controls. one of the possible reasons for this is that this gene was repressed under these conditions. the reporter gene analysis also demonstrated that the aat and glnb promoters were active under the conditions tested, although the aat promoter showed a higher activity than the glnb promoter. these observations show that a reporter system based on eyfp can be used for in vivo promoter analyses in a. amazonense.  genetic manipulation is fundamental for taking full advantage of the information generated by dna sequences. thus, in the present work, we described a series of tools that could assist genetic studies of the diazotrophic bacteria a. amazonense, a microorganism presenting potential for use as an agricultural inoculant. 
azospirillum amazonense has potential to be used as agricultural inoculant since it promotes plant growth without causing pollution, unlike industrial fertilizers. furthermore, glnk-specific a. amazonense mutants were generated utilizing the pk19mobsacb vector system. in this work, genetic tools that can support the study of a. amazonense were described.
 azospirillum amazonense has potential to be used as agricultural inoculant since it promotes plant growth without causing pollution, unlike industrial fertilizers. owing to this fact, the study of this species has gained interest. however, a detailed understanding of its genetics and physiology is limited by the absence of appropriate genetic tools for the study of this species.  conjugation and electrotransformation methods were established utilizing vectors with broad host-replication origins. two genes of interest - glnk and glnb, encoding pii regulatory proteins - were isolated. furthermore, glnk-specific a. amazonense mutants were generated utilizing the pk19mobsacb vector system. finally, a promoter analysis protocol based on fluorescent protein expression was optimized to aid genetic regulation studies on this bacterium.  in this work, genetic tools that can support the study of a. amazonense were described. these methods could provide a better understanding of the genetic mechanisms of this species that underlie its plant growth promotion. 