Genetic Modifications of Phosphate
Solubilizing Bacteria To Be Used as Agricultural Inoculants
Hilda Rodríguez1,2, Reynaldo Fraga1,
Tania Gonzalez1 and Yoav Bashan2
2Environmental Microbiology, The Center for Biological
Research of the Northwest, La Paz B.C.S. 23 000, MÉXICO
E-mail: hrodri@cibnor.mx
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Plant growth-promoting bacteria (PGPB) are soil and rhizosphere bacteria that can benefit plant growth by different mechanisms (1). Their use as natural biofertilizers is advantageous, not only from the economical, but also from the ecological point of view. A large proportion of phosphorous in soil is present in an insoluble form, and therefore, not available for plant nutrition. The ability of some microorganisms to convert insoluble P to an accessible form, like orthophosphate, is an important trait for a PGPB for increasing plant yields (2).
Introduction or
over-expression of genes involved in soil P solubilization in natural
rhizosphere bacteria is a very attractive approach to improve
microorganisms’ capacity as inoculants. Here, we present recent advances
in the manipulation of genes related to microbial P solubilization and its
relationship with the use of rhizobacteria as improved inoculants.
There are two components of P in soil,
organic and inorganic phosphates. Phosphorous can be released form organic
compounds in soil by means of 3 groups of enzymes: phosphatases, phytases, and
phosphonatases. The major role apparently corresponds to acid phosphatases and
phytases.
Several acid
phosphatase genes from Gram negative bacteria have been isolated and
characterized (3). These cloned genes represent an important source of material
for the genetic transfer of this trait to PGPB strains. Among rhizobacteria, we
isolated a gene from Burkholderia cepacia that facilitates phosphatase
activity. This gene codes for an outer membrane protein that enhances its
synthesis in the absence of soluble P in the medium and was suggested as being
involved in P transport to the cell.
The heterologous
expression of these genes in agriculturally-important bacterial strains is the
next step in this approach. We
transferred the napA phosphatase gene from the soil bacteria Morganella
morganii to Burkholderia cepacia IS-16, a strain used as an inoculant, using a
broad-host range vector (pRK293). An increase in the extracellular phosphatase
activity of the recombinant strain was achieved.
Insertion of the
transferred genes into the bacterial chromosome is advantageous for stability
and ecological safety. In our lab, a plasmid for the stable chromosomal
insertion of the phoC phosphatase gene from Morganella morganii was constructed
and we are currently attempting to insert this gene into Azospirillum spp. and Burkholderia
cepacia strains.
A few genes
involved in mineral phosphate solubilization (MPS) from different species, such
as Erwinia herbicola, Pseudomonas cepacia, Enterobacter agglomerans, and Rhanella
aquatilis have been isolated. These genes conferred to Escherichia coli the ability to
hydrolyze insoluble P substrates, and in some cases, they have been found to be
involved in the synthesis of the coenzyme pyrroloquinoline quinone (PQQ).
The PQQ synthetase
gene from Erwinia herbicola, isolated by Dr. Alan Goldstein
and associates (4), was subcloned in our lab in a broad-host range vector
(pKT230). The recombinant plasmid (pL230) was expressed in E. coli, and thereafter
transferred to the PGPB Burkholderia cepacia and Pseudomonas aeruginosa strains, using
tri-parental conjugation. Several of the exconjugants recovered in the
selection medium showed a larger-sized clearing halo in medium with tricalcium
phosphate as the sole P source. This indicates the heterologous expression of
this gene in the recombinant strains, which gave rise to improved MPS ability
of these PGPB.
Conclusions
Knowledge about the
genetics of P solubilization is still scant. However, the preliminary
achievements in the manipulation of these genes open a promising perspective
for obtaining PGPB strains with enhanced P solubilization capacity, and thus, a
more efficient use of microbes as agricultural inoculants