Identification and functional characterization of a fructose-inducible phosphotransferase system in <i>Azospirillum brasilense</i> Sp7

Plant growth-promoting rhizobacterium <i>Azospirillum brasilense</i> Sp7 utilizes fructose efficiently via a fructose phosphotransferase system (Fru-PTS). Its genome encodes two putative Fru-PTS, each consisting of FruB (EIIA), FruK (Pfk), and FruA (EIIBC) proteins. We compared the proteomes of <i>A. brasilense</i> Sp7 grown with malate or fructose as sole carbon source, and noticed upregulation of the constituent proteins of Fru-PTS1 only on fructose. Inactivation of <i>fruA</i> gene of both the Fru-PTS showed that Fru-PTS1 is the main PTS involved in fructose utilization. Overexpression of <i>fruA1</i> in <i>A. brasilense</i> Sp7 enhanced its growth on fructose showing improved consumption of fructose. This suggested that fructose utilization in <i>A. brasilense</i> Sp7 is limited due to the limitation of EIIBC component. A FruR-type regulator, encoded divergently to the Fru-PTS1 operon, was required for chemotaxis toward fructose. Although not an absolute necessity for the growth of fructose, FruR was required for the optimal growth of fructose. The <i>fruB1</i> promoter was activated by fructose and repressed by malate, but FruR does not seem to regulate its expression. A 27-nucleotide stem-loop structure located between the -125 and -99 promoter proximal region of <i>fruB1</i> was involved in fructose inducibility and malate repression. Fructose also upregulated several proteins involved in the biogenesis of a Type 6 secretion system. Here, we have shown that <i>A. brasilense</i> Sp7 was able to inhibit the growth of <i>Escherichia coli</i> and <i>Agrobacterium tumefaciens</i> in the presence of fructose, and that an intact T6SS was required for contact-dependent growth inhibition of the two Gram-negative bacteria.IMPORTANCE<i>Azospirillum brasilense</i>, a plant growth-promoting rhizobacterium, has limited ability to utilize carbohydrates and sugars. Although it is known to utilize fructose via a fructose phosphotransferase system (fructose-PTS), the genes involved in fructose utilization and the role of fructose in its biology were not well characterized. This study has shown that out of the two units of fructose-PTS encoded in its genome, fructose-PTS1 plays the major role in fructose utilization. Overexpression of the membrane component (EIIBC) improved the growth of <i>A. brasilense</i> on fructose. The ability of fructose to induce proteins of the Type 6 Secretion System (T6SS) enables <i>A. brasilense</i> to cause contact-dependent inhibition of the growth of <i>Escherichia coli</i> as well as <i>A. tumefaciens</i>. This is the first report on the fructose inducibility of T6SS in <i>A. brasilense</i>, which may provide a handle to control the growth of undesirable bacteria using T6SS of <i>A. brasilense</i> in a mixed culture.