Expression of the Rhizobium leguminosarum bv. trifolii pssA Gene, Involved in Exopolysaccharide Synthesis, Is Regulated by RosR, Phosphate, and the Carbon Source

Abstract

Rhizobium leguminosarum bv. trifolii pssA encodes a glucosyl-isoprenylphosphate (IP)-transferase involved in the first step of exopolysaccharide (EPS) synthesis. It was found that the pssA gene is an important target for regulation of this biosynthetic pathway. The data of this study indicate that pssA transcription is a very complex and mainly positively regulated process. A detailed analysis of a 767-bp-long pssA upstream region revealed the presence of several sequence motifs recognized by regulatory proteins that are associated with phosphate-, carbon-, and iron-dependent regulation. In addition, numerous inverted repeats of different lengths have been identified in this region. pssA transcription is directed from two distal P1 and proximal P3 promoters whose sequences demonstrate a significant identity to promoters recognized by RNA polymerase sigma factor 70 . Among rhizobial proteins, RosR seems to be a primary regulator that positively affects pssA expression. This protein binds to RosR box 1 located downstream of the P1 promoter. In addition, phosphate and the carbon source strongly affect pssA transcription. A significantly lower level of pssA expression was observed in both the wild-type strain growing under phosphate-rich conditions and the phoB mutant. In this regulation, the PhoB protein and Pho box 2 located upstream of the P3 promoter were engaged. pssA transcription is also significantly affected by glucose. Transcriptional analysis of a set of pssA-lacZ fusions expressed in Escherichia coli wild-type and cyaA and crp mutants confirmed that cyclic AMP (cAMP) receptor protein (CRP) and two cAMP-CRP boxes located upstream of the P1 are required for this upregulation. Moreover, the production of EPS was totally abolished in R. leguminosarum bv. trifolii mutant strains 4440 and 1012 containing a Tn5 insertion downstream of the P3 promoter and downstream of the P3 ؊35 hexamer, respectively. R hizobium leguminosarum bv. trifolii is a soil bacterium able to induce formation of nitrogen-fixing root nodules on its host plant, clover (Trifolium pratense). The establishment of symbiosis is a complex process involving an exchange of a series of signals between the plant and the bacterium; among them, flavonoids secreted from plant roots and bacterial Nod factors are the best characterized signals (1). In addition, acidic exopolysaccharide (EPS) secreted by rhizobia in large amounts plays a crucial role in the symbiotic interactions with legumes that form indeterminatetype nodules (e.g., Trifolium, Pisum, Vicia, and Medicago) (2). EPS-deficient mutants of R. leguminosarum bv. trifolii induce formation of small, only partially infected nodules on clover plants that are ineffective in nitrogen fixation (3-5). In free-living cells of rhizobia, EPS has several other functions such as nutrient gathering, protection against environmental stresses, attachment to abiotic and biotic surfaces, and biofilm formation, ensuring adaptation of these bacteria to changing environmental conditions (6). EPS produced by R. legu...

Genetic characterization of the Pss region and the role of PssS in exopolysaccharide production and symbiosis of Rhizobium leguminosarum bv. trifolii with clover

“…This protein contains a Cys 2 His 2 -type zinc-finger motif responsible for binding to a 22-bp-long sequence called the RosR-box. Previously, it was confirmed that RosR affected transcription of pssA and its own gene (Janczarek and Skorupska 2007;Janczarek and Urbanik-Sypniewska 2013).…”

“…These proteins are encoded by the pssC, pssD, and pssE genes located in a cluster named Pss-I (Król et al 2007). Mutations in the pssA and pssD genes totally abolished EPS synthesis (Borthakur et al 1988;Ivashina et al 1994;Janczarek and Urbanik-Sypniewska 2013), whereas a disruption of the pssC gene resulted in a decrease in EPS synthesis to 40 % of the amount produced by the wild-type strain (van Workum et al 1997). pssJ encodes a galactosyltransferase, which probably participates in the last step of the unit assembly (Breedveld et al 1993).…”

The response of the Rhizobium leguminosarum bv. trifolii wild-type and exopolysaccharide-deficient mutants to oxidative stress

“…Previously, we have established that the transcriptional regulator RosR of R. leguminosarum bv. trifolii positively regulates the pssA expression (Janczarek and Skorupska 2007;Janczarek and Urbanik-Sypniewska 2013). This gene encodes a protein, which besides its enzymatic activity is probably involved additionally in other cellular processes (Janczarek and Skorupska 2003).…”

Production of exopolysaccharide by Rhizobium leguminosarum bv. trifolii and its role in bacterial attachment and surface properties

“…EPS was isolated and quantified as described previously (Janczarek and Urbanik-Sypniewska 2013). Briefly, bacteria were grown in appropriate 79CA medium for up to 4 days at 28°C in a rotary shaker (160 rpm).…”

“…Besides flavonoids and Nod factors, rhizobial acidic exopolysaccharides (EPS) play a significant role in the establishment of an effective symbiosis especially with legumes that form indeterminate-type nodules (e.g. clover, pea, vetch, and alfalfa) (Rolfe et al 1996;Becker and Pühler 1998;Janczarek and Urbanik-Sypniewska 2013). This polysaccharide is indispensable for protection against host plant defence reactions and initiation and elongation of infection threads, special tubular structures via which rhizobia colonize root nodules (Cheng and Walker 1998;Jaszek et al 2014).…”