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Polysialic and colanic acids metabolism in Escherichia coli K92 is regulated by RcsA and RcsB.

Navasa N, Rodríguez-Aparicio L, Ferrero MÁ, Monteagudo-Mera A, Martínez-Blanco H - Biosci. Rep. (2013)

Bottom Line: Deletion of either rcsA or rcsB genes resulted in decreased expression of cps (CA biosynthesis cluster) at 19°C and 37°C, but only CA production was reduced at 19°C.Concerning PA, both deletions enhanced its synthesis at 37°C, which does not correlate with the reduced kps (PA biosynthesis cluster) expression observed in the rcsB mutant.Although RcsA and RcsB acted as negative regulators of PA synthesis at 37°C, their absence did not reestablish PA expression at low temperatures, despite the deletion of rcsB resulting in enhanced kps expression.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular, Área de Bioquímica y Biología Molecular, Universidad de León, Campus de Vegazana, 24071 León, Spain.

ABSTRACT
We have shown previously that Escherichia coli K92 produces two different capsular polymers known as CA (colanic acid) and PA (polysialic acid) in a thermoregulated manner. The complex Rcs phosphorelay is largely related to the regulation of CA synthesis. Through deletion of rscA and rscB genes, we show that the Rcs system is involved in the regulation of both CA and PA synthesis in E. coli K92. Deletion of either rcsA or rcsB genes resulted in decreased expression of cps (CA biosynthesis cluster) at 19°C and 37°C, but only CA production was reduced at 19°C. Concerning PA, both deletions enhanced its synthesis at 37°C, which does not correlate with the reduced kps (PA biosynthesis cluster) expression observed in the rcsB mutant. Under this condition, expression of the nan operon responsible for PA catabolism was greatly reduced. Although RcsA and RcsB acted as negative regulators of PA synthesis at 37°C, their absence did not reestablish PA expression at low temperatures, despite the deletion of rcsB resulting in enhanced kps expression. Finally, our results revealed that RcsB controlled the expression of several genes (dsrA, rfaH, h-ns and slyA) involved in the thermoregulation of CA and PA synthesis, indicating that RcsB is part of a complex regulatory mechanism governing the surface appearance in E. coli.

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CA production (measured as glucuronic acid) by E. coli K92, E. coli K92ΔrcsA and E. coli K92ΔrcsB growth in MM containing Xyl–Asn (A,C) or Glc-Pro (B,D) at 19°C (A,B) and 37° (C,D)**P<0.005, ***P<0.001 by Student's t test.
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Figure 3: CA production (measured as glucuronic acid) by E. coli K92, E. coli K92ΔrcsA and E. coli K92ΔrcsB growth in MM containing Xyl–Asn (A,C) or Glc-Pro (B,D) at 19°C (A,B) and 37° (C,D)**P<0.005, ***P<0.001 by Student's t test.

Mentions: We previously showed that E. coli K92 (WT) synthesizes CA as capsular polymers in a temperature-dependent manner [11,21]. It is well known that Rcs phosphorelay and the auxiliary protein RcsA act as positive regulators of CA synthesis [15]. To investigate the role of RcsA and RcsB in CA synthesis by E. coli K92, we performed gene deletion experiments to obtain E. coli K92ΔrcsA and E. coli K92ΔrcsB mutant strains lacking rcsA and rcsB genes, respectively. WT and both mutant strains were grown in Glc–Pro and Xyl–Asn MM at 19°C, the optimal growth temperature for synthesis of CA by E. coli K92 [11,12], and production of this polymer was determined after 120 h. Neither mutant showed any change in growth under the conditions tested (Figure 2) and both deletions resulted in a dramatically decreased CA production at 19°C (measured as glucuronic acid content) in the media tested (Figures 3A and 3B), but it was not completely abrogated. We ensured that all glucuronic acid detected belonged to a high molecular mass structure, such as CA polymer, through prior dialysis of supernatants using a 10 kDa membrane pore size. To assess the absence of CA capsules surrounding the bacteria, WT and E. coli K92ΔrcsA were grown on Glc–Pro MM agar plates at 19°C and the Burri method using China ink was applied. This staining technique revealed large amounts of capsular polymer produced by WT in contrast to the mutant strain (Figure 4).


Polysialic and colanic acids metabolism in Escherichia coli K92 is regulated by RcsA and RcsB.

Navasa N, Rodríguez-Aparicio L, Ferrero MÁ, Monteagudo-Mera A, Martínez-Blanco H - Biosci. Rep. (2013)

CA production (measured as glucuronic acid) by E. coli K92, E. coli K92ΔrcsA and E. coli K92ΔrcsB growth in MM containing Xyl–Asn (A,C) or Glc-Pro (B,D) at 19°C (A,B) and 37° (C,D)**P<0.005, ***P<0.001 by Student's t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3673037&req=5

Figure 3: CA production (measured as glucuronic acid) by E. coli K92, E. coli K92ΔrcsA and E. coli K92ΔrcsB growth in MM containing Xyl–Asn (A,C) or Glc-Pro (B,D) at 19°C (A,B) and 37° (C,D)**P<0.005, ***P<0.001 by Student's t test.
Mentions: We previously showed that E. coli K92 (WT) synthesizes CA as capsular polymers in a temperature-dependent manner [11,21]. It is well known that Rcs phosphorelay and the auxiliary protein RcsA act as positive regulators of CA synthesis [15]. To investigate the role of RcsA and RcsB in CA synthesis by E. coli K92, we performed gene deletion experiments to obtain E. coli K92ΔrcsA and E. coli K92ΔrcsB mutant strains lacking rcsA and rcsB genes, respectively. WT and both mutant strains were grown in Glc–Pro and Xyl–Asn MM at 19°C, the optimal growth temperature for synthesis of CA by E. coli K92 [11,12], and production of this polymer was determined after 120 h. Neither mutant showed any change in growth under the conditions tested (Figure 2) and both deletions resulted in a dramatically decreased CA production at 19°C (measured as glucuronic acid content) in the media tested (Figures 3A and 3B), but it was not completely abrogated. We ensured that all glucuronic acid detected belonged to a high molecular mass structure, such as CA polymer, through prior dialysis of supernatants using a 10 kDa membrane pore size. To assess the absence of CA capsules surrounding the bacteria, WT and E. coli K92ΔrcsA were grown on Glc–Pro MM agar plates at 19°C and the Burri method using China ink was applied. This staining technique revealed large amounts of capsular polymer produced by WT in contrast to the mutant strain (Figure 4).

Bottom Line: Deletion of either rcsA or rcsB genes resulted in decreased expression of cps (CA biosynthesis cluster) at 19°C and 37°C, but only CA production was reduced at 19°C.Concerning PA, both deletions enhanced its synthesis at 37°C, which does not correlate with the reduced kps (PA biosynthesis cluster) expression observed in the rcsB mutant.Although RcsA and RcsB acted as negative regulators of PA synthesis at 37°C, their absence did not reestablish PA expression at low temperatures, despite the deletion of rcsB resulting in enhanced kps expression.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular, Área de Bioquímica y Biología Molecular, Universidad de León, Campus de Vegazana, 24071 León, Spain.

ABSTRACT
We have shown previously that Escherichia coli K92 produces two different capsular polymers known as CA (colanic acid) and PA (polysialic acid) in a thermoregulated manner. The complex Rcs phosphorelay is largely related to the regulation of CA synthesis. Through deletion of rscA and rscB genes, we show that the Rcs system is involved in the regulation of both CA and PA synthesis in E. coli K92. Deletion of either rcsA or rcsB genes resulted in decreased expression of cps (CA biosynthesis cluster) at 19°C and 37°C, but only CA production was reduced at 19°C. Concerning PA, both deletions enhanced its synthesis at 37°C, which does not correlate with the reduced kps (PA biosynthesis cluster) expression observed in the rcsB mutant. Under this condition, expression of the nan operon responsible for PA catabolism was greatly reduced. Although RcsA and RcsB acted as negative regulators of PA synthesis at 37°C, their absence did not reestablish PA expression at low temperatures, despite the deletion of rcsB resulting in enhanced kps expression. Finally, our results revealed that RcsB controlled the expression of several genes (dsrA, rfaH, h-ns and slyA) involved in the thermoregulation of CA and PA synthesis, indicating that RcsB is part of a complex regulatory mechanism governing the surface appearance in E. coli.

Show MeSH
Related in: MedlinePlus