Comprehensive Alanine-scanning Mutagenesis ofEscherichia coliCsrA Defines Two Subdomains of Critical Functional Importance

Abstract

The RNA-binding protein CsrA (carbon storage regulator) of Escherichia coli is a global regulator of gene expression and is representative of the CsrA/RsmA family of bacterial proteins. These proteins act by regulating mRNA translation and stability and are antagonized by binding to small noncoding RNAs. Although the RNA target sequence and structure for CsrA binding have been well defined, little information exists concerning the protein requirements for RNA recognition. The three-dimensional structures of three CsrA/RsmA proteins were recently solved, revealing a novel protein fold consisting of two interdigitated monomers. Here, we performed comprehensive alanine-scanning mutagenesis on csrA of E. coli and tested the 58 resulting mutants for regulation of glycogen accumulation, motility, and biofilm formation. Quantitative effects of these mutations on expression of glgCA-lacZ, flhDC-lacZ, and pgaA-lacZ translational fusions were also examined, and eight of the mutant proteins were purified and tested for RNA binding. These studies identified two regions of the amino acid sequence that were critical for regulation and RNA binding, located within the first (␤ 1 , residues 2-7) and containing the last (␤ 5 , residues 40 -47) ␤-strands of CsrA. The ␤ 1 and ␤ 5 strands of opposite monomers lie adjacent and parallel to each other in the three-dimensional structure of this protein. Given the symmetry of the CsrA dimer, these findings imply that two distinct RNA binding surfaces or functional subdomains lie on opposite sides of the protein.

Overexpression of CsrA (BB0184) Alters the Morphology and Antigen Profiles ofBorrelia burgdorferi

“…The ORF BB0184 present in the linear chromosome of B. burgdorferi has been annotated as a homolog of CsrA/RsmA (32). In several bacterial species, CsrA/RsmA has been characterized as a small RNA binding protein capable of regulating multiple metabolic and virulence mechanisms via precise interactions with its cognate small RNA molecules CsrB and CsrC (7,53). Amino acid sequence analysis of borrelial CsrA indicated significant sequence conservation and the presence of two regions that have been biochemically shown to contain several critical residues (Fig.…”

“…Amino acid sequence analysis of borrelial CsrA indicated significant sequence conservation and the presence of two regions that have been biochemically shown to contain several critical residues (Fig. 1A) that mediate the interaction of CsrA with its cognate small RNA molecule in E. coli (53). An interesting feature of the borrelial CsrA in all three sequenced species (B. burgdorferi strain B31, Borrelia afzelii PKo, and Borrelia garinii PBi) is the presence of an additional 7 amino acids at the C terminus, even though the exact contribution of these residues to the function of CsrA Bb remains to be determined.…”

“…This observation indicated that csrA Bb was part of the flgK operon and is cotranscribed with its upstream neighbor, BB0183. Similarly, primer 1 (regF, within BB0182; Table 3) and primer 4 (csrAR, (53). (B) Overexpression of CsrA Bb inhibits glycogen synthesis in E. coli.…”

“…CsrA has been shown to control gene expression posttranscriptionally via specific and high-affinity binding to 5Ј untranslated regions of mRNA, by affecting translation or stability of RNA (6). Alternatively, the effects of CsrA can also be titrated by binding of the noncoding RNAs such as CsrB and CsrC, which have multiple binding sites to sequester CsrA (53). The B. burgdorferi CsrA (CsrA Bb ) has significant sequence similarity to CsrA proteins in other species, including the conserved domains that have been biochemically shown to be critical for the regulatory functions of CsrA in other eubacterial species (53).…”

“…Alternatively, the effects of CsrA can also be titrated by binding of the noncoding RNAs such as CsrB and CsrC, which have multiple binding sites to sequester CsrA (53). The B. burgdorferi CsrA (CsrA Bb ) has significant sequence similarity to CsrA proteins in other species, including the conserved domains that have been biochemically shown to be critical for the regulatory functions of CsrA in other eubacterial species (53). Hence, it is possible for CsrA Bb to have multiple functions in regulating gene expression in B. burgdorferi even though there is no sequence information or identity of its cognate small RNA molecule.…”