Characterization of rcsB and rcsC from Escherichia coli O9:K30:H12 and examination of the role of the rcs regulatory system in expression of group I capsular polysaccharides.
In Escherichia coli K-12, RcsC and RcsB are thought to act as the sensor and effector components, respectively, of a two-component regulatory system which regulates expression of the slime polysaccharide colanic acid (V. Stout and S. Gottesman, J. Bacteriol. 172:659-669, 1990). Here, we report the cloning and DNA sequence of a 4.3-kb region containing rcsC and rcsB from E. coli O9:K30:H12. This strain does not produce colanic acid but does synthesize a K30 (group I) capsular polysaccharide. The rcsB gene from E. coli K30 (rcsBK30) is identical to the rcsB gene from E. coli K-12 (rcsBK-12). rcsCK30 has 16 nucleotide changes, resulting in six amino acid changes in the predicted protein. To examine the function of the rcs regulatory system in expression of the K30 capsular polysaccharide, chromosomal insertion mutations were constructed in E. coli O9:K30:H12 to independently inactivate rcsBK30 and the auxiliary positive regulator rcsAK30. Strains with these mutations maintained wild-type levels of K30 capsular polysaccharide expression and still produced a K30 capsule, indicating that the rcs system is not essential for expression of low levels of the group I capsular polysaccharide in lon+ E. coli K30. However, K30 synthesis is increased by introduction of a multicopy plasmid carrying rcsBK30. K30 polysaccharide expression is also markedly elevated in an rcsBK30-dependent fashion by a mutation in rcsCK30, suggesting that the rcs system is involved in high levels of synthesis. To determine whether the involvement of the rcs system in E. coli K30 expression is typical of group I (K antigen) capsules, multicopy rcsBK30 was introduced into 22 additional strains with structurally different group I capsules. All showed an increase in mucoid phenotype, and the polysaccharides produced in the presence and absence of multicopy rcsBK30 were examined. It is has been suggested that E. coli strains with group I capsules can be subdivided based on K antigen structure. For the first time, we show that strains with group I capsules can also be subdivided by the ability to produce colanic acid. Group IA contains capsular polysaccharides (including K30) with repeating-unit structures lacking amino sugars, and expression of group IA capsular polysaccharides is increased by multicopy rcsBK30. Group IB capsular polysaccharides all contain amino sugars. In group IB strains, multicopy rcsBK30 activates synthesis of colanic acid.
Jayaratne
P
P
Department of Microbiology, University of Guelph, Ontario, Canada.
Keenleyside
W J
WJ
MacLachlan
P R
PR
Dodgson
C
C
Whitfield
C
C
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial; D000954:Antigens, Surface / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005809:Genes, Regulator; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016296:Mutagenesis; D010749:Phosphoprotein Phosphatases; D011134:Polysaccharides / Q000096:biosynthesis / Q000235:genetics*; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis / Q000235:genetics*; D011494:Protein Kinases; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics*
D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C030659:K antigens; D009097:Multienzyme Complexes; D011134:Polysaccharides; D011135:Polysaccharides, Bacterial; C489568:RcsB protein, E coli; D014157:Transcription Factors; C489728:rcsC protein, E coli; C074889:RcsB protein, Bacteria; C004275:colanic acid; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases