THERMOREGULATION OF KPSF, THE FIRST REGION-1 GENE IN THE KPS LOCUS FOR POLYSIALIC ACID BIOSYNTHESIS IN ESCHERICHIA-COLI K1

The kps locus for biosynthesis of the capsular polysialic acid virulen ce factor in Escherichia coli K1 contains at least two convergently tr anscribed operons, designated region 1 and regions 2 plus 3. On the ba sis of DNA sequence analysis, kpsF appeared to be a good candidate for the first gene of region 1 (M. J. Cieslewicz, S. M. Steenbergen, and E. R. Vimr, J. Bacteriol. 175:8018-8023, 1993). A preliminary indicati on that kpsF is required for capsule production is the capsule-negativ e phenotype of an aphT insertion in the chromosomal copy of kpsF. The present communication describes the isolation and phenotypic character ization of this mutant. Although transcription through kpsF was requir ed for capsule production, complementation analysis failed to indicate a clear requirement for the KpsF polypeptide. However, since E. coli contains at least two other open reading frames that could code for ho mologs of KpsF, the apparent dispensability of KpsF remains provisiona l. DNA sequence analysis of 1,100 bp upstream from the kpsF translatio nal start site did not reveal any open reading frames longer than 174 nucleotides, consistent with kpsF being the first gene of region 1. Si nce kpsF appeared to be the first gene of a region whose gene products are required for polysialic acid transport and because capsule produc tion is known to be thermoregulated, primer extension analyses were ca rried out with total RNA isolated from cells grown at permissive (37 d egrees C) and nonpermissive (20 degrees C) temperatures. The results r evealed a potentially complex kpsF promoter-like region that was trans criptionally silent at the nonpermissive temperature, suggesting that thermoregulation of region 1 may be exerted through variations in kpsF expression. Additional evidence supporting this conclusion vas obtain ed by demonstrating the effects of temperature on expression of the ge ne kpsE, immediately downstream of kpsF. Chloramphenicol acetyltransfe rase assays were carried out with constructs containing the kpsF 5' un translated region fused to a promoterless cat cassette, providing furt her evidence that kpsF is thermoregulated. Although the function of Kp sF is unclear, primary structure analysis indicated two motifs commonl y observed in regulatory proteins and homology with glucosamine syntha se from Rhizobium meliloti.