Escherichia coli rRNA contains 10 pseudouridines
of unknown function. They are made by synthases, each of
which is specific for one or more pseudouridines. Here
we show that the sfhB (yfiI) ORF of E.
coli is a pseudouridine synthase gene by cloning,
protein overexpression, and reaction in vitro with rRNA
transcripts. Gene disruption by miniTn10(cam) insertion
revealed that this synthase gene, here renamed rluD,
codes for a synthase which is solely responsible in vivo
for synthesis of the three pseudouridines clustered in
a stem-loop at positions 1911, 1915, and 1917 of 23S RNA.
The absence of RluD results in severe growth inhibition.
Both the absence of pseudouridine and the growth defect
could be reversed by insertion of a plasmid carrying the
rluD gene into the mutant cell, clearly linking
both effects to the absence of RluD. This is the first
report of a major physiological defect due to the deletion
of any pseudouridine synthase. Growth inhibition may be
due to the lack of one or more of the 23S RNA pseudouridines
made by this synthase since pseudouridines 1915 and 1917
are universally conserved and are located in proximity
to the decoding center of the ribosome where they could
be involved in modulating codon recognition.