MICROBIAL AND MAMMALIAN SULFUR METABOLISM 
Nicholas M. Kredich, M.D., Investigator 
\. Regulation of Cysteine Biosynthesis in Salmonella 
typhimurium. 
A. Interactions ofCysB protein with the cysJIH pro- 
moter. CysB protein is a tetramer of identical 36 
kDa subunits that serves as a transcriptional activa- 
tor for expression of the cysteine regulon, i.e., the 
genes required for de novo cysteine synthesis in Es- 
cherichia coli and Salmonella typhimurium. Tran- 
scriptional activation also requires acetylserine, 
which in its role as a signal of sulfur deprivation 
acts as an internal inducer for the cysteine regulon. 
In vitro studies in Dr. Kredich 's laboratory have 
shown that CysB protein binds to a portion of the 
cysJIH promoter, designated CBS-Jl, that extends 
from positions -80 to -35 relative to the transcrip- 
tion start site. Acetylserine stimulates binding three- 
to eightfold and is required for bound CysB protein 
to form a transcription initiation complex with RNA 
polymerase. These results show that the effects of 
CysB protein are similar to those of other positive 
regulatory proteins, where an interaction with RNA 
polymerase is postulated to occur just upstream of 
the RNA polymerase-binding site. Dr. Kredich and 
his associates have also examined the effects of sul- 
fide and cysteine on in vitro interactions between 
purified CysB protein and the cysJIH promoter. In 
vivo these compounds interfere with the ability of 
inducer to stimulate expression of genes of the cys- 
teine regulon. In vitro studies indicate that sulfide 
is an anti-inducer, which competes with the stimu- 
latory effects of acetylserine on CysB protein bind- 
ing and on transcription initiation. Cysteine had no 
measurable effects in vitro, and its in vivo effects 
are postulated to be secondary to conversion to 
sulfide via cysteine desulfhydrase. 
B. cysB autoregulation. CysB protein is encoded by 
the cysB gene. The transcription initiation start site 
for cysB has been identified by primer extension 
studies, and in vitro DNA-binding and DNase I pro- 
tection experiments show that CysB protein binds 
to the -10 region of the cysB promoter at a site des- 
ignated CBS-Bl. In transcription run-off experi- 
ments with cysB, CysB protein has been shown to 
prevent formation of a transcription initiation com- 
plex, presumably by binding to the promoter and 
blocking access to RNA polymerase. In contrast to 
the results obtained with the CBS-Jl site of the cys- 
JIH promoter, the inducer acetylserine inhibits 
binding of CysB protein to CBS-Bl and restores 
transcription initiation at the cysB promoter. These 
findings corroborate and expand earlier in vivo 
studies with cysB-lac fusions, which indicated that 
cysB is autoregulated. 
C. Interactions of CysB protein with the cysK pro- 
moter. Dr. Kredich's laboratory has found that 
there are two contiguous CysB protein-binding 
sites in the cysK promoter region. The first, desig- 
nated CBS-Kl, extends from position -78 to -39 
relative to the major transcription start site; the 
second, designated CBS-K2, extends from position 
-123 to -87. CysB protein binds readily to both 
sites in the absence of inducer, but inducer stimu- 
lates binding to CBS-Kl while inhibiting binding to 
CBS-K2. In this regard, CBS-Kl resembles CBS-Jl, 
and CBS-K2 behaves more like CBS-Bl. Removal of 
CBS-K2 by site-directed mutagenesis eliminates 
CysB protein binding to this region but does not af- 
fect binding to CBS-Kl or cysK promoter function, 
as assessed by in vivo expression and by in vitro 
transcription run-off assays. Upstream deletions ex- 
tending into CBS-Kl and certain point mutations in 
CBS-Kl diminish or totally abolish cysK promoter 
activity both in vivo and in vitro. Thus CBS-Kl has 
been identified as the site required for the positive 
regulatory effect of CysB protein and inducer. Un- 
like the situation with CBS-Bl, there is no evidence 
that binding to CBS-K2 inhibits cysK promoter ac- 
tivity, and the significance of this site is unknown. 
D. Catalytic mechanism of sulfite reductase 
hemoprotein. A large number of mutations have 
been introduced into the E. coli sulfite reductase 
hemoprotein in a collaborative study with Dr. Lewis 
Siegel (Duke University), which is designed to char- 
acterize amino acid residues involved in electron 
transfer between the Fe^S^ and siroheme prosthetic 
groups of this protein. In attempting to construct a 
high-level expression vector for large-scale purifica- 
tion of these mutant proteins, researchers in Dr. 
Kredich's laboratory have discovered that the cofac- 
tor siroheme is a limiting constituent for holoen- 
2yme production and that siroheme-deficient apo- 
en2yme is toxic to cells. This obstacle has been 
overcome recently by constructing a plasmid that 
contains both the structural gene for the apopro- 
tein {cysl) and the gene for the enzyme catalyzing 
the final step in siroheme synthesis {cysG). The c\xr- 
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