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The Conserved Cys-X1-X2-Cys Motif Present in the TtcA Protein Is Required for the Thiolation of Cytidine in Position 32 of tRNA from Salmonella enterica serovar Typhimurium. Gunilla Jäger, 2004.The modified nucleoside 2-thiocytidine (s2C) has so far been found in tRNA from organisms belonging to the phylogenetic domains Archaea and Bacteria . In the bacteria Escherichia coli and Salmonella enterica serovar Typhimurium, s2C is present in position 32 of only four tRNA species—  ,
 ,
 ,
and
 .
An in-frame deletion of an S . enterica gene (designated ttcA, for
"two-thio-cytidine") was constructed, and such a mutant has no
detectable s2C in its tRNA . The TtcA protein family is characterized
by the existence of both a PP-loop and a Cys-X1-X2-Cys
motif in the central region of the protein but can be divided into
two distinct groups based on the presence and location of additional
Cys-X1-X2-Cys motifs in terminal regions of the
sequence . Mutant analysis showed that both cysteines in this central
conserved Cys-X1-X2-Cys motif are required for
the formation of s2C . The
 ttcA1
mutant grows at the same rate as the congenic wild-type strain, and
no growth disadvantage caused by the lack of s2C was
observed in a mixed-population experiment . Lack of s2C32
did not reduce the selection rate at the ribosomal aminoacyl-tRNA
site (A-site) for
at any of its cognate CGN codons, whereas A-site selection at AGG by
was dependent on the presence of s2C32 . The presence of s2C32
in peptidyl-
or in peptidyl-
interfered with decoding in the A-site . The presence of s2C32
in
decreased the rate of translation of the CGA codon but not that of the CGU
codon .
Protein Content of Polyhedral Organelles Involved in Coenzyme B12-Dependent Degradation of 1,2-Propanediol in Salmonella enterica Serovar Typhimurium LT2. Gregory D. Havemann, 2003.Salmonella enterica forms polyhedral organelles during coenzyme B12-dependent growth on 1,2-propanediol (1,2-PD) . Previously, these organelles were shown to consist of a protein shell partly composed of the PduA protein, the majority of the cell's B12-dependent diol dehydratase, and additional unidentified proteins . In this report, the polyhedral organelles involved in B12-dependent 1,2-PD degradation by S . enterica were purified by a combination of detergent extraction and differential and density gradient centrifugation . The course of the purification was monitored by electron microscopy and gel electrophoresis, as well as enzymatic assay of B12-dependent diol dehydratase . Following one- and two-dimensional gel electrophoresis of purified organelles, the identities and relative abundance of their constituent proteins were determined by N-terminal sequencing, protein mass fingerprinting, Western blotting, and densitometry . These analyses indicated that the organelles consisted of at least 15 proteins, including PduABB'CDEGHJKOPTU and one unidentified protein . Seven of the proteins identified (PduABB'JKTU) have some sequence similarity to the shell proteins of carboxysomes (a polyhedral organelle involved in autotrophic CO2 fixation), suggesting that the S . enterica organelles and carboxysomes have a related multiprotein shell . In addition, S . enterica organelles contained four enzymes: B12-dependent diol dehydratase, its putative reactivating factor, aldehyde dehydrogenase, and ATP cob(I)alamin adenosyltransferase . This complement of enzymes indicates that the primary catalytic function of the S . enterica organelles is the conversion of 1,2-PD to propionyl coenzyme A (which is consistent with our prior proposal that the S . enterica organelles function to minimize aldehyde toxicity during growth on 1,2-PD) . The possibility that similar protein-bound organelles may be more widespread in nature than currently recognized is discussed .
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