Tumor necrosis factor (TNF)-

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Bacterial flagellar motors obtain energy for rotation from the membrane gradient of protons or, in some species, sodium ions. The molecular mechanism of flagellar rotation is not understood. MotA and MotB are integral membrane proteins that function in proton conduction and are believed to form the stator of the motor. Previous mutational studies identified two conserved proline residues in MotA (Pro 173 and Pro 222 in the protein from Escherichia coli) and a conserved aspartic acid residue in MotB (Asp 32) that are important for function. Asp 32 of MotB probably forms part of the proton path through the motor. To learn more about the roles of the conserved proline residues of MotA, we examined motor function in Pro 173 and Pro 222 mutants, making measurements of torque at high load, speed at low and intermediate loads, and solvent-isotope effects (D2O versus H2O). Proton conduction by wild-type and mutant MotA-MotB channels was also assayed, by a growth defect that occurs upon overexpression. Several different mutations of Pro 173 reduced the torque of the motor under high load, and a few prevented motor rotation but still allowed proton flow through the MotA-MotB channels. These and other properties of the mutants suggest that Pro 173 has a pivotal role in coupling proton flow to motor rotation and is positioned in the channel near Asp 32 of MotB. Replacements of Pro 222 abolished function in all assays and were strongly dominant. Certain Pro 222 mutant proteins prevented swimming almost completely when expressed at moderate levels in wild-type cells. This dominance might be caused by rotor-stator jamming, because it was weaker when FliG carried a mutation believed to increase rotor-stator clearance. We propose a mechanism for torque generation, in which specific functions are suggested for the proline residues of MotA and Asp32 of MotB.

We have investigated the organization and expression of the Caulobacter crescentus flbF gene because it occupies a high level in the flagellar gene regulatory hierarchy. The nucleotide sequence comprising the 3' end of theflaO operon and the adjacentflbF promoter and structural gene was determined, and the organization of transcription units within this sequence was investigated. We located the 3' ends of theflaO operon transcript by using a nuclease S1 protection assay, and the 5' end of theflbF transcript was precisely mapped by primer extension analysis. The nucleotide sequence upstream from the 5' end of the flbF transcript contains -10 and -35 elements similar to those found in promoters transcribed by C28 RNA polymerase in other organisms. Mutations that changed nucleotides in the -10 or -35 elements or altered their relative spacing resulted in undetectable levels of flbF transcript, demonstrating that these sequences contain nucleotides essential for promoter function. We identified a 700-codon open reading frame, downstream from theflbF promoter region, that was predicted to be theflbF structural gene. The amino-terminal half of the FlbF amino acid sequence contains eight hydrophobic regions predicted to be membrane-spanning segments, suggesting that the FlbF protein may be an integral membrane protein. The FlbF amino acid sequence is very similar to that of a transcriptional regulatory protein called LcrD that is encoded in the highly conserved low-calcium-response region of virulence plasmid pYV03 in Yersinia enterocolitica (A.-M. Viitanen, P. Toivanen, and M. Skurnik, J. Bacteriol. 172:3152-3162, 1990).The Caulobacter crescentus flagellum has been used as a model for investigating cell differentiation because its morphogenesis is under strict spatial and temporal control in the cell division cycle (for reviews, see references 35 and 48). Flagellum biosynthesis and function in C. crescentus is a complex process that requires at least 48 genes, and about half of these are contained in three major gene clusters (11). The C. crescentus flagellar genes (6, 7, 36, 38, 55), like those in Escherichia coli (23,24) and Salmonella typhimurium (26), can be arranged in a regulatory hierarchy in which trans-acting genes at one level are required for expression of genes at lower levels. The flbD and flbF genes occupy high levels in the C. crescentus regulatory hierarchy because they are required in trans for the expression of genes needed for the synthesis of the flagellar hook and filament (30,32,36,39). These genes that depend upon flbD and flbF for their transcription contain conserved cis-acting sequences that include ur54-type promoters, a sequence calledftr (30, 32, 33, 37), and another sequence that matches the consensus binding site for E. coli integration host factor protein (15). flbD is a member of the flaO operon (39), whose transcription in vivo depends upon a sequence similar to that of the -10 box of E. coli promoters that are transcribed by cr32 RNA polymerase (34,52). It is evident that the promot...

Tumor necrosis factor (TNF)-α is a major cytokine produced by alveolar macrophages in response to pathogen associated molecular patterns such as LPS. TNF-α secretion is regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation occurs by modulation of TNF-α mRNA stability via the binding of tristetraprolin (TTP) to the AU (adenosine/uridine)-rich elements (AREs) found in the 3’-untranslated region (3’-UTR) of TNF-α transcript. Phosphorylation plays important roles in modulating mRNA stability. Our results show that inhibition of PP2A by okadaic acid or siRNA significantly enhanced the stability of TNF-α mRNA. This result was associated with increased phosphorylation of p38 MAPK and MK-2. PP2A inhibition increased TTP phosphorylation and enhanced complex formation with chaperone protein 14-3-3. A functional consequence of TTP/14-3-3 complex formation appeared to be protection of TTP from dephosphorylation by inhibition of the binding of the catalytic unit of PP2A to phosphorylated TTP. Our data indicates that PP2A regulates TNF-α mRNA stability by modulating the phosphorylation state of members of the p38/MK-2/TTP pathway. This work was supported by grants from the National Institute of Health (7R01GM066839-03, to TPS).

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