Escherichia coli FtsH is an essential integral membrane protein that has an AAA-type ATPase domain at its C-terminal cytoplasmic part, which is homologous to at least three ATPase subunits of the eukaryotic 26S proteasome. We report here that FtsH is involved in degradation of the heat-shock transcription factor a32, a key element in the regulation of the E.coli heat-shock response. In the temperature-sensitive ftsHJ mutant, the amount of aY32 at a non-permissive temperature was higher than in the wild-type under certain conditions due to a reduced rate of degradation. In an in vitro system with purified components, FtsH catalyzed ATPdependent degradation of biologically active histidinetagged &32. FtsH has a zinc-binding motif similar to the active site of zinc-metalloproteases. Protease activity of FtsH for histidine-tagged a32 was stimulated by Zn2+ and strongly inhibited by the heavy metal chelating agent o-phenanthroline. We conclude that FtsH is a novel membrane-bound, ATP-dependent metalloprotease with activity for a32. These findings indicate a new mechanism of gene regulation in E.coli.

SummaryCspA was originally found as the major cold-shock protein in Escherichia coli, consisting of 70-aminoacid residues. It forms a ␤-barrel structure with five anti-parallel ␤-strands and functions as an RNA chaperone. Its dramatic but transient induction upon cold shock is regulated at the level of transcription, mRNA stability and translation. Surprisingly, E. coli contains a large CspA family, consisting of nine genes from cspA to cspI. Phylogenetic analysis of these gene products and the cold-shock domain of human YB-1 protein reveals that there are two major branches in the evolution of CspA homologues: one branch for CspF and CspH, and another for all the other known CspA homologues from both prokaryotes and eukaryotes. The locations of these genes on the E. coli chromosome suggest that the large CspA family probably resulted from a number of gene duplications and, after subsequent adaptation, resulted in specific groups of genes that respond to different environmental stresses; for example, cspA, cspB and cspG for cold-shock stress and cspD for nutritional deprivation. The E. coli CspA family will be discussed in terms of their structures and functions, and their gene structures and regulation.

Changes in oligosaccharide structures have been reported in certain types of malignant transformations and, thus, could be used for tumor markers in certain types of cancer. In the case of pancreatic cancer cell lines, a variety of fucosylated proteins are secreted into their conditioned media. To identify fucosylated proteins in the serum of patients with pancreatic cancer, we performed western blot analyses using Aleuria Aurantica Lectin (AAL), which is specific for fucosylated structures. An 40 kD protein was found to be highly fucosylated in pancreatic cancer and an N-terminal analysis revealed that it was the b chain of haptoglobin. While the appearance of fucosylated haptoglobin has been reported in other diseases such as hepatocellular carcinoma, liver cirrhosis, gastric cancer and colon cancer, the incidence was significantly higher in the case of pancreatic cancer. Fucosylated haptoglobin was observed more frequently at the advanced stage of pancreatic cancer and disappeared after an operation. A mass spectrometry analysis of haptoglobin purified from the serum of patients with pancreatic cancer and the medium from a pancreatic cancer cell line, PSN-1, showed that the a 1-3/a 1-4/a 1-6 fucosylation of haptoglobin was increased in pancreatic cancer. When a hepatoma cell line, Hep3B, was cultured with the conditioned media from pancreatic cancer cells, haptoglobin secretion was dramatically increased. These findings suggest that fucosylated haptoglobin could serve as a novel marker for pancreatic cancer. Two possibilities were considered in terms of the fucosylation of haptoglobin. One is that pancreatic cancer cells, themselves, produce fucosylated haptoglobin; the other is that pancreatic cancer produces a factor, which induces the production of fucosylated haptoglobin in the liver. ' 2005 Wiley-Liss, Inc.Key words: haptoglobin; pancreatic cancer; fucosylation; tumor marker; mass spectrometry; oligosaccharide; lectin; fucosyltransferase Pancreatic cancer is currently one of the leading causes of cancer-related deaths and the overall 5-year survival has been reported to be less than 5%.

mukB mutants of Escherichia coli are defective in the correct partitioning of replicated chromosomes. This results in the appearance of normal‐sized anucleate (chromosome‐less) cells during cell proliferation. Based on the nucleotide sequence of the mukB gene, the MukB protein of 177 kDa was predicted to be a filamentous protein with globular domains at the ends, and also having DNA binding and nucleotide binding abilities. Here we present evidence that the purified MukB protein possesses these characteristics. MukB forms a homodimer with a rod‐and‐hinge structure having a pair of large, C‐terminal globular domains at one end and a pair of small, N‐terminal globular domains at the opposite end; it tends to bend at a middle hinge site of the rod section. Chromatography in a DNA‐cellulose column and the gel retardation assay revealed that MukB possesses DNA binding activity. Photoaffinity cross‐linking experiments showed that MukB binds to ATP and GTP in the presence of Zn2+. Throughout the purification steps, acyl carrier protein was co‐purified with MukB.

The hsH gene is essential for cell viability in Escherichia coli. We cloned and sequenced the wild-typeftsH gene and the temperature-sensitive ftsHl(Ts) gene MATERIALS AND METHODS Bacterial strains, plasmids, phages, and media. The following E. coli K-12 strains were used in this study: Y16 [thr-J leu-6 thi-1 supE44 lacYl tonA21 ftsHl(Ts) ftsI372(Ts*)] (2, 27), AR1025 (thr-1 leu-6 thi-1 supE44 lacYl tonA21 recD1009 AftsJ: :kan), W3110 [IN(rnD-rmE)