No abstract

An Escherichia coli temperature sensitive mutant which produces spontaneously normal size anucleate cells at low temperature was isolated. The mutant is defective in a previously undescribed gene, named mukB, located at 21 min on the chromosome. The mukB gene codes for a large protein (approximately 180 kd). A 1534 amino acid protein (176,826 daltons) was deduced from the nucleotide sequence of the mukB gene. Computer analysis revealed that the predicted MukB protein has distinct domains: an amino‐terminal globular domain containing a nucleotide binding sequence, a central region containing two alpha‐helical coiled‐coil domains and one globular domain, and a carboxyl‐terminal globular domain which is rich in Cys, Arg and Lys. A 180 kd protein detected in wild‐type cell extracts by electrophoresis is absent in mukB null mutants. Although the null mutants are not lethal at low temperature, the absence of MukB leads to aberrant chromosome partitioning. At high temperature the mukB null mutants cannot form colonies and many nucleoids are distributed irregularly along elongated cells. We conclude that the MukB protein is required for chromosome partitioning in E. coli.

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.

We have identified a gene, cpdA, located at 66.2 min of the chromosome of Escherichia coli that encodes cyclic 3,5-adenosine monophosphate phosphodiesterase (cAMP phosphodiesterase, EC 3.1.4.17). The expression of ␤-galactosidase, which is a product of the lacZ gene, was repressed in cells that harbored multiple copies of the plasmid carrying the cpdA gene. Northern blotting showed that the transcription of the lacZ gene was inhibited in these cells. Multiple copies of the cpdA gene decreased the intracellular concentration of cAMP, which is a positive regulator for transcription of the lacZ gene. We found that the purified CpdA protein repressed in vitro transcription from the lacP1 promoter by decreasing cAMP. In addition, we showed that the CpdA protein hydrolyzed cAMP to 5-adenosine monophosphate and that its activity was activated by iron. Our results suggested that regulation of intracellular concentration of cAMP is dependent not only on synthesis of cAMP but also on hydrolysis of cAMP by cAMP phosphodiesterase.cAMP is an important cellular mediator in Escherichia coli. The role of cAMP in mediating glucose effects has been well investigated (1). The cAMP receptor protein (CRP) 1 is a regulatory protein, which binds cAMP and mediates transcriptional regulation at several promoters (2). The CRP-cAMP complex is a positive transcriptional regulator of a number of catabolic operons, including the lac operon in E. coli, and as such, plays a role in catabolite repression, whereby secondary carbon sources are not catabolized in the presence of glucose (3, 4). This complex is involved not only in positive regulation of several catabolic functions but also in regulation of flagellum synthesis (5), toxin production (6), minicell production (7), coupling of DNA replication and cell division (8), and many other functions that are not directly related to catabolism. CRP forms an active conformation only when it binds cAMP. Therefore, the concentration of active CRP-cAMP complex and the biological responses mediated by active CRP-cAMP are influenced by the intracellular concentration of cAMP.Another role of cAMP, which is independent of CRP transcription, has been reported. cAMP interacts directly with the DnaA protein and plays a role in the re-activation of DnaA, which is an essential element for initiation of DNA replication from the chromosome origin, oriC (9). Thus the cellular level of cAMP has some effects on controlling the initiation of DNA replication. Moreover, cAMP has a role in regulation of cell division in E. coli. Filamentation of growing cells is induced by elevated levels of cAMP (10). cAMP may affect cell division only indirectly through unidentified cAMP-dependent functions that are not obligatory (11).In E. coli, the intracellular concentration of cAMP has been thought to be mainly controlled by its own synthesis. Synthesis of cAMP is catalyzed by adenylate cyclase, encoded by the cya gene, and its activity is regulated transcriptionally (12, 13) and post-translationally (14, 15). However, the ex...

Recently, a large subfamily of nucleotide-binding and oligomerization domain-containing proteins that have an N-terminal pyrin-like domain and C-terminal leucine-rich repeats has been described. In this study, we identified PYNOD, a novel member of this family that lacks the leucine-rich repeats. We found that human PYNOD mRNA is expressed in various tissues and at high levels in heart, skeletal muscle and brain. It is also expressed in various cell lines, including haematopoietic cell lines. PYNOD oligomerizes and binds to ASC, an adaptor protein that plays a role in apoptotic and inflammatory signal transduction, and to caspase-1 and IL-1beta. PYNOD inhibits apoptosis-associated speck-like protein containing a CARD (ASC)-mediated NF-kappaB activation and apoptosis, and caspase-1-mediated IL-1beta maturation, and it does so in the presence and absence of constitutively active mutants of CARD12 and PYPAF1, which are enhancers of these processes. Thus, PYNOD is a novel regulator of apoptosis and inflammation.