Kunitoshi Yamanaka

Selective mRNA Degradation by Polynucleotide Phosphorylase in Cold Shock Adaptation in Escherichia coli

Upon cold shock, Escherichia coli cell growth transiently stops. During this acclimation phase, specific cold shock proteins (CSPs) are highly induced. At the end of the acclimation phase, their synthesis is reduced to new basal levels, while the non-cold shock protein synthesis is resumed, resulting in cell growth reinitiation. Here, we report that polynucleotide phosphorylase (PNPase) is required to repress CSP production at the end of the acclimation phase. A pnp mutant, upon cold shock, maintained a high level of CSPs even after 24 h. PNPase was found to be essential for selective degradation of CSP mRNAs at 15 degrees C. In a poly(A) polymerase mutant and a CsdA RNA helicase mutant, CSP expression upon cold shock was significantly prolonged, indicating that PNPase in concert with poly(A) polymerase and CsdA RNA helicase plays a critical role in cold shock adaptation.

Complex formation of MukB, MukE and MukF proteins involved in chromosome partitioning in Escherichia coli

mukF, mukE and mukB genes are essential for the process of chromosome partitioning in Escherichia coli. We have studied protein–protein interactions among MukB, MukE and MukF proteins by co‐immunoprecipitation and sucrose gradient sedimentation experiments, using mukFEB null cells harboring plasmids carrying the wild‐type or mutant‐type mukFEB operon. MukB forms a complex with MukF and MukE. Analysis of mutant MukB proteins suggested that MukF and MukE bind the C‐terminal globular domain of MukB. MukF is indispensable for an interaction between MukB and MukE; however, MukF itself is able to associate with MukB even in the absence of MukE. We have also found that MukF has a Ca2+‐binding activity. Although purified MukF was able to make a complex either with MukE or MukB, a complex consisting of the three Muk proteins was barely detected in vitro. However, increasing the Ca2+ or Mg2+ concentration in the reaction partially restored complex formation. This suggests that Ca2+ or Mg2+ may be required for the formation of a complex consisting of the three Muk proteins, and thus may participate in a particular step during chromosome partitioning.

Characterization of GTPase Activity of TrmE, a Member of a Novel GTPase Superfamily, from Thermotoga maritima

A gene encoding a putative GTP-binding protein, a TrmE homologue that is highly conserved in both prokaryotes and eukaryotes, was cloned from Thermotoga maritima, a hyperthermophilic bacterium. T. maritima TrmE was overexpressed in Escherichia coli and purified. TrmE has a GTPase activity but no ATPase activity. The GTPase activity can be competed with GTP, GDP, and dGTP but not with GMP, ATP, CTP, or UTP. K(m) and k(cat) at 70 degrees C were 833 microM and 9.3 min(-1), respectively. Our results indicate that TrmE is a GTP-binding protein with a very high intrinsic GTP hydrolysis rate. We also propose that TrmE homologues constitute a novel subfamily of the GTPase superfamily.

Identification and characterization of five cspA homologous genes from Myxococcus xanthus

Escherichia coli contains a large CspA family consisting of nine homologues, in which four are cold-shock inducible and one is stationary-phase inducible. Here, we demonstrate that Myxococcus xanthus possesses at least five CspA homologues, CspA to CspE. Hydrophobic residues forming a hydrophobic core, and aromatic residues, which are included in functional motifs RNP-1 and RNP-2 involved in binding to RNA and ssDNA, are well conserved. These facts suggest that M. xanthus CspA homologues have a similar structure and function as E. coli CspA. However, in contrast to the E. coli CspA family, the expression of M. xanthus csp genes as judged by primer extension analysis is not significantly regulated by temperature changes, except for cspB of which expression was reduced to less than 10% upon heat shock at 42 degrees C. Such constitutive expression of the csp genes may be important for M. xanthus, a soil-dwelling bacterium, to survive under conditions of exposure to various environmental changes in nature.