Nonsense mutations in cspA cause ribosome trapping leading to complete growth inhibition and cell death at low temperature in Escherichia coli.

CspA, the major cold shock protein of Escherichia coli, is dramatically induced immediately after cold shock. CspA production is transient and reduces to a low basal level when cells become adapted. Here we show that expression from multicopy plasmids of mutant cspA mRNAs bearing nonsense mutations in the coding region caused sustained high levels of the mutant mRNAs at low temperature, resulting in complete inhibition of cell growth ultimately leading to cell death. We demonstrate that the observed growth inhibition was caused by largely exclusive occupation of cellular ribosomes by the mutant cspA mRNAs. Such sequestration of ribosomes even occurs without a single peptide bond formation, implying that the robust translatability of the cspA mRNA is determined at the step of initiation. Further analysis demonstrated that the downstream box of the cspA mRNA was dispensable for the effect, whereas the upstream box of the mRNA was essential. Our system may offer a novel means to study sequence or structural elements involved in the translation of the cspA mRNA and may also be utilized to regulate bacterial growth at low temperature.

Posttranslational mechanisms regulate the mammalian circadian clock.

We have examined posttranslational regulation of clock proteins in mouse liver in vivo. The mouse PERIOD proteins (mPER1 and mPER2), CLOCK, and BMAL1 undergo robust circadian changes in phosphorylation. These proteins, the cryptochromes (mCRY1 and mCRY2), and casein kinase I epsilon (CKIepsilon) form multimeric complexes that are bound to DNA during negative transcriptional feedback. CLOCK:BMAL1 heterodimers remain bound to DNA over the circadian cycle. The temporal increase in mPER abundance controls the negative feedback interactions. Analysis of clock proteins in mCRY-deficient mice shows that the mCRYs are necessary for stabilizing phosphorylated mPER2 and for the nuclear accumulation of mPER1, mPER2, and CKIepsilon. We also provide in vivo evidence that casein kinase I delta is a second clock relevant kinase.

A sequence downstream of the initiation codon is essential for cold shock induction of cspB of Escherichia coli.

Cold shock induction of cspB has been shown to be primarily regulated at the mRNA level. Here, we demonstrate that the induction of cspB at low temperature also requires the translational cis-acting element called the downstream box (DB). Full induction of cspB at low temperature is achieved in the presence of both the Shine-Dalgarno sequence and DB. We propose that the DB sequence functions as a translational enhancer for the biosynthesis of CspB to bypass the inhibitory effect in translation caused by cold shock.

Translational enhancement by an element downstream of the initiation codon in Escherichia coli.

The translation initiation of Escherichia coli mRNAs is known to be facilitated by a cis element upstream of the initiation codon, called the Shine-Dalgarno (SD) sequence. This sequence complementary to the 3' end of 16 S rRNA enhances the formation of the translation initiation complex of the 30 S ribosomal subunit with mRNAs. It has been debated that a cis element called the downstream box downstream of the initiation codon, in addition to the SD sequence, facilitates formation of the translation initiation complex; however, conclusive evidence remains elusive. Here, we show evidence that the downstream box plays a major role in the enhancement of translation initiation in concert with SD.

CspA, CspB, and CspG, major cold shock proteins of Escherichia coli, are induced at low temperature under conditions that completely block protein synthesis.

CspA, CspB, and CspG, the major cold shock proteins of Escherichia coli, are dramatically induced upon temperature downshift. In this report, we examined the effects of kanamycin and chloramphenicol, inhibitors of protein synthesis, on cold shock inducibility of these proteins. Cell growth was completely blocked at 37 degrees C in the presence of kanamycin (100 microgram/ml) or chloramphenicol (200 microgram/ml). After 10 min of incubation with the antibiotics at 37 degrees C, cells were cold shocked at 15 degrees C and labeled with [35S]methionine at 30 min after the cold shock. Surprisingly, the synthesis of all these cold shock proteins was induced at a significantly high level virtually in the absence of synthesis of any other protein, indicating that the cold shock proteins are able to bypass the inhibitory effect of the antibiotics. Possible bypass mechanisms are discussed. The levels of cspA and cspB mRNAs for the first hour at 15 degrees C were hardly affected in the absence of new protein synthesis caused either by antibiotics or by amino acid starvation.

Differential thermoregulation of two highly homologous cold-shock genes, cspA and cspB, of Escherichia coli.

BACKGROUND: The major cold-shock protein in Escherichia coli is CspA, a 7.4 kDa protein. A CspA family has been found which consists of four additional proteins, CspB, CspC, CspD and CspE. The expression of cspB, unlike the other homologues, is cold-shock inducible like cspA. RESULTS: We examined the cold-shock induction of CspA and CspB at various temperatures. The cspA induction is observed by temperature shift from 37 to 30 degrees C and high levels of CspA production are observed between 24 and 10 degrees C. In contrast, CspB production occurs only by temperature shift to below 20 C, with maximum induction at 15 degrees C. Both cspA and cspB expressions were found to be induced at the level of transcription as determined by primer extension. CONCLUSIONS: These results show that cspA and cspB expressions are differentially regulated at low temperature indicating that E. coli contains at least two different biothermostats or thermoregulators that are likely to play important roles in cellular adaptation to low temperature. The cspB promoter shows sequence similarity to the cspA promoter. Furthermore, both cspA and cspB mRNAs have unusually long 5' untranslated regions (159 and 161 bases, respectively), both of which are able to form similar extensive secondary structures. These features are considered to contribute to the nature of the thermostats for cspA and cspB.

Family of the major cold-shock protein, CspA (CS7.4), of Escherichia coli, whose members show a high sequence similarity with the eukaryotic Y-box binding proteins.

The cspA is a gene of Escherichia coli, whose expression is specifically induced at low temperatures to a level of 13% of total protein synthesis. The CspA protein consisting of 70 amino acid residues has high sequence similarity with eukaryotic Y-box DNA-binding proteins. We found two independent clones from the Kohara miniset phage collection, which hybridized with a DNA fragment containing cspA. DNA sequencing of these clones confirmed that the two genes are highly homologous to cspA. One designated cspB is mapped at 35 min on the E. coli chromosome and encodes a 71-residue protein with 79% identity to CspA, while the other, cspC, is mapped at 40 min and encodes a 69-residue protein with 70% identity. In addition, a DNA sequence upstream of the clpA gene at 19 min published elsewhere contains an open reading frame for a 74-residue protein with 45% identity to CspA. All csp genes were fused in the coding regions with the lacZ gene, and the expression of beta-galactosidase was examined for these hybrid genes upon cold shock. A similar cold-shock induction to cspA was observed for cspB but not cspC and cspD. These results indicate that E. coli has a family of the cspA gene, some of which are induced by cold shock.

Specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum.

To obtain specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum, a discriminatory recombinant DNA library was constructed using selective fragments of the bacterial genome. Three renibacterial clones, pMAM29, pMAM46 and pMAM77, containing 149, 73, and 154 bp respectively, were isolated and characterized. The specificity of the probes was confirmed by dot-blot and Southern hybridization analyses. Bacterial hybridization experiments revealed that pMAM29 discriminates the R. salmoninarum genome from that of other fish pathogens such as Aeromonas salmonicida, Yersinia ruckeri, Flexibacter columnaris, Lactobacillus piscicola, Vibrio ordalii, Vibrio anguillarum and Aeromonas hydrophila. Thus, this probe may provide a new means to diagnose bacterial kidney disease in asymptomatic fish and ova.

Molecular cloning of Renibacterium salmoninarum DNA fragments.

A Renibacterium salmoninarum enriched recombinant DNA library was constructed to isolate DNA fragments which could be used as probes to detect gene sequences specific for the causative agent of bacterial kidney disease in salmonid fish. One fragment of 149 base pairs was isolated and its specificity and sequence determined. This probe may prove useful in the design of diagnostic tests for the disease in asymptomatic fish and ova.