Restart of exponential growth of cold-shocked Yersinia enterocolitica occurs after down-regulation of cspA1/A2 mRNA

The cellular content of major cold shock protein (MCSP) mRNA transcribed from the tandem gene duplication cspA1/A2 and growth of Yersinia enterocolitica were compared when exponentially growing cultures of this bacterium were cold shocked from 30 to 20, 15, 10, 5, or 0 degrees C, respectively. A clear correlation between the time point when exponential growth resumes after cold shock and the degradation of cspA1/A2 mRNA was found. A polynucleotide phosphorylase-deficient mutant was unable to degrade cspA1/A2 mRNA properly and showed a delay, as well as a lower rate, of growth after cold shock. For this mutant, a correlation between decreasing cspA1/A2 mRNA and restart of growth after cold shock was also observed. For both wild-type and mutant cells, no correlation of restart of growth with the cellular content of MCSPs was found. We suggest that, after synthesis of cold shock proteins and cold adaptation of the cells, MCSP mRNAs must be degraded; otherwise, they trap ribosomes, prevent translation of bulk mRNA, and thus inhibit growth of this bacterium at low temperatures.     

Mutation Analysis of the 5′ Untranslated Region of the Cold Shock cspA mRNA of Escherichia coli

The mRNA for CspA, a major cold shock protein in Escherichia coli, contains an unusually long (159 bases) 5' untranslated region (5'-UTR), and its stability has been shown to play a major role in cold shock induction of CspA. The 5'-UTR of the cspA mRNA has a negative effect on its expression at 37 degrees C but has a positive effect upon cold shock. In this report, a series of cspA-lacZ fusions having a 26- to 32-base deletion in the 5'-UTR were constructed to examine the roles of specific regions within the 5'-UTR in cspA expression. It was found that none of the deletion mutations had significant effects on the stability of mRNA at both 37 and 15 degrees C. However, two mutations (Delta56-86 and Delta86-117) caused a substantial increase of beta-galactosidase activity at 37 degrees C, indicating that the deleted regions contain a negative cis element(s) for translation. A mutation (Delta2-27) deleting the highly conserved cold box sequence had little effect on cold shock induction of beta-galactosidase. Interestingly, three mutations (Delta28-55, Delta86-117, and Delta118-143) caused poor cold shock induction of beta-galactosidase. In particular, the Delta118-143 mutation reduced the translation efficiency of the cspA mRNA to less than 10% of that of the wild-type construct. The deleted region contains a 13-base sequence named upstream box (bases 123 to 135), which is highly conserved in cspA, cspB, cspG, and cspI, and is located 11 bases upstream of the Shine-Dalgarno (SD) sequence. The upstream box might be another cis element involved in translation efficiency of the cspA mRNA in addition to the SD sequence and the downstream box sequence. The relationship between the mRNA secondary structure and translation efficiency is discussed.     

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.     

Complementation analysis of the cold-sensitive phenotype of the Escherichia coli csdA deletion strain

The cold shock response of Escherichia coli is elicited by downshift of temperature from 37 degrees C to 15 degrees C and is characterized by induction of several cold shock proteins, including CsdA, during the acclimation phase. CsdA, a DEAD-box protein, has been proposed to participate in a variety of processes, such as ribosome biogenesis, mRNA decay, translation initiation, and gene regulation. It is not clear which of the functions of CsdA play a role in its essential cold shock function or whether all do, and so far no protein has been shown to complement its function in vivo. Our screening of an E. coli genomic library for an in vivo counterpart of CsdA that can compensate for its absence at low temperature revealed only one protein, RhlE, another DEAD-box RNA helicase. We also observed that although not detected in our genetic screening, two cold shock-inducible proteins, namely, CspA, an RNA chaperone, and RNase R, an exonuclease, can also complement the cold shock function of CsdA. Interestingly, the absence of CsdA and RNase R leads to increased sensitivity of the cells to even moderate temperature downshifts. The correlation between the helicase activity of CsdA and the stability of mRNAs of cold-inducible genes was shown using cspA mRNA, which was significantly stabilized in the DeltacsdA cells, an effect counteracted by overexpression of wild-type CsdA or RNase R but not by that of the helicase-deficient mutant of CsdA. These results suggest that the primary role of CsdA in cold acclimation of cells is in mRNA decay and that its helicase activity is pivotal for promoting degradation of mRNAs stabilized at low temperature.     

The psychrotrophic bacterium Yersinia enterocolitica requires expression of pnp, the gene for polynucleotide phosphorylase, for growth at low temperature (5°C)

The psychrotrophic bacterium Yersinia enterocolitica is characterized by temperature-dependent adaptations. To investigate Y . enterocolitica genes involved in cold adaptation, a mutant restricted in its ability to grow at 5°C was isolated from a transposon mutant library. The transposon insertion site in this psychrotrophy-defective (PD) mutant mapped 16 bp upstream of an open reading frame whose predicted amino acid sequence showed 93% similarity with the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase), encoded by pnp . Expression of this gene was blocked in the PD mutant. However, the introduction of a second copy of pnp , including 0.33 kbp sequences upstream of its coding region, into the chromosome of the PD mutant restored pnp expression as well as the ability to grow at 5°C. Furthermore, the expression of pnp appeared to be temperature dependent: in the parental Y . enterocolitica strain, the levels of both pnp mRNA and PNPase were 1.6-fold higher at 5°C compared with 30°C. A similarly enhanced level of PNPase at 5°C was observed in the merodiploid recombinant strain, which indicates that the 0.33 kbp region upstream of pnp harboured a cold-inducible promoter. A putative cold shock promoter motif (ATTGG) was observed in this region.     

CspI, the ninth member of the CspA family of Escherichia coli, is induced upon cold shock

Escherichia coli contains the CspA family, consisting of nine proteins (CspA to CspI), in which CspA, CspB, and CspG have been shown to be cold shock inducible and CspD has been shown to be stationary-phase inducible. The cspI gene is located at 35.2 min on the E. coli chromosome map, and CspI shows 70, 70, and 79% identity to CspA, CspB, and CspG, respectively. Analyses of cspI-lacZ fusion constructs and the cspI mRNA revealed that cspI is cold shock inducible. The 5'-untranslated region of the cspI mRNA consists of 145 bases and causes a negative effect on cspI expression at 37 degrees C. The cspI mRNA was very unstable at 37 degrees C but was stabilized upon cold shock. Analyses of the CspI protein on two-dimensional gel electrophoresis revealed that CspI production is maximal at or below 15 degrees C. Taking these results together, E. coli possesses a total of four cold shock-inducible proteins in the CspA family. Interestingly, the optimal temperature ranges for their induction are different: CspA induction occurs over the broadest temperature range (30 to 10 degrees C), CspI induction occurs over the narrowest and lowest temperature range (15 to 10 degrees C), and CspB and CspG occurs at temperatures between the above extremes (20 to 10 degrees C).