No abstract

Escherichia coli contains operons called "addiction modules," encoding toxin and antitoxin, which are responsible for growth arrest and cell death. Here, we demonstrate that MazF toxin encoded by "mazEF addiction module" is a sequence-specific (ACA) endoribonuclease functional only for single-stranded RNA. MazF works as a ribonuclease independent of ribosomes, and is, therefore, functionally distinct from RelE, another E. coli toxin, which assists mRNA cleavage at the A site on ribosomes. Upon induction, MazF cleaves whole cellular mRNAs to efficiently block protein synthesis. Purified MazF inhibited protein synthesis in both prokaryotic and eukaryotic cell-free systems. This inhibition was released by MazE, the labile antitoxin against MazF. Thus, MazF functions as a toxic endoribonuclease to interfere with the function of cellular mRNAs by cleaving them at specific sequences leading to rapid cell growth arrest and cell death. The role of such endoribonucleases may have broad implication in cell physiology under various growth conditions.

CspA, the major cold-shock protein of Escherichia coli, is dramatically induced during the cold-shock response. The amino acid sequence of CspA shows 43% identity to the "cold-shock domain" of the eukaryotic Y-box protein family, which interacts with RNA and DNA to regulate their functions. Here, we demonstrate that CspA binds to RNA as a chaperone. First, CspA cooperatively binds to heat-denatured single-stranded RNA if it is larger than 74 bases, causing a supershift in gel electrophoresis. A minimal concentration of CspA at 2.7 ؋ 10 ؊5 M is absolutely required for this cooperative binding, which is sufficiently lower than the estimated cellular concentration of CspA (10 ؊4 M) in cold-shocked cells. No specific RNA sequences for CspA binding were identified, indicating that it has a broad sequence specificity for its binding. When the 142-base 5-untranslated region of the cspA mRNA was used as a substrate for ribonucleases A and T1, the addition of CspA significantly stimulated RNA hydrolysis by preventing the formation of RNase-resistant bands due to stable secondary structures in the 5-untranslated region. These results indicate that binding of CspA to RNA destabilizes RNA secondary structures to make them susceptible to ribonucleases. We propose that CspA functions as an RNA chaperone to prevent the formation of secondary structures in RNA molecules at low temperature. Such a function may be crucial for efficient translation of mRNAs at low temperatures and may also have an effect on transcription.

Almost all bacteria and many archaea contain genes whose expression inhibits cell growth and may lead to cell death when overproduced, reminiscent of apoptotic genes in higher systems. The cellular targets of these toxins are quite diverse and include DNA replication, mRNA stability, protein synthesis, cell-wall biosynthesis, and ATP synthesis. These toxins are co-expressed and neutralized with their cognate antitoxins from a TA (toxin-antitoxin) operon in normally growing cells. Antitoxins are more labile than toxins and are readily degraded under stress conditions, allowing the toxins to exert their toxic effect. Presence of at least 33 TA systems in Escherichia coli and more than 60 TA systems in Mycobacterium tuberculosis suggests that the TA systems are involved not only in normal bacterial physiology but also in pathogenicity of bacteria. The elucidation of their cellular function and regulation is thus crucial for our understanding of bacterial physiology under various stress conditions.

Multicopy single-stranded DNA (msDNA), a branched DNA-RNA molecule, has been shown in Escherichia coli B and clinical strain Cl-1 to be synthesized by reverse transcriptase. We report that 13% of the strains of the ECOR collection, a sample of 72 E. coli isolates representing the breadth of genetic variation of the species, produce msDNA. Three of the four major subspecific groups include msDNA-producing strains. Screening of 25 isolates that are genetically related to msDNA-producing clinical strains uncovered 22 additional msDNA-producing strains. A phylogenetic tree based on allelic variation detected electrophoretically at 20 enzyme-encoding loci revealed two major clusters and several deep branches composed of strains that synthesize msDNA. Although E. coli K-12 does not harbor msDNA, other closely related strains of the K-12 family do. The results support the hypothesis that msDNA-synthesizing systems, including reverse transcriptase genes, were acquired recently and independently in different lineages of E. coli.The recent discovery of bacterial reverse transcriptases (RTs) was engendered by the observation of a peculiar satellite DNA, now called multicopy single-stranded DNA (msDNA), in the soil bacterium Myxococcus xanthus (26). The satellite (msDNA-Mxl62) is unusual because it consists of a 162-base single DNA strand linked to a 77-base RNA (msdRNA) sequence by a 2',5'-phosphodiester linkage and occurs in hundreds of copies per cell (4). An msDNA of a similar structure (msDNA-Sa163) has been found in the closely related myxobacterium Stigmatella aurantiaca (5, 6). Recently it has been reported that M. xanthus contains another species of msDNA (msDNA-Mx65) in addition to msDNA-Mxl62 (3). Most recently, msDNA molecules with structural features similar to those of myxobacterial msDNAs have been found in clinical isolate Cl-1 (9), strain B (10), and several other clinical isolates (21) of Escherichia coli.In M. xanthus (8), E. coli clinical isolate Cl-1 (9), and E. coli B (10), the synthesis of msDNA depends on RT encoded by a sequence adjacent to the chromosomal region specifying msdRNA. Although the three bacterial RTs are substantially divergent in size and amino acid sequence, as inferred from the nucleotide sequences, they exhibit the common amino acid motifs of retroviral reverse transcriptases (22).The objective of the study reported here was to determine the frequency of occurrence and genetic relationships among strains that harbor msDNA synthesized by RT in natural populations of E. coli. To accomplish this, we examined the 72 strains of the ECOR collection (13), a sample of natural isolates chosen to represent the major subspecific groups of the E. coli species as a whole. These groups have been identified on the basis of allelic variation at enzyme-encoding genes detected by multilocus enzyme electrophoresis (14, 23) and include isolates recovered from a variety of human and animal hosts in diverse geographic areas (13). In addition, we characterized the multilocus genotypes of previously repor...

When exponentially growing Escherichia coli cell cultures were transferred from 37C to 100C or 15TC,

Escherichia coli K-12 contains at least 36 toxin genes, the expression of which causes growth inhibition and eventual death. These toxins are usually co-expressed with their cognate antitoxins in operons called toxin-antitoxin (TA) modules. Under normal growth conditions, toxins and antitoxins form stable complexes. However, stress-induced proteases preferentially eliminate unstable antitoxins, releasing free toxins to inhibit various cellular functions. TA systems have important roles in the physiology of cells in their natural habitats, including functions in biofilm formation and multidrug resistance. In this Review, we describe these TA systems in light of their functions and roles in the regulation of cell growth and death.

CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone, which is thought to facilitate translation at low temperature by destabilizing mRNA structures. Here we demonstrate that CspA, as well as homologous RNA chaperones CspE and CspC, are transcription antiterminators. In vitro, the addition of physiological concentrations of recombinant CspA, CspE, or CspC decreased transcription termination at several intrinsic terminators and also decreased transcription pausing. In vivo, overexpression of cloned CspC and CspE at 37°C was sufficient to induce transcription of the metY-rpsO operon genes nusA, infB, rbfA, and pnp located downstream of multiple transcription terminators. Similar induction of downstream metY-rpsO operon genes was observed at cold shock, a condition to which the cell responds by massive overproduction of CspA. The products of nusA, infB, rbfA, and pnp-NusA, IF2, RbfA, and PNP-are known to be induced at cold shock. We propose that the cold-shock induction of nusA, infB, rbfA, and pnp occurs through transcription antitermination, which is mediated by CspA and other cold shock-induced Csp proteins.CspA proteins ͉ cold shock ͉ transcription antitermination