C21orf57 is a human homologue of bacterial YbeY proteins

The product of the human C21orf57 (huYBEY) gene is predicted to be a homologue of the highly conserved YbeY proteins found in nearly all bacteria. We show that, like its bacterial and chloroplast counterparts, the HuYbeY protein is an RNase and that it retains sufficient function in common with bacterial YbeY proteins to partially suppress numerous aspects of the complex phenotype of an Escherichia coli ΔybeY mutant. Expression of HuYbeY in Saccharomyces cerevisiae, which lacks a YbeY homologue, results in a severe growth phenotype. This observation suggests that the function of HuYbeY in human cells is likely regulated through specific interactions with partner proteins similarly to the way YbeY is regulated in bacteria.National Institutes of Health (U.S.) (Grant GM31010)National Institutes of Health (U.S.) (Grant GM17151

The Stringent Response and Cell Cycle Arrest in Escherichia coli

The bacterial stringent response, triggered by nutritional deprivation, causes an accumulation of the signaling nucleotides pppGpp and ppGpp. We characterize the replication arrest that occurs during the stringent response in Escherichia coli. Wild type cells undergo a RelA-dependent arrest after treatment with serine hydroxamate to contain an integer number of chromosomes and a replication origin-to-terminus ratio of 1. The growth rate prior to starvation determines the number of chromosomes upon arrest. Nucleoids of these cells are decondensed; in the absence of the ability to synthesize ppGpp, nucleoids become highly condensed, similar to that seen after treatment with the translational inhibitor chloramphenicol. After induction of the stringent response, while regions corresponding to the origins of replication segregate, the termini remain colocalized in wild-type cells. In contrast, cells arrested by rifampicin and cephalexin do not show colocalized termini, suggesting that the stringent response arrests chromosome segregation at a specific point. Release from starvation causes rapid nucleoid reorganization, chromosome segregation, and resumption of replication. Arrest of replication and inhibition of colony formation by ppGpp accumulation is relieved in seqA and dam mutants, although other aspects of the stringent response appear to be intact. We propose that DNA methylation and SeqA binding to non-origin loci is necessary to enforce a full stringent arrest, affecting both initiation of replication and chromosome segregation. This is the first indication that bacterial chromosome segregation, whose mechanism is not understood, is a step that may be regulated in response to environmental conditions

Strain and plasmid list.

a<p>All strains are isogenic with MG1655 with the genotype F-<i>rph</i>-1.</p>b<p>The <i>dam</i> gene was cloned using the Gateway Cloning Technology and methods provided by the manufacturer (Invitrogen). The <i>dam</i> gene was amplified by PCR with Triplemaster (Eppendorf), primers 5′-GGGGACAAGT TTGTACAAAA AAGCAGGCTT CACAGCCGGA GAAGGTGTAA TTAGTTAGTC AGCATGAAGA AAAA and 5′-GGGGACCACT TTGTACAAGA AAGCTGGGTT TATTTTTTCG CGGGTGAAAC GACT and the chromosomal template DNA derived from <i>E. coli</i> K-12 MG1655 (Masterpure DNA purification kit, Epicentre). After purification (Qiagen PCR purification kit), the <i>dam</i> gene was inserted into vector pDONR201 and subcloned from this plasmid into the destination vector pSTL360 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000300#pgen.1000300-Dutra1" target="_blank">[64]</a>, a derivative of pBAD18 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000300#pgen.1000300-Guzman1" target="_blank">[65]</a> with an arabinose-regulated promoter. Plasmids were recovered by transformation into <i>E. coli</i> K-12 strain DH5α. The presence of intact <i>dam</i> in these plasmids was confirmed by DNA sequence analysis.</p

Release from stringent arrest.

<p>Wild type cells in M9 Glucose CAA medium were treated with serine hydroxamate for 1.5 h as previously described. Cells were then washed of serine hydroxamate, incubated in fresh medium, and samples were taken for flow cytometric analysis for DNA content and DAPI staining as previously described. (A) DNA content histograms and (B) corresponding fluorescent DAPI images of cultures incubated with serine hydroxamate for 1.5 h and of cultures at 15, 30, and 60 min after being released from serine hydroxamate. (C) Model for chromosome segregation patterns for 4N cells released from stringent arrest. Cohesion of sister chromosomes help enforce the sequence of segregation of chromosomes from the single nucleoid mass. Pairs of sister chromosomes held in cohesion at their termini segregate first at midcell. After release of cohesion, sisters separate at ¼ positions.</p

Flow cytometric analysis of cell cycle and effects of the stringent response.

<p>Cells were grown in 0.2% glucose, 0.4% glucose, or 0.4% glucose 0.2% casamino acids—M9 minimal media. DNA content for wild type and mutant cells before treatment (left panel), treated with serine hydroxamate (SHX) for 1.5 h (middle panel), or rifampicin or cephalexin for 4 h (right panel). Samples were taken for determination of DNA content by PicoGreen fluorescence (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000300#s4" target="_blank">Materials and Methods</a>). The fluorescent scale (x-axis) corresponds to chromosome equivalents as indicated. The population doubling times, T_d, for each growth condition are indicated.</p