An internal region of the RpoH heat shock transcription factor is critical for rapid degradation by the FtsH protease

AbstractThe proteolysis of regulatory proteins plays an important role in the control of gene expression. The Escherichia coli heat shock sigma factor RpoH (σ32) is highly unstable. Its instability is determined by interactions with the DnaK chaperone machine, RNA polymerase and the ATP-dependent protease FtsH. Bradyrhizobium japonicum expresses three RpoH proteins of which RpoH1 is highly stable. To determine which regions of E. coli RpoH determine protein lability, we generated a number of truncated versions and hybrid proteins. Truncation of N-terminal amino acids had no, and deletion of C-terminal amino acids only a minor effect on stability of RpoH. A major determinant of RpoH lability was mapped to a region of about 85 amino acids (residues 36–122) roughly comprising the sigma factor region 2. This is the first demonstration of an internal RpoH region being responsible for FtsH-mediated degradation

Noise in timing and precision of gene activities in a genetic cascade

Biological developmental pathways require proper timing of gene expression. We investigated timing variations of defined steps along the lytic cascade of bacteriophage λ. Gene expression was followed in individual lysogenic cells, after induction with a pulse of UV irradiation. At low UV doses, some cells undergo partial induction and eventually divide, whereas others follow the lytic pathway. The timing of events in cells committed to lysis is independent of the level of activation of the SOS response, suggesting that the lambda network proceeds autonomously after induction. An increased loss of temporal coherence of specific events from prophage induction to lysis is observed, even though the coefficient of variation of timing fluctuations decreases. The observed temporal variations are not due to cell factors uniformly dilating the timing of execution of the cascade. This behavior is reproduced by a simple model composed of independent stages, which for a given mean duration predicts higher temporal precision, when a cascade consists of a large number of steps. Evidence for the independence of regulatory modules in the network is presented

Phage Lambda CIII: A Protease Inhibitor Regulating the Lysis-Lysogeny Decision

The ATP-dependent protease FtsH (HflB) complexed with HflKC participates in post-translational control of the lysis-lysogeny decision of bacteriophage lambda by rapid degradation of lambda CII. Both phage-encoded proteins, the CII transcription activator and the CIII polypeptide, are required for efficient lysogenic response. The conserved CIII is both an inhibitor and substrate of FtsH. Here we show that the protease inhibitor CIII is present as oligomeric amphipathic α helical structures and functions as a competitive inhibitor of FtsH by preventing binding of the CII substrate. We identified single alanine substitutions in CIII that abolish its activity. We characterize a dominant negative effect of a CIII mutant. Thus, we suggest that CIII oligomrization is required for its function. Real-time analysis of CII activity demonstrates that the effect of CIII is not seen in the absence of either FtsH or HflKC. When CIII is provided ectopically, CII activity increases linearly as a function of the multiplicity of infection, suggesting that CIII enhances CII stability and the lysogenic response. FtsH function is essential for cellular viability as it regulates the balance in the synthesis of phospholipids and lipopolysaccharides. Genetic experiments confirmed that the CIII bacteriostatic effects are due to inhibition of FtsH. Thus, the early presence of CIII following infection stimulates the lysogenic response, while its degradation at later times ensures the reactivation of FtsH allowing the growth of the established lysogenic cell

Increased Bending Rigidity of Single DNA Molecules by H-NS, a Temperature and Osmolarity Sensor

Histonelike nucleoid structuring protein (H-NS) is an abundant prokaryotic protein participating in nucleoid structure, gene regulation, and silencing. It plays a key role in cell response to changes in temperature and osmolarity. Force-extension measurements of single, twist-relaxed λ-DNA-H-NS complexes show that these adopt more extended configurations compared to the naked DNA substrates. Crosslinking indicates that H-NS can decorate DNA molecules at one H-NS dimer per 15–20 bp. These results suggest that H-NS polymerizes along DNA, forming a complex of higher bending rigidity. These effects are not observed above 32°C or at high osmolarity, supporting the hypothesis that a direct H-NS-DNA interaction plays a key role in gene silencing. Thus, we propose that H-NS plays a unique structural role, different from that of HU and IHF, and functions as one of the environmental sensors of the cell

Integration host factor binds to a unique class of complex repetitive extragenic DNA sequences in Escherichia coli

Summary Interspersed repeated DNA sequences are characteristic features of both prokaryotic and eukaryotic genomes. REP sequences are defined as conserved repetitive extragenic palindromic sequences and are found in Escherichia coli, Salmonella typhimurium and other closely related enteric bacteria. These REP sequences may participate in the folding of the bacterial chromosome. In this work we describe a unique class of 28 conserved complex REP clusters, about 100bp long, in which two inverted REPs are separated by a singular integration host factor (IHF) recognition sequence. We term these sequences RIP (for repetitive IHF‐binding palindromic) elements and demonstrate that IHF binds to them specifically. It is estimated that there are about 70 RIP elements in E. coli. Our analysis shows that the RIP elements are evenly distributed around the bacterial chromosome. The possible function of the RIP element is discussed

Schematic model for CIII activity.

<p>(A) The role of the CIII/CII/FtsH/HflKC module in the lambda genetic decision. The FtsH and HflKC complex regulates CII and CIII levels and CIII controls FtsH/HflKC proteolytic activity. (B) A schematic drawing of the CIII mechanism of inhibition (based on the model presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000363#pone.0000363-Ito1" target="_blank">[16]</a>). In panel 1 in the absence of CIII, CII binds the FtsH protease and is translocated into the enzymatic cavity where it is first unfolded and then degraded. In panel 2, CIII binds the FtsH protease and prevents CII from binding. In panel 3, the CIII protein, which is found as an oligomer, is also subjected to degradation.</p

CIII levels effect on CII and Q activity <i>in vivo.</i>

<p>CII activity is reported by pE-<i>gfp</i> fusions (blue diamonds), whereas Q activity is reported by pR‘-tR‘-<i>gfp</i> fusions (red circles). Promoter activity is given as a function of time of infected cultures carrying the pCTCIII plasmid in the presence or absence of 0.1mM IPTG (B, D, F or A, C, E respectively) with λ<i>c+</i> (A, B), λ<i>cIII⁻</i> (C, D), and λ<i>cII⁻</i> (E, F). All measurements were carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000363#s4" target="_blank">materials and methods</a> at a MOI of 6.</p

Conservation of CIII in phage and bacteria.

<p>The multi sequences alignment was done using the ClustalX (version 1.81) program. Conserved or partially conserved residues are colored according to their biochemical properties. The essential region for CIII activity is underlined and the positions predicted coiled coil (by the MARCOIL) is marked above the sequence. (A) Alignment of CIII proteins from different phages and prophages (represented by the phage name or the bacterial strain carrying the prophage, respectively). CIII proteins were collected from the BLAST program by using the lambda CIII sequence as a query for the non-redundant database. A separate search using the HK022 CIII sequence was done. The HK022 CIII sequence is identical to that of HK097A and H-19B. A complete list of all the phages and prophages in which CIII homologs were recognized, is presented in the supplementary material. (B) Alignment of CIII and SpoVM proteins was carried out with the lambda CIII protein aligned with three SpoVM proteins from different Bacillus strains. (C) Alignment of CIII and YjhS hypothetical proteins that carry the DUF1737 and DUF303, was carried out. The three YjhS-like proteins were derived from different Shigella toxin carrying phages. The HP stands for hypothetical protein without a specific name.</p