A negative feedback loop is critical for recovery of RpoS after stress in Escherichia coli.

RpoS is an alternative sigma factor needed for the induction of the general stress response in many gammaproteobacteria. Tight regulation of RpoS levels and activity is required for bacterial growth and survival under stress. In Escherichia coli, various stresses lead to higher levels of RpoS due to increased translation and decreased degradation. During non-stress conditions, RpoS is unstable, because the adaptor protein RssB delivers RpoS to the ClpXP protease. RpoS degradation is prevented during stress by the sequestration of RssB by anti-adaptors, each of which is induced in response to specific stresses. Here, we examined how the stabilization of RpoS is reversed during recovery of the cell from stress. We found that RpoS degradation quickly resumes after recovery from phosphate starvation, carbon starvation, and when transitioning from stationary phase back to exponential phase. This process is in part mediated by the anti-adaptor IraP, known to promote RpoS stabilization during phosphate starvation via the sequestration of adaptor RssB. The rapid recovery from phosphate starvation is dependent upon a feedback loop in which RpoS transcription of rssB, encoding the adaptor protein, plays a critical role. Crl, an activator of RpoS that specifically binds to and stabilizes the complex between the RNA polymerase and RpoS, is also required for the feedback loop to function efficiently, highlighting a critical role for Crl in restoring RpoS basal levels.

Directed Screening for sRNA Targets in E. coli Using a Plasmid Library.

A large number of bacterial small regulatory RNAs (sRNAs) modulate gene expression by base pairing to a target mRNA, affecting its translation or stability. This posttranscriptional regulation has been shown to be essential and critical for bacterial physiology. One of the challenges of studying sRNA signaling is identifying the sRNA regulators of specific genes. Here, we describe a protocol for making an sRNA expression library and using this library to screen for sRNA regulators of genes of interest in E. coli. This library can be easily expanded and adapted to use in other bacteria.

A negative feedback loop is critical for recovery of RpoS after stress in Escherichia coli.

RpoS is an alternative sigma factor needed for the induction of the general stress response in many gammaproteobacteria. Tight regulation of RpoS levels and activity is required for bacterial growth and survival under stress. In Escherichia coli, various stresses lead to higher levels of RpoS due to increased translation and decreased degradation. During non-stress conditions, RpoS is unstable, because the adaptor protein RssB delivers RpoS to the ClpXP protease. RpoS degradation is prevented during stress by the sequestration of RssB by anti-adaptors, each of which is induced in response to specific stresses. Here, we examined how the stabilization of RpoS is reversed during recovery of the cell from stress. We found that RpoS degradation quickly resumes after recovery from phosphate starvation, carbon starvation, and when transitioning from stationary phase back to exponential phase. This process is in part mediated by the anti-adaptor IraP, known to promote RpoS stabilization during phosphate starvation via the sequestration of adaptor RssB. The rapid recovery from phosphate starvation is dependent upon a feedback loop in which RpoS transcription of rssB, encoding the adaptor protein, plays a critical role. Crl, an activator of RpoS that specifically binds to and stabilizes the complex between the RNA polymerase and RpoS, is also required for the feedback loop to function efficiently, highlighting a critical role for Crl in restoring RpoS basal levels.

An acetyltranferase moonlights as a regulator of the RNA binding repertoire of the RNA chaperone Hfq in Escherichia coli.

Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with their target mRNAs. In Escherichia coli and many other bacteria, this process is dependent on the RNA chaperone Hfq, a mediator for sRNA-mRNA annealing. YhbS (renamed here as HqbA), a putative Gcn5-related N-acetyltransferase (GNAT), was previously identified as a silencer of sRNA signaling in a genomic library screen. Here, we studied how HqbA regulates sRNA signaling and investigated its physiological roles in modulating Hfq activity. Using fluorescent reporter assays, we found that HqbA overproduction suppressed all tested Hfq-dependent sRNA signaling. Direct interaction between HqbA and Hfq was demonstrated both in vivo and in vitro, and mutants that blocked the interaction interfered with HqbA suppression of Hfq. However, an acetylation-deficient HqbA mutant still disrupted sRNA signaling, and HqbA interacted with Hfq at a site far from the active site. This suggests that HqbA may be bifunctional, with separate roles for regulating via Hfq interaction and for acetylation of undefined substrates. Gel shift assays revealed that HqbA strongly reduced the interaction between the Hfq distal face and low-affinity RNAs but not high-affinity RNAs. Comparative RNA immunoprecipitation of Hfq and sequencing showed enrichment of two tRNA precursors, metZWV and proM, by Hfq in mutants that lost the HqbA-Hfq interaction. Our results suggest that HqbA provides a level of quality control for Hfq by competing with low-affinity RNA binders.

Structure of phosphorylated-like RssB, the adaptor delivering σs to the ClpXP proteolytic machinery, reveals an interface switch for activation.

In enterobacteria such as Escherichia coli, the general stress response is mediated by σs, the stationary phase dissociable promoter specificity subunit of RNA polymerase. σs is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which is thought to be stimulated by the phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the structure of RssB bound to the IraD anti-adaptor, our new RssB structure with bound beryllofluoride reveals conformational differences and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the interdomain segmented helical linker. These are accompanied by masking of the α4-ß5-α5 (4-5-5) "signaling" face of the RssB receiver domain by its C-terminal domain. Critically, using hydrogen-deuterium exchange mass spectrometry, we identify σs-binding determinants on the 4-5-5 face, implying that this surface needs to be unmasked to effect an interdomain interface switch and enable full σs engagement and hand-off to ClpXP. In activated receiver domains, the 4-5-5 face is often the locus of intermolecular interactions, but its masking by intramolecular contacts upon phosphorylation is unusual, emphasizing that RssB is a response regulator that undergoes atypical regulation.

Lack of polyamines leads to cotranslational degradation of the general stress factor RpoS in Escherichia coli.

The specialized sigma factor RpoS mediates a general stress response in Escherichia coli and related bacteria, activating promoters that allow cells to survive stationary phase and many stresses. RpoS synthesis and stability are regulated at multiple levels. Translation of RpoS is positively regulated by multiple small RNAs in response to stress. Degradation of RpoS, dependent upon the adaptor protein RssB, is rapid during exponential growth and ceases upon starvation or other stresses, increasing accumulation of RpoS. E. coli carrying mutations that block the synthesis of polyamines were previously found to have low levels of RpoS, while levels increased rapidly when polyamines were added. We have used a series of reporters to examine the basis for the lack of RpoS in polyamine-deficient cells. The polyamine requirement was independent of small RNA-mediated positive regulation of RpoS translation. Mutations in rssB stabilize RpoS and significantly bypassed the polyamine deficit, suggesting that lack of polyamines might lead to rapid RpoS degradation. However, rates of degradation of mature RpoS were unaffected by polyamine availability. Codon optimization in rpoS partially relieved the polyamine dependence, suggesting a defect in RpoS translation in the absence of polyamines. Consistent with this, a hyperproofreading allele of ribosomal protein S12, encoded by rpsL, showed a decrease in RpoS levels, and this decrease was also suppressed by either codon optimization or blocking RpoS degradation. We suggest that rpoS codon usage leads it to be particularly sensitive to slowed translation, due to either lack of polyamines or hyperproofreading, leading to cotranslational degradation. We dedicate this study to Herb Tabor and his foundational work on polyamines, including the basis for this study.

Identification of Attenuators of Transcriptional Termination: Implications for RNA Regulation in Escherichia coli.

The regulatory function of many bacterial small RNAs (sRNAs) requires the binding of the RNA chaperone Hfq to the 3' portion of the sRNA intrinsic terminator, and therefore sRNA signaling might be regulated by modulating its terminator. Here, using a multicopy screen developed with the terminator of sRNA SgrS, we identified an sRNA gene (cyaR) and three protein-coding genes (cspD, ygjH, and rof) that attenuate SgrS termination in Escherichia coli. Analyses of CyaR and YgjH, a putative tRNA binding protein, suggested that the CyaR activity was indirect and the effect of YgjH was moderate. Overproduction of the protein attenuators CspD and Rof resulted in more frequent readthrough at terminators of SgrS and two other sRNAs, and regulation by SgrS of target mRNAs was reduced. The effect of Rof, a known inhibitor of Rho, was mimicked by bicyclomycin or by a rho mutant, suggesting an unexpected role for Rho in sRNA termination. CspD, a member of the cold shock protein family, bound both terminated and readthrough transcripts, stabilizing them and attenuating termination. By RNA sequencing analysis of the CspD overexpression strain, we found global effects of CspD on gene expression across some termination sites. We further demonstrated effects of endogenous CspD under slow growth conditions where cspD is highly expressed. These findings provided evidence of changes in the efficiency of intrinsic termination, confirming this as an additional layer of the regulation of sRNA signaling. IMPORTANCE Growing evidence suggests that the modulation of intrinsic termination and readthrough of transcription is more widespread than previously appreciated. For small RNAs, proper termination plays a critical role in their regulatory function. Here, we present a multicopy screen approach to identify factors that attenuate small RNA termination and therefore abrogate signaling dependent on the small RNA. This study highlights a new aspect of regulation of small RNA signaling as well as the modulation of intrinsic termination.

Multiple in vivo roles for the C-terminal domain of the RNA chaperone Hfq.

Hfq, a bacterial RNA chaperone, stabilizes small regulatory RNAs (sRNAs) and facilitates sRNA base-pairing with target mRNAs. Hfq has a conserved N-terminal domain and a poorly conserved disordered C-terminal domain (CTD). In a transcriptome-wide examination of the effects of a chromosomal CTD deletion (Hfq1-65), the Escherichia coli mutant was most defective for the accumulation of sRNAs that bind the proximal and distal faces of Hfq (Class II sRNAs), but other sRNAs also were affected. There were only modest effects on the levels of mRNAs, suggesting little disruption of sRNA-dependent regulation. However, cells expressing Hfq lacking the CTD in combination with a weak distal face mutation were defective for the function of the Class II sRNA ChiX and repression of mutS, both dependent upon distal face RNA binding. Loss of the region between amino acids 66-72 was critical for this defect. The CTD region beyond amino acid 72 was not necessary for distal face-dependent regulation, but was needed for functions associated with the Hfq rim, seen most clearly in combination with a rim mutant. Our results suggest that the C-terminus collaborates in various ways with different binding faces of Hfq, leading to distinct outcomes for individual sRNAs.

A fluorescence-based genetic screen reveals diverse mechanisms silencing small RNA signaling in E. coli.

As key players of gene regulation in many bacteria, small regulatory RNAs (sRNAs) associated with the RNA chaperone Hfq shape numerous phenotypic traits, including metabolism, stress response and adaptation, as well as virulence. sRNAs can alter target messenger RNA (mRNA) translation and stability via base pairing. sRNA synthesis is generally under tight transcriptional regulation, but other levels of regulation of sRNA signaling are less well understood. Here we used a fluorescence-based functional screen to identify regulators that can quench sRNA signaling of the iron-responsive sRNA RyhB in Escherichia coli The identified regulators fell into two classes, general regulators (affecting signaling by many sRNAs) and RyhB-specific regulators; we focused on the specific ones here. General regulators include three Hfq-interacting sRNAs, CyaR, ChiX, and McaS, previously found to act through Hfq competition, RNase T, a 3' to 5' exonuclease not previously implicated in sRNA degradation, and YhbS, a putative GCN5-related N-acetyltransferase (GNAT). Two specific regulators were identified. AspX, a 3'end-derived small RNA, specifically represses RyhB signaling via an RNA sponging mechanism. YicC, a previously uncharacterized but widely conserved protein, triggers rapid RyhB degradation via collaboration with the exoribonuclease PNPase. These findings greatly expand our knowledge of regulation of bacterial sRNA signaling and suggest complex regulatory networks for controlling iron homeostasis in bacteria. The fluorescence-based genetic screen system described here is a powerful tool expected to accelerate the discovery of novel regulators of sRNA signaling in many bacteria.

Exonuclease VII repairs quinolone-induced damage by resolving DNA gyrase cleavage complexes.

The widely used quinolone antibiotics act by trapping prokaryotic type IIA topoisomerases, resulting in irreversible topoisomerase cleavage complexes (TOPcc). Whereas the excision repair pathways of TOPcc in eukaryotes have been extensively studied, it is not known whether equivalent repair pathways for prokaryotic TOPcc exist. By combining genetic, biochemical, and molecular biology approaches, we demonstrate that exonuclease VII (ExoVII) excises quinolone-induced trapped DNA gyrase, an essential prokaryotic type IIA topoisomerase. We show that ExoVII repairs trapped type IIA TOPcc and that ExoVII displays tyrosyl nuclease activity for the tyrosyl-DNA linkage on the 5'-DNA overhangs corresponding to trapped type IIA TOPcc. ExoVII-deficient bacteria fail to remove trapped DNA gyrase, consistent with their hypersensitivity to quinolones. We also identify an ExoVII inhibitor that synergizes with the antimicrobial activity of quinolones, including in quinolone-resistant bacterial strains, further demonstrating the functional importance of ExoVII for the repair of type IIA TOPcc.

IgaA negatively regulates the Rcs Phosphorelay via contact with the RcsD Phosphotransfer Protein.

Two-component systems and phosphorelays play central roles in the ability of bacteria to rapidly respond to changing environments. In E. coli and related enterobacteria, the complex Rcs phosphorelay is a critical player in the bacterial response to antimicrobial peptides, beta-lactam antibiotics, and other disruptions at the cell surface. The Rcs system is unusual in that an inner membrane protein, IgaA, is essential due to its negative regulation of the RcsC/RcsD/RcsB phosphorelay. While it is known that IgaA transduces signals from the outer membrane lipoprotein RcsF, how it interacts with the phosphorelay has remained unknown. Here we performed in vivo interaction assays and genetic dissection of the critical proteins and found that IgaA interacts with the phosphorelay protein RcsD, and that this interaction is necessary for regulation. Interactions between IgaA and RcsD within their respective periplasmic domains of these two proteins anchor repression of signaling. However, the signaling response depends on a second interaction between cytoplasmic loop 1 of IgaA and a truncated Per-Arndt-Sim (PAS-like) domain in RcsD. A single point mutation in the PAS-like domain increased interactions between the two proteins and blocked induction of the phosphorelay. IgaA may regulate RcsC, the histidine kinase that initiates phosphotransfer through the phosphorelay, indirectly, via its contacts with RcsD. Unlike RcsD, and unlike many other histidine kinases, the periplasmic domain of RcsC is dispensable for the response to signals that induce the Rcs phosphorelay system. The multiple contacts between IgaA and RcsD constitute a poised sensing system, preventing potentially toxic over-activation of this phosphorelay while enabling it to rapidly and quantitatively respond to signals.

The Complex Rcs Regulatory Cascade.

RcsB, a response regulator of the FixJ/NarL family, is at the center of a complex network of regulatory inputs and outputs. Cell surface stress is sensed by an outer membrane lipoprotein, RcsF, which regulates interactions of the inner membrane protein IgaA, lifting negative regulation of a phosphorelay. In vivo evidence supports a pathway in which histidine kinase RcsC transfers phosphate to phosphotransfer protein RcsD, resulting in phosphorylation of RcsB. RcsB acts either alone or in combination with RcsA to positively regulate capsule synthesis and synthesis of small RNA (sRNA) RprA as well as other genes, and to negatively regulate motility. RcsB in combination with other FixJ/NarL auxiliary proteins regulates yet other functions, independent of RcsB phosphorylation. Proper expression of Rcs and its targets is critical for success of Escherichia coli commensal strains, for proper development of biofilm, and for virulence in some pathogens. New understanding of how the Rcs phosphorelay works provides insight into the flexibility of the two-component system paradigm.

Experimental Evolution of Escherichia coli K-12 at High pH and with RpoS Induction.

Experimental evolution of Escherichia coli K-12 W3110 by serial dilutions for 2,200 generations at high pH extended the range of sustained growth from pH 9.0 to pH 9.3. pH 9.3-adapted isolates showed mutations in DNA-binding regulators and envelope proteins. One population showed an IS1 knockout of phoB (encoding the positive regulator of the phosphate regulon). A phoB::kanR knockout increased growth at high pH. phoB mutants are known to increase production of fermentation acids, which could enhance fitness at high pH. Mutations in pcnB [poly(A) polymerase] also increased growth at high pH. Three out of four populations showed deletions of torI, an inhibitor of TorR, which activates expression of torCAD (trimethylamine N-oxide respiration) at high pH. All populations showed point mutations affecting the stationary-phase sigma factor RpoS, either in the coding gene or in genes for regulators of RpoS expression. RpoS is required for survival at extremely high pH. In our microplate assay, rpoS deletion slightly decreased growth at pH 9.1. RpoS protein accumulated faster at pH 9 than at pH 7. The RpoS accumulation at high pH required the presence of one or more antiadaptors that block degradation (IraM, IraD, and IraP). Other genes with mutations after high-pH evolution encode regulators, such as those encoded by yobG (mgrB) (PhoPQ regulator), rpoN (nitrogen starvation sigma factor), malI, and purR, as well as envelope proteins, such as those encoded by ompT and yahO Overall, E. coli evolution at high pH selects for mutations in key transcriptional regulators, including phoB and the stationary-phase sigma factor RpoS.IMPORTANCEEscherichia coli in its native habitat encounters high-pH stress such as that of pancreatic secretions. Experimental evolution over 2,000 generations showed selection for mutations in regulatory factors, such as deletion of the phosphate regulator PhoB and mutations that alter the function of the global stress regulator RpoS. RpoS is induced at high pH via multiple mechanisms.

Alternative pathways for Escherichia coli biofilm formation revealed by sRNA overproduction.

Small regulatory RNAs have major roles in many regulatory circuits in Escherichia coli and other bacteria, including the transition from planktonic to biofilm growth. We tested Hfq-dependent sRNAs in E. coli for their ability, when overproduced, to inhibit or stimulate biofilm formation, in two different growth media. We identify two mutually exclusive pathways for biofilm formation. In LB, PgaA, encoding an adhesion export protein, played a critical role; biofilm was independent of the general stress factor RpoS or CsgD, regulator of curli and other biofilm genes. The PgaA-dependent pathway was stimulated upon overproduction of DsrA, via negative regulation of H-NS, or of GadY, likely by titration of CsrA. In yeast extract casamino acids (YESCA) media, biofilm was dependent on RpoS and CsgD, but independent of PgaA; RpoS appears to indirectly negatively regulate the PgaA-dependent pathway in YESCA medium. Deletions of most sRNAs had very little effect on biofilm, although deletion of hfq, encoding an RNA chaperone, was defective in both LB and YESCA. Deletion of ArcZ, a small RNA activator of RpoS, decreased biofilm in YESCA; only a portion of this defect could be bypassed by overproduction of RpoS. Overall, sRNAs highlight different pathways to biofilm formation.

Structural and Functional Characterization of the LPS Transporter LptDE from Gram-Negative Pathogens.

Incorporation of lipopolysaccharide (LPS) into the outer membrane of Gram-negative bacteria is essential for viability, and is accomplished by a two-protein complex called LptDE. We solved crystal structures of the core LptDE complexes from Yersinia pestis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and a full-length structure of the K. pneumoniae LptDE complex. Our structures adopt the same plug and 26-strand ß-barrel architecture found recently for the Shigella flexneri and Salmonella typhimurium LptDE structures, illustrating a conserved fold across the family. A comparison of the only two full-length structures, SfLptDE and our KpLptDE, reveals a 21° rotation of the LptD N-terminal domain that may impart flexibility on the trans-envelope LptCAD scaffold. Utilizing mutagenesis coupled to an in vivo functional assay and molecular dynamics simulations, we demonstrate the critical role of Pro231 and Pro246 in the function of the LptD lateral gate that allows partitioning of LPS into the outer membrane.

Stress sigma factor RpoS degradation and translation are sensitive to the state of central metabolism.

RpoS, the stationary phase/stress sigma factor of Escherichia coli, regulates a large cohort of genes important for the cell to deal with suboptimal conditions. Its level increases quickly in the cell in response to many stresses and returns to low levels when growth resumes. Increased RpoS results from increased translation and decreased RpoS degradation. Translation is positively regulated by small RNAs (sRNAs). Protein stability is positively regulated by anti-adaptors, which prevent the RssB adaptor-mediated degradation of RpoS by the ClpXP protease. Inactivation of aceE, a subunit of pyruvate dehydrogenase (PDH), was found to increase levels of RpoS by affecting both translation and protein degradation. The stabilization of RpoS in aceE mutants is dependent on increased transcription and translation of IraP and IraD, two known anti-adaptors. The aceE mutation also leads to a significant increase in rpoS translation. The sRNAs known to positively regulate RpoS are not responsible for the increased translation; sequences around the start codon are sufficient for the induction of translation. PDH synthesizes acetyl-CoA; acetate supplementation allows the cell to synthesize acetyl-CoA by an alternative, less favored pathway, in part dependent upon RpoS. Acetate addition suppressed the effects of the aceE mutant on induction of the anti-adaptors, RpoS stabilization, and rpoS translation. Thus, the bacterial cell responds to lowered levels of acetyl-CoA by inducing RpoS, allowing reprogramming of E. coli metabolism.

Production of recombinant protein by a novel oxygen-induced system in Escherichia coli.

BACKGROUND: The SoxRS regulon of E. coli is activated in response to elevated dissolved oxygen concentration likely to protect the bacteria from possible oxygen damage. The soxS expression can be increased up to 16 fold, making it a possible candidate for recombinant protein expression. Compared with the existing induction approaches, oxygen induction is advantageous because it does not involve addition or depletion of growth factors or nutrients, addition of chemical inducers or temperature changes that can affect growth and metabolism of the producing bacteria. It also does not affect the composition of the growth medium simplifying the recovery and purification processes. RESULTS: The soxS promoter was cloned into the commercial pGFPmut3.1 plasmid creating pAB49, an expression vector that can be induced by increasing oxygen concentration. The efficiency and the regulatory properties of the soxS promoter were characterized by measuring the GFP expression when the culture dissolved oxygen concentration was increased from 30% to 300% air saturation. The expression level of recombinant GFP was proportional to the oxygen concentration, demonstrating that pAB49 is a controllable expression vector. A possible harmful effect of elevated oxygen concentration on the recombinant product was found to be negligible by determining the protein-carbonyl content and its specific fluorescence. By performing high density growth in modified LB medium, the cells were induced by increasing the oxygen concentration. After 3 hours at 300% air saturation, GFP fluorescence reached 109000 FU (494 mg of GFP/L), representing 3.4% of total protein, and the cell concentration reached 29.1 g/L (DW). CONCLUSIONS: Induction of recombinant protein expression by increasing the dissolved oxygen concentration was found to be a simple and efficient alternative expression strategy that excludes the use of chemical, nutrient or thermal inducers that have a potential negative effect on cell growth or the product recovery.

Reducing acetate excretion from E. coli K-12 by over-expressing the small RNA SgrS.

When exposed to the nonmetabolized glucose derivative alpha methyl glucoside (αMG), both Escherichia coli K-12 (JM109 and MG1655) and E. coli B (BL21) respond by reducing the concentration of the mRNA of the ptsG gene which is responsible for the biosynthesis of the glucose transporter EIICB(glu). This occurs through the over-expression of the noncoding small RNA SgrS, which interacts specifically with the mRNA of the ptsG gene and prevents its translation. However, when these bacteria are exposed to a glucose concentration of 40 g/L, over-expression of SgrS is observed only in E. coli B (BL21). Unlike E. coli K-12 (JM109 and MG1655), which are affected by high glucose concentration and produce higher levels of acetate, E. coli B (BL21) is not affected. Based on this information, it was assumed that over-expression of SgrS enables E. coli B (BL21) to reduce its acetate excretion by controlling the glucose transport. When SgrS was over-expressed in both E. coli K-12 strains from a multicopy plasmid, it was possible to reduce their acetate excretion levels to those seen in E. coli B. This observation opens a new approach towards controlling bacterial metabolism through the use of noncoding RNA.

Adenovirus with hexon Tat-protein transduction domain modification exhibits increased therapeutic effect in experimental neuroblastoma and neuroendocrine tumors.

Adenovirus serotype 5 (Ad5) is widely used as an oncolytic agent for cancer therapy. However, its infectivity is highly dependent on the expression level of coxsackievirus-adenovirus receptor (CAR) on the surfaces of tumor cells. Furthermore, infected cells overproduce adenovirus fiber proteins, which are released prior to cell lysis. The released fibers block CAR on noninfected neighboring cells, thereby preventing progeny virus entry. Our aim was to add a CAR-independent infection route to Ad5 to increase the infectivity of tumor cells with low CAR expression and prevent the fiber-masking problem. We constructed Ad5 viruses that encode the protein transduction domain (PTD) of the HIV-1 Tat protein (Tat-PTD) in hypervariable region 5 (HVR5) of the hexon protein. Tat-PTD functions as a cell-penetrating peptide, and Tat-PTD-modified Ad5 showed a dramatic increased transduction of CAR-negative cell lines compared to unmodified vector. Moreover, while tumor cell infectivity was severely reduced for Ad5 in the presence of fiber proteins, it was only marginally reduced for Tat-PTD-modified Ad5. Furthermore, because of the sequence alteration in the hexon HVR, coagulation factor X-mediated virus uptake was significantly reduced. Mice harboring human neuroblastoma and neuroendocrine tumors show suppressed tumor growths and prolonged survival when treated with Tat-PTD-modified oncolytic viruses. Our data suggest that modification of Ad5 with Tat-PTD in HVR5 expands its utility as an oncolytic agent.

The RpoS-mediated general stress response in Escherichia coli.

Under conditions of nutrient deprivation or stress, or as cells enter stationary phase, Escherichia coli and related bacteria increase the accumulation of RpoS, a specialized sigma factor. RpoS-dependent gene expression leads to general stress resistance of cells. During rapid growth, RpoS translation is inhibited and any RpoS protein that is synthesized is rapidly degraded. The complex transition from exponential growth to stationary phase has been partially dissected by analyzing the induction of RpoS after specific stress treatments. Different stress conditions lead to induction of specific sRNAs that stimulate RpoS translation or to induction of small-protein antiadaptors that stabilize the protein. Recent progress has led to a better, but still far from complete, understanding of how stresses lead to RpoS induction and what RpoS-dependent genes help the cell deal with the stress.