Bacteria often cope with environmental stress by inducing alternative sigma (σ) factors, which direct RNA polymerase to specific promoters, thereby inducing a set of genes called a regulon to combat the stress. To understand the conserved and organism-specific functions of each σ, it is necessary to be able to predict their promoters, so that their regulons can be followed across species. However, the variability of promoter sequences and motif spacing makes their prediction difficult. We developed and validated an accurate promoter prediction model for Escherichia coli σE, which enabled us to predict a total of 89 unique σE-controlled transcription units in E. coli K-12 and eight related genomes. σE controls the envelope stress response in E. coli K-12. The portion of the regulon conserved across genomes is functionally coherent, ensuring the synthesis, assembly, and homeostasis of lipopolysaccharide and outer membrane porins, the key constituents of the outer membrane of Gram-negative bacteria. The larger variable portion is predicted to perform pathogenesis-associated functions, suggesting that σE provides organism-specific functions necessary for optimal host interaction. The success of our promoter prediction model for σE suggests that it will be applicable for the prediction of promoter elements for many alternative σ factors.

The heat-shock response (HSR), a universal cellular response to heat, is crucial for cellular adaptation. In Escherichia coli, the HSR is mediated by the alternative factor, 32 . To determine its role, we used genome-wide expression analysis and promoter validation to identify genes directly regulated by 32 and screened ORF overexpression libraries to identify 32 inducers. We triple the number of genes validated to be transcribed by 32 and provide new insights into the cellular role of this response. Our work indicates that the response is propagated as the regulon encodes numerous global transcriptional regulators, reveals that 70 holoenzyme initiates from 12% of 32 promoters, which has important implications for global transcriptional wiring, and identifies a new role for the response in protein homeostasis, that of protecting complex proteins. Finally, this study suggests that the response protects the cell membrane and responds to its status: Fully 25% of 32 regulon members reside in the membrane and alter its functionality; moreover, a disproportionate fraction of overexpressed proteins that induce the response are membrane localized. The intimate connection of the response to the membrane rationalizes why a major regulator of the response resides in that cellular compartment.[Keywords: Heat-shock response; 32; transcription; microarray] Supplemental material is available at http://www.genesdev.org. Received March 13, 2006; revised version accepted April 25, 2006. When cells are shifted from low to high temperature, synthesis of the heat-shock proteins (hsps) is rapidly and selectively induced. The heat-shock response (HSR), was first identified by Ritossa (1963), who showed that exposure to heat lead to transient changes in the puffing pattern of salivary chromosomes in Drosophila; Tissieres et al. (1974) demonstrated that these changes reflected the transient induction of several proteins. Initially, hsp function was unclear; however, experiments in several organisms revealed that many hsps were chaperones that promote protein folding (Pelham 1986;Beckmann et al. 1990;Gaitanaris et al. 1990;Skowyra et al. 1990). These studies not only suggested that a major function of the HSR is to maintain the protein folding state of the cell, but also indicated that some of these chaperones, such as Hsp70 and Hsp90, are present in all organisms Craig 1984, 1987). Thus, both the HSR and some hsps are universally conserved among organisms.In Escherichia coli, 32 , an alternative factor, controls the HSR by directing RNA polymerase to transcribe hsps (Yamamori and Yura 1980;Grossman et al. 1984;Taylor et al. 1984;Cowing et al. 1985). Synthesis of hsps is induced upon temperature upshift and repressed upon temperature downshift (Lemaux et al. 1978;Yamamori et al. 1978;Straus et al. 1987 Straus et al. , 1989Taura et al. 1989), thereby allowing a rapid cellular response to changes in temperature.32 is controlled by negative feedback loops controlling its activity (Straus et al. 1989;Blaszczak et al. 1999) and stabili...

Fifty-seven tannins and related compounds, including gallotannins, ellagitannins, and condensed and complex tannins, were evaluated for their cytotoxicities against human tumor cell lines, including malignant melanoma, lung carcinoma, ileocecal adenocarcinoma, epidermoid carcinoma, malignant melanoma, and medulloblastoma cell lines. Among them, chebulagic acid [1], geraniin [2], sanguiin H-11 [3], 4,5-di-O-galloylquinic acid [12], 1,3,4,5-tetra-O-galloylquinic acid [15], 1(beta)-O-galloylpedunculagin [24], furosin [29], castalagin [38], sanguiin H-2 [34], vescalagin [39], grandinin [40], phyllyraeoidin A [42], (-)-epicatechin 3-O-gallate [50], cinnamtannin B2 [55], and acutissimin A [56] exhibited moderate selective cytotoxicity against PRMI-7951 melanoma cells with ED50 values in the range of 0.1-0.8 microgram/ml. Selective cytotoxicities against the melanoma cells were also observed for strictinin [22], pedunculagin [23], eugeniin [25], elaeocarpusin [28], punicacortein C [37], casuarinin [41], sanguiin H-6 [43], procyanidin B-2 3,3'-di-O-gallate [51], procyanidin C-1 3,3',3"-tri-O-gallate [52], and cinnamtannin B1 [54] with ED50 values of 1-4 micrograms/ml. All of the tannins were found to be inactive (greater than 10 micrograms/ml) against lung carcinoma (A-549), ileocecal adenocarcinoma (HCT-8), epidermoid carcinoma of nasopharnyx (KB), and medulloblastoma (TE-671) tumor cells.

Nine tannins, including gallo- and ellagitannins, were evaluated as potential inhibitors of HIV replication. 1,3,4-Tri-O-galloylquinic acid [1], 3,5-di-O-galloyl-shikimic acid [2], 3,4,5-tri-O-galloylshikimic acid [3], punicalin [6], and punicalagin [7] inhibited HIV replication in infected H9 lymphocytes with little cytotoxicity. Two compounds, punicalin and punicacortein C [8], inhibited purified HIV reverse transcriptase with ID50 of 8 and 5 microM, respectively. Further studies with H9 lymphocytes indicated that chebulagic acid [5] and punicalin did not inactivate virus directly. However, 1,3,4-tri-O-galloylquinic acid and 3,5-di-O-galloylshikimic acid were more effective inhibitors under those conditions. All tannins appear to inhibit virus-cell interactions. Thus, inspite of their anti-RT activity, the mechanism by which tannins inhibit HIV may not be associated with this enzyme.

The E -directed envelope stress response maintains outer membrane homeostasis and is an important virulence determinant upon host infection in Escherichia coli and related bacteria.E is activated by at least two distinct mechanisms: accumulation of outer membrane porin precursors and an increase in the alarmone ppGpp upon transition to stationary phase. Expression of the E regulon is driven from a suite of approximately 60 E -dependent promoters. Using green fluorescent protein fusions to each of these promoters, we dissected promoter contributions to the output of the regulon under a variety of in vivo conditions. We found that the E promoters exhibit a large dynamic range, with a few strong and many weak promoters. Interestingly, the strongest promoters control either transcriptional regulators or functions related to porin homeostasis, the very functions conserved among E. coli and its close relatives. We found that (i) the strength of most promoters is significantly affected by the presence of the upstream (؊35 to ؊65) region of the promoter, which encompasses the UP element, a binding site for the C-terminal domain of the ␣-subunit of RNA polymerase; (ii) ppGpp generally activates E promoters, and (iii) E promoters are responsive to changing E holoenzyme levels under physiological conditions, reinforcing the idea that the E regulon is extremely dynamic, enabling cellular adaptation to a constantly changing environment.

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BackgroundEscherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli.ResultsBecause the redox enzymes can reduce the disulfide that forms on proteins, we first tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coliL-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI and ΔcysJ) and the L-cysteine synthase gene (ΔcysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (ΔcysC or ΔcysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coliL-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell.ConclusionsIn this work, we showed that Grx1 and NrdH reduce SSC to L-cysteine, and the generated sulfite is then utilized as the sulfur source to produce additional L-cysteine molecule through the sulfate pathway in E. coli. We also found that co-overexpression of NrdH, CysI, and CysK increases L-cysteine production. Our results propose that the enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from SSC is a novel method for improvement of L-cysteine production.

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