PubMed Health⌕ Search

Biomedical subjects

Jeffrey Green

Publications and source records attributed to Jeffrey Green.

At least 37 records · Page 2Linked to original sources
PubMed2004

Post-transcriptional regulation of bacterial motility by aconitase proteins.

Escherichia coli and Bacillus subtilis aconitases can act as iron and oxidative stress-responsive post-transcriptional regulators. Here, it is shown that a Salmonella enterica serovar Typhimurium LT2 acnB mutant exhibits impaired binding to the surface of J774 macrophage-like cells. Proteomic analyses were used to investigate further the binding defect of the acnB mutant. These revealed that the levels of the flagellum protein FliC were much lower for the acnB mutant. This strain was correspondingly less motile and possessed fewer flagella than either the parental strain or the acnA and acnAB mutants. The acnB lesion did not alter fliC transcription, nor did apo-AcnB select the fliC transcript from a library of S. enterica transcripts; thus, the effect of AcnB on FliC is indirect. Evidence is presented to show that apo-AcnB regulates FliC synthesis via interaction with the ftsH transcript to decrease the intracellular levels of FtsH. The lower levels of FtsH protease activity then influence sigma32, DnaK and, ultimately, FliC production.

ATP-Dependent Proteases↗
PubMed2004

Adaptation of the endogenous Salmonella enterica serovar Typhi clyA-encoded hemolysin for antigen export enhances the immunogenicity of anthrax protective antigen domain 4 expressed by the attenuated live-vector vaccine strain CVD 908-htrA.

Bacterial live-vector vaccines aim to deliver foreign antigens to the immune system and induce protective immune responses, and surface-expressed or secreted antigens are generally more immunogenic than cytoplasmic constructs. We hypothesize that an optimum expression system will use an endogenous export system to avoid the need for large amounts of heterologous DNA encoding additional proteins. Here we describe the cryptic chromosomally encoded 34-kDa cytolysin A hemolysin of Salmonella enterica serovar Typhi (ClyA) as a novel export system for the expression of heterologous antigens in the supernatant of attenuated Salmonella serovar Typhi live-vector vaccine strains. We constructed a genetic fusion of ClyA to the reporter green fluorescent protein and showed that in Salmonella serovar Typhi CVD 908-htrA, the fusion protein retains biological activity in both domains and is exported into the supernatant of an exponentially growing live vector in the absence of detectable bacterial lysis. The utility of ClyA for enhancing the immunogenicity of an otherwise problematic antigen was demonstrated by engineering ClyA fused to the domain 4 (D4) moiety of Bacillus anthracis protective antigen (PA). A total of 11 of 15 mice immunized intranasally with Salmonella serovar Typhi exporting the protein fusion manifested fourfold or greater rises in serum anti-PA immunoglobulin G, compared with only 1 of 16 mice immunized with the live vector expressing cytoplasmic D4 (P = 0.0002). In addition, the induction of PA-specific gamma interferon and interleukin 5 responses was observed in splenocytes. This technology offers exceptional versatility for enhancing the immunogenicity of bacterial live-vector vaccines.

Animals↗
PubMed2004

Regulation of Escherichia coli hemolysin E expression by H-NS and Salmonella SlyA.

The Escherichia coli hlyE gene (also known as clyA or sheA) codes for a novel pore-forming toxin. Previous work has shown that the global transcription factors FNR and CRP positively regulate hlyE expression by binding at the same site. Here in vivo transcription studies reveal that FNR occupies the hlyE promoter more frequently than CRP, providing a mechanism for the moderate upregulation of hlyE expression in response to two distinct environmental signals (oxygen and glucose starvation). It has been reported that H-NS interacts with two large regions of the hlyE promoter (PhlyE), one upstream of the -35 element and one downstream of the -10 element. Here we identify two high-affinity H-NS sites, H-NS I, located at the 3' end of the extended upstream footprint, and H-NS II, located at the 5' end of the extended downstream footprint. It is suggested that these high-affinity sites initiate the progressive formation of higher order complexes, allowing a range of H-NS-mediated regulatory effects at PhlyE. Finally, the identification of a SlyA binding site that overlaps the H-NS I site in PhlyE suggests a mechanism to explain how SlyA overproduction enhances hlyE expression by antagonizing the negative effects of H-NS.

Bacterial Proteins↗
PubMed2003

Mechanism of oxygen sensing by the bacterial transcription factor fumarate-nitrate reduction (FNR).

The facultative anaerobe Escherichia coli adopts different metabolic modes in response to the availability of oxygen. The global transcriptional regulator FNR (fumarate-nitrate reduction) monitors the availability of oxygen in the environment. Binding as a homodimer to palindromic sequences of DNA, FNR carries a sensory domain, remote from the DNA binding helix-turn-helix motif, which responds to oxygen. The sensing mechanism involves the transformation of a [4Fe-4S](2+) cluster into a [2Fe-2S] form in vitro on reaction with oxygen. Evidence is presented to show that this process proceeds by at least two steps, the first, an oxidative one, being the formation, on reaction with O(2), of a [3Fe-4S](1+) cluster as an intermediate accompanied by the production of hydrogen peroxide. This is followed by a slower, non-redox, pseudo-first order step in which the [3Fe-4S](1+) form converts to a [2Fe-2S](2+) cluster. This must be accompanied by a substantial protein conformational change since the four cysteine ligands that bind the two forms of the FeS clusters have different spatial disposition. Hydrogen peroxide is also an oxidant of the [4Fe-4S](2+), causing a similar cluster transformation to a [2Fe-2S] form. Either the hydrogen peroxide formed on reaction with oxygen can be recycled by intracellular catalase or it can be used to oxidize further Fe-S clusters. In both cases, the efficacy of oxygen sensing by FNR will be increased.

DNA-Binding Proteins↗
PubMed2003

FNR-mediated regulation of hyp expression in Escherichia coli.

The hypA-E operon is involved in the maturation of all three NiFe hydrogenases in Escherichia coli. Two hyp promoters have been described; a sigma54-dependent promoter upstream of hypA, and a sigma70-dependent promoter (PhypA) within the hypA coding region. Here it is shown that the oxygen-responsive transcription factor FNR regulates PhypA under anaerobic conditions only. PhypA does not possess a canonical FNR recognition sequence, but two FNR half-sites are present. Studies using PHYPA::lacZ fusions carrying lesions in one or both FNR half-sites indicated that although some residual anaerobic activity was retained by the promoter containing only the downstream FNR half-site, both half-sites are required for maximal PhypA activity in vivo. In vitro gel retardation analysis suggested that the primary interaction occurs at the downstream FNR half-site. Possible explanations for these observations and the implications for other FNR-regulated promoters are discussed.

Anaerobiosis↗
PubMed2003

DNA target sequence and FNR-dependent gene expression.

FNR proteins are global transcription regulators that respond to fluctuations in environmental oxygen. They recognise a DNA target consisting of an inverted repeat, TTGATN(1)N(2)N(3)N(4)ATCAA (where N(1-4) represents a non-conserved tetrad, NCT). Analysis of 68 known and predicted FNR sites from the Escherichia coli K12 genome revealed a bias toward A or T at positions N(2) and N(3) of the NCT. The effect of the NCT sequence on FNR-dependent transcription in vivo was assessed using a series of class II and class I model promoters with different NCT sequences. Changing the NCT sequence did not affect basal activity but altered anaerobic induction by as much as an order of magnitude. Thus, the NCT sequence is a fundamental component in setting the dynamic range of the FNR switch.

Base Sequence↗
PubMed2003

PhoP-responsive expression of the Salmonella enterica serovar typhimurium slyA gene.

The SlyA protein of Salmonella enterica serovar Typhimurium is a member of the MarR family of transcription regulators and is required for virulence and survival in professional macrophages. Isolated SlyA protein was able to bind a specific DNA target without posttranslational modification. This suggested that SlyA might not be activated by directly sensing an external signal but rather that the intracellular concentration of SlyA is enhanced in appropriate environments through the action of other transcription factors. Analysis of slyA transcription reveals the presence of a promoter region located upstream of the previously recognized SlyA repressed promoter. The newly identified upstream promoter region did not respond to SlyA but was activated by Mg(II) starvation in a PhoP-dependent manner. We present here evidence for a direct link between two transcription factors (PhoP and SlyA) crucial for Salmonella virulence.

Bacterial Proteins↗
PubMed2003

Transcription regulation by tandem-bound FNR at Escherichia coli promoters.

FNR is an Escherichia coli transcription factor that regulates the transcription of many genes in response to anaerobiosis. We have constructed a series of artificial FNR-dependent promoters, based on the melR promoter, in which a consensus FNR binding site was centered at position -41.5 relative to the transcription start site. A second consensus FNR binding site was introduced at different upstream locations, and promoter activity was assayed in vivo. FNR can activate transcription from these promoters when the upstream FNR binding site is located at many different positions. However, sharp repression is observed when the upstream-bound FNR is located near positions -85 or -95. This repression is relieved by the FNR G74C substitution mutant, previously identified as being defective in transcription repression at the yfiD promoter. A parallel series of artificial FNR-dependent promoters, carrying a consensus FNR binding site at position -61.5 and a second upstream DNA site for FNR, was also constructed. Again, promoter activity was repressed by FNR when the upstream-bound FNR was located at particular positions.

Base Sequence↗
PubMed2002

NO sensing by FNR: regulation of the Escherichia coli NO-detoxifying flavohaemoglobin, Hmp.

Nitric oxide (NO) is a signalling and defence molecule of major importance in biology. The flavohaemoglobin Hmp of Escherichia coli is involved in protective responses to NO. Because hmp gene transcription is repressed by the O(2)-responsive regulator FNR, we investigated whether FNR also senses NO. The [4Fe-4S](2+) cluster of FNR is oxygen labile and controls protein dimerization and site-specific DNA binding. NO reacts anaerobically with the Fe-S cluster of purified FNR, generating spectral changes consistent with formation of a dinitrosyl-iron-cysteine complex. NO-inactivated FNR can be reconstituted, suggesting physiological relevance. FNR binds at an FNR box within the hmp promoter (P(hmp)). FNR samples inactivated by either O(2) or NO bind specifically to P(hmp), but with lower affinity. Dose-dependent up-regulation of P(hmp) in vivo by NO concentrations of pathophysiological relevance is abolished by fnr mutation, and NO also modulates expression from model FNR-regulated promoters. Thus, FNR can respond to not only O(2), but also NO, with major implications for global gene regulation in bacteria. We propose an NO-mediated mechanism of hmp regulation by which E.coli responds to NO challenge.

Anaerobiosis↗
PubMed2002

Activation of cancer-specific gene expression by the survivin promoter.

BACKGROUND: Survivin, a member of the IAP (inhibitor of apoptosis) gene family, appears to be overexpressed in common cancers but not in corresponding normal adult tissues. To investigate whether the survivin promoter controls cancer cell-specific gene expression, we determined whether the survivin gene promoter could regulate reporter gene expression in cancer cell lines and xenografts. METHODS: Survivin protein levels were determined in human and murine cancer cell lines and in normal tissues of adult C57BL/6 mice by Western blot analysis. A reporter construct in which a portion of the survivin gene promoter was used to drive transcription of a human secreted alkaline phosphatase (SEAP) gene was transiently transfected into cancer cells, and promoter activity was extrapolated from SEAP activity. A2780 human ovarian cancer cells were transfected with this construct, and stable transfectants were injected into the intrabursal ovarian space of immunodeficient mice. Tumor growth was monitored, and plasma SEAP levels were used as a measure of survivin promoter activity in vivo. RESULTS: Survivin protein was detected in all cancer cell lines examined but not in most normal adult mouse tissues. After transfection, the survivin promoter was more active in all cancer cell lines than in normal ovarian surface epithelial cells or mouse 3T3 cells. After 0.8 x 10(6) stable transfectant cells were injected into the intrabursal cavity of mouse ovaries, plasma SEAP activity was detected within 24 hours, and the activity increased with time and tumor growth. CONCLUSION: Transfection experiments indicate that survivin protein expression in cancer tissue appears to be regulated, at least in part, transcriptionally. Thus, the survivin promoter may be useful in controlling gene expression in cancer cells.

Alkaline Phosphatase↗
PubMed2002

Interaction of the Salmonella typhimurium transcription and virulence factor SlyA with target DNA and identification of members of the SlyA regulon.

The SlyA protein from Salmonella typhimurium is a transcription factor that contributes to virulence. It is shown that a slyA mutant is attenuated in the presence of murine macrophages compared with the parent strain. Moreover, after growth in minimal medium, survival of the slyA mutant was reduced. Altered levels of flagellin (fliC), PagC, IroN, and outer membrane proteins suggest that the slyA mutation affects the surface properties of Salmonella. The isolated SlyA protein is a cofactor-free homodimer that recognizes five sites within the promoter region of the slyA gene. One of these sites contained a near perfect inverted repeat TTAGCAAGCTAA. The other four sites contained related sequences. Occupation of the SlyA sites in the slyA promoter prevented open-complex formation, consistent with the pattern of slyA::lacZ expression parental and slyA mutant strains. By combining the footprinting data with potential SlyA binding sites recovered from a pool of random DNA sequences, a consensus was defined and used to probe the NIH Salmonella unfinished genomes data base. These searches revealed the presence of consensus SlyA sites upstream of omp, ispA, xseB, slyA, and a gene encoding a protein with homology to a hemagglutinin. Accordingly, transcription of an omp::lacZ fusion was reduced in a slyA mutant. Given the difficulties in obtaining a comprehensive picture of intracellular gene expression, the definition of the DNA sequence recognized by a transcription factor (SlyA) that is essential for survival in the macrophage environment should allow a complete regulon of genes with altered expression upon exposure to macrophages to be determined once the S. typhimurium genome annotation is complete.

Animals↗
PubMed2002

Adipose tissue: a vital in vivo role in mammary gland development but not differentiation.

Development and differentiation of the mammary gland occurs by means of critical stromal-epithelial interactions. Although many studies have attempted to understand these complex interactions, it has been difficult to demonstrate the essential role of adipose tissue in the development and function of the mammary gland. By using the A-ZIP/F-1 transgenic mice lacking in white adipose tissue (WAT), we have studied the role of adipocytes in mammary gland development and differentiation. In the absence of WAT, rudimentary mammary anlagen form but are unable to grow and branch normally, resulting in a few, short, severely distended ducts. However, during pregnancy, a tremendous amount of epithelial cell division and alveolar cell formation occurs even in the absence of adipocytes, illustrating that adipose tissue is not required for mammary gland differentiation. Mammary gland transplantation revealed that epithelial cells from these transgenic mice possess the potential for normal growth and differentiation when placed into a normal stromal environment. These experiments clearly demonstrate that the absence of adipocytes in the mammary gland results in disruption of stromal-epithelial interactions that prevent normal mammary gland development. The rudimentary epithelial anlage, however, contain mammary stem cells, which are fully capable of alveolar differentiation.

Adipocytes↗
PubMed2002

E. coli aconitase B structure reveals a HEAT-like domain with implications for protein-protein recognition.

The major bifunctional aconitase of Escherichia coli (AcnB) serves as either an enzymic catalyst or a mRNA-binding post-transcriptional regulator, depending on the status of its iron sulfur cluster. AcnB represents a large, distinct group of Gram-negative bacterial aconitases that have an altered domain organization relative to mitochondrial aconitase and other aconitases. Here the 2.4 A structure of E. coli AcnB reveals a high degree of conservation at the active site despite its domain reorganization. It also reveals that the additional domain, characteristic of the AcnB subfamily, is a HEAT-like domain, implying a role in protein protein recognition. This domain packs against the remainder of the protein to form a tunnel leading to the aconitase active site, potentially for substrate channeling.

Aconitate Hydratase↗
PubMed2002

AcnC of Escherichia coli is a 2-methylcitrate dehydratase (PrpD) that can use citrate and isocitrate as substrates.

Escherichia coli possesses two well-characterized aconitases (AcnA and AcnB) and a minor activity (designated AcnC) that is retained by acnAB double mutants and represents no more than 5% of total wild-type aconitase activity. Here it is shown that a 2-methylcitrate dehydratase (PrpD) encoded by the prpD gene of the propionate catabolic operon (prpRBCDE) is identical to AcnC. Inactivation of prpD abolished the residual aconitase activity of an AcnAB-null strain, whereas inactivation of ybhJ, an unidentified acnA paralogue, had no significant effect on AcnC activity. Purified PrpD catalysed the dehydration of citrate and isocitrate but was most active with 2-methylcitrate. PrpD also catalysed the dehydration of several other hydroxy acids but failed to hydrate cis-aconitate and related substrates containing double bonds, indicating that PrpD is not a typical aconitase but a dehydratase. Purified PrpD was shown to be a monomeric iron-sulphur protein (M(r) 54000) having one unstable [2Fe-2S] cluster per monomer, which is needed for maximum catalytic activity and can be reconstituted by treatment with Fe(2+) under reducing conditions.

Aconitate Hydratase↗
PubMed2002

Expression of the Escherichia coli yfiD gene responds to intracellular pH and reduces the accumulation of acidic metabolic end products.

The YfiD protein of Escherichia coli has been reported to be an acid-inducible protein. Here it is shown that expression of a yfiD::lac reporter fusion is enhanced up to 3 small middle dot5-fold during acidic growth. The anaerobic transcription factor FNR was confirmed as the major regulator of yfiD expression, and ArcA was found to enhance anaerobic yfiD expression, probably by displacing a repressing FNR dimer in the -93 small middle dot5 region of the promoter. Moreover, the pyruvate sensor PdhR was shown to act as a minor anaerobic repressor of yfiD expression. On the basis of its strong homology to the C-terminal region of pyruvate formate-lyase (PFL) it was predicted that YfiD would be a radical-containing enzyme. The YfiD radical was found to be introduced by the PFL-activase enzyme, but unlike PFL, AdhE did not deactivate radicalized YfiD. The extent of radical activation of YfiD was enhanced by low intracellular pH, and thus it was concluded that both yfiD expression and YfiD activity are affected by growth at low pH. The yfiD mutant strain JRG4033 excreted increased levels of organic acids compared to the parental strain when grown in chemostat culture under oxygen-starved conditions, consistent with the acid-inducibility of yfiD expression and the recently reported ability of YfiD to rescue the activity of oxygenolytically cleaved PFL.

Acetyltransferases↗
PubMed2002

Escherichia coli aconitases and oxidative stress: post-transcriptional regulation of sodA expression.

Escherichia coli possesses two aconitases, a stationary-phase enzyme (AcnA), which is induced by iron and oxidative stress, and a major but less stable enzyme (AcnB), synthesized during exponential growth. In addition to the catalytic activities of the holo-proteins, the apo-proteins function as post-transcriptional regulators by site-specific binding to acn mRNAs. Thus, it has been suggested that inactivation of the enzymes could mediate a rapidly reacting post-transcriptional component of the bacterial oxidative stress response. Here it is shown that E. coli acn mutants are hypersensitive to the redox-stress reagents H(2)O(2) and methyl viologen. Proteomic analyses further revealed that the level of superoxide dismutase (SodA) is enhanced in acnB and acnAB mutants, and by exposure to methyl viologen. The amounts of other proteins, including thioredoxin reductase, 2-oxoglutarate dehydrogenase, succinyl-CoA synthetase and chaperone proteins, were also affected in the acn mutants. The altered patterns of sodA expression were confirmed in studies with sodA-lacZ reporter strains. Quantitative Northern blotting indicated that AcnA enhances the stability of the sodA transcript, whereas AcnB lowers its stability. Direct evidence that the apo-proteins have positive (AcnA) and negative (AcnB) effects on SodA synthesis was obtained from in vitro transcription-translation experiments. It is suggested that the aconitase proteins of E. coli serve as a protective buffer against the basal level of oxidative stress that accompanies aerobic growth by acting as a sink for reactive oxygen species and by modulating translation of the sodA transcript.

Aconitate Hydratase↗
PubMed2002

Transcription activation by FNR: evidence for a functional activating region 2.

The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia via the assembly-disassembly of oxygen-labile iron-sulfur clusters. Previous work identified patches of surface-exposed amino acids (designated activating regions 1 and 3 [AR1 and AR3, respectively]) of FNR which allow it to communicate with RNA polymerase (RNAP) and thereby activate transcription. Previously it was thought that FNR lacks a functional activating region 2 (AR2), although selecting for mutations that compensate for defective AR1 or a miscoordinated iron-sulfur cluster can reactivate AR2. Here we show that the substitution of two surface-exposed lysine residues (Lys49 and Lys50) of FNR impaired transcription from class II (FNR box centered at -41.5) but not class I (FNR box centered at -71.5) FNR-dependent promoters. The degree of impairment was greater when a negatively charged residue (Glu) replaced either Lys49 or Lys50 than when uncharged amino acid Ala was substituted. Oriented heterodimers were used to show that only the downstream subunit of the FNR dimer was affected by the Lys-->Ala substitutions at a class II promoter. Site-directed mutagenesis of a negatively charged patch ((162)EEDE(165)) within the N-terminal domain of the RNAP alpha subunit that interacts with the positively charged AR2 of the cyclic AMP receptor protein suggested that Lys49 and Lys50 of FNR interact with this region of the alpha subunit of RNAP. Thus, it was suggested that Lys49 and Lys50 form part of a functional AR2 in FNR.

Amino Acid Substitution↗