Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of sigma32 in vivo

The heat shock response of Escherichia coli is regulated by the cellular level and the activity of sigma32, an alternative sigma factor for heat shock promoters. FtsH, a membrane-bound AAA-type metalloprotease, degrades sigma32 and has a central role in the control of the sigma32 level. The ftsH null mutant was isolated, and establishment of the DeltaftsH mutant allowed us to investigate control mechanisms of the stability and the activity of sigma32 separately in vivo. Loss of the FtsH function caused marked stabilization and consequent accumulation of sigma32 ( approximately 20-fold of the wild type), leading to the impaired downregulation of the level of sigma32. Surprisingly, however, DeltaftsH cells express heat shock proteins only two- to threefold higher than wild-type cells, and they also show almost normal heat shock response upon temperature upshift. These results indicate the presence of a control mechanism that downregulates the activity of sigma32 when it is accumulated. Overproduction of DnaK/J reduces the activity of sigma32 in DeltaftsH cells without any detectable changes in the level of sigma32, indicating that the DnaK chaperone system is responsible for the activity control of sigma32 in vivo. In addition, CbpA, an analogue of DnaJ, was demonstrated to have overlapping functions with DnaJ in both the activity and the stability control of sigma32.     

Effects of the cys mutations on structure and function of the ATP-dependent HslVU protease in Escherichia coli. The Cys287 to Val mutation in HslU uncouples the ATP-dependent proteolysis by HslvU from ATP hydrolysis

To define the role of the Cys residues in the ATP-dependent HslVU protease, mutagenesis was performed to replace either Cys261 or Cys287 in HslU with Val and Cys159 in HslV with Ser or Ala. Whereas HslU/C261V could hydrolyze ATP and support the ATP-dependent proteolytic activity of HslV as well as the wild-type HslU, HslU/C287V could not hydrolyze ATP. Nevertheless, HslU/C287V could support the HslV-mediated proteolysis by forming the HslVU complex in the presence of ATP but not its absence, indicating that ATP binding but not its hydrolysis is essential for proteolysis. Whereas treatment of N-ethylmaleimide (NEM) resulted in dissociation of the oligomeric HslU into monomers, the C261V mutation, but not C287V, prevented the NEM effect. These results suggest that Cys261 is involved in oligomerization and that Cys287 is related to the ATPase function of HslU. NEM also dissociated the dodecameric HslV into monomers, and this effect could be prevented by either the C159S or C159A mutation, suggesting the involvement of Cys159 in oligomerization of HslV. Moreover, either mutation abolished both the basal and HslU-activated proteolytic activity of HslV and its ability to activate the HslU ATPase and to form the HslVU complex, indicating that Cys159 is essential for the proteolytic activity of HslV and its interaction with HslU. These results suggest that the Cys residues play an important role in maintaining the structure and function of the HslVU protease.     

Serum antibodies to secreted proteins in patients infected with Escherichia coli O157 and other VTEC

OBJECTIVES: The purpose of this study was to survey the efficiency of visible light curing units in dental practices across Australia. METHODS: Survey forms were distributed to representatives of 3M Health Care to complete when visiting dentists in their working areas. The information collected included the type and age of the unit, curing times used, history of maintenance, replacement of components, and the light intensity reading. RESULTS: Of the 214 light curing units surveyed, approximately 27% recorded a light intensity of 200 mW cm-2 or less, a level regarded as inadequate to cure a 2-mm thick increment of composite resin. An additional 26% registered an output of between 201 and 399 mW m-2. This level would be considered acceptable with additional curing time; however, 44% of practitioners were curing for 20 s or less. A negative correlation was found between the age of the unit and the intensity recorded. Nearly 50% of respondents had never checked the light output of their unit. CONCLUSIONS: The results indicate that just over one-half of the light curing units surveyed were not functioning satisfactorily. An obvious reduction in intensity was noted with the older units. There is a substantial lack of awareness among dentists of the need for maintenance and regular checking of the light intensity of these units.     

Characterization of the roles of hemolysin and other toxins in enteropathy caused by alpha-hemolytic Escherichia coli linked to human diarrhea

Escherichia coli strains producing alpha-hemolysin have been associated with diarrhea in several studies, but it has not been clearly demonstrated that these strains are enteropathogens or that alpha-hemolysin is an enteric virulence factor. Such strains are generally regarded as avirulent commensals. We examined a collection of diarrhea-associated hemolytic E. coli (DHEC) strains for virulence factors. No strain produced classic enterotoxins, but they all produced an alpha-hemolysin that was indistinguishable from that of uropathogenic E. coli strains. DHEC strains also produced other toxins including cytotoxic necrotizing factor 1 (CNF1) and novel toxins, including a cell-detaching cytotoxin and a toxin that causes HeLa cell elongation. DHEC strains were enteropathogenic in the RITARD (reversible intestinal tie adult rabbit diarrhea) model of diarrhea, causing characteristic enteropathies, including inflammation, necrosis, and colonic cell hyperplasia in both small and large intestines. Alpha-hemolysin appeared to be a major virulence factor in this model since it conferred virulence to nonpathogenic E. coli strains. Other virulence factors also appear to be contributing to virulence. These findings support the epidemiologic link to diarrhea and suggest that further research into the role of DHEC and alpha-hemolysin in enteric disease is warranted.     

Suc-Phe-Leu-Phe-SBzl--a new substrate for functional study of Escherichia coli ATP-dependent Lon-proteinase and its modified forms

A new efficient substrate, Suc-Phe-Leu-Phe-SBzl, was proposed for studying the function of the Escherichia coli ATP-dependent Lon protease and its modified forms. The kinetic parameters of hydrolysis of the substrate were determined. The esterase activity of protease Lon was found to be nucleotide-regulated.     

Predictable deuteration of recombinant proteins expressed in Escherichia coli

Carrageenan was used to study inflammation-induced changes in spinal nociception and its brain stem modulation in the pentobarbitone-anesthetized rat. Carrageenan was administered intraplantarly into one hindpaw 2 h before the start of electrophysiological single unit recordings of wide-dynamic range (WDR) neurons of the spinal dorsal horn. Carrageenan produced a significant leftward shift in the stimulus-response function for mechanical stimuli, whereas that for noxious heat stimuli was short of statistical significance. Conditioning electrical stimulation in the rostroventromedial medulla (RVM) significantly attenuated noxious heat-evoked, but not mechanically evoked, responses to spinal dorsal horn WDR neurons in the control (contralateral) side. However, in the carrageenan-treated side RVM stimulation had no significant effect on mechanically or noxious heat-evoked responses. Following direct spinal administration of neuropeptide FF (NPFF), noxious heat-evoked responses, but not mechanically evoked responses, were attenuated by RVM-stimulation also in the carrageenan-treated side. This selective NPFF-induced enhancement of brain stem-spinal inhibition was not reversed by naloxone. The results indicate that carrageenan-induced inflammation significantly changes the response properties of spinal nociceptive neurons and their brain stem-spinal modulation. During inflammation, NPFF in the spinal cord produces a submodality-selective potentiation of the antinociceptive effect induced by brain stem-spinal pathways, independent of naloxone-sensitive opioid receptors.     

Recombinant expression of rat and human Gro proteins in Escherichia coli

A full-length rat gro cDNA containing the signal sequence was inserted to a plasmid/phage vector pTD-lacs which had the Escherichia coli alkaline phosphatase leader sequence down-stream of the lac promoter. After removal of the gro signal sequence by site-directed mutagenesis, the vector was introduced to E. coli JM109. The cells grown in the presence of isopropyl beta-D-thiogalactopyranoside were found to contain the recombinant mature rat Gro protein in the periplasmic space. The protein was released from the cells by osmotic shock, and could be purified to homogeneity from the periplasmic fluid by a single-step procedure using reverse phase high performance liquid chromatography. By similar procedures, recombinant human Gro alpha could be obtained. In each case, about 10 mg of purified cytokine were obtained from 1 litre of bacterial culture.     

Genetic analysis of prokaryotic and eukaryotic DNA-binding proteins in Escherichia coli

This report describes an Escherichia coli genetic system that permits bacterial genetic methods to be applied to the study of essentially any prokaryotic or eukaryotic site-specific DNA binding protein. It consists of two parts. The first part is a set of tools that facilitate construction of customized E.coli strains bearing single copy lacZYA reporters that are repressed by a specific target protein. The second part is a pair of regulatable protein expression vectors that permit in vivo production of the target protein at levels appropriate for genetic experiments. When expressed in a properly designed reporter strain, the target protein represses the lac genes, resulting in an E.coli phenotype that can be quantitatively measured or exploited in large scale genetic screens or selections. As a result, large plasmid-based libraries of protein genes or pools of mutagenized variants of a given gene may be examined in relatively simple genetic experiments. The strain construction technique is also useful for generating E.coli strains bearing reporters for other types of genetic systems, including repression-based and activation-based systems in which chimeric proteins are used to examine interactions between foreign protein domains.     

Affinity-assisted in vivo folding of a secreted human peptide hormone in Escherichia coli

We show that coexpression of a specific binding protein in Escherichia coli can significantly improve the relative yields of correctly folded human insulin-like growth factor I (IGF-I). A glutathione redox buffer was used during growth to allow formation and breakage of disulfide bonds in the periplasm of the bacterial host. Both the binding protein and the peptide hormone were produced as affinity fusions, which allowed purification of the in vivo formed heterodimer by alternative affinity purification methods. The use of affinity-assisted in vivo folding has general implications for expression, folding, and purification of recombinant proteins.     

Scanning force microscopy of Escherichia coli RNA polymerase.sigma54 holoenzyme complexes with DNA in buffer and in air

Scanning force microscopy (SFM) was used to visualize complexes of Escherichia coli RNA polymerase.sigma54 (RNAP.sigma54) and a 1036 base-pair linear DNA fragment containing the glnA promoter. In order to preserve the native hydration state of the protein-DNA complexes, the samples were injected directly into the SFM fluid cell and imaged in buffer. With this protocol, an apparent bending angle of 26(+/-34) degrees was determined for the specific complexes at the promoter. The bending angle of the unspecifically bound RNAP.sigma54 showed a somewhat broader distribution of 49(+/-48) degrees, indicating the existence of conformational differences as compared to the closed complex. In about two-thirds of the closed complexes, the RNA polymerase holoenzyme was located in a lateral position with respect to the DNA and the bend of the DNA was pointing away from the protein. This conformation was consistent with the finding that for the complexes at the promoter, the apparent contour length was reduced by only about 6 nm in buffer as compared to the free DNA. From these results we conclude that in the closed complex of RNAP. sigma54, the DNA was not wrapped around the polymerase, and we present a model for the trajectory of the DNA with respect to the RNA polymerase. The images acquired in buffer were compared to samples that were washed with water and then dried before imaging. Two artefacts of the washing and drying process were detected. First, extensive washing of the sample reduced the number of the specific complexes bound at the promoter (closed complex of RNAP.sigma54) from about 70% to 30%. This is likely to be a result of sliding of the RNAP.sigma54 holoenzyme along the DNA induced by the washing process. Second, the apparent DNA shortening of the contour length of RNAP.sigma54-DNA complexes at the promoter as compared to the contour length of the free DNA was 22 nm for the dried samples as opposed to only 6 nm for the undried samples imaged in buffer. This suggests an artefact of the drying process.     

Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli

Four Rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (AAV). The two largest proteins, Rep68 and Rep78, have been shown in vitro to perform several activities related to AAV DNA replication. The Rep78 and Rep68 proteins are likely to be involved in the targeted integration of the AAV DNA into human chromosome 19, and the full characterization of these proteins is important for exploiting this phenomenon for the use of AAV as a vector for gene therapy. To obtain sufficient quantities for facilitating the characterization of the biochemical properties of the Rep proteins, the AAV rep open reading frame was cloned and expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP). Recombinant MBP-Rep68 and MBP-Rep78 proteins displayed the following activities reported for wild-type Rep proteins when assayed in vitro: (i) binding to the AAV inverted terminal repeat (ITR), (ii) helicase activity, (iii) site-specific (terminal resolution site) endonuclease activity, (iv) binding to a sequence within the integration locus for AAV DNA on human chromosome 19, and (v) stimulation of radiolabeling of DNA containing the AAV ITR in a cell extract. These five activities have been described for wild-type Rep produced from mammalian cell extracts. Furthermore, we recharacterized the sequence requirements for Rep binding to the ITR and found that only the A and A' regions are necessary, not the hairpin form of the ITR.     

Opposing roles of the holliday junction processing systems of Escherichia coli in recombination-dependent adaptive mutation

Aspects of the molecular mechanism of "adaptive" mutation are emerging from one experimental system: reversion of an Escherichia coli lac frameshift mutation carried on a conjugative plasmid. Homologous recombination is required and the mutations resemble polymerase errors. Reports implicating a role for conjugal transfer proteins suggested that the mutation mechanism is ordinary replication error occurring during transfer synthesis, followed by conjugation-like recombination, to capture the replicated fragment into an intact replicon. Whereas conjugational recombination uses either of two systems of Holliday junction resolution, we find that the adaptive lac reversions are inhibited by one resolution system and promoted by the other. Moreover, temporary absence of both resolution systems promotes mutation. These results imply that recombination intermediates themselves promote the mutations.