Stress Responses as a Tool To Detect and Characterize the Mode of Action of Antibacterial Agents

Single-copy gene fusions between the lacZ reporter gene and Escherichia coli strains containing promoters induced by cold shock (cspA), cytoplasmic stress (ibp), or protein misfolding in the cell envelope (P3rpoH) were constructed and tested to determine their ability to detect antibacterial agents while simultaneously providing information on their cellular targets. Antibiotics that affect prokaryotic ribosomes selectively induced the cspA::lacZ or ibp::lacZ gene fusion, depending on their mode of action. The membrane-damaging peptide polymyxin B induced both the P3rpoH::lacZ and ibp::lacZ fusions, while the β-lactam antibacterial agent carbenicillin activated only the P3rpoH promoter. Nalidixic acid, a compound that causes DNA damage, downregulated β-galactosidase synthesis from P3rpoH but had little effect on expression of the reporter enzyme from either the cspA or ibp promoter. All model antibiotics could be identified over a wide range of sublethal concentrations with signal-to-noise ratios between 2 and 11. A blue halo assay was developed to rapidly characterize the modes of action of antibacterial agents by visual inspection, and this assay was used to detect chloramphenicol secreted into the growth medium of Streptomyces venezuelae cultures. This simple system holds promise for screening natural or combinatorial libraries of antimicrobial compounds.     

Growth, Survival and Characterization of cspA in Salmonella enteritidis Following Cold Shock

Salmonella enteritidis is a major foodborne microbial pathogen that can grow and survive at low temperatures for a considerable period of time. Increased survival was evidenced from a frozen S. enteritidis culture when treated at 10°C prior to freezing. Western blot analysis with Escherichia coli CspA antibody and analysis of radiolabeled proteins from S. enteritidis cultures after cold shock at 10°C and 5°C showed increased expression of a 7.4-kDa major cold shock protein, CS7.4, similar in size to that reported for E. coli. Cloning followed by nucleotide sequence analysis of the cspA gene from S. enteritidis showed a 100% nucleotide sequence identity in the promoter elements (−35 and −10) and the amino acid sequence encoded by the open reading frame (ORF) with the E. coli cspA gene. However, the differences in the nucleotide sequences between E. coli and S. enteritidis cspA genes in the putative repressor protein binding domain, the fragment 7, and in various segments throughout the upstream 0.642-kbp DNA may contribute to the expression of CS7.4 at less stringent temperatures in S. enteritidis. As in E. coli, the actual role of CS7.4 in protecting S. enteritidis from the damaging effects of cold or freezing temperatures is not yet understood. Received: 14 March 1997 / Accepted: 10 July 1997     

Expression of ferritin-like protein in Listeria monocytogenes after cold and freezing stress

The cold shock protein family consists of the transfer of the foodborne pathogen Listeria monocytogenes from 37 to 4 and ?20?°C and was characterized by the sharp induction of a low molecular mass protein. This major cold shock protein ferritin-like protein (Flp) has an important role in regulation of various microbial physiological processes. Flp have a molecular mass of about 18?kDa, as observed on SDS?CPAGE. The purification procedure including ammonium sulfate fractionation was used. Monospecific polyclonal antibodies raised in rabbits against the purified new Flp immunostained a single 18-kDa Flp band in extracts from different cytoplasmic proteins blotted onto nitrocellulose. A 411-bp cDNA fragment that corresponds to an internal region of an flp gene was obtained by RT-PCR. Our result indicated a surexpression of major cold shock protein and an important increase in flp mRNA amount after a downshift temperature especially at ?20?°C.     

Heat shock mRNA accumulation during recovery from cold shock in Drosophila melanogaster

Heat shock protein synthesis is induced in response to a variety of chemical and physical stresses. Among these are heating above normal growing temperatures, treatment with heavy metals, amino acid analogues, steroid hormones and a variety of other chemicals (CRC Crit. Rev. Biochem. 18, 239–280). We have shown previously that heat shock proteins are also synthesized during recovery from prolonged 0°C treatment in Drosophila larval salivary glands. In this paper we describe the cold treatments which induce heat shock protein synthesis in more detail, and show that heat shock mRNA does not accumulate during the cold treatment, but rather during the recovery period when the larvae are returned to 25°C. The implications of these results for the regulation of heat shock mRNA levels, and for the role of heat shock proteins in recovery from cold shock are discussed.     

Expression of a heat-inducible gene in transfected mosquito cells

The evolutionary conservation of the heat shock response suggests that plasmids containing promoters from Drosophila heat shock protein (hsp) genes will be useful in the development of gene transfer procedures for cell lines representing a variety of insect species. Conditions for induction of endogenous hsp genes and for expression of the chloramphenicol acetyltransferase (CAT) gene regulated by the Drosophila hsp 70 promoter were examined in Aedes albopictus (mosquito) cells. Five hsps, ranging in size from 27,000 to 90,000 D, were induced in A. albopictus cells during incubation at 41°C in medium containing [³⁵S]methionine. Relative synthesis of these proteins at 37 and 41°C indicated that Aedes hsp 66 is homologous to Drosophila hsp 70. Detection of CAT activity in transfected mosquito cells was enhanced 10-fold under heat shock conditions (6 h, 41°C) based on maximal expression of hsp 66, relative to conditions defined for expression of hsp 70 in Drosophila cells. Analysis of the endogenous heat shock response may be essential to the optimal use of plasmids containing the Drosophila hsp 70 promoter with other insect cell types.     

Cold adaptive potential of chironomids overwintering in a glacial stream

Insects inhabiting cold streams must either tolerate or avoid freezing to survive. The present study reports the strategy adopted by fourth‐instar larvae of two chironomid species [Pseudodiamesa branickii (Nowicki) and Diamesa cinerella (Meigen)] overwintering in a glacial stream (in the Italian Alps). The cold adaptive potential of both species under acute cold stress is investigated down to –30 °C. Supercooling points, lower lethal temperatures (LLTs), haemolymph thermal hysteresis, whole body content of sugars and polyols, and the expression of heat shock protein (HSP) genes (hsc70 and hsp70) expression are estimated. Comparable thermal hysteresis (> 2 °C) is measured in the two species, both of which accumulate glucose and sucrose as the main cryoprotectants. According to the supercooling points (= –6.37 and –6.85 °C, respectively) and LLT₁₀₀ (= –16.2 and –14.7 °C, respectively), P. branickii and D. cinerella can both be considered as freeze tolerant. However, the cumulative proportion of individual freezing values and the LLT₅₀ (–9.14 and –6.13 °C, respectively) suggest that P. branickii is more cold hardy than D. cinerella, whereas the gene expression data (i.e. an absence of up‐regulation of hsp70 in D. cinerella) suggest that D. cinerella is more cold hardy than P. branickii. These findings are discussed in relation to the validity of the different metabolic indicators for defining the level of cold hardiness of a species, even in relation to its cold stenothermy. The results are also discussed in relation to climate warming, which represents a serious threat for species from glacier‐fed streams.