ALBERT

Notes: Abstract The Escherichia coli modABCD operon, which encodes a high-affinity molybdate uptake system, is transcriptionally regulated in response to molybdate availability by ModE. Here we describe a highly effective enrichment protocol, applicable to any gene with a repressor role, and establish its application in the isolation of transposon mutations in modE. In addition we show that disruption of the ModE C-terminus abolishes derepression in the absence of molybdate, implying this region of ModE controls the repressor activity. Finally, a mutational analysis of a proposed molybdate binding motif indicates that this motif does not function in regulating the repressor activity of ModE.

Notes: Expression of the Escherichia coli dmsABC operon that encodes a molybdenum-containing DMSO/TMAO reductase is increased in response to anaerobiosis and repressed by nitrate. These changes are mediated by the transcription factors Fnr and NarL respectively. Interestingly, modC strains that are defective in molybdate uptake exhibit impaired anaerobic induction and no nitrate-dependent repression of the dmsABC operon. To determine if the molybdate-responsive transcription factor ModE is involved in this process, a set of dmsA–lacZ operon fusions were constructed and analysed. The pattern of dmsA–lacZ expression in response to anaerobiosis and nitrate addition was identical in both modC and modE strains, thus suggesting a regulatory role for ModE. In vitro studies confirmed that ModE bound the dmsA promoter at a high-affinity site typical of other E. coli ModE operator sites. Mutations in this site abolished ModE binding in vitro and displayed the same phenotype as a modE mutation. In contrast to previously characterized ModE operator sites, which either overlap or are located immediately upstream of the ModE-regulated promoter, the ModE site is centred 52.5 bp downstream of the major dmsA transcript start site. We identified a putative integration host factor (IHF) binding site in the intervening sequence, and in vitro studies confirmed that IHF bound this site with high affinity. Using himA mutants, we confirmed that IHF plays a role in the molybdate-dependent regulation of dmsA–lacZ expression in vivo. This study provides the first example in which ModE affects gene regulation in concert with another transcription factor.

Notes: The effect of environmental parameters on secretion, stability and activity of Listeria monocytogenes listeriolysin O (LLO) was examined. Increasing the initial glucose concentration in proteose peptone medium resulted in reduced LLO activity and lower final pH. Yield of LLO was substantially increased when the medium was buffered with ß-glycerophosphate. Extended storage of active LLO in growth medium adjusted to pH 5.3 resulted in loss of only 10% LLO activity; however, up to 90% activity was lost during storage atpH 〈 4.5. In contrast, LLO activity was maximal at pH between 4.0 and 5.5. When the initial pH of culture medium was adjusted to give a final pH after growth of 5.3, as little as 10% of the maximum LLO activity was obtained. The results suggest that the degree of acidity may control both secretion and stability of LLO.

Notes: Abstract— This study is concerned with the influence of a single-peak overload and the overload ratio on the subsequent rate of growth of a fatigue crack in steels. Retardation increases with increasing overload ratio.The crack opening load was also measured during all tests. It is shown that the Elber's crack closure concept is not able to explain the effect of overloads. The importance of the material yield stress was evaluated by testing steels of different strength. It seems that the residual stress state induced by the overload is the major factor causing retardation. Two models are analyzed.

Notes: Abstract— This work is concerned with the determination of the fatigue threshold ΔKth for an aluminium alloy. No dependency of ΔKth with grain size was observed. Crack closure concepts were applied in order to explain the effect of mean stress and environment. The opening load Pop was determined by the compliance method. Results show no significant variation of Pop with environment (air or vacuum) but partially explain the influence of mean stress on crack growth rates. Fractographic analysis (SEM) of fracture surfaces and evolution of compliance diagrams suggest that a change of mechanism in crack propagation occurs when stage I changes to stage II growth.

Notes: IcsA of Shigella flexneri is required for intercellular spread and is located in the outer membrane at one pole of the bacterium, where it catalyses the polymerization of host-cell actin. The formation of the actin tail provides the force to move the bacterium in a unidirectional manner through the host-cell cytoplasm. We have previously demonstrated that rough lipopolysaccharide (LPS) mutants of S. flexneri 2a are avirulent and cannot form plaques in tissue-culture monolayers. This inability to form plaques is associated with non-polar localization of IcsA and loss of host-cell membrane-protrusion formation ('fireworks'). To define the minimal LPS structure required for fireworks formation, we constructed a strain of S. flexneri (BS497) that contains a mutation in rfc, encoding the O side-chain polymerase, and a strain, BS520, that possesses a defective O side-chain ligase due to a mutation in rfaL. BS497 produces a LPS that consists of a core with one repeat unit of the O side-chain, while BS520 produces a LPS consisting of a complete core with no O side-chain. BS497 remained invasive but did not form fireworks or plaques in tissue-culture monolayers and was negative in the Serény test. BS520 was invasive, generated reduced numbers of short fireworks, and made tiny plaques, but it was negative in the Serény test. Analysis of BS497 with anti-lcsA antibody demonstrated that IcsA was distributed over the entire cell surface. The distribution of IcsA on the surface of BS520 was predominantly unipolar, with some trail-back of IcsA label along the sides of the bacterium. A similar pattern was seen when infected monolayers were stained for polymerized actin. These results suggest that both the presence and the length of the O side-chain are important in the proper localization or maintenance of IcsA at the pole which subsequently affects the ability to form actin tails and produce fireworks. This reduced ability to form actin tails and fireworks results in a decreased ability of Shigella to move into adjacent host cells. To determine if the sugar composition of the O side-chain is important in the ability to form fireworks, the rfb region of S. flexneri2a was replaced with the rfb region from Escherichia coll serotype O8 or O25. Both hybrids were invasive, formed plaques, and gave positive Serény reactions. These results suggest that, unlike LPS length, the sugar composition of the O side-chain is not a critical requirement for the proper localization of IcsA and efficient intercellular movement.