Molecular Microbiology

Moradas‐Ferreira, P.; Costa, V.; Piper, P.; Mager, W.

doi: 10.1046/j.1365-2958.1996.403940.xpmid: 8820636

There is rapidly expanding interest into the protective systems against reactive oxygen species (ROS) in the eukaryotic cell, now that the links between oxidative damage, various disease states, and ageing, are firmly established in higher organisms. Yeast molecular genetics should be able to provide powerful insight into these mechanisms; this potential is now starting to be exploited. A number of primary antioxidant activities and systems of metal‐ion homeostasis or detoxification have now been demonstrated to contribute to oxidative‐stress protection in yeast. Also, evidence is emerging that the oxidative‐stress response of this organism is complex, involving separate transcription‐factor responses to peroxide, superoxide anion and metal ions.

Herrick, John; Kohiyama, Masamichi; Atlung, Tove; Hansen, Flemming G.

doi: 10.1046/j.1365-2958.1996.432956.xpmid: 8820637

This review concerns the mechanisms which control initiation of chromosome replication in enterobacteria with respect to cell growth. Initiation control is commonly separated into positive and negative regulatory mechanisms. Four main points are advanced concerning these different aspects of initiation control. (i) The average concentration of the initiator protein DnaA is proportional to the origin concentration, i.e. the origin per cell mass ratio and, thus, inversely proportional to the very often used term of the ‘initiation mass’. (ii) The time of initiation of chromosome replication in the cell cycle is set by DnaA protein accumulating to a threshold level, which in concert with a number of other factors allows for a co‐operative formation of the initiation complex. (iii) The time of initiation is not determined by the interaction with these other factors or by the transient interaction between newly replicated origins (oriC ) and the cell surface. (iv) The aberrant initiation phenotype observed in various mutants, including dnaA (ts) mutants, might be due to a defective pre‐initiation DnaA–oriC interaction or it might be due to a defect in the protection of newly initiated origins from reinitiation. Many of these points are discussed and evaluated in view of recent developments concerning the regulation of chromosome replication in Escherichia coli

Díaz, Eduardo; Munthali, Mathildah; Lünsdorf, Heinrich; Höltje, Joachim‐Volker; Timmis, Kenneth N.

doi: 10.1046/j.1365-2958.1996.399929.xpmid: 8820638

The holin function Ejh of the pneumococcal bacteriophage EJ‐1 has been characterized. It shows structural features similar to, and functionally complemented, the prototype member of the holin family. In Escherichia coli and Pseudomonas putida the Ejh product caused cellular death, and changes in cell morphology could be accounted for by lesions in the cytoplasmic membrane. Expression of ejh resulted in the inhibition of growth in a variety of phylogenetically distant bacterial genera, suggesting a broad spectrum of action. Concomitant expression of the ejh and ejl (encodes a lysin) genes led to lysis of E. coli and P. putida cells. Remarkably, the Ejl lysin was able to attack murein from bacteria lacking choline in their sacculi, which suggests that pneumococcal lysins have a broader substrate specificity than previously assumed. Furthermore, the Ejh holin was able to trigger activity of the major pneumococcal autolysin cloned and expressed in E. coli, and this raised new questions about the regulation of this model autolysin. A new function for holins in systems where the phage lysin is supposed to be associated with the membrane is proposed.

Garcia, Marie‐Isabelle; Gounon, Pierre; Courcoux, Pascale; Labigne, Agnès; Le Bouguénec, Chantal

doi: 10.1046/j.1365-2958.1996.394935.xpmid: 8820639

The afa‐3 gene cluster determines the formation of an afimbrial adhesive sheath that is expressed by uropathogenic as well as diarrhoea‐associated Escherichia coli strains. It contains six genes (afaA–afaF ), among which the afaE3 gene is known to code for the structural AfaE‐III adhesin (previously designated AFA‐III), whereas no role has yet been identified for the afaD gene product. The afa‐3 gene cluster is closely related to the daa operon that codes for an adhesin, the F1845 adhesin, which is highly related to the AfaE‐III adhesin; however, unlike the AfaE‐III adhesin, F1845 is a fimbrial adhesin. Reported in this work is the construction of chimeras between the afa‐3 and daa operons. Analyses of the phenotypes conferred by these afa‐3/daa chimeric clusters allowed us to conclude that the biogenesis of a fimbrial or an afimbrial adhesin is fully determined by the amino acid sequence of the AfaE‐III and F1845 adhesins. Moreover, the role of the AfaD product in the biosynthesis of the afimbrial sheath was assessed by immunogold and immunofluorescence experiments. The AfaD and the AfaE‐III products were purified and used to raise rabbit and mouse antisera. Similar to AfaE‐III, AfaD was found to be a surface‐exposed protein as well as an adhesin; both AfaD and AfaE‐III are concomittantly expressed by the bacterial cell. These results demonstrate, for the first time, that the afimbrial adhesive sheath expressed by pathogenic E. coli is composed of two adhesins.

Wang, Hui; Matsumura, Philip

doi: 10.1046/j.1365-2958.1996.393934.xpmid: 8820640

The cheA gene encodes two overlapping polypeptides with a common carboxyl terminus: CheAL and CheAS. CheAL plays a central role in the Escherichiacoli chemotaxis signalling pathway by autophosphorylation and transferring the phosphate to both CheY and CheB. On the other hand, the physiological functions of CheAS remain unknown. We have observed that overproduction of CheAS in wild‐type cells increased counterclockwise‐biased flagellar rotation, and this effect is dependent on the presence of CheZ. CheZ specifically facilitates CheY‐phosphate (CheY‐P) dephosphorylation and generates a smooth swimming signal. A physical interaction was detected between CheZ and CheAS in wild‐type cell lysates by immunoprecipitation. The CheAS/CheZ interaction does not require other chemotaxis components, as we could form the complex using purified CheAS and CheZ proteins. The ability of CheAS to bind to CheZ depends on its being in the reduced state. We found that under non‐reducing conditions, CheAS appears to form intermolecular disulphide bonds and loses the ability to bind to CheZ. Finally, the CheAS/CheZ complex formed in vitro shows a greater dephosphorylating activity on CheY‐P than does free CheZ.

Nieto, Jose M.; Bailey, Marc J. A.; Hughes, Colin; Koronakis, Vassilis

doi: 10.1046/j.1365-2958.1996.446951.xpmid: 8820641

Expression of the Escherichia coli hlyCABD operon encoding synthesis, maturation and export of haemolysin toxin was strongly dependent upon a 35 bp DNA sequence, spanning the element GGCGGTAG, located 2 kbp upstream. When the hly operon was placed under the control of the inducible tac promoter, expression remained dependent upon this element, when transcribed in its native orientation 3′ of the promoter. The increase in ptac‐directed transcription was strongest for the distal, export genes of the hly operon, and was particularly striking when ptac and the element were placed far upstream. The element did not influence transcript stability, and we suggest that it is a key component of a novel regulatory mechanism may suppresses transcription polarity within operons. The mechanism that be of widespread importance in bacterial gene expression because the 8 bp element is present in many Gram‐negative species as an upstream component of operons encoding the production of toxins and the surface assembly of polysaccharides and components required for the conjugal transfer of DNA. We name it the ops element for operon polarity suppressor.

Alfano, James R.; Bauer, David W.; Milos, Timothy M.; Collmer, Alan

doi: 10.1046/j.1365-2958.1996.415946.xpmid: 8820642

Pseudomonas syringae pv. syringae, like many plant pathogenic bacteria, secretes a ‘harpin’ protein that can elicit the hypersensitive response (HR), a defensive cellular suicide, in non‐host plants. The harpin‐encoding hrpZ gene is located in an operon that also encodes Hrp secretion pathway components and is part of the functional cluster of hrp genes carried on cosmid pHIR11 that enables saprophytic bacteria like Escherichia coli and Pseudomonas fluorescens to elicit the HR in tobacco leaves. We have constructed functionally non‐polar hrpZ deletion mutations, revealing that HrpZ is necessary for saprophytic bacteria carrying pHIR11 to elicit a typical HR, whereas it only enhances the elicitation activity of P. s. syringae. Partial deletion mutations revealed that the N‐terminal 153 amino acids of HrpZ can enable E. coli MC4100‐(pHIR11) to elicit a strong HR. hrpZ subclone products comprising the N‐terminal 109 amino acids and C‐terminal 216 amino acids, respectively, of the 341 amino acid protein were isolated and found to elicit the HR. P. fluorescens (pHIR11 hrmA ::TnphoA) mutants do not elicit the HR, but cell fractionation and immunoblot analysis revealed that they produce and secrete wild‐type levels of HrpZ. Therefore, elicitor activity resides in multiple regions of HrpZ, P. syringae produces elicitor(s) in addition to HrpZ, and HrpZ is essential but not sufficient for HR elicitation by saprophytic bacteria carrying pHIR11.

Gordia, Sylvie; Gutierrez, Claude

doi: 10.1046/j.1365-2958.1996.418945.xpmid: 8820643

The transcription of the osmotically inducible gene osmC of Escherichia coli is initiated by two overlapping promoters, osmCp1 and osmCp2. The existence of these two promoters was confirmed by site‐directed mutagenesis. osmC transcription is regulated by the growth phase. In a medium of low osmotic pressure, expression of osmC is induced at the onset of decelerating phase and continues during the beginning of stationary phase. At elevated osmotic pressure, the induction occurs somewhat earlier during growth. Both promoters are repressed during early exponential phase. osmCp2 is induced during entry into stationary phase. Transcription from osmCp1, which is approximately 10‐fold lower than that of osmCp2 in rich medium, starts during the mid‐log phase and stops in early stationary phase. In the absence of σS, the stationary‐phase sigma factor encoded by rpoSosmCp2 expression is much reduced while expression of osmCp1 is unaffected. As a consequence, the regulation of osmC as a function of growth is at least partially independent of σS

Ames, Peter; Yu, Yong A.; Parkinson, John S.

doi: 10.1046/j.1365-2958.1996.408930.xpmid: 8820644

The serine chemoreceptor Tsr and other methyl‐accepting chemotaxis proteins (MCPs) control the swimming behaviour of Escherichia coli by generating signals that influence the direction of flagellar rotation. MCPs produce clockwise (CW) signals by stimulating the autophosphorylation activity of CheA, a cytoplasmic histidine kinase, and counter‐clockwise signals by inhibiting CheA. CheW couples CheA to chemoreceptor control by promoting formation of MCP/CheW/CheA ternary complexes. To identify MCP structural determinants essential for CheA stimulation, we inserted fragments of the tsr coding region into an inducible expression vector and used a swimming contest called ‘pseudotaxis’ to select for transformant cells carrying CW‐signalling plasmids. The shortest active fragment we found, Tsr (350–470), stimulated CheA in a CheW‐dependent manner, as full‐length Tsr molecules do. It spans a highly conserved ‘core’ (370–420) that probably specifies the CheA and CheW contact sites and other determinants needed for stimulatory control of CheA. Tsr (350–470) also carries portions of the left and right arms flanking the core, which probably play roles in regulating MCP signalling state. However, this Tsr fragment lacks all of the methylation sites characteristic of MCP molecules, indicating that methylation segments are not essential for generating receptor output signals.

Pope, Margaret K.; Green, Brian D.; Westpheling, Janet

doi: 10.1046/j.1365-2958.1996.414933.xpmid: 8820645

Mutants of Streptomyces coelicolor blocked at the earliest visible stage of morphological differentiation are called bld mutants. These mutants fail to form aerial hyphae on rich medium and most are defective in antibiotic production. One striking feature of these mutants is that, with the exception of bldB, their morphological defect is carbon‐source dependent. In our investigation of catabolite control in Streptomyces, we identified mutants that were resistant to glucose repression and were also bld. The existence of these new bld mutants led us to examine the catabolite control phenotype of the previously described bld mutants which were not known to contain defects in carbon regulation. We report here that all of the characterized bld mutants of S.coelicolor are defective in the regulation of galP1, and that at least one of the bld mutants, bldB, is globally deregulated for carbon utilization. Complementation of the morphological defect of bldA and bldB mutants with a cloned copy of the wild‐type bld gene simultaneously restored normal regulation of galP1, indicating that both aspects of the mutant phenotype are caused by the same lesion. We suggest a new interpretation for the role of the bld genes in development in Streptomyces. We suggest that the primary defect in bld mutants is in the regulation of carbon utilization, not specifically in the activation of genes whose products regulate the development pathway as previously suggested. We speculate that the inability of bld mutants to initiate morphogenesis is a secondary consequence of their inability to sense and/or signal starvation.