Devorah Friedberg

Hierarchy in the expression of the locus of enterocyte effacement genes of enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) elicit changes in host cell morphology and cause actin rearrangement, a phenotype that has commonly been referred to as attaching/effacing (AE) lesions. The ability of EPEC to induce AE lesions is dependent upon a type III protein secretion/translocation system that is encoded by genes clustered in a 35.6 kb DNA segment, named the locus of enterocyte effacement (LEE). We used transcriptional fusions between the green fluorescent protein (gfp) reporter gene and LEE genes rorf2, orf3, orf5, escJ, escV and eae, together with immunoblot analysis with antibodies against Tir, intimin, EspB and EspF, to analyse the genetic regulation of the LEE. The expression of all these LEE genes was strictly dependent upon the presence of a functional integration host factor (IHF). IHF binds specifically upstream from the ler (orf1) promoter and appears to activate expression of ler, orf3, orf5 and rorf2 directly. The ler‐encoded Ler protein was involved in activating the expression of escJ, escV, tir, eae, espB and espF. Expression of both IHF and Ler was needed to elicit actin rearrangement associated with AE lesions. In conclusion, IHF directly activates the expression of the ler and rorf2 transcriptional units, and Ler in turn mediates the expression of the other LEE genes.

Thermoregulated expression of virulence genes in enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhoea in young children. The locus of enterocyte effacement (LEE) pathogenicity island comprises a cluster of operons encoding a type III secretion system and related proteins that are associated with EPEC virulence. The LEE1 operon encodes Ler that positively regulates the LEE2, LEE3, LEE4, LEE5 and espG transcriptional units. The LEE operons are repressed at 27 degrees C and expressed at 37 degrees C. This paper describes a regulatory cascade of the thermoregulation of LEE operons. LEE1 including ler is repressed by H-NS at 27 degrees C but not at 37 degrees C. In contrast, the expression of the LEE2, LEE3, LEE4, LEE5 and espG transcriptional units is repressed by H-NS at both 27 degrees C and 37 degrees C. Upon shifting the culture temperature from 27 degrees C to 37 degrees C, Ler is synthesized and in turn activates the expression of LEE2, LEE3, LEE4 and espG by releasing the H-NS mediated repression. In the case of LEE5, Ler acts both by alleviating the H-NS mediated repression and by an additional mechanism, as yet to be defined.

Controlled gene expression utilising Lambda phage regulatory signals in a cyanobacterium host

SummaryThis study presents plasmid systems that utilize regulatory signals of bacteriophage Lambda to accomplish regulated expression of cloned genes in an A. nidulans R2 derivative strain. An operator-promoter region and the temperature-sensitive repressor gene cI857 of bacteriophage Lambda were employed. Linked to a cyanobacterial replicon, the plasmid vectors efficiently transformed Anacystis and were stably maintained within this host. The cat structural gene, encoding chloramphenicol acetyltransferase, was used to demonstrate that expression can be regulated by temperature shift. We have identified in extracts from the vector bearing Anacystis, a protein similar in size and immunology to the Lambda repressor. The systems described should allow controlled expression of adventitious genes in the cyanobacterial host.

A new hybrid plasmid capable of transforming Escherichia coli and Anacystis nidulans

We have constructed a hybrid plasmid, pDF30, by combining the 8-kb pDF3 plasmid derived from the cyanobacterium Anacystis nidulans 6311 with the Escherichia coli vector pBR325. pDF30 transforms, replicates and confers chloramphenicol resistance (Cmr) and ampicillin resistance (Apr) on both A. nidulans and E. coli. The level of resistance to ampicillin in A. nidulans transformants, although low, is above background resistance and an Apr activity was demonstrated in cell-free extracts of A. nidulans that harbored pDF30. pDF30 is stably maintained within E. coli and can be isolated intact from A. nidulans after several months of subculture under chloramphenicol selection and by this criterion is stable in vivo. The unique restriction sites for XhoI, SalI and EcoRI can be used for molecular cloning of chromosomal genes in E. coli and A. nidulans. Until now pDF3 is phenotypically cryptic in both A. nidulans and E. coli. Examination of the polypeptides encoded on and expressed by pDF30 in E. coli minicells revealed only gene products from pBR325 but none from pDF3.

Plasmids in two cyanobacterial strains

The cyanobacteria are an heterogenous group of photosynthetic prokaryotes [I]. An extremely wide variation of DNA base composition and genome size was shown among cyanobacteria of different subgroups or within the same subgroup [2-41. The presence of extrachromosomal DNA elements may contribute even more to this divergence. Although such DNA elements have been encountered in several cyanobacteria [S-7], until recently, only in the case of cyanobacteria Agmenellum quadruplicatum, these elements were shown to be covalently closed circular (CCC) DNA (plasmids) [6]. While the present study was in progress, the presence of two different plasmids in several closely related cyanobacteria species was reported [8]. Characterization of the cyanobacterial plasmids may provide useful information regarding: (a) The heterogeneity between and within the subgroups; (b) The understanding of the intermediary position that this group occupies in the evolution of the photosynthetic world between the prokaryotes and eukaryotic plants [l]; (c) Development of genetic tools, as most genetic techniques proved to be inadequate in cyanobacteria [9]. Here two different cyanobacterial strains were found to harbor plasmids. One is the unicellular strain Anacystis sp. 63 11 which possesses a plant type, oxygen evolving photosynthetic system [l] and the other is the filamentous Oscillatoti limnetica which in addition to the oxygen evolving system, displays also the bacterial type anoxygenic photosynthesis [lo]. Both plasmids are presented by electron micro-

Effect of oxygen on the cyanobacterium Oscillatoria limnetica

Oscillatoria limnetica grown photoautotrophically under aerobic or anaerobic conditions contained a single superoxide dismutase (SOD) of identical electrophoretic mobility in both cases. Its activity was cyanide resistant and H2O2 sensitive, implicating Fe-SOD. The enzyme level was high in aerobically and low in anaerobically growing cells. Anaerobically grown cells were more sensitive than aerobic to photooxidation, as expressed by bleaching of phycocyanin and disintegration of the trichomes.

Growth of host dependent Bdellovibrio in host cell free system.

A particulate, subcellular fraction of Escherichia coli was shown to promote the growth of host dependent (H-D) Bdellovibrio in the absence of host cells. The growth promoting activity was enhanced by both cations and trypsin, and destroyed by pronase. During the axenic growth unipolar spheres appear in the elongating Bdellovibrio forms. Thymidine monophosphate was more readily incorporated than thymidine into the Bdellovibrio DNA during growth in the host free system.

Sulfonylurea-resistant mutants and natural tolerance of cyanobacteria

The herbicide sulfometuron methyl (SM) inhibited the growth of the cyanobacterium Synechococcus sp. PCC7942, but not of Synechocystis sp. PCC6714. The inhibitory effect was alleviated by the simultaneous addition of valine, leucine and isoleucine. SM resistant mutants were isolated from Synechococcus 7942, two types of which were further analysed. In these mutants, SM3/20 and SM2/32, the activity of acetolactate synthase (ALS) — a key enzyme in the biosynthesis of branched-chain amino acids —appeared 2600- and 300-fold, respectively, more resistant to SM than that of their wild type. Strain SM2/32 also exhibited a low level of ALS activity. Although the growth of the latter mutant was extremely inhibited by valine, the sensitivity of its ALS activity to feed-back inhibition by the amino acid was unaltered. At high concentrations valine inhibited growth of the wild type strains and of the mutant SM3/20. Isoleucine alleviated the valine-induced growth inhibition. Unlike that of Synechococcus 7942, the ALS activity of Synechocystis was found to tolerate high concentrations (100-fold) of the herbicide. The study confirms that the SM mutations are correlated with a cyanobacterial ilv gene.

Mutations in the 5’ flanking region of rbcL inhibit the growth of Synechococcus PCC7942 in air-level of CO 2 and alter the carboxysome structure

Cyanobacteria undergo a syndrome of changes during adaptation from high to low-CO2 concentration in their environment. As a result they can concentrate inorganic carbon (Ci) within the cell and grow photoautotrophically under air level of CO2 (1). To elucidate the molecular basis of the adaptation process we have used mutants of Synechococcus PCC7942, which do not grow in air-level of CO2 but grow normally at 5% CO2 in air (2,3).