Use of a two-hybrid assay to study the assembly of a complex multicomponent protein machinery: bacterial septosome differentiation.

The ability of each of the nine Escherichia coli division proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL, FtsW, FtsI, FtsN) to interact with itself and with each of the remaining eight proteins was studied in 43 possible combinations of protein pairs by the two-hybrid system previously developed by the authors' group. Once the presumed interactions between the division proteins were determined, a model showing their temporal sequence of assembly was developed. This model agrees with that developed by other authors, based on the co-localization sequence in the septum of the division proteins fused with GFP. In addition, this paper shows that the authors' assay, which has already proved to be very versatile in the study of prokaryotic and eukaryotic protein interaction, is also a powerful instrument for an in vivo study of the interaction and assembly of proteins, as in the case of septum division formation.

Mutations arise independently of transcription in non-dividing bacteria.

It has been proposed that transcription introduces a bias into the random process of mutation. Although this hypothesis is supported by experimental data for mutations arising during active bacterial growth, the role of transcription in mutagenesis in non-dividing bacteria is entirely hypothetical. In the present study, we tested the hypothesis of a possible role of transcription in a non-dividing E. coli K12 strain. In this strain (BD010), a mutated trpB allele (trpB9578), placed under stringent transcriptional control, was tested for the appearance of prototrophic revertants on synthetic medium lacking tryptophan. The number of phenotypic revertants which appeared in the absence of trp transcription was compared to that observed when the mutated gene was continuously transcribed. Our results showed that transcription of trpB is not mutagenic under conditions of tryptophan starvation, and that the frequency of TrpB+ reversion is solely a function of the duration of starvation.

A two-hybrid system based on chimeric operator recognition for studying protein homo/heterodimerization in Escherichia coli.

The development of a convenient and promising alternative to the various two-hybrid methods that are used to study protein-protein interactions is described. In this system, a lambdoid chimeric operator is recognized by a hybrid repressor formed by two chimeric monomers whose C-terminal domains are composed of heterologous proteins (or protein domains). Only if these proteins efficiently dimerize in vivo is a functional repressor formed able to bind the chimeric operator and shut off the synthesis of a downstream reporter gene. This new approach was tested with several interacting proteins ranging in size from less than 100 to more than 800 amino acids and, to date, no size or topology limit has been detected.

Two-hybrid assay: construction of an Escherichia coli system to quantify homodimerization ability in vivo.

A hybrid system which takes advantage of the properties of the lambda repressor allows detection of protein-protein interactions. Fusion of the cI N-terminal domain to a heterologous protein will result in a functional lambda repressor, able to strongly bind to its operator and conferring immunity to lambda infection only when the heterologous protein dimerizes efficiently. In this paper, construction of a recombinant plasmid which allows detection of the activity of the lambda chimeric repressor formed by the N-terminal part of cI fused with a heterologous protein is reported. This construct is interesting due to its potential to be integrated in any target gene of the bacterial host, thus permitting this hybrid assay to be performed, not only in Escherichia coli strains, but in every bacterial genus where the reporter gene can be expressed. In addition, because of its modular construction, this plasmid can be easily modified to be exploitable in many experimental situations, such as in the detection of promoter region activity.

FtsZ dimerization in vivo.

A hybrid assay, based on the properties of the lambda repressor, was developed to detect FtsZ dimerization in Escherichia coli in vivo. A gene fusion comprising the N-terminal end of the lambda cI repressor gene and the complete E. coli ftsZ gene was constructed. The fused protein resulted in a functional lambda repressor and was able to complement the thermosensitive mutant ftsZ84. Using the same strategy, a series of 10 novel mutants of FtsZ that are unable to dimerize was selected, and a deletion analysis of the protein was carried out. Characterization of these mutants allowed the identification of three separate FtsZ portions: the N-terminal of about 150 amino acids; the C-terminal of about 60 amino acids, which corresponds to the less conserved portion of the protein; and a central region of about 150 residues. Mutants belonging to this region would define the dimerization domain of FtsZ.

A spontaneous Escherichia coli K12 mutant which inhibits the excision-reintegration process of Mu gem2ts.

Escherichia coli K12 strains lysogenic for Mu gem2ts with the prophage inserted in a target gene (i.e., lacZ::Mu gem2ts lysogenic strains) revert to Lac+ by prophage precise excision with a relatively high frequency (about 1 x 10(-6)). The revertants obtained are still lysogens with the prophage inserted elsewhere in the bacterial chromosome. We have observed that, with the time of storage in stabs, bacterial cultures lysogenic for Mu gem2ts lose the ability to excise the prophage. The mutation responsible for this effect was co-transducible with the gyrB gene. After the removal of the prophage by P1 vir transduction from these strains, one randomly chosen clone, R3538, was further analyzed. It shows an increment of DNA supercoiling of plasmid pAT153, used as a reporter, and a reduced beta-galactosidase activity. On the other hand, R3538 is totally permissive to both lytic and lysogenic cycles of bacteriophage Mu.

A temperature-sensitive strain of Histoplasma capsulatum has an altered delta 9-fatty acid desaturase gene.

We have isolated and characterized the delta 9-desaturase gene (Ole1), which codes for a key enzyme involved in regulating membrane fluidity in animal cells and microorganisms, from two strains of Histoplasma capsulatum, one that is temperature-tolerant (G217B) and the other temperature-susceptible (Downs). These pathogenic fungi are dimorphic in that they undergo a morphologic transition from the mycelial to yeast-like form when the temperature of incubation is switched from 25 to 37 degrees C or when they infect a susceptible host. The coding sequences of the two genes, both containing an intron of 93 nucleotides, are virtually identical and analogous to the delta 9-desaturase gene of Saccharomyces cerevisiae and those of the rat, mouse and human. Ole1 transcription of the thermotolerant G217B and thermosensitive Downs strains is similar in yeast phase cells and during the temperature shift down from 34, 37, or 40 to 25 degrees C (yeast-to-mycelia transition). Nevertheless, the delta 9-desaturase gene is transcriptionally inactive in mycelia of G217B at 25 degrees C while it is actively transcribed in the Downs strain at the same temperature. These results are in agreement with the finding that membranes of the Downs strain have a higher level of oleic acid. The differential expression of delta 9-desaturase genes is discussed in relationship to differences in thermosensitivity in the fungal isolates and in regulating the level of expression of heat shock genes.

The Histoplasma capsulatum cdc2 gene is transcriptionally regulated during the morphologic transition.

To understand the molecular mechanisms that control the reversible morphologic transition from mycelia to yeast in dimorphic fungi, we have isolated and characterized a cdc2 gene from Histoplasma capsulatum. This organism is a dimorphic pathogenic fungus that grows as a filamentous saprobic mold in soil and as a unicellular pathogenic yeast in human tissue. The cloned gene, whose protein product has a high degree of homology with other members of the cdc2 family, is split into four exons and three introns of 95, 52 and 85 nucleotides. Analyses of cDNA clones confirm the presence of the eukaryotic splice donor (GT) and acceptor (AG) sites. The spliced gene codes for a protein of 324 amino acids (aa) with a predicted molecular mass of 36.9 kDa. The H. capsulatum cdc2 product has 71% aa identity with Saccharomyces cerevisiae and 70% with Schizosaccharomyces pombe. The deduced protein contains the sequence, PSTAIRE, that is normally found in most p34cdc2 proteins. H. capsulatum cdc2 is transcriptionally regulated during the morphologic mycelium<==>yeast transitions and is more actively transcribed in the yeast than in the mycelial phase.

The bacteriophage Mu gem gene: a positive regulator of the C operon required for normal levels of late transcription.

The gem product of bacteriophage Mu modulates synthesis of various host proteins and alters the host chromosome topology. To elucidate the role of the gem gene in Mu development, we analyzed the behavior of several mutants in this gene. The results, obtained with two Mu gem- phages, show that (1) phage growth is significantly delayed and inhibited, (2) early transcription is normal but late transcription is delayed and reduced, (3) DNA replication appears normal, and (4) the Mu C gene, whose product positively regulates Mu late genes, is one of the gem target sites. Transcription of a C promoter-lacZ fusion, carried by the pPH91 plasmid, is stimulated both after infection with Mu gem+ or Mu gem3 and is strains lysogenic for the same phages in the presence of viral immunity. These data suggest that the primary role of the gem product is modulation of gene expression. This control could be carried out by direct interaction with transcription factors or by changing DNA supercoiling.