Essential and non-essential interactions in interactome networks: the Escherichia coli division proteins FtsQ-FtsN interaction.

The Escherichia coli division protein FtsQ, which plays a central role in the septosome assembly, interacts with several protein partners of the division machinery. Its interaction with FtsB and FtsL allows the formation of the trimeric complex connecting the early cytoplasmic cell division proteins with the late, essentially periplasmic, ones. Little is known about the interactions that FtsQ contracts with other divisome components, besides the fact that all are localized in its periplasmic domain. In this domain, two independent subdomains, both involved in FtsQ, FtsI and FtsN interactions, were also identified. The study of FtsQ interaction-defective mutants constituted a basis to investigate the biological significance of its interactions. However, in the case of interactions where two independent sites are involved, mutation(s) in one domain can be suppressed by the presence of the still-functional second interaction region. To ascertain the biological role of these interactions, it is therefore necessary to select double mutants, where both sites are impaired. This paper describes the behaviour of FtsQ double mutants that have lost the ability to interact with FtsN, which is the last component in the hierarchy of divisome assembly, and is necessary to guarantee its stability and function.

Reversal of Mu gem2ts-induced mutations.

Mutations induced by the integration of a Mu gem2ts mutant prophage can revert at frequencies around 1 x 10(-6), more than 10(4)-fold higher than that obtained with Mu wild-type. Several aspects characterize Mu gem2ts precise excision: (i) the phage transposase is not involved; (ii) the RecA protein is not necessary; and (iii) revertants remain lysogenic with the prophage inserted elsewhere in the host genome. In addition, prophage re-integration seems to be non-randomly distributed, whereas Mu insertion into the host genome is a transposition event without any sequence specificity. In this paper, we describe that the site of re-integration somehow depends on the original site of insertion. Two alternative models are proposed to explain the strong correlation between donor and receptor sites.

Deletions induced by heat treatment of E. coli K12 lysogenic for lambda prophages.

We have investigated the production of prophage deletions in heat-induced lambda lysogens of E. coli K12. Our results are indicative of a direct action of the heat-induced prophage in producing deletions. The temperature of 40 degrees used for the experiments may be critical to prove this effect. The phage function involved in deletion formation is not known.

A method for DNA extraction from the desert cyanobacterium chroococcidiopsis and its application to identification of ftsZ

A method was developed for extraction of DNA from Chroococcidiopsis that overcomes obstacles posed by bacterial contamination and the presence of a thick envelope surrounding the cyanobacterial cells. The method is based on the resistance of Chroococcidiopsis to lysozyme and consists of a lysozyme treatment followed by osmotic shock that reduces the bacterial contamination by 3 orders of magnitude. Then DNase treatment is performed to eliminate DNA from the bacterial lysate. Lysis of Chroococcidiopsis cells is achieved by grinding with glass beads in the presence of hot phenol. Extracted DNA is further purified by cesium-chloride density gradient ultracentrifugation. This method permitted the first molecular approach to the study of Chroococcidiopsis, and a 570-bp fragment of the gene ftsZ was cloned and sequenced.

Mu gem2ts DNA integration is not necessary for induction of synchrony of cell division in Escherichia coli K12.

The gem2ts mutant of bacteriophage Mu induced synchrony of cell division on bacteria surviving infection. Induction of synchronous growth could also be observed as a response to the entire infected bacterial population, as in the case of infection of hic mutants, a peculiar class of gyrB alleles. After Mu wild-type or Mu gem2ts infection of hic mutants, there was a lack of viral DNA integration and replication, while phage gene expression (including that of A gene, coding for the transposase) seemed to be quite normal. These data indicate that the mechanism of bacterial synchronization induced by Mu gem2ts does not require integration nor replication of the phage DNA.

Global changes in gene expression in Escherichia coli K12 induced by bacteriophage Mu Gem protein.

We have studied the growth properties of some Mu lysogens with respect to the non-lysogenic strain and have observed that the division time in minimal medium was increased over 4-fold when the bacteria carried the prophage mutated in the gem gene (Mu gem3). Since this phage gene has previously been shown to be involved in modulation of expression of host genes, we have analysed the proteins extracted from lysogens and non-lysogens as a rapid assay of global gene expression. The pattern of proteins extracted showed marked quantitative variations between non-lysogens, lysogens for wild-type Mu and lysogens for phage Mu gem3. These effects were no longer as evident when the strains were grown in rich medium. This dramatic change in the physiological state of the lysogenic strain versus the non-lysogenic in particular growth conditions extends the concept of lysogeny. For many years, the prophage has been considered only as a potentially lethal factor, while here it also appears as a genetic element capable of profoundly modifying host biology.

Synchronous division induced in Escherichia coli K12 by phage Mu: analysis of DNA topology and gene expression during the cell cycle.

Bacteriophage Mu mutants in gene gem (Mu gemts2) induce cycles of synchronous divisions after infection of a bacterial population in steady-state conditions. In this paper, two classes of gyrB mutants, synchronizable and non-synchronizable, are described. The existence of the non-synchronizable class suggests that the gyrase B subunit is involved with Gem in the process of synchronization. Cyclical variations in DNA topology of a resident multicopy plasmid occur during synchronous growth and correlate with a modulation of the chromosomal lacZ gene expression. Transcription data for the cell division genes, ftsZ and envA, obtained studying first steps in synchronous growth after infection, show that synthesis of the two mRNA is not constant. The specific mRNA of envA seems to be stimulated soon after infection, whereas the two transcripts initiating upstream from ftsZ apparently decrease to a basal level. In both cases, however, synthesis of the mRNA virtually doubles at the time of cell division.

Mu gem3 as a tool to investigate the influence of chromosome supercoiling on gene expression in Escherichia coli K12.

We have developed a rapid method to investigate the influence of chromosome supercoiling on gene expression in Escherichia coli K12. This method exploits the ability of the gem3 mutant of the bacteriophage Mu, even in the prophagic state in immune cells, to induce relaxation of the host chromosome. The experiments can thus be performed under physiological conditions, and without the use of the drugs. In theory, this method can be applied to any bacterial gene. Here, we report the results obtained with four DNA replication and three cell division genes.

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.

Prokaryotic division interactome: setup of an assay for protein-protein interaction mutant selection.

A method which enables selection of protein mutants impaired in their ability to interact with their normal protein partners is presented here. The method is the two-phage two-hybrid assay adapted for mutant selection. In the two-phage assay, the interaction between two proteins enables the formation of a functional hybrid lambdoid repressor that shuts down expression of a reporter gene governed by a chimeric promoter/operator region. To adapt the assay to interaction mutant selection, antibiotic resistance was used as a reporter gene. In this case, the interaction between the two proteins resulted in antibiotic sensitivity, whereas the loss of interaction conferred resistance to the bacterial strain. Therefore, turning on reporter gene expression highlights the loss of interaction due to a mutation in one of the genes for the two protein partners, and leads to direct selection of the mutants regardless of the mutant phenotype. In this paper, application of this method to isolation of interaction mutants in proteins involved in Escherichia coli K12 cytokinesis is reported.

FtsQ interaction mutants: a way to identify new antibacterial targets.

FtsQ is a highly conserved component of the divisome that plays a central role in the assembly of early and late cell division proteins. The biological activity of this protein is still largely unknown, but its ability to interact with many components of the divisome was described by both two-hybrid assays and co-immunoprecipitation experiments. This paper describes the behaviour of ftsQ point mutants, created by random mutagenesis without regard to their phenotype, in which FtsQ is impaired in its ability to interact with its Escherichia coli division partners. Our results allow the identification of FtsQ residues involved in the interaction with other partner proteins and the determination of the biological significance of these interactions. The knowledge derived by this study could constitute not only the basis for understanding how these proteins assemble in the divisome, but also a starting point for the design of new antibacterial drugs that disrupt the bacterial division machinery.

Three functional subdomains of the Escherichia coli FtsQ protein are involved in its interaction with the other division proteins.

FtsQ, an essential protein for the Escherichia coli divisome assembly, is able to interact with various division proteins, namely FtsI, FtsL, FtsN, FtsB and FtsW. In this paper, the FtsQ domains involved in these interactions were identified by two-hybrid assays and co-immunoprecipitations. Progressive deletions of the ftsQ gene suggested that the FtsQ self-interaction and its interactions with the other proteins are localized in three periplasmic subdomains: (i) residues 50-135 constitute one of the sites involved in FtsQ, FtsI and FtsN interaction, and this site is also responsible for FtsW interaction; (ii) the FtsB interaction is localized between residues 136 and 202; and (iii) the FtsL interaction is localized at the very C-terminal extremity. In this third region, the interaction site for FtsK and also the second site for FtsQ, FtsI, FtsN interactions are located. As far as FtsW is concerned, this protein interacts with the fragment of the FtsQ periplasmic domain that spans residues 67-75. In addition, two protein subdomains, one constituted by residues 1-135 and the other from 136 to the end, are both able to complement an ftsQ null mutant. Finally, the unexpected finding that an E. coli ftsQ null mutant can be complemented, at least transiently, by the Streptococcus pneumoniae divIB/ftsQ gene product suggests a new strategy for investigating the biological significance of protein-protein interactions.

General method for the isolation of conditional lethal mutants in any required region of the virus genome: its application to the 'semi-essential' region of phage Mu.

A general method is presented that enables viral mutants to be isolated in any required region of their genome. An application of the method is reported relating to the isolation of conditional lethal mutants in the "semi-essential' region of phage Mu. Some properties are described of one mutant found in this way, Mucts62SEEam8 Apl, which is typical of a class of mutants that were isolated.

Mu DNA reintegration upon excision: evidence for a possible involvement of nucleoid folding.

Mutations induced by the integration of a Mugem2ts prophage can revert at frequencies around 1x10(-6). In these revertant clones, the prophage excised from its original localization is not lost but reintegrated elsewhere in the host genome. One of the most intriguing aspects of this process is that the prophage reintegration is not randomly distributed: there is a strong correlation between the original site of insertion (the donor site) and the target site of the phage DNA migration (the receptor site). In this paper, it is shown that in the excision-reintegration process mediated by Mugem2ts, the position of the initial prophage site strongly influences the location of the reintegration site. In addition, for each donor site, the receptor site is a discrete DNA region within which the excised Mu DNA can reintegrate and the two sites implicated in phage DNA migration must be located on the same DNA molecule. These data suggest the involvement of nucleoid folding in the excision-reintegration process.

Regulation of the bacteriophage Mu gem operon.

The gem operon of bacteriophage Mu, responsible for the complex phenomenon of phage conversion, is included in the so called "semiessential early" region of phage DNA. Unlike the other early genes of the phage which are transcribed from the pe promoter, expression of the gem operon is driven by its own promoter, which escapes the control of the repressor. In fact, the transcript corresponding to gem was detected in immune lysogens by using a combined reverse transcription and a subsequent amplification of the resulting cDNA. The transcription initiation site from pgem was determined by primer extension mapping experiments and localized at 8217 bp from the left end of phage DNA. Two elements which could perform the negative control of gem were also identified. The first is a phage product, GemB, which presumably interferes with gem expression at a posttranscriptional level, whereas the second is a structural element, an inverted repeat immediately downstream of pgem, which acts as a terminator for the transcripts starting from pe. These transcripts could regulate gem expression by interfering with the initiation of transcription from pgem.

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.

[Ligase activity of lig- bacteria infected with mu bacteriophage at non-permissive temperatures]. / Expression à température non permissive d'une activité ligasique chez des bactéries lig-infectées par le phage mu.

Infection of bacteria E. coli lig ts7, which contain a thermosensitive ligase by phage Mu results in a decrease in the sensitivity to the lethal action of ultraviolet irradiation and stimulation of lysogenization among the surviving cells. These observations suggest the existence of a phage coded ligase which has the character of an integrase.