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.

Characterisation of the MutS and MutL Proteins from the Pseudomonas avellanae Mismatch Repair (MMR) System.

The identification and analysis of the Pseudomonas avellanae mismatch repair system (MMR) were performed via sequencing and cloning the mutS and mutL genes and then analyzing the characteristics of the corresponding proteins studying their function and biological role in an E. coli heterologous system. In these studies, the P.

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.

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.

Division protein interaction web: identification of a phylogenetically conserved common interactome between Streptococcus pneumoniae and Escherichia coli.

The ability of each of the 11 Streptococcus pneumoniae division proteins to interact with itself and with each of the remaining proteins was studied in 66 combinations of protein pairs, using a bacterial two-hybrid system. Interactions (homo- or hetero-dimerizations) were detected between 37 protein pairs, whereas 29 protein pairs did not interact. In some cases, positive interactions of the S.

The Escherichia coli FtsK functional domains involved in its interaction with its divisome protein partners.

FtsK is a multifunctional protein involved in both cell division and chromosome segregation. As far as its role in cell division is concerned, FtsK is among the first divisome proteins that localizes at mid-cell, after FtsZ, FtsA and ZipA, and is required for the recruitment of the other divisome components. The ability of FtsK to interact with several cell division proteins, namely FtsZ, FtsQ, FtsL and FtsI, by the two-hybrid assay was already shown by our group.

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.

Streptococcus pneumoniae DivIVA: localization and interactions in a MinCD-free context.

To clarify the function of DivIVA in Streptococcus pneumoniae, we localized this protein in exponentially growing cells by both immunofluorescence microscopy and immunoelectron microscopy and found that S. pneumoniae DivIVA (DivIVA(SPN)) had a unique localization profile: it was present simultaneously both as a ring at the division septum and as dots at the cell poles. Double-immunofluorescence analysis suggested that DivIVA is recruited to the septum at a later stage than FtsZ and is retained at the poles after cell separation.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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)).

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.

H-NS regulation of virulence gene expression in enteroinvasive Escherichia coli harboring the virulence plasmid integrated into the host chromosome.

We have previously shown that integration of the virulence plasmid pINV into the chromosome of enteroinvasive Escherichia coli and of Shigella flexneri makes these strains noninvasive (C. Zagaglia, M. Casalino, B.

A novel illegitimate recombination event: precise excision and reintegration with the Mu gem mutant prophage.

The bacteriophage Mu is known to insert its DNA more or less randomly within the Escherichia coli chromosome, as do transposable elements, but unlike the latter, precise excision of the prophage, thereby restoring the original sequence, is not observed with wild-type Mu, although it has been reported with certain defective mutants. We show here that the mutant prophage Mu gem2ts can excise precisely from at least three separate loci -- malT, lac and thyA (selected as Mal+, Lac+ and Thy+, respectively). This excision occurs under permissive conditions for phage development, is observed in fully immune (c+) lysogens, and is independent of RecA and of Mu transposase.

The Mu gem operon: its role in gene expression, recombination and cell cycle.

Two genes, gemA and gemB, belong to the gem operon located in the semi-essential early region of bacteriophage Mu. The product of gemA modulates the expression of various host genes, including cell division and DNA replication genes. In addition, GemA is also responsible for decreasing host DNA gyrase activity and for DNA relaxation.