Frédéric Boccard

Macrodomain organization of the Escherichia coli chromosome

We have explored the Escherichia coli chromosome architecture by genetic dissection, using a site‐specific recombination system that reveals the spatial proximity of distant DNA sites and records interactions. By analysing the percentages of recombination between pairs of sites scattered over the chromosome, we observed that DNA interactions were restricted to within subregions of the chromosome. The results indicated an organization into a ring composed of four macrodomains and two less‐structured regions. Two of the macrodomains defined by recombination efficiency are similar to the Ter and Ori macrodomains observed by FISH. Two newly characterized macrodomains flank the Ter macrodomain and two less‐structured regions flank the Ori macrodomain. Also the interactions between sister chromatids are rare, suggesting that chromosome segregation quickly follows replication. These results reveal structural features that may be important for chromosome dynamics during the cell cycle.

Bacterial strategies to overcome insect defences

Recent genetic and molecular analyses have revealed how several strategies enable bacteria to persist and overcome insect immune defences. Genetic and genomic tools that can be used with Drosophila melanogaster have enabled the characterization of the pathways that are used by insects to detect bacterial invaders and combat infection. Conservation of bacterial virulence factors and insect immune repertoires indicates that there are common strategies of host invasion and pathogen eradication. Long-term interactions of bacteria with insects might ensure efficient dissemination of pathogens to other hosts, including humans.

Prevalence of local immune response against oral infection in a Drosophila/Pseudomonas infection model

Pathogens have developed multiple strategies that allow them to exploit host resources and resist the immune response. To study how Drosophila flies deal with infectious diseases in a natural context, we investigated the interactions between Drosophila and a newly identified entomopathogen, Pseudomonas entomophila. Flies orally infected with P. entomophila rapidly succumb despite the induction of both local and systemic immune responses, indicating that this bacterium has developed specific strategies to escape the fly immune response. Using a combined genetic approach on both host and pathogen, we showed that P. entomophila virulence is multi-factorial with a clear differentiation between factors that trigger the immune response and those that promote pathogenicity. We demonstrate that AprA, an abundant secreted metalloprotease produced by P. entomophila, is an important virulence factor. Inactivation of aprA attenuated both the capacity to persist in the host and pathogenicity. Interestingly, aprA mutants were able to survive to wild-type levels in immune-deficient Relish flies, indicating that the protease plays an important role in protection against the Drosophila immune response. Our study also reveals that the major contribution to the fly defense against P. entomophila is provided by the local, rather than the systemic immune response. More precisely, our data points to an important role for the antimicrobial peptide Diptericin against orally infectious Gram-negative bacteria, emphasizing the critical role of local antimicrobial peptide expression against food-borne pathogens.

Complete genome sequence of the entomopathogenic and metabolically versatile soil bacterium Pseudomonas entomophila

Pseudomonas entomophila is an entomopathogenic bacterium that, upon ingestion, kills Drosophila melanogaster as well as insects from different orders. The complete sequence of the 5.9-Mb genome was determined and compared to the sequenced genomes of four Pseudomonas species. P. entomophila possesses most of the catabolic genes of the closely related strain P. putida KT2440, revealing its metabolically versatile properties and its soil lifestyle. Several features that probably contribute to its entomopathogenic properties were disclosed. Unexpectedly for an animal pathogen, P. entomophila is devoid of a type III secretion system and associated toxins but rather relies on a number of potential virulence factors such as insecticidal toxins, proteases, putative hemolysins, hydrogen cyanide and novel secondary metabolites to infect and kill insects. Genome-wide random mutagenesis revealed the major role of the two-component system GacS/GacA that regulates most of the potential virulence factors identified.

DNA dynamics vary according to macrodomain topography in the E. coli chromosome.

The organization of the Escherichia coli chromosome has been defined genetically as consisting of four insulated macrodomains and two less constrained regions. Here we have examined the movement of chromosomal loci by tracking fluorescent markers in time‐lapse microscopy during a complete cell cycle. Analysing the positioning, the segregation pattern and the motility of markers allowed us to show that the dynamic behaviour of loci belonging to various macrodomains and less constrained regions is radically different. In macrodomains constraints on mobility are apparent whereas in non‐structured regions, markers exhibited a greater motility that may explain their ability to interact with flanking macrodomains. Following replication, duplicated markers belonging to macrodomains show a colocalization step and this landmark is not apparent in non‐structured regions. Chromosome segregation occurs in three steps: first, the origin‐proximal half of the chromosome consisting of the Ori macrodomain and the two non‐structured region segregates concomitantly in a short period of time. Second, the Right and Left macrodomains segregate progressively following the genetic map. Third, the Ter macrodomain is rapidly segregated before division, after a significant period of colocalization. Macrodomain territories defined as cellular spaces occupied by the different macrodomains can be identified.

The MatP/matS Site-Specific System Organizes the Terminus Region of the E. coli Chromosome into a Macrodomain

The organization of the Escherichia coli chromosome into insulated macrodomains influences the segregation of sister chromatids and the mobility of chromosomal DNA. Here, we report that organization of the Terminus region (Ter) into a macrodomain relies on the presence of a 13 bp motif called matS repeated 23 times in the 800-kb-long domain. matS sites are the main targets in the E. coli chromosome of a newly identified protein designated MatP. MatP accumulates in the cell as a discrete focus that colocalizes with the Ter macrodomain. The effects of MatP inactivation reveal its role as main organizer of the Ter macrodomain: in the absence of MatP, DNA is less compacted, the mobility of markers is increased, and segregation of Ter macrodomain occurs early in the cell cycle. Our results indicate that a specific organizational system is required in the Terminus region for bacterial chromosome management during the cell cycle.

CONSTRUCTION OF A SERIES OF PSAM2-BASED INTEGRATIVE VECTORS FOR USE IN ACTINOMYCETES

We have developed vectors which allowed integration of cloned DNA at a single site in the chromosome of Streptomyces lividans 66. These vectors made use of (1) an Escherichia coli replicon, (2) a thiostrepton (Th)- and a streptomycin/spectinomycin-resistance gene for selection in Streptomyces, (3) a 3.5-kb fragment of the Streptomyces integrative plasmid pSAM2 containing its xis and int genes as well as its attachment site, attP, to direct the integration of the vectors at the chromosomal pSAM2 attachment site attB, (4) the origin of transfer of the IncP broad-host-range plasmid RK2 which allowed the mobilization of the vectors from E. coli to S. lividans, and (5) the Th-inducible tipA promoter to permit regulated transcription of cloned genes. We demonstrated that pPM927, a plasmid which contained all of these elements, was able to transfer cloned fragments from E. coli to S. lividans by conjugation, stably integrate into the chromosome, and express cloned genes from the tipA promoter. Furthermore, since pPM927 contained the pBR322 replicon, cloned fragments could be conveniently recovered from the S. lividans chromosome for analysis in E. coli by cleavage of genomic DNA isolated from transformed strains, intramolecular ligation and transformation. Since we have shown that the pSAM2 attB site forms part of a conserved prokaryotic tRNA gene, these integrative vectors are potentially useful tools for analysis and expression of genes in diverse bacteria.

Organization and nucleotide sequence analysis of a ribosomal RNA gene cluster from Streptomyces ambofaciens

The Streptomyces ambofaciens genome contains four rRNA gene clusters. These copies are called rrnA, B, C and D. The complete nucleotide (nt) sequence of rrnD has been determined. These genes possess striking similarity with other eubacterial rRNA genes. Comparison with other rRNA sequences allowed the putative localization of the sequences encoding mature rRNAs. The structural genes are arranged in the order 16S-23S-5S and are tightly linked. The mature rRNAs are predicted to contain 1528, 3120 and 120 nt, for the 16S, 23S and 5S rRNAs, respectively. The 23S rRNA is, to our knowledge, the longest of all sequenced prokaryotic 23S rRNAs. When compared to other large rRNAs it shows insertions at positions where they are also present in archaebacterial and in eukaryotic large rRNAs. Secondary structure models of S. ambofaciens rRNAs are proposed, based upon those existing for other bacterial rRNAs. Positions of putative transcription start points and of a termination signal are suggested. The corresponding putative primary transcript, containing the 16S, 23S and 5S rRNAs plus flanking regions, was folded into a secondary structure, and sequences possibly involved in rRNA maturation are described. The G + C content of the rRNA gene cluster is low (57%) compared with the overall G + C content of Streptomyces DNA (73%).

A MatP–divisome interaction coordinates chromosome segregation with cell division in E. coli

Initiation of chromosome segregation in bacteria is achieved by proteins acting near the origin of replication. Here, we report that the precise choreography of the terminus region of the Escherichia coli chromosome is also tightly controlled. The segregation of the terminus (Ter) macrodomain (MD) involves the structuring factor MatP. We characterized that migration of the Ter MD from the new pole to mid‐cell and its subsequent persistent localization at mid‐cell relies on several processes. First, the replication of the Ter DNA is concomitant with its recruitment from the new pole to mid‐cell in a sequential order correlated with the position on the genetic map. Second, using a strain carrying a linear chromosome with the Ter MD split in two parts, we show that replisomes are repositioned at mid‐cell when replication of the Ter occurs. Third, we demonstrate that anchoring the Ter MD at mid‐cell depends on the specific interaction of MatP with the division apparatus‐associated protein ZapB. Our results reveal how segregation of the Ter MD is integrated in the cell‐cycle control.

The integrated conjugative plasmid pSAM2 of Streptomyces ambofaciens is related to temperate bacteriophages.

Streptomyces ambofaciens ATCC23877 and derivatives contain the 11‐kb element pSAM2 present in an integrated state or as a free and integrated plasmid. This element, able to integrate site‐specifically in the genome of different Streptomyces species, is conjugative and mobilizes chromosomal markers. Besides these plasmid functions, we have shown that the site‐specific recombination system of pSAM2 presents strong similarities with that of several temperate phages. The integration event is promoted by a site‐specific recombinase of the integrase family. The int gene encoding this integrase is closely linked to the plasmid attachment site (attP). A small open reading frame (ORF) overlaps the int gene and the predicted protein exhibits similarities with Xis proteins involved in phages excision. The integrated copy of pSAM2 in strain ATCC23877 is flanked by att sequences (attL and attR). Another att sequence (attX) is present in this strain and attX and attL are the boundaries of a 42‐kb fragment (xSAM1) absent, as well as pSAM2, from S.ambofaciens DSM40697. Sequences partially similar to pSAM2 int gene are found near the chromosomal integration zone in both S.ambofaciens strains. The possible origin of pSAM2, an element carrying plasmid as well as phage features, is discussed.