Genome-wide identification of genes required for alternative peptidoglycan cross-linking in Escherichia coli revealed unexpected impacts of β-lactams.

The D,D-transpeptidase activity of penicillin-binding proteins (PBPs) is the well-known primary target of β-lactam antibiotics that block peptidoglycan polymerization. β-lactam-induced bacterial killing involves complex downstream responses whose causes and consequences are difficult to resolve. Here, we use the functional replacement of PBPs by a β-lactam-insensitive L,D-transpeptidase to identify genes essential to mitigate the effects of PBP inactivation by β-lactams in actively dividing bacteria.

Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei.

Casposons are a group of bacterial and archaeal DNA transposons encoding a specific integrase, termed casposase, which is homologous to the Cas1 enzyme responsible for the integration of new spacers into CRISPR loci. Here, we characterized the sequence motifs recognized by the casposase from a thermophilic archaeon Aciduliprofundum boonei. We identified a stretch of residues, located in the leader region upstream of the actual integration site, whose deletion or mutagenesis impaired the concerted integration reaction.

Novel Families of Archaeo-Eukaryotic Primases Associated with Mobile Genetic Elements of Bacteria and Archaea.

Cellular organisms in different domains of life employ structurally unrelated, non-homologous DNA primases for synthesis of a primer for DNA replication. Archaea and eukaryotes encode enzymes of the archaeo-eukaryotic primase (AEP) superfamily, whereas bacteria uniformly use primases of the DnaG family. However, AEP genes are widespread in bacterial genomes raising questions regarding their provenance and function.

topIb, a phylogenetic hallmark gene of Thaumarchaeota encodes a functional eukaryote-like topoisomerase IB.

Type IB DNA topoisomerases can eliminate torsional stresses produced during replication and transcription. These enzymes are found in all eukaryotes and a short version is present in some bacteria and viruses. Among prokaryotes, the long eukaryotic version is only observed in archaea of the phylum Thaumarchaeota.

A highly divergent archaeo-eukaryotic primase from the Thermococcus nautilus plasmid, pTN2.

We report the characterization of a DNA primase/polymerase protein (PolpTN2) encoded by the pTN2 plasmid from Thermococcus nautilus. Sequence analysis revealed that this protein corresponds to a fusion between an N-terminal domain homologous to the small catalytic subunit PriS of heterodimeric archaeal and eukaryotic primases (AEP) and a C-terminal domain related to their large regulatory subunit PriL. This unique domain configuration is not found in other virus- and plasmid-encoded primases in which PriS-like domains are typically fused to different types of helicases.

[Enigmatic archaeal viruses].

Viruses infecting microorganisms of the third domain of life, Archaea, are still poorly characterized: to date, only about fifty of these viruses have been isolated. Their hosts are hyperthermophilic, acidothermophilic, and extreme halophilic or methanogenic archaea. Their morphotypes are highly diverse and their gene content is very specific.

Massive activation of archaeal defense genes during viral infection.

Archaeal viruses display unusually high genetic and morphological diversity. Studies of these viruses proved to be instrumental for the expansion of knowledge on viral diversity and evolution. The Sulfolobus islandicus rod-shaped virus 2 (SIRV2) is a model to study virus-host interactions in Archaea.

Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon.

The 2 465 177 bp genome of Sulfolobus islandicus LAL14/1, host of the model rudivirus SIRV2, was sequenced. Exhaustive comparative genomic analysis of S. islandicus LAL14/1 and the nine other completely sequenced S.

Cis-regulatory logic in archaeal transcription.

For cellular fitness and survival, gene expression levels need to be regulated in response to a wealth of cellular and environmental signals. TFs (transcription factors) execute a large part of this regulation by interacting with the basal transcription machinery at promoter regions. Archaea are characterized by a simplified eukaryote-like basal transcription machinery and bacteria-type TFs, which convert sequence information into a gene expression output according to cis-regulatory rules.

Solution structure of an archaeal DNA binding protein with an eukaryotic zinc finger fold.

While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1.

Structure and function of AvtR, a novel transcriptional regulator from a hyperthermophilic archaeal lipothrixvirus.

The structural and functional analysis of the protein AvtR encoded by Acidianus filamentous virus 6 (AFV6), which infects the archaeal genus Acidianus, revealed its unusual structure and involvement in transcriptional regulation of several viral genes. The crystal structure of AvtR (100 amino acids) at 2.6-Å resolution shows that it is constituted of a repeated ribbon-helix-helix (RHH) motif, which is found in a large family of bacterial transcriptional regulators.

Evidence for a Xer/dif system for chromosome resolution in archaea.

Homologous recombination events between circular chromosomes, occurring during or after replication, can generate dimers that need to be converted to monomers prior to their segregation at cell division. In Escherichia coli, chromosome dimers are converted to monomers by two paralogous site-specific tyrosine recombinases of the Xer family (XerC/D). The Xer recombinases act at a specific dif site located in the replication termination region, assisted by the cell division protein FtsK.

Two novel families of plasmids from hyperthermophilic archaea encoding new families of replication proteins.

Thermococcales (phylum Euryarchaeota) are model organisms for physiological and molecular studies of hyperthermophiles. Here we describe three new plasmids from Thermococcales that could provide new tools and model systems for genetic and molecular studies in Archaea. The plasmids pTN2 from Thermococcus nautilus sp.

Structure, function, and targets of the transcriptional regulator SvtR from the hyperthermophilic archaeal virus SIRV1.

We have characterized the structure and the function of the 6.6-kDa protein SvtR (formerly called gp08) from the rod-shaped virus SIRV1, which infects the hyperthermophilic archaeon Sulfolobus islandicus that thrives at 85 degrees C in hot acidic springs. The protein forms a dimer in solution.

Escherichia coli physiology in Luria-Bertani broth.

Luria-Bertani broth supports Escherichia coli growth to an optical density at 600 nm (OD(600)) of 7. Surprisingly, however, steady-state growth ceases at an OD(600) of 0.3, when the growth rate slows down and cell mass decreases.

A novel archaeal regulatory protein, Sta1, activates transcription from viral promoters.

While studying gene expression of the rudivirus SIRV1 in cells of its host, the hyperthermophilic crenarchaeon Sulfolobus, a novel archaeal transcriptional regulator was isolated. The 14 kDa protein, termed Sulfolobus transcription activator 1, Sta1, is encoded on the host chromosome. Its activating effect on transcription initiation from viral promoters was demonstrated in in vitro transcription experiments using a reconstituted host system containing the RNA polymerase, TATA-binding protein (TBP) and transcription factor B (TFB).

Conjugal immunity of Streptomyces strains carrying the integrative element pSAM2 is due to the pif gene (pSAM2 immunity factor).

Mechanisms of conjugal immunity preventing redundant exchange between two cells harbouring the same conjugative element have been reported in diverse bacteria. Such a system does exist for pSAM2, a conjugative and integrative element of Streptomyces. The apparition of the conjugative free form of pSAM2 in the donor strain during mating can be considered as the initial step of transfer.

Precise excision of bacteriophage Mu DNA.

The temperate bacteriophage Mu is a transposable element that can integrate randomly into bacterial DNA, thereby creating mutations. Mutants due to an integrated Mu prophage do not give rise to revertants, as if Mu, unlike other transposable elements, were unable to excise precisely. In the present work, starting with a lacZ::Muc62(Ts) strain unable to form Lac+ colonies, we cloned a lacZ+ gene in vivo on a mini-Mu plasmid, under conditions of prophage induction.

The GemA protein of phage Mu and the GyrB gyrase subunit of Escherichia coli: the search for targets and interactions leading to the reversion of Mu-induced mutations.

The mutant bacteriophage Mugem2(Ts), known to synchronize the division of infected cells, to relax DNA supercoiling and, as prophage, to give rise to precisely excised revertants, has been thought to overexpress the gemA-mor operon, and genetic evidence suggests that the B subunit of DNA gyrase (GyrB) is the target of action of GemA. In two different double hybrid tests presented here, we find no evidence of GemA-GyrB protein-protein interaction. We do observe a GemA-GemA interaction, however, indicating that GemA can dimerize.

KorSA from the Streptomyces integrative element pSAM2 is a central transcriptional repressor: target genes and binding sites.

pSAM2, a 10.9-kb mobile integrative genetic element from Streptomyces ambofaciens, possesses, as do a majority of Streptomyces conjugative plasmids, a kil-kor system associated with its transfer. The kor function of pSAM2 was attributed to the korSA gene, but its direct role remained unclear.

Replicase, excisionase, and integrase genes of the Streptomyces element pSAM2 constitute an operon positively regulated by the pra gene.

pSAM2 is a site-specific integrative element from Streptomyces ambofaciens. The pra gene described earlier as an activator of pSAM2 replication is shown here to be also involved in the activation of its integration and excision. This was evidenced with derivatives of pSAM2 mutant B3 in which the pra gene was placed under the control of the inducible tipAp promoter.

Complete conversion of antibiotic precursor to pristinamycin IIA by overexpression of Streptomyces pristinaespiralis biosynthetic genes.

A Streptomyces pristinaespiralis strain, which produces a streptogramin antibiotic pristinamycin II (PII) as a mixture of two biologically active molecules PIIB and PIIA, was genetically engineered to produce exclusively PIIA. The snaA,B genes, which encode a PIIA synthase that performs oxidation of the precursor (PIIB) to the final product (PIIA), were integrated in the chromosome of S. pristinaespiralis using an integrative derivative of the pSAM2 genetic element from Streptomyces ambofaciens.

Characterization of pra, a gene for replication control in pSAM2, the integrating element of Streptomyces ambofaciens.

pSAM2 is a genetic element found integrated in Streptomyces ambofaciens (B2) and additionally in a replicating form in two mutants B3 and B4. The presence of the pSAM2 replicating form in these mutants was the result of mutations located on pSAM2 in the pra locus, named pra3 and pra4, respectively. The pra gene is not directly involved in replication, but its inactivation led to the disappearance of the pSAM2 free form; therefore, it was considered as a replication regulator.

Transfer functions of the conjugative integrating element pSAM2 from Streptomyces ambofaciens: characterization of a kil-kor system associated with transfer.

pSAM2 is an 11-kb integrating element from Streptomyces ambofaciens. During matings, pSAM2 can be transferred at high frequency, forming pocks, which are zones of growth inhibition of the recipient strain. The nucleotide sequences of the regions involved in pSAM2 transfer, pock formation, and maintenance have been determined.