Pierre Alexandre Kaminski

Identification of a nifA-like regulatory gene of Azospirillum brasilense Sp7 expressed under conditions of nitrogen fixation and in the presence of air and ammonia.

A gene bank of Azospirillum lipoferum Br17 constructed in the vector λGEM11 was screened with a Bradyrhizobium japonicum nifA gene probe. A 7.3 kb EcoRI fragment carrying a nifA‐like gene was thereby isolated and subsequently used to screen a gene bank of Azospirillum brasilense Sp7 constructed in pUC18. Two EcoRI fragments of 5.6 kb and 3.6 kb covering the nifA‐homology region were found. Mutants with Nif‐phenotype were obtained by site‐directed Tn5 mutagenesis of the 5.6 kb fragment and subsequent recombination into the A. brasilense Sp7 genome. The mutations were clustered into two loci located at each extremity of the fragment. One of these loci corresponded to nifA and the other to nifB. The nucleotide sequence of nifA of A. brasilense Sp7 was determined. Comparison of the deduced amino acid sequences of NifA of A. brasilense Sp7 and NifA of B. Japonicum, Rhizobium leguminosarum biovar trifolii and Klebsiella pneumoniae confirmed that it was a nifA‐like gene. Construction of a nifA‐lacZ fusion and mapping of the RNA transcriptional start site showed that the nifA‐like gene was expressed from an unidentified promoter, under conditions of nitrogen fixation and in the presence of oxygen and ammonia.

Functional cloning, heterologous expression, and purification of two different N-deoxyribosyltransferases from Lactobacillus helveticus.

Lactobacillus helveticus contains two types of N-deoxyribosyltransferases: DRTase I catalyzes the transfer of 2′-deoxyribose between purine bases exclusively whereas DRTase II is able to transfer the 2′-deoxyribose between two pyrimidine or between pyrimidine and purine bases. An Escherichia colistrain, auxotrophic for guanine and unable to use deoxyguanosine as source of guanine, was constructed to clone the corresponding genes. By screening a genomic bank for the production of guanine, the L. helveticus ptd and ntd genes coding for DRTase I and II, respectively, were isolated. Although the two genes have no sequence similarity, the two deduced polypeptides display 25.6% identity, with most of the residues involved in substrate binding and the active site nucleophile Glu-98 being conserved. Overexpression and purification of the two proteins shows that DRTase I is specific for purines with a preference for deoxyinosine (dI) > deoxyadenosine > deoxyguanosine as donor substrates whereas DRTase II has a strong preference for pyrimidines as donor substrates and purines as base acceptors. Purine analogues were substrates as acceptor bases for both enzymes. Comparison of DRTase I and DRTase II activities with dI as donor or hypoxanthine as acceptor and colocalization of the ptd and add genes suggest a specific role for DRTase I in the metabolism of dI.

Involvement of fixLJ in the regulation of nitrogen fixation in Azorhizobium caulinodans

A gene bank of Azorhizobium caulinodans DNA constructed in the bacteriophage λGEM11 was screened with Rhizobium meliloti fixL and fixJ genes as probes. One positive recombinant phage, ORSλL, was isolated. The nucleotide sequence of a 3.7 kb fragment was established. Two open reading frames of 1512 bp and 613bp were identified as fixL and fixJ. Kanamycin cartridges were inserted into the cloned fixL and fixJ genes and recombined into the host genome. The resulting mutants were Nif− Fix−, suggesting that the two genes were required for symbiotic nitrogen fixation and for nitrogen fixation in the free‐living state. Using pnifH–lacZ and pnifA–lacZ fusions, it was shown that the FixLJ products controlled the expression of nifH and nifA in bacteria grown in the free‐living state.

Regulation of nitrogen fixation in Azorhizobium caulinodans : identification of a fixK-like gene, a positive regulator of nifA

The nucleotide sequence of a 1 kb fragment upstream of Azorhizobium caulinodans fixL was established. An open reading frame of 744 bp was identified as a fixK homologue. A kanamycin cartridge was inserted into the cloned fixK‐1ike gene and recombined Into the host genome. The resulting mutant was Nif− Fix− suggesting that FixK was required for nitrogen fixation both in symbiotic conditions and in the free‐living state. Using a pfixK–lacZ fusion, the FixLJ products were shown to control the expression of fixK. Using a pnifA–lacZ fusion, the FixK product was shown to regulate positively the transcription of nifA in bacteria grown in the free‐living state. In addition, a double ntrC–fixL mutant was constructed and was shown to be completely devoid of nitrogenase activity. A model of regulation, based on these data, is presented and might explain the unusual ability of A. caulinodans to fix nitrogen both under symbiotic conditions and in the free‐living state.

Characterisation of the glnK-amtB operon and the involvement of AmtB in methylammonium uptake in Azorhizobium caulinodans

Abstract This work reports the characterisation of the Azorhizobium caulinodans amtB gene, the deduced protein sequence of which shares similarity to those of several ammonium transporters. amtB is located downstream from glnK, a glnB-like gene. It is cotranscribed with glnK from an NtrC- and σ54-dependent promoter. glnK and amtB insertion mutant strains have been isolated. Methylammonium uptake was assayed in these strains and in other mutant strains in which the regulation of nitrogen metabolism is impaired. Our data suggest that the AmtB protein is an ammonium transporter, which is mainly regulated by NtrC in response to nitrogen availability.

Extracellular nucleotide catabolism by the group B Streptococcus ectonucleotidase NudP increases bacterial survival in blood

Background: Ectonucleotidases regulate extracellular nucleotide concentration. Results: The NudP ecto-5′-nucleotidase of Streptococcus agalactiae has specific substrate specificities necessary for survival in blood and organ colonization. Conclusion: Extracellular nucleotide catabolism is involved in the control of Group B streptococcal pathogenesis. Significance: Bacterial pathogens exploit different enzymatic specificities to subvert extracellular nucleotide signaling. Streptococcus agalactiae (Group B Streptococcus) is a commensal of the human intestine and vagina of adult women but is the leading cause of invasive infection in neonates. This Gram-positive bacterium displays a set of virulence-associated surface proteins involved in the interaction with the host, such as adhesion to host cells, invasion of tissues, or subversion of the immune system. In this study, we characterized a cell wall-localized protein as an ecto-5′-nucleoside diphosphate phosphohydrolase (NudP) involved in the degradation of extracellular nucleotides which are central mediators of the immune response. Biochemical characterization of recombinant NudP revealed a Mn2+-dependent ecto-5′-nucleotidase activity on ribo- and deoxyribonucleoside 5′-mono- and 5′-diphosphates with a substrate specificity different from that of known orthologous enzymes. Deletion of the gene coding the housekeeping enzyme sortase A led to the release of NudP into the culture supernatant, confirming that this enzyme is anchored to the cell wall by its non-canonical LPXTN motif. The NudP ecto-5′-nucleotidase activity is reminiscent of the reactions performed by the mammalian ectonucleotidases CD39 and CD73 involved in regulating the extracellular level of ATP and adenosine. We further demonstrated that the absence of NudP activity decreases bacterial survival in mouse blood, a process dependent on extracellular adenosine. In vivo assays in animal models of infection showed that NudP activity is critical for virulence. These results demonstrate that Group B Streptococcus expresses a specific ecto-5′-nucleotidase necessary for its pathogenicity and highlight the diversity of reactions performed by this enzyme family. These results suggest that bacterial pathogens have developed specialized strategies to subvert the mammalian immune response controlled by the extracellular nucleotide signaling pathways.

In Vivo Reshaping the Catalytic Site of Nucleoside 2′-Deoxyribosyltransferase for Dideoxy- and Didehydronucleosides via a Single Amino Acid Substitution

Nucleoside 2′-deoxyribosyltransferases catalyze the transfer of 2-deoxyribose between bases and have been widely used as biocatalysts to synthesize a variety of nucleoside analogs. The genes encoding nucleoside 2′-deoxyribosyltransferase (ndt) from Lactobacillus leichmannii and Lactobacillus fermentum underwent random mutagenesis to select variants specialized for the synthesis of 2′,3′-dideoxynucleosides. An Escherichia coli strain, auxotrophic for uracil and unable to use 2′,3′-dideoxyuridine, cytosine, and 2′,3′-dideoxycytidine as a source of uracil was constructed. Randomly mutated lactobacilli ndt libraries from two species, L. leichmannii and L. fermentum, were screened for the production of uracil with 2′,3′-dideoxyuridine as a source of uracil. Several mutants suitable for the synthesis of 2′,3′-dideoxynucleosides were isolated. The nucleotide sequence of the corresponding genes revealed a single mutation (G → A transition) leading to the substitution of a small aliphatic amino acid by a nucleophilic one, A15T (L. fermentum) or G9S (L. leichmannii), respectively. We concluded that the “adaptation” of the nucleoside 2′-deoxyribosyltransferase activity to 2,3-dideoxyribosyl transfer requires an additional hydroxyl group on a key amino acid side chain of the protein to overcome the absence of such a group in the corresponding substrate. The evolved proteins also display significantly improved nucleoside 2′,3′-didehydro-2′,3′-dideoxyribosyltransferase activity.

Probing the Active Site of the Deoxynucleotide N-Hydrolase Rcl Encoded by the Rat Gene c6orf108

Rcl is a potential anti-angiogenic therapeutic target that hydrolyzes the N-glycosidic bond of 2′-deoxyribonucleoside 5′-monophosphate, yielding 2-deoxyribose 5-phosphate and the corresponding base. Its recently elucidated solution structure provided the first insight into the molecular basis for the substrate recognition. To facilitate the development of potent and specific inhibitors of Rcl, the active site was probed by site-directed mutagenesis and by the use of substrate analogs. The nucleobase shows weak interactions with the protein, and the deoxyribose binding pocket includes the catalytic triad Tyr-13, Asp-69, and Glu-93 and the phosphate binding site Ser-87 and Ser-117. The phosphomimetic mutation of Ser-17 to Glu prevents substrate binding and, thus, abolishes the activity of Rcl. The synthetic ligand-based analysis of the Rcl binding site shows that substitutions at positions 2 and 6 of the nucleobase as well as large heterocycles are well tolerated. The phosphate group at position 5 of the (deoxy)ribose moiety is the critical binding determinant. This study provides the roadmap for the design of small molecules inhibitors with pharmacological properties.

Phosphodeoxyribosyltransferases, Designed Enzymes for Deoxyribonucleotides Synthesis

Background: The nucleoside deoxyribosyltransferase family contains hydrolases and transferases with different substrate specificities. Results: Chimeras exchange deoxyribose 5-(mono, di, and tri)-phosphate between natural bases and analogues. Conclusion: Comparison of the structures and catalytic mechanisms of members of the nucleoside deoxyribosyltransferase family allows the design of unprecedented enzymes. Significance: Phosphodeoxyribosyltransferases open the road to new deoxyribonucleotides synthetic pathways. A large number of nucleoside analogues and 2′-deoxynucleoside triphosphates (dNTP) have been synthesized to interfere with DNA metabolism. However, in vivo the concentration and phosphorylation of these analogues are key limiting factors. In this context, we designed enzymes to switch nucleobases attached to a deoxyribose monophosphate. Active chimeras were made from two distantly related enzymes: a nucleoside deoxyribosyltransferase from lactobacilli and a 5′-monophosphate-2′-deoxyribonucleoside hydrolase from rat. Then their unprecedented activity was further extended to deoxyribose triphosphate, and in vitro biosyntheses could be successfully performed with several base analogues. These new enzymes provide new tools to synthesize dNTP analogues and to deliver them into cells.

PbsP, a cell wall‐anchored protein that binds plasminogen to promote hematogenous dissemination of group B Streptococcus

Streptococcus agalactiae (Group B Streptococcus or GBS) is a leading cause of invasive infections in neonates whose virulence is dependent on its ability to interact with cells and host components. We here characterized a surface protein with a critical function in GBS pathophysiology. This adhesin, designated PbsP, possesses two Streptococcal Surface Repeat domains, a methionine and lysine‐rich region, and a LPXTG cell wall‐anchoring motif. PbsP mediates plasminogen (Plg) binding both in vitro and in vivo and we showed that cell surface‐bound Plg can be activated into plasmin by tissue plasminogen activator to increase the bacterial extracellular proteolytic activity. Absence of PbsP results in a decreased bacterial transmigration across brain endothelial cells and impaired virulence in a murine model of infection. PbsP is conserved among the main GBS lineages and is a major plasminogen adhesin in non‐CC17 GBS strains. Importantly, immunization of mice with recombinant PbsP confers protective immunity. Our results indicate that GBS have evolved different strategies to recruit Plg which indicates that the ability to acquire cell surface proteolytic activity is essential for the invasiveness of this bacterium.