Etude du rôle de la région terminale du chromosome dans le positionnement, la ségrégation du chromosome et le contrôle de la division cellulaire chez Escherichia coli

Escherichia coli, as the majority of bacteria, has a unique circular chromosome. Faithfull cell proliferation requires that a least one copy of the chromosome is transmitted to sister cells prior to cell division. A strict temporal and spatial coupling of chromosome segregation with cell division is thus required to ensure the accurate separation of the two fully replicated chromosomes. The terminal region of the chromosome (ter) is the last one to be replicated and segregated, and moves from the pole to the middle of the cell where the division septum is formed, at the end of the cell cycle. Loci of the ter region display an extended cohesion period. This extended cohesion is controlled by the MatP protein, which binds specific matS sites restricted to the ter region. MatP binds DNA as a dimer and forms tetramers via its N-terminal and C-terminal domains respectively. Tetramerisation is stimulated by binding to DNA and pairs remote matS sites. MatP also interacts with ZapB, a component of the divisome, the protein machinery that contributes to septum formation. While tetramerisation of MatP appears important for compacting the ter region, its interaction with ZapB, which is localized at the septum via ZapA and FtsZ, is involved in the positioning and the extended cohesion of this region. The linkage of the ter region with the divisome is required for the success of many later events of the cell cycle : (i) the active, ordered and progressive segregation of the ter region by FtsK, a component of the divisome, (ii) resolution of chromosome dimers via the site-specifique recombination XerCD/dif, activated by FtsK, (iii) the resolution of intercatenation links by TopoIV and (iv) the positive regulation of divisome assembly in the absence of the negative regulators MinCDE and SlmA. During my thesis, I first studied the role of MatP in the chromosome management. By using pairs of loci tagged with parSp1 and parSpMT1 sites recognized by cognate ParB-XFP proteins, we directly analysed chromosome positioning and orientation in the cell. We show that MatP is required for normal positioning and orientation of the whole chromosome at the end of the cell cycle. The localisation of SlmA in wt and Delta matP strains proves that inactivation of MatP leads to inaccuracy of nucleoid positioning accompanied by defects in SlmA localisation, and thus induces division inhibition. Take together, these results show that MatP, SlmA and their interplay are important for chromosome management and control of cell division in E. coli. In collaboration with O. Espeli's team, we have used genomic and molecular biology methods to characterize TopoIV regulation during the E. coli cell cycle. We show that at the dif site, TopoIV binging and cleavage are enhanced by the presence of the XerCD recombinases and MatP. This enhancement of TopoIV activity at dif promotes decatenation of fully replicated chromosomes and ensure, through interaction with other processes, accurate separation of sister chromosomes. These results provide insight into the protein network dedicated to the final step of chromosome management during the cell cycle, and how the chromosome management is linked to cell division.Escherichia coli, comme la majorité des bactéries, possède un unique chromosome circulaire. Au moins une copie du chromosome doit être transmise à chacune des cellules filles avant la division cellulaire afin d'assurer une prolifération cellulaire correcte. Une couplage spatio-temporel précis de la ségrégation avec la division cellulaire est donc nécessaire pour assurer la bonne répartition des deux chromosomes après réplication. La région terminale du chromosome (ter) est la dernière à être répliquée et ségrégée, et migre du pôle vers le centre de la cellule au moment de la mise en place du septum de division, à la fin du cycle cellulaire. Les loci de la région ter présentent une période de cohésion post-réplicative étendue. Cette cohésion étendue est contrôlée par la protéine MatP, qui se fixe spécifiquement au niveau des sites matS, présents uniquement dans ter. MatP se fixe à l'ADN sous forme de dimère, via son domaine N-terminal, et tétramérise via son domaine C-terminal. La tétramérisation est stimulée par la liaison à l'ADN et permet le pontage de deux sites matS distants. MatP interagit aussi avec ZapB, un composant du divisome, la machinerie protéique participant à la formation du septum. Alors que la tétramérisation de MatP semble importante pour la compaction de la région ter, son interaction avec ZapB, qui est localisée au septum via ZapA et FtsZ, participe au positionnement et à la cohésion étendue de cette région. Le couplage de la région ter avec le divisome est essentiel pour le bon déroulement de nombreux évènements tardifs du cycle cellulaire : (i) la ségrégation active, ordonnée et progressive de la région ter par FtsK, un composant du divisome, (ii) la résolution des dimères de chromosomes via la recombinaison spécifique de site XerCD/dif, activée par FtsK, (iii) la résolution des liens d'intercaténation par la TopoIV et (iv) la régulation positive de l'assemblage du divisome en absence des régulateurs négatifs MinCDE et SlmA. Pendant ma thèse, je me suis tout d'abord intéressée au rôle de MatP dans la structuration globale du chromosome. En utilisant un système permettant de visualiser deux loci marqués avec un site parSp1 et un site parSpMT1, reconnu par ParBp1 et ParBpMT1 spécifiquement, nous avons analysé le positionnement et l'orientation du chromosome dans la cellule. Nous avons montré que MatP est nécessaire au positionnement et à l'orientation de tout le chromosome à la fin du cycle cellulaire. La localisation de SlmA dans des souches wt et DeltamatP prouve que l'inactivation de MatP, induisant une mauvais positionnement du chromosome, s'accompagne d'une défaut de localisation de SlmA, et induit donc une inhibition de la division. Ces résultats pris ensemble montre que MatP, SlmA et leur communication à travers la structuration globale du chromosome sont importants pour le management du chromosome et le contrôle de la division cellulaire. En collaboration avec l'équipe d'Olivier Espeli, nous avons utilisé des méthodes de génomiques et de biologie moléculaire pour caractériser la régulation de la TopoIV au cours du cycle cellulaire d'E. coli. Nous avons montré qu'au site dif, les activités de fixation et de clivage de la TopoIV sont améliorées par la présence des recombinases XerCD et de MatP. L'amélioration de l'activité de la TopoIV favorise la décaténation des chromosomes nouvellement répliqués et assure, en lien avec d'autres processus, la séparation précise des chromosomes frères. Ces résultats permettent de mieux comprendre le réseau d'interactions dédiées au management du chromosome à la fin du cycle cellulaire, et l'influence du management du chromosome sur le contrôle de la division cellulaire

Study of the role of the ter region in chromosome positioning, chromosome segregation and control of cell division in Escherichia coli

Escherichia coli, comme la majorité des bactéries, possède un unique chromosome circulaire. Au moins une copie du chromosome doit être transmise à chacune des cellules filles avant la division cellulaire afin d'assurer une prolifération cellulaire correcte. Une couplage spatio-temporel précis de la ségrégation avec la division cellulaire est donc nécessaire pour assurer la bonne répartition des deux chromosomes après réplication. La région terminale du chromosome (ter) est la dernière à être répliquée et ségrégée, et migre du pôle vers le centre de la cellule au moment de la mise en place du septum de division, à la fin du cycle cellulaire. Les loci de la région ter présentent une période de cohésion post-réplicative étendue. Cette cohésion étendue est contrôlée par la protéine MatP, qui se fixe spécifiquement au niveau des sites matS, présents uniquement dans ter. MatP se fixe à l'ADN sous forme de dimère, via son domaine N-terminal, et tétramérise via son domaine C-terminal. La tétramérisation est stimulée par la liaison à l'ADN et permet le pontage de deux sites matS distants. MatP interagit aussi avec ZapB, un composant du divisome, la machinerie protéique participant à la formation du septum. Alors que la tétramérisation de MatP semble importante pour la compaction de la région ter, son interaction avec ZapB, qui est localisée au septum via ZapA et FtsZ, participe au positionnement et à la cohésion étendue de cette région. Le couplage de la région ter avec le divisome est essentiel pour le bon déroulement de nombreux évènements tardifs du cycle cellulaire : (i) la ségrégation active, ordonnée et progressive de la région ter par FtsK, un composant du divisome, (ii) la résolution des dimères de chromosomes via la recombinaison spécifique de site XerCD/dif, activée par FtsK, (iii) la résolution des liens d'intercaténation par la TopoIV et (iv) la régulation positive de l'assemblage du divisome en absence des régulateurs négatifs MinCDE et SlmA. Pendant ma thèse, je me suis tout d'abord intéressée au rôle de MatP dans la structuration globale du chromosome. En utilisant un système permettant de visualiser deux loci marqués avec un site parSp1 et un site parSpMT1, reconnu par ParBp1 et ParBpMT1 spécifiquement, nous avons analysé le positionnement et l'orientation du chromosome dans la cellule. Nous avons montré que MatP est nécessaire au positionnement et à l'orientation de tout le chromosome à la fin du cycle cellulaire. La localisation de SlmA dans des souches wt et DeltamatP prouve que l'inactivation de MatP, induisant une mauvais positionnement du chromosome, s'accompagne d'une défaut de localisation de SlmA, et induit donc une inhibition de la division. Ces résultats pris ensemble montre que MatP, SlmA et leur communication à travers la structuration globale du chromosome sont importants pour le management du chromosome et le contrôle de la division cellulaire. En collaboration avec l'équipe d'Olivier Espeli, nous avons utilisé des méthodes de génomiques et de biologie moléculaire pour caractériser la régulation de la TopoIV au cours du cycle cellulaire d'E. coli. Nous avons montré qu'au site dif, les activités de fixation et de clivage de la TopoIV sont améliorées par la présence des recombinases XerCD et de MatP. L'amélioration de l'activité de la TopoIV favorise la décaténation des chromosomes nouvellement répliqués et assure, en lien avec d'autres processus, la séparation précise des chromosomes frères. Ces résultats permettent de mieux comprendre le réseau d'interactions dédiées au management du chromosome à la fin du cycle cellulaire, et l'influence du management du chromosome sur le contrôle de la division cellulaire.Escherichia coli, as the majority of bacteria, has a unique circular chromosome. Faithfull cell proliferation requires that a least one copy of the chromosome is transmitted to sister cells prior to cell division. A strict temporal and spatial coupling of chromosome segregation with cell division is thus required to ensure the accurate separation of the two fully replicated chromosomes. The terminal region of the chromosome (ter) is the last one to be replicated and segregated, and moves from the pole to the middle of the cell where the division septum is formed, at the end of the cell cycle. Loci of the ter region display an extended cohesion period. This extended cohesion is controlled by the MatP protein, which binds specific matS sites restricted to the ter region. MatP binds DNA as a dimer and forms tetramers via its N-terminal and C-terminal domains respectively. Tetramerisation is stimulated by binding to DNA and pairs remote matS sites. MatP also interacts with ZapB, a component of the divisome, the protein machinery that contributes to septum formation. While tetramerisation of MatP appears important for compacting the ter region, its interaction with ZapB, which is localized at the septum via ZapA and FtsZ, is involved in the positioning and the extended cohesion of this region. The linkage of the ter region with the divisome is required for the success of many later events of the cell cycle : (i) the active, ordered and progressive segregation of the ter region by FtsK, a component of the divisome, (ii) resolution of chromosome dimers via the site-specifique recombination XerCD/dif, activated by FtsK, (iii) the resolution of intercatenation links by TopoIV and (iv) the positive regulation of divisome assembly in the absence of the negative regulators MinCDE and SlmA. During my thesis, I first studied the role of MatP in the chromosome management. By using pairs of loci tagged with parSp1 and parSpMT1 sites recognized by cognate ParB-XFP proteins, we directly analysed chromosome positioning and orientation in the cell. We show that MatP is required for normal positioning and orientation of the whole chromosome at the end of the cell cycle. The localisation of SlmA in wt and Delta matP strains proves that inactivation of MatP leads to inaccuracy of nucleoid positioning accompanied by defects in SlmA localisation, and thus induces division inhibition. Take together, these results show that MatP, SlmA and their interplay are important for chromosome management and control of cell division in E. coli. In collaboration with O. Espeli's team, we have used genomic and molecular biology methods to characterize TopoIV regulation during the E. coli cell cycle. We show that at the dif site, TopoIV binging and cleavage are enhanced by the presence of the XerCD recombinases and MatP. This enhancement of TopoIV activity at dif promotes decatenation of fully replicated chromosomes and ensure, through interaction with other processes, accurate separation of sister chromosomes. These results provide insight into the protein network dedicated to the final step of chromosome management during the cell cycle, and how the chromosome management is linked to cell division

Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome

International audienceCatenation links between sister chromatids are formed progressively during DNA replica-tion and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chro-matin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle

Role of the <i>dif</i> site for the management of circular chromosomes.

<p>A) Southern blot analysis of Topo IV cleavage at the <i>dif</i> and 2.56 Mb sites in the <i>mukB</i> mutant grown in minimal medium at 22°C. B) Southern blot analysis of Topo IV cleavage at the <i>dif</i> and 2.56 Mb sites in the <i>seqA</i> mutant grown in minimal medium at 37°C. C) Southern blot analysis of Topo IV cleavage at the <i>dif</i> and 1.9 Mb sites in the <i>matP</i> mutant grown in LB at 37°C. D) Colony Forming Unit (CFU) analysis of the WT and <i>nalR</i> strains deleted for the <i>dif</i> site, the <i>xerC</i> and <i>matP</i> genes in the presence of ciprofloxacin. E) Colony Forming Unit (CFU) analysis of the WT, <i>parEts</i> and <i>gyrBts</i> strains deleted for the <i>matP</i> at a semi permissive temperature (38°C). F) Southern blot analysis of the Topo IV cleavage at the <i>dif</i> and 1.9Mb sites in cells with a circular or linearized chromosome. G) Phenotypes observed during exponential growth in LB in the <i>matP</i> mutant strains with circular or linear chromosome (DNA is labeled with DAPI, green). Scale bar is 5μm.</p

Topo IV binding pattern of replicating chromosome.

<p>A) Circos plot of the ChIP-seq experiments for ParC-flag and ParE-flag. The IP / input ratio over the entire <i>E</i>. <i>coli</i> genome is presented for three independent experiments, one IP on the <i>parC-flag</i> strain and two IPs on the <i>parE-flag</i> strain. From the center to the outside, circles represent: genomic coordinates, macrodomain map, position of tRNA genes and ribosomal operons, ParE-Flag 1 ChIP-seq (untreated data, orange), ParE-Flag 1 ChIP-seq (filtered data, red), ParE-Flag 2 ChIP-seq (untreated data, orange), ParE-Flag 2 ChIP-seq (filtered data, red), ParC-Flag ChIP-seq (untreated data, orange), ParC-Flag ChIP-seq (filtered data, red), position of the 19 validated Topo IV binding sites. The right panels represent magnifications for four specific Topo IV binding sites, position 1.25 Mb, position 1.58 Mb (<i>dif</i>), position 1.85 Mb and position 2.56Mb. The three first rows correspond to filtered IP/Input ratio for ParC-Flag, ParE-Flag1 and ParE-Flag2 IPs, the fourth and fifth rows correspond respectively to the forward and reverse raw read numbers of the <i>parC-flag</i> experiment. The position and orientation of genes are illustrated at the bottom of each panel. B) Sliding averages of the IP (blue, left Y axis), Input (red, left Y axis) and IP/input (green, right Y axis) data for the <i>parC-flag</i> experiment over 60 kb regions along the genome. To facilitate the reading, <i>oriC</i> is positioned at 0 and 4.639 Mb. C) Analysis of Topo IV binding during the bacterial cell cycle. Marker frequency analysis was used to demonstrate the synchrony of the population at each time point. Stars represent the position of the selected Topo IV sites. D) IP/input ratio for 7 regions presenting specific Topo IV enrichment during S and G2 phases. For each genomic position the maximum scale is set to the maximum IP/Input ratio observed.</p

Targeting Topo IV cleavage sites along the <i>E</i>. <i>coli</i> genome.

<p>A) Circos plot of the ParC-Flag Chipseq and ParC-Flag 1 NorflIP experiments. From the center to the outside, circles represent: genomic coordinates, macrodomain map, Fis binding sites in mid exponential phase, % of bases bound by Fis per 20 kb windows of genomic DNA, H-NS binding sites in mid exponential phase, % of bases bound by H-NS per 20 kb windows of genomic DNA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006025#pgen.1006025.ref033" target="_blank">33</a>], ParC-Flag ChIP-seq (depleted regions blue, IP/input <1), ParC-Flag ChIP-seq (enriched regions red, IP/input >1), ParC-Flag 1 NorflIP (depleted regions blue, IP/input <1), ParC-Flag 1 NorflIP (enriched regions red, IP/input >1), gene expression data (RNA-seq results performed in the ChIP-seq and NorflIP conditions). For visualization purpose, the maximum scale of RNAseq data has been fixed to 500 reads which approximately corresponds to the 400 transcription units that were the most expressed (the distribution of read counts scaled from 0 to 30 000). B) Correlation between the localization of Topo IV cleavages and chromatin markers. The NUST [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006025#pgen.1006025.ref032" target="_blank">32</a>] hypergeometric test was used to compare Topo IV and chromatin markers localization. The set of 172 validated Topo IV cleavage sites was used. The number of common localizations over the total number of chromatin marker localization is indicated. The P value of a Fisher’s exact test is indicated. C) Genome browser magnifications of the panel A’s pink and yellows regions. Mid log phase Fis and H-NS binding sites are respectively indicated with burgundy and black boxes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006025#pgen.1006025.ref033" target="_blank">33</a>]. D) Magnification of the 2.56 Mb Topo IV binding and cleavage site that overlaps a Fis binding site. The position of the deleted Topo IV site is marked by vertical lines (<i>frt</i>). Southern blot analysis of Topo IV cleavage at the 2.56 Mb locus, in the <i>nalR strain</i>, the <i>nalR</i> strain with a deletion of the Topo IV cleavage and binding site and the deletion of <i>fis</i>. E) Same as D for the 1.92 Mb Topo IV cleavage site. Southern blot analysis of Topo IV cleavage at the 1.92 Mb locus, in the WT, the <i>nalR strain</i>, the <i>nalR</i> strain with a deletion of the Topo IV cleavage and Fis binding site and the <i>nalR</i> strain with <i>fis</i> deletion. The cleavage was also analyzed following a 20 min treatment with rifampicin (rif).</p

Topo IV cleavage at the Topo IV binding sites.

<p>A) Norfloxacin mediated DNA cleavage revealed by Southern blot with a radiolabeled probe near the <i>dif</i> site, the 1.25 Mb, 1.85 Mb, 2.56 Mb and the 3.24 Mb site. The size of the expected fragment generated by Topo IV cleavage is marked by an arrow. Topo IV cleavage can be differentiated from gyrase cleavages because of their presence in a <i>nalR</i> strain. B) Genome browser image of a 15kb region representative of Topo IV cleavage frequency (purple). These cleavage sites are not correlated with Topo IV enrichment in the ChIP-seq experiments described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006025#pgen.1006025.g001" target="_blank">Fig 1</a> (red). C) Circos plot of the NorflIP experiments. From the center to the outside, circles represent: genomic coordinates, macrodomain map, position of tRNA genes and ribosomal operons, ParC-Flag 1 NorflIP (untreated data, orange), ParC-Flag 1 NorflIP (filtered data, purple), ParE-Flag NorflIP(filtered data, purple), ParC-Flag 2 NorflIP (filtered data, purple), validated TopoIV sites present in the ParC-Flag 1, ParE-Flag and ParC-Flag 2 experiments. For visualization purpose, the maximum scale of NorflIP data has been fixed to an IP/input ratio of 10. D) Peak calling procedure, dedicated to DNA cleavage mediated by TopoIV in the presence of norfloxacin (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006025#pgen.1006025.s006" target="_blank">S6 Fig</a>), revealed 571 sites in total, in three experiments. Venn diagrams of common Topo IV cleavage sites in two experiments. About 200 common sites are observed in each pair of experiments. E) Genome browser zooms on the <i>dif</i>, 1.85, 1.92 and 2.56 Mb regions for Topo IV cleavage (purple) and Topo IV binding revealed by ChIP-seq (red). F) DNA cleavage mediated by TopoIV in the presence of norfloxacin revealed by Southern blot with a radiolabeled probe at 0.02, 1.92 Mb and the 3.2 Mb sites. G) Cleavage experiments performed on synchronized cultures, revealed a replication dependency (AS asynchronous, NR not replicating, S20 20 min after the initiation of replication (IR), S40 40 min after IR, S60 60 min after IR. H) Distribution of the ParC-Flag 1 NorflIP validated sites on the genome by 50 kb bins.</p

Determinants of Topo IV activity at <i>dif</i>.

<p>A) Southern blot analysis of the Topo IV cleavage at the <i>dif</i> site. Genomic DNA extracted from WT, Δ<i>dif</i>::<i>Tc</i>, Δ<i>dif</i>, Δ<i>xerD</i> box, Δ<i>xerC</i> box, Δ<i>xerD</i>, and Δ<i>xerC</i> strains was digested by <i>Pst</i>I; the size of the fragment generated by Topo IV cleavage at <i>dif</i> is marked by an arrow. The average percentage of cleavage observed in two independent experiments is presented. B) Southern blot analysis of the Topo IV cleavage at the <i>dif</i> site. Genomic DNA extracted from WT, Δ<i>xerC</i>, Δ<i>xerC pUCxerC</i>, Δ<i>xerC pUCxerCK172A</i>, Δ<i>xerC pUCxerCK172Q</i> strains was digested with <i>Pst</i>I; the size of the cleaved fragment in <i>dif</i> is marked by an arrow. C) Topo IV cleavage at the 1.9Mb site in the WT, Δ<i>xerC and</i> Δ<i>dif</i>. D) Plating of <i>parEts</i>, <i>parEts xerC</i>, <i>parEts xerD and parEts recN</i> mutants at 30 and 37°C. E) Colony Forming Unit (CFU) analysis of the WT and <i>nalR</i> strains deleted for the <i>dif</i> site, the <i>xerC</i>, <i>xerD</i> genes or the C-terminal domain of FtsK in the presence of ciprofloxacin. F) EMSA on a 250 bp CY3 probe containing <i>dif</i> (green) and a 250 bp CY5 control probe (red). The amount of Topo IV, XerC or XerD proteins present in each line is indicated above the gel. G) Quantification of Topo IV EMSA presented in C, data are an average of three experiments. H) Southern blot analysis of Topo IV cleavage at <i>dif</i> and position 1.92Mb in a strain overexpressing the C-terminal domain of ParC.</p

IARC monographs: 40 years of evaluating carcinogenic hazards to humans.

BackgroundRecently, the International Agency for Research on Cancer (IARC) Programme for the Evaluation of Carcinogenic Risks to Humans has been criticized for several of its evaluations, and also for the approach used to perform these evaluations. Some critics have claimed that failures of IARC Working Groups to recognize study weaknesses and biases of Working Group members have led to inappropriate classification of a number of agents as carcinogenic to humans.ObjectivesThe authors of this Commentary are scientists from various disciplines relevant to the identification and hazard evaluation of human carcinogens. We examined criticisms of the IARC classification process to determine the validity of these concerns. Here, we present the results of that examination, review the history of IARC evaluations, and describe how the IARC evaluations are performed.DiscussionWe concluded that these recent criticisms are unconvincing. The procedures employed by IARC to assemble Working Groups of scientists from the various disciplines and the techniques followed to review the literature and perform hazard assessment of various agents provide a balanced evaluation and an appropriate indication of the weight of the evidence. Some disagreement by individual scientists to some evaluations is not evidence of process failure. The review process has been modified over time and will undoubtedly be altered in the future to improve the process. Any process can in theory be improved, and we would support continued review and improvement of the IARC processes. This does not mean, however, that the current procedures are flawed.ConclusionsThe IARC Monographs have made, and continue to make, major contributions to the scientific underpinning for societal actions to improve the public's health