Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus

In most bacteria, the ferric uptake regulator (Fur) is a global regulator that controls iron homeostasis and other cellular processes, such as oxidative stress defense. In this work, we apply a combination of bioinformatics, in vitro and in vivo assays to identify the Caulobacter crescentus Fur regulon. A C. crescentus fur deletion mutant showed a slow growth phenotype, and was hypersensitive to H(2)O(2) and organic peroxide. Using a position weight matrix approach, several predicted Fur-binding sites were detected in the genome of C. crescentus, located in regulatory regions of genes not only involved in iron uptake and usage but also in other functions. Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis. Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency. Furthermore, several genes that are regulated via small RNAs in other bacteria were found to be directly regulated by Fur in C. crescentus. In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.

Prediction of transcriptional regulatory interactions in bacteria : a comparative genomics approach

Exponential growth of genomic data in the last two decades has made manual analyses impractical for all but trial studies. As genomic analyses have become more sophisticated, and move toward comparisons across large datasets, computational approaches have become essential. One of the most important biological questions is to understand the mechanisms underlying gene regulation. Genetic regulation is commonly investigated and modelled through the use of transcriptional regulatory network (TRN) structures. These model the regulatory interactions between two key components: transcription factors (TFs) and the target genes (TGs) they regulate. Transcriptional regulatory networks have proven to be invaluable scientific tools in Bioinformatics. When used in conjunction with comparative genomics, they have provided substantial insights into the evolution of regulatory interactions. Current approaches to regulatory network inference, however, omit two additional key entities: promoters and transcription factor binding sites (TFBSs). In this study, we attempted to explore the relationships among these regulatory components in bacteria. Our primary goal was to identify relationships that can assist in reducing the high false positive rates associated with transcription factor binding site predictions and thereupon enhance the reliability of the inferred transcription regulatory networks. In our preliminary exploration of relationships between the key regulatory components in Escherichia coli transcription, we discovered a number of potentially useful features. The combination of location score and sequence dissimilarity scores increased de novo binding site prediction accuracy by 13.6%. Another important observation made was with regards to the relationship between transcription factors grouped by their regulatory role and corresponding promoter strength. Our study of E.coli ��70 promoters, found support at the 0.1 significance level for our hypothesis | that weak promoters are preferentially associated with activator binding sites to enhance gene expression, whilst strong promoters have more repressor binding sites to repress or inhibit gene transcription. Although the observations were specific to �70, they nevertheless strongly encourage additional investigations when more experimentally confirmed data are available. In our preliminary exploration of relationships between the key regulatory components in E.coli transcription, we discovered a number of potentially useful features { some of which proved successful in reducing the number of false positives when applied to re-evaluate binding site predictions. Of chief interest was the relationship observed between promoter strength and TFs with respect to their regulatory role. Based on the common assumption, where promoter homology positively correlates with transcription rate, we hypothesised that weak promoters would have more transcription factors that enhance gene expression, whilst strong promoters would have more repressor binding sites. The t-tests assessed for E.coli �70 promoters returned a p-value of 0.072, which at 0.1 significance level suggested support for our (alternative) hypothesis; albeit this trend may only be present for promoters where corresponding TFBSs are either all repressors or all activators. Nevertheless, such suggestive results strongly encourage additional investigations when more experimentally confirmed data will become available. Much of the remainder of the thesis concerns a machine learning study of binding site prediction, using the SVM and kernel methods, principally the spectrum kernel. Spectrum kernels have been successfully applied in previous studies of protein classification [91, 92], as well as the related problem of promoter predictions [59], and we have here successfully applied the technique to refining TFBS predictions. The advantages provided by the SVM classifier were best seen in `moderately'-conserved transcription factor binding sites as represented by our E.coli CRP case study. Inclusion of additional position feature attributes further increased accuracy by 9.1% but more notable was the considerable decrease in false positive rate from 0.8 to 0.5 while retaining 0.9 sensitivity. Improved prediction of transcription factor binding sites is in turn extremely valuable in improving inference of regulatory relationships, a problem notoriously prone to false positive predictions. Here, the number of false regulatory interactions inferred using the conventional two-component model was substantially reduced when we integrated de novo transcription factor binding site predictions as an additional criterion for acceptance in a case study of inference in the Fur regulon. This initial work was extended to a comparative study of the iron regulatory system across 20 Yersinia strains. This work revealed interesting, strain-specific difierences, especially between pathogenic and non-pathogenic strains. Such difierences were made clear through interactive visualisations using the TRNDifi software developed as part of this work, and would have remained undetected using conventional methods. This approach led to the nomination of the Yfe iron-uptake system as a candidate for further wet-lab experimentation due to its potential active functionality in non-pathogens and its known participation in full virulence of the bubonic plague strain. Building on this work, we introduced novel structures we have labelled as `regulatory trees', inspired by the phylogenetic tree concept. Instead of using gene or protein sequence similarity, the regulatory trees were constructed based on the number of similar regulatory interactions. While the common phylogentic trees convey information regarding changes in gene repertoire, which we might regard being analogous to `hardware', the regulatory tree informs us of the changes in regulatory circuitry, in some respects analogous to `software'. In this context, we explored the `pan-regulatory network' for the Fur system, the entire set of regulatory interactions found for the Fur transcription factor across a group of genomes. In the pan-regulatory network, emphasis is placed on how the regulatory network for each target genome is inferred from multiple sources instead of a single source, as is the common approach. The benefit of using multiple reference networks, is a more comprehensive survey of the relationships, and increased confidence in the regulatory interactions predicted. In the present study, we distinguish between relationships found across the full set of genomes as the `core-regulatory-set', and interactions found only in a subset of genomes explored as the `sub-regulatory-set'. We found nine Fur target gene clusters present across the four genomes studied, this core set potentially identifying basic regulatory processes essential for survival. Species level difierences are seen at the sub-regulatory-set level; for example the known virulence factors, YbtA and PchR were found in Y.pestis and P.aerguinosa respectively, but were not present in both E.coli and B.subtilis. Such factors and the iron-uptake systems they regulate, are ideal candidates for wet-lab investigation to determine whether or not they are pathogenic specific. In this study, we employed a broad range of approaches to address our goals and assessed these methods using the Fur regulon as our initial case study. We identified a set of promising feature attributes; demonstrated their success in increasing transcription factor binding site prediction specificity while retaining sensitivity, and showed the importance of binding site predictions in enhancing the reliability of regulatory interaction inferences. Most importantly, these outcomes led to the introduction of a range of visualisations and techniques, which are applicable across the entire bacterial spectrum and can be utilised in studies beyond the understanding of transcriptional regulatory networks.

Global transcriptional response of Caulobacter crescentus to iron availability

BACKGROUND: In the alpha subclass of proteobacteria iron homeostasis is controlled by diverse iron responsive regulators. Caulobacter crescentus, an important freshwater α-proteobacterium, uses the ferric uptake repressor (Fur) for such purpose. However, the impact of the iron availability on the C. crescentus transcriptome and an overall perspective of the regulatory networks involved remain unknown. RESULTS: In this work we report the identification of iron-responsive and Fur-regulated genes in C. crescentus using microarray-based global transcriptional analyses. We identified 42 genes that were strongly upregulated both by mutation of fur and by iron limitation condition. Among them, there are genes involved in iron uptake (four TonB-dependent receptor gene clusters, and feoAB), riboflavin biosynthesis and genes encoding hypothetical proteins. Most of these genes are associated with predicted Fur binding sites, implicating them as direct targets of Fur-mediated repression. These data were validated by β-galactosidase and EMSA assays for two operons encoding putative transporters. The role of Fur as a positive regulator is also evident, given that 27 genes were downregulated both by mutation of fur and under low-iron condition. As expected, this group includes many genes involved in energy metabolism, mostly iron-using enzymes. Surprisingly, included in this group are also TonB-dependent receptors genes and the genes fixK, fixT and ftrB encoding an oxygen signaling network required for growth during hypoxia. Bioinformatics analyses suggest that positive regulation by Fur is mainly indirect. In addition to the Fur modulon, iron limitation altered expression of 113 more genes, including induction of genes involved in Fe-S cluster assembly, oxidative stress and heat shock response, as well as repression of genes implicated in amino acid metabolism, chemotaxis and motility. CONCLUSIONS: Using a global transcriptional approach, we determined the C. crescentus iron stimulon. Many but not all of iron responsive genes were directly or indirectly controlled by Fur. The iron limitation stimulon overlaps with other regulatory systems, such as the RpoH and FixK regulons. Altogether, our results showed that adaptation of C. crescentus to iron limitation not only involves increasing the transcription of iron-acquisition systems and decreasing the production of iron-using proteins, but also includes novel genes and regulatory mechanisms

Comparative genomics reveals `novel' Fur regulated sRNAs and coding genes in diverse proteobacteria

Ferric uptake regulator (Fur) is a transcriptional regulator controlling the expression of genes involved in iron homeostasis and plays an important role in pathogenesis. Fur-regulated sRNAs/CDSs were found to have upstream Fur Binding Sites (FBS). We have constructed a Positional Weight Matrix from 100 known FBS (19 nt) and tracked the `Orphan' FBSs. Possible Fur regulated sRNAs and CDSs were identified by comparing their genomic locations with the `Orphan' FBSs identified. Thirty-eight `novel' and all known Fur regulated sRNAs in nine proteobacteria were identified. In addition, we identified high scoring FBSs in the promoter regions of the 304 CDSs and 68 of them were involved in siderophore biosynthesis, iron-transporters, two-component system, starch/sugar metabolism, sulphur/methane metabolism, etc. The present study shows that the Fur regulator controls the expression of genes involved in diverse metabolic activities and it is not limited to iron metabolism alone. (C) 2012 Elsevier B.V. All rights reserved.

Status, biology, and ecology of fur seals: Proceedings of an International Symposium and Workshop Cambridge, England, 23-27 April 1984

The 1984 International Symposium and Workshop on the Biology of Fur Seals originated in informal talks in 1981. However, the scope and focus of the symposium remained unclear until an informal workshop was held in San Diego in June 1983. This meeting synthesised data on the foraging and pup attendance activities of six species of fur seals, and attempted to formulate a coherent framework for the adaptations associated with their maternal strategies (Gentry et al. 1986). During the workshop it was clear that comparative data on many key aspects of fur seal biology and ecology were missing. This absence of data applied not only to less well known species, for some of which considerable unpublished data existed, but also to better known species for which research in some areas had either been neglected or unreported. The value of applying the comparative method to seals, especially comparisons integrating physiology, ecology, and reproductive biology, was amply demonstrated by the results of the 1983 workshop (Gentry and Kooyman 1986). However, we were also aware that many other problems outside the area of maternal strategies could benefit from comparative data, such as recovery of populations from the effects of harvesting. Therefore, to accommodate the range of potential research, we organized this symposium to produce an up-to-date synthesis of relevant information for all species of fur seals. It was also clear that fur seal research could benefit from increased communication and collaboration among its practitioners. To foster the spread of ideas, we held oral presentations on some topics of current research and techniques and organized workshops on specific topics, in addition to providing opportunities for informal talks among participants. Thanks to generous support from the British Antarctic Survey, the National Marine Fisheries Service of the United States, and the Scientific Committee on Antarctic Research, the International Fur Seal Symposium was held at the British Antarctic Survey, Cambridge, England, 23-27 April 1984. The 36 participants are shown in Figure 1. A list of Symposium participants and authors is presented in Appendix 1 of the Proceedings. (PDF file contains 220 pages.)

Management of northern fur seals on the Pribilof Islands, Alaska, 1786-1981

This paper includes information about the Pribilof Islands since their discovery by Russia in 1786 and the population of northern fur seals, Cailorhinus ursinus, that return there each summer to bear young and to breed. Russia exterminated the native population of sea Oilers, Enhydra lulris, here and nearly subjected the northern fur seal to the same fate before providing proper protection. The northern fur seal was twice more exposed to extinction following the purchase of Alaska and the Pribilof Islands by the United States in 1867. Excessive harvesting was stopped as a result of strict management by the United States of the animals while on land and a treaty between Japan, Russia, Great Britain (for Canada), and the United States that provided needed protection at sea. In 1941, Japan abrogated this treaty which was replaced by a provisional agreement between Canada and the United States that protected the fur seals in the eastern North Pacific Ocean. Japan, the U.S.S.R., Canada, and the United States again insured the survival of these animals with ratification in 1957 of the "Interim Convention on the Conservation of North Pacific Fur Seals," which is still in force. Under the auspices of this Convention, the United States launched an unprecedented manipulation of the resource through controlled removal during 1956-68 of over 300,000 females considered surplus. The biological rationale for the reduction was that production of fewer pups would result in a higher pregnancy rate and increased survival, which would, in turn, produce a sustained annual harvest of 55,000-60,000 males and 10,000-30,000 females. Predicted results did not occur. The herd reduction program instead coincided with the beginning of a decline in the number of males available for harvest. Suspected but unproven causes were changes in the toll normally accounted for by predation, disease, adverse weather, and hookworms. Depletion of the animals' food supply by foreign fishing Heets and the entanglement of fur seals in trawl webbing and other debris discarded at sea became a prime suspect in altering the average annual harvest of males on the Pribilof Islands from 71,500 (1940-56) to 40,000 (1957-59) to 36,000 (1960) to 82,000 (1961) and to 27,347 (1972-81). Thus was born the concept of a research control area for fur seals, which was agreed upon by members of the Convention in 1973 and instituted by the United States on St. George Island beginning in 1974. All commercial harvesting of fur seals was stopped on St. George Island and intensive behavioral studies were begun on the now unharvested population as it responds to the moratorium and attempts to reach its natural ceiling. The results of these and other studies here and on St. Paul Island are expected to eventually permit a comparison between the dynamics of unharvested and harvested populations, which should in turn permit more precise management of fur seals as nations continue to exploit the marine resources of the North Pacific Ocean and Bering Sea. (PDF file contains 32 pages.)

Eine Chance fur eine positive Bestandsentwicklung in der Ostsee? Hoffnungswoller Dorschjahrgang in der Ostee bestagtigt

Der Stratified Random Survey, der vom 22. 10. bis 3.11. 2004 in der Arkonasee, Kieler und Mecklenburger Bucht durchgef

Fangmengenbegrenzungen in der Garnelenfischerei - eine Chance fur weitere Biefangreduzierungen

Aiming for price stabilisation Danish, German and Dutch brown shrimp fisheries agreed on weekly catch limitations for the years 1998 and 1999. This resulted in fishing effort reduction of 18 % of the total number of fishing trips in 1998 and up to 24 % in summer. In that period highest abundance of young plaice occurs in the Wadden Sea which is the fishing area of the brown shrimp fleets of Germany and the Netherlands. Consequently as a side effect a reduction of the total annual by-catch especially of young plaice must have occurred. According to formerly conducted EU-studies and investigations the by-catch reduction due to the agreed catch limitations should have led to survival of millions of young plaice. They give a potential of some extra catch in coming years which is 2,5 % of the total TAC of plaice in the North Sea. Compared to the German TAC in year 2000 the gain equals 44 %. The catch limitations effect on by-catch reduction in 1998 was in the same order of magnitude of the one achievable by technical measures in net selection applied in that fishery and research. A combination of both could substantially reduce traditional by-catch levels in brown shrimp fisheries.Aiming for price stabilisation Danish, German and Dutch brown shrimp fisheries agreed on weekly catch limitations for the years 1998 and 1999. This resulted in fishing effort reduction of 18 % of the total number of fishing trips in 1998 and up to 24 % in summer. In that period highest abundance of young plaice occurs in the Wadden Sea which is the fishing area of the brown shrimp fleets of Germany and the Netherlands. Consequently as a side effect a reduction of the total annual by-catch especially of young plaice must have occurred. According to formerly conducted EU-studies and investigations the by-catch reduction due to the agreed catch limitations should have led to survival of millions of young plaice. They give a potential of some extra catch in coming years which is 2,5 % of the total TAC of plaice in the North Sea. Compared to the German TAC in year 2000 the gain equals 44 %. The catch limitations effect on by-catch reduction in 1998 was in the same order of magnitude of the one achievable by technical measures in net selection applied in that fishery and research. A combination of both could substantially reduce traditional by-catch levels in brown shrimp fisheries.