Transcriptomic analysis of Vibrio vulnificus in response to iron concentration and Fur protein
ABSTRACT: V. vulnificus is a marine bacteria that causes diseases in both mammals and fish. In both hosts, the iron concentration represents a key factor that greatly influences the virulence of this bacterium. To further define the gene repertoire that is regulated by iron concentration and Fur protein (the main transcriptional regulator in response to iron concentration) in V. vulnificus, we obtained a mutant in Fur and used DNA microarray technology to monitor the expression of the entire gene repertoire in response to iron. Global transcriptomic response was reconstructed by comparing the transcriptional profiles of the wild-type (R99) and Fur mutant strains in poor and rich iron conditions. To identify the genes that were under control of Fur, we compared the transcriptomic profile of the wild-type strain with the profile of a mutant strain in Fur protein; in contrast, to identify the genes that were under control of iron, we compared the transcriptomic profile of the wild-type strain grown in iron-rich conditions with the profile of the wild-type strain grown in iron-restricted conditions. For each one of the four samples, three replicates were performed, and RNA was sampled in the mid-log phase of growth (wild-type, 6h; Fur mutant, 6h; wild-type+iron, 5h; wild-type-iron,9h).
Project description:In a proteomic analysis of rpoS-deficient Vibrio vulnificus versus the wild type, one of the down-regulated proteins in the rpoS mutant strain was identified as a Fur protein, a ferric uptake regulator. The expression of a fur::luxAB fusion was significantly influenced by sigma factor S, the rpoS gene product, and positively regulated by Fur under iron-limited conditions.
Project description:Vibrio vulnificus infections have been associated with iron overload and preexisting liver disease. Iron may play a major role in the pathogenesis of V. vulnificus infections. Many virulence genes, as well as genes involved in the transport of iron by bacteria, are regulated by iron, with increased expression under low-iron conditions. In Escherichia coli and Vibrio cholerae, transcriptional regulation by iron depends on the fur gene. We utilized Southern hybridization under low- and high-stringency conditions with both E. coli and V. cholerae fur gene probes to demonstrate that there are fur-homologous sequences in the DNAs of V. vulnificus, Vibrio fischeri, and Aeromonas sp. but not in the DNAs of the other bacterial species tested. We developed a restriction map and cloned the fur-homologous sequence from V. vulnificus. The hybridizing clone of V. vulnificus chromosomal DNA complemented a V. cholerae fur mutant. DNA sequence analysis confirmed the presence of a 149-amino-acid open reading frame that was 77% homologous to E. coli Fur and 93% homologous to V. cholerae Fur. Primer extension localized a single promoter for the V. vulnificus fur gene. Northern (RNA) blot analysis and beta-galactosidase assays of an operon fusion to lacZ suggested that there was not significant regulation of transcription of V. vulnificus fur by iron or the E. coli Fur protein. We used marker exchange to construct a V. vulnificus fur deletion mutant and confirmed its phenotype by observing overexpression of iron-regulated outer membrane proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fur deletion mutant of V. vulnificus will be helpful in future studies of the role of iron in V. vulnificus pathogenesis.
Project description:In many bacteria, the ferric uptake regulator (Fur) protein plays a central role in the regulation of iron uptake genes. Because iron figures prominently in the agriculturally important symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, we wanted to assess the role of Fur in the interaction. We identified a fur mutant by selecting for manganese resistance. Manganese interacts with the Fur protein and represses iron uptake genes. In the presence of high levels of manganese, bacteria with a wild-type copy of the fur gene repress iron uptake systems and starve for iron, whereas fur mutants fail to repress iron uptake systems and survive. The B. japonicum fur mutant, as expected, fails to repress iron-regulated outer membrane proteins in the presence of iron. Unexpectedly, a wild-type copy of the fur gene cannot complement the fur mutant. Expression of the fur mutant allele in wild-type cells leads to a fur phenotype. Unlike a B. japonicum fur-null mutant, the strain carrying the dominant-negative fur mutation is unable to form functional, nitrogen-fixing nodules on soybean, mung bean, or cowpea, suggesting a role for a Fur-regulated protein or proteins in the symbiosis.
Project description:Vibrio vulnificus is a halophilic, marine pathogen that has been associated with septicemia and serious wound infections in patients with iron overload and preexisting liver disease. For V. vulnificus, the ability to acquire iron from the host has been shown to correlate with virulence. V. vulnificus is able to use host iron sources such as hemoglobin and heme. We previously constructed a fur mutant of V. vulnificus which constitutively expresses at least two iron-regulated outer membrane proteins, of 72 and 77 kDa. The N-terminal amino acid sequence of the 77-kDa protein purified from the V. vulnificus fur mutant had 67% homology with the first 15 amino acids of the mature protein of the Vibrio cholerae heme receptor, HutA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for HupA, the heme receptor of V. vulnificus. DNA sequencing of hupA demonstrated a single open reading frame of 712 amino acids that was 50% identical and 66% similar to the sequence of V. cholerae HutA and similar to those of other TonB-dependent outer membrane receptors. Primer extension analysis localized one promoter for the V. vulnificus hupA gene. Analysis of the promoter region of V. vulnificus hupA showed a sequence homologous to the consensus Fur box. Northern blot analysis showed that the transcript was strongly regulated by iron. An internal deletion in the V. vulnificus hupA gene, done by using marker exchange, resulted in the loss of expression of the 77-kDa protein and the loss of the ability to use hemin or hemoglobin as a source of iron. The hupA deletion mutant of V. vulnificus will be helpful in future studies of the role of heme iron in V. vulnificus pathogenesis.
Project description:The ability of Vibrio vulnificus to acquire iron from the host has been shown to correlate with virulence. Many iron transport genes are regulated by iron, and in V. vulnificus, transcriptional regulation by iron depends on the fur gene. The N-terminal amino acid sequence of a 72-kDa iron-regulated outer membrane protein purified from a V. vulnificus fur mutant had 53% homology with the first 15 amino acids of the mature protein of the Vibrio cholerae vibriobactin receptor, ViuA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for VuuA, the vulnibactin receptor of V. vulnificus. Analysis of the DNA sequence of the vuuA promoter region demonstrated a sequence identical to the upstream Fur box of V. cholerae viuA. Northern blot analysis showed that the transcript was strongly regulated by iron. The amino acid sequence of VuuA was 74% identical to the sequence of V. cholerae ViuA and was homologous to those of several TonB-dependent outer membrane receptors. An internal deletion of the V. vulnificus vuuA gene resulted in the loss of expression of the 72-kDa protein and the loss of the ability to use transferrin or vulnibactin as a source of iron. This mutant showed reduced virulence in an infant mouse model. Introduction of a plasmid containing the complete viuA coding sequence and 342 bp of upstream DNA into the mutant restored ferric vulnibactin and ferric transferrin utilization to the mutant.
Project description:Iron is an important nutrient for the survival and growth of many organisms. In order to survive, iron uptake from the environment must be strictly regulated and maintained to avoid iron toxicity. The ferric uptake regulator protein (Fur) regulates genes involved in iron homeostasis in many bacteria, including phytopathogens. However, to date, the role played by Fur in the biology of Pectobacterium carotovorum subsp. brasiliense (Pcb1692), an important pathogen of potatoes, has not yet been studied. To this end, we used the lambda recombineering method to generate a fur mutant strain of Pcb1692 and assessed the virulence and fitness of the mutant strain. The results showed that production of siderophores in Pcb1692?fur increased compared to the Pcb1692 wild-type and the complemented strain Pcb1692?fur-pfur. However, production of N-acyl homoserine lactone (AHLs), biofilm formation, exopolysaccharide (EPS) production, virulence on potato tubers and swimming motility, were all significantly decreased in Pcb1692?fur compared to the wild-type and complemented Pcb1692?fur-pfur strains. The Pcb1692?fur mutant also demonstrated significant sensitivity to oxidative stress when exposed to H2O2. Consistent with phenotypic results, qRT-PCR results demonstrated that Fur down-regulates genes which encode proteins associated with: iron uptake (HasA-extracellular heme-binding protein and Ferrodoxin-AED-0004132), stress response (SodC-superoxide dismutase), plant cell wall degrading enzymes (PrtA and CelV) and motility (FlhC and MotA). We conclude that the ferric uptake regulator protein (Fur) of Pcb1692 regulates traits that are important to host-pathogens interactions.
Project description:In Escherichia coli, the small regulatory noncoding RNA (sRNA) RyhB and the global ferric uptake regulator (Fur) mediate iron acquisition and storage control. Iron is both essential and potentially toxic for most living organisms, making the precise maintenance of iron homeostasis necessary for survival. While the roles of these regulators in iron homeostasis have been well studied in a nonpathogenic E. coli strain, their impact on the production of virulence-associated factors is still unknown for a pathogenic E. coli strain. We thus investigated the roles of RyhB and Fur in iron homeostasis and virulence of the uropathogenic E. coli (UPEC) strain CFT073. In a murine model of urinary tract infection (UTI), deletion of fur alone did not attenuate virulence, whereas a ?ryhB mutant and a ?fur ?ryhB double mutant showed significantly reduced bladder colonization. The ?fur mutant was more sensitive to oxidative stress and produced more of the siderophores enterobactin, salmochelins, and aerobactin than the wild-type strain. In contrast, while RyhB was not implicated in oxidative stress resistance, the ?ryhB mutant produced lower levels of siderophores. This decrease was correlated with the downregulation of shiA (encoding a transporter of shikimate, a precursor of enterobactin and salmochelin biosynthesis) and iucD (involved in aerobactin biosynthesis) in this mutant grown in minimal medium or in human urine. iucD was also downregulated in bladders infected with the ?ryhB mutant compared to those infected with the wild-type strain. Our results thus demonstrate that the sRNA RyhB is involved in production of iron acquisition systems and colonization of the urinary tract by pathogenic E. coli.
Project description:Magnetospirillum gryphiswaldense strain MSR-1 has the unique capability of taking up large amounts of iron and synthesizing magnetosomes (intracellular magnetic particles composed of Fe(3)O(4)). The unusual high iron content of MSR-1 makes it a useful model for studying biological mechanisms of iron uptake and homeostasis. The ferric uptake regulator (Fur) protein plays a key role in maintaining iron homeostasis in many bacteria. We identified and characterized a fur-homologous gene (MGR_1314) in MSR-1. MGR_1314 was able to complement a fur mutant of E. coli in iron-responsive manner in vivo. We constructed a fur mutant strain of MSR-1. In comparison to wild-type MSR-1, the mutant strain had lower magnetosome formation, and was more sensitive to hydrogen peroxide and streptonigrin, indicating higher intracellular free iron content. Quantitative real-time RT-PCR and chromatin immunoprecipitation analyses indicated that Fur protein directly regulates expression of several key genes involved in iron transport and oxygen metabolism, in addition it also functions in magnetosome formation in M. gryphiswaldense.
Project description:The ferric uptake regulator (Fur) is a transcription factor and the main regulator of iron acquisition in prokaryotes. When bound to ferric iron, Fur recognizes its DNA binding site and generally executes its function by repressing transcription of its target genes. Due to its importance in virulence, the Fur regulon is well studied for several model bacteria. In our previous work, we used computational predictions and microarray to gain insights into Fur-regulation in Aliivibrio salmonicida, and have identified a number of genes and operons that appear to be under direct control of Fur. To provide a more accurate and deeper global understanding of the biological role of Fur we have now generated an A. salmonicida fur knock-out strain and used RNA-sequencing to compare gene expression between the wild-type and fur null mutant strains.An A. salmonicida fur null mutant strain was constructed. Biological assays demonstrate that deletion of fur results in loss of fitness, with reduced growth rates, and reduced abilities to withstand low-iron conditions, and oxidative stress. When comparing expression levels in the wild-type and the fur null mutant we retrieved 296 differentially expressed genes distributed among 18 of 21 functional classes of genes. A gene cluster encoding biosynthesis of the siderophore bisucaberin represented the highest up-regulated genes in the fur null mutant. Other highly up-regulated genes all encode proteins important for iron acquisition. Potential targets for the RyhB sRNA was predicted from the list of down-regulated genes, and significant complementarities were found between RyhB and mRNAs of the fur, sodB, cysN and VSAL_I0422 genes. Other sRNAs with potential functions in iron homeostasis were identified.The present work provides by far the most comprehensive and deepest understanding of the Fur regulon in A. salmonicida to date. Our data also contribute to a better understanding of how Fur plays a key role in iron homeostasis in bacteria in general, and help to show how Fur orchestrates iron uptake when iron levels are extremely low.
Project description:Iron is essential for the growth and survival of many organisms. Intracellular iron homeostasis must be maintained for cell survival and protection against iron toxicity. The ferric uptake regulator protein (Fur) regulates the high-affinity ferric uptake system in many bacteria. To investigate the function of the fur gene in Xanthomonas vesicatoria (Xv), we generated a fur mutant strain, fur-m, by site-directed mutagenesis. Whereas siderophore production increased in the Xv fur mutant, extracellular polysaccharide production, biofilm formation, swimming ability and quorum sensing signals were all significantly decreased. The fur mutant also had significantly reduced virulence in tomato leaves. The above-mentioned phenotypes significantly recovered when the Xv fur mutation allele was complemented with a wild-type fur gene. Thus, Fur either negatively or positively regulates multiple important physiological functions in Xv.