No description available.

Staphylococcus aureus (S. aureus) is a significant pathogen that causes a wide variety of disease in humans and animals. Methicillin resistant S. aureus (MRSA) isolates carrying mecC, the gene that confers resistance to the antibiotic, have been isolated from humans but also from diverse animal species covering livestock, domestic and wild animals throughout Europe. Many of the known MRSA mecC isolates have been whole-genome sequenced by our group to gain insight into the evolution and epidemiology of these emerging lineages. For microbes and humans alike, iron is an essential cofactor in many biochemical reactions and S. aureus requires iron for colonisation and subsequent pathogenesis. The success of S. aureus is partly attributed to its ability to exploit the host iron pool. It does this through multiple iron uptake mechanisms, including at least two high-affinity iron scavenging siderophores (staphyloferrins A and B) and an iron-regulated surface determinant (Isd) pathway for haem-iron acquisition. Here I describe the identification of a novel locus encoding a siderophore-like non-ribosomal peptide synthetase (NRPS), directly downstream of the SCCmec insertion site in mecC S. aureus isolates. A homologous region was identified in Streptococcus equi 4047 (S. equi) which encodes a NRPS termed 'equibactin' that is involved in iron acquisition. I have therefore named the NRPS product 'staphylobactin' in MRSA, and the aim of this study was to determine the function of the staphylobactin biosynthesis cluster: is this region involved in iron acquisition and how might it be regulated? Analysis of the prevalence of isolates containing the staphylobactin locus showed it to be present in a large number of mecC strains in our collection but also identified homologues in other staphylococcus isolates. The region is highly conserved in all S. aureus isolates belonging to clonal complex (CC) 130 (broad host range lineage), suggesting that staphylobactin might impact on S. aureus's ability to infect a broad range of host species. The staphylobactin gene cluster contains 14 coding sequences, stbB-F, F1, G-M and O. Bioinformatic analysis results in predictions of domain and gene functions associated with iron acquisition. I hypothesized that staphylobactin might have been acquired to compensate for the lack of another siderophore, such as staphyloferrin B, but the staphyloferrin B biosynthesis cluster and transport is present in nearly all S. aureus strains, ruling out this model. Unlike the equibactin locus, however, the staphylobactin locus lacks a homolog for the iron-dependent regulator eqbA. Instead, expression of this locus appears to be regulated by MntR, a DtxR-like regulator. The staplylobactin gene cluster is flanked by direct repeats which suggest staphylobactin could have been gained by horizontal gene transfer. In order to study the role of the staphylobactin gene cluster, deletion mutants of MntR, the staphylobactin locus and staphyloferrins A and B, were generated using the pIMAY two step gene deletion procedure in the previously un-manipulated mecC S. aureus CC130 strains - a challenging protocol that required significant optimization given the difficulties with manipulating this bacterium. Analysis of the MntR mutant suggests that the staphylobactin operon is regulated by MntR, acting as a positive regulator, in an iron-dependent manner. By RT-PCR, I found that expression of the staphylobactin NRPS genes is increased when cultures are grown in the absence of iron, suggesting an involvement with iron acquisition. Genomic inactivation of the staphyloferrins resulted in a mutant severely incapacitated for growth in serum and transferrin as the sole iron source, and addition of iron reversed this phenotype. However, deletion of staphylobactin alone or in addition to the staphyloferrins, lacked an iron-dependent growth defect, and numerous assays failed to identify a clear role for staphylobactin in iron metabolism. Therefore, further experiments are needed to elucidate the function of this siderophore like NRPS. Analysis of the same sequenced CC130 mecC isolates from our strain collection in which the staphylobactin locus was found, led to the identification of a novel Von Willebrand (vwb) gene. In order to investigate possible reasons for these isolates to infect a wide range of host species, wild-type and vwb deletion mutant strains, along with the novel vwb expressed in lactococcus, were tested using a coagulation assay and were able to clot plasma from a broad range of host species. Thus the specificity of vWbp proteins can be used to infer the host specificity and evolutionary history of the S. aureus strains that harbour them. Although I was unable to generate definitive evidence revealing the biological role for the staphylobactin locus this study has generated valuable tools for further studies and thoroughly tested a number of hypotheses concerning its role in cation metabolism.

Rhodococcus, a soil bacterium, displays a diverse range of metabolic capabilities with a number of potential practical applications. To exploit the metabolic potential of Rhodococcus, their basic physiology, genetics, and especially the acquisition of essential nutrients like iron, must be understood. R. erythropolis strain IGTS8 releases a small compound called a siderophore, that scavenges ferric iron from the environment. To learn more about the genetic control of iron acquisition, mutant(s) defective in siderophore production were isolated. Mutants were generated, by inserting a mutagenic plasmid, pJCS506, into the bacterial cell using electroporation. The plasmid, which cannot replicate in these bacterial cells, randomly inserts into the R. erythropolis genome producing mutations. The potential mutants were detected by screening on a chrome azurol S plate, which detects siderophore production. Colonies that failed to produce siderophore were tested by liquid assays. The strain N5-59 was confirmed as a non-siderophore producing mutant by liquid assays.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Phillip E. Klebba / Siderophore uptake systems are one the most prominent methods of Fe³+-iron acquisition in Gram negative bacteria. The catecholate siderophore enterobactin is synthesized and utilized by many members of Enterobacteriaceae as well as several of the ESKAPE pathogens. The outer membrane (OM) transporter of ferric enterobactin (FeEnt), FepA is a ligand-gated porin (LGP) that requires interaction with the inner membrane (IM) protein TonB in order to accomplish active transport. TonB is thought to transduce the electrochemical energy created by the proton gradient across the IM to LGPs like FepA in the OM, to promote siderophore transport through their occluded channels. However, we do not yet have a clear picture of either how TonB transfers energy to FepA, or what kind of conformational changes occur in the occluding domain of FepA to allow ligand passage. The experiments described herein investigate these two questions, building on previously outlined models and observations. Using fluorescence labeling of strategically substituted cysteines in the surface loops of FepA, we unraveled a hierarchy of loop motion during binding of FeEnt to FepA. Additionally, by rendering parts of the FepA protein immobile as a result of engineered disulfide bonds, I identified residues or regions within its occluding domain that may normally unfold to open a size-specific channel for FeEnt. I also elucidated the role of the peptidoglycan polymer beneath the OM a framework for protein-protein interactions between IM and OM proteins. This includes the proposed interaction between a rotating TonB and FepA, or other LGPs, that may transfer kinetic energy to the OM transporter. The role of iron in microbial survival and pathogenesis makes iron-uptake pathways an attractive target for therapeutic intervention. Using the FeEnt-FepA uptake system as a model, we used a fluorescence based high-throughput screening method to identify novel small molecule inhibitors of TonB action in E. coli. The approach used can be potentially adopted to screen bigger chemical libraries as well as used to find inhibitors of ESKAPE pathogens that use FeEnt such as, Acinetobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa. Finally, we discoverd a TonB-dependent OM transporter of heme/hemoglobin called HutA in the oligotrophic bacterium Caulobacter crescentus.

N5-59, a siderophore deficient mutant strain of Rhodococcus erythropolis strain IGTS8 (IGTS8) was investigated to learn more about how this poorly characterized bacterium acquires iron. N5-59 cells were starved for iron and then lysed to release any intracellular siderophore. No intracellular siderophore was detected indicating that N5-59 is not defective in the export or release of siderophore but is probably unable to synthesize siderophore. In a cross-feeding bioassay, growth of N5-59 (in an iron depleted medium) was greatly enhanced by the addition of exogenous siderophore from IGTS8 and other Rhodococcus species indicating that N5-59 is not defective in siderophore uptake. A DNA hybridization probe was prepared using DNA flanking the site of insertional mutation in strain N5-59. This probe was then used to clone a 6 kilobase pair, PstI restriction fragment from the chromosome IGTS8. This cloned DNA is expected to contain the intact gene(s) that was interrupted in N5-59.

No description available.

Many gram-negative bacteria produce and secrete siderophores under iron-deficient conditions. Siderophores are low molecular weight compounds (600-1500 Daltons), which chelate ferric iron with an extremely high affinity, and the complex is actively transported across the outer and inner membranes of gram-negative bacteria. There are two main classes of siderophores: catechol and hydroxamate. Catechol-type siderophores chelate ferric iron via hydroxyl groups, and hydroxamate-type siderophores chelate ferric iron via a carbonyl group with an adjacent nitrogen. Rhizobia fix atmospheric nitrogen symbiotically in leguminous plants using the iron-containing enzyme nitrogenase. To satisfy their iron requirements, many rhizobia are known to produce siderophores. Rhizobium leguminosarum Strain IARI 312 is known to infect pigeon pea plants. R. leguminosarum Strain IARI 312 produces both a catechol-type and a hydroxamate-type siderophore when grown under iron deficient conditions. The catechol-type siderophore has been purified and chemically characterized, and is consistent with that of enterobactin.

Iron is an essential nutrient for most bacteria because enzymes like nitrate reductase and cytochromes use it as a cofactor. However, in most aerobic, neutral pH environments, iron is essentially insoluble and not easily available for bacteria to use. Many bacteria respond to this problem by releasing small organic compounds called siderophores that bind and effectively solubilize iron so that it can be transported into the cell for growth. The focus of this study was to learn more about the iron acquisition and especially the transport of iron by the soil bacterium Rhodococcus erythropolis. To fulfill this aim, mutant strains of the bacteria were screened for those that overproduce siderophore. Often, a bacterium will over produce siderophore to compensate for a defect in transport. One such mutant, R187-12, was further analyzed by cloning the region of the chromosome containing the defective gene responsible for over production of siderophore into a plasmid vector. The DNA sequence of this region was determined and analyzed for the presence of similar genes encoding transport proteins.

No description available.

Mastitis resulting from mammary gland infection is a common and painful disease associated with lactation. In addition to the impact on human and animal health, mastitis causes substantial economic losses in the dairy industry. Staphylococcus aureus is a frequent cause of mastitis worldwide. Despite significant progress in understanding S. aureus pathogenesis in general, much remains to be learned regarding virulence factors relevant in the context of mastitis. In mammary gland infections, it is not fully understood which metal acquisition systems are required for S. aureus survival. To help understand molecular mechanisms by which S. aureus might acquire essential metals, such as iron, within lactating mammary glands, S. aureus mutants were tested for growth defects in vitro. A low-iron media (TMM) was created and supplemented with differing iron sources relevant to mastitis infection such as host iron-binding proteins lactoferrin and transferrin. Mutants were grown in the various iron sources to determine which genes were involved with iron acquisition for each specific media tested. Results show that a double knock-out (∆htsA/sirA::ba) involved with two iron siderophore receptors and the ATPase, fhuC::ba, which powers those receptors are essential for growth in media supplemented with human lactoferrin, while mutants involved with the iron siderophore Staphyloferrin B (sbnE::ba) and its specific receptor (sirA::ba) proved important for growth in bovine lactoferrin. Additionally, S. aureus mutants were grown in bovine and human milk. Significant growth defects in human milk were found for mutants involved with zinc (znuBC::ba) and manganese (psaA::ba) acquisition. Iron limitations leading to growth defects were also found in ∆htsA/sirA::ba and fhuC::ba grown in human milk. Growth defects in bovine milk were seen for psaA::ba but not zinc genes. Growth of the fhuC mutant was shown to be significant, but not the double knock-out, indicating that iron acquisition in bovine milk does not involve the SirABC or HtsABC siderophore receptors. A mutant involved in purine synthesis, purH::ba, was also shown to have a significant growth defect in bovine milk. The importance of S. aureus metal acquisition has been well established, but there is a significant need to research these multifaceted processes further. Increased understanding of how metal acquisition facilitates bacterial survival in the lactating mammary gland can provide therapeutic targets for more effective mastitis prevention and treatment.