Iron acquisition by the haem-binding Isd proteins in Staphylococcus aureus: studies of the mechanism using magnetic circular dichroism.

The bacterium Staphylococcus aureus is responsible for numerous hospital-acquired infections ranging from superficial wound lesions to more severe infections such as pneumonia, osteomyelitis and septicaemia and, in some cases, death. The Isd (iron-regulated surface determinant) proteins expressed by S. aureus and select other bacteria are anchored to the bacterial cell wall and membrane and are involved in extracting haem from haemoglobin as an iron source. Our knowledge of the overall haem-scavenging mechanism on the bacterial surface is limited. A detailed description of the haem-binding properties in the transport pathway is critical to our understanding of the mechanism for haem-iron scavenging in S. aureus. Our work involves using a combination of techniques to characterize both the dynamic and steady-state haem-binding properties of these proteins. UV-visible absorption and MCD (magnetic circular dichroism) spectroscopy provide diagnostic spectral data sensitive to the axial ligands, the spin state and oxidation state of the central haem-iron. Electrospray MS provides stoichiometric information on the numbers of haems bound, the effect of haem binding on the overall folding of each protein and kinetic information about the rate of haem binding. Together, these data allow us to address the outstanding questions regarding the mechanism of haem transport via the Isd protein chain in S. aureus.

Demonstration of the iron-regulated surface determinant (Isd) heme transfer pathway in Staphylococcus aureus.

In this study, we report experimental results that provide the first complete challenge of a proposed model for heme acquisition by Staphylococcus aureus via the Isd pathway first put forth by Mazmanian, S. K., Skaar, E. P., Gaspar, A. H., Humayun, M., Gornicki, P., Jelenska, J., Joachmiak, A., Missiakas, D. M., and Schneewind, O. (2003) Science 299, 906-909. The heme-binding NEAT domains of Isd proteins IsdA, IsdB (domain 2), IsdC, and HarA/IsdH (domain 3), and the heme-binding IsdE protein, were overexpressed and purified in apo (heme-free) form. Absorption and magnetic circular dichroism spectral data, together with electrospray ionization mass spectrometry were used to unambiguously identify that heme transfers from NEAT-A through NEAT-C to IsdE. Heme transfer was demonstrated to occur in a unidirectional fashion in the sequence NEAT-B2 --> NEAT-A --> NEAT-C --> IsdE or, alternatively, initiating from NEAT-H3 instead of NEAT-B2: NEAT-H3 --> NEAT-A --> NEAT-C --> IsdE. Under the conditions of our experiments, only NEAT-H3 and NEAT-B2 could transfer bidirectionally, which is in the reverse direction as well, and only with each other. Whereas apo-IsdE readily accepted heme from holo-NEAT-C, it would not accept heme from holo-NEAT-A. Heme transfer to IsdE requires the presence of holo-NEAT-C, in agreement with the proposal that IsdC serves as the central conduit of the heme transfer pathway. These experimental findings corroborate the heme transfer model first proposed by the Schneewind group. Our data show that heme transport from the wall-anchored IsdH/IsdB proteins proceeds directly to IsdE at the membrane and, for this to occur, we propose that specific protein-protein interactions must take place.

Heme binding in the NEAT domains of IsdA and IsdC of Staphylococcus aureus.

Absorption, magnetic circular dichroism (MCD), and electrospray mass spectral (ESI-MS) data are reported for the heme binding NEAr iron Transporter (NEAT) domains of IsdA and IsdC, two proteins involved in heme scavenging by Staphylococcus aureus. The mass spectrometry data show that the NEAT domains are globular in structure and efficiently bind a single heme molecule. In this work, the IsdA NEAT domain is referred to as NEAT-A, the IsdC NEAT domain is referred to as NEAT-C, heme-free NEAT-C is NEAT-A and NEAT-C are inaccessible to small anionic ligands. Reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-A results in coordination by histidine and opens access, allowing for CO axial ligation, yielding 6-coordinate low-spin Fe(II) heme. In contrast, reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-C results in loss of the heme from the binding site of the protein due to the absence of a proximal histidine. The absorption and MCD data for NEAT-A closely match those previously reported for the whole IsdA protein, providing evidence that heme binding is primarily a property of the NEAT domain.

A novel, intracellular antifreeze protein in an antarctic bacterium, Flavobacterium xanthum.

One strain of Antarctic bacteria, Flavobacterium xanthum IAM12026, has a highly active antifreeze protein (AFP) in the intracellular space. The cell-free extract from strain IAM12026 after culturing at 4 degree C for 7 days in TSB medium, had activity of 0.04 degree C at a concentration of 0.7 mg/ml. The ice crystals formed do not have distinct facets without typically rounded shape and the changes of their morphology during the course of the thermal hysteresis (TH) measurement. The ice crystal 'burst' occurring at the end-point of the TH is dendritic with hexagonal symmetry. Also, this activity was not affected by the treatment of dialysis and the addition of EDTA. Furthermore, this cell-free extract had high levels of ice recrystallization-inhibiting (RI) activity like those of Fish AFPs. The AFP (FlAFP) was homogeneity purified using chromatography. A relative molecular mass of approximately 59,000 was calculated from gel filtration and SDS-PAGE data. The thermal stability of FlAFP was below 50 degree C, and TH value was absent above 60 degree C. The TH value of FlAFP was activated at 5.2 degree C by the addition of 0.5 M malate. This activation was decreased with increasing protein concentration. To our knowledge this is the first report on the high level of TH and RI activities of bacterial intracellular AFP.

Cloning and expression of afpA, a gene encoding an antifreeze protein from the arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2.

The Arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 secretes an antifreeze protein (AFP) that promotes survival at subzero temperatures. The AFP is unusual in that it also exhibits a low level of ice nucleation activity. A DNA fragment with an open reading frame encoding 473 amino acids was cloned by PCR and inverse PCR using primers designed from partial amino acid sequences of the isolated AFP. The predicted gene product, AfpA, had a molecular mass of 47.3 kDa, a pI of 3.51, and no previously known function. Although AfpA is a secreted protein, it lacked an N-terminal signal peptide and was shown by sequence analysis to have two possible secretion systems: a hemolysin-like, calcium-binding secretion domain and a type V autotransporter domain found in gram-negative bacteria. Expression of afpA in Escherichia coli yielded an intracellular 72-kDa protein modified with both sugars and lipids that exhibited lower levels of antifreeze and ice nucleation activities than the native protein. The 164-kDa AFP previously purified from P. putida GR12-2 was a lipoglycoprotein, and the carbohydrate was required for ice nucleation activity. Therefore, the recombinant protein may not have been properly posttranslationally modified. The AfpA sequence was most similar to cell wall-associated proteins and less similar to ice nucleation proteins (INPs). Hydropathy plots revealed that the amino acid sequence of AfpA was more hydrophobic than those of the INPs in the domain that forms the ice template, thus suggesting that AFPs and INPs interact differently with ice. To our knowledge, this is the first gene encoding a protein with both antifreeze and ice nucleation activities to be isolated and characterized.

Purification and characterization of uridine phosphorylase from the ice-nucleating bacterium, Pantoea agglomerans NBRC12686.

The ice-nucleating bacterium, Pantoea agglomerans NBRC12686 responds to a decrease in temperature with the induction of proteins, which are classified as cold-induced proteins. When the temperature of the strain NBRC12686 culture was lowered from 30 degree C to 12 degree C, the viability after freezing treatment significantly improved. By the use of SDS-polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC), we analyzed the cold acclimation response in strain NBRC12686. After a shift from 30 degree C to 12 degree C, several proteins and saccharides were synthesized. After 48 h of cold acclimation, the induction level of proteins increased. In addition, ribose-1-phosphate was fractionated by HPLC using a TSK gel Sugar AXG column. Cell-free extracts were prepared from a cold acclimation culture (30 degree C to 12 degree C) and a non-cold acclimation culture (30 degree C), and then subjected to SDS-PAGE. A protein of approximately 29.7-kDa was present in the cold acclimation culture but was not present in the non-cold acclimation culture. The 29.7-kDa protein was purified by various chromatographies. We found that apparent molecular mass of the protein was approximately 119-kD constructed of 4 subunits of 29.7-kDa each. Based on the analysis of the N-terminal amino acid sequences of proteins, the 29.7-kDa protein had 83 percent identity with that of uridine phosphorylase (UPase) obtained from Escherichia coli K-12. We confirmed that the 29.7-kDa protein was novel, judged by molecular mass different from the already-known UPase or cryoprotectants. The cryoprotective activity of UPase of 29.7-kDa protein for LDH was approximately 30 percent at 5.0 microgram per ml of the protein. Furthermore, UPase had a high level of cryoprotective activity even after treating at 70 degree C for 30 min, but had no activity after treating at 100 degree C. We could elucidate that UPase from strain NBRC12686 had a cryoprotective activity as well as an enzyme activity, and it seems that UPase works in two different mechanisms for freezing tolerance.

Production of two types of ice crystal-controlling proteins in Antarctic bacterium.

Pseudomonas fluorescens KUAF-68, which was isolated from Antarctica, had both ice-nucleating protein and antifreeze protein activities in the culture broth. We found that both proteins were separately produced based on the results of column chromatography, SDS-PAGE analysis and Southern hybridization. The activity of the ice-nucleating protein was stimulated by the addition of glycine (0.020 N%), whereas the activity of the antifreeze protein was stimulated by the addition of L-asparagine (0.025 N%). This is the first report on the production of two types of ice crystal-controlling proteins in one bacterial strain.

Properties of a novel extracellular cell-free ice nuclei from ice-nucleating Pseudomonas antarctica IN-74.

Some ice-nucleating bacterial strains, including Pantoea ananatis (Erwinia uredovora), Pseudomonas fluorescens, and Pseudomonas syringae isolates, were examined for the ability to shed ice nuclei into the growth medium. A novel ice-nucleating bacterium, Pseudomonas antarctica IN-74, was isolated from Ross Island, Antarctica. Cell-free ice nuclei from P. antarctica IN-74 were different from the conventional cell-free ice nuclei and showed a unique characterization. Cell-free ice nuclei were purified by centrifugation, filtration (0.45 microm), ultrafiltration, and gel filtration. In an ice-nucleating medium in 1 liter of cell culture, maximum growth was obtained with the production of 1.9 mg of cell-free ice nuclei. Ice nucleation activity in these cell-free ice nuclei preparations was extremely sensitive to pH. It was demonstrated that the components of cell-free ice nuclei were protein (33%), saccharide (12%), and lipid (55%), indicating that cell-free ice nuclei were lipoglycoproteins. Also, carbohydrate and lipid stains showed that cell-free ice nuclei contained both carbohydrate and lipid moieties.

Purification and properties of an ice-nucleating protein from an ice-nucleating bacterium, Pantoea ananatis KUIN-3.

An ice-nucleating protein (INP) from the extracellular ice-nucleating matter (EIM) of Pantoea ananatis (Erwinia uredovora) KUIN-3 was purified and characterized. The EIM produced by the strain KUIN-3 was purified by ultrafiltration, sucrose density-gradient ultracentrifugation and gel filtration. The INP was purified using of column chromatography on hydroxyapatite and Superdex 200 in the nondenaturing detergent of 0.1% (w/v) Triton X-100. The purified INP was composed of one subunit of 117 kDa according to SDS-PAGE. It has become apparent that the INP was the ice-nucleating lipoglycoprotein based on the reaction of carbohydrate stain and lipid stain with the INP. It was inhibited by p-mercuribenzoate and N-bromosuccinimide. The activity of the INP gradually decreased from 65 degrees C. The pH stability was held between pH 7.0 and pH 11.0. The INP had a lower ice-nucleating temperature below pH 6.0. It has become apparent that the INP consisted of the class C structure in the EIM based on its freezing difference spectrum in D2O versus H2O.