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Loyola University Chicago

Department of Biology

Domenic Castignetti

 

  Professor
Ph.D., 1980, University of Massachusetts
Microbial physiology and biochemistry
Phone: 773.508.3638
Fax: 773.508.3646
E-Mail: dcastig@luc.edu

RESEARCH INTERESTS

Siderophores are low molecular weight, avid ferric ion-binding compounds synthesized by fungi and bacteria. They sequester ferric ion from environments where its concentration is critically low. Without siderophores, microbes in such environments would cease growth due to lack of iron. Siderophores are employed by microbes to supply iron in environments such as soil, water and clinical infections.

We are examining the fate of siderophores in the environment, specifically, how are siderophores degraded and return to the natural carbon and nitrogen cycles. We have isolated a soil bacterium (a Mesorhizobium loti) that utilizes the siderophore deferrioxamine B (DFB) as its sole source of carbon and is thus able to return the molecule to the carbon cycle. Work has begun to elucidate the mechanism and the enzymes by which the bacterium is able to use DFB as a carbon source. The catalyst responsible for the initial breakdown of DFB, which we call DFB hydrolase, may be a single enzyme or an enzyme consortium. Biochemical investigations revealed that the enzyme begins the dismantling of DFB by generating its constituent units, termed monohydroxamates. Molecular biology studies have resulted in "knocking out" DFB metabolism in M. loti mutants and tagging the responsible genes with a transposon (Tn5:OT182). We are in the process of cloning and sequencing these tagged genes in order to better understand what genes may be responsible for the degradation of DFB by M. loti.

Our personnel are also investigating the metabolism of nitrogen by cave bacteria. Our colleague, Dr. Hazel Barton of the University of Northern Kentucky, has provided isolates of pseudomonads (a type of bacterium) from a cave in Kentucky. We have examined these isolates for their ability to denitrify (the process of reducing nitrate or nitrite to nitrous oxide or nitrogen gas) and determined that only 5 of the 24 isolates were capable of denitrification even though the key gene of denitrificaiton (nirS or nirK) was present in 6 of the isolates. Work is ongoing to determine if other nitrogen metabolism genes, such as those for ammonification, are present in the isolates.

 

REPRESENTATIVE PUBLICATIONS

K.A. Allard, J. Dao, P. Sanjeevaiah, K. McCoy-Simandle, C. H. Chatfield, D. S. Crumrine, D. Castignetti and N. P. Cianciotto. 2009. Purification of Legiobactin and the Importance of This Siderophore in Lung Infection by Legionella pneumophila. Infect. Immun. 77: 2887-2895.

Thomas, M. and D. Castignetti. 2009. Examination of anthrax lethal factor inhibition by siderophores, small hydroxamates and protamine. J. Microbiol., Immunol. Infect. 2009. 42:284-289.

Morton, J., K. Marsh, M. Frawley and D. Castignetti. 2007. The response of a siderophore-degrading bacterium (Mesorhizobium loti) to iron-deprivation: evidence of siderophore and iron-repressible protein synthesis. Adv. Biol. Res. (in press).

Pierwola, A., T. Krupinski, P. Zalupski, M. Chiarelli and D. Castignetti. 2004. Degradation pathway and generation of monohydroxamic acids from the trihydroxamate siderophore deferrioxamine B. Appl. Environ. Microbiol. 70: 831-836.

Thupvong, T., A. Wiideman, D. Dunn, K. Oreschak, B. Jankowicz, J. Doering and D. Castignetti. 1999. Sequence heterogeneity of the ferripyoverdine uptake (fpvA), but not the ferric uptake regulator (fur), genes among strains of the fluorescent pseudomonads Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas. BioMetals. 12:265-274.

Zaya, N., A. Roginsky, J. Williams and D. Castignetti. 1998. Evidence that a deferrioxamine B degrading enzyme is a serine protease. Canadian Journal of Microbiology. 44:521-527, 1998.


Fig. Non-denaturing gel of fractions from Sephadex C-25 column. Lanes 1and 4 were inactive fractions (no DFB hydrolase activity) while lanes 2 and 3 were active DFB hydrolase fractions. Note the absence of the top band in lanes 1 and 4 versus lanes 2 and 3. The top band represents DFB hydrolase, estimated to be about 110,000 daltons based on the non-denaturing (lanes 5-8) PAGE standards. Size of the standards for the non-denaturing lanes are 14,200, 29,000, 45,000, 66,000, 132,000, 272,000 and 545,000 (alpha-lactalbumin, carbonic anhydrase, chicken egg albumin, bovine serum albumin [monomer at 66K and dimer at 132K], jack bean urease [272K and 545K]). A number of these bands, however, are difficult to see as they are either in the dye front or run as charged isomers. The heavy band in lanes 5-8 is bovine serum albumin monomer (66 kDa) while the next band above is its the dimer, 132 kDa. In lane 5, the band below the bovine serum albumin 66 kDa band is the 45,000 kDa chicken egg albumin and the band below it is the 29 kDa carbonic anhydrase. Analysis of the standards and the unknown bands indicates that the higher molecular weight band in lanes 2 and 3 (DFB hydrolase) has a molecular weight of about 110 kDa. The contaminant band has a molecular weight of about 66 kDa.



Loyola

Loyola University Chicago · 1032 W. Sheridan Road, Chicago,IL 60660
Phone: 773.508.3620 · Fax: 773.508.3646 · E-mail: biologydept@luc.edu

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