Personal Information

1966-68	Tel Aviv University Microbiology.& Biochemistry. M.Sc.
1969-1974	Tel Aviv University Microbiology Ph.D.

Academic And Professional Experience

1997-01	Tel Aviv University Vice President and Dean for Research and Development
1995-97	Director, The Laura-Schwarz-Kipp Institute of Biotechnology
1993-94	Sabbatical at the Department of Biochemistry, Karolinska Institute, Stockholm, Sweden
1993-	Professor, Tel Aviv University
1990-92	Chairman Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Faculty of Life Sciences.
1989-90	Chairman Department of Biotechnology, Tel Aviv University, Faculty of Life Sciences.
1986-88	Chairman Department of Microbiology, Tel Aviv University, Faculty of Life Sciences.

Research Interests

Current Research Projects

A. A novel bacterial route for the synthesis of glutathione: A multifunctional fused protein (GshF) integrates the two primary catalytic activities.

Glutathione, -L-glutamyl-L-cysteinylglycine (GSH) is the major low molecular weight intracellular thiol peptide in virtually all eukaryotes ranging from protozoa to plants. Within the prokaryotes it is found primarily in Gram-negative bacteria and much less commonly in Gram-positive bacteria. GSH plays a central role in maintaining the cellular thiol–redox balance. It functions as an enzyme cofactor, in the protection of cells against oxidative damage, in protein folding and in providing reducing equivalents for key reductuive enzymes such as ribonucleotide reductase. GSH synthesis occurs via a highly conserved two-step ATP-dependent process. Glutamate and cysteine are ligated by the enzyme glutamate cysteine ligase, encoded by gshA. The product, -glutamylcysteine is combined with glycine to form GSH in a reaction catalysed by glutathione synthetase encoded by gshB. In most organisms the genes encoding the GshA and GshB proteins are unlinked. An interesting variation on this theme occurs in a small number of bacteria, such as Listeria monocytogenes, which possesses an open reading frame (ORF) that is predicted to encode a GshA-like protein fused to an ATP-grasp super family member. The principal goal of our research is to determine whether the GshA-ATP-grasp ORFs present in Listeria monocytogenes and some other bacteia are responsible for the synthesis of glutathione. The research will focus on L. monocytogenes and Streptococcus mutans as the model organisms for which there are well developed molecular biology tools. It will employ analytical systems for detection and quantification of biological thiols, biochemical and physiological studies of recombinant fusion proteins, molecular genetic tools for mutant construction and gene expression, and structural studies of wild type and mutant proteins.

These studies will provide new insights into some fundamental aspects of the biosynthesis of glutathione in Gram-positive bacteria. One key issue is whether Listeria, clostridia, streptococci and enterococci synthesize glutathione or obtain it from their milieu. A second is whether glutathione synthesis in L. monocytogenes occurs via a single enzyme and represents a “primitive” form of a non-ribosomal multifunctional peptide synthetase. A third concerns the evolution of the different types of glutathione synthetase genes in Gram-postive bacteria. Finally, because this research focuses on characterizing novel biochemical and structural features of a unique protein that is found in some clinically important bacterial pathogens, for which there is no mammalian counterpart, it may prove valuable in identifying new targets for antimicrobial drug development.

colonize and propagate in its host. However, the natures of the signal transudation pathways that operate in S. aureus in response to changing oxygen tension are not well known.

     In this research project we plan to characterize the regulatory mechanisms used by S. aureus to adapt to changes in oxygen tension during the transition from aerobic to anaerobic growth. For this purpose we have chosen to analyze the oxygen-dependent regulation of the genes coding for ribonucleotide reductase (RNR). RNRs are essential enzymes that catalyze the reduction of ribonucleotides to deoxy- ribonucleotides for DNA replication and repair. S. aureus contains two ribonucleotide reductase gene clusters, nrdEF which determines an aerobic enzyme, and nrdDG which determines an anaerobic enzyme. We have recently shown that nrdDG is essential for anaerobic growth. Studies will be performed to a) biochemically characterize the two RNRs and determine the nature of their electron transport systems, b) analyze the Transcriptional regulation of the nrdEF and nrdDG gene clusters in terms of oxygen tension, c) identify the oxygen sensing and signal transduction systems that control nrd gene expression, d) construct reporter fusions to nrd and to virulence determinant genes to compare their regulation of expression during induction of anaerobic growth, and e) create nrd gene disruptions to establish viability in a mouse model. Attainment of these goals will, it is anticipated, provide new insights into the regulation mechanisms operating in the S. aureus aerobic-anaerobic interface. They may also validate whether the anaerobic RNR, which does not exist in the mammalian host, may serve as a valuable target for development of anti staphylococcal agents.

While many of the properties of ribonucleotide reductases have been elucidated, in particular their mechanism of action and allosteric regulation, important questions remain regarding the way in which these systems are regulated at the gene level. Streptomyces determine two RNRs, a class Ia oxygen-dependent enzyme and a class II oxygen-independent enzyme, either of which is sufficient to support aerobic growth. However, the physiological circumstances in which Streptomyces employs these two systems, in vegetative growth and during morphological development, are unclear. Streptomycetes are considered to be strict aerobes that can survive for long periods in the absence of oxygen. In an attempt to define specific roles for the individual RNR systems, we examined the effect of mutations in the RNR systems on the ability of S. coelicolor to recover growth following oxygen starvation. The figure presented below shows the growth of M145, M145ΔnrdJ::apr and M145ΔnrdB::apr on solid medium following a period of three days incubation in an anaerobic chamber and subsequent exposure to aerobic conditions. No growth was visible on the plates during the anaerobic incubation. When the plates were exposed to air for one day, M145 and M145ΔnrdB::apr consistently showed substantial growth whereas M145ΔnrdJ::apr, which lacks a functional oxygen-independent RNR, showed no detectable growth. Exposure of plates to air for a further 3 days showed that M145ΔnrdJ::apr had recovered growth to the same extents as the parent M145. The groqwth curves show that similar results were obtained in experiments performed in liquid culture. The nrdJ mutant strain exhibited a pronounced lag in growth after anaerobic incubation.

In our studies we explore the molecular mechanisms that control the expression of the class Ia and class II RNR genes, we are studying other regulatory factors involved in controlling the activity of the RNR system, and we anticipate that it will lead to new insights into their respective roles for growth and differentiation. The knowledge gained may prove useful in the engineering of industrial streptomycetes strains for the improved production of valuable secondary metabolites.

D. Studies on control of bacterial gene expression by “riboswitch” mechanisms.

“Riboswitches” are structured domains that usually reside in the noncoding regions of mRNAs, where they bind metabolites and control gene expression. Like their protein counterparts, these RNA gene control elements form highly specific binding pockets for the target metabolite and undergo allosteric changes in structure. Numerous classes of riboswitches are present in bacteria and they comprise a common and robust metabolite-sensing system (Winkler, WC., and Breaker, RR., (2005) Annu. Rev. Microbiol. 59: 487–517).

We have recently shown the presence of a consensus B12 genetic control element in the approximately 350-nucleotide 5'-untranslated leader region (UTR) of the Streptomyces coelicolor nrdABS mRNA (a Class I ribonucleotide reductase (RNR) system) and speculated that its function is to enable B12 to control nrdABS expression (Borovok et al. 2004, Mol. Microbiol. 54:1022-1035). The B12 element has been one of a growing number of genetic control elements, riboswitches, that modulate gene expression in bacteria through binding of small molecules (such as vitamins, amino acids, and purines) to the 5'-UTR of mRNA to generate alternative secondary structures. The RNA sensor element embedded in the leader sequences binds the metabolite, causing repression or activation of their cognate genes. Our studies have demonstrated that the B12 riboswitch is indeed an important control element in transcriptional regulation of the Streptomyces class Ia RNR genes (Borovok et al. 2006. J. Bacteriol. 188:2512-2520). We have noted that B12 may function in a similar way to control the S. coelicolor B12-dependent and B12-independent methionine synthetases since the S. coelicolor (and some other streptomycetes) metE gene encoding the B12-independent isozyme contains a B12 riboswitch in the 5'-UTR. The Streptomyces B12-dependent class II RNR is the primary RNR system in vegetative growth and functions to enable efficient growth recovery after oxygen deprivation. The class Ia RNR system may then function as a backup system when the class II RNR is inactive, for example, when B12 biosynthesis is limiting due to insufficient availability of cobalt or B12 biosynthetic precursors releasing the B12 riboswitch from bound ligand.

Selected Publications

• Aharonowitz, Y., and G. Cohen, (1981) The microbial production of pharmaceuticals. Scientific American, Sept. 140-152.
  
  
• Shiffman,D., Cohen,G., Aharonowitz,Y.,Palissa,H., von Dohren,H., Kleinkauf, H., and Mevarech,M., (1990) Nucleotide sequence of the Isopenicillin N synthase gene (pcbC) of the gram-negative Flavobacterium sp. SC 12,154. Nucleic Acid Research 18: 660.
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• Cohen, G., Shiffman, D., Mevarech, M., and Aharonowitz,Y., (1990) The microbial Isopenicillin N synthase genes: structure, function, diversity and evolution. Trends in Biotechnology, 8:105-111.
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• Abramov,S., Aharonowitz,Y., Harnik,M., Lamed,R., and Freeman,A., (1990) Continuous stereospecific delta 4-3-Keto-steroid reduction by PAAH bead entrapped Clostridium paraputrificum cells. Enz. Microb. Technology, 12: 982-988.
  
  
• Landan,G., Cohen,G., Aharonowitz,Y., Shauli,Y., Graur,D., and Shiffman,D., (1990) Evolution of isopenicillin N synthase genes may have involved horizontal gene transfer. Molec. Biol. Evol. 7: 399-406
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• Landman,O., Shiffman,D., Av-Gay,Y., Aharonowitz,Y., and Cohen,G., (1991). High level expression in Escherichia coli of isopenicillin N synthase genes from Flavobacterium and Streptomyces and recovery of active enzyme from inclusion bodies. FEMS Microbiol. Lett. 84:239-244
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• Schwecke,T., Aharonowitz,Y., Palissa,H., von Dohren,H., Kleinkauf, H., van Liempt,H., (1992). Enzymatic characterization of the multifunctional enzyme d-(L-a-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus. Eur. J. Biochem 205:687-694
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• Av-Gay,Y., Aharonowitz,Y., Cohen,G., (1992). Streptomyces contain a 7.0 kDa cold-shock protein. Nucleic Acid Research. 20:5478
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• Aharonowitz, Y., Cohen,G., and Martin J.F., (1992), Penicillin and cephalosporin biosynthetic genes: structure, organization, regulation and evolution. Annu. Rev. Microbiol. 46: 461-495
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• Aharonowitz, Y., Av-Gay,Y., Schreiber, Cohen G., (1993) Characterization of a thioredoxin-like disulfide reductase from Streptomyces clavuligerus and its possible role in b-lactam antibiotic biosynthesis. J.Bacteriol. 175: 623-629
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• Newton, J., Fahey, R.C., Cohen,G., and Aharonowitz, Y., (1993) Low molecular weight thiols in Streptomycetes and their potential role as antioxidants. J .Bacteriol. 175:2734-2742
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• Aharonowitz,Y., Bergmeyer,H., Cantoral, J., Cohen,G., Demain, A.L., Fink,U., Kinghorn, J., H. Kleinkauf, MacCabe,A., Palissa,H., Pfeifer,E.,Schwecke,T., van Limpt,H., von Dohren,H., Wolfe,S., and Zhang,J.,(1993). d-(L-a-aminoadipyl)-L-cysteinyl-D-valine synthetase, themultienzyme integrating the four primary reactions in b-lactam biosynthesis, as a model peptide synthetase. Bio/technology 11:807-810.
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• Cohen, G., Janko, M., Mislovati,R., Argaman, A., Schreiber, R., Av-Gay, Y., and Aharonowitz, Y., (1993) Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequence, expression and organization of the genes. J.Bacteriol. 175:5159-5167
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• Cohen,G., Argaman, A., Schreiber, R., Mislovati, M., and Aharonowitz, Y., (1994) The thioredoxin system of Streptomyces clavuligerusand its possible role in penicillin biosynthesis. J. Bacteriol. 176: 973-984
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• Newton, G. L., Bewley, G. A., Dwyer, T. J., Horn, R., Aharonowitz, Y., Cohen, G., Davies, J. E., Faulkner, D. J., and Fahey, R. C., (1995) The structure of U17 isolated from Streptomyces clavuligerusand its properties as an antioxidant thiol. Eur. J. Biochem 230:821-825.
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• Cohen, G., and Aharonowitz, Y., (1995) Molecular genetics of antimicrobials: a case study of beta-lactam antibiotics. Darby, G.K., Hunter, P.A., Russel, A. D., eds. 50 Years of Microbials, Soc. Gen. Microbiol, Symp. 53. 139-163. Cambridge University Press. UK.
  
  
• Borovok. I., Landman, O., Kreisberg, R., Aharonowitz, Y., and Cohen, G., (1996). The ferrous active site of isopenicillin N synthase: genetic and sequence analysis of the endogenous ligands. Biochemistry, 35:1981-1987
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• Newton, G., Arnold, K., Price, M. S., Sherrill, C., Delcardayre, S. B., Aharonowitz, Y., Cohen, G., Davies, J. E., and Fahey, R. C., and Davis, C., (1996) Distribution of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes. J. Bacteriol. 178:1990-1995
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• Gal-Mor, O., Borovok, I., Av-Gay, Y., Cohen, G., And Aharonowitz, Y., (1998). Gene organization in the trxA/B -oriC region of the Streptomyces coelicolor chromosome and comparison with other bacteria. Gene, 217: 83-90
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• Kreisberg-Zakarin, R., Brorovok, I., Yanko, M., Aharonowitz, Y., and Cohen G., (1999). Recent advances in the structure and Function of Isopenicillin N synthase. Antonie van Leeuwenhoek 75: 33-39
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• Kreisberg-Zakarin, R., Brorovok, I., Yanko, M., Frolow, F., Aharonowitz, Y., and Cohen, G., 2000. Structure-function studiesn of the non-heme iron active site of isopenicillin N synthase: some implications for catalysis. Biophysical Chemistry. 86: 109-118
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• Masalha, M., Borovok, I., Schreiber, R., Aharonowitz Y., and Cohen., G., (2001) Analysis of Transcription of the Staphylococcus aureus Aerobic Class Ib and Anaerobic Class III Ribonucleotide Reductase Genes in Response to Oxygen. J Bacteriol. 183: 7260-7272
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• Paget, M. S. B., Molle, M., Cohen, G., Aharonowitz, Y.,and Buttner M. J.,(2001) Defining the disulphide stress response in Streptomyces coelicolor A3(2): identification of the sR regulon. Molecular Microbiology. 42: 1007-1020
  
  
• Ilya Borovok , Rachel Kreisberg-Zakarin1, Michaela Yanko, Rachel Schreiber, Margarita Myslovati, Fredrik Aslund, Arne Holmgren, Gerald Cohen and Yair Aharonowitz (2002) Streptomyces contain class Ia and class II ribonucleotide reductases expression analysis of the genes in vegetative growth. Microbiology. 148: 391-404
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• Dafna Ben-Bashat, Yael Meller, Yair Aharonowitz, David utnick, Shmuel Carmeli, and Gil Navon. (2002) Excretion of a Phosphorus-Containing Carbohydrate by Streptomyces sp. A50. J. Natural Products. 64: 1538-1540
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• Uziel Orit, Ilya Borovok, Rachel Schreiber, Gerald Cohen and Yair Aharonowitz. (2004) Transcriptional regulation of the Staphylococcus aureus thioredoxin and thioredoxin reductase genes in responde to oxygenand disulfide stress. Journal of Bacteriology 186: 326-334
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• James K. Lithgow, Emma J. Hayhurst, Gerald Cohen, Yair haronowitz, and Simon J. Foster (2004) Role of a Cysteine Synthase in Staphylococcus aureus. J. Bacteriol. 186: 1579-1590
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• Borovok, I., Gorovitz, B., Yanku, M., Schreiber, R., Gust, B., Chater, K., Aharonowitz, Y., Cohen, G., (2004) Alternative oxygen-dependent and oxygen-independent ribonucleotide reductases in Streptomyces: cross-regulation and physiological role in response to oxygen limitation. Molecular Microbiology 54:1022-1035.
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• Gopal, S., Borovok, I., Ofer, A., Yanku, M., Cohen, G., Goebel, W., Kreft, J.,and Aharonowitz, Y., (2005). A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis. J. Bacteriol. 187: 3839-3847
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• Borovok, I., Gorovitz, B., Schreiber, R., Aharonowitz, Y., Cohen, G., (2006) Coenzyme B12 Controls Transcription of the Streptomyces Class Ia Ribonucleotide Reductase nrdABS Operon via a Riboswitch Mechanism. J. Bacteriol. 188: 2512-2520
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• Zhang, S., Borovok, I., Aharonowitz, Y., Sharan, R., Bafna, V. (2006) A sequence-based filtering method for ncRNA identification and its application to searching for riboswitch elements. Bioinformatics. 2006 Jul 15;22(14):e557-65.

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• Grinberg, I., Shteinberg, T., Gorovitz, B., Aharonowitz, Y., Cohen, G., Borovok, I. (2006) The Streptomyces coelicolor NrdR protein contains Zn-finger and ATP-cone domains and regulates transcription of the ribonucleotide reductase operons. J. Bacteriol. 188:7635-7644.

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• Torrents, E. I. Grinberg, B. Gorovitz-Harris, H. Lundström, I. Borovok, Y. Aharonowitz, B. Sjöberg and Cohen, G. (2007) NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes. J Bacteriol. 189:5012-5021.

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• Makhlin J, Kofman T, Borovok I, Kohler C, Engelmann S, Cohen G, Aharonowitz, y. (2007) Staphylococcus aureus ArcR controls expression of the arginine deiminase operon. J Bacteriol. 189:5976-5986.

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• Grinberg I, Shteinberg T, Hassan Q, Aharonowitz Y, Borovok I, and Cohen, G. (2009) Functional analysis of the Streptomyces coelicolor NrdR ATP-cone domain: Role in nucleotide binding, oligomerization and DNA interactions. J. Bacteriol. 191: 1169-1179.

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• Malki L, Yanku M, Borovok I, Cohen G, Mevarech M, Aharonowitz, Y. (2009) Identification and characterization of gshA, a gene encoding the glutamate-cysteine ligase in the halophilic archeon Haloferax volcanii. J Bacteriol. 191: 5196-5204
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• Pöther DC, Liebeke M, Hochgräfe F, Antelmann H, Becher D, Lalk M, Lindequist U, Borovok I, Cohen G, Aharonowitz Y, Hecker M. (2009) Diamide triggers mainly S-Thiolations in the cytoplasmic proteomes of Bacillus subtilis and Staphylococcus aureus. J Bacteriol. 191:7520-7530.

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• Rabinovitch I, Yanku M, Yeheskel A, Cohen G, Borovok I, Aharonowitz Y. (2010) Staphylococcus aureus NrdH redoxin is a reductant of the class Ib ribonucleotide reductase. J Bacteriol. 192:4963-4972.

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• Ofer A, Kreft J, Logan DT, Cohen G, Borovok I, Aharonowitz Y. (2011) Implications of the Inability of Listeria monocytogenes EGD-e To Grow Anaerobically Due to a Deletion in the Class III NrdD Ribonucleotide Reductase for Its Use as a Model Laboratory Strain. J Bacteriol. 193:2931-2940.
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Patents

• Jensen,S.E., Westlake,D.W.S., Leskiw, B.K., Aharonowitz,Y., and Mevarech,M., Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus. Application for Canadian Letter Patent, Aug. 10.1987. serial No 544122
  
  European patent, 88306779.5; 22.07.88
  
  
• Aharonowitz,Y. et.al. ACV reductase system, the set of ACV reductase genes and a method for influencing and using the expression of these genes for increasing antibiotic production.
  
  European Patent Application EP 0-462-674-A1 Date of filling 18.06.91
  
  
• Aharonowitz, Y., et al. 1994. Oxido reductase enzyme system obtained from P. chrysogenum. Oxido reductase enzyme system obtainable from P. chrysogenum, the set of genes encoding the same and the use of oxido reductase enzyme systems or genes encoding the same for increasing antibiotic production
  
  US Patent 5,328,839 July 12 1994.
  
  US Patent 5,652,132 July 29 1997.
  
  US Patent 5,753,435 May 19 1998.
  
  
• Aharonowitz, et al., 2004. Recombinant Staphylococcus thioredoxin reductase and inhibitors thereof useful as antimicrobial agents.
  
  U.S. Patent No. 6,767,536 , July 27, 2004