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MP052 Cloning and characterization of a Lon homologous protease from Bartonella henselae
Wissenschaftliches Programm 55. DGHM-Tagung 29. September-1. Oktober 2003 in Dresden Abstracts - Poster
G~~ G ES~LI~C,
3
Molecular Characterization of the Arginine Deiminase Operon of Streptococcus suis GrQning, p.1; Goethe, R}; Winterhoff, N.1; Rhode, M.~; Valentin-Weigand, P} ~Tieraerztliche Hochschute Hannover; Mikrobiologie, Zentrum f. Infektionsmedizin 2Gesellschaft f[Jr Biotechnologische Forschung ( GBF); Mikrobiologie
Streptococcus
(S.) suis is an important cause of infectious diseases in young pigs such as meningitis, septicemia and bronchopneumonia and is known as a zoonotic agent in humans. Little is known about pathogenesis and virulence factors of S. suis, and, therefore, our aim is to identify new enviromentaldependent virulence factors. Recently, we identified a temperature-induced arginine deiminase (AdiS) of S. suis. Here, we could show that the adiS gene is located on an operon, together with genes which belong to the arginine deiminase system (ADS). The operon shows high homologies to genes of S. pyogenes. The ADS is also found in other bacteria and supports their survival in anaerobic or acidic enviroments, e. g. in deeper tissues or phagocytic cells of the host. The operon consists of four genes, adiS, off2, octS, and ckS, with identities of 80,2% (adiS), 81,2 (octS) and 70,1% (ckS) with the respective genes in S. pyogenes. Upstream of adiS and octS we found putative promotor structures. Putative stemloop structures were located downstream of the orf2, octS and ckS gene, which might be of importance in regulation of the operon. Northern Analyses showed that the ADS was inducible by a temperature shift from 32°C/37°C to 42°C and, in additon, by incubation at 10% CO2 versus aerobic conditions. We also determined the arginine deiminase (AD) activity, and could show that all tested strains had an increased AD-activity after a temperature shift from 37°C to 42°C. Immuno electron microscopy revealed that, after temperature shift to 42°C, AdiS was expressed on the streptococcal surface in association with the capsular polysaccharides. In conclusion, we have identified a novel stress regulated operon that codes for surface-associated proteins of the ADS and may, thus, facilitate survival of S. suis in the host.
Cloning and characterization of a Lon homologous protease from Bartonella henselae Zimmermann, R. 1, Koenig, J.1; Sander, A. 1 iUniversity of Freiburg; Institute of Medical Microbiology and Hygiene Introduction:
Bartonella henselae is the infectious agent of cat scratch disease, bacillary angiomatosis and bacillary peliosis hepatis. The reservoirs for this bacterium are cats which can transmit the pathogen to humans. Depending on the environmental conditions the availability and activity of cellular proteins must be properly adjusted by the bacteria. Protein activity is
http://www.dghm.org
273
Wissenschaftliches Programm 55. DGHM-Tagung 29. September-1. Oktober 2003 in Dresden Abstracts - Poster regulated by a variety proteolytic processing.
of
mechanisms,
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~
s
including
Methods and Data: We successfully cloned and characterized a Ion protease gene homolog from B. henselae. The nucleotide sequence encodes a protein of 807 amino acids with a predicted molecular mass of 90.4 kDa. The deduced amino acid sequence showed a high identity and homology to respective proteins from Brucella melitensis, Rhizobium and Agrobacterium species. The conserved amino acid sequence of a Walker A and B motif for ATP binding and the serine in the putative active site was detected. An open reading frame homologous to the ATP-depending protease gene xlpX was found before and a homologous to the DNA binding protein gene hu after the Ion gene. We could demonstrate that the transcription of the Ion gene of B. henselae was induced after heat shock and ethanol exposition. The transcriptional start site was determined, and the promoter showed sequence similarities to the sigma32-factor dependent polymerases. The Ion gene was heterologous expressed in E. coil TB1. The protein was purified to apparent homogeneity and the characteristic ATP-dependent proteolytic activity is under investigation. Our B. henselae Ion mutant, with a KanR Transposon inserted into the Ion gene, showed a retarded growth compared to the WT strain and exhibited a higher UV sensitivity. Conclusions: The gene homologous to a Ion-protease was characterized, and the protein was purified. Additionally our Ion-mutant is more sensitive to UV treatment. The precise role of the Ion protease in B. henselae is under further investigation.
The aspartate ammonia-lyase of Actinobacillus pleuropneumoniae is upregulated in an ex-vivo model. Jacobsen, 1.1; Trost, M.2; Gerlach, G.F} ITier~rztliche Hochschule Hannover; Zentrum for Infektionsmedizin, Institut fElr Mikrobiologie und Tierseuchen 2Gesellschaft few Biotechnologische Forschung ( GBF); Zellbiologie Introduction: Actinobacillus (A.) pleuropneurnoniae is the etiological agent of a porcine respiratory disease characterized by acute infection and subsequent sequestration of affected tissues. The pathogen is able to survive on the surface of lung epithelia, in the tonsils, and in the anaerobic environment of encapsulated sequesters. In order to identify putative virulence associated factors we used an ex-vivo model modulating A. pleuropneumoniae protein expression by the addition of bronchoalveolar lavage fluid (BALF) to culture media.
http://www.dghm.org
274
G~~ G ES~LI~C,
3
Molecular Characterization of the Arginine Deiminase Operon of Streptococcus suis GrQning, p.1; Goethe, R}; Winterhoff, N.1; Rhode, M.~; Valentin-Weigand, P} ~Tieraerztliche Hochschute Hannover; Mikrobiologie, Zentrum f. Infektionsmedizin 2Gesellschaft f[Jr Biotechnologische Forschung ( GBF); Mikrobiologie
Streptococcus
(S.) suis is an important cause of infectious diseases in young pigs such as meningitis, septicemia and bronchopneumonia and is known as a zoonotic agent in humans. Little is known about pathogenesis and virulence factors of S. suis, and, therefore, our aim is to identify new enviromentaldependent virulence factors. Recently, we identified a temperature-induced arginine deiminase (AdiS) of S. suis. Here, we could show that the adiS gene is located on an operon, together with genes which belong to the arginine deiminase system (ADS). The operon shows high homologies to genes of S. pyogenes. The ADS is also found in other bacteria and supports their survival in anaerobic or acidic enviroments, e. g. in deeper tissues or phagocytic cells of the host. The operon consists of four genes, adiS, off2, octS, and ckS, with identities of 80,2% (adiS), 81,2 (octS) and 70,1% (ckS) with the respective genes in S. pyogenes. Upstream of adiS and octS we found putative promotor structures. Putative stemloop structures were located downstream of the orf2, octS and ckS gene, which might be of importance in regulation of the operon. Northern Analyses showed that the ADS was inducible by a temperature shift from 32°C/37°C to 42°C and, in additon, by incubation at 10% CO2 versus aerobic conditions. We also determined the arginine deiminase (AD) activity, and could show that all tested strains had an increased AD-activity after a temperature shift from 37°C to 42°C. Immuno electron microscopy revealed that, after temperature shift to 42°C, AdiS was expressed on the streptococcal surface in association with the capsular polysaccharides. In conclusion, we have identified a novel stress regulated operon that codes for surface-associated proteins of the ADS and may, thus, facilitate survival of S. suis in the host.
Cloning and characterization of a Lon homologous protease from Bartonella henselae Zimmermann, R. 1, Koenig, J.1; Sander, A. 1 iUniversity of Freiburg; Institute of Medical Microbiology and Hygiene Introduction:
Bartonella henselae is the infectious agent of cat scratch disease, bacillary angiomatosis and bacillary peliosis hepatis. The reservoirs for this bacterium are cats which can transmit the pathogen to humans. Depending on the environmental conditions the availability and activity of cellular proteins must be properly adjusted by the bacteria. Protein activity is
http://www.dghm.org
273
Wissenschaftliches Programm 55. DGHM-Tagung 29. September-1. Oktober 2003 in Dresden Abstracts - Poster regulated by a variety proteolytic processing.
of
mechanisms,
%G~r~GESEtl•Q
~
s
including
Methods and Data: We successfully cloned and characterized a Ion protease gene homolog from B. henselae. The nucleotide sequence encodes a protein of 807 amino acids with a predicted molecular mass of 90.4 kDa. The deduced amino acid sequence showed a high identity and homology to respective proteins from Brucella melitensis, Rhizobium and Agrobacterium species. The conserved amino acid sequence of a Walker A and B motif for ATP binding and the serine in the putative active site was detected. An open reading frame homologous to the ATP-depending protease gene xlpX was found before and a homologous to the DNA binding protein gene hu after the Ion gene. We could demonstrate that the transcription of the Ion gene of B. henselae was induced after heat shock and ethanol exposition. The transcriptional start site was determined, and the promoter showed sequence similarities to the sigma32-factor dependent polymerases. The Ion gene was heterologous expressed in E. coil TB1. The protein was purified to apparent homogeneity and the characteristic ATP-dependent proteolytic activity is under investigation. Our B. henselae Ion mutant, with a KanR Transposon inserted into the Ion gene, showed a retarded growth compared to the WT strain and exhibited a higher UV sensitivity. Conclusions: The gene homologous to a Ion-protease was characterized, and the protein was purified. Additionally our Ion-mutant is more sensitive to UV treatment. The precise role of the Ion protease in B. henselae is under further investigation.
The aspartate ammonia-lyase of Actinobacillus pleuropneumoniae is upregulated in an ex-vivo model. Jacobsen, 1.1; Trost, M.2; Gerlach, G.F} ITier~rztliche Hochschule Hannover; Zentrum for Infektionsmedizin, Institut fElr Mikrobiologie und Tierseuchen 2Gesellschaft few Biotechnologische Forschung ( GBF); Zellbiologie Introduction: Actinobacillus (A.) pleuropneurnoniae is the etiological agent of a porcine respiratory disease characterized by acute infection and subsequent sequestration of affected tissues. The pathogen is able to survive on the surface of lung epithelia, in the tonsils, and in the anaerobic environment of encapsulated sequesters. In order to identify putative virulence associated factors we used an ex-vivo model modulating A. pleuropneumoniae protein expression by the addition of bronchoalveolar lavage fluid (BALF) to culture media.
http://www.dghm.org
274