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MP049 Differential fluorescence induction in Streptococcus agalactiae
Wissenschaftliches Programm 55. DGHM-Tagung 29. September-l. Oktober 2003 in Dresden Abstracts - Poster
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Differential fluorescence induction in
Streptococcus agalactiae Gleich, U.1; Pohl, B.2; Weber-Heynemann, j.2; Spellerberg, B. 1
1University of UIm; Department of Medical Microbiology and Hygiene 2University Hospital of the RWTH Aachen; Institute of Medical Microbiology Introduction: Contact with human tissues or body fluids can modulate gene expression and virulence of bacterial pathogens. Streptococci are responsible for a variety of infections in human and animal hosts. Streptococcus agalactiae causes invasive diseases in neonates and immunocompromised adult patients as well as bovine mastitis. Methods and Data: To identify genes that are activated in the human host, we constructed a green fluorescent reporter gene construct in the streptococcal shuttle vector pAT28. A copy of the EGFP gene lacking a promoter was inserted downstream of the multiple cloning site of pAT28. Insertion of the promoter of the CAMP- factor gene cfb resulted in a strong green fluorescence of S. agalactiae strains transformed with this construct. To screen for promoters that are differentially activated, a plasmid pool was created by insertion of a random chromosomal digest of S. agalactiae upstream of EGFP. This system allows the isolation of differentially activated genes by FACS. To isolate S. agalactiae genes that are activated upon contact with human serum, the plasmid pool was transformed into the S. agalactiae strain 090R and grown to midlogarithmic phase in the presence of pooled human serum. Clones that were activated under these conditions were selected and retested in regular growth medium without the stimulus. Differentially activated genes were selected for further analysis. To confirm differential activation of a gene, real time RT-PCR analysis was carried out. This procedure led to the isolation of one clone on three different occasions harboring a fragment of a recently identified regulator of S. agalactiae. Activation of this gene by human serum was dose dependent and could be destroyed by heatinactivation of the serum. Conclusion: The DFI technique can be successfully applied for the isolation of S, agalactiae genes that are differentially activated by contact with human tissue or body fluids.
http://www.dghm.org
271
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a o
~ o
Differential fluorescence induction in
Streptococcus agalactiae Gleich, U.1; Pohl, B.2; Weber-Heynemann, j.2; Spellerberg, B. 1
1University of UIm; Department of Medical Microbiology and Hygiene 2University Hospital of the RWTH Aachen; Institute of Medical Microbiology Introduction: Contact with human tissues or body fluids can modulate gene expression and virulence of bacterial pathogens. Streptococci are responsible for a variety of infections in human and animal hosts. Streptococcus agalactiae causes invasive diseases in neonates and immunocompromised adult patients as well as bovine mastitis. Methods and Data: To identify genes that are activated in the human host, we constructed a green fluorescent reporter gene construct in the streptococcal shuttle vector pAT28. A copy of the EGFP gene lacking a promoter was inserted downstream of the multiple cloning site of pAT28. Insertion of the promoter of the CAMP- factor gene cfb resulted in a strong green fluorescence of S. agalactiae strains transformed with this construct. To screen for promoters that are differentially activated, a plasmid pool was created by insertion of a random chromosomal digest of S. agalactiae upstream of EGFP. This system allows the isolation of differentially activated genes by FACS. To isolate S. agalactiae genes that are activated upon contact with human serum, the plasmid pool was transformed into the S. agalactiae strain 090R and grown to midlogarithmic phase in the presence of pooled human serum. Clones that were activated under these conditions were selected and retested in regular growth medium without the stimulus. Differentially activated genes were selected for further analysis. To confirm differential activation of a gene, real time RT-PCR analysis was carried out. This procedure led to the isolation of one clone on three different occasions harboring a fragment of a recently identified regulator of S. agalactiae. Activation of this gene by human serum was dose dependent and could be destroyed by heatinactivation of the serum. Conclusion: The DFI technique can be successfully applied for the isolation of S, agalactiae genes that are differentially activated by contact with human tissue or body fluids.
http://www.dghm.org
271