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Infection and Immunity, January 2001, p. 392-399, Vol. 69, No. 1
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.1.392-399.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Identification and Disruption of Two Discrete Loci Encoding Hyaluronic Acid Capsule Biosynthesis Genes hasA, hasB, and hasC in Streptococcus uberis

Philip N. Ward,1 Terence R. Field,1 William G. F. Ditcham,1 Emmanuelle Maguin,2 and James A. Leigh1,*

Institute for Animal Health, Compton Laboratory, Compton, Berkshire RG20 7NN, United Kingdom,1 and Laboratoire de Genetique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas, France2

Received 24 May 2000/Returned for modification 31 July 2000/Accepted 9 October 2000

The hyaluronic acid capsule of Streptococcus uberis has been implicated in conferring resistance to phagocytosis by bovine neutrophils. Construction of a bank of random insertion mutants of S. uberis (strain 0140J) was achieved using the pGh9::ISS1 mutagenesis system (22). Phenotypic screening of approximately 5,000 clones enabled the isolation of 11 acapsular mutants. Southern hybridization indicated that two mutants carried a lesion within a group of genes similar to those involved in the assembly of the hyaluronic acid capsule found in the group A Streptococcus (GAS) has operon. The DNA sequence flanking the points of insertion confirmed the presence of homologues of GAS hasA and hasB in S. uberis. The DNA sequence flanking the ISS1 insertion in another mutant identified a homologue of hasC in S. uberis. The GAS hasABC operon structure was not conserved in S. uberis, and two discrete loci comprising homologues of either hasAB or hasC were identified. Disruption of S. uberis hasA or hasC resulted in the complete cessation of hyaluronic acid capsule production. Correspondingly, these mutants were found to have lost their resistance to phagocytosis by bovine neutrophils. The bactericidal action of bovine neutrophils on S. uberis 0140J was shown unequivocally to depend upon the capsule status of the bacterium.

* Corresponding author. Mailing address: Institute for Animal Health, Compton Laboratory, Compton, Berkshire RG20 7NN, United Kingdom. Phone: 44 (0) 1635 578411. Fax: 44 (0) 1635 577243. E-mail: james.leigh{at}bbsrc.ac.uk.

Infection and Immunity, January 2001, p. 392-399, Vol. 69, No. 1
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.1.392-399.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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