Agr and sar are known regulatory loci of Staphylococcus aureus that control the production of several extracellular and cell-wall-associated proteins. A pleiotropic insertional mutation in S. aureus, designated sae, that leads to the production of drastically diminished levels of alpha- and beta-hemolysins and coagulase and slightly reduced levels of protein A has been described. The study of the expression of the genes coding for these exoproteins in the sae::Tn551 mutant (carried out in this work by Northern blot analyses) revealed that the genes for alpha- and beta-hemolysins (hla and hlb) and coagulase (coa) are not transcribed and that the gene for protein A (spa) is transcribed at a somewhat reduced level. These results indicate that the sae locus regulates these exoprotein genes at the transcriptional level. Northern blot analyses also show that the sae mutation does not affect the expression of agr or sar regulatory loci. An sae::Tn551 agr::tetM double mutant has been phenotypically characterized as producing reduced or null levels of alpha-, beta-, and delta-hemolysins, coagulase, and high levels of protein A. Northern blot analyses carried out in this work with the double mutant revealed that hla, hlb, hld, and coa genes are not transcribed, while spa is transcribed at high levels. The fact that coa is not expressed in the sae agr mutant, as in the sae parental strain, while spa is expressed at the high levels characteristic of the agr parental strain, suggests that sae and agr interact in a complex way in the control of the expression of the genes of several exoproteins.

A Tn551 insertional pleiotropic mutant defective in the production of several exoproteins was isolated from Staphylococcus aureus 196E and characterized. The pleiotropism of the mutant was due to a single insertion of the transposon as evidenced by Southern blot hybridization and by the transfer of its phenotype by transduction to S. aureus ISP479. The mutants showed diminished or null levels of alpha- and beta-hemolysis, DNase, coagulase, and protein A in the supernatants of broth cultures. Production of proteases, lipase, staphylokinase, or enterotoxin A was not modified. The mutants did synthesize the cell-bound form of protein A and also the extracellular form of this protein coded by pRIT11, which lacks the COOH-terminal segment of the molecule. These observations suggest that the sae locus does not involve a positive regulatory gene acting at the transcriptional level. The phenotype of the mutant was different from that of other insertional mutants affecting exoprotein synthesis, such as agr, xpr, or sar. This new mutation has been designated sae (for S. aureus exoprotein expression).

Sae is a regulatory locus that activates the production of several exoproteins in Staphylococcus aureus. A 3.4-kb fragment of a S. aureus genomic library, screened with a probe adjacent to the transposon insertion of a sae::Tn551 mutant, was cloned into a bifunctional vector. This fragment was shown to carry the sae locus by restoration of exoprotein production in sae mutants. The sae locus was mapped to the SmaI-D fragment of the staphylococcal chromosome by pulse-field electrophoresis. Sequence analysis of the cloned fragment revealed the presence of two genes, designated saeR and saeS, encoding a response regulator and a histidine protein kinase, respectively, with high homology to other bacterial two-component regulatory systems.

Global regulatory locus sae consists of a two-component signal transduction system coded by saeR and saeS genes that upregulates the transcription of several exoproteins. Northern analysis carried out in this study reveals the synthesis at late and post-exponential phases of a cotranscript of saeR and saeS structural genes of about 2.4 kb. This transcript is diminished in the isogenic agr:: tetM mutant. Likewise, transcriptional fusion experiments show that sae expression is downregulated in the agr null mutant. Complementation analyses with plasmids carrying fragments of about 1.2 or 0.2 kbp upstream of saeR-saeS genes, which restore fully or only partially, respectively, the wild-type phenotype to the sae mutant, are in agreement with two initiation start points of transcription revealed by primer extension experiments. This work, as well as previous studies, reveals a complex hierarchical regulatory network involving several loci that control the expression of virulence determinants in S. aureus.

A vaccine was developed against bovine mastitis based on inactivated, highly encapsulated Staphylococcus aureus cells; a crude extract of Staph. aureus exopolysaccharides; and inactivated, unencapsulated Staph, aureus and Streptococcus spp. cells. This vaccine was tested on 30 heifers during a 7-mo period. The 30 heifers were randomly assigned to three groups of 10 heifers each. The prepartum group received two injections of the vaccine at 8 and 4 wk before calving, and the postpartum group received two injections at 1 and 5 wk after calving. The control group received two injections of a placebo at 8 and 4 wk before calving. The vaccine or the placebo was administered subcutaneously in the brachiocephalicus muscle of the neck. The frequencies of intramammary infections caused by Staph. aureus were reduced from 18.8% for heifers in the control group to 6.7 and 6.0% for heifers in the prepartum and postpartum groups, respectively. This protective effect was maintained for at least 6 mo. The relative risk of mastitis caused by Staph. aureus was 0.31 and 0.28 for heifers in the prepartum and postpartum groups, respectively, compared with that for heifers in the control group. The results of the trial indicated the effectiveness of the vaccine in decreasing the incidence of intrammammary infections caused by Staph. aureus. A slight but nonsignificant increase occurred in fat production in the milk of vaccinated cows. The vaccine had no observable effect on somatic cell count or streptococcal infections.