HtrA is the unique surface housekeeping protease in Lactococcus lactis and is required for natural protein processing

Abstract

SummaryWe identified an exported protease in Lactococcus lactis ssp. lactis strain IL1403 belonging to the HtrA/ DegP family. Inactivation of the chromosomal gene (htrA Ll ) encoding this protease (HtrA Ll ) results in growth thermo-sensitivity at very high temperatures (above 378C for L. lactis). The role of htrA Ll in extracellular proteolysis under normal growth conditions was examined by testing the stability of different exported proteins (i.e. fusions, a heterologous pre-pro-protein or a native protein containing repeats), having different locations. In the wild-type (wt) strain, degradation products, including the Cterminal protein ends, were present in the medium, indicating that proteolysis occurs during or after export to the cell surface; in one case, degradation was nearly total. In contrast, proteolysis was totally abolished in the htrA strain for all five proteins tested, and the yield of full-length products was significantly increased. These results suggest that HtrA Ll is the sole extracellular protease that degrades abnormal exported proteins. In addition, our results reveal that HtrA Ll is needed for the pro-peptide processing of a natural pro-protein and for maturation of a native protein. We propose that in lactococci, and possibly in other Gram-positive organisms with small sizedgenomes, a single surface protease, HtrA, is totally responsible for the housekeeping of exported proteins.

VimA is part of the maturation pathway for the major gingipains of Porphyromonas gingivalis W83

“…However, proteolytic activity is induced at high temperature. HtrA has also been shown to degrade misfolded proteins; it can play a role in oxidative stress resistance and can regulate growth at high temperature (az-Torres & Russell, 2001;Cortes et al, 2002;Foucaud-Scheunemann & Poquet, 2003;Lipinska et al, 1990;Lyon & Caparon, 2004;Pallen & Wren, 1997;Poquet et al, 2000). In addition, HtrA is involved in the maturation of proteins, including cysteine protease (Poquet et al, 2000;Foucaud-Scheunemann & Poquet, 2003;Lyon & Caparon, 2004).…”

Peptidoglycan N-acetylglucosamine deacetylation decreases autolysis in Lactococcus lactis

“…No difference was found in the intensity of the AcmA activity band on micrococci used as substrate between the pgdA mutant and wild-type IL6288 or the overexpressing strain (data not shown). Breakdown products of AcmA, which retained activity in the zymogram (Poquet et al, 2000;Steen et al, 2003), were barely detected in IL6288 and were present in similar amounts in its derivative mutants, indicating no difference in AcmA degradation among the tested strains.…”

“…Different mechanisms of PGH control have been proposed, including proteolytic maturation (Buist et al, 1998;Poquet et al, 2000;Shockman, 1992), interaction with a specific modifier protein (Lazarevic et al, 1992), regulation by the membrane proton motive force (Calamita et al, 2001;Kemper et al, 1993), and interaction with secondary cell wall polymers such as teichoic acids (Palumbo et al, 2006;Wecke et al, 1997). Finally, structural modifications of the peptidoglycan substrate by O-acetylation (Pfeffer et al, 2006) or de-N-acetylation (Atrih et al, 1999) may control the action of PGHs.…”

Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria

“…PA-fusion proteins can be produced by L. lactis PA1001, a MG1363 derived strain lacking acmA and htrA genes, and containing the nisRK genes needed for nisin induced expression. The housekeeping protease HtrA has been shown to degrade the AcmA cell-wall binding domain [25][26][27] and strains lacking htrA are more eYcient in production of PA containing proteins. Lack of the acmA gene causes the bacteria to grow in strings, since the hydrolase needed for cell separation is absent [25].…”