cereus KF1, a strain isolated from artisanal kefir produced in Vietnam, were included. For flagellin extraction,

we set up a protocol involving the use of 5 M LiCl. LiCl is a chaotropic agent which destabilizes both hydrophobic/electrostatic interactions and hydrogen bonds, leading to the extraction of noncovalent surface-associated proteins (Sánchez et al., 2008). LiCl (5 M) solution was always supplemented with EDTA 5 mM and PMSF 1 mM, the absence of the latter leading to complete proteolysis of the flagellins (data not shown). As shown in Fig. 1a, incubations of 30 min at 4 °C led to the extraction of prominent bands with observed molecular masses of 28–60 kDa. All bands were identified as Bacillus flagellins by MS, or by tandem MS (MS/MS) in cases that needed a more powerful analytic technique (Table 2). A single flagellin product was

INCB024360 concentration detected in all the cases except for B. cereus ATCC Sorafenib 14579 and B. cereusN, in which two bands identified as flagellins were observed. These flagellins could be extracted from SDS gels and renaturalized as described (Peant & LaPointe, 2004); they were able to bind mucin, as shown in a previous work (data not shown) (Sánchez et al., 2009a). The size of the different flagellins varied from approximately 30 to 60 kDa (Fig. 1). In the B. cereus group, flagellins comprise a set of variable proteins in terms of sequence and molecular masses. This is due to 3-mercaptopyruvate sulfurtransferase the fact that the domains D2 and D3 are highly variable, producing potentially infinite variants (Beatson et al., 2006). The gene coding from the B. cereus CH flagellin of 45 kDa was amplified using its total DNA as template. Primer sequences were deduced from protein sequence AAZ22698, to which B. cereus CH flagellin showed the higher degree of identity. This gene was cloned into the blunt-end NaeI site of pNZ8110, the resulting ligation being electroporated in L. lactis NZ9000. In this way we isolated a Lactococcus clone producing the recombinant flagellin, which was denominated L. lactis ssp. cremoris NZ9000-CH. Surface-associated protein and secreted protein profiles were obtained and, surprisingly, two bands of around 30 and 25 kDa were identified in

the supernatant fraction of the induced cultures (data not shown). After MS analysis, these bands were properly identified as the B. cereus CH flagellin (Table 1). These results suggested that flagellin was proteolyzed on the Lactococcus surface, an aberrant form of the flagellin being released into the bacterial environment. It is known that the housekeeping protease HtrA from L. lactis, is targeted to the cell surface, where it degrades abnormal proteins, somehow monitoring the quality of surface proteins (Lyon & Caparon, 2004). We thought of the possibility that the recombinant protein was recognized as aberrant, being degraded by the action of this enzyme (or other surface-associated proteases) and released to the surrounding media.