TatCy and TatAy-dependent YwbN secretion
In an approach to identify Tat-dependent B. subtilis preproteins other than PhoD, the 46 kDa YwbN preprotein was tagged with the c-Myc epitope (EQKLISEEDLN), as described in Supplementary material. YwbN was selected, because its signal peptide conforms to the most stringent criteria for predictions of genuine RR-signal peptides of E. coli (Jongbloed et al., 2000; 2002). The YwbN-myc encoding gene was placed under control of the xylose-inducible xylA promoter, and the resulting xylA-ywbN-myc cassette (X-ywbN) was integrated into the amyE gene of B. subtilis 168. To test YwbN-myc production, an overnight culture of the resulting strain 168 X-ywbN was diluted in fresh medium, grown for 3 h without and, subsequently, for 3 h with 1% xylose. As revealed by Western blotting and immunodetection with c-Myc specific antibodies, YwbN-myc was produced at detectable levels (Fig. 1). Both precursor and mature forms of YwbN-myc were identified in cells, and mature YwbN-myc was present in the medium (Fig. 1A). An unidentified, extracellular protein (protein X), migrating close to mature YwbN-myc during SDS-PAGE, cross-reacts with c-Myc specific antibodies. The detection of protein X depends on the batch of c-Myc antibody used.
Figure 1. Tat-dependent YwbN secretion. B. subtilis strains 168 (wt), 168 X-ywbN, tatCd-tatCy X-ywbN, and total-tat2 X-ywbN (A), and strains 8G5 X-ywbN (wt) and ΔsipSU X-ywbN (B) were analysed as described in the Experimental procedures section. Precursor and mature forms of YwbN-myc are indicated. Protein X, extracellular B. subtilis protein, which is cross-reactive with c-Myc specific antibodies.
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To test whether YwbN is secreted Tat-dependently, the X-ywbN cassette was introduced into B. subtilis strains tatCd-tatCy, lacking both tatC genes, and total-tat2, lacking all tat genes. To study the effects of these mutations, cells of strains 168 X-ywbN, tatCd-tatCy X-ywbN, and total-tat2 X-ywbN were analysed as described above. Western blotting showed significantly reduced levels of mature YwbN-myc in cellular fractions of tat mutants lacking both TatC or all known Tat components (Fig. 1A). Moreover, YwbN-myc was absent from the medium. These observations show that YwbN secretion is dependent on a functional Tat pathway.
To verify whether processing of preYwbN-myc to the mature form was catalysed by signal peptidases (SPases) that are required for the processing of genuine secretory proteins, the X-ywbN cassette was introduced in B. subtilis strains lacking one or more of the five chromosomal SPase-encoding sip genes. Single sip mutations did not affect processing of preYwbN-myc and secretion of mature YwbN-myc into the medium (not shown). However, mature YwbN-myc was completely absent from the medium of a strain lacking SipS and SipU (Fig. 1B). Moreover, cellular fractions showed reduced levels of a mature product. These results show that translocation of pre-Y wbN-myc across the membrane and release of the mature protein into the medium depends on a functional Tat pathway and either SipS or SipU.
Previous analyses showed that TatCd is specifically required for PhoD secretion (Jongbloed et al., 2000). Therefore, we investigated whether one or both TatC components are involved in YwbN secretion. For this purpose, the X-ywbN cassette was introduced into B. subtilis strains carrying tatCd or tatCy mutations. The resulting strains were analysed as described above. Like the double tatC and total-tat mutants, the tatCy mutant did not secrete mature YwbN-myc into the medium, and only small amounts of mature YwbN-myc were detectable in cells (Fig. 2A). In contrast, significant amounts of mature YwbN-myc were present in cellular and medium fractions of the tatCd mutant and the parental strain 168. These observations show that TatCy, but not TatCd, has a critical role in YwbN secretion.
Figure 2. TatAy and TatCy-dependent YwbN secretion. B. subtilis strains 168 (wt), tatCd, tatCy, tatCd-tatCy, and total-tat2 (A), 168 (wt), tatAc1, tatAd, tatAy, tatAd-tatAc2, tatAc1-tatAy, tatAd-tatAy, and total-tatA (B), and 168 (wt), tatAy (pGDL48; empty vector), tatAy (pCAy; tatAy), tatAy (pCCy; tatCy), and tatAy (pCACy; tatAyCy) (C), all containing the X-ywbN cassette, were analysed as described in the Experimental procedures section. Precursor and mature forms of YwbN-myc are indicated. Protein X, extracellular B. subtilis protein.
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To investigate whether one or more TatA components are involved in YwbN secretion, single and multiple tatA mutants were constructed. The X-ywbN cassette was introduced into the resulting mutants (Table 1; see: Supplementary material, TableS2), and YwbN-myc secretion analysed. As shown by Western blotting, YwbN-myc was absent from the media of all strains lacking tatAy (Fig. 2B; note that small variations in extracellular YwbN levels of TatAyCy-containing strains can occur). In contrast, tatAc and tatAd single or double mutants secreted significant amounts of YwbN-myc into the medium. Consistently, tatAy mutant cells contained relatively small amounts of mature YwbN-myc compared to cells with an intact tatAy gene. These findings indicate that TatAy, but not TatAc or TatAd, is required for YwbN secretion.
Table 1. Plasmids and strains.
| ||Relevant properties||Reference|
| pX ||Vector for the integration of genes in the amyE locus of B. subtilis; integrated genes transcribed from the xylA promoter; carries the xylR gene; 7.5 kb; Apr; Cmr||Kim et al. (1996)|
| pXYNm1||pX-derivative; carries ywbN-myc downstream of the xylA promoter; 8.9 kb; Apr; Cmr||This work|
| pGDL48 ||Contains multiple cloning site to place genes under the control of the erythromycin promoter; 6.8 kb; Apr; Kmr||Tjalsma et al. (1998)|
| pCAy||pGDL48-derivative containing the tatAy gene; 7.0 kb; Apr; Kmr||This work|
| pCCy||pGDL48-derivative containing the tatCy gene; 7.5 kb; Apr; Kmr||This work|
| pCACy||pGDL48-derivative containing the tatAy-tatCy operon; 7.7 kb; Apr; Kmr||This work|
| B. subtilis|
| 168||trpC2||Kunst et al. (1997)|
| tatAc||trpC2; tatAc::Em; Emr, previously referred to as ΔtatAc||Jongbloed et al. (2002)|
| tatAc1||trpC2; tatAc::Km; Kmr||This work|
| tatAc2||trpC2; tatAc::Tc; Tcr||This work|
| tatAd||trpC2; tatAd::Km; Kmr||This work|
| tatAy||trpC2; tatAy::Em; Emr||This work|
| tatAd-tatAc2||trpC2; tatAd::Km; tatAc::Tc; Kmr; Tcr||This work|
| tatAc1-tatAy||trpC2; tatAc::Km; tatAy::Em; Kmr; Emr||This work|
| tatAd-tatAy||trpC2; tatAd::Km; tatAy::Em; Kmr; Emr||This work|
| total-tatA||trpC2; tatAd::Km; tatAc::Tcr; tatAy::Emr; Kmr; Tcr; Emr||This work|
| tatCd||trpC2; tatCd::Km; Kmr; previously referred to as ΔtatCd||Jongbloed et al. (2000)|
| tatCy||trpC2; tatCy::Sp; Spr; previously referred to as ΔtatCy||Jongbloed et al. (2000)|
| tatCd-tatCy||trpC2; tatCd::Km; tatCy::Sp; Kmr; Spr; previously referred to as ΔtatCd-ΔtatCy||Jongbloed et al. (2000)|
| tatAdCd||trpC2; tatAd-tatCd::Km; Kmr||This work|
| tatAyCy||trpC2; tatAy-tatCy::Sp; Spr; previously referred to as ΔtatAyCy||Jongbloed et al. (2002)|
| tatAdCd-tatAc2||trpC2; tatAd-tatCd::Km; tatAc::Tc; Kmr; Tcr||This work|
| tatAc-tatAyCy||trpC2; tatAc::Em; tatAy-tatCy::Sp; Emr; Spr; previously referred to as ΔtatAc-ΔtatAyCy||Jongbloed et al. (2002)|
| tatAyCy-tatAc2||trpC2; tatAy-tatCy::Sp; tatAc::Tc; Spr; Tcr||This work|
| total-tat2||trpC2; tatAc::Em; tatAy-tatCy::Sp; tatAd-tatCd::Km; Emr Spr; Kmr||This work|
| 8G5||like 168; tyr; his; nic; ura; rib; met; ade||Tjalsma et al. (1998)|
| ΔsipSU||like 168; tyr; his; nic; ura; rib; met; ade; sipS; sipU; previously referred to as ΔSU||Tjalsma et al. (1998)|
| all strains designated X-ywbN||amyE::xylA-ywbN-myc; Cmr||This work|
As tatCy is located downstream of tatAy, the replacement of the latter might have polar effects on tatCy transcription and thus TatCy production. To test which gene(s) of the tatAy-tatCy region can restore YwbN export in the tatAy mutant, a trans complementation analysis was performed with the tatAy, tatCy, or tatAy plus tatCy genes. As shown in Fig. 2C, introduction of the plasmid-borne tatAy-tatCy region in the tatAy mutant did not only restore secretion of YwbN-myc, but also resulted in significantly increased extracellular levels of this protein (compare also the relative levels of YwbN and protein X with those in Fig. 1A). In contrast, plasmid-borne copies of tatAy or tatCy did not restore YwbN-myc secretion, showing that the expression of tatCy is affected in tatAy mutants. These findings show that TatAy and TatCy are indispensable for YwbN secretion.
TatAy and TatCy are sufficient for YwbN secretion
We next raised the question whether TatAy and TatCy are sufficient for the export of YwbN. To answer this question, tatAd-tatCd double and tatAd-tatCd tatAc triple mutants were constructed. As controls, tatAy-tatCy double and tatAy-tatCy tatAc triple mutants were constructed. Next, the X-ywbN cassette was introduced in these strains and YwbN-myc secretion was analysed. As shown by Western blotting, the simultaneous disruption of tatAd-tatCd (not shown), or tatAd-tatCd and tatAc(Fig. 3), did not block YwbN-myc secretion into the medium. Consistently, cells of the tatAd-tatCd (not shown) and tatAd-tatCd tatAc mutants contained significant amounts of mature YwbN-myc (Fig. 3). As expected, the tatAy-tatCy double and the tatAy-tatCy tatAc triple mutants did not secrete YwbN-myc (not shown). These results show that TatAy and TatCy are sufficient for YwbN secretion.
Figure 3. TatAc, TatAd and TatCd are dispensable for YwbN secretion. B. subtilis strains 168 X-ywbN (wt), total-tat2 X-ywbN, and tatAdCd-tatAc2 X-ywbN were analysed as described in the Experimental procedures section. Precursor and mature forms of YwbN-myc are indicated.
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TatAd and TatCd are sufficient for PhoD secretion
Previous studies showed that TatCd is of major importance for PhoD secretion (Jongbloed et al., 2000), and that TatAd also fulfils a role in membrane targeting and/or secretion of this protein (Pop et al., 2002; 2003). To address the question whether TatAd and TatCd are sufficient for PhoD secretion, the export of PhoD by the tatAy-tatCy tatAc triple mutant was studied. The mutant was grown under conditions of phosphate starvation, inducing phoD expression, and PhoD secretion was analysed by 2D gel electrophoresis. Figure 4 shows that mature PhoD was present in the media of both the tatAy-tatCy tatAc mutant and the parental strain 168. Moreover, the secretion of proteins lacking (genuine) RR-signal peptides was not affected by the tat mutation, as exemplified by the ‘control spots’ of WprA, LytD, XynD, YnfF, YwtD and Pel. This shows that TatAd and TatCd are sufficient for PhoD secretion. In conclusion, our present observations show that minimal Tat translocases of B. subtilis are composed of specific TatA and TatC molecules.
Figure 4. TatAc, TatAy and TatCy are dispensable for PhoD secretion. B. subtilis strains 168 X-ywbN (wt) and tatAyCy-tatAc2 X-ywbN were analysed as described in the Experimental procedures section. Only the relative positions of the PhoD, LytD, Pel, WprA, XynD, YnfF and YwtD spots are shown.
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