- Research article
- Open Access
The Yersinia enterocolitica type three secretion chaperone SycO is integrated into the Yop regulatory network and binds to the Yop secretion protein YscM1
© Dittmann et al; licensee BioMed Central Ltd. 2007
- Received: 29 November 2006
- Accepted: 05 July 2007
- Published: 05 July 2007
Pathogenic yersiniae (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica) share a virulence plasmid encoding a type three secretion system (T3SS). This T3SS comprises more than 40 constituents. Among these are the transport substrates called Yops (Yersinia outer proteins), the specific Yop chaperones (Sycs), and the Ysc (Yop secretion) proteins which form the transport machinery. The effectors YopO and YopP are encoded on an operon together with SycO, the chaperone of YopO. The characterization of SycO is the focus of this study.
We have established the large-scale production of recombinant SycO in its outright form. We confirm that Y. enterocolitica SycO forms homodimers which is typical for Syc chaperones. SycO overproduction in Y. enterocolitica decreases secretion of Yops into the culture supernatant suggesting a regulatory role of SycO in type III secretion. We demonstrate that in vitro SycO interacts with YscM1, a negative regulator of Yop expression in Y. enterocolitica. However, the SycO overproduction phenotype was not mediated by YscM1, YscM2, YopO or YopP as revealed by analysis of isogenic deletion mutants.
We present evidence that SycO is integrated into the regulatory network of the Yersinia T3SS. Our picture of the Yersinia T3SS interactome is supplemented by identification of the SycO/YscM1 interaction. Further, our results suggest that at least one additional interaction partner of SycO has to be identified.
- Deletion Mutant
- Yersinia Enterocolitica
- Secretion Efficiency
- Transport Machinery
- Enterocolitica Strain
An ever increasing number of Gram-negative bacteria is known to utilize a type three secretion system (T3SS), mostly pathogens that manipulate their host cells by injecting effector proteins . One of the characteristics of T3SSs is the existence of specific energy-independent chaperones which assist several of the transport substrates. Chaperones assisting the effectors (class I chaperones) are distinguished from those assisting the pore forming translocators (class II chaperones) . The mode of interaction between class I chaperones and their respective substrates is well-investigated, mainly due to crystal structures of several chaperone/substrate complexes [3–5]. Typically, a region of 50–80 amino acids following the putative N-terminal secretion signal is wrapped around a chaperone dimer thereby preventing tertiary structure formation of this substrate region. The rest of the substrate, however, is folded [4–8]. The chaperone/substrate complexes are recognized by a T3SS-specific ATPase , which is able to displace the chaperone from its substrate and further to act as an unfoldase unraveling the substrate .
Despite these recent discoveries, the substrate recognition process has remained enigmatic [11, 12]. It has been suggested that the chaperone/substrate complexes may constitute a three-dimensional secretion signal . This hypothesis is supported by the intriguing fact that the structures of all classI chaperones share a common fold even across the genus barrier and despite low sequence similarity.
The T3SS of pathogenic Yersinia spp. is among the most intensively studied T3SSs with the structures of the homodimeric chaperones SycE [13–15], SycH , SycT [17, 18], and the atypical SycN/YscB heterodimer  being solved. According to phylogenetic analyses and experimental results, the Yersinia T3SS holds at least one additional classI chaperone, SycO [19, 20].
Here, we focus on characterization of Y. enterocolitica SycO, encoded by a gene termed orf155 until recently. In Y. enterocolitica, sycO is organized in an operon together with yopO (ypkA in Y. pseudotuberculosis and Y. pestis) and yopP (yopJ in Y. pseudotuberculosis and Y. pestis). Within this operon, sycO is located upstream of the yopO gene. A nonpolar deletion mutant of orf1, the sycO orthologue in Y. pseudotuberculosis, secreted YpkA and YopJ in amounts comparable to the wildtype and was fully virulent. Further, orf1 was not found to be involved in temperature and calcium-dependent control of Yop expression and secretion . Because of these results and the use of the infrequent "GTG" start codon of orf1, Galyov et al.  proposed that orf1 might not be translated. Later, the corresponding orf7 of Y. pestis was discussed as a potential Syc-like chaperone due to the typical size (15.7 kDa), isoelectric point (pI of 4.39), and amphipathic character of the putative translation product . However, in Y. enterocolitica the induction of apoptosis by YopP in macrophages was shown to be independent of orf155, and it was found that secretion of YopO into the culture supernatant was apparently not restricted in the absence of orf155 . Only recently, a chaperone function of the orf155 product on the effector YopO was reported. Consequently, the gene product of orf155 was termed SycO and the gene was renamed sycO .
Here, we characterize recombinantly produced SycO as a homodimer. We demonstrate the potential of SycO to influence Yop expression and secretion, and identify a novel interaction partner of SycO.
Recombinant production of SycO
To examine SycO in vitro, we recombinantly produced SycO in E. coli and purified it to near homogeneity. For this purpose, we followed a protocol previously established for production of SycH and SycT to generate the proteins in their outright form without an affinity tag [7, 18]. The sycO sequence of Y. enterocolitica strain WA-314  was amplified by PCR, thereby changing the start codon from "GTG" to "ATG" to ensure maximal expression. The fragment was cloned into vector pWS . Expression was performed in E. coli BL21 (DE3) pLysS. SycO was produced in abundance and its solubility was optimized by expression at 27°C overnight. A purification protocol was established combining ammonium sulfate precipitation followed by MonoQ-based anion exchange chromatography and a final gel filtration step. The yield was approximately 15 mg of purified protein per liter of culture with a homogeneity of about 95%.
Recombinant SycO forms homodimers
Overproduction of SycO represses Yop secretion in Y. enterocolitica
Strikingly, analysis of the pellet fractions revealed a decrease of all Yops including YopO within the cells overproducing SycO (Fig. 2C). It was recently demonstrated, that secretion of YopO into the culture supernatant does not strictly rely on SycO [20, 23]. Thus, excess of SycO could increase the secretion efficiency of YopO. The finding that the amount of secreted YopO was constant despite of reduced intracellular levels suggests that YopO secretion efficiency was increased by excess of SycO.
On closer inspection of YopP secretion, it became evident that deletion of yopO generated a polar effect resulting in reduced YopP secretion (Fig. 2A). Interestingly, SycO overproduction did not cause as pronounced a reduction of YopP secretion in the yopO mutant as in the parental strain. This observation might point to an interrelation between YopO/SycO and YopP and hence to an explanation for the operon structure.
Taken together, these results suggest that SycO overproduction interferes with the T3SS regulatory network. Strikingly, this effect is neither mediated by an interaction of SycO with its effector YopO nor by an interaction with YopP which is also encoded within the same operon.
As a further control of specificity, we analyzed the influence of SycO overproduction on the Sec-pathway by monitoring beta-lactamase-mediated resistance to carbenicillin. We found no influence of SycO overproduction on the level of carbenicillin resistance even at concentrations above 1 mg/ml of carbenicillin (data not shown).
Differential effect of SycO overproduction on accumulation of Yops in the Yersinia cytosol in the presence of calcium ions
SycO interacts with the T3SS regulatory protein YscM1
Cross-linking experiments were performed to verify the YscM1/SycO interaction. However, the data did not help to interpret the results from native gel electrophoresis since YscM1 and SycO are very similar in size and both form homodimers (, Fig. 1). As a consequence, the major cross-linking products could not be unambiguously assigned to heterocomplexes (data not shown).
Overproduction of SycO in the yscM1 and yscM2 deletion mutants
In summary, these results show that neither YscM1 nor YscM2 is required to establish the SycO-mediated shutdown of Yop expression and secretion. However, the yscM1 deletion mutant exhibits a phenotype suggesting that SycO and YscM1 act together in the regulation of YopO and YopP.
In this study, we demonstrate that Y. enterocolitica SycO behaves as a homodimer in solution. This is in full agreement with a recent study from Letzelter et al.  who also applied gel filtration analysis and cross-linking techniques with the same outcome. Homodimer formation is typical for T3SS chaperones of effectors, and SycO dimerization is thus plausible in the light of the recent discovery that SycO serves as a chaperone for YopO . Now, that we have established a protocol for the large-scale production of native SycO we aim at generating high-resolution structural data to characterize SycO on the molecular level.
We have further presented evidence that the functions of SycO are not restricted to chaperoning YopO. We could show that overproduction of SycO causes an apparent repression of Yop secretion and expression. Two principle explanations for this phenomenon seem plausible. On the one hand, SycO could interfere with the regulatory network involved in Yop expression, thereby causing down-regulation of Yop expression and consequently reducing the amount of secreted Yops. On the other hand, SycO could interfere with some component of the T3SS transport machinery. In particular, T3SS chaperones are capable of interacting with the specific ATPase of the transport machinery  irrespective of effector-binding to the chaperones. Thus, abundant SycO could compete with chaperone/Yop complexes and thereby reduce the efficiency of Yop secretion. Since a shutdown of secretion typically results in feedback inhibition of Yop expression, this scenario could explain the phenotype equally well. This latter hypothesis is challenged by the observation that overproduction of SycH in Y. pestis causes a slight increase of Yop expression and secretion as well as a relaxation of the Calcium-dependent control of Yop secretion . Similarly, moderate overproduction of SycH induces Yop production in Y. enterocolitica and Y. pseudotuberculosis . Even the dramatically elevated SycH levels that we applied could not provoke an effect comparable to that of SycO. Therefore, an interference of SycO with the Yop regulatory network is a more likely explanation. Since YopO and YopP proved to be dispensable for mediating the effect caused by SycO overproduction, we speculated on a role of the Yop regulatory proteins YscM1 and YscM2 in this context. Clearly, neither YscM1 nor YscM2 was required to establish the SycO phenotype. Presumably, at least one further interaction partner of SycO has to be identified. This will help to understand whether SycO overexpression acts at the level of transcription or translation.
Nevertheless, we could demonstrate an interaction between YscM1 and SycO in vitro. In support of such an interaction, overproduction of SycO in the yscM1 deletion strain influenced YopO and YopP secretion and intracellular accumulation of YopO. The YscM proteins are involved in the post-transcriptional regulation of Yop expression. In addition, they are T3S substrates themselves and chaperoned by SycH, another actor in the Yop expression network [25, 36, 37]. Recently, binding of SycE to YscM1 and YscM2, and binding of SycD to YscM2 was demonstrated [30, 31]. What could be the meaning of this YscM promiscuity? An unspecific interaction of both YscM1 and YscM2 with the acidic T3SS chaperones seems unlikely, given that the isoelectric point of YscM1 is 6.05 whereas the pI of YscM2 is 9.79. We propose that the YscM proteins function as an interface, integrating information on whether T3SS chaperones are loaded with Yops, and transducing these signals into repression or derepression of Yop translation.
This study supports the recently established view that SycO is a typical T3SS chaperone of class I that increases solubility and secretion efficiency of the effector YopO . Beyond that, our results suggest that SycO is part of the T3SS regulatory network. This finding highlights the need for a complete T3SS interactome to understand the complex T3SS machinery.
Yersinia enterocolitica WA-314 sycO was amplified by PCR using primers 5'-AAAAAACATATG ATTAACTCAACCTTTACTGAGC-3' and 5'-ACTCGAGGTCGAC TCAATAACCGATTGAGTAGATTGAGTAAG-3', thereby introducing NdeI and SalI restriction sites, respectively. The NdeI-SalI cleaved PCR product was introduced into expression vector pWS  resulting in plasmid pWS-sycO. Y. enterocolitica WA-314 secB was cloned into pWS after generation of a PCR product using primers 5'-CATATG TCAGAACAAAACAACACCGAG-3' and 5'-GTCGAC TCAATGGTGATGGTGATGGTGGGCATCCTGACGTTGTTCAGC-3', and subcloning of the PCR product into pGEM-T (Promega). The resulting plasmid was termed pWS-secB. Construction of plasmid pWS-sycH was conducted in an analogous manner using primers 5'-CATATG CGCACTTACAGTTCATTAC-3' and 5'-GTCGAC TTAAACCAGTAAATGAGATGATG-3'. Deletion of yscM1 and yscM2, respectively, in Y. enterocolitica WA-314 was introduced applying the phage lambda Red recombinase technique as previously established for Y. enterocolitica . The kanamycin gene of plasmid pACYC177 (New England Biolabs) was amplified with 50-nucleotide-homology extensions corresponding to the flanks of the yscM1 coding region using primers 5'- AATAAATAACTTAGAATATCGTAGAGATAATTATAGCGACAGGAGACTCGTCACTGACACCCTCATCAGTG-3' and 5'- ATCAACCTGGGGGTATTATCGCCTCAATATACAGTAATATATTATCGCGTCAAGTCAGCGTAATGCTC-3'. Analogously, primers 5'-TGGTGGTTTAGTTTGTGTTTATTTTAAAATATACATGACATATCGACGTTTCACTGACACCCTCATCAGTG-3' and 5'- TGAAAAATTGAATTTTTAGTTTTAAGGTAACTTATCATTAAGTTGTGATACGTCAAGTTGTGATACGTCAAGTCAGCGTAATGCTC-3' were used in the case of yscM2. Construction of yopO and yopP deletion mutants was described previously .
Culturing of yersiniae
Induction of the T3SS of Y. enterocolitica WA-314 was accomplished essentially as described . In brief, yersiniae overnight cultures grown at 27°C in brain heart infusion broth (BHI; Difco) were diluted 1:20 in BHI supplemented with 0.2 mM CaCl2 and cultured for 2 h at 37°C. Then, EGTA was added to a final concentration of 5 mM and MgCl2 to a final concentration of 10 mM for induction of Yop secretion. Alternatively, for repression of Yop secretion yersiniae were cultivated for 4 h at 37°C in BHI supplemented with 5 mM CaCl2. Overproduction of SycO and SecB was induced by addition of 1 mM IPTG at the time of EGTA supplementation.
Expression and purification of recombinant SycO
The strategy for production of recombinant SycO was essentially as described for SycT . Plasmid pWS-sycO was transformed into BL21 (DE3) pLysS (Stratagene). A 100 ml overnight culture was grown in LB medium supplemented with 1% glucose at 37°C and diluted 1:50 in 5 liter LB medium. The culture was further grown in a BIOSTAT B fermenter (B.Braun Biotech International) at 37°C under conditions of constant aeration (10 l/min), pH (7.2) and stirring (200 rpm). At an OD600 of 0.7 the temperature was decreased to 27°C and subsequently SycO expression was induced by adding 0.2 mM IPTG. Bacteria were cultivated for another 18 h, harvested by centrifugation and frozen at -20°C. The bacterial pellet from 1.25 liter of culture was resuspended in 80 ml of Buffer A (50 mM Tris-HCl pH7.6, 100 mM NaCl, 2 mM DTT) supplemented with 4 mM PMSF and 250 U Benzonase (Merck Biosciences). The sample was passed through a French press three times (1000 psi) and centrifuged at 20,000 g for 40 min. The supernatant was adjusted to 20% (NH4)2SO4 and gently stirred for 2 h at 4°C. The SycO precipitate was obtained by centrifugation (20,000 g, 4°C), washed with precipitation buffer and resuspended in buffer A. The sample was then loaded on a MonoQ column (HiTrap Q FF, 5 ml; Amersham Biosciences) equilibrated with buffer A. The column was washed with 10 ml of buffer A, then elution was conducted by applying a 100 ml linear gradient of 0–100% elution buffer (50 mM Tris-HCl pH 7.6, 1 M NaCl, 1 mM DTT). SycO eluted in the range of 280–370 mM NaCl.
Cross-linking reagent DSP (dithiobis [succinimidylpropionate]) was purchased from Pierce. For cross-linking of SycO, 10 μl reaction volume containing 1.3 mM DSP and 3 μg/μl recombinant SycO in phosphate buffered saline (PBS) was incubated for 30 min at 22°C. The reaction was stopped by adding 0.4 μl of a 1 M Tris-HCl pH 7.5 solution and was incubated for another 15 min at 22°C. 5 μl of SDS-PAGE loading buffer containing 0.5% 2-mercaptoethanol were added and the sample was heated for 5 min at 95°C before loading.
Native gel electrophoresis
Tris-buffered continuous native gel electrophoresis was performed as previously described . Gels were stained with Coomassie Brilliant Blue or were alternatively electro-blotted.
Analytical gel filtration chromatography
Analytical gel filtration was performed with a SMART system (Amersham Biosciences) as described . Recombinant SycO (50 μg) and the monomeric 29 kDa protein carbonic anhydrase (50 μg) were mixed in a volume of 50 μl of PBS and loaded on a Superdex 75 PC 3.2/30 column (Amersham Biosciences) equilibrated with PBS. Fractions of 50 μl were collected and analyzed by SDS-PAGE. Marker proteins for column calibration were purchased from Sigma (bovine serum albumine, 67 kDa; carbonic anhydrase, 29 kDa; cytochrome c, 12.4 kDa).
We thank Gabriele Liegl and Emilia Sieger for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 594 Teilprojekt B6).
- Troisfontaines P, Cornelis GR: Type III secretion: more systems than you think. Physiology (Bethesda). 2005, 20: 326-339.View ArticleGoogle Scholar
- Cornelis GR, Van Gijsegem F: Assembly and function of type III secretory systems. Annu Rev Microbiol. 2000, 54: 735-774. 10.1146/annurev.micro.54.1.735.View ArticlePubMedGoogle Scholar
- Stebbins CE, Galan JE: Maintenance of an unfolded polypeptide by a cognate chaperone in bacterial type III secretion. Nature. 2001, 414: 77-81. 10.1038/35102073.View ArticlePubMedGoogle Scholar
- Birtalan SC, Phillips RM, Ghosh P: Three-dimensional secretion signals in chaperone-effector complexes of bacterial pathogens. Mol Cell. 2002, 9: 971-980. 10.1016/S1097-2765(02)00529-4.View ArticlePubMedGoogle Scholar
- Schubot FD, Jackson MW, Penrose KJ, Cherry S, Tropea JE, Plano GV, Waugh DS: Three-dimensional structure of a macromolecular assembly that regulates type III secretion in Yersinia pestis . J Mol Biol. 2005, 346: 1147-1161. 10.1016/j.jmb.2004.12.036.View ArticlePubMedGoogle Scholar
- Luo Y, Bertero MG, Frey EA, Pfuetzner RA, Wenk MR, Creagh L, Marcus SL, Lim D, Sicheri F, Kay C, Haynes C, Finlay BB, Strynadka NC: Structural and biochemical characterization of the type III secretion chaperones CesT and SigE. Nat Struct Biol. 2001, 8: 1031-1036. 10.1038/nsb717.View ArticlePubMedGoogle Scholar
- Neumayer W, Groll M, Lehmann V, Antoneka U, Kähler S, Heesemann J, Wilharm G: Yersinia enterocolitica type III secretion chaperone SycH. Recombinant expression, purification, characterisation, and crystallisation. Protein Expr Purif. 2004, 35: 237-247. 10.1016/j.pep.2004.02.017.View ArticlePubMedGoogle Scholar
- Wilharm G, Lehmann V, Neumayer W, Trèek J, Heesemann J: Yersinia enterocolitica type III secretion: evidence for the ability to transport proteins that are folded prior to secretion. BMC Microbiol. 2004, 4: 27-10.1186/1471-2180-4-27.PubMed CentralView ArticlePubMedGoogle Scholar
- Gauthier A, Finlay BB: Translocated intimin receptor and its chaperone interact with ATPase of the type III secretion apparatus of enteropathogenic Escherichia coli . J Bacteriol. 2003, 185: 6747-6755. 10.1128/JB.185.23.6747-6755.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Akeda Y, Galan JE: Chaperone release and unfolding of substrates in type III secretion. Nature. 2005, 437: 911-915. 10.1038/nature03992.View ArticlePubMedGoogle Scholar
- Cornelis GR: How Yops find their way out of Yersinia . Mol Microbiol. 2003, 50: 1091-1094. 10.1046/j.1365-2958.2003.03812.x.View ArticlePubMedGoogle Scholar
- Ramamurthi KS, Schneewind O: Substrate recognition by the Yersinia type III protein secretion machinery. Mol Microbiol. 2003, 50: 1095-1102. 10.1046/j.1365-2958.2003.03777.x.View ArticlePubMedGoogle Scholar
- Birtalan S, Ghosh P: Structure of the Yersinia type III secretory system chaperone SycE. Nat Struct Biol. 2001, 8: 974-978. 10.1038/nsb1101-974.View ArticlePubMedGoogle Scholar
- Evdokimov AG, Tropea JE, Routzahn KM, Waugh DS: Three-dimensional structure of the type III secretion chaperone SycE from Yersinia pestis . Acta Crystallogr D Biol Crystallogr. 2002, 58: 398-406. 10.1107/S090744490200015X.View ArticlePubMedGoogle Scholar
- Trame CB, McKay DB: Structure of the Yersinia enterocolitica molecular-chaperone protein SycE. Acta Crystallogr D Biol Crystallogr. 2003, 59: 389-392. 10.1107/S0907444902020826.View ArticlePubMedGoogle Scholar
- Phan J, Tropea JE, Waugh DS: Structure of the Yersinia pestis type III secretion chaperone SycH in complex with a stable fragment of YscM2. Acta Crystallogr D Biol Crystallogr. 2004, 60: 1591-1599. 10.1107/S0907444904017597.View ArticlePubMedGoogle Scholar
- Buttner CR, Cornelis GR, Heinz DW, Niemann HH: Crystal structure of Yersinia enterocolitica type III secretion chaperone SycT. Protein Sci. 2005, 8: 1993-2002. 10.1110/ps.051474605.View ArticleGoogle Scholar
- Locher M, Lehnert B, Krauss K, Heesemann J, Groll M, Wilharm G: Crystal structure of the Yersinia enterocolitica type III secretion chaperone SycT. J Biol Chem. 2005, 280: 31149-31155. 10.1074/jbc.M500603200.View ArticlePubMedGoogle Scholar
- Ghosh P: Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev. 2004, 68: 771-795. 10.1128/MMBR.68.4.771-795.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Letzelter M, Sorg I, Mota LJ, Meyer S, Stalder J, Feldman M, Kuhn M, Callebaut I, Cornelis GR: The discovery of SycO highlights a new function for type III secretion effector chaperones. EMBO J. 2006, 25: 3223-3233. 10.1038/sj.emboj.7601202.PubMed CentralView ArticlePubMedGoogle Scholar
- Galyov EE, Hakansson S, Wolf-Watz H: Characterization of the operon encoding the YpkA Ser/Thr protein kinase and the YopJ protein of Yersinia pseudotuberculosis . J Bacteriol. 1994, 176: 4543-4548.PubMed CentralPubMedGoogle Scholar
- Perry RD, Straley SC, Fetherston JD, Rose DJ, Gregor J, Blattner FR: DNA sequencing and analysis of the low-Ca2+-response plasmid pCD1 of Yersinia pestis KIM5. Infect Immun. 1998, 66: 4611-4623.PubMed CentralPubMedGoogle Scholar
- Trülzsch K, Roggenkamp A, Aepfelbacher M, Wilharm G, Ruckdeschel K, Heesemann J: Analysis of chaperone-dependent Yop secretion/translocation and effector function using a mini-virulence plasmid of Yersinia enterocolitica . Int J Med Microbiol. 2003, 293: 167-177. 10.1078/1438-4221-00251.View ArticlePubMedGoogle Scholar
- Heesemann J, Laufs R: Construction of a mobilizable Yersinia enterocolitica virulence plasmid. J Bacteriol. 1983, 155: 761-767.PubMed CentralPubMedGoogle Scholar
- Cambronne ED, Cheng LW, Schneewind O: LcrQ/YscM1, regulators of the Yersinia yop virulon, are injected into host cells by a chaperone-dependent mechanism. Mol Microbiol. 2000, 37: 263-273. 10.1046/j.1365-2958.2000.01974.x.View ArticlePubMedGoogle Scholar
- Wulff-Strobel CR, Williams AW, Straley SC: LcrQ and SycH function together at the Ysc type III secretion system in Yersinia pestis to impose a hierarchy of secretion. Mol Microbiol. 2002, 43: 411-423. 10.1046/j.1365-2958.2002.02752.x.View ArticlePubMedGoogle Scholar
- Trülzsch K, Sporleder T, Igwe EI, Rüssmann H, Heesemann J: Contribution of the major secreted Yops of Yersinia enterocolitica O:8 to pathogenicity in the mouse infection model. Infect Immun. 2004, 72: 5227-5234. 10.1128/IAI.72.9.5227-5234.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Bergman T, Hakansson S, Forsberg A, Norlander L, Macellaro A, Backman A, Bolin I, Wolf-Watz H: Analysis of the V antigen lcrGVH-yopBD operon of Yersinia pseudotuberculosis : evidence for a regulatory role of LcrH and LcrV. J Bacteriol. 1991, 173: 1607-1616.PubMed CentralPubMedGoogle Scholar
- Forsberg A, Wolf-Watz H: Genetic analysis of the yopE region of Yersinia spp.: identification of a novel conserved locus, yerA, regulating yopE expression. J Bacteriol. 1990, 172: 1547-1555.PubMed CentralPubMedGoogle Scholar
- Swietnicki W, O'Brien S, Holman K, Cherry S, Brueggemann E, Tropea JE, Hines HB, Waugh DS, Ulrich RG: Novel protein-protein interactions of the Yersinia pestis type III secretion system elucidated with a matrix analysis by surface plasmon resonance and mass spectrometry. J Biol Chem. 2004, 279: 38693-38700. 10.1074/jbc.M405217200.View ArticlePubMedGoogle Scholar
- Schmid A, Dittmann S, Grimminger V, Walter S, Heesemann J, Wilharm G: Yersinia enterocolitica type III secretion chaperone SycD: recombinant expression, purification and characterization of a homodimer. Protein Expr Purif. 2006, 49: 176-182. 10.1016/j.pep.2006.04.012.View ArticlePubMedGoogle Scholar
- Stainier I, Iriarte M, Cornelis GR: YscM1 and YscM2, two Yersinia enterocolitica proteins causing downregulation of yop transcription. Mol Microbiol. 1997, 26: 833-843. 10.1046/j.1365-2958.1997.6281995.x.View ArticlePubMedGoogle Scholar
- Wilharm G, Neumayer W, Heesemann J: Recombinant Yersinia enterocolitica YscM1 and YscM2: homodimer formation and susceptibility to thrombin cleavage. Protein Expr Purif. 2003, 31: 167-172. 10.1016/S1046-5928(03)00183-9.View ArticlePubMedGoogle Scholar
- Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 2000, 97: 6640-6645. 10.1073/pnas.120163297.PubMed CentralView ArticlePubMedGoogle Scholar
- Wilharm G, Lehmann V, Krauss K, Lehnert B, Richter S, Ruckdeschel K, Heesemann J, Trülzsch K: Yersinia enterocolitica type III secretion depends on the proton motive force but not on the flagellar motor components MotA and MotB. Infect Immun. 2004, 72: 4004-4009. 10.1128/IAI.72.7.4004-4009.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Pettersson J, Nordfelth R, Dubinina E, Bergman T, Gustafsson M, Magnusson KE, Wolf-Watz H: Modulation of virulence factor expression by pathogen target cell contact. Science. 1996, 273: 1231-1233. 10.1126/science.273.5279.1231.View ArticlePubMedGoogle Scholar
- Cambronne ED, Schneewind O: Yersinia enterocolitica type III secretion: yscM1 and yscM2 regulate yop gene expression by a posttranscriptional mechanism that targets the 5' untranslated region of yop mRNA. J Bacteriol. 2002, 184: 5880-5893. 10.1128/JB.184.21.5880-5893.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Trček J, Wilharm G, Jacobi CA, Heesemann J: Yersinia enterocolitica YopQ: strain-dependent cytosolic accumulation and post-translational secretion. Microbiology. 2002, 148: 1457-1465.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.