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Journal of Bacteriology, September 2004, p . 6320-6324, Vol . 186, No . 18

The Secretion Signal of YopN, a Regulatory Protein of the Yersinia enterocolitica Type III Secretion Pathway{dagger}

John W . Goss, Joseph A . Sorg, Kumaran S . Ramamurthi, Hung Ton-That, and Olaf Schneewind*

Committee on Microbiology, University of Chicago, Chicago, Illinois

Received 2 April 2004/ Accepted 7 June 2004


 

  ABSTRACT

 
The type III secretion signal of Yersinia enterocolitica YopN was mapped using a gene fusion approach . yopN codons 1 to 12 were identified as critical for signal function . Several synonymous mutations that abolish secretion of hybrid proteins withoutaltering the codon specificity of yopN mRNA were identified.


 

  TEXT

 
Many gram-negative bacteria employ a type III secretion mechanismto transport virulence factors across the bacterial envelopeand, in some cases, even into host cells during infection [15]. The 70-kb virulence plasmid of yersiniae harbors ysc genes, whose products are assembled to generate the type III machinery [10], as well as genes that encode substrates for the secretionpathway [29] . Some substrates play a role in the injection ofproteins into host cells [16, 17], whereas others travel thetype III pathway into host cells [33] . Yersinia type III secretion is regulated by environmental signals [for example, low calcium] that promote specific transport reactions [21, 29] . Four Yersiniagenes ensure proper activation of the type III machinery andthe fidelity of transport into host cells: yopN, sycN, tyeA,and yscB [18-20, 40] . SycN and YscB bind to the injection substrateYopN and promote its initiation into the type III pathway [7,12] . TyeA binds to the C-terminal portion of YopN, an interactionthat prevents type III transport of YopN and of other injectionsubstrates [7, 8].

The first approximately 15 amino acids of Yop proteins are sufficient to direct the type III secretion of fused reporter proteins[6, 24, 35, 36] . Single amino acid replacements at each positionof these secretion signals failed to detect residues that arecritically important for function [3, 35], and no discernible similarity was identified among the first 15 amino acid residuesof all Yop proteins [2, 3, 5] . Several frameshift mutations,altering the reading frame of secretion signals but not thatof the fused reporter gene, did not abolish secretion [3, 22].This unusual experimental result led to the RNA signal hypothesis,i.e., a property of yop mRNA may be responsible for the initiationof these polypeptides into the type III pathway [4].

yopQ harbors a minimal secretion signal within the first 10 codons [30, 31] . The minimal secretion signal of yopQ does nottolerate frameshift mutations; however, the phenotype of suchmutations can be suppressed by fusion of additional downstreamsequences with linear signal dimensions, typically the first15 codons . The function of the minimal secretion signal of yopQcan be abrogated by synonymous mutations that alter mRNA sequencewithout affecting codon specificity or amino acid incorporationinto the polypeptide [31] . Such mutations have pointed to codon3, isoleucine, as a critical element in substrate recognitionfor yopQ [31] . In silico analysis of the presumed secretionsignals for all Yop proteins revealed the presence of isoleucinecodons in twelve yop genes, whereas two genes, lcrV and yopN,represent an exception to this rule [28].

To identify the secretion signal of yopN we generated hybrid proteins by fusing DNA sequence specifying the yopN promoter, upstream untranslated yopN mRNA sequence, and portions of the yopN coding sequence to npt, encoding the cytoplasmic reporterprotein neomycin phosphotransferase [Npt] [32] . Unless YopN-Npthybrids encompass the secretion signal for the type III pathway,fusion proteins would be expected to reside in the bacterialcytoplasm . Gene sequences for yopN-npt fusions were cloned onthe low-copy-number plasmid pHSG576 [38] . An NdeI restrictionsite was introduced downstream of the yopN untranslated region.Oligonucleotides specifying the desired sequence to be fusedto npt were annealed and inserted between the NdeI and KpnIrestriction sites [6] . Recombinant plasmids were transformedinto Yersinia enterocolitica strain W22703 [11] . To measureprotein transport, organisms were induced by growing bacterialcultures for 3 h at 37°C in calcium-depleted medium [M9medium, containing 42 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl,18.6 mM NH4Cl, 0.01 mg of FeSO4/ml, 0.001% thiamine, 1 mM MgSO4, 0.4% glucose, and 1% Casamino Acids], a condition known to trigger type III secretion [25, 27] . The cultures were then centrifuged,and the extracellular medium was separated with the supernatant[S] from the bacterial sediment in the pellet fraction [P].Proteins in both fractions were precipitated with trichloroaceticacid, washed in acetone, and suspended in sample buffer priorto separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.After electrotransfer of proteins onto polyvinylidene difluoride[PVDF] membrane, YopN-Npt hybrids were detected by immunoblottingusing specific rabbit antibody, horseradish peroxidase-conjugatedsecondary antibody, and chemiluminescence . As a control forproper fractionation of type III secretion substrates, the bacteriasecreted YopE into the extracellular medium while chloramphenicolacetyltransferase remained in the bacterial cytoplasm [9] [Fig. 1] . The hybrid YopN1-15-Npt, generated through fusion of the first 15 codons of yopN to the npt reporter, was mostly secretedinto the extracellular medium, as 82% of YopN1-15-Npt was foundin the culture supernatant after centrifugation [Fig. 1A] . Fusionof shorter coding sequences of yopN to npt led to a reductionin the amount of secreted protein, as 68% of YopN1-14-Npt, 43%of YopN1-13-Npt, 38% of YopN1-12-Npt, and 18% of YopN1-11-Npt was observed in culture supernatants . Further truncation ofthe yopN gene sequence, generated via fusion of yopN codons1 to 10 to npt, failed to promote type III secretion of YopN1-10-Npt [Fig . 1A] . These results suggest that the minimal secretionsignal of yopN is encoded by codons 1 to 12, as the resultingYopN1-12-Npt hybrid is initiated into the type III pathway withabout a twofold-reduced rate compared to that of wild-type YopN[86%] [8] or YopN1-15-Npt [82%] . We wondered whether the minimalsecretion signal of yopN is able to tolerate deletions of codonsat the 5' end without loss of signaling function . To test this,codon 2 [{Delta}2], codons 2 to 3 [{Delta}2-3], 2 to 4 [{Delta}2-4], 2 to 5 [{Delta}2-5], 2 to 6 [{Delta}2-6], 2 to 7 [{Delta}2-7], 2 to 8 [{Delta}2-8], or 2 to 9 [{Delta}2-9] wereomitted from fused yopN1-12-npt . All deletions of codons fromthe 5' end of the yopN coding sequence eliminated the functionof the type III secretion signal, and the YopN1-12-Npt hybridscould not be found in culture supernatants [Fig . 1B] . Togetherthe results depicted in Fig . 1 suggest that the mRNA sequence of yopN codons 1 to 12 or amino acids 1 to 12 of YopN function as a secretion signal able to initiate hybrid reporter proteinsinto the type III pathway and that the boundaries of this linearelement cannot tolerate truncations.


 

FIG . 1 . Defining the minimal secretion signal of yopN. Successive 3' yopN truncations were fused to the reporter gene npt, and recombinant genes were cloned into low-copy-number plasmids and then transformed into Y . enterocolitica W22703 . Yersinia cultures were grown in M9 minimal media supplemented with Casamino Acids and were induced for type III secretion by temperature shift to 37°C and depletion of calcium ions . Protein in the extracellular culture supernatant [S] or the bacterial cell pellet [P] were precipitated with trichloroacetic acid and were analyzed by immunoblotting . [A] The percent amount of secreted YopN-Npt hybrids [i.e., the amount of secreted protein divided by the total amount of immunoreactive material and then multiplied by 100] is indicated underneath each panel . As a control for proper fractionation, the secretion of YopE, a type III secretion substrate, and chloramphenicol acetyltransferase, a cytosolic protein, was monitored with specific antiserum [{alpha}YopE or {alpha}CAT] . [B] To determine the 5' boundaries of the yopN secretion signal, codon 2 [{Delta}2], codons 2 to 3 [{Delta}2-3], and so forth were deleted and the secretion of YopN-Npt hybrids were measured . WT, wild type.

 
A mutant mRNA was generated that carried 15 nucleotide substitutions of the yopN secretion signal, each of which was predicted to not alter the codon specificity of the translational hybrid.When yopN1-12-npt was expressed in Y . enterocolitica W22703,the hybrid polypeptide synthesized from wobble mRNA was notsecreted [Fig . 2] . The properties of the yopN secretion signaldescribed here were examined with another reporter [portionsof the bla gene encoding the amino acid sequence of mature,secreted ß-lactamase], and the resulting hybrid proteinsdisplayed similar transport properties [data not shown] . SynonymousmRNA mutations with similar phenotypes have been generated inthe minimal secretion signal of yopQ [31] . Considering theseearlier results, the data shown in Fig . 2 suggest that at leasta portion of the minimal secretion signal of yopN may be decoded by a property of its mRNA nucleotide sequence . We sought toascertain that yopN1-12 and yopN1-12 wobble indeed generated the same polypeptide sequence . Y . enterocolitica strain W22703 cells, expressing plasmid-encoded yopN1-12-npt hybrids, werelysed in a French pressure cell, and cytoplasmic protein wasprepurified by ion exchange chromatography on a MonoQ column[Fig . 3A and B] . Following separation on SDS-PAGE, proteinswere electrotransferred to PVDF membrane and stained with CoomassieBrilliant Blue, and amino acid sequences were determined byEdman degradation [Fig . 3] . The chromatography data in Fig.3B, D, and F quantify phenylthiohydantoin amino acyl residuesduring each of the 11 sequencing cycles . The eluted compoundof YopN1-12-Npt following the first Edman degradation cyclewas threonine [T] and not the initiator methionine [M] [Fig.3B and D] . Every phenylthiohydantoin amino acyl eluting afterthe initial cycle matched the residues predicted by the sequenceof mRNA codons [Fig. 3B and D] . Thus, these results suggestthat yopN1-12 and yopN1-12 wobble indeed generated the identicalpolypeptide sequence . A simple explanation for the position of threonine at the amino acyl end of YopN1-12-Npt is deformylationand amino methionyl peptidase cleavage of YopN by f-Met peptidedeformylase [Def] and methionine amino peptidase [MAP], respectively[23] . Is deformylation and removal of methionyl caused by thesecretion defect of YopN1-12-Npt, perhaps because the polypeptidenow resides in the bacterial cytoplasm and is substrate forDef and MAP [26], or does methionyl removal also occur priorto the secretion of wild-type YopN? To address this question,YopN was purified from the extracellular medium of Y . enterocoliticastrain 8081 cultures by using reversed-phase high-performanceliquid chromatography, and the peptide was then subjected toEdman degradation . Again, the first eluted residue of YopN followingthe initial Edman degradation cycle was threonine [T] and notmethionine [M] . Forsberg and colleagues made a similar observationfor YopN of Yersinia pseudotuberculosis [14] . These data thereforesuggest that YopN may be modified by Def and MAP prior to theinitiation of YopN into the type III secretion pathway . Theseresults and previously published observations support the notionthat type III substrates can travel the pathway in a posttranslationalmanner [1, 13, 34, 37].


 

FIG . 2 . Synonymous mutations in the minimal secretion signal of yopN . [A] Two signal fusions with the npt reporter gene were generated that carried either the wild-type [WT] mRNA sequence or a variant with 15 synonymous mutations that altered the nucleotide sequence of yopN1-12 without affecting its encoded protein sequence . [B] Plasmid with cloned recombinant genes, yopN1-12-npt or yopN1-12wobble-npt, was transformed into Y . enterocolitica strain W22703 . Yersinia organisms were induced for type III secretion, cultures were fractionated, and YopN-Npt secretion was measured as described in the legend to Fig . 1.

 

 

FIG . 3 . Amino acid sequence analysis of peptides encoded by yopN1-12 secretion signals . [A] YopN1-12-Npt, synthesized from wild-type [WT] yopN1-12 secretion signal, was purified from bacterial extracts generated via French pressure cell by chromatography on a MonoQ column . The crude lysate [L, for load] and eluate [M, for MonoQ] were subjected to SDS-PAGE and electrotransfer to PVDF membrane . The membranes were analyzed by immunoblotting with {alpha}NPT or were stained with Coomassie brilliant blue . The arrow points to the species that was subjected to Edman degradation . [B] Edman degradation profile reveals the elution of phenylthiohydantoin residues per sequencing cycle of the sample generated in panel A . High concentrations of glycine that eluted during each cycle were interpreted as sample contamination by electrotransfer buffer . [C] YopN1-12-Npt, synthesized from the yopN1-12 wobble variant [see Fig. 2], was purified from bacterial extracts generated via French pressure cell by chromatography on a MonoQ column . The crude lysate [L] and eluate [M] were subjected to SDS-PAGE and electrotransfer to PVDF membrane . The membranes were analyzed by immunoblotting with {alpha}NPT or were stained with Coomassie brilliant blue . The arrow points to the species that was subjected to Edman degradation . [D] Edman degradation profile reveals the elution of phenylthiohydantoin residues per sequencing cycle of the sample depicted in panel C . [E] YopN was purified by reversed-phase high-performance liquid chromatography [HPLC] on a C18 column from the supernatant of centrifuged Y . enterocolitica strain 8081 cultures, separated on SDS-PAGE, and stained with Coomassie Brilliant Blue . The arrow points to the species that was subjected to Edman degradation . Molecular size markers [Marker] were included for size measurements . [F] Edman degradation profile reveals the elution of phenylthiohydantoin residues per sequencing cycle of the sample shown in panel E . MW, molecular size.

 
To identify the nucleotide properties of yopN that are involved in secretion signaling, variants of yopN1-12 were assembled from annealed oligonucleotides, cloned in frame to generate yopN1-12-npt [see the supplementary material] . Most nucleotidepositions of yopN1-12 tolerated transversions, i.e., replacementof purine nucleotides with pyrimidine nucleotides or reciprocalsubstitutions, without a measurable loss of secretion signalfunction [a fivefold reduction in protein transport was viewedas a significant loss of signaling] [Fig . 4] . Using these criteria,single-nucleotide substitutions at seven positions [A4U, U11A,A14U, U20A, A21U, U25A, and C30G] of the 36-nucleotide sequencewere sensitive to mutation . Many of the mutations analyzed inFig . 4 introduced changes in codon specificity [amino acid substitutions]without causing a loss of function . With the exception of codon7, where mutations at two positions affected signaling, onlyone of the three nucleotide positions at each of the codons2, 4, 5, 9, and 10 was sensitive to mutagenesis . Because eachcodon tolerated at least one nonsynonomous nucleotide substitution,it appears that no single codon or amino acid of the yopN signalis absolutely essential for substrate recognition by the typeIII machinery . The mutational analysis described here identifiedsome, but certainly not all, of the relevant nucleotide positionsthat may play a role in secretion signaling of yopN1-12 . Thereason for this limitation resides in the chemical nature ofthe changes that were introduced by the transversion mutations.Uracil was typically replaced with adenine or guanine was replacedwith cytidine and vice versa to preserve the general uracil-and adenine-rich sequence character of yop secretion signals.More dramatic changes, for example, replacing uracil with guanineor replacing adenine with cytidine, may identify additionalnucleotide positions that are sensitive to mutagenesis.


 

FIG . 4 . Mutations within the minimal secretion signal of yopN . To identify nucleotide positions that are important for the function of the secretion signal of yopN, nucleotide transversions [purine and/or pyrimidine substitutions] were introduced into yopN1-12. The variant signals were fused to npt, yopN1-12-npt was cloned on low-copy-number vectors, and recombinant plasmids were then transformed into Y . enterocolitica strain W22703 . In the event that a stop codon would have been introduced by the transversion [codon 9], an alternate nucleotide was used for mutagenesis . Bacteria were induced for type III secretion, cultures were fractionated, and YopN-Npt secretion was measured as described in the legend to Fig. 1.

 
The experiment depicted in Fig . 2, reporting a secretion defectfor yopN1-12 wobble, introduced 15 nucleotide substitutionsinto a signal that is comprised of 36 nucleotides . Our concernwas that such a large number of mutations may have altered a general property of yopN mRNA, presumably affecting secretion in a manner that may not be pertinent to substrate recognition by the type III pathway . To analyze the yopN1-12 secretion signal for synonymous mutations that alter nucleotide positions without affecting the specificity of individual codons, we tested all possible nucleotide triplets at each of the 11 positions thatwere accessible to mutagenesis . The data for this experimentare summarized in Table 1 . Synonymous mutations at codons 2, 3, 4, 5, 6, 8, 9, 11, and 12 did not affect secretion signaling. However, two synonymous mutations at codon 7 eliminated secretion signaling . The wobble [X] position of the CUX leucine triplet must be occupied by a purine base, either adenyl or guanidyl,to provide for secretion signaling, as neither cytidyl nor uridylwas tolerated at this position [Table 1] . The central nucleotide position of leucine codon 7, i.e., the uridyl in CUA, was also identified as important for secretion signaling, because U20A eliminated its function [Fig . 4] . In contrast, the first nucleotideposition [cytidyl] at yopN codon 7 could be replaced with uridyl,thereby representing a synonymous mutation, or with guanidyl,changing the codon specificity to valine, each of which didnot produce a defect in secretion signaling . Replacement of the first nucleotide [C in CUA] with adenyl presumably may not affect secretion signaling, because the yopN gene of Y . enterocolitica strain 8081 harbors an AUA triplet at codon 7, incorporating isoleucine at position 7 of the YopN polypeptide [Fig . 3E and F] [10, 39] . Three nucleotide triplets at Y . enterocoliticastrain W22703 yopN codon 10, GGC, GGA, and GGU, promoted typeIII secretion, whereas the GGG triplet caused a significantreduction in secretion signaling.


 

TABLE 1 . Synonymous mutations in the yopN1-12 secretion signal and their effect on the secretion of YopN1-12-Npt

 
Conclusions. Systematic mutagenesis of all nucleotides in the yopN minimalsecretion signal identified 7 positions [A4U, U11A, A14U, U20A,A21U, U25A, and C30G] out of 36 nucleotides that were sensitiveto mutation . One conclusion of this experiment is that the minimalsecretion signal of yopN is astonishingly resilient to codonchanges or amino acid substitutions . In fact, each of the 11amino acids following the initiator methionine could be changedwithout loss of function . Further, three nucleotide positions that were critical for function showed that synonymous mutations at codons 7 or 10 can eliminate secretion of hybrid reporter proteins without altering the codon specificity of yopN mRNA.

 


 

  ACKNOWLEDGMENTS

 
J.S . acknowledges support from Molecular Cell Biology TrainingGrant T32GM007183 awarded by NIH/NIGMS to the University ofChicago . This work was supported by U.S . Public Health ServiceGrant AI42797 to O.S.

J . W . Goss and J . A . Sorg contributed equally to this work.


 

  FOOTNOTES

 
* Corresponding author . Mailing address: Committee on Microbiology, The University of Chicago, 920 E . 58th St., Chicago, IL 60637 . Phone: [773] 834-9060 . Fax: [773] 834-8150 . E-mail: oschnee@bsd.uchicago.edu.

 

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.


 

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