INTRODUCTION

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The human-pathogenic yersiniae Yersinia pestis, which causes plague, and Yersinia pseudotuberculosis and Yersinia enterocolitica, which primarily cause gastrointestinal disease, have a ca. 70-kb plasmid that encodes a complex virulence property called the low-Ca²⁺ response (LCR) (35, 45, 87, 88, 120, 121). The LCR was discovered in Y. pestis growing in vitro, where the bacteria respond to the absence of Ca²⁺ at 37°C by the strong expression and secretion of a virulence protein called V antigen (now also called LcrV). In certain media, this is accompanied by a growth response termed restriction in which the yersiniae undergo an orderly metabolic shutdown and cease growth (23, 25, 42, 51, 119). It is now known that under these in vitro LCR-inductive conditions, the yersiniae maximally induce the transcription, translation, and secretion of a set of virulence proteins called Yops (Yersinia outer proteins) in addition to LcrV. The operons encoding these proteins and other similarly regulated operons on the LCR plasmid have been referred to as the LCR stimulon (LCRS) (83, 107). Millimolar concentrations of Ca²⁺ permit a full growth yield at 37°C, weaker expression of LcrV and Yops, and essentially no secretion of these proteins (83, 107). There is only very weak, basal expression of Yops and LcrV at environmental temperatures and no secretion: the LCR is designed to function within a mammal. In addition to the presence or absence of Ca²⁺, other environmental inputs, such as Mg²⁺, Cl, Na⁺, glutamate, nucleotides, and anaerobiosity, modulate the LCR (23, 42, 60, 118, 119). The molecular basis of these effects has not been determined, but these elements of environmental modulation could be important in adjusting virulence protein expression and secretion in response to the wide range of niches that yersiniae are expected to encounter during an infection (106).

It is believed that the absence of Ca²⁺ mimics an unidentified signal that yersiniae receive when they are in contact with a mammalian cell (33, 83). The LCR plasmid encodes a type III secretion system called Ysc, for Yop secretion (68), that is dedicated to the secretion of Yops, LcrV, and some regulatory proteins in the LCR; cell contact causes this system to be locally activated at the interface between the bacterium and the eukaryotic cell. Environmentally regulated inner and outer gates of the Ysc (LcrG and LcrE [also called YopN], respectively) then open, permitting the secretion of negative regulatory proteins (a key one being LcrQ, also called YscM). This allows full transcriptional activation of LCRS operons by an AraC-like activator protein, LcrF. Yops are secreted locally, without processing. The secretion mechanism recognizes two signals: one in the first 45 nucleotides of the yop mRNA and one related to a domain that has been found for some Yops to bind a specific Yop chaperone (Syc), also encoded by the LCR plasmid (29, 112). Some Yops (YopB, YopD, and YopK) serve as components of a mechanism for targeting effector Yops directly into the eukaryotic cell. The effector Yops (YopE, YopH, YpkA [Yersinia protein kinase], YopM, and probably YopJ) then act on their intracellular target molecules and derange cellular signaling and cytoskeletal functions. LcrV has a bifunctional role in the LCR: it is a regulatory protein, acting at the levels of Yop secretion and targeting, and it has a role as a potent antihost protein (77, 79, 83). LcrV is the only LCRS protein that is secreted in large amounts into the surrounding medium by yersiniae in contact with eukaryotic cells (79). It is the only LCRS protein that has been shown to have an effect when given by itself to mice (77); all others require delivery by the Ysc machinery from yersiniae in intimate contact with mammalian cells.

The overall effect of the LCR is a profound immunosuppression, resulting from the paralysis of innate defenses at the site of infection and the failure to mobilize an effective cell-mediated immune response. Y. pestis, and also the enteropathogenic yersiniae in immunocompromised individuals, grows unchecked in the lymphoid system in a fulminant disease that has a high mortality if not treated with appropriate antibiotics (26, 83). In contrast, without the LCR plasmid, these bacteria are completely avirulent (33, 83).

It is now apparent that several other important pathogens have virulence systems with many striking similarities to the LCR; however, the LCR is the best characterized of these and remains a prototype for investigations at the forefront of molecular pathogenesis. The fact that the LCR was plasmid borne greatly facilitated its characterization, which began in the early years of the existence of molecular genetic techniques. The early studies delineated a large cluster of genes necessary for the Ca²⁺ dependence of Y. pestis growth (47, 87, 116, 117). Subsequent studies with the enteropathogenic yersiniae as well as Y. pestis resulted in the following picture for the layout of genes involved in the LCR (83). The Ca²⁺ dependence region turned out to encode LCR regulatory proteins and the enormously complex Ysc type III secretion mechanism, which is comprised of at least 22 gene products. This LCR cluster now includes the immediately adjacent Yop-targeting and secretion control operon lcrGVH-yopBD and is 25.7 kb in size. Only part of this had been sequenced for Y. pestis (lcrDR, lcrGVH-yopB', yscN'OPQRS, ysc[A]B-F, and lcrF [7, 37, 49, 53, 76, 82, 84, 89]), but the similarities of LCR-related genes among the three species of human-pathogenic yersiniae have been so high that information from one species has been assumed to apply to the others (83). There are Yops within the LCR cluster (e.g., YopB, YopD, and LcrE [which proved to be the same as YopN] and YscH, which apparently is a Yop [3]), but the effector Yops were found to be scattered outside the cluster (104). The only Yop genes in Y. pestis that had been sequenced were yopM and yopE, with the divergently transcribed SycE-encoding gene (40, 61).

There are two other LCR-associated genes outside the LCR cluster. YlpA is a lipoprotein studied only for Y. enterocolitica; it is a member of the LCRS group, but a knockout of its gene did not affect virulence in an intravenous mouse model (30). Although not a true LCR member, YadA is an adhesin whose gene is activated for transcription by LcrF (99). It is enormously important for virulence of Y. enterocolitica, and in both Y. enterocolitica and Y. pseudotuberculosis, it serves as an adhesin that can promote the productive contact with eukaryotic cells, resulting in Ysc activation and Yop targeting (see, e.g., reference 15). Interestingly, its gene in Y. pestis EV76 has a frameshift mutation that would effectively abolish expression of the protein (98).

The high homology of the LCR cluster and yop genes from the three pathogenic Yersinia species includes a similar genetic organization. In addition, the replication and partitioning functions of the family of LCR plasmids are also highly conserved (6, 12, 32, 68, 83, 87, 88, 108). Sequencing of the replication region of the Y. enterocolitica LCR plasmid pYVe439-80 demonstrated that it possessed all the essential components of an IncFIIA replicon (108). Plasmid pYVe439-80 is maintained at an estimated seven copies per cell (32). Although the replicon region of this plasmid has 68% homology with the IncFIIA replicon of plasmid R100, the two plasmids are compatible. This may be due to differences in the nucleotide sequences in the stem-loop structures of the antisense RNA encoded by copA (64, 108). The observed incompatibility of LCR plasmids with derivatives of the F plasmid appears to be due to a shared partitioning system (6, 12, 40).

Despite all the similarities within the LCR plasmid family, there are also significant differences. Plasmids from Y. pseudotuberculosis and Y. pestis are more closely related than those from Y. enterocolitica. Y. enterocolitica plasmid regions outside the LCRS, replication, and partitioning function regions do not hybridize to analogous regions from Y. pseudotuberculosis or Y. pestis LCR plasmids (88). In addition, the location of the replicon in relation to the LCR cluster and the organization of the yopE-yadA region on Y. enterocolitica plasmids differ from those of LCR plasmids from the other two yersiniae (12, 32, 68, 83). DNA sequencing has revealed two distinct types of lcrV that differ in a hypervariable region between amino acids (aa) 225 and 232 in the translated protein. Serotype O:8 Y. enterocolitica strains synthesize one type, while Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serotypes O:3, O:9, and O:5,27 produce the other (94). Motin et al. (76) identified a species-specific difference in the lcrV genes from Y. pestis and Y. enterocolitica. These differences likely account for the ability of some anti-LcrV antibodies to protect against infections with Y. pestis and Y. pseudotuberculosis but not against Y. enterocolitica infections (75, 94). Finally, the more stringent growth restriction observed under LCRS-inducing conditions for Y. pestis compared to that for the enteropathogenic yersiniae appears to be due to undetermined differences in the LCR plasmid itself (104). Consequently, it is likely that additional significant differences may reside within the LCR family of plasmids.

To identify new potential virulence factors, discover any further differences between Y. pestis LCRS genes and those previously sequenced for the enteropathogenic yersiniae, and identify other undefined traits of this family of virulence plasmids, we have sequenced one entire LCR plasmid. We chose to sequence the LCR plasmid pCD1 of Y. pestis KIM5 (for Kurdistan Iran man) because it has several advantages over other strains. Derivatives of Y. pestis KIM have been directly demonstrated to retain high-virulence characteristics in both mammals and fleas (8, 22, 52, 83). These derivatives are widely used in research and thus are more genetically characterized than other strains. The vast majority of investigations on the regulation, physiological characteristics, and virulence properties of the Y. pestis LCRS have been performed with derivatives of strain KIM. Finally, nearly all previous DNA sequence information on the Y. pestis LCRS components (~16 kb) has been derived from pCD1 of strain KIM5 (7, 22, 37, 49, 61, 82-84, 89).

Our analysis of the DNA sequence of pCD1 has revealed a set of LCRS genes very similar to those sequenced for the enteropathogenic yersiniae, a potential new Yop and Yop chaperone, two new insertion sequences (ISs), the IncFIIA replication region, and SopABC partitioning functions. We have also identified IS element remnants scattered throughout the plasmid that suggest that pCD1 has undergone numerous insertional events as well as genetic recombinations and rearrangements during its history.

	MATERIALS AND METHODS

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Bacterial strains, media, and plasmid isolation. Y. pestis KIM5 is conditionally avirulent due to deletion of the 102-kb pgm locus; it possesses all three prototypical Y. pestis plasmids, i.e., the 9.5-kb pPCP1, ~70-kb pCD1, and ~100-kb pMT1 (83). Plasmid pCD1 was isolated from Y. pestis KIM5 by alkaline lysis followed by precipitation with polyethylene glycol (13, 55). Since pCD1 has no selectable marker, a mixture of pCD1 and pBR322 was transformed into Escherichia coli HB101 (47). Transformants were selected for the ampicillin resistance encoded by pBR322 (5). Five hundred transformants were transferred to nitrocellulose membranes and hybridized against pCD1 radioactively labeled by nick translation. One transformant containing both pCD1 and pBR322 was identified. This isolate was cured of pBR322 by fusaric acid selection (17). pCD1 appears to be stably maintained in E. coli HB101, and this transformant has been stored in buffered glycerol at 70°C. Plasmid DNA from E. coli HB101(pCD1) cells grown in Luria broth was isolated by alkaline lysis (13) followed by further purification with polyethylene glycol (55). This purified pCD1 DNA was used in subsequent sequencing.

DNA sequencing. Libraries were prepared from nebulized, size-fractionated plasmid DNA (63) in the M13 Janus vector (24). DNA templates were purified from random library clones (81), and sequences were collected by using dye-terminator-labeled fluorescent cycle sequencing Prism reagents and ABI377 automated sequencers (Applied Biosystem Division of Perkin-Elmer). Sequences were assembled into contigs by the SeqMan II program (DNASTAR), and clones were selected for sequencing from the opposite end to fill in coverage, resolve ambiguities, and close gaps (24). Final coverage was about eightfold.

View larger version (35K): [in this window] [in a new window]   FIG. 1.   Genetic organization of the LCR plasmid, pCD1, of Y. pestis KIM5. The map shows significant ORFs and transposable elements as well as replication and partitioning regions. For genes on the outside of the map, transcription proceeds clockwise. Genes on the inside are transcribed from the complementary strand. The oriR and sopC regions are not transcribed. The placement of transposable elements (solid boxes) does not indicate the direction of transcription. A d indicates a defective element, while a p indicates a partial element. Most IS remnants are not denoted on this figure.

Annotation. We identified open reading frames (ORFs) encoding at least 50 aa, using Geneplot or GeneQuest (DNASTAR) programs to display start codons (including GUG), stop codons, and codon usage statistics plots for each reading frame. Codon usage analysis helped to predict ORFs. It was assessed in the program by second- and third-order statistical comparisons (20) with a matrix built from all available sequences for Yersinia species. Although this matrix was more useful than one derived from E. coli genes, it was necessarily constructed from a relatively small data set and is no doubt imperfect. Generally, in the absence of experimental data, the start codon farthest upstream was used to annotate the ORF start (14). ORFs with products smaller than 50 aa were included if codon usage statistics showed a high score. ORF amino acid sequences were searched against SWISS-PROT 34 by using the BLOSUM26 matrix, with the DeCypher II System (TimeLogic Inc.). In the first pass, the best hit was automatically saved as an annotation for each ORF, and then known genes and putative functions were assigned for individual ORFs by inspection of the search output.

Nucleotide sequence accession number. The entire sequence of pCD1 from Y. pestis KIM5 has been deposited in the GenBank database and assigned accession number AF074612.

	RESULTS AND DISCUSSION

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General description. From our DNA sequencing, we have constructed a genetic map of the Y. pestis KIM5 pCD1 plasmid (Fig. 1), which is 70,509 nucleotides in length. Table 1 lists significant ORFs and their primary characteristics. Of the 61 ORFs in Table 1 (excluding the ylpA and 'yadA pseudogenes), 8 have GTG starts (repA, yscD, yscJ, yscW, Orf7, Orf42, Orf43, and Orf74) and 3 have TTG starts (Orf54, Orf61, and Orf73). We have omitted most IS element remnants and partial ORFs that appear to be nonfunctional due to IS-related events or other deletions and rearrangements.

Replication and partition functions. Sequencing confirmed that pCD1 has an IncFIIA replication system (Fig. 2). For the IncFIIA resistance plasmid R1 and its close relatives (R100 and R6), a quite detailed analysis of this replication system has been performed. RepA (formerly RepA1) is required for replication at the origin of replication (oriR). Transcription from P_repA is repressed by CopB (RepB in Fig. 2 is its homologue); constitutive transcription from the P_copB promoter (P_repB in Fig. 2) results in a long mRNA encoding RepB, Tap, and RepA that is the source of most of the RepA protein in E. coli (110). Posttranscriptional expression of RepA is controlled by copA, which lies within the leader region of RepA mRNA and encodes an antisense RNA transcribed in the opposite direction compared to RepA mRNA transcription. CopA and its RepA mRNA target site, termed CopT, possess complex secondary stem-loop structures that form a loop-loop RNA-RNA complex. This interaction completely blocks translation of the adjacent gene, tap, which encodes a 24-aa protein. Translation of tap is required for translation of repA (16, 64).

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Genetic organization of the replication and partitioning regions of pCD1. (A) The replication region contains four genes (repB, copA, tap, and repA) and an origin of replication (oriR). Black boxes indicate the promoter regions for repB and repA; the promoter region for copA is not shown. (B) The par region possesses two putative trans-acting genes (sopA and sopB) and a putative DNA binding site (sopC). Arrowheads within the promoter region show the locations and orientations of short, imperfect repeats. The six 45-bp direct repeats that compose sopC are shown as open boxes, with the locations of five 16-bp inverted repeat structures designated by arrowheads. RBS, ribosome binding site. For both panels, arrows indicate the directions of transcription of individual genes, while locations within pCD1 are indicated by numbers that follow the numbering system in Fig. 1.

Insertion elements. Several mobile genetic elements have been found in the pathogenic yersiniae, and most of them are present on LCR plasmids as well as on the chromosome (27, 38, 40, 70, 78, 80, 90). ISs known to be associated with the LCR plasmid of Y. pestis include IS100 and IS285 (38, 83, 87). Additional elements are found on the LCR plasmid of Y. enterocolitica but are not present on the Y. pestis plasmid (40, 78). Sequence analysis of pCD1 from Y. pestis KIM5 revealed the presence of three complete insertion elements and numerous partial IS elements. Complete and partial IS elements with >85% identity at the DNA sequence level were considered to be the same as previously described IS elements. For the remaining elements, the element with the highest database match at the amino acid sequence level was considered the closest relative. Only complete IS elements were given new IS number designations.

View larger version (28K): [in this window] [in a new window]   FIG. 3.   Diagrammatic representation of four clusters of intact, defective, and partial IS elements in pCD1. A p or p indicates that only part of the IS element is present. A d indicates an IS element that is likely defective. Arrows show the presence of inverted repeats within IS elements. Each type of IS element is represented by a unique design that is maintained in each of the four panels. In panel D, the yadA'/'yadA pseudogene is represented as yadA. Numbers correspond to nucleotide positions in Fig. 1.

Virulence-related genes. The LCR genes previously sequenced for Y. enterocolitica and Y. pseudotuberculosis were all present in Y. pestis (with the exception of yscM2, which appears to be unique to Y. enterocolitica [101]). Within previously described operons, the gene order is conserved and the ORFs generally have the same length (three exceptions are discussed below). As anticipated, the homology is high: usually 95% and often 98 to 100% identity. Two Ysc components, YscG and YscE, and one effector Yop, YopJ, had 94% identity (95% similarity) to the corresponding proteins of Y. enterocolitica, due to differences scattered within the predicted proteins. Detailed studies will be required to determine if these differences translate into significant functional differences such as arise from the heterogeneity in LcrV (see the introduction). The largest difference between effector Yop proteins of Y. pestis and Y. enterocolitica was in YopM (93% identity [94% similarity] to YopM of Y. enterocolitica), discussed below.

Previously studied loci. The YpkA- and YopJ-encoding operon in Y. pestis KIM5, like that in Y. pseudotuberculosis (44), also has a small ORF (Orf7) upstream, but in Y. pestis, this ORF is shorter by 7 residues and is spaced from ypkA by 17, compared to 6 bp. In these two yersiniae these ORFs are identical up to the sequence encoding the C-terminal 10 residues (17 in Y. pseudotuberculosis), where they become very different. An in-frame deletion within this ORF in Y. pseudotuberculosis did not affect expression or secretion of YpkA or YopJ or virulence in mice, leaving it without any obvious role in the LCR or in virulence, and it was speculated that this ORF in fact is not translated (44). The Orf7 product is interesting because it has properties very much like those of the Yop chaperones (Sycs). Its molecular mass of 15,747 Da is similar to the sizes of other described Sycs (111), and like other Sycs, it is predicted to be acidic (pI of 4.39) and to have an amphipathic character. Intriguingly, the database search revealed some homology of this predicted ORF to an ORF upstream of the gene encoding the Avr-like protein HrmA in Pseudomonas syringae pv. syringae (a plant pathogen that secretes virulence proteins by a type III secretion mechanism) (1, 2). These similarities suggest that we should revisit the role of Orf7 to reassess its expression and possible role as a Syc for YopJ, which now is believed to be targeted into eukaryotic cells and has been shown to cause apoptosis of macrophages by Y. pseudotuberculosis and Y. enterocolitica (71, 72). Deletion of this ORF might not have had an effect on virulence, because abolishing YopJ itself did not affect virulence in mice infected orally by Y. pseudotuberculosis (44).

Potential virulence-related new ORFs. Fourteen ORFs are not obviously associated with IS elements and either have products with no significant similarity to proteins in the database with known functions or have features suggesting a virulence-related role. These are ORFs that deserve future study as potentially having virulence or virulence-accessory functions.

Summary. Our analysis of the pCD1 DNA sequence has identified an IncFIIA replicon and Sop-like partitioning system necessary for plasmid maintenance. We noted the insertion sites of IS100, Tn1000p, IS21p, and several partial IS285 elements as well as two new IS elements, IS1616 and IS1617. In addition, there are numerous IS element remnants clustered in four regions of the 70-kb plasmid. We found no evidence for the existence of yopL, and, in Y. pestis, ylpA and yadA are pseudogenes. Although regulatory and secretory components of the LCR constitute a contiguous LCR cluster, elements suggesting that this region is a PAI were not identified. Genes for effector Yops are scattered throughout the plasmid and have widely varying GC contents, indicative of multiple gene acquisition events. This observation coupled with the presence of IS remnants from only distantly related microorganisms suggests that a very complex history of DNA acquisition, insertions, deletions, and rearrangements was required for assembly of pCD1.