Genomic Rearrangements at rrn Operons in Salmonella

Corresponding author: Stanley Maloy, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182-4614., smaloy{at}sciences.sdsu.edu (E-mail)

Communicating editor: A. SONENSHEIN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Most Salmonella serovars are general pathogens that infect a variety of hosts. These "generalist" serovars cause disease in many animals from reptiles to mammals. In contrast, a few serovars cause disease only in a specific host. Host-specific serovars can cause a systemic, often fatal disease in one species yet remain avirulent in other species. Host-specific Salmonella frequently have large genomic rearrangements due to recombination at the ribosomal RNA (rrn) operons while the generalists consistently have a conserved chromosomal arrangement. To determine whether this is the result of an intrinsic difference in recombination frequency or a consequence of lifestyle difference between generalist and host-specific Salmonella, we determined the frequency of rearrangements in vitro. Using lacZ genes as portable regions of homology for inversion analysis, we found that both generalist and host-specific serovars of Salmonella have similar tolerances to chromosomal rearrangements in vitro. Using PCR and genetic selection, we found that generalist and host-specific serovars also undergo rearrangements at rrn operons at similar frequencies in vitro. These observations indicate that the observed difference in genomic stability between generalist and host-specific serovars is a consequence of their distinct lifestyles, not intrinsic differences in recombination frequencies.

THE species Salmonella enterica consists of >2500 serovars, which share 96–99% sequence similarity (EDWARDS et al. 2002 ). Most of these serovars are generalist pathogens, infecting a wide range of hosts. For example, S. enterica serovar Typhimurium and S. enterica serovar Enteritidis can infect a variety of hosts from reptiles to humans, causing many different symptoms (PASCOPELLA et al. 1995 ). However, a few serovars are usually able to infect only a specific host. These include S. enterica serovar Typhi, which infects only humans, and S. enterica serovar Pullorum, which causes disease only in fowl. These host-specific serovars cause a systemic typhoid-like disease (BARROW et al. 1994 ).

By performing pulsed-field gel electrophoresis (PFGE) on independent isolates of multiple Salmonella serovars, Liu and Sanderson found that isolates of generalist serovars obtained from a variety of hosts and from different locations around the world (BELTRAN et al. 1991 ) shared a conserved chromosomal arrangement (Fig 1A; LIU and SANDERSON 1995A , LIU and SANDERSON 1995B , LIU and SANDERSON 1998 ). In stark contrast, the host-specific serovars had numerous, large-scale genomic rearrangements (LIU and SANDERSON 1995A , LIU and SANDERSON 1995B , LIU and SANDERSON 1996 , LIU and SANDERSON 1998 ). Different isolates of the same host-specific serovar have distinct genomic arrangements, implying that strains with rearranged chromosomes predominate within subpopulations.

View larger version (11K): In this window In a new window Download PPT slide   Figure 1. Two different rrn arrangements of Salmonella. (A) Arrangement of Typhimurium, which is the same arrangement as E. coli. All natural isolates of generalist Salmonella serovars analyzed to date have this arrangement. The DNA between the rrn operons has been previously labeled Ceu fragments A, B, C, D, E, F, and G. (B) Arrangement of Typhi strain TYT3521. The Ceu fragments are rearranged due to recombination at the rrn operons. Different strains of a host-specific serovar often have various rrn arrangements.

The rearrangements appear to be the result of homologous recombination at the ribosomal RNA (rrn) operons, including inversions and levitation of significant portions of the genome relative to the genome organization of the generalist serovars (Fig 1). Inversions can result from homologous recombination between two regions of homology in opposite orientation (Fig 2A). Levitations can arise by recombination between regions of homology in the same orientation leading to excision, followed by reinsertion by homologous recombination at another site (Fig 2B). Both of these events result in the reorganization of large fragments of the chromosome (HUGHES 1999 ).

View larger version (15K): In this window In a new window Download PPT slide   Figure 2. Two different ways chromosomal rearrangements can occur. (A) Two regions of homology in opposite orientation can recombine to yield an inversion in the chromosome and two hybrid rrn operons. (B) Regions of homology in direct orientation can recombine to generate a transient circular DNA fragment that can subsequently recombine with the chromosome at a different site, yielding a levitation of the rrn interval and three hybrid rrn operons.

The rrn operons provide regions of homology required for chromosome rearrangements (ANDERSON and ROTH 1979 ; HILL et al. 1990 ). The number and location of rrn operons are conserved among the enteric bacteria (KRAWIEC and RILEY 1990 ). An rrn operon consists of genes coding for 16S, 23S, and 5S rRNA and a spacer region between the 16S and 23S genes. While the spacer region can be variable, the other genes are at least 99.5% identical between the seven operons within an organism (LEHNER et al. 1984 ; CHRISTENSEN et al. 2000 ). Salmonella and Escherichia coli each have seven rrn operons located at noncontiguous sites that center around the origin of replication (oriC; ELLWOOD and NOMURA 1982 ). Transcription of the rrn operons is oriented in the same direction as the leading strand of DNA replication.

The discrepancy between the conserved rrn arrangements found in generalist Salmonella serovars and the variable rrn arrangements found in host-specific serovars may be explained by two alternative hypotheses. The first possibility is that this is due to a "mechanistic difference" between the generalist and host-specific serovars—i.e., host-specific serovars have the ability to rearrange their genomes while the generalists do not. A second possibility is that the difference in chromosome stability is a result of the "lifestyle difference" between these serovars—i.e., all Salmonella have the ability to undergo chromosomal rearrangements but because of differences in their physiology or ecology, the rearrangements are tolerated, selected for, or induced in the host-specific serovars while the generalist serovars maintain a consistent genome arrangement. If the "mechanistic" hypothesis is correct, we would predict that genomic rearrangements would occur at a much higher frequency in host-specific serovars compared to generalists in vitro. If the "lifestyle" hypothesis is correct, genomic rearrangements would potentially occur at a similar frequency in all serovars in vitro and rearrangements would be observed only in vivo; i.e., the frequency of rearrangements in vivo would be dependent upon the particular aspect of the host-specific lifestyle, which encourages the maintenance of the altered gene order established by the recombination events.

We assayed for inversions using three approaches: (i) a genetic selection for inversions at portable regions of homology similar to a method previously described (SEGALL and ROTH 1989 ), (ii) a PCR method for determining the rrn arrangement (HELM and MALOY 2001 ), and (iii) a genetic selection for inversions between rrn operons. We assayed inversion frequencies in a generalist serovar (Typhimurium) and a host-specific serovar (Typhi). Both generalist and host-specific Salmonella serovars incurred inversions at a low frequency in vitro. These results indicate that generalist and host-specific Salmonella have similar recombination frequencies, implying that the variation in chromosome arrangement is a result of the lifestyle differences between the serovars.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Bacterial strains, growth conditions, and media:
The strains used in this study are shown in Table 1 and plasmids used are shown in Table 2. Unless otherwise noted, bacteria were grown on rich medium [Luria-Bertani (LB)] at 37° (BERTANI 1951 ). Minimal media consisted of E medium supplemented with 0.2% glucose as a carbon source or E medium with no nitrogen source supplemented with 0.2% proline as the sole nitrogen source and 0.6% succinate as a carbon source (MALOY and ROTH 1983 ). When lactose was used as a sole carbon source, E medium with no citrate was supplemented with 0.2% lactose. His, Trp, and Cys auxotrophs were supplemented with 0.1 M histidine, tryptophan, or cysteine, respectively. Ampicillin (Amp) was added at a final concentration of 90 µg/ml in LB to maintain Amp-resistant plasmids. Kanamycin SO₄ (Kan) was added at final concentrations of 50 µg/ml in LB and 125 µg/ml in minimal medium except during selection for inversions at rrn operons when Kan concentration was 25 µg/ml in LB. Chloramphenicol (Cam) was added at final concentrations of 20 µg/ml in LB and 10 µg/ml in minimal medium. Gentamycin SO₄ (Gen) was added to LB at final concentrations of 10 µg/ml for Typhimurium and 5 µg/ml for Typhi.

Transduction:
The high-frequency transducing mutant of phage P22 (HT105/1 int-201) was used as previously described (MALOY 1990 ). To construct the MudJ strains, multiplicities of infection (MOI) ranging from 0.1 to 0.5 were used for transductions from Typhimurium into Typhimurium. MOI from 0.5 to 2 were used when transducing from Typhimurium into Typhi due to the homeologous recombination barrier between the two serovars (ZAHRT et al. 1994 ). To select for inversions at rrn operons, MOI between 1 and 2 were used. Transductions were performed by mixing a phage lysate with an overnight culture followed by expression at 37° for 1 hr prior to plating on selective media.

Construction of MudJ strains:
Typhi and Typhimurium derivatives with two MudJ markers in opposite orientation were constructed with the lacZ in each MudJ disrupted by a selectable insertion. One copy of lacZ had a Gen^R insertion and the second copy of lacZ had a Cam^R insertion, with the relative positions of the two insertions separated by 1845 bp in different copies of the lacZ gene. To construct the insertions, Cam or Gen was cloned into lacZ from pUR278 and then transduced into an LT2 strain containing a MudJ insertion, yielding either MudJ(lacZ::Gen) or MudJ(lacZ::Cam). These markers were then transduced into Typhimurium or Typhi.

The genotypes and construction of the plasmids used in this study are shown in Table 2. The lacZ::Cam and lacZ::Gen insertions in MudJ were constructed as follows. pPC253 was digested with XbaI and PstI, which cut at sites on lacZ flanking the Cam insert, and pPC254 was digested with PvuII, which cuts at two sites on lacZ flanking the Gen insert. Gen or Cam fragments were gel purified and electroporated into strain MST4880. This strain carries hisD9953::MudJ (Lac⁺) in an sbcE recB recD background (FIGUEROA-BOSSI et al. 1997 ) to promote homologous recombination via a recE-like activity and eliminate the effect of Exonuclease V. Cam^R or Gen^R electroporants were selected and then screened for Amp^S and loss of Lac⁺. Strains with MudJ(lacZ::Gen) and MudJ(lacZ::Cam) were transduced into LT2 strains containing intact MudJ fusions to create the following strains: MST4882, LT2 hisD9953::MudJ(lacZ::Gen); MST4883, LT2 putA::MudJ(lacZ::Cam); and MST4885, LT2 trpED2490::MudJ(lacZ::Cam). The insertions were then transduced into both LT2 and Typhi strain TYT4076 to yield strains with two MudJ fusions in opposite orientation. Fig 3 indicates the relative locations of the MudJ insertions in Typhi and Typhimurium.

View larger version (10K): In this window In a new window Download PPT slide   Figure 3. Locations of MudJ fusions in lacZ inversion assay. (A) The chromosomal locations of his and put. MST4884 and TYT4026 have one lacZ inserted in each of these genes in Typhimurium and Typhi, respectively. (B) The chromosomal locations of his and trp. MST4886 and TYT4027 have one lacZ inserted in each of these genes in Typhimurium and Typhi, respectively.

Genetic tests for inversions at lacZ:
Strains with the two disrupted lacZ fusions were tested for inversions by selecting for growth on lactose as a sole carbon source on minimal medium supplemented with cysteine, tryptophan, and histidine. For these studies all three supplements were added to all media, even when not required, for consistency. Inversions were confirmed via P22 transduction as previously described (SEGALL and ROTH 1989 ).

Bottleneck assays:
To test whether rrn rearrangements occur in the presence of a genetic bottleneck, we used two independent isolates each of Typhimurium and Typhi and a PCR assay, which allows rapid mapping of the rrn arrangement of strains (HELM and MALOY 2001 ). TYT3052 and TYT3060 are independent isolates of Typhimurium, and TYT4076 and TYT3521 are independent clinical isolates of Typhi. Strains were streaked on rich medium, and 24 colonies from each strain were randomly isolated and restreaked. Every day a single colony from each restreak was randomly picked and restreaked. This was repeated daily for 60 days, with genomic DNA isolated from each of the 96 samples at intervals for PCR analysis. The rrn arrangement of each sample was compared to the parent colony.

Construction of rrn inversion selection strains:
The -Red recombinase system (DATSENKO and WANNER 2000 ) was used to insert Cam- and Kan-resistant cassettes directly adjacent to rrn operons in Pullorum and Typhimurium. Primers designed to anneal to DNA sequences bordering the rrn operons in Typhi (PARKHILL et al. 2001 ) yield equivalent PCR products in multiple Salmonella serovars, including Typhimurium and Pullorum (HELM and MALOY 2001 ). We used these sequences to design oligonucleotides to insert Cam and Kan cassettes at the two ends of rrnH in Typhimurium and rrnG/H in Pullorum via Red swaps (Table 3). The resulting PCR products were electroporated into Pullorum or Typhimurium strains carrying pKD46 (Table 2). Each recipient received a single antibiotic resistance marker. Cam or Kan insertions were verified using PCR primers that anneal to DNA flanking the rrn operons (HELM and MALOY 2001 ) combined with primers that anneal to Cam or Kan cassettes (DATSENKO and WANNER 2000 ). The Cam and Kan insertions were then backcrossed via P22 transduction. The resulting strains TYT3890 and TYT3893 are shown in Fig 4. MST4887 was constructed by transducing DNA from TYT3890 into Typhimurium, selecting for both markers, with subsequent confirmation by PCR. MST4887 is a useful transduction donor because while DNA modified by Pullorum methylases is strongly restricted by Typhimurium, there is little restriction of Typhimurium methylase-modified DNA by Pullorum or Typhi (data not shown).

View larger version (13K): In this window In a new window Download PPT slide   Figure 4. Chromosomal locations of CamR and KanR markers in rrn inversion selection strains.

Selection for inversions at rrn operons and calculation of inversion index:
Transductions were performed to select for inversions at rrn operons. If the strain being tested for inversions had intact rrnG and rrnH operons, such as LT2 or TYT4076, the transduction donor was MST4887 (Fig 4). If the strain being tested had rrnG/H and rrnH/G, such as TYT3521, the transduction donor was MST3893 (Fig 4). In all cases, the strain being tested was the transduction recipient. Transductants were selected on LB Kan plates. After 48 hr the transductants were replica plated onto LB Kan Cam plates. The rrn arrangement of colonies resistant to both markers was confirmed via PCR (HELM and MALOY 2001 ). Primers that yield a product for rrnG, rrnH, rrnG/H, and rrnH/G were used for each Cam^R Kan^R colony.

The inversion index was calculated by dividing the number of confirmed inversions by the total number of Kan^R colonies. Note that transduction is predicted to occur at a much higher frequency if the selected marker is flanked on both sides by substantial homology. Hence, if the donor and the recipient have the same rrn arrangement the inheritance of a single marker will occur at a higher frequency than if a rrn inversion has occurred. For example, the transduction of Kan^R occurs 10-fold more frequently when TYT3893 is a donor and TYT4076 is the recipient than when TYT3521 is the recipient (data not shown). For this reason, the number of Kan^R Cam^R inversions observed relative to the total number of Kan^R colonies does not equal the actual inversion frequency, but the inversion index represents the relative ability of a strain to invert compared to another strain tested under similar conditions.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Inversion frequency at lacZ:
Selection for recombination between mutant lacZ genes in opposite orientation on the Typhimurium chromosome demonstrated that inversions can occur between certain "permissive" intervals but do not occur between other "nonpermissive" intervals (SEGALL et al. 1988 ; SEGALL and ROTH 1989 ). In an attempt to determine whether the Typhi genome is more tolerant of inversions than is the Typhimurium genome, we designed strains with two mutant MudJ elements placed in opposite orientation in the same genetic loci of the two serovars. Within each MudJ element, the lacZ gene contained a single selectable insertion, rendering the strain Lac^-. The relative distance of the insertion in one copy of lacZ was 1.85 kb from the position of the insertion in the second copy of lacZ. MST4884 (Typhimurium) and TYT4026 (Typhi) each had a hisD::lacZ insertion (at 44.7 Cs) and a putA::lacZ insertion (at 25.6 Cs), a permissive interval. MST4886 (Typhimurium) and TYT4027 (Typhi) each had a hisD::lacZ insertion and a trpED::lacZ insertion (at 38.0 Cs), a nonpermissive interval (Fig 3). To screen for inversions these strains were plated on lactose as a sole carbon source. Because Lac⁺ colonies can be the result of events in addition to inversions, potential inversions were verified by transduction as previously described (SEGALL and ROTH 1989 ). No difference in the frequency of Lac⁺ inversions at either interval in the two serovars was detected (Table 4). The inversion frequency of Typhi and Typhimurium was within the same order of magnitude in the permissive his-put interval. No inversions were found in the nonpermissive interval in either serovar.

Rearrangements at rrn operons over time:
Although PFGE experiments clearly demonstrated that different isolates of host-specific Salmonella had chromosomal rearrangements relative to one another, chromosomal rearrangements had not been observed to occur within a single strain. One possible explanation for this discrepancy could be that rrn inversions occur at a high frequency but are outcompeted by the parental strain. Isolation of strains with inversions during bottlenecks accompanying host-specific infections could evade this competition, allowing inversion strains to predominate (ANDERSSON and HUGHES 1996 ). To determine whether rrn rearrangements accumulate when forced through bottlenecks in vitro, we used a PCR assay to determine the typical chromosomal arrangement in populations of two clinical isolates of Typhimurium and two clinical isolates of Typhi following many subcultures grown on laboratory medium. On the first day 24 colonies were randomly selected from each of the four strains and streaked for isolated colonies on rich medium. On the second day a single colony was randomly selected from each of these cultures and restreaked on rich media. This process was repeated daily for 60 days. At intervals through day 60 chromosomal DNA was isolated from colonies and analyzed via PCR to determine the rrn arrangement of each sample compared to the original parent. All of the colonies tested at each stage of the experiment had the same rrn arrangement as the parent strain. Thus, a genetic bottleneck of this magnitude does not lead to the predominance of rrn rearrangements under standard lab conditions.

Selection of inversions at rrn operons:
Taken together, the lacZ inversion experiments and the rrn PCR experiments led to two conclusions: (i) using portable regions of homology, the genome stability of Typhi and Typhimurium was indistinguishable and (ii) if rearrangements occur at rrn operons under these conditions, the frequency is too low to detect by the PCR assay. Although the PCR assay is quite sensitive, it is not as sensitive as a genetic selection. Therefore, three strains were constructed to provide a genetic selection for inversions between rrn operons (Fig 4). A Cam^R marker was inserted adjacent to one side of an rrn operon and a Kan^R marker was inserted adjacent to other side of the rrn operon. Transducing phage were grown on each Cam^R Kan^R donor. If the arrangement of the rrn operons in a recipient is inverted relative to the donor, the flanking homologies on either side of the rrn operon will be separated. Hence, the antibiotic resistance markers will be too far away to be coinherited from a single transducing particle. The only way a recipient can coinherit both antibiotic resistant markers from a single transducing particle is if an inversion in the recipient places the two rrn operons in the same arrangement as in the donor strain (Fig 5). We assayed for inversions between rrnG and rrnH because this is a common inversion in natural isolates (LIU and SANDERSON 1996 , LIU and SANDERSON 1998 ; HELM and MALOY 2001 ). Furthermore, an inversion at these join points does not cause a substantial shift in either the relative distance between the origin and terminus of replication or the relative distance of genes within the inverted region compared to the origin of replication (Fig 1) and, hence, does not affect gene dosage (LIU and SANDERSON 1995A , LIU and SANDERSON 1995B ).

View larger version (18K): In this window In a new window Download PPT slide   Figure 5. Method of selection for inversions at rrn operons. (A) Selection for inversions between rrnG and rrnH in Typhimurium LT2. A single transducing particle cannot simultaneously cotransduce CamR and KanR into LT2 unless an inversion occurred between rrnG and rrnH. (B) Selection for inversions between rrnG/H and rrnH/G in Typhi TYT3521. A single transducing particle cannot simultaneously cotransduce both markers into TYT3521 unless an inversion occurred between rrnG/H and rrnH/G.

In Salmonella, rrnG and rrnH are in opposite orientation (Fig 1). A recombination event between two regions of homology in opposite orientation will have two results (Fig 2A). First, the region of the chromosome between the homologous regions will be inverted. The region between rrnG and rrnH consists of approximately half the Salmonella chromosome, including the replication terminus, a region previously designated I-Ceu fragment A (Fig 1; LIU and SANDERSON 1998 ). Second, the recombination process will generate two hybrid rrn operons at the expense of the two original rrn operons. In this case, rrnG and rrnH will be lost and rrnG/H and rrnH/G will be produced, where rrnG/H consists of the 5' end of rrnG and the 3' end of rrnH, and rrnH/G consists of the 5' end of rrnH and the 3' end of rrnG.

For this study we tested three strains, LT2, TYT4076, and TYT3521. LT2 is a Typhimurium strain with an intact rrnG and rrnH. TYT4076 is a Typhi strain, which, like Typhimurium, has an intact rrnG and rrnH. TYT3521 is a Typhi strain that has an inversion resulting in rrnG/H and rrnH/G (Fig 5). To test for inversions in LT2 and TYT4076, the donor strain MST4887, which has Cam and Kan markers flanking rrnG/H, was used (Fig 4). In the case of LT2 and TYT4076, only a cell with an inversion between rrnG and rrnH will inherit both markers from a single transducing particle (Fig 5A). To test for inversions in TYT3521, the donor strain TYT3893, which has Cam and Kan markers flanking rrnH was used (Fig 4). In the case of TYT3521, only a cell with an inversion between rrnG/H and rrnH/G will inherit both markers from a single transducing particle (Fig 5B).

The Poisson distribution can be used to calculate the probability that the observed inversions are due to inheritance of two independent transducing particles, one carrying the Cam marker and another carrying the Kan marker. The analysis predicts that the frequency of such double transducing events would be <0.4% at the highest MOI used in this study, well below the observed frequency of Cam^R Kan^R transductants. The frequency of repair of two unlinked auxotrophic markers was tested using the same phage lysate used for the inversion studies and the same MOI. The observed frequency of repair of the unlinked auxotrophic markers was 0.2%, supporting the Poisson distribution calculation. Moreover, if the inversion assay required two transducing fragments, it would be very sensitive to the MOI used, but in both Typhi and Typhimurium, the inversion index changes <2-fold when the MOI is increased >10-fold (data not shown).

The results of the selection for rrn inversions are shown in Table 5. The results indicate that: (i) inversions at rrn operons occur in both Typhimurium and Typhi, even though rearrangements are not observed in natural isolates of Typhimurium (LIU and SANDERSON 1995A , LIU and SANDERSON 1995B ; HELM and MALOY 2001 ), and (ii) the rearrangements occur at approximately equal frequencies in both host-specific and generalist serovars, suggesting that under standard laboratory conditions Typhi and Typhimurium have similar potential for rrn rearrangements.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this study we sought to discern why chromosomal rearrangements at rrn operons are so prevalent in host-specific Salmonella while generalist serovars exhibit extreme stability. Two alternative hypotheses could account for this difference. First, there could be an inherent recombinational difference between generalist and host-specific serovars. Second, all Salmonella may have the ability to undergo recombination at rrn operons at a similar frequency, but the differences in lifestyle between generalist and host-specific serovars favor accumulation of rearrangements in host-specific serovars. If the first hypothesis is correct, it should be possible to experimentally observe a difference in the frequency of rearrangements between generalists and host-specific serovars in vitro. If the second hypothesis is correct, all Salmonella serovars would exhibit the same frequency of rearrangement in vitro. To test these alternative hypotheses, we used multiple approaches to compare the genomic stability of generalist serovar, Typhimurium, and host-specific serovar, Typhi.

Given the inherent limits of each method, these approaches provided independent tests of the alternative predictions. The first approach used mutant lacZ genes located at identical sites on the Typhimurium and Typhi chromosomes to assay the frequency of inversions between portable regions of homology. The propensity for inversions was indistinguishable in both serovars: nearly equal inversion frequencies were detected in the permissive interval of both serovars, while no inversions were detected in the nonpermissive interval of either serovar. These results indicate that under standard lab conditions the Typhi chromosome was not more permissive for inversions than the Typhimurium chromosome.

Although the frequency of inversions between portable regions of homology was indistinguishable in Typhi and Typhimurium, it remained possible that there was a difference in the frequency of rearrangements at rrn operons. To test for rrn rearrangements, we assayed for products generated following PCR with primers that flank each of the rrn operons. The frequency of rrn rearrangements was followed over 60 passages of individual colonies. Despite the imposed bottlenecks and the many generations, no rrn rearrangements were observed in either Typhi or Typhimurium. Hence, given the sensitivity of PCR detection and the limited sample size tested, the results indicate that the majority of both Typhi and Typhimurium populations retain the parental rrn arrangement during prolonged growth in laboratory medium.

An alternative possibility was that the frequency of rearrangements at rrn operons is simply too low for detection by the PCR assay. We reasoned that a genetic selection for inversions would provide a simple and exquisitely sensitive assay for the frequency of these events. The selection method we developed provides an assay for the frequency of inversions between rrnG and rrnH. The results clearly indicate that Typhimurium experiences inversions at rrnG and rrnH, and during growth in vitro these inversions occur at a frequency similar to that observed in Typhi. Again, this data indicates that under standard laboratory conditions, the frequency of inversion between rrn operons is essentially identical in Typhi and Typhimurium.

Taken together, these results indicate that during growth under standard lab conditions the Typhi and Typhimurium genomes undergo rearrangements at an equivalent low frequency, and thus the genomes of generalist and host-specific Salmonella appear to be equally stable. This finding coupled with the observation that natural Typhimurium isolates have a uniform rrn rearrangement strongly suggests that the disparity in the frequency of chromosome rearrangements observed in nature is due to differences that occur during the course of infection. Either some disparity between the in vivo environments promotes chromosomal rearrangements in host-specific serovars or chromosomal rearrangements are less disadvantageous for host-specific serovars. The variation in chromosome stability of the generalist vs. host-specific serovars could be due to differences in their population biology, differences in their response to stress conditions encountered in the host, or differences in the response of the host to the infection. These possibilities should be directly testable using the genetic selection described in this manuscript.

	ACKNOWLEDGMENTS

We thank Nello Bossi for sharing strains and advice, Patti Fields for providing clinical isolates from the Centers for Disease Control and Prevention, John Roth for advice on the genetic bottleneck experiment, and Rob Edwards for assistance and for critically reading this manuscript. We are indebted to Abe Eisenstark and Ken Sanderson for incisive discussions about chromosome rearrangements. This work was supported by grant AG 2001-35201-09950 from the U.S. Department of Agriculture.

Manuscript received September 19, 2002; Accepted for publication January 10, 2003.

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