Molecular Basis of the Indole-Negative Reaction in Shigella Strains: Extensive Damages to the tna Operon by Insertion Sequences

PCR sequencing of the tna operon.

The entire tna operon, including the three structural genes (tnaL, tnaA, and tnaB) and the promoter region, was sequenced for each strain, with exceptions where only the tnaB gene could be amplified. Primers for PCR and sequencing were based on the E. coli K-12 sequence (GenBank accession numbers AE000447 and AE000448 ) and designed to give overlapping amplicons of approximately 1 kb in length. PCR products were purified using a Wizard PCR purification system (Promega, Madison, Wis.) or a MO BIO PCR Clean-Up kit (MO BIO Laboratories, Solana Beach, Calif.). Samples were sequenced using dye terminator technology (Perkin-Elmer Cetus, Norwalk, Conn.) through the Sydney University and Prince Alfred Hospital Macromolecular Analysis Centre and an automated 377 DNA sequencer (Applied Biosystems, Burwood, Victoria, Australia).

DNA sequences were assembled and edited by using the programs PHRED, PHRAP, and CONSED (10). Further analysis was undertaken using programs available from the Australian National Genomic Information Service at the University of Sydney. Sequence comparisons were done using MULTICOMP (20).

The strains representing 23 indole-negative serotypes were described previously (19). They are referred to as D1, B1, and F1A for S. dysenteriae 1, S. boydii 1, and S. flexneri 1A, respectively, and so forth.

Indole-negative property in all Shigella strains is due to damage by IS-mediated insertion and or deletion.

All cluster 1 strains except D7 are indole negative. Sequencing revealed that the indole-negative strains have at least one IS1 sequence inserted in tnaL at base 55 (Fig. 1), which disrupted the tnaL gene. In D3 there is an additional insertion in the tnaA gene in the opposite orientation. In D3, D4, D11, D12, and D13, the original insertion sequence (IS) was flanked by a nine-base direct repeat (GACAATAAG), characteristic of IS1 insertion (14). In B2, B4, B14, F6, and F6A, the insertion is followed by a 1,001-bp deletion to base 777 of the tnaA gene. The insertion and deletion damages both tnaL and tnaA.

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FIG. 1.

Schematic diagram of the tna operon and genetic changes in Shigella strains. The map of the operon at the top is based on E. coli K-12. Below are shown 12 forms of the operon that can be distinguished in indole-negative Shigella serotypes. Data for D1 and S. sonnei are partly based on the incomplete genome sequences from the Sanger Centre. Blank regions indicate that PCR failed to amplify a fragment from that region while dotted lines indicate deletions with a defined end or ends. Triangles indicate IS insertions with base position marked below. Positions are given as the base pair in the gene, with negative values indicating the position relative to the start of the next gene or major feature (see the text). The name of the IS is also indicated if not IS1. Solid arrowheads at the ends of the IS indicate repeats of chromosomal DNA if present. For deletions the number of the first base is shown, with lengths in parentheses.

A third group of strains comprising B1, B3, B6, B8, B10, B18, and D5 have one partial 192-bp IS1 sequence followed by a full IS1 sequence. All except B3 and D5 have a deletion of 49 bp including 21 bp of the tnaL gene. The tnaA gene and part of tnaB from B3 could not be amplified. B3 also has an IS insertion in the tnaB gene. In D5 there has been a further IS insertion close to that inserted in tnaL at base 55; an IS insertion in the tnaL/tnaA intergenic region was found by PCR using an IS primer and a tnaL/tnaA intergenic region primer. Sequencing from the tnaA end revealed a junction between an IS1 insertion and the intergenic region at base 29. The IS1 insertion was in the opposite orientation to that at base 55. However, the structural arrangement in the region between the IS insertions at tnaL and the intergenic region could not be determined, and as it is unique to D5, is not needed to follow the course of the changes. Also we were not able to amplify the tnaA gene or the 5′ part of the tnaB gene. The boundaries of this deletion were not determined.

Although the IS elements present in the three groups of cluster 1 strains have a different configuration at position 55, it is most likely that the initial insertion event occurred in the common ancestor before the groups diverged. The complete IS1 sequences were nearly identical, with some base substitutions, as indicated in Fig. 2.

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FIG. 2.

Evolution of Shigella cluster 1 strains. A tree based on housekeeping gene sequences (19) is presented. Changes in housekeeping genes (19) and in the tna operon (this study) are indicated at the nodes. For example 2HK (3) means that three changes in two housekeeping genes supported that branch. Events in the tna operon are marked by the gene name with changes in parentheses. Regions that failed to amplify a product are treated as unknown events and shown as tna(unk). Changes that occurred in individual strains only are marked for the tna operon but not housekeeping genes, leading to the collapse of most external branches.

Cluster 3 has only one consistently indole-negative strain, B12. An IS1 sequence was found inserted two bases before the σ⁷⁰ binding site of the promoter region, separating it from the cyclic AMP receptor protein binding site, which seems to be sufficient to disrupt the expression of the tna operon. All three structural genes were intact.

Three of the five outlier strains, D1, D10, and SS, are indole negative. We sequenced only part of tnaB gene of D1, as the rest could not be amplified. The D1 strain has a six-base in-frame deletion in the tnaB gene but in a different position (base 649 to 654) from that of the cluster 1 strains (see below). Analysis of the genome sequence of the D1 strain sequenced by the Sanger Centre revealed that it too had the 6-bp deletion, and also an IS2 inserted at base 91 of tnaB and the tna operon up to the IS2 insertion site in tnaB is deleted.

No product could be amplified from S. sonnei. Analysis of the genome sequence of the S. sonnei strain sequenced by the Sanger Centre indicates that the entire operon is deleted starting from 374 bp upstream of tnaL to 486 bp downstream of tnaB and is replaced by an IS1 element that probably mediated the deletion.

Two insertion sequences were found in D10, one at the end of thdF, the gene immediately upstream of the tna operon, and one at base 333 of the tnaB gene. The insertion at base 333 also led to the deletion of six bases. Interestingly, both insertions were due to ISSfl7 of the IS5 family (4, 14). We were able to obtain a PCR product from base 575 of tnaA but unable to PCR amplify a product from the start of the tna operon to that segment of tnaA, suggesting that there had been some restructuring which we did not resolve.

Deletions in the tnaB gene.

Apart from the IS insertions and associated deletions described above, several other events lead to further damage to the tna operon (Fig. 1). A single base deletion at position 19 of the tnaB gene was observed in seven strains: D3, D4, D6, D9, D11, D12, and D13, which generated a stop codon at bases 52 to 54. An in-frame six-base deletion of bases 706 to 711 of the tnaB gene was observed in strains B1, B3, B6, B8, B10, B18, and D5. As described above, a different six-base deletion of bases 649 to 654 in the tnaB gene was observed in the outlier strain D1. B14 has a base substitution in tnaB at position 149, which generates a stop codon.

Sequence of events in cluster 1 strains.

Variation observed in this study and multilocus sequence data from housekeeping genes allow us to look at the evolution of the cluster 1 strains. Cluster 1 strains were grouped into three subgroups in a previous study of eight housekeeping genes (19), but there were only a few base changes to support the branching pattern. When all the data are considered together it is clear that the subgroups are well supported, and we refer to them here as subclusters 1a, 1b, and 1c (Fig. 2). Subcluster 1a consists of D3, D4, D6, D9, D11, D12, and D13, supported by three mutations in housekeeping genes and two in the tnaB gene. Subcluster 1b consisting of B2, B4, B14, F6, and F6a is supported by three unique base changes in housekeeping genes and the IS-mediated 1,001-bp deletion of the 5′ end of the tna operon. Subcluster 1c, consisting of B1, B3, B6, B8, B10, B18, and D5, is supported by two base changes in housekeeping genes, a six-base deletion in tnaB, and a double IS insertion at base 55 of the tnaL gene. Further branching within each subcluster is evident. No reverse or parallel changes were observed, giving confidence to the tree presented in Fig. 2.

D7, the only indole-positive strain in cluster 1, is at the base of cluster 1 and in most genes studied appears to be almost unchanged from the common ancestor of cluster 1, with the tna operon still functional and only one housekeeping gene base change not present in all cluster 1 strains (19). The IS insertion at base 55 of tnaL is present in all cluster 1 strains except D7, indicating that it occurred early in the development of this group of serotypes. It is interesting that the D7 line did not expand while the other 3 lineages (subclusters) expanded dramatically to give multiple serotypes.

Concluding comments.

Analysis of the tna operon of strains representing each of the 25 Shigella serotypes that are consistently indole negative revealed at least nine IS-related events that damaged the tna operon, leading to a block in conversion of tryptophan to indole. These events are shown to be independent for cluster 1, cluster 3, and the three indole-negative outlier strains, supporting our previous conclusion that these lineages have independent origins. Further, genetic changes in this operon in cluster 1 strains are consistent with a tree based on housekeeping genes, giving strong support for recognition of three subclusters in cluster 1.

The loss of catabolic functions in Shigella strains could be due to niche adaptation. Shigella strains occupy a very different niche from commensal E. coli and could be expected to shed functions not needed for life inside a eukaryotic cell (13), as a trade-off for optimal fitness (5). The parallel loss of a biochemical trait is indicative that the loss is likely to have an adaptive advantage (5, 6). This phenomenon of gene decay is also seen in other clones adapting to a new niche, for example the recently sequenced Salmonella enterica serovar Typhi genome has many pseudogenes (17) as does the Yersinia pestis genome (18). Both are clones adapted to live in a very specific part of the species niche (note that Y. pestis is a clone of Yersinia pseudotuberculosis [2]). However, Shigella strains of E. coli give us additional evidence, as there are several independent lineages and parallel loss is clearly evident for many traits.

With regard to reasons for frequent loss of the tryptophanase pathway in Shigella lineages, little is known of the effect of tryptophan utilization on host or host-pathogen interaction. Indole can act as an extracellular signal, activating a number of genes, and is toxic at high concentrations (23). In E. coli, the presence of tryptophanase has been shown to affect adherence to human epithelial cells and biofilm formation (8), showing that the enzyme can have effects on relevant functions. Four of the seven Shigella lineages within E. coli have lost the indole trait (D1, D10, S. sonnei, and the major part of cluster 1). In addition, one serotype of cluster 2 has lost it and several cluster 3 serotypes are often indole negative (9), suggesting that they are losing the trait. These include serotype 2, prevalent in both developed and developing countries. It appears that conversion of tryptophan to indole is deleterious for Shigella forms, such that those without it have an advantage. The most important lineages are affected. Cluster 1 can be viewed as very successful with expansion to have the most serotypes among the three clusters. S. sonnei is the major form in developed countries, while D1 is the major cause of epidemic-level disease in developing countries (1). However, it must be stated that any effect of tryptophan utilization and indole production on Shigella fitness or virulence is yet to be determined. Also, selection for loss is apparently not as strong as for loss of lactose fermentation, lysine decarboxylation, and motility, uniformly absent in Shigella, and for which the negative selection pressures are better understood (13). In the case of lysine decarboxylation, it has been shown that the property is very deleterious for Shigella (7, 15). The introduction of cadA, the gene encoding lysine decarboxylase, into S. flexneri 2a resulted in attenuation of virulence by inhibition of iron-regulated enterotoxins.

There are also several mutations and insertions in tnaB, and for cluster 1, where we have several subclusters and strong support for the tree as shown in Fig. 2, we can see that these occurred in strains that already lacked tnaA function. This could be due to a functional tnaB gene being deleterious in the absence of tnaA. The tryptophanase operon (tna) is induced by tryptophan and high levels of tryptophan are ordinarily required to obtain maximum tna operon induction (25). The operon would be induced under conditions where there was significant level of tryptophane in the environment and “better” substrates for catabolism were not available. It is possible that under such circumstances the accumulation of tryptophane in the absence of tryptophanase is deleterious. Only a few cluster 1 strains, B2, F6A, F6, and B4, have no deletion, insertion, or frameshift mutation in tnaB, indicating that there is selection for loss of function.

IS elements are known to mediate various genetic rearrangements including inversions and deletions. Clearly IS elements were involved in most of the events affecting function of the tna operon in indole-negative Shigella strains. It is also evident that IS insertion lead to loss of other properties in Shigella. The loss of motility was found to be due to an IS insertion in the flhD operon in one case, and insertion of an IS element impaired the flagellin gene in another (21). This is perhaps not surprising as the S. flexneri 2A genome contains more than 300 IS elements and half of the invasion plasmid genome consists of IS sequences (4, 22). The IS content in the S. flexneri 2a genome is more than sevenfold greater than that of the nonpathogenic E. coli K-12 and enterohemorrhagic E. coli 0157:H7 (12, 24). IS transposition seems to be a major force in the evolution of the Shigella phenotypes. The high level of IS transposition could be viewed as a mutator trait leading to acceleration of the adaptation under selection pressure for loss of function in a range of genes.

Nucleotide sequence accession numbers.

The nucleotide sequences reported in this study have been deposited in the GenBank database (accession numbers AY746464 to AY746494 ).