- Research article
- Open Access
Glutamine synthetase sequence evolution in the mycobacteria and their use as molecular markers for Actinobacteriaspeciation
BMC Evolutionary Biologyvolume 9, Article number: 48 (2009)
Although the gene encoding for glutamine synthetase (glnA) is essential in several organisms, multiple glnA copies have been identified in bacterial genomes such as those of the phylum Actinobacteria, notably the mycobacterial species. Intriguingly, previous reports have shown that only one copy (glnA1) is essential for growth in M. tuberculosis, while the other copies (glnA2, glnA3 and glnA4) are not.
In this report it is shown that the glnA1 and glnA2 encoded glutamine synthetase sequences were inherited from an Actinobacteria ancestor, while the glnA4 and glnA3 encoded GS sequences were sequentially acquired during Actinobacteria speciation. The glutamine synthetase sequences encoded by glnA4 and glnA3 are undergoing reductive evolution in the mycobacteria, whilst those encoded by glnA1 and glnA2 are more conserved.
Different selective pressures by the ecological niche that the organisms occupy may influence the sequence evolution of glnA1 and glnA2 and thereby affecting phylogenies based on the protein sequences they encode. The findings in this report may impact the use of similar sequences as molecular markers, as well as shed some light on the evolution of glutamine synthetase in the mycobacteria.
Gene duplication is a common occurrence in bacterial genomes and may result from evolutionary pressures exerted on the organism by the niche it occupies, thereby enabling adaptation to changing environments [1–3]. Glutamine synthetases (GS; glutamate ammonia ligase EC 3.6.2) are enzymes present in most living organisms where they are involved in the ATP-dependant synthesis of glutamine from glutamate and ammonium. There are two main GS families, namely GSI, which is further subdivided into a GSIβ and the less common GSIα, and GSII. Both the GSI and GSII enzymes are found in prokaryotes, while the GSI enzyme is largely absent in eukaryotes. Various studies have shown that the genes encoding the various GS sub-types are widely distributed in various organisms and encode proteins that have very conserved catalytic and structurally important regions. This finding suggests that all the GS families diverged from a single ancestral sequence through duplication events prior to the divergence of prokaryotes and eukaryotes [4–7]. The GS sub-classes are distinguishable from each other by specific insertion sequences and mechanisms of regulation . The GSIβ sub-type is subjected to post-translational modification by adenylylation of a conserved tyrosine residue by an adenylyltransferase , while GSIα and GSII activity may mainly be regulated through feedback mechanisms. The enzymes also appear to differ in structure; the GS I enzymes form dodecamers , while GSII molecules are octamers . The DNA and protein sequences of GS have thus been used as molecular markers in the construction of the phylogenetic relationships between evolutionary diverse prokaryotic and eukaryotic organisms [6, 11]. These sequences are considered useful as phylogenetic markers due to their higher degree of sequence variation in comparison with other markers, such as 16S rRNA , which are very similar in ecologically related organisms.
Organisms belonging to the phylum Actinobacteria have adapted to occupy a wide variety of ecological niches and include species that are major antibiotic producers, as well as various human, animal and plant pathogens. The genome sequence of M. tuberculosis, a member of the Actinobacteria, revealed that this important human pathogen has four glnA gene copies that may encode GSIβ (glnA1 and glnA4) and GSII (glnA2 and glnA3) enzymes . Of the four glnA gene copies, it has been shown that glnA1 encodes the main and essential GS in M. tuberculosis , while the other glnA sequences (glnA2, glnA3 and glnA4) encode functional, but non-essential GS enzymes . Although these glnA sequences have been shown to encode enzymes that catalyse glutamine synthesis, their evolution and importance in M. tuberculosis is not well understood. Evidence has been presented that suggests that M. tuberculosis GSIβ (encoded by glnA1) may have evolved to perform other specialised functions not present in non-tuberculosis causing mycobacteria and may play a role in enabling M. tuberculosis to survive during infection and growth in the human host [16, 17]. These functions may include the synthesis of poly-L-glutamic acid, a cell wall constituent unique to M. tuberculosis that might play a role in maintaining cell wall homeostasis .
These observations suggest that M. tuberculosis might have been subjected to varying environmental pressures that may have influenced GS sequence evolution. This hypothesis questions the retention of potentially non-essential and/or non-functional sequences in the mycobacterial genome. Furthermore, if such sequences are retained, do they evolve at the same rate as the organism, but with enough changes over time, thereby enabling its use as a marker of evolution? In this report we attempted to study the evolution of the Actinobacteria, with specific reference to the Mycobacteriae, through a comparison of the GS sequences present in these genomes. The GS sequence data was used to construct Actinobacteria phylogenies, which were compared to phylogenies constructed from 16S rRNA and cytidine triphosphate (CTP) synthase genes. Through these comparisons it was determined that the GS sequences may undergo adaptive or reductive evolution due to the different evolutionary pressures exerted by the ecological niche the organism occupies. These differences may lead to subtle differences in phylogenetic reconstructions, although broad phylogenies could be defined.
Distribution of glnA sequences in the Actinobacteria
The distribution and similarity of GS protein sequences in all the available genomes of organisms defined as members of the phylum Actinobacteria  were detected through a BLAST sequence comparison of the M. tuberculosis glnA1, glnA2, glnA3 and glnA4- protein sequences (Table 1). Protein sequence data has been preferred to DNA sequences, since the various Actinobacteria genomes may differ with respect to G/C content that may result in skewing of sequence alignments. Protein sequences of high similarity (>60%) to the M. tuberculosis glnA1 and glnA2 encoded protein sequences could be detected in all the Actinobacteria genomes (Table 1), with Symbiobacterium thermophilum being the only exception, where only a single GS sequence with greater similarity to the glnA1-encoded M. tuberculosis GSIβ (50% similarity) was observed. The genome of S. thermophilum, a high G+C gram positive organism belonging to an as yet undefined taxon situated just outside the phylum Actinobacteria, was included due to its close relationships to the actinobacterial ancestor [19, 20]. It was observed that the glnA1 and glnA2 sequences were situated in close proximity to each other in many genomes, but that considerable variance in the distribution and similarity of GS sequences similar to that M. tuberculosis glnA3 and glnA4 sequences was observed. Some Actinobacteria genomes contained an additional glnA protein sequence similar to the M. tuberculosis glnA4 protein sequence. However, this sequence was less conserved than the glnA1 and glnA2 sequences. Only the mycobacteria and some other closely related actinomycetes, such as Frankia and Rhodococcus species, contained sequences similar to the four glnA-encoded GS sequences (summarised in Figure 1). An exception was observed in that sequences similar to glnA3 and glnA4 were absent in the genomes of M. leprae and M. ulcerans, which had glnA sequences similar to glnA1 and glnA2 only. It is well known that M. leprae and M. ulcerans have undergone major reductive evolution [21, 22] and as such may have lost these genes. Since the distribution of the glnA sequences (as seen in Figure 1) reflects the evolution of phylum Actinobacteria as defined by 16S phylogenetic analysis , it might be argued that there was a sequential acquisition of first glnA4 and later glnA3, rather than a loss of these genes from an actinomycete progenitor. In order to prove that glnA3 and glnA4 were lost in these two mycobacterial species specifically, rather than being separately acquired in different members of the mycobacteria, the chromosomal regions containing the glnA3 and glnA4 genes in M. tuberculosis were compared to the corresponding chromosomal regions of M. leprae and M. ulcerans (Figure 2). It was observed that the chromosomal regions of M. leprae and M. ulcerans contained copies of glnA3 in the form of pseudogenes situated in gene clusters corresponding to that of the M. tuberculosis H37Rv chromosome. In M. ulcerans it was observed that the glnA3 sequence had been disrupted by an insertion element (Figure 2). A copy of glnA4 can be observed in a gene cluster similar to that found on the M. tuberculosis chromosome, suggesting that both sequences have been retained from the mycobacterial ancestor during mycobacterial speciation, but that they have become non-functional through the evolutionary process in some members of the genus Mycobacterium.
Origins of the glnA4 and glnA3sequences
The sequence annotations of the M. tuberculosis glnA genes suggest that glnA1 and glnA3 encode GSI enzymes and glnA2 and glnA4 GSII enzymes, which together with the results summarised in Figure 1, suggest that the glnA4 and glnA3 GS sequences were acquired either through sequential duplication of a GSI and GSII sequence, or through separate lateral genetic transfer events. Therefore the ancestry of the glnA sequences was investigated through a phylogenetic analysis of all the glnA sequences present in the phylum Actinobacteria (Table 1). The simplified tree shown in Figure 3 (see additional file 1) indicates that, consistent with previous reports, the glnA-encoded protein sequences may have been derived from a common ancestral GS sequence . The sequence phylogeny further shows that the glnA2, glnA3 and glnA4-encoded sequences are clustered on a separate branch from the glnA1-encoded sequence, indicating that these sequence are related and may share a common ancestor.
This finding was unexpected, since the glnA4-encoded GS sequence has a conserved tyrosine residue in the adenylylation region of the GS sequence, suggesting that it may rather be derived from glnA1 and would encode a GSIβ enzyme. Therefore the structural relationships between the GS protein sequences encoded by the four M. tuberculosis glnA genes were investigated by aligning the glnA1 (Rv2220; 478 amino acids), glnA2 (Rv2222; 446 amino acids), glnA3 (Rv1878; 450 amino acids) and glnA4 (Rv2860c; 457 amino acids) -protein sequences according to maximum probability of amino acid identities (Figure 4). Inspection of the aligned protein sequences of the four M. tuberculosis glnA sequences (Figure 4) showed differences in functional regions that separate the GSI and GSII protein families. This data reflects a low level of similarity between the GS sequences due to the low level of sequence conservation in regions containing putative functional domains, notably those that might be involved in the formation of the GS-catalytic site . Furthermore, the protein sequences encoded by glnA2, glnA3 and glnA4 lack the insert sequence that is used to identify GSIβ sequences . In addition, the tyrosine residue in the glnA1 protein sequence involved in post-translational regulation of GSIβ through adenylylation  is situated in a run of amino acids that is not conserved in the other three proteins. Therefore the tyrosine residue present in the glnA4-encoded GS sequence might not be subjected to post-transcriptional regulation by adenylylation, which indicates that the protein sequences encoded by the glnA3 and glnA4 genes are of the type II GS family. This observation supports the phylogenetic analysis which indicated that the glnA3 and glnA4 protein sequences are related to or may have been derived from the glnA2 protein sequence.
Alignment scores of the GS sequences (calculated as a percentage of amino acid identities per GS sequence length, Table 1) showed that the glnA3 and glnA4 protein sequences were dissimilar to those encoded by the glnA1 and glnA2 genes. From the alignment scores it is evident that the protein sequences encoded by glnA1 and glnA2 are most similar (32.4% – 32.7%, Table 1), while the sequence encoded by glnA3 shows the lowest similarity to the protein sequences encoded by glnA1, glnA2 and glnA4 (less than 23%; Table 1). Because it was expected that recent gene duplicates would share a high degree of similarity, the low level of glnA4 and glnA3 sequence conservation in comparison to the glnA1 and glnA2 sequences suggests that these sequences either may have undergone rapid evolution after duplication, or have been derived from separate lateral gene transfer events during the speciation of the later actinobacteria. Therefore the glnA3 and glnA4-encoded protein sequences were compared to all available microbial genomes on the NCBI BLAST server. Sequences with similarity to the glnA4 sequence were detected in members of the proteobacteria, such as Nitrococcus mobilis (61% similarity) and Acidiphilum cryptum (54% similarity). Both these organisms had an additional GSI copy, although it had lower similarity to the glnA1-encoded GS of M. tuberculosis (50% and 51% similarity respectively). The similarity of these sequences to the glnA4 sequence was confirmed by a protein sequence BLAST of the N. mobilis protein sequence against all the genomes of the Actinobacteria. Higher protein sequence similarity to the glnA4 sequence (see Table 1) were observed in all cases, with the sequence of A. cellulolyticus (YP_873609) being the most similar (63% identity). In organisms where a glnA4 sequence is absent (see Figure 1), no sequences of significant similarity could be detected. However, it could not be conclusively shown whether these sequences were similar enough to suggest that the presence of the glnA3 and glnA4 sequences could be due to a lateral transfer event. The comparison of the chromosomal regions on which the glnA4 gene is found showed remarkable consistency even in more distantly related actinobacteria, while the same was not true for the glnA3 gene. For instance, the gene arrangement surrounding the glnA4 gene remained the same in M. tuberculosis as in K. radiotolerans, while very few genes of significant similarity surround the glnA3 locus. These observations suggest that the genomic region containing the glnA4 gene was inherited from the Actinobacteria progenitor, rather than being transferred from an organism outside the phylum. The ancestry of the glnA3 gene is more difficult to explain, since a similar sequence could not be detected, suggesting that the glnA3 gene arose through a duplication event, but may be undergoing reductive evolution.
Actinobacteria GS sequences as phylogenetic markers
The lower level of GSIβ sequence conservation observed in comparison to the GSII sequence between species (Table 1) was surprising, since GSIβ may be the major GS of M. tuberculosis and other Actinobacteria [14, 15, 25]. Since this observation suggests that the GSIβ and GSII sequences evolve differently, Actinobacteria phylogenies based on the GSIβ and GSII sequences were compared to phylogenies based on 16S rRNA sequences . Since the glnA3 and glnA4 protein sequences might be undergoing reductive evolution, they were excluded from the phylogeny. Figure 5 shows that the Actinobacteria phylogeny based on the glnA2-encoded GSII sequence reflects the 16S rRNA phylogeny, while shifts are observed in the phylogeny based on the glnA1-encoded GSIβ sequence. In the GSII sequence phylogeny, organisms are clustered according to suborders, such as the Micrococcineae (B. linens, Arthrobacter, L. xyli, and Janibacter), Corynebacterineae (Corynebacteria sp., Mycobacterium sp., Rhodococcus and N. farcinica), Streptomycineae (Streptomyces sp.), Streptosporangineae (T. fusca) and the Frankineae (A. cellulolyticus, Frankia sp). Exceptions were observed in that K. radiotolerans (Frankineae), P. acnes and Nocardiodes sp. (Propionibacterineae) were dispersed amongst the Micrococcineae. However, bootstrap values below 50 were obtained for these branches making a true interpretation of the inter-relatedness of these organisms impossible. In the phylogenetic tree based on the GSIβ sequence, bootstrap values above 50 were obtained at some of the nodes, but the clustering of organisms to defined Actinobacteria suborders were not observed.
The differences in the GS phylogenies are most marked in the mycobacteria. Although the slow-growing and fast-growing mycobacteria are clustered in two separate lineages, only the GSII sequence phylogeny reflects the suggested 16S rRNA phylogeny . For instance, the GSI phylogeny put members of the M. tuberculosis complex (M. tuberculosis, M. microtti and M. africanum) in different lineages with M. ulcerans and M. avium as M. tuberculosis complex ancestors. This differs from the GSII phylogeny, which clusters the M. tuberculosis complex and puts M. leprae and M. avium just outside the complex similar to what is observed in 16S rRNA phylogenetic analyses. The branch depth reflects the small amount of variation between the sequences, and the synonymous to nonsynonymous substitution ratio (Figure 5) indicates that there is a selective constraint that preserves the accumulation of amino acid changes over time. However, most of the sequence variation within these sequences occurred outside important functional GS domains. Since phylogenies are not absolute, the results suggest that using GS as a marker in phylogenetic reconstructions gives a broad definition of phylogeny, although subtle differences between trees are observed.
GSIβ remains conserved between species
Since the sequence encoded by the glnA1 locus is the major GS of M. tuberculosis, it is expected to undergo little evolutionary change over time. However, the genetic conservation of the gene was studied to assess whether it is subject to gradual changes over time. The glnA1 gene (1434 bp) and its 5' and 3' regions were PCR amplified from purified genomic DNA of 54 clinical M. tuberculosis isolates. These strains were selected on the basis that they were genotyped by IS6110 insertion mapping in a previous study and included highly prevalent and less prevalent strain families as defined in a high tuberculosis incidence community . These clinical isolates are genetically diverse and encompassed the broad M. tuberculosis strain families that are grouped according to IS6110 banding pattern identities exceeding 65%. The glnA1 sequence data obtained in this manner was compared with the corresponding sequences of the M. tuberculosis H37Rv reference strain, M. tuberculosis CDC1551 and M. tuberculosis 210 (clinical isolate) through BLAST. The glnA1 sequences were 100% similar in all respects and no mutations, deletions or insertions were found in any of the M. tuberculosis glnA1 loci, showing that the glnA1 sequence undergoes no evolutionary change within M. tuberculosis.
Glutamine synthetase has long been considered a good molecular marker for evolutionary studies because, similar to the 16S rRNA gene, it is a universally present and essential component of most living organisms and therefore may be constrained to evolve at a slow rate [4, 28]. In addition, the GS sequence is long enough to be used together with other sequences, such as 16S rRNA, to obtain a higher degree of confidence in phylogenetic analyses . However, multiple copies of GS encoding genes have been observed in the genomes of some organisms, notably M. tuberculosis (which has four GS encoding genes) . Of these sequences, only the glnA1 gene (encoding a GSIβ) has been shown to be essential for M. tuberculosis growth, while the other sequences are not . To further understand the evolution of GS and the use of duplicated proteins as evolutionary markers, it was attempted to reconstruct Actinobacteria speciation by using GS sequences as phylogenetic markers. Through this study insight was gained into the possible evolutionary scenario of the glnA genes in the mycobacteria.
Through sequence comparisons it was shown that most members of phylum Actinobacteria had at least one copy of both the glnA1 and glnA2 genes and that the protein sequences these genes encode are conserved between species. Symbiobacterium thermophilum was an exception having only one glnA gene similar to the glnA1 sequence. Since S. thermophilum may be closely related to the Actinobacteria ancestor , the absence of the glnA2 gene may indicate that glnA2 (which is present outside of the phylum Actinobacteria) was either not passed down from the Symbiobacterium ancestor, or may have been lost from this organism. Previous studies have shown that the GSI and GSII sequences are duplicated derivatives of an ancient GS sequence , which suggests that S. thermophilum may have lost the glnA2 sequence during speciation. It remains to be investigated if other members of the Symbiobacterium species may have retained a glnA2 gene. It is interesting to note that in many cases, the glnA1 and glnA2 genes were situated in close proximity to each other. This arrangement has been observed in the genomes of other organisms , which suggests that these GS enzymes may be functionally linked. In support of this observation it has been demonstrated that the synthesis of the GSII enzyme was up regulated while the synthesis of GSI was reduced significantly during nitrogen starvation in the Frankia , therefore suggesting a synergistic role of both enzymes under different conditions. The close proximity of the coding genes for the two GS enzymes also suggests that the chromosomal region containing the glnA copies may be conserved. The genomic region containing the glnA2 sequence has been studied in M. tuberculosis and C. glutamicum and in both cases it was shown that the glnA2 gene was situated adjacent to and transcriptionally linked to the glnE gene [15, 32]. The glnE gene encodes the adenylyltransferase involved in the post-translational regulation of GSIβ, and deletion of this gene is fatal owing to disturbances caused from the resulting unchecked GS function . Therefore it is possible that disruptions in the chromosomal region containing the glnA2 sequence may be under negative selection pressure.
The distribution and ancestry of the other GS-encoding genes (apart from glnA1 and glnA2) have not yet been described. The relationships between the glnA proteins were investigated by generating a phylogeny of all Actinobacteria GS sequences. Through this phylogeny it was revealed that the glnA3 and glnA4 protein sequences are most closely related to the glnA2 protein sequence. Our results suggested that the genes might have been derived from either serial duplications of the glnA2 gene, or from separate lateral gene transfer events with glnA4 being the first and glnA3 the most recent acquisition. Analysis of the functional regions of the GS sequences confirmed the possibility, since it was noted that glnA2, glnA3 and glnA4 encode GSII enzymes. We attempted to establish whether these sequences may have entered the Actinobacteria genomes through other mechanisms, such as lateral gene transfer. No clear conclusion could be reached other than that similar sequences were present in some members of the γ-proteobacteria. It is known that lateral gene transfer between mycobacterial species and members of the proteobacteria has occurred . However, these transferred elements are usually related to virulence  or pathogenicity . Since GS is involved in central metabolism, no definite conclusion could be made.
The evolutionary history of species within the genus Mycobacterium has been investigated using the DNA sequence encoding 16S rRNA . Intriguingly, in comparison to this, subtle differences were observed in the mycobacterial phylogeny based on the GSIβ protein sequence, although the phylogeny based on the GSII sequence reflected the proposed mycobacterial speciation more closely. This observation suggests that, although the coding sequences are constricted as measured by synonymous to non-synonymous substitution rates, change in the GSIβ and GSII sequences may be influenced by environmental pressure. The greater similarity between the GSII sequences may suggest that this sequence remains more conserved and undergoes change at a different rate to the GSIβ sequence. The greater conservation between the GSII sequences indicates that this enzyme might have played a more important role in the early Actinobacteria species, although it may have become redundant in some of the later mycobacteria. In this respect, it is interesting to note that deletions of the glnA2 sequence lead to attenuation of M. bovis in guinea pigs , whilst the same result was not observed in mice infected with M. tuberculosis strains with glnA2 disruptions . From the analysis of actinobacterial genomes containing sequences similar to the glnA sequence, it seems that the glnA3 and glnA4 duplication event may have occurred independently, since some Actinobacteria genomes contain either glnA3, glnA4 or both, together with the glnA1 and glnA2 sequences. However, some bacteria, such as M. leprae and M. ulcerans, might have had a copy of glnA3 and glnA4, which was lost due to transposon insertions or deletions, suggesting that a lack of glnA3, glnA4 or both genes might also be due to reductive evolution such as is observed in the genomes of M. leprae and M. ulcerans [21, 39]. If it is accepted that some of the mycobacteria have lost the glnA3 and glnA4 sequences, this could indicate the redundancy of the GS encoded by these sequences, since if they had a function besides glutamine synthesis they might have been under different evolutionary pressure to be retained in the genome.
The influence of evolutionary pressures on such a critical metabolic enzyme may be explained by adaptive evolution of GS due to pressures exerted by the distinct ecological niches these organisms occupy. Adaptive evolution may lead to functional promiscuity whereby an enzyme can exert other functions, whilst still using the same active site as for the original singular activity . In this respect, it has been shown that the GSIβ enzyme may be exported in great quantities by M. tuberculosis and M. bovis (also the BCG sub-strains) and that it might be involved in the formation of poly-L-glutamic acid, a cell wall constituent unique to these two mycobacterial species . Evidence has been presented that these functions might be essential for M. tuberculosis survival in vivo , and that the GSIβ enzyme may have functions that contribute to the virulence of these important human pathogens, which cannot be substituted by the GSIβ from non-pathogenic mycobacteria (such as M. smegmatis) . The ability of the GSI sequence to undergo evolutionary specialisation may be the underlying reason why this enzyme has been functionally replaced by the more evolutionary stable GSII sequence in eukaryotes. It was suggested that the GSII enzyme is present in eukaryotes due to lateral transfer from endosymbionts early in the eukaryote evolution and, that in some cases, these eukaryotes had other GS-enzymes that were functionally replaced by GSII . Indeed, a remnant of GSI, lengsin, has been observed in the vertebrate eye lens [42, 43]. Lengsin has a dodecameric structure and conserved GSI functionally important regions, but is not catalytically active and has undergone significant evolutionary change in the N-terminal region and probably specialised to play a role in lens homeostasis and transparency.
In conclusion, the specialisation of critical metabolic enzymes may have implications for the use of such enzymes as molecular markers for evolution. Although diversity in these protein sequences may be useful for discriminating between closely related species that show little variance in the 16S rRNA sequences , adaptive evolution of these sequences may skew phylogenies.
Sequence retrieval and multiple sequence alignments
Mycobacterium tuberculosis glnA1, glnA2, glnA3 and glnA4 protein sequences were retrieved from Genolist (Pasteur Institute)  and compared to the Actinobacteria genome databases on the NCBI microbial genomes BLAST server . Glutamine synthetase protein sequences were retrieved and compared through multiple sequence alignment using ClustalW 1.8 software at the European Bioinformatics Institute [44, 46]. The alignments were manually checked for errors using BioEdit 5.0.9 . For phylogenetic reconstructions, some alignments were manually edited during which unaligned regions (inserts) were removed. BLAST searches against the genomes of M. africanum, M. marinum and M. microtti were carried out on the Sanger Institute website  by using the function TBLASTN.
The edited GS protein sequences were subjected to phylogenetic analysis using the neighbour joining algorithm (PAUP 4.0*; Phylogenetic Analysis Using Parsimony (*Other Methods) Version 4b10. Sinauer Associates, Sunderland, Massachusetts). A 1000 subsets were generated for bootstrap resampling of the data to establish a degree of statistical support for nodes within each phylogenetic reconstruction . A consensus tree was generated using the program contree (PAUP 4.0*) in combination with the majority rule formula. The GS protein sequence of Symbiobacterium thermophylum was selected as out-group to assign roots due the closer relation of this organism to the Actinobacteria ancestor . Only branches which occurred in > 50% of the bootstrap trees were included in the final tree and all branches with a zero branch length were collapsed. Overall topology of the trees were confirmed using PhyML 3.0  (data not shown). Synonymous (Ks) and non-synonymous (Ka) substitutions were calculated using DnaSP software . In these calculations, the glnA1 or glnA2 DNA sequence of C. efficiens was selected as the out-group.
M. tuberculosis clinical isolate DNA preparation and glnA1sequencing
DNA was isolated from M. tuberculosis clinical isolates representative of the various strain families  and genotypically classified through the internationally standardised IS-3' fingerprinting method . The Southern-blot autoradiographs were normalised and the IS-3' bands were assigned using GelCompar software (version 4.1). Assignments were visually checked by two independent persons and bands with a >20% intensity than the other bands were scored as representing the IS6110-mediated evolutionary events . This DNA was used as template for the PCR amplification of glnA1 using the primers listed in Table 2. PCR reactions were carried out in a GeneAmp 2500 PCR-system (Perkin Elmer) with an initial enzyme activation and DNA denaturing step of 15 min 92°C, followed by 30 cycles at 92°C (2 min); Tm (Table 3, 30 sec) and 72°C (1 min) and a final 7 min elongation step at 72°C. PCR products were purified using the Promega SV-miniprep system and submitted for direct automated DNA sequencing (Central Analytical Facility, Stellenbosch University, South Africa). Full-length glnA1 sequences were assembled from sequencing data using DnaMan software and compared to each other through multiple sequence alignment using ClustalW 1.8 software .
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The authors would like to thank the CSIR (Dr C. Kenyon) and the MRC for financial assistance, Dr N. Gey van Pittius and Dr R. Warren for advice in preparing the manuscript.
DH carried out all experimental work, interpretation of data and drafted the manuscript. PvH and IJFW were responsible for initiating the project and revising the manuscript for intellectual content.