Glutamine synthetase sequence evolution in the mycobacteria and their use as molecular markers for Actinobacteria speciation

  • Don Hayward1Email author,

    Affiliated with

    • Paul D van Helden1 and

      Affiliated with

      • Ian JF Wiid1

        Affiliated with

        BMC Evolutionary Biology20099:48

        DOI: 10.1186/1471-2148-9-48

        Received: 03 December 2008

        Accepted: 26 February 2009

        Published: 26 February 2009

        Abstract

        Background

        Although the gene encoding for glutamine synthetase (gln A) 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 (gln A1) is essential for growth in M. tuberculosis, while the other copies (gln A2, gln A3 and gln A4) are not.

        Results

        In this report it is shown that the gln A1 and gln A2 encoded glutamine synthetase sequences were inherited from an Actinobacteria ancestor, while the gln A4 and gln A3 encoded GS sequences were sequentially acquired during Actinobacteria speciation. The glutamine synthetase sequences encoded by gln A4 and gln A3 are undergoing reductive evolution in the mycobacteria, whilst those encoded by gln A1 and gln A2 are more conserved.

        Conclusion

        Different selective pressures by the ecological niche that the organisms occupy may influence the sequence evolution of gln A1 and gln A2 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.

        Background

        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 [13]. 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 [47]. The GS sub-classes are distinguishable from each other by specific insertion sequences and mechanisms of regulation [5]. The GSIβ sub-type is subjected to post-translational modification by adenylylation of a conserved tyrosine residue by an adenylyltransferase [8], 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 [9], while GSII molecules are octamers [10]. 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 [12], 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 (gln A1 and gln A4) and GSII (gln A2 and gln A3) enzymes [13]. Of the four glnA gene copies, it has been shown that gln A1 encodes the main and essential GS in M. tuberculosis [14], while the other glnA sequences (gln A2, gln A3 and gln A4) encode functional, but non-essential GS enzymes [15]. 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 gln A1) 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 [18].

        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.

        Results

        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 [19] were detected through a BLAST sequence comparison of the M. tuberculosis gln A1, gln A2, gln A3 and gln A4- 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 gln A1 and gln A2 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 gln A1-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 gln A1 and gln A2 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 gln A3 and gln A4 sequences was observed. Some Actinobacteria genomes contained an additional glnA protein sequence similar to the M. tuberculosis gln A4 protein sequence. However, this sequence was less conserved than the gln A1 and gln A2 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 gln A3 and gln A4 were absent in the genomes of M. leprae and M. ulcerans, which had glnA sequences similar to gln A1 and gln A2 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 [19], it might be argued that there was a sequential acquisition of first gln A4 and later gln A3, rather than a loss of these genes from an actinomycete progenitor. In order to prove that gln A3 and gln A4 were lost in these two mycobacterial species specifically, rather than being separately acquired in different members of the mycobacteria, the chromosomal regions containing the gln A3 and gln A4 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 gln A3 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 gln A3 sequence had been disrupted by an insertion element (Figure 2). A copy of gln A4 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.
        Table 1

        GlnA protein sequence distribution and similarity in the Actinobacteria

         

        Sequence accesion number, length (amino acids) and percentage similarity

        Organism

        gln A1

        gln A2

        gln A3

        gln A4

        Acidothermus cellulolyticus 11B

        YP_872682

        (474 aa) 72%

        YP_872678

        (453 aa) 68%

        YP_872678

        (453 aa) 30%

        YP_873609

        (446 aa) 61%

        Arthrobacter sp. FB24

        YP_947504

        (474 aa) 63%

        YP_947491

        (446 aa) 65%

        YP_831086

        (446 aa) 29%

        YP_831086

        (446 aa) 31%

        Bifidobacterium longum NCC2705

        NP_696248

        (478 aa) 62%

        NP_696466

        (445 aa) 60%

        NP_696466

        (445 aa) 27%

        NP_696466

        (445 aa) 29%

        Brevibacterium linens BL2

        ZP_00378605

        (474 aa) 62%

        ZP_00378066

        (452 aa) 62%

        ZP_00378066

        (452 aa) 29%

        ZP_00381218

        (454 aa) 56%

        Corynebacterium diphtheriae NCTC 13129

        NP_939986

        (478 aa) 67%

        NP_940011

        (446 aa) 64%

        NP_940011

        (446 aa) 25%

        NP_940011

        (466 aa) 28%

        C. efficiens YS-314

        NP_738714

        (477 aa) 70%

        NP_738737

        (516 aa) 66%

        NP_738737

        (516 aa) 29%

        NP_738737

        (516 aa) 29%

        C. glutamicum ATCC 13032

        YP_226455

        (477 aa) 70%

        YP_226471

        (446 aa) 65%

        YP_226471

        (446 aa) 29%

        YP_226471

        (446 aa) 29%

        C. jeikeium K411

        YP_250482

        (500 aa) 71%

        YP_250455

        (448 aa) 71%

        YP_250455

        (448 aa) 29%

        YP_250455

        (448 aa) 29%

        Frankia sp. EAN1pec

        YP_001506114

        (474 aa) 66%

        YP_001506110

        (452 aa) 65%

        YP_001510745

        (496 aa) 38%

        YP_001505022

        (470 aa) 56%

        Janibacter sp. HTCC2649

        ZP_00994949

        (474 aa) 66%

        ZP_00995601 (445 aa) 70%

         

        ZP_00995688 (446 aa) 42%

         

        ZP_00997071

        (461 aa) 59%

        Kineococcus radiotolerans SRS30216

        YP_001363019

        (474 aa) 68%

        YP_001363024

        (447 aa) 65%

        YP_001363024

        (447 aa) 31%

        YP_001361387

        (460 aa) 61%

        Leifsonia xyli subsp. xyli str. CTCB07

        YP_062980

        (474 aa) 62%

        YP_061977

        (445 aa) 63%

        YP_061977

        (445 aa) 28%

        YP_061977

        (445 aa) 32%

        Mycobacterium avium 104

        YP_881471

        (478 aa) 90%

        YP_881448

        (446 aa) 94%

        YP_882016

        (450 aa) 80%

        YP_882894

        (468 aa) 78%

        M. bovis AF2122/97

        NP_855893

        (478 aa) 100%

        NP_855895

        (446 aa) 100%

        NP_855562

        (450 aa) 100%

        NP_856530

        (457 aa) 100%

        M. bovis BCG str. Pasteur 1173P2

        YP_978326

        (478 aa) 100%

        YP_978328

        (446 aa) 100%

        YP_978005

        (450 aa) 100%

        YP_978966

        (475 aa) 100%

        M. leprae TN

        NP_301707

        (478 aa) 91%

        NP_302123

        (448 aa) 93%

        NP_302123

        (448 aa) 27%

        NP_302123

        (448 aa) 29%

        M. smegmati s str. MC2 155

        YP_888567

        (478 aa) 84%

        YP_888571

        (446 aa) 88%

        YP_887864

        (453 aa) 64%

        YP_886932

        (457 aa) 74%

        M. sp. KMS

        YP_939366

        (478 aa) 85%

        YP_939374

        (446 aa) 89%

        YP_936250

        (437 aa) 47%

        YP_938091

        (455 aa) 74%

        M. tuberculosis CDC1551

        NP_336749

        (478 aa) 00%

        NP_336751

        (446 aa) 100%

        NP_336385

        (450 aa) 100%

        NP_337439

        (457 aa) 100%

        M. tuberculosis F11

        ZP_01685137

        (478 aa) 100%

        ZP_01685139

        (446 aa) 100%

        ZP_01684789

        (450 aa) 100%

        ZP_01685769

        (462 aa) 100%

        M. tuberculosis H37Rv

        NP_216736

        (478 aa) 100%

        NP_216738

        (446 aa) 100%

        NP_216394

        (450 aa) 100%

        NP_217376

        (457 aa) 100%

        M. ulcerans Agy99

        YP_905364

        (478 aa) 90%

        YP_905360

        (446 aa) 93%

        YP_905360

        (446 aa) 27%

        YP_905360

        (446 aa) 30%

        M. gilvum PYR-GCK

        YP_001134193

        (478 aa) 84%

        YP_001134174

        (446 aa) 89%

        YP_001134583

        (453 aa) 66%

        YP_001135323

        (469 aa) 74%

        M. vanbaalenii PYR-1

        YP_954385

        (478 aa) 85%

        YP_954396

        (446 aa) 88%

        YP_953732

        (442 aa) 64%

        YP_953098

        (459 aa) 72%

        Nocardia farcinica IFM 10152

        YP_117877

        (478 aa) 77%

        YP_117870

        (446 aa) 83%

        YP_117870

        (446 aa) 28%

        YP_117870

        (446 aa) 31%

        Nocardioides sp. JS614

        YP_923487

        (474 aa) 71%

        YP_923242

        (455 aa) 66%

        YP_923242

        (455 aa) 28%

        YP_923778

        (464 aa) 59%

        Propionibacterium acnes KPA171202

        YP_055385

        (473 aa) 66%

        YP_055378

        (468 aa) 63%

        YP_055378

        (468 aa) 30%

        YP_055378

        (468 aa) 30%

        Rhodococcus sp. RHA1

        YP_701142

        (478 aa) 81%

        YP_701152

        (446 aa) 84%

        YP_701692

        (433 aa) 47%

        YP_705251

        (451 aa) 33%

        Salinispora tropica CNB-440

        YP_001160144

        (474 aa) 70%

        YP_001160151

        (451 aa) 68%

        YP_001160151

        (451 aa) 29%

        YP_001160151

        (451 aa) 30%

        Streptomyces avermitilis MA-4680

        NP_827182

        (469 aa) 70%

        NP_827131

        (453 aa) 69%

        NP_827131

        (453 aa) 29%

        NP_827901

        (454 aa) 65%

        S. coelicolor A3(2)

        NP_626450

        (469 aa) 71%

        NP_626490

        (453 aa) 69%

        NP_626490

        (453 aa) 28%

        NP_625889

        (462 aa) 64%

        Symbiobacterium thermophilum IAM 14863

        YP_074027

        (471 aa) 50%

        YP_074027

        (471 aa) 33%

        YP_074027

        (471 aa) 28%

        YP_074027

        (471 aa) 32%

        Thermobifida fusca YX

        YP_289049

        (474 aa) 68%

        YP_289043

        (453 aa) 68%

        YP_289043

        (453 aa) 27%

        YP_289043

        (453 aa) 28%

        Marine actinobacterium PHSC20C1

        ZP_01131573

        (478 aa) 64%

        ZP_01129622

        (445 aa) 62%

        ZP_01129567

        (416 aa) 27%

        ZP_01129199

        (455 aa) 70%

        Gln A protein sequences distribution in the Actinobacteria. The percentage similarity to the M. tuberculosis GS sequences is indicated by means of amino acid identity. The accession number and amino acid length of the protein sequence is indicated for each sequence.

        http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-9-48/MediaObjects/12862_2008_Article_962_Fig1_HTML.jpg
        Figure 1

        The distribution of glnA sequences within the genomes of different actinobacterial species reflects the evolutionary history of the phylumActinobacteriaas derived from 16S rRNA phylogenetic analyses and indicates that theglnA3 andglnA4 sequences were acquired in a serial fashion. *(The gln A3 and gln A4 sequences are present as pseudogenes in the genomes of M. leprae and M. ulcerans.)

        http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-9-48/MediaObjects/12862_2008_Article_962_Fig2_HTML.jpg
        Figure 2

        The chromosomal regions ofM. lepraeandM. ulceranssimilar to that ofM. tuberculosiscontaining theglnA3 andglnA4 sequences show that these GS encoding sequences were disrupted by insertions (glnA3,M. ulcerans) or deletions (glnA3,M. leprae;glnA4,M. ulcerans). Similar genes are indicated in the same colour and the percentage amino acid identity to the M. tuberculosis H37Rv reference sequence is indicated between brackets. Open arrows indicate no significant similarity to sequences in the corresponding chromosomal regions.

        Origins of the glnA4 and glnA3 sequences

        The sequence annotations of the M. tuberculosis glnA genes suggest that gln A1 and gln A3 encode GSI enzymes and gln A2 and gln A4 GSII enzymes, which together with the results summarised in Figure 1, suggest that the gln A4 and gln A3 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 [4]. The sequence phylogeny further shows that the gln A2, gln A3 and gln A4-encoded sequences are clustered on a separate branch from the gln A1-encoded sequence, indicating that these sequence are related and may share a common ancestor.
        http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-9-48/MediaObjects/12862_2008_Article_962_Fig3_HTML.jpg
        Figure 3

        Phylogenetic analysis of the all the actinobacterial glnA protein sequences showed that theglnA3 andglnA4 protein sequences are closer related to theglnA2 protein sequence that to that ofglnA1. (Distances not drawn to scale).

        This finding was unexpected, since the gln A4-encoded GS sequence has a conserved tyrosine residue in the adenylylation region of the GS sequence, suggesting that it may rather be derived from gln A1 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 gln A1 (Rv2220; 478 amino acids), glnA2 (Rv2222; 446 amino acids), gln A3 (Rv1878; 450 amino acids) and gln A4 (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 gln A 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 [23]. Furthermore, the protein sequences encoded by gln A2, gln A3 and gln A4 lack the insert sequence that is used to identify GSIβ sequences [5]. In addition, the tyrosine residue in the gln A1 protein sequence involved in post-translational regulation of GSIβ through adenylylation [24] is situated in a run of amino acids that is not conserved in the other three proteins. Therefore the tyrosine residue present in the gln A4-encoded GS sequence might not be subjected to post-transcriptional regulation by adenylylation, which indicates that the protein sequences encoded by the gln A3 and gln A4 genes are of the type II GS family. This observation supports the phylogenetic analysis which indicated that the gln A3 and gln A4 protein sequences are related to or may have been derived from the gln A2 protein sequence.
        http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-9-48/MediaObjects/12862_2008_Article_962_Fig4_HTML.jpg
        Figure 4

        Multiple protein sequence alignment of theM. tuberculosisglnA encoded sequences shows the amount of variation between these proteins. Identical amino acid sequences are blocked; the insert sequence distinguishing GSIβ are in bold type and the active site tyrosine (position 429) is indicated in red.

        Alignment scores of the GS sequences (calculated as a percentage of amino acid identities per GS sequence length, Table 1) showed that the gln A3 and gln A4 protein sequences were dissimilar to those encoded by the gln A1 and gln A2 genes. From the alignment scores it is evident that the protein sequences encoded by gln A1 and gln A2 are most similar (32.4% – 32.7%, Table 1), while the sequence encoded by gln A3 shows the lowest similarity to the protein sequences encoded by gln A1, gln A2 and gln A4 (less than 23%; Table 1). Because it was expected that recent gene duplicates would share a high degree of similarity, the low level of gln A4 and gln A3 sequence conservation in comparison to the gln A1 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 gln A3 and gln A4-encoded protein sequences were compared to all available microbial genomes on the NCBI BLAST server. Sequences with similarity to the gln A4 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 gln A1-encoded GS of M. tuberculosis (50% and 51% similarity respectively). The similarity of these sequences to the gln A4 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 gln A4 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 gln A4 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 gln A3 and gln A4 sequences could be due to a lateral transfer event. The comparison of the chromosomal regions on which the gln A4 gene is found showed remarkable consistency even in more distantly related actinobacteria, while the same was not true for the gln A3 gene. For instance, the gene arrangement surrounding the gln A4 gene remained the same in M. tuberculosis as in K. radiotolerans, while very few genes of significant similarity surround the gln A3 locus. These observations suggest that the genomic region containing the gln A4 gene was inherited from the Actinobacteria progenitor, rather than being transferred from an organism outside the phylum. The ancestry of the gln A3 gene is more difficult to explain, since a similar sequence could not be detected, suggesting that the gln A3 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 [19]. Since the gln A3 and gln A4 protein sequences might be undergoing reductive evolution, they were excluded from the phylogeny. Figure 5 shows that the Actinobacteria phylogeny based on the gln A2-encoded GSII sequence reflects the 16S rRNA phylogeny, while shifts are observed in the phylogeny based on the gln A1-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.
        http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-9-48/MediaObjects/12862_2008_Article_962_Fig5_HTML.jpg
        Figure 5

        Dendograms of aligned actinobacterial GSIβ (encoded byglnA1) and GSII (encoded byglnA2) sequences constructed using PAUP 4.0 with the GS sequence ofBifidobacterium longumas out-group (*). Percentage bootstrap support values are shown. The ratio of nonsynonymous (Ka) to synonymous mutations (Ks) in the GS sequences of the mycobacteria and C. diphteria were computed using the GS sequences in C. efficiens, and is shown between brackets.

        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 [26]. 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 gln A1 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 glnA 1 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 IS 6110 insertion mapping in a previous study and included highly prevalent and less prevalent strain families as defined in a high tuberculosis incidence community [27]. These clinical isolates are genetically diverse and encompassed the broad M. tuberculosis strain families that are grouped according to IS 611 0 banding pattern identities exceeding 65%. The gln A1 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 gln A1 sequences were 100% similar in all respects and no mutations, deletions or insertions were found in any of the M. tuberculosis gln A1 loci, showing that the gln A1 sequence undergoes no evolutionary change within M. tuberculosis.

        Discussion

        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 [29]. 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) [13]. Of these sequences, only the gln A1 gene (encoding a GSIβ) has been shown to be essential for M. tuberculosis growth, while the other sequences are not [15]. 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 gln A1 and gln A2 genes and that the protein sequences these genes encode are conserved between species. Symbiobacterium thermophilum was an exception having only one gln A gene similar to the gln A1 sequence. Since S. thermophilum may be closely related to the Actinobacteria ancestor [19], the absence of the gln A2 gene may indicate that gln A2 (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 [4], which suggests that S. thermophilum may have lost the gln A2 sequence during speciation. It remains to be investigated if other members of the Symbiobacterium species may have retained a gln A2 gene. It is interesting to note that in many cases, the gln A1 and gln A2 genes were situated in close proximity to each other. This arrangement has been observed in the genomes of other organisms [30], 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 [31], 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 gln A2 sequence has been studied in M. tuberculosis and C. glutamicum and in both cases it was shown that the gln A2 gene was situated adjacent to and transcriptionally linked to the gln E gene [15, 32]. The gln E 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 [33]. Therefore it is possible that disruptions in the chromosomal region containing the gln A2 sequence may be under negative selection pressure.

        The distribution and ancestry of the other GS-encoding genes (apart from gln A1 and gln A2) have not yet been described. The relationships between the gln A proteins were investigated by generating a phylogeny of all Actinobacteria GS sequences. Through this phylogeny it was revealed that the gln A3 and gln A4 protein sequences are most closely related to the gln A2 protein sequence. Our results suggested that the genes might have been derived from either serial duplications of the gln A2 gene, or from separate lateral gene transfer events with gln A4 being the first and gln A3 the most recent acquisition. Analysis of the functional regions of the GS sequences confirmed the possibility, since it was noted that gln A2, gln A3 and gln A4 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 [34]. However, these transferred elements are usually related to virulence [35] or pathogenicity [36]. 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 [26]. 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 gln A2 sequence lead to attenuation of M. bovis in guinea pigs [37], whilst the same result was not observed in mice infected with M. tuberculosis strains with gln A2 disruptions [38]. From the analysis of actinobacterial genomes containing sequences similar to the glnA sequence, it seems that the gln A3 and gln A4 duplication event may have occurred independently, since some Actinobacteria genomes contain either gln A3, gln A4 or both, together with the gln A1 and gln A2 sequences. However, some bacteria, such as M. leprae and M. ulcerans, might have had a copy of gln A3 and gln A4, which was lost due to transposon insertions or deletions, suggesting that a lack of gln A3, gln A4 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 gln A3 and gln A4 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 [40]. 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 [14]. Evidence has been presented that these functions might be essential for M. tuberculosis survival in vivo [18], 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) [38]. 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 [41]. 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.

        Conclusion

        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 [28], adaptive evolution of these sequences may skew phylogenies.

        Methods

        Sequence retrieval and multiple sequence alignments

        Mycobacterium tuberculosis gln A1, gln A2, gln A3 and gln A4 protein sequences were retrieved from Genolist (Pasteur Institute) [44] and compared to the Actinobacteria genome databases on the NCBI microbial genomes BLAST server [45]. 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 [47]. 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 [48] by using the function TBLASTN.

        Phylogenetic trees

        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 [49]. 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 [19]. 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 [50] (data not shown). Synonymous (Ks) and non-synonymous (Ka) substitutions were calculated using DnaSP software [51]. In these calculations, the gln A1 or gln A2 DNA sequence of C. efficiens was selected as the out-group.

        M. tuberculosis clinical isolate DNA preparation and glnA1 sequencing

        DNA was isolated from M. tuberculosis clinical isolates representative of the various strain families [52] and genotypically classified through the internationally standardised IS-3' fingerprinting method [53]. 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 IS 6110 -mediated evolutionary events [54]. 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 gln A1 sequences were assembled from sequencing data using DnaMan software and compared to each other through multiple sequence alignment using ClustalW 1.8 software [44].
        Table 2

        GlnA protein sequence similarity in M. tuberculosis

         

        gln A1

        gln A2

        gln A3

        gln A4

        gln A1

        -------

        32.5

        17.1

        22.3

        gln A2

        32.5

        ------

        19.9

        30.2

        gln A3

        17.1

        19.9

        ------

        24.3

        gln A4

        22.3

        30.2

        24.3

        ------

        Alignment similarities of the M. tuberculosis gln A2, gln A3 and gln A4 protein sequences to each other showed that these sequences are largely unrelated.

        Table 3

        PCR primer sequences and priming sites

        Name

        Sequence (5'-3')

        Product size:

        Pair Tm (°C)

        Genome Coordinates

        glnA Up F

        AGATGGACACGGTGGAGT

        796 bp

        55

        2486860

        glnA Up R

        CTTTACTGTATCCGCGGC

          

        2487605

        AI FI

        CACGGTCAGTAACGTCTGC

        550 bp

        55

        2487524

        AI RI

        TCCACCTCGTAGAAGGAGC

          

        2488081

        AI FII

        TTCGATTCGGTGAGCTTC

        574 bp

        57

        2488029

        AI RII

        GCCGCTTGTAGGAGTTCA

          

        2488602

        AI FIII

        ACGACGAGACGGGTTATG

        294 bp

        54

        2488483

        AI RIII

        ATCAGCATGGCCGAGAAC

          

        2488768

        AI FIV

        TGGTCTATAGCCAGCgcA

        597 bp

        56

        2488633

        AI RIV

        GAGATGATTGCCAAGCGG

          

        2489229

        Polymerase chain reaction primers used to amplify the gln A1-locus of M. tuberculosis, including its' 5'- and 3' surrounding regions, as overlapping PCR fragments, which facilitated the assembly of the full target region for sequencing (2369 bp).

        Declarations

        Acknowledgements

        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.

        Authors’ Affiliations

        (1)
        DST/NRF Centre for Excellence in Biomedical Tuberculosis Research, US/MRC Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics, Faculty of Health Sciences –, Stellenbosch University

        References

        1. Yamanaka K, Fang L, Inouye M: The CspA family in Escherichia coli: multiple gene duplication for stress adaptation. Mol Microbiol 1998, 27: 247–255.View ArticlePubMed
        2. Tekaia F, Dujon B: Pervasiveness of gene conservation and persistence of duplicates in cellular genomes. J Mol Evol 1999, 49: 591–600.View ArticlePubMed
        3. Riehle MM, Bennett AF, Long AD: Genetic architecture of thermal adaptation in Escherichia coli. Proc Natl Acad Sci USA 2001, 98: 525–530.View ArticlePubMed
        4. Kumada Y, Benson DR, Hillemann D, Hosted TJ, Rochefort DA, Thompson CJ, Wohlleben W, Tateno Y: Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes. Proc Natl Acad Sci USA 1993, 90: 3009–3013.View ArticlePubMed
        5. Brown JR, Masuchi Y, Robb FT, Doolittle WF: Evolutionary relationships of bacterial and archaeal glutamine synthetase genes. J Mol Evol 1994, 38: 566–576.View ArticlePubMed
        6. Mathis R, Gamas P, Meyer Y, Cullimore JV: The presence of GSI–like genes in higher plants: support for the paralogous evolution of GSI and GSII genes. J Mol Evol 2000, 50: 116–122.PubMed
        7. Tateno Y: Evolution of glutamine synthetase genes is in accordance with the neutral theory of molecular evolution. Jpn J Genet 1994, 69: 489–502.View ArticlePubMed
        8. Reitzer LJ, Magasanik B: Expression of glnA in Escherichia coli is regulated at tandem promoters. Proc Natl Acad Sci USA 1985, 82: 1979–1983.View ArticlePubMed
        9. Rahman RN, Fujiwara S, Takagi M, Imanaka T: Sequence analysis of glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 and comparison of the enzymatic characteristics of native and recombinant GDHs. Mol Gen Genet 1998, 257: 338–347.View ArticlePubMed
        10. Llorca O, Betti M, Gonzalez JM, Valencia A, Marquez AJ, Valpuesta JM: The three–dimensional structure of an eukaryotic glutamine synthetase: functional implications of its oligomeric structure. J Struct Biol 2006, 156: 469–479.View ArticlePubMed
        11. Brown JR, Doolittle WF: Archaea and the prokaryote–to–eukaryote transition. Microbiol Mol Biol Rev 1997, 61: 456–502.PubMed
        12. Benson DR, Stephens DW, Clawson ML, Silvester WB: Amplification of 16S rRNA genes from Frankia strains in root nodules of Ceanothus griseus, Coriaria arborea, Coriaria plumosa, Discaria toumatou, and Purshia tridentata. Appl Environ Microbiol 1996, 62: 2904–2909.PubMed
        13. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE III, et al.: Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998, 393: 537–544.View ArticlePubMed
        14. Harth G, Zamecnik PC, Tang JY, Tabatadze D, Horwitz MA: Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly–L–glutamate/glutamine cell wall structure, and bacterial replication. Proc Natl Acad Sci USA 2000, 97: 418–423.View ArticlePubMed
        15. Harth G, Maslesa–Galic S, Tullius MV, Horwitz MA: All four Mycobacterium tuberculosis glnA genes encode glutamine synthetase activities but only GlnA1 is abundantly expressed and essential for bacterial homeostasis. Mol Microbiol 2005, 58: 1157–1172.View ArticlePubMed
        16. Miller BH, Shinnick TM: Evaluation of Mycobacterium tuberculosis genes involved in resistance to killing by human macrophages. Infect Immun 2000, 68: 387–390.View ArticlePubMed
        17. Harth G, Horwitz MA: Inhibition of Mycobacterium tuberculosis glutamine synthetase as a novel antibiotic strategy against tuberculosis: demonstration of efficacy in vivo. Infect Immun 2003, 71: 456–464.View ArticlePubMed
        18. Harth G, Horwitz MA: An inhibitor of exported Mycobacterium tuberculosis glutamine synthetase selectively blocks the growth of pathogenic mycobacteria in axenic culture and in human monocytes: extracellular proteins as potential novel drug targets. J Exp Med 1999, 189: 1425–1436.View ArticlePubMed
        19. Gao B, Gupta RS: Conserved indels in protein sequences that are characteristic of the phylum Actinobacteria. Int J Syst Evol Microbiol 2005, 55: 2401–2412.View ArticlePubMed
        20. Ueda K, Yamashita A, Ishikawa J, Shimada M, Watsuji TO, Morimura K, Ikeda H, Hattori M, Beppu T: Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalism. Nucleic Acids Res 2004, 32: 4937–4944.View ArticlePubMed
        21. Eiglmeier K, Parkhill J, Honore N, Garnier T, Tekaia F, Telenti A, Klatser P, James KD, Thomson NR, Wheeler PR, et al.: The decaying genome of Mycobacterium leprae. Lepr Rev 2001, 72: 387–398.PubMed
        22. Cole ST, Eiglmeier K, Parkhill J, James KD, Thomson NR, Wheeler PR, Honore N, Garnier T, Churcher C, Harris D, et al.: Massive gene decay in the leprosy bacillus. Nature 2001, 409: 1007–1011.View ArticlePubMed
        23. Almassy RJ, Janson CA, Hamlin R, Xuong NH, Eisenberg D: Novel subunit–subunit interactions in the structure of glutamine synthetase. Nature 1986, 323: 304–309.View ArticlePubMed
        24. Fink D, Falke D, Wohlleben W, Engels A: Nitrogen metabolism in Streptomyces coelicolor A3(2): modification of glutamine synthetase I by an adenylyltransferase. Microbiology 1999, 145 (Pt 9) : 2313–2322.PubMed
        25. Jakoby M, Tesch M, Sahm H, Kramer R, Burkovski A: Isolation of the Corynebacterium glutamicum glnA gene encoding glutamine synthetase I. FEMS Microbiol Lett 1997, 154: 81–88.View ArticlePubMed
        26. Gey van Pittius NC, Sampson SL, Lee H, Kim Y, van Helden PD, Warren RM: Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT–6 (esx) gene cluster regions. BMC Evol Biol 2006, 6: 95.View Article
        27. Warren RM, Sampson SL, Richardson M, Spuy GD, Lombard CJ, Victor TC, van Helden PD: Mapping of IS6110 flanking regions in clinical isolates of Mycobacterium tuberculosis demonstrates genome plasticity. Mol Microbiol 2000, 37: 1405–1416.View ArticlePubMed
        28. Palys T, Berger E, Mitrica I, Nakamura LK, Cohan FM: Protein–coding genes as molecular markers for ecologically distinct populations: the case of two Bacillus species. Int J Syst Evol Microbiol 2000, 50 (Pt 3) : 1021–1028.PubMed
        29. Clawson ML, Bourret A, Benson DR: Assessing the phylogeny of Frankia–actinorhizal plant nitrogen–fixing root nodule symbioses with Frankia 16S rRNA and glutamine synthetase gene sequences. Mol Phylogenet Evol 2004, 31: 131–138.View ArticlePubMed
        30. Yooseph S, Sutton G, Rusch DB, Halpern AL, Williamson SJ, Remington K, Eisen JA, Heidelberg KB, Manning G, Li W, et al.: The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families. PLoS Biol 2007, 5: e16.View ArticlePubMed
        31. Tsai YL, Benson DR: Physiological characteristics of glutamine synthetases I and II of Frankia sp. strain CpI1. Archives of Microbiology 1989, 152: 382–386.View Article
        32. Jakoby M, Tesch M, Sahm H, Kramer R, Burkovski A: Isolation of the Corynebacterium glutamicum glnA gene encoding glutamine synthetase I. FEMS Microbiol Lett 1997, 154: 81–88.View ArticlePubMed
        33. Parish T, Stoker NG: glnE is an essential gene in Mycobacterium tuberculosis. J Bacteriol 2000, 182: 5715–5720.View ArticlePubMed
        34. Becq J, Gutierrez MC, Rosas–Magallanes V, Rauzier J, Gicquel B, Neyrolles O, Deschavanne P: Contribution of horizontally acquired genomic islands to the evolution of the tubercle bacilli. Mol Biol Evol 2007, 24: 1861–1871.View ArticlePubMed
        35. Rosas I, Salinas E, Martinez L, Calva E, Cravioto A, Eslava C, mabile–Cuevas CF: Urban dust fecal pollution in Mexico City: antibiotic resistance and virulence factors of Escherichia coli. Int J Hyg Environ Health 2006, 209: 461–470.View ArticlePubMed
        36. Nishio Y, Nakamura Y, Usuda Y, Sugimoto S, Matsui K, Kawarabayasi Y, Kikuchi H, Gojobori T, Ikeo K: Evolutionary process of amino acid biosynthesis in Corynebacterium at the whole genome level. Mol Biol Evol 2004, 21: 1683–1691.View ArticlePubMed
        37. Collins DM, Wilson T, Campbell S, Buddle BM, Wards BJ, Hotter G, De Lisle GW: Production of avirulent mutants of Mycobacterium bovis with vaccine properties by the use of illegitimate recombination and screening of stationary–phase cultures. Microbiology 2002, 148: 3019–3027.PubMed
        38. Lee S, Jeon BY, Bardarov S, Chen M, Morris SL, Jacobs WR Jr: Protection elicited by two glutamine auxotrophs of Mycobacterium tuberculosis and in vivo growth phenotypes of the four unique glutamine synthetase mutants in a murine model. Infect Immun 2006, 74: 6491–6495.View ArticlePubMed
        39. Stinear TP, Seemann T, Pidot S, Frigui W, Reysset G, Garnier T, Meurice G, Simon D, Bouchier C, Ma L, et al.: Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer. Genome Res 2007, 17: 192–200.View ArticlePubMed
        40. Copley SD: Enzymes with extra talents: moonlighting functions and catalytic promiscuity. Curr Opin Chem Biol 2003, 7: 265–272.View ArticlePubMed
        41. Robertson DL, Tartar A: Evolution of glutamine synthetase in heterokonts: evidence for endosymbiotic gene transfer and the early evolution of photosynthesis. Mol Biol Evol 2006, 23: 1048–1055.View ArticlePubMed
        42. Wyatt K, White HE, Wang L, Bateman OA, Slingsby C, Orlova EV, Wistow G: Lengsin is a survivor of an ancient family of class I glutamine synthetases re–engineered by evolution for a role in the vertebrate lens. Structure 2006, 14: 1823–1834.View ArticlePubMed
        43. Grassi F, Moretto N, Rivetti C, Cellai S, Betti M, Marquez AJ, Maraini G, Ottonello S: Structural and functional properties of lengsin, a pseudo–glutamine synthetase in the transparent human lens. Biochem Biophys Res Commun 2006, 350: 424–429.View ArticlePubMed
        44. ClustalW 1.8 (European Bioinformatics Institute) [http://​www.​ebi.​ac.​uk/​clustalw/​] 2007.
        45. NCBI genomic BLAST [http://​www.​ncbi.​nlm.​nih.​gov/​sutils/​genom_​table.​cgi] 2007.
        46. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position–specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22: 4673–4680.View ArticlePubMed
        47. Hall TA: BioEdit: a user–friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. 95–98.
        48. Welcome Trust Sanger Institute [http://​www.​sanger.​ac.​uk/​DataSearch/​blast.​shtml] 2008.
        49. Felsenstein J: Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985, 39: 783–793.View Article
        50. Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 2003, 52: 696–704.View ArticlePubMed
        51. Rozas J, Sanchez–DelBarrio JC, Messeguer X, Rozas R: DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 2003, 19: 2496–2497.View ArticlePubMed
        52. Richardson M, Carroll NM, Engelke E, Spuy GD, Salker F, Munch Z, Gie RP, Warren RM, Beyers N, van Helden PD: Multiple Mycobacterium tuberculosis strains in early cultures from patients in a high–incidence community setting. J Clin Microbiol 2002, 40: 2750–2754.View ArticlePubMed
        53. van Embden JD, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B, Hermans P, Martin C, McAdam R, Shinnick TM, et al.: Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 1993, 31: 406–409.PubMed
        54. de Boer AS, Kremer K, Borgdorff MW, de Haas PE, Heersma HF, van Soolingen D: Genetic heterogeneity in Mycobacterium tuberculosis isolates reflected in IS6110 restriction fragment length polymorphism patterns as low–intensity bands. J Clin Microbiol 2000, 38: 4478–4484.PubMed

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        © Hayward et al. 2009

        This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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