A gonococcal homologue of meningococcal γ-glutamyl transpeptidase gene is a new type of bacterial pseudogene that is transcriptionally active but phenotypically silent

Background It has been speculated that the γ-glutamyl transpeptidase (ggt) gene is present only in Neisseria meningitidis and not among related species such as Neisseria gonorrhoeae and Neisseria lactamica, because N. meningitidis is the only bacterium with GGT activity. However, nucleotide sequences highly homologous to the meningococcal ggt gene were found in the genomes of N. gonorrhoeae isolates. Results The gonococcal homologue (ggt gonococcal homologue; ggh) was analyzed. The nucleotide sequence of the ggh gene was approximately 95 % identical to that of the meningococcal ggt gene. An open reading frame in the ggh gene was disrupted by an ochre mutation and frameshift mutations induced by a 7-base deletion, but the amino acid sequences deduced from the artificially corrected ggh nucleotide sequences were approximately 97 % identical to that of the meningococcal ggt gene. The analyses of the sequences flanking the ggt and ggh genes revealed that both genes were localized in a common DNA region containing the fbp-ggt (or ggh)-glyA-opcA-dedA-abcZ gene cluster. The expression of the ggh RNA could be detected by dot blot, RT-PCR and primer extension analyses. Moreover, the truncated form of ggh-translational product was also found in some of the gonococcal isolates. Conclusion This study has shown that the gonococcal ggh gene is a pseudogene of the meningococcal ggt gene, which can also be designated as Ψggt. The gonococcal ggh (Ψggt) gene is the first identified bacterial pseudogene that is transcriptionally active but phenotypically silent.


Background
Two members of the gram-negative diplococci, Neisseria meningitidis and Neisseria gonorrhoeae, are particularly associated with pathological infections. N. meningitidis is specialized for the mucosa of the nasopharynx and causes meningitis and septicemia. N. gonorrhoeae is adapted for the mucosa of the urogenital tract and causes gonorrhoea and pelvic inflammatory diseases. Both species colonize only humans and share a great deal of relatedness at the nucleotide level [1]. This high degree of relatedness is reflected in the many common genetic, biochemical and antigenic features of the two bacteria.
γ-Glutamyl transpeptidase (also called γ-glutamyl aminopeptidase) (EC2.3.2.2; GGT) catalyzes the hydrolysis of γ-glutamyl compounds, and is found in a variety of bacteria such as Escherichia coli [2] and Helicobacter pylori [3,4]. To distinguish N. meningitidis from N. gonorrhoeae, GGT activity is used as one of the identification markers for N. meningitidis because N. meningitidis is positive for this activity but N. gonorrhoeae and related species, e.g., Neisseria lactamica, are not [5]. In fact, the detection of GGT activity is applied for the identification of N. meningitidis in the Gonochek II enzymatic identification system (E-Y Laboratories Inc., U.S.A.) [6][7][8][9]. From these empirical facts, it was believed that the gene encoding for GGT should exist only in N. meningitidis, but this has not been proven yet [3].
Recent remarkable progress in the sequencing of various genomes has led to the detection of nucleotide sequences that appear to be phenotypically silent, termed pseudogenes. The pseudogenes are defined as DNA sequences of formerly functional genes rendered nonfunctional by mutations and usually identified by their disrupted open reading frames (ORFs). Pseudogenes have been identified in a variety of eukaryotes, including insects [10], plants [11], and particularly vertebrates [10,12], but are relatively few in the bacterial genomes. Notable exceptions are intracellular bacterial parasites such as Rickettsia prowazekii and Mycobacterium leprae [13], which seem to have lost many genes due to obtaining nutritional supplies from the host cells. Cryptic genes such as the cel operon in E. coli [14][15][16] and the flagellar master operon in the genus Shigella [17][18][19] seem to be a kind of pseudogenes, but are different from pseudogenes because the cryptic genes completely retain intact ORFs, which can be occasionally activated by rare genetic events such as mutation, recombination, insertion of elements. As a whole, compared to the pseudogenes in eukaryotes, relatively few pseudogenes have been reported in bacterial genomes [20].
In this study, a gonococcal ggh gene, which is highly homologous to the meningococcal ggt gene, was found to be pseudogene. Sequence analyses of the flanking regions of both the ggt and ggh genes suggest that both genes were derived from a gene in a common ancestor, and subsequently diversified.

Results
The gonococcal ggh gene was highly homologous to the meningococcal ggt gene Since GGT activity was detected only in N. meningitidis among the related species, it was speculated that the corresponding gene also existed only in N. meningitidis. However, by BLAST search, the nucleotide sequences highly homologous to the meningococcal ggt gene were found in the genome of N. gonorrhoeae FA1090 [Gen-Bank:NC_002946]. The overall nucleotide sequence of the meningococcal ggt homologue was approximately 95 % identical to that of the meningococcal ggt gene (data not shown and additional file). Eleven N. gonorrhoeae clinical strains were analyzed by PCR, and the corresponding DNA fragments were amplified in all of these strains ( Figure 1B), indicating that this gene was generally present in N. gonorrhoeae. To analyze whether ggt homologues existed in the genomes of the other neisserial strains, Southern blotting was performed ( Figure 1C). DNA fragments that hybridized with the meningococcal ggt gene were found in the meningococcal and gonococcal genomes ( Figure 1C lanes 1-3) but not in the other neisserial genomes ( Figure 1C lanes 4-11). These results suggested that the meningococcal ggt homologue was present only in N. gonorrhoeae among the neisserial species examined. The putative gene in N. gonorrhoeae was named ggh (ggt gonococcal homologue).

Variations of the nucleotide sequences of ggh genes among clinical isolates
To characterize the gonococcal ggh gene, the ggh genes were amplified from the chromosomal DNA of 11 N. gonorrhoeae strains and sequenced. The nucleotide sequences of the ggt genes from 7 N. meningitidis strains and of the ggh genes from the 11 N. gonorrhoeae strains were aligned, and the distance matrix calculated from these data was displayed as a phylogenetic tree (Figure 2A). The results revealed that the nucleotide sequences of the gonococcal ggh genes were more divergent than those of the meningococcal ggt genes ( Figure 2A). Alignment of the nucleotide sequences of the 11 gonococcal ggh genes also showed that the mutations in the ggh gene consisted of the following four polymorphisms: 1) a 6-base insertion (named Type I), 2) an ochre mutation (Type II), 3) a 7-base deletion (Type III), 4) a 46-base insertion (Type IV) ( Figure 2B and Table 2). In addition, all of the 11 ggh genes had onenucleotide substitutions compared to the ggt genes in the same 25 sites ( Figure 2B and additional file), with only 2 exceptions: a one-nucleotide variation in NIID109 (48 th base A to G) and another in NIID105 (213 th base G to A) ( Table 2). The one-base substitutions in the common sites of the ggh genes strongly suggested that reconstruction of the ggh gene would have occurred at an early stage after speciation (See Discussion).

Putative amino acid sequences in hypothetical coding region of the ggh genes
Due to the ochre (Type II) and 7-base deletion (Type III) mutations, the ORF in each ggh gene was completely disrupted by the formation of 8 or 20 stop codons ( Figure  3A). In fact, none of the gonococcal isolates showed any GGT activity (data not shown and [5,9]), indicating that there was no expression of functional GGT-like protein in N. gonorrhoeae. All of these results showed that the gonococcal ggh gene was a pseudogene of the functional ggt gene in N. meningitidis. On the other hand, if the two types of mutations (Types II and III) in the ggh genes were The presence of a meningococcal ggt gene homologue in N. gonorrhoeae The scale bar represents uncorrected distances, and a fit parameter is also shown. B. Alignment of the nucleotide sequences containing 4 kinds of differences in the ggt and ggh genes of N. meningitidis strains H44/76 and H114/90; N. gonorrhoeae strains ATCC49226, NIID54, NIID103 and NIID106, respectively. Sequence identity is represented as *, polymorphism among the sequences of the 6 strains is indicated by the appropriate letter, and the absence of a base is shown by a hyphen (-). The nucleotide substitution for an ochre (Type II) mutation is shown in bold. Boxes at the Type I and Type III mutations indicate the tandem repeat. The tetranucleotide repeat in type I is also shown as a gray box. The newly deduced start codon of the meningococcal GGT is shown in underlined bold.
artificially corrected, the hypothetical amino acid sequences were approximately 97 % identical to those of the meningococcal ggt genes and were highly conserved among the gonococcal ggh genes ( Figure 3B). This result also supported the idea that the ggh and ggt genes were derived from a common ancestral gene and that the translational inactivation of the gonococcal ggh gene was solely due to the ochre (Type II) and the frame-shift mutations caused by the 7-base deletion (Type III).

The genetic organization of the ggt-and ggh-flanking regions in the genomes of N. meningitidis and N. gonorrhoeae
By using the information in the database for N. meningitidis strain MC58 [21], N. gonorrhoeae strain FA1090 and N. lactamica (the neisserial species most closely related to the above two species) ST-640 strain, the flanking regions of the meningococcal ggt and the gonococcal ggh genes were further analyzed. The ggt and ggh genes were both localized in the identical gene cluster of fbp-ggt (or ggh)-glyA-opcA-dedA-abcZ in the genomes of N. meningitidis and N. gonorrhoeae, respectively ( Figure 4). The fbp-glyA-dedA-  "+" or "-" denotes the presence or absence of the mutation, respectively. *Mutation type corresponds to the categories in Figure 2B.
A. Positions of stop codons in the hypothetical ORF in the ggh gene  Figure 2B is removed and the site of Type III deletion is replaced by the letter B. The site of the ochre (Type II) mutation is shown as X. The identical amino acid sequences between N. meningitidis and N. gonorrhoeae are shown in a black box and amino acid sequences common to only N. gonorrhoeae are shown in a gray box.
abcZ locus was also found in the genome of N. lactamica but lacked the ggt and opcA homologues (Figure 4). This highly conserved genetic organization implied that a DNA island containing an original ggt gene was first incorporated into an ancestor's genome of the above three species and subsequently diversified after the speciation (see Discussion).

The expression of the ggh gene in N. gonorrhoeae
The hitherto identified bacterial pseudogenes are not expressed transcriptionally or translationally [20,[22][23][24]. To examine the ggh transcriptional expression, dot blot analysis was first performed and RNA that hybridized with the ggt probe was detected in the total RNAs of 4 N. gonorrhoeae strains ( Figure 5A). The transcriptional expression was also confirmed by RT-PCR, and the products were amplified with all 3 sets of primers from total RNA of all 4 N. gonorrhoeae strains tested ( Figure 5B). These results strongly suggested that the full-length ggh gene transcript was expressed in N. gonorrhoeae. Primer exten-sion analysis further revealed that the gonococcal ggh RNA was transcribed from the same starting point as the meningococcal ggt mRNA ( Figure 5C and 5D). All of these results indicated that the gonococcal ggh gene was transcriptionally active though it was a pseudogene.
To further study the translational expression of the truncated GGT-like protein in N. gonorrhoeae, Western blotting was performed with anti-meningococcal GGT rabbit antiserum [25]. When the same amounts of the whole cell extracts were analyzed ( Figure 6B), approximately 15-kDa bands were detected in the extracts of NIID103 and NIID106 N. gonorrhoeae strains ( Figure 6A lanes 5 and 7).
Because the 15-kDa protein was not observed in the ∆ggh background of NIID103 and NIID106 ( Figure 6A lanes 6  and 8), the 15-kDa protein was thought to be the ggh gene product whose translation was terminated at the 145th codon ( Figure 3B). However, the 15-kDa protein was not found in the extracts of the ATCC49226 and NIID54 N. gonorrhoeae strains ( Figure 6A, lanes 3 and 4) and seemed Genetic organization of the genes around the ggt and ggh genes in N. meningitidis strain MC58 [21], N. gonorrhoeae strain FA1090 [47] and N. lactamica strain ST-640 [48] Figure 4 Genetic organization of the genes around the ggt and ggh genes in N. meningitidis strain MC58 [21], N. gonorrhoeae strain FA1090 [47] and N. lactamica strain ST-640 [48]. Black arrows and the open bar indicate coding and non-coding genes, respectively.
Transcriptional expression of the ggh genes Figure 5 Transcriptional expression of the ggh genes. A. Dot blot analysis using the meningococcal ggt gene as a probe. One to 16 micrograms of RNAs isolated from H44/76, HT1089 (H44/76 ∆ggt::spc) [39], ATCC49226, NIID54, NIID103 and NIID106 were subjected to this analysis. B. RT-PCR to detect the transcripts of the gonococcal ggh genes. The schematic figure in the box depicts the position of the primers used in this experiment (see Table 1). RT-PCR was performed without reverse transcriptase (RTase) (lanes 1 to 5) or with RTase (lanes 6 to 10).

Discussion
In this study, it was shown that the gonococcal ggh gene is a member of bacterial pseudogenes, and is transcribed but not properly translated so that active ggh protein product is not produced. 11Fh-mtTFA [26], OsMu4-2 [27], NA88-A [28], Makorin1-p1 [29], Dnm3a2 [30] and pseudoNOS [31] genes are known to be transcriptionally active eukaryotic pseudogenes. In neisseriae, the gonococcal porA and ΨopcB genes have been reported as neisserial pseudogenes [1,32,33]. The porA pseudogene contains mutations in the promoter and the coding regions, and is not translated [24]. While some hypothetical bacterial pseudogenes with repetitive runs of A and T are speculated to be potentially expressed by transcriptional slippage [34], the expression, including the transcription, has not been proven yet. To our knowledge, the gonococcal ggh gene is the first identified bacterial pseudogene that is transcriptionally active.
The 15-kDa derivative of the putative ggh protein product is detected in the NIID103 and NIID106 N. gonorrhoeae strains but not in the ATCC49226 and NIID54 N. gonorrhoeae strains ( Figure 6A). Since the predicted amino acid sequences of the putative 15-kDa proteins seem to be similar among the 4 gonococcal strains, the reason why the 15-kDa protein was not detected in ATCC49226 and NIID54 is not clear. The 15-kDa protein might be degraded in ATCC49226 and NIID54 backgrounds but not in NIID103 and NIID106 backgrounds. It seems unlikely that the 15-kDa protein encoded by the ggh gene has an essential function for N. gonorrhoeae because the 15-kDa protein is not always detected in any of gonococcal strains ( Figure 6A).
Why does the gonococcal ggh gene still retain the transcriptional activity? There are some examples in which RNAs transcribed from pseudogenes have some biological functions: antisense RNA expressed from the pseudoNOS gene hybridizes with nNOS (nitric oxide synthase) mRNA, resulting in the suppression of the nNOS gene expression in the neurons of the snail Lymnaea stagnalis [31]. RNA of Makorin1-1p, a pseudogene of Makorin1, regulates the Makorin1 mRNA stability, which is important for the correct formation of the kidneys and bone in mice [29]. Some eukaryotic genes may be duplicated and one of the plural genes may be subsequently reconstructed due to its redundancy, resulting in a pseudogene. However, since a bacterial pseudogene generally does not have a functional counterpart (wild-type gene) in a single organism, the ggh RNA does not seem to have the same kind of biological function as the pseudoNOS and Makorin1-1p genes. In fact, we could not find any prominent phenotype for a ∆ggh gonococcal mutant (unpublished data). However, we cannot exclude the possibility that the ggh RNA has some biological function(s) in other milieus such as the urogenital tract and further analyses will be required to address this possibility.
The ggt and ggh genes are located in the fbp-ggt (ggh)-glyA-opcA-dedA-abcZ common gene cluster in the genomes of both N. meningitidis and N. gonorrhoeae (Figure 4) [23]. The genome of N. lactamica lacks ggt and opcA homologues in the fbp-glyA-dedA-abcZ locus (Figure 4 and [23]). It would not be the result of chance that the two ggt (or ggh) and opcA genes are located in the same respective sites of the fbp-glyA-dedA-abcZ gene locus of both N. meningitidis and N. gonorrhoeae but not in that of N. lactamica. Moreover, it is also unlikely that a nonfunctional ggh gene was horizontally transferred into the gonococcal fbp-glyA-opcA-dedA-abcZ gene cluster since such a pseudogene could not have been sustained due to the lack of selection [23]. Therefore, it seems more probable that the fbp-ggt-glyA-opcA-dedA-abcZ gene cluster was present in an ancestor of the three neisserial species, and has been subsequently diversified independently among the three species, as shown for the opcA gene [23]. During the diversification, the meningococcal ggt gene has been maintained in an active state while the gonococcal ggh gene has been reconstructed by insertion, deletion and substitutions, resulting in the translational inactivation. In N. lactamica, the ggt and opcA homologues might have been lost because of their dispensability for N. lactamica (see below).
It is also interesting that the ggh gene has not been fully deleted from the gonococcal genome. The kinds and sites of mutations in the ggh genes are relatively few and highly conserved, respectively, among the gonococcal isolates ( Figure 2B, Table 2 and additional file). It is also noted that, while in general the RNA polymerase-binding sites and SD regions of pseudogenes are highly degraded, there are also a few exceptions in species such as Y. pestis that could have emerged in recent evolutionary times [35]. Since the ribosome-binding sites (Shine-Dalgarno regions) of the gonococcal ggh gene are identical to those of the meningococcal ggt gene and the RNA polymerasebinding sites are almost completely conserved (with a one-nucleotide difference) ( Figure 5D), it is speculated that the reconstruction of the ggh gene may have occurred in relatively recent evolutionary times. From the evolutional viewpoint, the drastic deletion of the approximately 2-kb DNA region containing the ggh gene in N. gonorrhoeae may not have been likely to occur in such a short period. Alternatively, deletion of the 2-kb DNA region may not be more advantageous for gonococcal evolution than reconstruction involving short deletions, insertions and substitutions.
Western blotting with anti-meningococcal GGT rabbit antiserum [25] (A) and Coomassie Brilliant Blue staining (B) of the whole cell extracts after SDS-PAGE The maintenance of a functional ggt gene in N. meningitidis would have some advantages for its survival. N. meningitidis causes meningitis, which is due to the meningococcal invasion into the human central nervous system, including cerebrospinal fluid (CSF) [36][37][38]. It has been shown that meningococcal GGT has a physiological function of acquiring cysteine from environmental γglutamyl-cysteinyl peptides under cysteine-limited environments such as the CSF [39]. Almost all (98.8 %) meningococcal isolates from humans are positive for GGT activity [40]. All of these results suggest that the GGT activity is important for N. meningitidis but not for N. gonorrhoeae. The dispensability of GGT activity for N. gonorrhoeae seems to be consistent with the fact that a cysteine-limited milieu such as the CSF in humans is not a natural gonococcal habitat. However, it is not very likely that the milieu of CSF exerts selective pressure for an active ggt gene because human CSF is not a relevant milieu for human-to-human spread of meningococcus [41]. We believe that the meningococcal GGT must have some unknown essential function(s) for N. meningitidis and further studies will elucidate the function(s).

Conclusion
Our data on the ggh gene indicate that the ggh gene in N. gonorrhoeae is a pseudogene of the functional ggt gene in N. meningitidis. To our knowledge, the ggh gene is the first reported bacterial pseudogene that is transcriptionally active but phenotypically silent. Our findings may also contribute to understanding the speciation of N. meningitidis and N. gonorrhoeae.

Isolation of chromosomal DNA, PCR, Southern blotting and dot blotting
Isolation of chromosomal DNA, PCR, Southern blotting and dot blotting were performed as described in our previous report [43].

Nucleotide sequence determination and analyses
The ggt and ggh genes were amplified with a set of primers (ggt-3 and ggt-4) by PCR, and the resulting products were purified using High Pure PCR Product Purification Kit (Roche) as templates for sequencing. The sequencing was performed as described previously [25]. Primers used for sequencing are listed in Table 1. Raw data from the ABI sequencer were assembled with the program DNASIS ver. 3.2 (HITACHI, Japan). The sequence alignment was performed with GENETYX-MAC ver.11 (GENETYX, Japan). Phylogenetic analyses were performed by constructing a distance matrix of nucleotide mismatches using the web site of the Belozersky Institute at Moscow State University [44] and visualized by Split decomposition analysis with the program SPLITSTREE, version 3.2 [45].
Construction of ∆ggh::spc N. gonorrhoeae mutants HT1195 (NIID103 ∆ggh::spc) and HT1196 (NIID106 ∆ggh::spc), in which a spectinomycin resistance gene (spc) was inserted into the ggh gene, were constructed as follows: A 2-kb fragment containing the ggh gene of NIID103 or NIID106 was amplified by PCR and cloned in the SmaI site of pUC18 (Takara Bio) to construct pHT412 or pHT413, respectively. A blunted 1-kb fragment containing the spc gene [25] was inserted into the EcoRV sites of pHT412 and pHT413, respectively. The EcoRV sites are located at 277 bp and 1642 bp (NIID103 [DDBJ:AB175025]) and at 271 bp and 1590 bp (NIID106 [DDBJ:AB175027]) downstream from the transcriptional start point of each ggh gene, respectively (see Figure 5D). The resulting plasmids were named pHT414 and pHT415, respectively. Five hundred nanograms of the plasmids linearized by digestion with EcoRI were transformed into NIID103 and NIID106, respectively, as described previously [43]. Spectinomycin-resistant clones were selected on GC agar plates containing 75 µg/ml spectinomycin.

RT-PCR
Bacteria grown on GC agar plates were suspended in 20 ml of GC broth to an OD 600 of 0.1 and continuously cultured to mid-log phase (OD 600 of ~ 0.6) at 37°C with shaking. The total RNA was isolated from the harvested bacteria as previously described [46] with an additional treatment with DNase I. RT-PCR was performed using one step RT-PCR Kit Ver. 1.1 (Takara Bio, Japan) with approximately 2 µg of total RNA according to the manufacturer's instructions. The products were visualized by electrophoresis in a 2 % agarose gel followed by ethidium bromide staining.