Comparative sequence analysis of acid sensitive/resistance proteins in Escherichia coli and Shigella flexneri

The molecular basis for the survival of bacteria under extreme conditions in which growth is inhibited is a question of great current interest. A preliminary study was carried out to determine residue pattern conservation among the antiporters of enteric bacteria, responsible for extreme acid sensitivity especially in Escherichia coli and Shigella flexneri. Here we found the molecular evidence that proved the relationship between E. coli and S. flexneri. Multiple sequence alignment of the gadC coded acid sensitive antiporter showed many conserved residue patterns at regular intervals at the N-terminal region. It was observed that as the alignment approaches towards the C-terminal, the number of conserved residues decreases, indicating that the N-terminal region of this protein has much active role when compared to the carboxyl terminal. The motif, FHLVFFLLLGG, is well conserved within the entire gadC coded protein at the amino terminal. The motif is also partially conserved among other antiporters (which are not coded by gadC) but involved in acid sensitive/resistance mechanism. Phylogenetic cluster analysis proves the relationship of Escherichia coli and Shigella flexneri. The gadC coded proteins are converged as a clade and diverged from other antiporters belongs to the amino acid-polyamine-organocation (APC) superfamily.


Background:
Microbes are not always boned to have the favorable condition for their survival. So as to tackle the unfavorable conditions, they adopt certain mechanisms to overcome it. All enteric pathogens are required to bypass the acidic environment of stomach before infecting the intestinal mucosa, where luminal pH approaches neutrality.
[1] Enteric micro-organisms have developed several inducible mechanisms for surviving transient periods of extreme acid stress.
[2] Though such acid resistance mechanism is found in Enterobacteriaceae family but it is not the characteristic feature of all microbes of the family. Escherichia coli and Shigella flexneri have been reported to possess the acid resistance mechanism [3] where gadA and gadB genes code for the isoforms of glutamate decarboxylase (GAD). The gad system is based on the coordinated action of these two homologues of glutamate decarboxylase and of a specific glutamate/gamma-aminobutyrate antiporter (GadC) [4], in which glutamate is internalized and converted to γaminobutyrate (consuming an intracellular proton) that is subsequently exchanged for another extracellular glutamate via a membrane-located antiporter.
[5] Gale and Epps [6] as well as others [7, 8] demonstrated that there are a variety of decarboxylases that respond to low pH. The putative glutamate/GABA antiporter which is encoded by the gadC gene is responsible for importing the glutamate inside the cell and simultaneously exporting the GABA to the acidic environment. This helps for neutralization and survival in the acidic environment. The acid sensitivity inner membrane antiporter protein plays a pivotal role in the acid resistance indirectly, it is also found that mutation in the inner membrane antiporter protein makes the organism acid sensitive as neither intake of glutamate nor export of GABA takes place, which pave an acidic environment where the microbes will undergo death phase. This specific GABA antiporter belongs to the amino acid -polyamineorganocation (APC) super family. Gad A, B, C, hde AB, all are essential for the expression of acid resistance strains and mutations in any of these regions may block gludependent systems.
[9] These genes encode a glutamatedependent acid resistance mechanism that is optimally active under conditions in which it is needed to maintain viability. [10] Present study deals with gadC encoded inner membrane antiporter due to its importance in transporting glutamate across inner membrane through gadC and making favourable environment for surviving in extreme condition.
[9] Here we tried to decipher, is there any evidence hidden in the antiporter protein, of Escherichia coli K-12, O157:H7 and Shigella flexneri? Because this mechanism was not found in any other Enterobacteriaceae. We suspect that there must be some sequence conservation which was not detected in other enterobacteriaceae. GadC of Listeria monocytogenes has a motif FHLVFFLLLGG that corresponds to the Shigella flexneri GadC FSLVFFLLLGG and is considered to play an important role in the recognition of the glutamate.
[5] Here we also address this pattern in the rest of the gadC coded acid sensitive/resistance proteins and other antiporters of APC super family as well.
A multiple sequence alignment was done by using Clustal X Ver.1.83 [12], the gap opening was set at 10.00, the gap extension at 0.20 with 30% delay divergent sequences and Gonnet series weight matrix was used. From the multiple sequence alignment, the guide tree was derived. To justify the confidence of the clades, re-sampling method (bootstrap) was used with 10000 trails. Web logo (ver 2.8.2) was used to identify the conserved pattern in the gadC coded antiporters of Enterobacteriaceae. Alignments were analysed and phylogenetic relationships among the sequences were established using different procedures: Neighbour-Joining (NJ) [13], Fast Minimum Evolution (FastME) [14] Unweighted Pair Group Method with Arithmetic Mean (UPGMA). [15] The final tree was displayed by using MEGA 3.1 [16], the nodes and clades of gadC antiporters were traced out by visual examination.

Results and discussion:
Tracing the gadC cluster among the antiporters A preliminary multiple sequence alignment was carried out among all antiporters of enteric bacteria belonging to the APC superfamily. Based on the multiple sequence alignment and tree construction with 10000 bootstrap trials Figure 1 shows that the gadC coded proteins form a separate cluster from other antiporters which belong to APC super family.
The similar trend was also observed in phylogenies obtained by using different methods (NJ, UPGMA and FastME). The antiporter sequence of Rhodopirellula baltica (NP_864077) which belongs to proteobacter was used as an out-group. The convergence of gadC coded antiporters stands separately from other antiporters which comprises of Listeria monocytogenes, Clostridium perfringens, Lactococcus lactis, E. coli, S. flexneri and S. dysenteriae.

Evolutionary distance between antiporters
From Figure 2, it is clear that ten major proteins coded by gadC forms the root of the tree (0.0) which corresponds to the gadC cluster (shown in Figure 1). The out group used showed maximum deviation (0.90) and 100% confident divergence from other antiporters from other operational taxonomical units ( The root (0.0) and the closely related cluster (0.43-0.47) have the conserved LVFFLLLGCC motif. The conservation goes on decreases with respect to other clusters or distantly related antiporters and reveals that electrochemical-potential-driven transporters essential for the expression of acid resistance, could not be detected in other family members of the Enterobacteriaceae. [17] In contrast to neutralophilic bacteria such as Salmonella typhimurium, E. coli and Shigella have acid resistance systems which are unique.
[2] Moreover the acid resistance in E. coli and Shigella species is similarly regulated. [18] Hence our main focus lies on gadC; the gadC clade was analyzed separately so as to determine the relationship between E. coli and S. flexneri because studies conducted by Lin et al. [3] showed that the toxic strain of E. coli, H10407 did adapt well at pH 4.3, although not quite as well as S. typhimurium UK1. It was also considered possible that two other strains of E. coli and S. flexneri might respond better to an acid shock at a less acidic pH. Therefore a separate sequence analysis was carried out between E. coli strains and S. flexneri. Careful analysis of multiple sequence alignment of E. coli [P63235], E. coli 0157:H7 [P58229], E. coli O6 [Q8FHG6], showed that these organisms have 98-99% homology with their closely related clades of phylogeny (Figure 3). This high similarity may be due to the two glutamate decarboxylases, encoded by gadA and gadB, with gadB forming part of an operon with the antiporter determinant gadC. These homologues obviously resulted from a gene duplication event, given that they share 98% and 99% similarity at the DNA and protein levels respectively [19] as shown in

Distinct pattern conservation of glutamate binding region
Multiple alignment of the gadC coded acid sensitive antiporter showed many conserved residue patterns in a regular interval at the N-terminal region and as the alignment approached the C-terminal end, the number of conserved residues decreased, indicating that the Nterminal region of this protein has a much active role when compared to the carboxyl terminal end. The motif FSLVFFLLLGG is considered to play an important role in the recognition of the glutamate and our alignment analysis ( Figure 4) confirmed that the motif FHLVFFLLLGG was well conserved within the entire gadC coded proteins (at the amino terminal). It proved that the FHLVFFLLLGG motif is not only unique for Shigella flexineri but also for the other gadC coded bacteria such as Escherichia coli, E. coli O157:H7, E. coli O6, Shigella sonnei Ss046, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Listeria monocytogenes.
We also extended our analysis to address the pattern conservation among the other antiporters (which are not coded by gadC) involved in acid sensitive/resistance mechanism. Amazingly we found that the pattern is still partially conserved for the acid sensitive/ resistance mechanism ( Figure 5). This pattern conservation also depicts that the function is highly dependant on the pattern used for the acid resistance. The motif 'FHLVFFLLLGG' was well conserved with the entire gadC coded proteins at the amino terminal where the binding residue could be found with in the first and second transmembrane helices.
[5] The partial conservation of this motif among the other antiporters (not coded by gadC) is due to the poor acid resistance. The strong motif conservation could be the reason for the extreme acid resistance of E. coli and S. flexneri. Our pattern analysis shows the relationship of Escherichia coli and Shigella flexneri. This can be correlated with the claims of Waterman and Small (2003) [19], for a strong-link between the possession of the gadC genes and the expression of stationary-phase acid resistance. This also correlates with the epidemiological data that associated these species with having a lower infective dose compared to other enteric pathogens and confirms the close evolutionary relationship between Escherichia coli and Shigella flexneri amongst the Enterobacteriaceae.
The overall analyses presented herein clearly confirm and adds support to the claim that Shigella species possess acid resistance because they are essentially E. coli [20] in agreement with the taxonomic criteria indicate that Shigella and Escherichia are actually the same genus [21] and have identical virulence determinants that cause clinically indistinguishable disease.