The genome of Mycobacterium tuberculosis
H37Rv contains several stress response genes, which contribute to pathogenicity.1
Among these, rv0899
encodes a 326-residue membrane protein (Rv0899) that is involved in conferring adaptation of M. tuberculosis
to acidic environments.2,3
gene and its two neighbors rv0900
, also encoding predicted membrane proteins, are found in pathogenic mycobacteria associated with tuberculosis (M. tuberculosis
, M. bovis
) and other diseases (M. marinum
, M. ulcerans
, M. kansasii
), but are absent from non-pathogenic mycobacteria, suggesting that they may be important for pathogenicity and, thus, may be attractive candidates for the development of chemotherapeutic agents.
Rv0899 contains three independently structured domains.4
The N-terminus (~ residues 1–80) includes a membrane-anchoring sequence of 20 hydrophobic amino acids (~ residues 28–50) that is required for membrane translocation but is not cleaved.5
The central region (~ residues 81–195) contains two consecutive repeats of the BON (Bacterial OsmY and Nodulation) domain (pfam04972), a conserved, putative lipid binding sequence found in some bacterial osmotic shock protection proteins, secretins, haemolysins, channels, and nodulation specificity proteins.6
Finally, the C-terminus (residues 196–326) contains an OmpA-like domain (pfam00691), a periplasmic peptidoglycan-binding structure found in several types of bacterial membrane proteins, including in the C-terminus of the outer membrane protein OmpA from E. coli
Owing to its strong homology with E. coli
OmpA, Rv0899 was originally annotated as OmpATb in the published genome sequence of M. tuberculosis
and was proposed to be an outer membrane porin.2,5,7–9
However, the three-dimensional structure shows that Rv0899 does not form a membrane-spanning β-barrel and, thus, is not compatible with porin function.4,10
Recent studies also show that Rv0899 is not a porin, but rather, that it is encoded by an operon (also known as ammonia release facilitator, arf
, operon), which includes rv0899
, and which is required for fast ammonia secretion, rapid pH neutralization and growth of M. tuberculosis
in acidic environments.3
However, the mechanism whereby Rv0899, Rv0900, and Rv0901 contribute to this function is not known.
The C-terminus of Rv0899 adopts the typical α/β structure of peptidoglycan-binding domains in the OmpA-like superfamily, reflecting association with the peptidoglycan layer. However, theα/β fold of the central domain, with three parallel/antiparallel α-helices packed against a six-stranded parallel/antiparallel β-sheet, was unprecedented in the Protein Data Bank (PDB) and, thus, provided limited insights about function.
A simple BLAST (Basic Local Alignment Search Tool) search of the protein databases for sequences similar to Rv0899 also provides little insights about the function of the central domain, because it is dominated by hits with strong homology to the C-terminus, but little homology to the rest of the protein. This reflects the strong sequence conservation and ubiquitous nature of bacterial OmpA-like domains, which are widespread in outer membrane proteins (e.g. OmpA inserts in the outer membrane as a β-barrel), as well as outer membrane lipoproteins (e.g. Pal is bound to the outer membrane through a lipid anchor), and inner membrane proteins (e.g. MotB inserts in the inner membrane through a transmembrane helix).11,12
In contrast, the central BON-containing domain of Rv0899 has only weak similarity to database sequences. Thus, matches to this region are obscured by numerous, much more pronounced matches to the OmpA-like region. Furthermore, performing the BLAST search with individual domains as query (i.e. only BON or only OmpA-like) yields sequences with homology primarily restricted to the specific query region, obscuring sequences that cover the entire length of Rv0899.
To overcome this problem and gain further insights about the potential function of Rv0899, a detailed iterative search of the NCBI (National Center for Biotechnology Information) database was performed to identify other Rv0899-like proteins with homology spanning the entire Rv0899 sequence, including the transmembrane, BON and OmpA-like domains. This analysis uncovers a family of Rv0899-like proteins in bacteria with functions in nitrogen metabolism, adaptation to nutrient poor environments, and/or establishing symbiosis with the host organism.