Evolutionary Link between the Mycobacterial Plasmid pAL5000 Replication Protein RepB and the Extracytoplasmic Function Family of   Factors (original) (raw)

2012, Journal of Bacteriology

Mycobacterial plasmid pAL5000 represents a family of plasmids found mostly in the Actinobacteria. It replicates using two plasmid-encoded proteins, RepA and RepB. While BLAST searches indicate that RepA is a replicase family protein, the evolutionary connection of RepB cannot be established, as no significant homologous partner (E < 10 ؊3) outside the RepB family can be identified. To obtain insight into the structure-function and evolutionary connections of RepB, an investigation was undertaken using homology modeling, phylogenetic, and mutational analysis methods. The results indicate that although they are synthesized from the same operon, the phylogenetic affinities of RepA and RepB differ. Thus, the operon may have evolved through random breaking and joining events. Homology modeling predicted the presence of a three-helical helix-turn-helix domain characteristic of region 4 of extracytoplasmic function (ECF) factors in the C-terminal region of RepB. At the N-terminal region, there is a helical stretch, which may be distantly related to region 3 of factors. Mutational analysis identified two arginines indispensable for RepB activity, one each located within the C-and N-terminal conserved regions. Apart from analyzing the domain organization of the protein, the significance of the presence of a highly conserved A/T-rich element within the RepB binding site was investigated. Mutational analysis revealed that although this motif does not bind RepB, its integrity is important for efficient DNA-protein interactions and replication to occur. The present investigation unravels the possibility that RepB-like proteins and their binding sites represent ancient DNA-protein interaction modules. T he pAL5000 family (23) consists of a group of plasmids that are found mostly in bacteria belonging to the phylum Actinobacteria. Several of these plasmids have been found to exist in various actinomycetal bacteria, such as Rhodococcus (16, 23, 31, 34, 35, 49), corynebacteria, propionibacteria (26), brevibacteria (1, 37), and mycobacteria (44), and in several bifidobacterial organisms (36, 46). The preponderance of these plasmids in the Actinobacteria suggests that they may have originated within this ancient phylum (63). However, similar plasmids have been reported for Neisseria (29) and Salmonella spp., which belong to the phylum Proteobacteria. It may be that although these plasmids originated in the Actinobacteria, they were transferred subsequently to unrelated bacteria. The pAL5000 family is distantly related to the ColE2 plasmids (11), which are found in various enteric bacteria such as Escherichia coli. The common feature shared by these two groups is that they both encode a replicase family protein known as RepA, which is conserved in evolution. The major difference, however, lies in the fact that whereas the ColE2 plasmids replicate using only RepA (22), pAL5000-like plasmids require RepB in addition to RepA. The two proteins are produced from a bicistronic operon in a translationally coupled manner (5). An interesting feature of the pAL5000 family of plasmids is that while RepA happens to be conserved, RepBs are highly divergent. No significant database match for RepB with homologs outside the pAL5000 family can be recovered using BLAST searches. However, conserved domain database (CDD) searches reveal that some members of the rhodococcal and bifidobacterial lineages possess a C-terminal domain corresponding to factor region 4 (40). Considering the lack of information regarding the mechanism of replication of pAL5000, studies were initiated to understand the domain structure of RepB using homology modeling, mutational, and phylogenetic analysis tools. The results presented in this study reveal that RepB-like proteins constitute a novel branch within the factor family of transcription factors. MATERIALS AND METHODS Bacterial strains and plasmids. E. coli strain XL1 Blue was used for the routine manipulation of plasmid DNA. For the overexpression of repB and its mutants using the T7 RNA polymerase (56)-based expression system, E. coli BL21(DE3) was used. Mycobacterial transformations were performed by using Mycobacterium smegmatis strain mc 2 155. Chemicals. Ni 2ϩ-nitrilotriacetic acid (NTA) agarose, used for the affinity chromatographic preparation of 6ϫHis-tagged protein, was purchased from Qiagen (Valencia, CA). Other chemicals for protein expression, purification, and analysis, of the highest grade of purity, were obtained from SRL Laboratories, India. Radiochemicals were purchased from BRIT (Mumbai, India). Restriction enzymes and DNA-modifying enzymes such as polynucleotide kinase were purchased from New England BioLabs (NEB). Phylogenetic analysis. CLUSTAL W (60)-based alignments of multiple sequences were performed by using MEGA 4.0 software (58). Pairwise and multiple-alignment penalties of 10 and 0.1 were used for gap opening and extension, respectively, or as stated. The weight matrices chosen were