The critical role of embC in Mycobacterium tuberculosis

Abstract

Arabinan polymers are major components of the cell wall in Mycobacterium tuberculosis and are involved in maintaining its structure, as well as playing a role in host-pathogen interactions. In particular, lipoarabinomannan (LAM) has multiple immunomodulatory effects. In the nonpathogenic species Mycobacterium smegmatis, EmbC has been identified as a key arabinosyltransferase involved in the incorporation of arabinose into LAM, and an embC mutant is viable but lacks LAM. In contrast, we demonstrate here that in M. tuberculosis, embC is an essential gene under normal growth conditions, suggesting a more crucial role for LAM in the pathogenic mycobacteria. M. tuberculosis EmbC has an activity similar to that of M. smegmatis EmbC, since we were able to complement an embC mutant of M. smegmatis with embC(Mtb), confirming that it encodes a functional arabinosyltransferase. In addition, we observed that the size of LAM produced in M. smegmatis was dependent on the level of expression of embC(Mtb). Northern analysis revealed that embC is expressed as part of a polycistronic message encompassing embC and three upstream genes. The promoter region for this transcript was identified and found to be up-regulated in stationary phase but down-regulated during hypoxia-induced nonreplicating persistence. In conclusion, we have identified one of the key genes involved in LAM biosynthesis in M. tuberculosis and confirmed its essential role in this species.

FIG. 1.

Demonstration of the essentiality of embC in M. tuberculosis. (A) The genetic organization of the wild-type embC region is shown. BamHI sites are indicated (BHI); the probe used for Southern analysis is shown as a solid bar. The region present in the complementing vector is indicated. (B) Map of the deletion. (C) Southern analysis of deletion DCOs isolated in the merodiploid background. Genomic DNA was digested with BamHI and hybridized to the probe. Lane MW, molecular mass marker (sizes are in kilobase pairs). Lane WT, wild-type genomic DNA. Lanes 1 to 6, genomic DNAs from Del-int strains (deletion DCOs with integrated embC).

FIG. 2.

Analysis of LAM/LM from M. smegmatis mutants. LAM/LM was extracted from M. smegmatis and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (A) Wild-type strain. (B) ΔembC strain with pVV16. (C) ΔembC strain with pMSembC (P_hsp60-EmbC_Msm). (D) ΔembC strain with pEMPTY25 (P_Ag85a-EmbC_Mtb). (E) ΔembC strain with pMTembC (P_hsp60-EmbC_Mth). M_W, molecular mass marker (masses are in kilodaltons).

FIG. 3.

Identification of the promoter for embC. (A) Promoter activities of p3791, p3792, and p3793 in M. tuberculosis. The regions upstream of Rv3790, Rv3791, Rv3792, and embC were cloned into the pSM128 reporter vector. β-Galactosidase activity from 10-ml static cultures was determined after 10 days. Results are the mean ± standard deviation of three individual transformants, each assayed in duplicate and are given in nanomoles of O-nitrophenyl-β-d-galactopyranoside produced per minute per milligram of total protein. (B) Northern blot analysis. Twelve micrograms of total RNA from M. tuberculosis grown in 7H9 medium was separated on a 1% agarose gel and transferred to a positively charged nylon membrane. The blot was hybridized to the two probes indicated, probe A for the embC transcript and probe B for the Rv3790 transcript.

FIG. 4.

P_embC activity and LAM production in M. tuberculosis. (A) Promoter activity assays. M. tuberculosis transformants carrying P_embC were grown in 10-ml static cultures (filled symbols) or on 7H10-OADC plates (open symbols), and β-galactosidase activity was measured. Results are the mean ± standard deviation of three individual transformants assayed in duplicate and are given in nanomoles of O-nitrophenyl-β-d-galactopyranoside produced per minute per milligram of total protein. (B) LM/LAM profile during growth. LM and LAM were extracted from liquid cultures after 15, 40, 55, 70, 85, and 158 days of static culture and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (C) Response of P_embC to stress conditions and drug treatment. P_embC activity was measured in M. tuberculosis after exposure to ethambutol (Eth; 0.5 μg ml⁻¹), ofloxacin (Ofl; 0.2 μg ml⁻¹), or hydrogen peroxide (H₂O₂; 10 mM for 30 min) or after 2 weeks under hypoxic conditions (Hyp). Results are the mean ± standard deviation of three individual transformants assayed in duplicate and are given in nanomoles of O-nitrophenyl-β-d-galactopyranoside produced per minute per milligram of total protein. C, control.