A Rifampin-Hypersensitive Mutant Reveals Differences between Strains of Mycobacterium smegmatis and Presence of a Novel Transposon, IS 1623

Abstract

Rifampin is a front-line antibiotic for the treatment of tuberculosis. Infections caused by rifampin-and multidrug-resistant Mycobacterium tuberculosis strains are difficult to treat and contribute to a poor clinical outcome. Rifampin resistance most often results from mutations in rpoB. However, some drug-resistant strains have rpoB alleles that encode the phenotype for susceptibility. Similarly, non-M. tuberculosis mycobacteria exhibit higher levels of baseline resistance to rifampin, despite the presence of rpoB alleles that encode the phenotype for susceptibility. To identify other genes involved in rifampin resistance, we generated a library of Mycobacterium smegmatis mc 2 155 transposon insertion mutants. Upon screening this library, we identified one mutant that was hypersensitive to rifampin. The transposon insertion was localized to the arr gene, which encodes rifampin ADP ribosyltransferase, an enzyme able to inactivate rifampin. Sequence analysis revealed differences in the arr alleles of M. smegmatis strain mc 2 155 and previously described strain DSM 43756. The arr region of strain mc 2 155 contains a second, partial copy of the arr gene plus a novel insertion sequence, IS1623.Mycobacterial infections, including tuberculosis (TB) and leprosy, are bacterial diseases of global importance. The World Health Organization estimates that the worldwide incidences of TB increased 0.4% in 2001, to 8.5 million new cases (28). Control of TB is complicated by its ease of transmission, difficulties in administering the long-course chemotherapy regimens, and the appearance of strains that are multidrug resistant (MDR), which is defined as resistance to the two front-line anti-TB drugs, isoniazid and rifampin. Rifampin is a broad-spectrum antibiotic that inhibits bacterial DNA-dependent RNA polymerase activity. Resistance to rifampin is most often caused by mutations in rpoB, which encodes the ␤ subunit of RNA polymerase (20,25). In rifampin-resistant clinical isolates of Mycobacterium tuberculosis, an estimated 96% of the rpoB mutations map to an 81-bp region (codons 507 to 533) near the middle of the gene (20). Mutations at codons 531, 526, and 516 are the most common (13). A variety of assays that detect these sequence polymorphisms have been developed (3, 23) and allow the rapid determination of the drug susceptibilities of clinical M. tuberculosis isolates. In addition, an estimated 90% of rifampin-resistant clinical isolates are also isoniazid resistant, such that rifampin resistance is a positive indicator of MDR TB. However, about 4% of rifampin-resistant clinical isolates of M. tuberculosis have no mutations in the 81-bp core region or elsewhere in the rpoB gene (3,8,14,20). In addition, some mycobacteria, particularly atypical mycobacteria, such as Mycobacterium smegmatis, Mycobacterium avium, and Mycobacterium intracellulare, are resistant to rifampin, even though they possess sensitive RNA polymerase (9,14,20). These findings indicate that other genes can contribute to rifampin resistance. To identify s...

Roles of Lsr2 in Colony Morphology and Biofilm Formation ofMycobacterium smegmatis

“…The method used to localize and identify the transposon-disrupted gene has been described previously (1,2). Briefly, total chromosomal DNA of the transposon insertion mutant was cleaved with BamHI and then self-ligated with T4 DNA ligase and transformed into competent E. coli DH5␣ pir116 cells.…”

Rifamycin antibiotic resistance by ADP-ribosylation: Structure and diversity of Arr

“…In the opportunistic pathogen Mycobacterium smegmatis, however, rifampin is an ineffective drug because of the presence of a chromosomally encoded rifampin ADPribosyltransferase (Arr-ms). Arr-ms is a small enzyme (Ϸ16 kDa) with no sequence similarity to known protein ADPribosyltransferases such as ART, PARP, and bacterial toxins (6,29). Paradoxically, a homologue of Arr, Arr-2 (55% identical with Arr-ms), also is present in several transposons and integrons found in Gram-negative pathogenic bacteria such as P. aeruginosa (30), Escherichia coli (31), Klebsiella pneumoniae (32), and Acinetobacter baumannii (33).…”

Antibacterial activity of rifamycins for M. smegmatis with comparison of oxidation and binding to tear lipocalin

“…The transposon insertion in RHS 234 disrupts the arr gene, which encodes rifampin ADP ribosyltransferase, the enzyme that inactivates rifampin. RHS 234 is a rifampin-hypersensitive mutant that in initial studies may mimic the drug sensitivity of Mycobacterium tuberculosis [5]. However, carefully controlled studies are needed for all major rifamycins to compare effectiveness.…”