Identification of differentially expressed mRNA in prokaryotic organisms by customized amplification libraries (DECAL): The effect of isoniazid on gene expression in Mycobacterium tuberculosis

Abstract

Understanding the effects of the external environment on bacterial gene expression can provide valuable insights into an array of cellular mechanisms including pathogenesis, drug resistance, and, in the case of Mycobacterium tuberculosis, latency. Because of the absence of poly(A) ؉ mRNA in prokaryotic organisms, studies of differential gene expression currently must be performed either with large amounts of total RNA or rely on amplification techniques that can alter the proportional representation of individual mRNA sequences. We have developed an approach to study differences in bacterial mRNA expression that enables amplification by the PCR of a complex mixture of cDNA sequences in a reproducible manner that obviates the confounding effects of selected highly expressed sequences, e.g., ribosomal RNA. Differential expression using customized amplification libraries (DECAL) uses a library of amplifiable genomic sequences to convert total cellular RNA into an amplified probe for gene expression screens. DECAL can detect 4-fold differences in the mRNA levels of rare sequences and can be performed on as little as 10 ng of total RNA. DECAL was used to investigate the in vitro effect of the antibiotic isoniazid on M. tuberculosis, and three previously uncharacterized isoniazid-induced genes, iniA, iniB, and iniC, were identified. The iniB gene has homology to cell wall proteins, and iniA contains a phosphopantetheine attachment site motif suggestive of an acyl carrier protein. The iniA gene is also induced by the antibiotic ethambutol, an agent that inhibits cell wall biosynthesis by a mechanism that is distinct from isoniazid. The DECAL method offers a powerful new tool for the study of differential gene expression.

iniBAC induction Is Vitamin B12- and MutAB-dependent in Mycobacterium marinum

“…For instance, the acyl carrier protein kasA has been found to be up-regulated in response to isoniazid (10). Another set of genes that was found to be highly induced by sublethal concentrations of ethambutol and isoniazid in M. tuberculosis and Mycobacterium bovis BCG is the iniBAC operon (11,12). This operon was found to be induced specifically by these two cell wall targeting antibiotics but not by general cell wall stress caused by disruption of the membrane integrity through the activity of granulysin or lysozyme (12).…”

Identification of Iron-Responsive, Differential Gene Expression in the CyanobacteriumSynechocystissp. Strain PCC 6803 with a Customized Amplification Library

“…Nonetheless, an absolute requirement when using such membranes is the removal of the abundant rRNA from total RNA before labeling to avoid high background which may interfere with signal analysis. Recently, a new method called differential expression using customized amplification library (DECAL) has been used in global comparisons of gene expression in Mycobacterium tuberculosis (1). DECAL accomplishes this by first creating a customized amplification library (CAL) of genomic DNA that has been manipulated for optimal performance during analysis.…”

Drug-Resistance inMycobacterium Tuberculosis:Molecular Basis and Genotypic Detection

“…Five different mutations were detected in this codon altering its first or third bases (AtGà GtG, ctG, AtA, Atc, Att) and resulting in three amino acid shifts (Metà Val, leu, ile) (54). Several additional loci encoding proteins that may participate in the response of M. tuberculosis to EMB treatment have been identified (2) and a an extensive study investigated more genes as related to the development of eMB resistance in tubercle bacilli (46). however, one fourth of eMB r strains still lacked any known mutation linked to the eMB resistance (46), implying multiple molecular pathways to its development, and some of them are yet to be discovered.…”