Expression of Mycobacterium leprae genes from a Streptococcus mutans promoter in Escherichia coli K-12.

Abstract

Genomic libraries of Mycobacterium leprae DNA partially digested with Pst I were constructed in the expression vector pYA626, which contains the promoter region from the Streptococcus mutans gene encoding aspartate asemialdehyde dehydrogenase, which is very efficiently expressed in Escherichia coli. We have detected several clones that complement a mutation in the citrate synthase gene of E. coli. Southern blot analysis demonstrated that the complementing DNA was M. leprae DNA. Sodium dodecyl sulfate/polyacrylamide gel analysis of polypeptides produced by minicells containing the citrate synthase-complementing recombinant molecules demonstrated the production of a 46-kDa polypeptide. When the citrate synthase-complementing fragment was cloned in pYA626 in the reverse orientation, the recombinant molecule was no longer able to complement the mutation in the citrate synthase gene and no longer produced the 46-kDa polypeptide. When the DNA fragment was cloned in the Pst I site of pHC79, so as to allow expression from the (3-lactamase promoter, the resulting recombinant failed to complement the mutation in the E. coli citrate synthase gene yet still produced the 46-kDa polypeptide, but in one-fourth the amounts than when expressed from the S. mutans asd promoter. This demonstrates that M. leprae translational sequences can be recognized by E. coli translational machinery. Promoter expression vectors can be used to obtain expression of protein antigens to be used for early diagnosis of leprosy or components of a vaccine and proteins that are targets of potential antileprosy drugs.Leprosy, an age-old chronic disease with a wide spectrum of manifestations, including gross skin disfigurement and peripheral nerve loss, afflicts over 15 million people in the world today (1). Its causative agent, Mycobacterium leprae, was shown to be associated with the disease by Gerhard Armauer Hansen in the early 1870s (2). Even so, M. leprae has been extremely difficult to study because of its inability to be cultivated in the laboratory. In the early 1960s, Shepard successfully cultivated M. leprae in the footpads of mice (3). Significant quantities of the organism became available for research upon the discovery that M. leprae produced a systemic infection in the nine-banded armadillo, Dasypus novemcinctus (4, 5).We had previously screened genomic libraries ofM. leprae DNA cloned in both plasmid and cosmid vectors and had not observed any complementation of a variety of mutations in amino acid, purine, and vitamin biosynthetic pathways or carbohydrate catabolic pathways in Escherichia coli K-12 (6). We cloned M. leprae DNA in the expression vector pYA626 and were able to demonstrate the expression of M. leprae polypeptides in minicells containing recombinant M. leprae molecules (6). In this manuscript, we describe the complementation of a mutation in the citrate synthase (EC 4.1.3.7) gene of E. coli K-12 by cloned M. leprae DNA that is expressed from the asd promoter of pYA626.

Glutamate and cyclic AMP regulate the expression of galactokinase in Mycobacterium smegmatis

“…The main reasons for this lack of information are the slow growth rate of the organism, poorly developed gene transfer systems and very little understanding of the genetic being made to the mechanisms of gene regulation in mYcobacteria* It has been shown that mYc0-bacterial promoters function poorly in Escherichia COli (Bashyam et al-9 1996 ;Clark-Curtiss et al7 1985 ; Das number of mycobacterial metabolic genes have been cloned by complementation of the mutational defects in the corresponding genes of E. coli (Anderson et al, 1993;Garbe et al, 1990;Jacobs et al, 1986;Stelandre et al, 1992) and mycobacteria (Hinshelwood & Stoker, 1992), very little is known about the regulatory mechanisms involved. More studies are needed to understand the basic features of the regulation of mycobacterial gene expression.…”

Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product

“…As many mycobacterial genes are expressed poorly in E. coli (27,57), DNA hybridization was used to search for a recA-like gene from M. tuberculosis rather than screening by functional complementation. The ability of this gene, expressed from a plasmid lac promoter, to complement the different functions of RecA in E. coli strains with various recA alleles has been investigated.…”

The contribution of Charles C. Shepard to leprosy research: from the mouse footpad model to new DNA technology

“…It was Shep's policy to retain a few strains of M. leprae by serial passage in mice, particularly as «standard» strains for drug testing. Some of these had been serially passaged in mice for [16][17][18][19][20][21][22][23][24] years , without any detectable change in pathogenicity or growth patterns in mice . Fi nally the stability and survival of M. leprae outside the body was assessed using the foot-pad model, on bacilli recovered from dried nasal discharges.…”

“…From then onwards a series of rapidly advancing technical publications by Roy & Josephine Curtiss and colleagues (19,22) have resulted on the specific genome size of M. leprae distinguishing it from other species of mycobacteria as well as genomic libraries of M. leprae resulting basically using E coli K-12 as the host vec tor. It is pleasing to see that one of these publications (22) is dedicated to Shepard .…”