Characterization of P55, a multidrug efflux pump in Mycobacterium bovis and Mycobacterium tuberculosis.

The Mycobacterium bovis P55 gene, located downstream from the gene that encodes the immunogenic lipoprotein P27, has been characterized. The gene was identical to the open reading frame of the Rv1410c gene in the genome of Mycobacterium tuberculosis H37Rv, annotated as a probable drug efflux protein. Genes similar to P55 were present in all species of the M. tuberculosis complex and other mycobacteria such as Mycobacterium leprae and Mycobacterium avium. By Western blotting, P55 was located in the membrane fraction of M. bovis. When transformed into Mycobacterium smegmatis after cloning, P55 conferred aminoglycoside and tetracycline resistance. The levels of resistance to streptomycin and tetracycline conferred by P55 were decreased in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the pump inhibitors verapamil and reserpine. M. smegmatis cells expressing the plasmid-encoded P55 accumulated less tetracycline than the control cells. We conclude that P55 is a membrane protein implicated in aminoglycoside and tetracycline efflux in mycobacteria.

WhiA, a protein of unknown function conserved among gram-positive bacteria, is essential for sporulation in Streptomyces coelicolor A3(2).

The whiA sporulation gene of Streptomyces coelicolor A3(2), which plays a key role in switching aerial hyphae away from continued extension growth and toward sporulation septation, was cloned by complementation of whiA mutants. DNA sequencing of the wild-type allele and five whiA mutations verified that whiA is a gene encoding a protein with homologues in all gram-positive bacteria whose genome sequence is known, whether of high or low G+C content. No function has been attributed to any of these WhiA-like proteins. In most cases, as in S. coelicolor, the whiA-like gene is downstream of other conserved genes in an operon-like cluster. Phenotypic analysis of a constructed disruption mutant confirmed that whiA is essential for sporulation. whiA is transcribed from at least two promoters, the most downstream of which is located within the preceding gene and is strongly up-regulated when colonies are undergoing sporulation. The up-regulation depends on a functional whiA gene, suggesting positive autoregulation, although it is not known whether this is direct or indirect. Unlike the promoters of some other sporulation-regulatory genes, the whiA promoter does not depend on the sporulation-specific sigma factor encoded by whiG.

A response regulator-like protein that functions at an intermediate stage of sporulation in Streptomyces coelicolor A3(2).

whiI is one of several loci originally described as essential for sporulation in Streptomyces coelicolor A3(2). We have characterized whiI at the molecular level. It encodes an atypical member of the response regulator family of proteins, lacking at least two of the residues strongly conserved in the conventional phosphorylation pocket. It is not adjacent to a potential sensor kinase gene. Fifteen mutant alleles of whiI were sequenced, revealing, among others, six mutations affecting conserved amino acids, several frameshift mutations and one mutation in the promoter. The whiI promoter is specifically transcribed by the sporulation-specific sigmaWhiG-containing form of RNA polymerase. Transcription of whiI is temporally controlled, reaching a maximum level coincident with the formation of spores. Further transcriptional studies suggested that WhiI is involved directly or indirectly in repressing its own expression and that of another sigmaWhiG-dependent sporulation-specific regulatory gene, whiH.

Molecular cloning and characterization of Tap, a putative multidrug efflux pump present in Mycobacterium fortuitum and Mycobacterium tuberculosis.

A recombinant plasmid isolated from a Mycobacterium fortuitum genomic library by selection for gentamicin and 2-N'-ethylnetilmicin resistance conferred low-level aminoglycoside and tetracycline resistance when introduced into M. smegmatis. Further characterization of this plasmid allowed the identification of the M. fortuitum tap gene. A homologous gene in the M. tuberculosis H37Rv genome has been identified. The M. tuberculosis tap gene (Rv1258 in the annotated sequence of the M. tuberculosis genome) was cloned and conferred low-level resistance to tetracycline when introduced into M. smegmatis. The sequences of the putative Tap proteins showed 20 to 30% amino acid identity to membrane efflux pumps of the major facilitator superfamily (MFS), mainly tetracycline and macrolide efflux pumps, and to other proteins of unknown function but with similar antibiotic resistance patterns. Approximately 12 transmembrane regions and different sequence motifs characteristic of the MFS proteins also were detected. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the levels of resistance to antibiotics conferred by plasmids containing the tap genes were decreased. When tetracycline accumulation experiments were carried out with the M. fortuitum tap gene, the level of tetracycline accumulation was lower than that in control cells but was independent of the presence of CCCP. We conclude that the Tap proteins of the opportunistic organism M. fortuitum and the important pathogen M. tuberculosis are probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.

katGI and katGII encode two different catalases-peroxidases in Mycobacterium fortuitum.

It has been suggested that catalase-peroxidase plays an important role in several aspects of mycobacterial metabolism and is a virulence factor in the main pathogenic mycobacteria. In this investigation, we studied genes encoding for this protein in the fast-growing opportunistic pathogen Mycobacterium fortuitum. Nucleotide sequences of two different catalase-peroxidase genes (katGI and katGII) of M. fortuitum are described. They show only 64% homology at the nucleotide level and 55% identity at the amino acid level, and they are more similar to catalases-peroxidases from different bacteria, including mycobacteria, than to each other. Both proteins were found to be expressed in actively growing M. fortuitum, and both could also be expressed when transformed into Escherichia coli and M. aurum. We detected the presence of a copy of IS6100 in the neighboring region of a katG gene in the M. fortuitum strain in which this element was identified (strain FC1). The influence of each katG gene on isoniazid (isonicotinic acid hydrazide; INH) susceptibility of mycobacteria was checked by using the INH-sensitive M. aurum as the host. Resistance to INH was induced when katGI was transformed into INH-sensitive M. aurum, suggesting that this enzyme contributes to the natural resistance of M. fortuitum to the drug. This is the first report showing two different genes encoding same enzyme activity which are actively expressed within the same mycobacterial strain.

Isolation by genetic labeling of a new mycobacterial plasmid, pJAZ38, from Mycobacterium fortuitum.

In a two-step mating experiment with recipient strains of Mycobacterium smegmatis, the Mycobacterium fortuitum cryptic plasmid pJAZ38 was isolated. Plasmid pJAZ38 was genetically labeled by cointegration formation mediated by the kanamycin-resistant mycobacterial transposon Tn611. The region responsible for replication of pJAZ38 was located and sequenced. This region showed homology with the Mycobacterium avium plasmid pLR7 and the Mycobacterium scrofulaceum plasmid pMSC262, a family of plasmids which have been found to be widespread throughout the mycobacteria. Further experiments showed pJAZ38 to be stably inherited in the absence of selection pressure and compatible with the most commonly used mycobacterial replicon, pAL5000. In contrast to pLR7 and pMSC262, pJAZ38 was able to replicate in M. smegmatis mc(2)155, making it a useful tool for mycobacterial genetics.

Aminoglycoside 2'-N-acetyltransferase genes are universally present in mycobacteria: characterization of the aac(2')-Ic gene from Mycobacterium tuberculosis and the aac(2')-Id gene from Mycobacterium smegmatis.

The genus Mycobacterium comprises clinically important pathogens such as M. tuberculosis, which has reemerged as a major cause of morbidity and mortality world-wide especially with the emergence of multidrug-resistant strains. The use of fast-growing species such as Mycobacterium smegmatis has allowed important advances to be made in the field of mycobacterial genetics and in the study of the mechanisms of resistance in mycobacteria. The isolation of an aminoglycoside-resistance gene from Mycobacterium fortuitum has recently been described. The aac(2')-Ib gene is chromosomally encoded and is present in all isolates of M. fortuitum. The presence of this gene in other mycobacterial species is studied here and genes homologous to that of M. fortuitum have been found in all mycobacterial species studied. In this report, the cloning of the aac(2')-Ic gene from M. tuberculosis H37Rv and the aac(2')-Id gene from M. smegmatis mc(2)155 is described. Southern blot hybridizations have shown that both genes are present in all strains of this species studied to date. In addition, the putative aac(2')-Ie gene has been located in a recent release of the Mycobacterium leprae genome. The expression of the aac(2')-Ic and aac(2')-Id genes has been studied in M. smegmatis and only aac(2')-Id is correlated with aminoglycoside resistance. In order to elucidate the role of the aminoglycoside 2'-N-acetyltransferase genes in mycobacteria and to determine whether they are silent resistance genes or whether they have a secondary role in mycobacterial metabolism, the aac(2')-Id gene from M. smegmatis has been disrupted in the chromosome of M. smegmatis mc(2)155. The disruptant shows an increase in aminoglycoside susceptibility along with a slight increase in the susceptibility to lysozyme.

Construction of a family of Mycobacterium/Escherichia coli shuttle vectors derived from pAL5000 and pACYC184: their use for cloning an antibiotic-resistance gene from Mycobacterium fortuitum.

The pSUM vectors, a family of Mycobacterium/Escherichia coli shuttle cloning vectors derived from the pSU series of vectors are described. They all contain the pACYC184 origin of replication enabling them to replicate in E. coli, the mycobacterial pAL5000 origin of replication and a kanamycin-resistance (KmR) gene which allows selection in both E. coli and mycobacteria. They carry a multiple cloning site (MCS) to facilitate cloning and the lacZ alpha reporter gene for screening inserts in E. coli. A library of total DNA from a strain of Mycobacterium fortuitum was constructed with one of these vectors, pSUM36, and a mycobacterial gentamicin-resistance (GmR) gene was cloned by selecting for GmR in Mycobacterium smegmatis.

Characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase gene from Mycobacterium fortuitum.

A novel gene encoding an aminoglycoside 2'-N-acetyltransferase (AAC) was cloned from Mycobacterium fortuitum. DNA sequencing results identified an open reading frame that we have called aac(2')-Ib encoding a putative protein with a predicted molecular mass of 24,800 Da. The deduced AAC(2')-Ib protein showed homology to the AAC(2')-Ia from Providencia stuartii. This is the second member of a subfamily of AAC(2')-I enzymes to be identified. No homology was found with other acetyltransferases, including all of the AAC(3) and AAC(6') proteins. The aac(2')-Ib gene cloned in a mycobacterial plasmid and introduced in Mycobacterium smegmatis conferred resistance to gentamicin, tobramycin, dibekacin, netilmicin, and 6'-N-ethylnetilmicin. DNA hybridization with an intragenic probe of aac(2')-Ib showed that this gene was present in all 34 strains of M. fortuitum tested. The universal presence of the aac(2')-Ib gene in M. fortuitum was not correlated with any aminoglycoside resistance phenotype, suggesting that this gene may play a role in the secondary metabolism of the bacterium.