Comparison of the Construction of Unmarked Deletion Mutations in Mycobacterium smegmatis, Mycobacterium bovis Bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by Allelic Exchange

Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organisms Mycobacterium bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains of M. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organism Mycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism.     

Saturation and specificity of the Lon protease of Escherichia coli.

Lon is an ATP-dependent protease of Escherichia coli. The lon mutation has a pleiotropic phenotype: UV sensitivity, mucoidy, deficiency for lysogenization by bacteriophage lambda and P1, and lower efficiency in the degradation of abnormal proteins. All of these phenotypes are correlated with the loss of protease activity. Here we examine the effects of overproduction of one Lon substrate, SulA, and show that it protects two other substrates from degradation. To better understand this protection, we mutagenized the sulA gene and selected for mutants that have partially or totally lost their ability to saturate the Lon protease and thus can no longer protect another substrate. Some of the SulA mutants lost their ability to protect RcsA from degradation but could still protect the O thermosensitive mutant protein (Ots). All of the mutants retained their capacity to induce cell division inhibition. It was also found that deletion of the C-terminal end of SulA affected its activity but did not affect its susceptibility to Lon. We propose that Lon may have more than one specificity for peptide cleavage.     

结核分枝杆菌中毒素-抗毒素系统的鉴定

【目的】鉴定结核分枝杆菌基因组上MazF同源蛋白基因与其上游基因是否组成毒素-抗毒素系统,阐明毒素蛋白的作用机理,并初步探讨毒素-抗毒素系统在营养缺乏时的表达调控。【方法】在大肠杆菌和耻垢分枝杆菌中将MazF同源蛋白单独表达或与其对应的抗毒素蛋白共同表达,鉴定MazF同源蛋白对细菌生长的抑制作用以及其对应的抗毒素蛋白能否消除这种生长抑制;通过体外RNA切割实验,检测MazF同源蛋白是否具有RNA切割活性;检测正常生长条件下和饥饿条件下毒素-抗毒素系统的启动子活性,探讨其在应激条件下的表达调控。【结果】结核分枝杆菌MazF同源蛋白中,Rv0659c、Rv1495和Rv1942c不具有抑制细菌生长的毒素蛋白活性,Rv1991c、Rv2801c、Rv1102c和mtPemK能够抑制细菌生长,而且它们的抑制作用可以分别被其对应的抗毒素Rv1991a、Rv2801a、Rv1103c和mtPemI解除。Rv1991c、Rv2801c和Rv1102c具有RNA切割活性,mtPemK则不能切割RNA。Rv1991a-1991c和Rv2801a-2801c系统的启动子在饥饿条件下活性显著升高。【结论】结核分枝杆菌基因组上Rv1991a-1991c、Rv2801a-2801c、Rv1103c-1102c和mtPemI-mtPemK是毒素-抗毒素系统。毒素蛋白Rv1991c、Rv2801c和Rv1102c通过切割RNA发挥抑菌或杀菌活性,mtPemK具体作用机理目前还不清楚。Rv1991a-1991c和Rv2801a-2801c系统可能参与结核分枝杆菌在营养匮乏条件下的生长调控。     

Cloning, expression, and characterization of the lon gene of Erwinia amylovora: evidence for a heat shock response.

The gene encoding the Lon protease of Erwinia amylovora has been cloned by complementation of an Escherichia coli lon mutant. Analysis of the determined nucleotide sequence of the lon gene revealed extensive homology to the nucleotide sequences of cloned lon genes from E. coli, Myxococcus xanthus, and Bacillus brevis. The predicted amino acid sequence of the E. amylovora Lon protease was 94, 59, and 54% identical to the predicted amino acid sequences of the Lon proteases of E. coli, M. xanthus, and B. brevis, respectively. The -10 and -35 promoter regions of the cloned lon gene had extensive homology to the respective consensus sequences of E. coli heat shock promoters. Promoter mapping of the lon gene located the start site 7 bases downstream of the -10 region. Cloning of the lon promoter upstream of a cat reporter gene demonstrated that expression of the E. amylovora lon gene was inducible by a heat shock. This is the first demonstration of a heat shock-regulated gene in E. amylovora. Site-directed mutagenesis of the -10 region of the lon promoter confirmed that the heat shock expression of the E. amylovora lon gene may be mediated by a sigma 32-like factor. Insertional inactivation of the E. amylovora chromosomal lon gene confirmed that the lon gene was not essential for either vegetative growth or infection of apple seedlings. E. amylovora lon mutants had increased sensitivity to UV irradiation and elevated levels of extracellular polysaccharide, suggesting comparable roles for the Lon proteases in both E. amylovora and E. coli.     

The Influence of Mycobacterium tuberculosis Sigma Factors on the Promotion Efficiency of ptpAt Promoter in Mycobacterium smegmatis

It was found in a previous study that Mycobacterium tuberculosis protein tyrosine phosphatase ptpAt promoter is a highly active promoter in slow-growing species of mycobacteria, such as M. tuberculosis and M. bovis BCG, but inert in fast-growing mycobacterial species, such as M. smegmatis. This difference is presumed to be due to the differences between sigma factors systems of slow-growing pathogenic mycobacteria and the fast-growing saprophyte M. smegmatis. Therefore, we constructed a series of plasmids, named pOLYG-13x, which can express various M. tuberculosis sigma factors and also contain a P(ptpAt)-gfp reporter gene construct. By inducing different sigma factor genes of M. tuberculosis in M. smegmatis, we were able to explore the influences of various sigma factors on the expression efficiency of the ptpAt promoter. The result show that of the 10 sigma factors evaluated, only sigF and sigL were able to weakly drive the ptpAt promoter in M. smegmatis and other sigma factors were unable to drive the promoter.     

A genomic view of sugar transport in Mycobacterium smegmatis and Mycobacterium tuberculosis

We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast- and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively.     

The mycosins of Mycobacterium tuberculosis H37Rv: a family of subtilisin-like serine proteases

There is little information regarding the role of proteolysis in Mycobacterium tuberculosis and no studies on the potential involvement of proteases in the pathogenesis of tuberculosis. We identified five M. tuberculosis genes (mycP1-5) that encode a family of serine proteases (mycosins-1 to 5), ranging from 36 to 47% identity. Each protein contains a catalytic triad (Asp, His, Ser) within highly conserved sequences, typical of proteases of the subtilisin family. These genes are also present in M. bovis BCG and other virulent mycobacteria, but only one homologue (mycP3) was detected in M. smegmatis. The mycosins have N-terminal signal sequences and C-terminal transmembrane anchors, and the localisation of the mycosins to the membrane/cell wall was verified by Western blot analysis of heterologously expressed proteins in cellular fractions of M. smegmatis. In M. tuberculosis, all the mycosins were expressed constitutively during growth in broth. Mycosins-2 and 3 were also expressed constitutively in M. bovis BCG, but no expression of mycosin-1 was detected. Mycosin-2 was modified by cleavage in all three mycobacterial species. The multiplicity and constitutive expression of these proteins suggests that they have an important role in the biology of M. tuberculosis.     

Instability of the acetamide-inducible expression vector pJAM2 in Mycobacterium tuberculosis

The Escherichia coli-mycobacterium shuttle vector pJAM2 has been used to inducibly express genes in mycobacteria. The vector carries the promoter region from the highly inducible acetamidase gene of Mycobacterium smegmatis which is used to drive expression of heterologous genes. We used pJAM2 to over-express the Mycobacterium tuberculosis gene Rv2868c, a homologue of gcpE. In M. smegmatis the plasmid was stable, but the promoter region was readily deleted when the parental vector or recombinant plasmids were transformed into M. tuberculosis. We mapped the deletion by sequencing and found that it encompassed the entire acetamidase promoter and adjacent sequence totalling approximately 7.3 kb and occurred very soon after introduction into M. tuberculosis. This is the first report of instability of a vector carrying the acetamidase promoter in M. tuberculosis.     

The Thermoplasma acidophilum Lon protease has a Ser-Lys dyad active site.

A gene with significant similarity to bacterial Lon proteases was identified during the sequencing of the genome of the thermoacidophilic archaeon Thermoplasma acidophilum. Protein sequence comparison revealed that Thermoplasma Lon protease (TaLon) is more similar to the LonB proteases restricted to Gram-positive bacteria than to the widely distributed bacterial LonA. However, the active site residues of the protease and ATPase domain are highly conserved in all Lon proteases. Using site-directed mutagenesis we show here that TaLon and EcLon, and probably all other Lon proteases, contain a Ser-Lys dyad active site. The TaLon active site mutants were fully assembled and, similar to TaLon wild-type, displayed an apparent molar mass of 430 kDa upon gelfiltration. This would be consistent with a hexameric complex and indeed electron micrographs of TaLon revealed ring-shaped particles, although of unknown symmetry. Comparison of the ATPase activity of Lon wild-type from Thermoplasma or Escherichia coli with respective protease active site mutants revealed differences in Km and V values. This suggests that in the course of protein degradation by wild-type Lon the protease domain might influence the activity of the ATPase domain.     

Investigating the function of the putative mycolic acid methyltransferase UmaA: divergence between the Mycobacterium smegmatis and Mycobacterium tuberculosis proteins

Mycolic acids are major and specific lipid components of the cell envelope of mycobacteria that include the causative agents of tuberculosis and leprosy, Mycobacterium tuberculosis and Mycobacterium leprae, respectively. Subtle structural variations that are known to be crucial for both their virulence and the permeability of their cell envelope occur in mycolic acids. Among these are the introduction of cyclopropyl groups and methyl branches by mycolic acid S-adenosylmethionine-dependent methyltransferases (MA-MTs). While the functions of seven of the M. tuberculosis MA-MTs have been either established or strongly presumed nothing is known of the roles of the remaining umaA gene product and those of M. smegmatis MA-MTs. Mutants of the M. tuberculosis umaA gene and its putative M. smegmatis orthologue, MSMEG0913, were created. The lipid extracts of the resulting mutants were analyzed in detail using a combination of analytical techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proton nuclear magnetic resonance spectroscopy, and chemical degradation methods. The M. smegmatis mutants no longer synthesized subtypes of mycolates containing a methyl branch adjacent to either trans cyclopropyl group or trans double bond at the "proximal" position of both alpha- and epoxy-mycolates. Complementation with MSMEG0913, but not with umaA, fully restored the wild-type phenotype in M. smegmatis. Consistently, no modification was observed in the structures of mycolic acids produced by the M. tuberculosis umaA mutant. These data proved that despite their synteny and high similarity umaA and MSMEG0913 are not functionally orthologous.     

A protein secretion pathway critical for Mycobacterium tuberculosis virulence is conserved and functional in Mycobacterium smegmatis

The Snm protein secretion system is a critical determinant of Mycobacterium tuberculosis virulence. However, genes encoding components of this pathway are conserved among all mycobacteria, including the nonpathogenic saprophyte Mycobacterium smegmatis. We show that the Snm system is operational in M. smegmatis and that secretion of its homologous ESAT-6 and CFP-10 substrates is regulated by growth conditions. Importantly, we show that Snm secretion in M. smegmatis requires genes that are homologous to those required for secretion in M. tuberculosis. Using a gene knockout strategy in M. smegmatis, we have also discovered four new gene products that are essential for Snm secretion, including the serine protease mycosin 1. Despite the evolutionary distance between M. smegmatis and M. tuberculosis, the M. smegmatis Snm system can secrete the M. tuberculosis ESAT-6 and CFP-10 proteins, suggesting that substrate recognition is also conserved between the two species. M. smegmatis, therefore, represents a powerful system to study the multicomponent Snm secretory machine and to understand the role of this conserved system in mycobacterial biology.     

The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein

Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF) sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress.     

Regulatory role of C-terminal residues of SulA in its degradation by Lon protease in Escherichia coli

The SulA protein is a cell division inhibitor in Escherichia coli, and is specifically degraded by Lon protease. To study the recognition site of SulA for Lon, we prepared a mutant SulA protein lacking the C-terminal 8 amino acid residues (SA8). This deletion protein was accumulated and stabilized more than native SulA in lon(+) cells in vivo. Moreover, the deletion SulA fused to maltose binding protein was not degraded by Lon protease, and did not stimulate the ATPase or peptidase activity of Lon in vitro, probably due to the much reduced interaction with Lon. A BIAcore study showed that SA8 directly interacts with Lon. These results suggest that SA8 of SulA was recognized by Lon protease. The SA8 peptide, KIHSNLYH, specifically inhibited the degradation of native SulA by Lon protease in vitro, but not that of casein. A mutant SA8, KAHSNLYH, KIASNLYH, or KIHSNAYH, also inhibited the degradation of SulA, while such peptides as KIHSNLYA did not. These results show that SulA has the specified rows of C-terminal 8 residues recognized by Lon, leading to facilitated binding and subsequent cleavage by Lon protease both in vivo and in vitro.     

ATP-dependent degradation of SulA, a cell division inhibitor, by the HslVU protease in Escherichia coli.

HslVU is an ATP-dependent protease consisting of two multimeric components, the HslU ATPase and the HslV peptidase. To gain an insight into the role of HslVU in regulation of cell division, the reconstituted enzyme was incubated with SulA, an inhibitor of cell division in Escherichia coli, or its fusion protein with maltose binding protein (MBP). HslVU degraded both proteins upon incubation with ATP but not with its nonhydrolyzable analog, ATPgammaS, indicating that the degradation of SulA requires ATP hydrolysis. The pulse-chase experiment using an antibody raised against MBP-SulA revealed that the stability of SulA increased in hsl mutants and further increased in lon/hsl double mutants, indicating that SulA is an in vivo substrate of HslVU as well as of protease La (Lon). These results suggest that HslVU in addition to Lon plays an important role in regulation of cell division through degradation of SulA.     

A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease.

A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease has been identified. This pin (proteolysis inhibition) gene was selected for its ability to support plaque formation by a lambda Ots vector at 40 degrees C. Southern blot experiments indicated that this T4 gene is included within the 4.9-kilobase XbaI fragment which contains gene 49. Subcloning experiments showed that T4 gene 49.1 (designated pinA) is responsible for the ability of the Ots vector to form plaques at 40 degrees C. Deficiencies in Lon protease activity are the only changes known in E. coli that permit lambda Ots phage to form plaques efficiently at 40 degrees C. lon+ lysogens of the lambda Ots vector containing pinA permitted a lambda Ots phage to form plaques efficiently at 40 degrees C. Furthermore, these lysogens, upon comparison with similar lysogens lacking any T4 DNA, showed reduced levels of degradation of puromycyl polypeptides and of canavanyl proteins. The lon+ lysogens that contained pinA exhibited other phenotypic characteristics common to lon strains, such as filamentation and production of mucoid colonies. Levels of degradation of canavanyl proteins were essentially the same, however, in null lon lysogens which either contained or lacked pinA. We infer from these data that the T4 pinA gene functions to block Lon protease activity; pinA does not, however, appear to block the activity of proteases other than Lon that are involved in the degradation of abnormal proteins.