Identification of genes involved in the sequestration of iron in mycobacteria: the ferric exochelin biosynthetic and uptake pathways

Mycobacteria produce two siderophores, mycobactin and exochelin. Mycobacterium smegmatis mutants defective in the production of exochelin were isolated using agar medium containing chrome azural S for the sensitive detection of siderophores. Cosmids of genomic libraries from M. smegmatis and Mycobacterium bovis BCG were screened for complementation of the mutation. Subcloning of the complementing M. smegmatis cosmid identified a 4.3 kb fragment required for restoring exochelin biosynthesis. Sequencing of the DNA revealed four open reading frames whose genes were named fxuA. fxuB, fxuC, and fxbA. FxuA, FxuB, and FxuC share amino acid sequence homology with the iron permeases FepG, FepC. and FepD from Escherichia coli. respectively. Deletion analysis identified fxbA as the gene required to restore exochelin biosynthesis in our mutant. Although fxbA does not share amino acid sequence homology with any of the published sequences for siderophore biosynthetic genes, it does show limited homology with the phosphoribosyl-glycineamide formyltransferases (GAR enzymes) and methionyl-tRNA formyltransferase over a limited region of the sequence, suggesting that fxbA may code for an enzyme which adds a formyl group in the synthesis of exochelin. A fusion of fxbA with the E. coli lacZ gene demonstrated regulation of gene expression by iron. The ability of M. smegmatis mutants to produce mycobactin in the absence of exochelin supports the hypothesis that exochelin is not a precursor of mycobactin and suggests that the siderophores have independent biosynthetic pathways. In addition, complementation of the M.     

Effect of IS900 Gene of Mycobacterium paratuberculosis on Mycobacterium smegmatis

Mycobacterium paratuberculosis is mycobactin dependent and contains multiple copies of the IS900 gene that encodes for p43 (46.5K protein). The correlation between the two characteristics has been investigated. A 3.2-kb BamHI fragment from M. paratuberculosis containing the 1.451 kb IS900 gene was cloned in Escherichia coli and Mycobacterium smegmatis with pcDNA II and pNEZ6.3 plasmids, respectively. Surprisingly, the recombinant M. smegmatis grew poorly and slower in 7H9 broth supplemented with OADC (12 day) compared with M. smegmatis wild type or to M. smegmatis transformed with pNEZ6.3 (2 day). The growth rate of the recombinant M. smegmatis was restored by the addition of 2.4 μM ferric mycobactin J to the media. There was no effect on the growth rate of E. coli recombinants. Western blot analysis with p43-specific anti-peptide antibodies resulted in the expression of 46.5K and a cleaved form of 33.5K protein bands in the recombinant E. coli. There was no expression in the recombinant M. smegmatis. A lower expression of 33.5K protein band was detected in the native M. paratuberculosis protein. The nucleotide sequence of the 3.2-kb fragment confirmed the presence of p43-encoded ORF. There was no additional encoding sequence in the fragment. This suggests that the IS900 gene and/or its encoding products are involved in mycobactin dependency and possibly the slow growth rate of M. paratuberculosis. Received: 12 May 1998 / Accepted: 6 July 1998     

Cell wall proteome analysis of <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis> strain MC2 155

Background  

The usually non-pathogenic soil bacterium Mycobacterium smegmatis is commonly used as a model mycobacterial organism because it is fast growing and shares many features with pathogenic mycobacteria. Proteomic studies of M. smegmatis can shed light on mechanisms of mycobacterial growth, complex lipid metabolism, interactions with the bacterial environment and provide a tractable system for antimycobacterial drug development. The cell wall proteins are particularly interesting in this respect. The aim of this study was to construct a reference protein map for these proteins in M. smegmatis.     

The studies of ParA and ParB dynamics reveal asymmetry of chromosome segregation in mycobacteria

Active segregation of bacterial chromosomes usually involves the action of ParB proteins, which bind in proximity of chromosomal origin (oriC) regions forming nucleoprotein complexes – segrosomes. Newly duplicated segrosomes are moved either uni‐ or bidirectionally by the action of ATPases – ParA proteins. In Mycobacterium smegmatis the oriC region is located in an off‐centred position and newly replicated segrosomes are segregated towards cell poles. The elimination of M. smegmatis ParA and/or ParB leads to chromosome segregation defects. Here, we took advantage of microfluidic time‐lapse fluorescent microscopy to address the question of ParA and ParB dynamics in M. smegmatis and M. tuberculosis cells. Our results reveal that ParB complexes are segregated in an asymmetrical manner. The rapid movement of segrosomes is dependent on ParA that is transiently associated with the new pole. Remarkably in M. tuberculosis, the movement of the ParB complex is much slower than in M. smegmatis, but segregation as in M. smegmatis lasts approximately 10% of the cell cycle, which suggests a correlation between segregation dynamics and the growth rate. On the basis of our results, we propose a model for the asymmetric action of segregation machinery that reflects unequal division and growth of mycobacterial cells.     

乙酰化修饰降低Ku蛋白的DNA结合活性

【目的】研究乙酰化修饰对Ku蛋白活性的影响。【方法】利用耻垢分枝杆菌为表达菌株,转入Ku蛋白表达质粒,纯化具有乙酰化修饰的Ku蛋白和无乙酰化的Ku蛋白突变体,比较两类蛋白的生化活性;分析氧化压力和酸性环境下耻垢分枝杆菌细胞内Ku蛋白乙酰化水平的变化。【结果】Ku蛋白过量表达的耻垢分枝杆菌比转入空质粒的对照菌株生长缓慢;乙酰化Ku蛋白比未发生乙酰化Ku蛋白修复断裂DNA的活性降低、DNA结合活性降低;氧化压力和酸性压力环境下,耻垢分枝杆菌细胞内Ku蛋白数量降低,乙酰化Ku蛋白数量变化不大。【结论】乙酰化修饰能够调节Ku蛋白的DNA结合活性,从而调节非同源末端连接修复系统的活性;Ku蛋白乙酰化程度升高是耻垢分枝杆菌对不良生长环境的反应。     

Polyphosphate Deficiency Affects the Sliding Motility and Biofilm Formation of <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

Inorganic polyphosphate (polyP) is a ubiquitous linear polymer of hundreds of orthophosphate (Pi) residues linked by ATP-like, high-energy, phosphoanhydride bonds. The gene Rv1026 in Mycobacterium tuberculosis encodes a putative exopolyphosphatase which progressively hydrolyzes the terminal residues of polyP to liberate Pi. Rv1026 was cloned into the expressive plasmid pMV261. The resulting plasmid pRv1026 and the plasmid pMV261 were transformed into M. smegmatis strain mc²155 by electroporation. The recombinant M. smegmatis (pRv1026) showed relatively decreased polyP concentration and a phenotype different from the M. smegmatis (pMV261) in sliding motility and biofilm formation. The surfactant Tween 80 can enhance this effect on the sliding motility and biofilm formation of M. smegmatis. There are four different peaks between the gas chromatography of cellular wall fatty acid of the M. smegmatis (pRv1026) and the M. smegmatis (pMV261). These results indicate that polyP deficiency can affect the fatty acid composition of cellular wall and these alteration of cell wall might elucidate the reductive ability of strains to slide and form biofilm. This investigation provides novel recognition about the role of Rv1026, which provides novel clues for further study on the physiological role of Rv1026 in M. tuberculosis.     

Upregulation of a histone-like protein in dormant Mycobacterium smegmatis

The aerobic saprophyte Mycobacterium smegmatis, like its pathogenic counterpart M. tuberculosis, has the ability to adapt to anaerobiosis by shifting down to a dormant state. Here, we report the identification and molecular genetic characterisation of the first dormancy-induced protein in M. smegmatis. Comparative SDS-polyacrylamide gel electrophoresis of protein extracts of aerobically growing and dormant anaerobic M. smegmatis cultures revealed the upregulation of a 27-kDa protein in the dormant state. Peptide sequencing showed that the induced protein is a homologue of the histone-like protein Hlp, predicted by the M. tuberculosis genome project. The corresponding hlp gene was cloned from M. smegmatis and sequenced. Disruption of the hlp gene eliminated the histone-like protein but did not affect the viability of the dormant culture. Received: 3 June 1998 / Accepted: 22 September 1998     

Effect of 2,3-Dinitrilo-1,4-dithia-9,10-anthraquinone onMycobacterium smegmatis

2,3-Dinitrilo-1,4-dithia-9,10-anthraquinone (DDA) is an effective inhibitor of respiration of intact cells ofMycobacterium smegmatis in the presence of glucose, glycerol, pyruvate, acetate and other citric acid cycle intermediates or substrates associate d with this cycle (glutamate, asparagine). DDA inhibits the incorporation of both¹⁴C-leucine and¹⁴C-adenine into appropriate macromolecules ofM. smegmatis (TCA-precipitable fractions), and causes a drop in the incorporated activity ofU-¹⁴C-glycine or its degradation products in all the cell fractions studied (lipids, RNA, DNA, proteins). DDA suppresses the growth ofM. smegmatis probably through an interference with the cell energy-carbon metabolism.     

Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis

The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.     

Translation enhancement by optimized downstream box sequences in <Emphasis Type="Italic">Escherichia coli </Emphasis>and<Emphasis Type="Italic"> Mycobacterium smegmatis</Emphasis>

The effect on translation of downstream box sequences optimized for binding to Mycobacterium smegmatis and Escherichia coli 16S rRNA in the absence of a Shine--Dalgarno (SD) region was investigated. The relative translational efficiency of each construct in either M. smegmatis or E. coli was determined. Eradication of the SD region in the absence of a downstream box abolished the translation activity. In contrast, optimized downstream box constructs resulted in a 13- and 18-fold increase in protein synthesis, relative to non-optimized DB controls in E. coli and M. smegmatis, respectively.     

Roles of the three Mycobacterium smegmatis katG genes for peroxide detoxification and isoniazid susceptibility

Three different katG sequences (katGI, katGII and katGIII) were identified in the Mycobacterium smegmatis genome. The contributions of the three katG genes to survival of the bacterium were examined by constructing disruptants of these three genes. The katGIII sequence did not produce a functional catalase‐peroxidase. Analyses of peroxidase activity and mRNA expression revealed that in wild type M. smegmatis, expression dominance between KatGI and KatGII was switched in the exponential and stationary growth phases. Susceptibility of the M. smegmatis gene disruptants to hydrogen peroxide (H₂O₂) was tested in two growth phases. In the exponential phase, the katGI‐null strain was more susceptible to H₂O₂ than the katGII‐null strain, indicating that KatGI plays a more important role in survival than KatGII in this growth phase. In contrast, in the stationary phase, growth of the katGII‐null strain was inhibited at lower concentrations of H₂O₂. These results suggest that M. smegmatis has two types of catalase‐peroxidases, expressions of which are controlled under different gene regulatory systems. Isoniazid (INH) susceptibilities of the katG‐null strains were also examined and it was found that katGI is a major determinant of M. smegmatis susceptibility to INH.

     

Metabolism of cyclic AMP in non-pathogenic Mycobacterium smegmatis

The intracellular concentration of cyclic AMP reached a maximum in 3.5-day old cultures of Mycobacterium smegmatis grown in the presence of glycerol as the main source of carbon. Glucose-grown cells exhibited decreased cyclic AMP levels at all stages of growth. When M. smegmatis cells were incubated with various metabolites, pyruvate increased whereas glucose, citric acid, succinic acid and lactic acid decreased intracellular cyclic AMP levels. No cyclic AMP was detected in the incubation medium. The presence of a cyclic AMP-binding protein was demonstrated in cellfree extracts of M. smegmatis.     

Transformation of distinct mycobacterial species by shuttle vectors derived from theMycobacterium fortuitum pAL5000 plasmid

Mycobacterium tuberculosis H37Ra,M. smegmatisATCC 607,M. smegmatis MC²155,M. aurum A +,M. aurum A11, and one representative strain ofM. flavescens were transformed by electroporation with plasmid pMY10 and cosmid pDC100. Plasmid pMY 10 contained the origin of replication of pAL5000, the origin of replication of pBR322, a kanamycin resistance gene, and the origin of transfer of the Inc plasmid RK2; the cosmid pDC100 contained the pHC79 SS cosmid, the origin of replication of pAL5000, and a kanamycin resistance gene. The efficiency of transformation varied with the recipient cells used and was in decreasing order: 7×10⁵ forM. smegmatis MC²155, 6×10³ forM. tuberculosis H37Ra, 10³ forM. aurum, 50 forM. smegmatis ATCC 607, and 5 forM. flavescens. A rapid protocol for plasmid extraction from mycobacteria was developed.The satisfactory transformation of the nonvirulentM. tuberculosis strain H37Ra was of interest for future studies on cloning of virulence genes, while the satisfactory transformation ofM. aurum was of interest for future studies on the genetics of drug resistance because these bacteria are sensitive to drugs specifically used in the treatment of tuberculosis and leprosy. However, neither vector was stably maintained inM. smegmatis, indicating that further investigations are still necessary to resolve this difficulty.     

L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria

Recombinant bacteriophages provide efficient delivery systems for introducing reporter genes into specific bacterial hosts. We have constructed mycobacteriophage L5 recombinants carrying the firefly luciferase gene inserted into the tRNA region of the phage genome. Infection of Mycobacterium smegmatis by these phages results in expression of the luciferase gene and light emission. Fortuitously, the luciferase gene is expressed continuously in lysogens surviving infection. Synthesis of luciferase from a mycobacterial promoter created by cloning enables the detection of extremely small numbers of M. smegmatis cells. These reporter phages can be used to discriminate between drug-sensitive and drug-resistant strains of M. smegmatis, and may provide tools for the rapid identification and classification of antimycobacterial agents.     

Presence of mycobacteriophage I3-like DNA sequences in the genome of its host Mycobacterium smegmatis

The genomes of phage I3 and its host Mycobacterium smegmatis have been compared. From thermal melting studies the GC contents of DNA from mycobacteriophage I3 and its host M. smegmatis were found to be 66%. A new method, based only on the initial rates of reassociation, has been developed for calculating the DNA homology. Analysis of DNA reassociation kinetics suggested the presence of one equivalent of the phage I3 genome within the M. smegmatis genome. Southern analysis revealed the presence of almost all of the phage I3 specific sequences within the host genome.