Phosphate is an essential but often growth-limiting nutrient for bacteria. At low concentrations of phosphate in the growth medium, bacteria induce high-affinity uptake systems for phosphate, and this is usually the ABC-type phosphate specific transport system Pst. In the fully sequenced genomes of pathogenic species of mycobacteria, several copies of the genes encoding for the Pst system (pstSCAB) have been identified and some of these genes have been shown to be virulence factors. The reasons for the presence of multiple copies of pst genes in pathogenic mycobacteria are not understood, and phosphate transport by these bacteria, as well as the gene regulation involved, is poorly characterised. The fast-growing M. smegmatis contains only a single copy of the pst operon, but we recently identified a gene locus containing three genes, phnDCE, which encode for a putative ABC-type phosphate/phosphonate transport system, and a gene, phnF, which encodes for a putative transcriptional regulator of the HutC subfamily of GntR like regulators. To identify a function for the PhnDCE transport system and to characterise high-affinity phosphate transport in M. smegmatis, we created allelic exchange mutants in phnD and pstS, as well as a phnD pstS double deletion mutant. All three mutants failed to grow in minimal medium containing 10 mM phosphate, while the wildtype was able to grow in the presence of micromolar phosphate concentrations. No differences were observed in complex growth medium. Steady-state levels of [��P]-phosphate uptake were approximately 25% lower in all mutant strains as compared to the wildtype. Kinetics of phosphate uptake in the wildtype strain when grown at low phosphate concentrations (50 [mu]M P[i]) were biphasic, suggesting the presence of two inducible transport systems with apparent K[m] values of 16 [mu]M P[i] and 64 [mu]M P[i], respectively. Analysis of the kinetics of phosphate transport in the mutant strains led us to the proposition that the Pst system has an apparent Km value of ca. 16 [mu]M P[i], and the Phn system has an apparent Km of ca. 60 [mu]M P[i]. A third inducible phosphate transport system, which was active in the double mutant strain, had an apparent K[m] of ca. 90 [mu]M P[i]. Uptake of phosphate in all strains was not inhibited by the presence of excess phosphonates or phosphite, suggesting that all three transport systems were specific for phosphate. The study of phosphate transport in the presence of various metabolic inhibitors revealed that uptake by the Phn and Pst systems is driven by ATP-hydrolysis, consistent with ABC-type transport, while the third, unidentified transport system may be driven by the proton motive force. We showed that phnDCE formed an operon, and that the promoter area of the operon lies within 200 bp of the start of phnD. To investigate the regulation of the phn and pst genes, β-galacosidase activities of strains carrying transcriptional lacZ-fusions of the pstSCAB, phnDCE and phnF promoter areas, and levels of mRNA of the phn and pst genes were studied. All genes were induced when phosphate concentrations fell below a threshold value of 30 [mu]M, which coincided with a shift in the growth characteristics of M. smegmatis. Expression of the pst operon appeared to be controlled directly by the PhoPR two-component regulatory system, while the phn operon may be under direct or indirect control by PhoPR. To identify a role for PhnF in the regulation of phn gene expression, we created a phnF deletion mutant. PhnF appeared to repress transcription of phnDCE and phnF under phosphate-replete conditions. We identified two putative binding sequences for PhnF in the intergenic region between phnD and phnF with the sequence TGGTATAGACCA, which is similar to the proposed recognition consensus for HutC-like transcriptional regulators. Using site-directed mutagenesis of these sequences, we demonstrated that they are required for the repression of phnDCE and phnF. To prove PhnF binding to these potential binding sites, we attempted to express the M. smegmatis PhnF protein in E. coli, but could not obtain soluble recombinant protein. Electrophoretic mobility shift assays of the phnDCE promoter fragment using cell-free crude extracts of M. smegmatis were not successful. We propose that Pst and Phn both constitute high-affinity phosphate specific transport systems of M. smegmatis, and that a third inducible phosphate transport system is present in this bacterium. PhnF is required for repression of phnDCE and phnF transcription under phosphate-replete conditions, while induction of the pst operon, and possibly the phn operon, under phosphate-limited conditions involves the PhoPR system.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2010. / Title from PDF title page (viewed on June 28, 2010). Includes bibliographical references.

Transcription is a major step in expression of genes of a given organism. Due to environmental constrains this step must be regulated in the favour of the sustenance and growth of the organism. Here comes the relevance of transcription factors, mostly proteins which regulate transcription. One such important group of transcription factors is the zinc finger proteins. It is well known that in eukaryotes the C2H2 zinc finger domain containing proteins are the largest group of transcription factors while in prokaryotes the largest group of transcription factors are represented by helix-turn-helix motif containing proteins. Till now only two C2H2 zinc finger domain proteins-Ros and Muc have been found in alpha proteobacteria which are also transcription factors. In eukaryotes the second largest group of zinc finger proteins have their zinc ion coordinated by four cysteine residues- the C4 zinc finger proteins. They make the nuclear hormone receptor superfamily of proteins. They have also been shown to act as transcription factors. But in eubacteria no such proteins have been described in details except an isolated report of crystal structure of a C-terminal zinc finger domain protein- Jann_2411 from Jannaschia sp. Though a lot of transcription factors have been described in mechanistic details in Escherichia coli and Bacillus subtilis, the list of well described mycobacterial transcription factors is short. Given this fact and the lack of any known zinc finger domain transcription factor in actinobacteria we wanted to see whether M. smegmatis genome also encode any homologue of Jann_2411 and if does whether they have ability to modulate transcription. To meet our aim we did BLASTP search against the genome of M. smegmatis using Jann_2411 as query. We found four C-terminal zinc finger domain proteins –Msmeg_0118. Msmeg_3613, Msmeg_3408 and Msmeg_1531, which we named as Mycobacterial single zinc finger protein (Mszfp) and numbered- Mszfp1, Mszfp2, Mszfp3 and Mszfp4, respectively. Mszfp1 and Mszfp2 were chosen for study as they were the top most hits. In this thesis:- Chapter1 introduces zinc finger proteins, transcription and several levels of control of transcription process in eubacteria. In chapter2 we characterised Mszfp1 biophysically and probed its secondary structure content and oligomeric state in the native and demetallated conditions. We have also shown that this conserved hypothetical protein is expressed throughout the growth phase of M. smegmatis, regulated by SigA and SigB. We have also showed that Mszfp1 is a DNA binding protein in the native state and the demetallated protein has altered DNA binding ability. It was noted that on over expression Mszfp1 affects colony morphology and biofilm forming ability, of M. smegmatis. In chapter3 the ability of Mszfp1 to bind to RNA polymerase of M. smegmatis has been explored. It was found that Mszfp1 can activate transcription by interacting with CTD/NTD of α subunit and domain 4 of σA like CRP on type II CRP activated promoter. In chapter4 similar to Mszfp1 the biophysical study of Mszfp2 has been carried out. It was found that Mszfp2 is also a predominantly alpha helical protein with oligomeric structure having DNA binding ability. Similar to Mszfp1 Mszfp2 on over expression changes the colony morphology. Chapter5 deals with the RNA polymerase binding ability of Mszfp2 and its ability to activate transcription by interacting with CTD/NTD of α subunit but not the σA. In chapter6 we have presented a glimpse of the possible biophysical properties of Mszfp3 and Mszfp4 and given a snapshot of distribution of homologues of Mszfps among other actinobacteria. We have also put forward a hypothesis about the origin of C4 and CCHC zinc finger domains. Chapter7 is the summary of the work embedded in the earlier chapters. In Appendix I is described the making of a bacteria (Bacillus licheniformis) driven heat engine. Appendix II describes an effort to study the visco-elastic properties of Mycobacterium smegmatis cells.

No description available.

Chemotherapy of tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (M. tuberculosis), is successful against actively-growing bacilli but ineffective against dormant/persistent organisms, found mainly in a protective lipid-laden granuloma, possibly necessitating the use of lipophilic antibiotics. In vitro, these bacilli are encased in lipid-rich biofilms. In this study, the antimycobacterial activity of one such agent, clofazimine, and its nanoparticle formulation, have been investigated against Mycobacterium smegmatis (M. smegmatis), as a surrogate for M. tuberculosis, by determining the bacteriostatic and bactericidal activities of the native (NC) and spray-dried (SDC) preparations of this agent on planktonic and biofilm populations, as well as their effects on biofilm formation and its lipid compositions, specifically free mycolic acid (FM) content. Both preparations were comparable, being bacteriostatic for rapidly-proliferating bacilli, bactericidal for slow-growing, biofilm-producing sessile bacteria, but ineffective against non-replicating, biofilm-encased M. smegmatis organisms. However, similar studies in M. tuberculosis are required. / Dissertation (MSc)--University of Pretoria, 2013. / Immunology / Unrestricted

<p>Normal healthy people are not susceptible to tuberculosis (TB) but immunocompromised and HIV positive patients are at high risk of TB. The treatment regimen (rifampin, isoniazid and amikacin) for TB patients is 6-9 months for normal patients but if <i>Mycobacterium tuberculosis</i> (Mtb) becomes multidrug resistant, it takes 20-30 months to treat. According to the World Health Organization in 2018, there were about half a million new cases among which 78% were multidrug resistant TB. This antibiotic resistance is due in part to its ability to survive in the macrophage in our body by entering a non-replicating persistent state. Mtb also contains efflux pumps that increase antibiotic tolerance by pumping out the drugs. Therefore, if the efflux pump activity can be blocked by using efflux pump inhibitors, then it might increase antibiotic susceptibility of the pathogen. In our study, we used <i>Mycobacterium smegmatis</i> (Msm) as a model organism for Mtb and subjected it to a combination of three stresses (low oxygen, low pH and low nutrients) that mimic the physiological stresses in the human body and report that these conditions produced a non-replicating state in Msm. This is the first report of the use of this combination of stresses to produce a non-replicating state in Msm. Our results show that non-replicating Msm became completely tolerant to isoniazid and displayed increased tolerance to rifampin and clarithromycin by nearly 2-fold when compared to log-phase cells. Moreover, the efflux pump inhibitor verapamil decreased the antibiotic tolerance of the nonreplicating Msm to the antibiotics by 6-10 fold and the efflux pump inhibitor piperine decreased tolerance to the antibiotics by 2-4 fold. Also, in this study we attempted to construct a gene knockout mutant lacking two potential ATP-binding cassette transporters to study their functions as drug exporters. However, we were unable to obtain homologous recombination mutants. Further studies on efflux pump inhibitors could potentially enable greater understanding of antibiotic tolerance mechanisms in non-replicating, drug tolerant Mtb and enable the development of novel therapies that shorten treatment time for tuberculosis.</p>

<p>  </p> <p>Phages are viruses that infect bacteria and use its host machinery to replicate. They are one of the most ubiquitous and diverse biological entities in the biosphere with a long evolutionary history. There is renewed interest in phage therapeutics due to increased levels of antimicrobial resistance (AMR) and decreased numbers of newly developed antibiotics. Additionally, phages have applications in food safety, water quality, biocontrol, and vaccines. While many phage genomes have been sequenced and annotated, proteomic and lipidomic profiles of phage have barely been explored. The aim of this research is to measure the protein and lipid dynamics of phages with different infection cycles at different timepoints in the host’s growth cycle using mass spectrometry (MS). The data produced from this experiment can verify genomic annotation, discover novel proteins and lipids, and inspire future mechanistic and functional studies. For this study, Krili and PotatoSplit, lytic cluster O and lysogenic cluster A3 mycobacteriophages, were used to infect <em>Mycobacterium smegmatis</em> during the mid-exponential growth stage. Samples were taken in triplicate at hours 0.5 (immediately after infection), 3 (early exponential), 7 (late exponential), and 10 (early stationary). The samples were washed, mechanically lysed, quantified on BCA assay, lipid extracted, protein digested and desalted. For MS analysis, the samples were analyzed using a HPLC-ESI-MS/MS. Protein data was processed using MaxQuant and lipid data using MS-DIAL and MS-FINDER. Normalized data was statistically analyzed on MetaboAnalyst, correlated on JMP, and functionally analyzed using DAVID. The results showed that the protein and lipid expression of both phages differed with time in comparison to the control (uninfected <em>Mycobacterium smegmatis</em>) and to each other. Additionally, phage protein-lipid correlations suggest simultaneous phage infection and bacterial defense mechanisms particularly during late infection. Temporal and correlation data produced from this experiment can be used for future targeted -omics research. As one of the first experiments of its kind, the MS workflow used in this study can provide a scaffold for future phage temporal proteomic and lipidomic analysis. </p>

Despite the fact that E. coli supports limited posttranslational modification, this bacterium has been universally used as the expression system of choice. Expression of modified proteins in E. coli may lead to expression of recombinant proteins that lack essential immunomodulatory or catalytic components essentials for infectious processes. Previously in our laboratory, pMptb#28 plasmid containing a 4.8 kb insert from M. paratuberculosis has been identified which expressed 16 kDa recombinant protein in E. coli and 19 kDa recombinant protein in Mycobacterium smegmatis. The objective of this study is to identify the ORF sequence, investigate possible posttranslational modification and characterize the protein forms in the two hosts. Earlier in the study, the genome sequence for M. paratuberculosis was not available and therefore sequencing both the 5' and 3' ends of the 4.8 kb insert did not help in the identification of the ORF. However, unidirectional Exonuclease deletion resulted in identification of subclones containing possible ORF sequence. Later on, the publication of the M. paratuberculosis genome sequence along with BLAST analysis of sequences from the subclones resulted in the identification of 486 bp ORF with significant identity to that from M. tuberculosis and M. leprae. Cloning of the 486 ORF coding sequence in E. coli resulted in the expression of 16 kDa protein similar to the calculated predicted size of translated peptide. Cloning of the 486 bp ORF coding sequence in M. smegmatis resulted in the expression of 19 kDa protein similar to that from M. paratuberculosis. The 16/19 kDa forms of the same protein were verified using rabbit anti-M. paratuberculosis antibodies adsorbed in E. coli and M. smegmatis lysates. The size of the 19 kDa proteins was not reduced following treatment with deglycosylation enzymes in absence of any enzyme inhibitors. The 19 kDa product was confirmed not be a glycoprotein when failed to react with ConA stain. The 16/19 kDa forms of the protein were evaluated against T-lymphocytes from Crohn's disease patients and normal controls. T- proliferation assay included controls such as PHA and PPD from M. paratuberculosis. There was not a significant difference between the two forms of the protein (16/19 kDa) against T-cell response from both populations. Overall, the study identified the ORF of the 19 kDa non-glycoprotein from M. paratuberculosis. Moreover, this is the first study which reports that the zoonotic M. paratuberculosis supports posttranslational modification similar to M. tuberculosis and M. leprae pathogens. Although the posttranslational modification component in this 19 kDa nonglycoprotein did not affect T- cell response, the finding is significant toward glycoproteins from M. paratuberculosis and their role in the pathogenesis of this bacterial infection in animals and humans.

Mycothiol (MSH), a low-molecular- weight thiol, is a primary reducing agent and essential for the survival of mycobacteria. The full pathway of MSH biosynthesis and detoxification includes various promising drug targets. Several metalloenzymes are involved in this pathway, such as a deacetylase (MshB) and mycothiol S-conjugate amidase (MCA). MshB catalyzes the deacetylation of GlcNAc-Ins to form GlcN-Ins and acetate. Mycothiol S-conjugate amidase (MCA) cleaves the amide bond of mycothiol S-conjugates of various drugs and toxins. The identification of the native co-factor is critical for the design of potent and effective inhibitors. Therefore, in this study, we identified the possible native co-factors of MshB and MCA from M. smegmatis and M. tuberculosis. To reach our aim, we used a pull-down method to rapidly purify halo-MshB and halo-MCA under anaerobic conditions. Our data indicates that the metal bound to MshB and MCA anaerobically purified from E. coli grown in minimal medium is mainly Fe(II), while proteins purified under aerobic conditions contain bound Zn (II) and Fe(II) that varies with the metal content of the medium. For a further clarification of the metal ion preferences of MshB and MCA, we determined the MshB and MCA affinity for Zn(II) to be in the picomolar range and Ms MshB affinity for Fe(II) in nanomolar range. These results indicate that MshB and MCA can be found bound with either iron or zinc and this is independent to their affinities for these metal ions. / Master of Science

Les mycobactéries sont classées parmi les bactéries contenant des acides mycoliques dans leur paroi et un haut GC% dans leur génome. Elles peuvent être isolées à partir du sol ou d'environnement d'eau douce où vivent aussi les protozoaires libres. Plusieurs études ont montré une possibilité de co-isolement des mycobactéries et des amibes à partir de ces sources environnementales. Il a été montré également que la plupart des mycobactéries de l'environnement ont la capacité à survivre dans les trophozoites et les kystes d'amibes et dans certaines cellules eucaryotes, y compris les macrophages. Les manipulations génétiques des mycobactéries en général et des mycobactéries du complexe Mycobacterium tuberculosis en particulier sont compliquées et aucune étude de modification génétique des mycobactéries (pathogènes ou non pathogènes) n'avait été réalisée dans notre laboratoire avant notre travail de thèse. Dans notre travail de thèse, nous avons montré que les amibes ou d'autres organismes phagocytaires peuvent servir comme sources et lieu de transfert des gènes chez les mycobactéries. Ce transfert des gènes peut avoir contribué à l'adaptation des mycobactéries à un mode de vie intracellulaire. Nous avons développé ensuite deux systèmes de coculture: Mycobacterium smegmatis-Acanthamoeba polyphaga et Mycobacterium gilvum-A. polyphaga et nous avons clarifié le spectre des interactions des mycobactéries à croissance rapide avec les amibes. Ce modèle d'interaction mycobactéries-amibes a été utilisé pour tester l'hypothèse contraire au paradigme dominant que l'addition des gènes réduit la virulence des bactéries. / Mycobacteria are mycolic-acid containing, high GC% bacterial organisms which can be recovered from soil and fresh water environments where free-living protozoa also live. Co-isolation of mycobacteria and amoeba collected from such environmental sources has been reported. Several experiments further demonstrated the ability of most environmental mycobacteria to survive in the amoebal trophozoites and cysts and in some eukaryotic cells including macrophages. Genetic modification of mycobacteria in general and mycobacteria belonging to Mycobacterium tuberculosis complex in particular are complicated and no studies using genetic modification of mycobacteria (pathogenic or non-pathogenic) had been performed in our laboratory prior to our work. In our thesis work, we showed that amoebae or other phagocytic organisms can serve as sources and places for gene transfers in mycobacteria. Gene transfers may have contributed to the adaptation of mycobacteria to an intracellular lifestyle. In addition, we developed two co-culture systems: Mycobacterium smegmatis-Acanthamoeba polyphaga and Mycobacterium gilvum-A. polyphaga and we clarified the spectrum of rapid-growing mycobacteria and amoeba interactions. This model of mycobacteria-amoeba interactions was then used to test another hypothesis according to which unlike the prevailing paradigm, the addition of genes does not reduce the virulence of bacteria. For the first time in our laboratory we modified two species of the M. tuberculosis complex, M. tuberculosis H37Rv and Mycobacterium bovis BCG to observe the effect of these changes on their pathogenicity and survival.