scholarly journals Transcriptional Analysis of the Principal Cell Division Gene, ftsZ, of Mycobacterium tuberculosis

2005 ◽  
Vol 187 (7) ◽  
pp. 2540-2550 ◽  
Author(s):  
Sougata Roy ◽  
Parthasarathi Ajitkumar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Division ◽  
Growth Conditions ◽  
Principal Cell ◽  
Transcriptional Analysis ◽  
Upstream Region ◽  
Reading Frame ◽  
Multiple Promoters ◽  
Promoter Fusion

ABSTRACT Multiple promoters drive the expression of the principal cell division gene, ftsZ, in bacterial systems. Primer extension analysis of total RNA from Mycobacterium tuberculosis and a Mycobacterium smegmatis transformant containing 1.117 kb of the upstream region of M. tuberculosis ftsZ and promoter fusion studies identified six ftsZ transcripts and their promoters in the ftsQ open reading frame and ftsQ-ftsZ intergenic region. The presence of multiple promoters reflects the requirement to maintain a high basal level of, or to differentially regulate, FtsZ expression during different growth conditions of the pathogen in vivo.

scholarly journals The Escherichia coli cysG promoter belongs to the ‘extended −10’ class of bacterial promoters

1993 ◽  
Vol 296 (3) ◽  
pp. 851-857 ◽  
Author(s):  
T Belyaeva ◽  
L Griffiths ◽  
S Minchin ◽  
J Cole ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Point Mutations ◽  
Growth Conditions ◽  
Upstream Region ◽  
E Coli ◽  
Run Off ◽  
A Site ◽  
Specific Sequences

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5′-TGN-3′ motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the ‘extended -10’ class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.

scholarly journals The Largest Open Reading Frame (pks12) in the Mycobacterium tuberculosis Genome Is Involved in Pathogenesis and Dimycocerosyl Phthiocerol Synthesis

2003 ◽  
Vol 71 (7) ◽  
pp. 3794-3801 ◽  
Author(s):  
Tatiana D. Sirakova ◽  
Vinod S. Dubey ◽  
Hwa-Jung Kim ◽  
Michael H. Cynamon ◽  
Pappachan E. Kolattukudy
Keyword(s):  
Fatty Acids ◽  
Mycobacterium Tuberculosis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoblot Analysis ◽  
Open Reading Frame ◽  
Pcr Analysis ◽  
Reading Frame ◽  
Sds Page

ABSTRACT The cell wall lipids in Mycobacterium tuberculosis are probably involved in pathogenesis. The largest open reading frame in the genome of M. tuberculosis H37Rv, pks12, is unique in that it encodes two sets of domains needed to produce fatty acids. A pks12-disrupted mutant was produced, and disruption was confirmed by both PCR analysis and Southern blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a 430-kDa protein band present in the wild type was missing in the mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) and liquid chromatography (LC)-MS analysis of tryptic peptides showed that 54 peptides distributed throughout this protein matched the pks12-encoded sequence. Biochemical analysis using [1-14C]propionate as the radiotracer showed that the pks12 mutant was deficient in the synthesis of dimycocerosyl phthiocerol (DIM). SDS-PAGE, immunoblot analysis of proteins, and analysis of fatty acids showed that the mutant can produce mycocerosic acids. Thus, the pks12 gene is probably involved in the synthesis of phthiocerol, the diol required for DIM synthesis. Growth of the pks12 mutant was attenuated in mouse alveolar macrophage cell line MH-S, and the virulence of the mutant in vivo was highly attenuated in a murine model. Thus, pks12 probably participates in DIM production and its expression is involved in pathogenesis.

scholarly journals Transcriptional Analysis of the grlRA Virulence Operon from Citrobacter rodentium

2010 ◽  
Vol 192 (14) ◽  
pp. 3722-3734 ◽  
Author(s):  
Marija Tauschek ◽  
Ji Yang ◽  
Dianna Hocking ◽  
Kristy Azzopardi ◽  
Aimee Tan ◽  
...  
Keyword(s):  
Core Promoter ◽  
Regulatory Protein ◽  
Transcriptional Analysis ◽  
Upstream Region ◽  
Citrobacter Rodentium ◽  
Dnase I Footprinting ◽  
Intestinal Pathogens ◽  
Enterocyte Effacement

ABSTRACT The locus for enterocyte effacement (LEE) is the virulence hallmark of the attaching-and-effacing (A/E) intestinal pathogens, namely, enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium. The LEE carries more than 40 genes that are arranged in several operons, e.g., LEE1 to LEE5. Expression of the various transcriptional units is subject to xenogeneic silencing by the histone-like protein H-NS. The LEE1-encoded regulator, Ler, plays a key role in relieving this repression at several major LEE promoters, including LEE2 to LEE5. To achieve appropriate intracellular concentrations of Ler in different environments, A/E pathogens have evolved a sophisticated regulatory network to control ler expression. For example, the LEE-encoded GrlA and GrlR proteins work as activator and antiactivator, respectively, of ler transcription. Thus, control of the transcriptional activities of the LEE1 (ler) promoter and the grlRA operon determines the rate of transcription of all of the LEE-encoded virulence factors. To date, only a single promoter has been identified for the grlRA operon. In this study, we showed that the non-LEE-encoded AraC-like regulatory protein RegA of C. rodentium directly stimulates transcription of the grlRA promoter by binding to an upstream region in the presence of bicarbonate ions. In addition, in vivo and in vitro transcription assays revealed a σ70 promoter that is specifically responsible for transcription of grlA. Expression from this promoter was strongly repressed by H-NS and its paralog StpA but was activated by Ler. DNase I footprinting demonstrated that Ler binds to a region upstream of the grlA promoter, whereas H-NS interacts specifically with a region extending from the grlA core promoter into its coding sequence. Together, these findings provide new insights into the environmental regulation and differential expressions of the grlR and grlA genes of C. rodentium.

scholarly journals Expression and Characterization of a Novel Structural Protein of Human Cytomegalovirus, pUL25

1999 ◽  
Vol 73 (5) ◽  
pp. 3800-3809 ◽  
Author(s):  
Maria Concetta Battista ◽  
Giovanna Bergamini ◽  
Maria Cristina Boccuni ◽  
Fabio Campanini ◽  
Alessandro Ripalti ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Transcriptional Analysis ◽  
Structural Protein ◽  
Native Form ◽  
Flag Epitope ◽  
Reading Frame ◽  
Tegument Proteins ◽  
Infected Cells

ABSTRACT Human cytomegalovirus (HCMV) UL25 has recently been found to encode a new structural protein that is present in both virion and defective viral particles (C. J. Baldick and T. Shenk, J. Virol. 70:6097–6105, 1996). In the present work a polyclonal antibody was raised against a prokaryotic pUL25 fusion protein in order to investigate the biosynthesis and localization of the UL25 product (pUL25) during HCMV replication in human fibroblasts. Furthermore, pUL25 was transiently expressed in its native form and fused to the FLAG epitope, in COS7 and U373MG cells, in order to compare the properties of the isolated protein and that produced during infection. Immunoblotting analysis revealed a group of polypeptides, ranging from 80 to 100 kDa, in both transfected and infected cells; in vivo labeling experiments with infected cells demonstrated they are posttranslationally modified by phosphorylation. The transcriptional analysis of the UL25 open reading frame combined with the study of pUL25 biosynthesis showed true late kinetics for this protein in infected human fibroblasts. By indirect immunofluorescence both recombinant and viral pUL25 were detected exclusively in the cytoplasm of transfected or infected cells. Interestingly, pUL25 was shown to localize in typical condensed structures in the perinuclear region as already observed for other HCMV tegument proteins. Colocalization of ppUL99 in the same vacuoles suggests that these structure are endosomal cisternae, which are proposed to be a preferential site of viral particle envelopment. Our data suggest that pUL25 is most likely a novel tegument protein and possibly plays a key role in the process of envelopment.

scholarly journals Variable Expression Patterns of Mycobacterium tuberculosis PE_PGRS Genes: Evidence that PE_PGRS16 and PE_PGRS26 Are Inversely Regulated In Vivo

2006 ◽  
Vol 188 (10) ◽  
pp. 3721-3725 ◽  
Author(s):  
Veerabadran Dheenadhayalan ◽  
Giovanni Delogu ◽  
Maurizio Sanguinetti ◽  
Giovanni Fadda ◽  
Michael J. Brennan
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Expression Profile ◽  
Expression Patterns ◽  
Constitutive Expression ◽  
Growth Conditions ◽  
Variable Expression

ABSTRACT Evaluation of expression of 16 PE_PGRS genes present in Mycobacterium tuberculosis under various growth conditions demonstrated constitutive expression of 7 genes, variable expression of 7 genes, and no expression of 2 genes. An inverse expression profile for genes PE_PGRS16 and PE_PGRS26 was observed to occur in macrophages and in mice infected with M. tuberculosis. Variable expression of PE_PGRS proteins could have implications for their role in the immunopathogenesis of tuberculosis.

scholarly journals The serine/threonine kinase PknB of Mycobacterium tuberculosis phosphorylates PBPA, a penicillin-binding protein required for cell division

Microbiology ◽  
2006 ◽  
Vol 152 (2) ◽  
pp. 493-504 ◽  
Author(s):  
Arunava Dasgupta ◽  
Pratik Datta ◽  
Manikuntala Kundu ◽  
Joyoti Basu
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Division ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Peptidoglycan Biosynthesis ◽  
Division Site ◽  
Class B ◽  
First Time

A cluster of genes encoded by ORFs Rv0014c–Rv0018c in Mycobacterium tuberculosis encodes candidate cell division proteins RodA and PBPA, a pair of serine/threonine kinases (STPKs), PknA and PknB, and a phosphatase, PstP. The organization of genes encompassing this region is conserved in a large number of mycobacterial species. This study demonstrates that recombinant PBPA of M. tuberculosis binds benzylpenicillin. Knockout of its counterpart in M. smegmatis resulted in hindered growth and defective cell septation. The phenotype of the knockout (PBPA-KO) could be restored to that of the wild-type upon expression of PBPA of M. tuberculosis. PBPA localized to the division site along with newly synthesized peptidoglycan, between segregated nucleoids. In vivo coexpression of PBPA and PknB, in vitro kinase assays and site-specific mutagenesis substantiated the view that PknB phosphorylates PBPA on T362 and T437. A T437A mutant could not complement PBPA-KO. These studies demonstrate for the first time that PBPA, which belongs to a subclass of class B high-molecular-mass PBPs, plays an important role in cell division and cell shape maintenance. Signal transduction mediated by PknB and PstP likely regulates the positioning of this PBP at the septum, thereby regulating septal peptidoglycan biosynthesis.

scholarly journals Structural and Functional Analysis of Rv3214 from Mycobacterium tuberculosis, a Protein with Conflicting Functional Annotations, Leads to Its Characterization as a Phosphatase

2006 ◽  
Vol 188 (10) ◽  
pp. 3589-3599 ◽  
Author(s):  
Harriet A. Watkins ◽  
Edward N. Baker
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Functional Annotation ◽  
Sequence Similarity ◽  
Phosphoglycerate Mutase ◽  
Phosphopantetheinyl Transferase ◽  
Ph Optimum ◽  
Reading Frame ◽  
Functional Annotations ◽  
The Many

ABSTRACT The availability of complete genome sequences has highlighted the problems of functional annotation of the many gene products that have only limited sequence similarity with proteins of known function. The predicted protein encoded by open reading frame Rv3214 from the Mycobacterium tuberculosis H37Rv genome was originally annotated as EntD through sequence similarity with the Escherichia coli EntD, a 4′-phosphopantetheinyl transferase implicated in siderophore biosynthesis. An alternative annotation, based on slightly higher sequence identity, grouped Rv3214 with proteins of the cofactor-dependent phosphoglycerate mutase (dPGM) family. The crystal structure of this protein has been solved by single-wavelength anomalous dispersion methods and refined at 2.07-Å resolution (R = 0.229; Rfree = 0.245). The protein is dimeric, with a monomer fold corresponding to the classical dPGM α/β structure, albeit with some variations. Closer comparisons of structure and sequence indicate that it most closely corresponds with a broad-spectrum phosphatase subfamily within the dPGM superfamily. This functional annotation has been confirmed by biochemical assays which show negligible mutase activity but acid phosphatase activity with a pH optimum of 5.4 and suggests that Rv3214 may be important for mycobacterial phosphate metabolism in vivo. Despite its weak sequence similarity with the 4′-phosphopantetheinyl transferases (EntD homologues), there is little evidence to support this function.

scholarly journals Increased pathology in lungs of mice after infection with an α-crystallin mutant of Mycobacterium tuberculosis: changes in cathepsin proteases and certain cytokines

Microbiology ◽  
2006 ◽  
Vol 152 (1) ◽  
pp. 233-244 ◽  
Author(s):  
Julie N. Stewart ◽  
Hilda N. Rivera ◽  
Russell Karls ◽  
Frederick D. Quinn ◽  
Jesse Roman ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Granulomatous Inflammation ◽  
Serum Levels ◽  
Growth Conditions ◽  
High Serum ◽  
Pcr Analysis ◽  
Intracellular Growth ◽  
Cathepsin Proteases ◽  
The Matrix

Latency and reactivation are a significant problem that contributes to the incidence, transmission and pathogenesis of tuberculosis. The mechanisms involved in these processes, at the level of both the bacillus and the host, are poorly understood. In Mycobacterium tuberculosis the α-crystallin (acr) gene has been linked to latency, because it is highly expressed during hypoxic growth conditions. Deletion of the acr gene in M. tuberculosis H37Rv (Δacr strain) was previously shown to reduce the intracellular growth of bacilli in macrophages; however, its impact on pathogenesis in vivo was unknown. This study demonstrated that infection of C57BL6 mice with Δacr results in lung bacillary loads 1-2 log units higher in comparison to parental H37Rv. Haematoxylin/eosin staining of lungs revealed exacerbated pathology characterized by extensive obliteration of alveolar air spaces by granulomatous inflammation. RT-PCR analysis and immunostaining of lungs showed that infection with either H37Rv or Δacr results in the differential expression of lysosomal cathepsin proteases. A slight increase in the expression of the matrix-degrading acidic-type cathepsins B, D and H was noted in Δacr-infected mice and was associated with clusters of macrophages within lung granulomas. Δacr-infected mice also showed high serum levels of TNF-α, IFN-γ and G-CSF, suggesting that Acr may play a role in modulating the host response to infection.

scholarly journals Mycobacterium tuberculosis Cells Growing in Macrophages Are Filamentous and Deficient in FtsZ Rings

2006 ◽  
Vol 188 (5) ◽  
pp. 1856-1865 ◽  
Author(s):  
Ashwini Chauhan ◽  
Murty V. V. S. Madiraju ◽  
Marek Fol ◽  
Hava Lofton ◽  
Erin Maloney ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Low Frequency ◽  
Sole Source ◽  
Growth Conditions ◽  
Ring Formation ◽  
Structural Element ◽  
Slow Growth Rate ◽  
Z Ring

ABSTRACT FtsZ, a bacterial homolog of tubulin, forms a structural element called the FtsZ ring (Z ring) at the predivisional midcell site and sets up a scaffold for the assembly of other cell division proteins. The genetic aspects of FtsZ-catalyzed cell division and its assembly dynamics in Mycobacterium tuberculosis are unknown. Here, with an M. tuberculosis strain containing FtsZTB tagged with green fluorescent protein as the sole source of FtsZ, we examined FtsZ structures under various growth conditions. We found that midcell Z rings are present in approximately 11% of actively growing cells, suggesting that the low frequency of Z rings is reflective of their slow growth rate. Next, we showed that SRI-3072, a reported FtsZTB inhibitor, disrupted Z-ring assembly and inhibited cell division and growth of M. tuberculosis. We also showed that M. tuberculosis cells grown in macrophages are filamentous and that only a small fraction had midcell Z rings. The majority of filamentous cells contained nonring, spiral-like FtsZ structures along their entire length. The levels of FtsZ in bacteria grown in macrophages or in broth were comparable, suggesting that Z-ring formation at midcell sites was compromised during intracellular growth. Our results suggest that the intraphagosomal milieu alters the expression of M. tuberculosis genes affecting Z-ring formation and thereby cell division.

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