Identification and characterization of the regulatory elements of the inducible acetamidase operon from Mycobacterium smegmatis

The highly inducible acetamidase promoter from Mycobacterium smegmatis has been used as a tool in the study of mycobacterial genetics. The 4.2 kb acetamidase operon contains four putative open reading frames (ORFs) (amiC, amiA, amiD, and amiS) upstream of the 1.2 kb acetamidase ORF (amiE). In this article, using electrophoretic mobility shift assay and promoter probe analyses with a lacZ reporter system, we show the position of three putative operators within the acetamidase operon in M. smegmatis. Results from these studies reinforce previous findings about the involvement of multiple promoters in the regulation of acetamidase gene expression. Each of the identified operators are positioned upstream of the respective promoter reported in previous studies. We also found that the crude cell lysate of M. smegmatis containing potential regulators, obtained from bacteria grown under inducing or noninducing conditions, binds to specific operators. The binding affinity of each operator with its cognate regulator is significantly different from the other. This supports not only the previous model of acetamidase gene regulation in M. smegmatis but also explains the role of these operators in controlling the expression of respective promoters under different growth conditions.     

Sequence-divergent chordopoxvirus homologs of the o3 protein maintain functional interactions with components of the vaccinia virus entry-fusion complex

Composed of 35 amino acids, O3 is the smallest characterized protein encoded by vaccinia virus (VACV) and is an integral component of the entry-fusion complex (EFC). O3 is conserved with 100% identity in all orthopoxviruses except for monkeypox viruses, whose O3 homologs have 2 to 3 amino acid substitutions. Since O3 is part of the EFC, high conservation could suggest an immutable requirement for interaction with multiple proteins. Chordopoxviruses of other genera also encode small proteins with a characteristic predicted N-terminal α-helical hydrophobic domain followed by basic amino acids and proline in the same relative genome location as that of VACV O3. However, the statistical significance of their similarity to VACV O3 is low due to the large contribution of the transmembrane domain, their small size, and their sequence diversity. Nevertheless, trans-complementation experiments demonstrated the ability of a representative O3-like protein from each chordopoxvirus genus to rescue the infectivity of a VACV mutant that was unable to express endogenous O3. Moreover, recombinant viruses expressing O3 homologs in place of O3 replicated and formed plaques as well or nearly as well as wild-type VACV. The O3 homologs expressed by the recombinant VACVs were incorporated into the membranes of mature virions and, with one exception, remained stably associated with the detergent-extracted and affinity-purified EFC. The ability of the sequence-divergent O3 homologs to coordinate function with VACV entry proteins suggests the conservation of structural motifs. Analysis of chimeras formed by swapping domains of O3 with those of other proteins indicated that the N-terminal transmembrane segment was responsible for EFC interactions and for the complementation of infectivity.     

Vaccinia Virus H7 Protein Contributes to the Formation of Crescent Membrane Precursors of Immature Virions

Crescent membranes are the first viral structures that can be discerned during poxvirus morphogenesis. The crescents consist of a lipoprotein membrane and an outer lattice scaffold, which provides uniform curvature. Relatively little is known regarding the composition of the crescent membrane or its mode of formation. Here, we show that the H7 protein, which is conserved in all vertebrate poxviruses but has no discernible functional motifs or nonpoxvirus homologs, contributes to the formation of crescents and immature virions. Synthesis of the 17-kDa H7 protein was dependent on DNA replication and occurred late during vaccinia virus infection. Unlike many late proteins, however, H7 was not incorporated into mature virions or localized in cellular organelles. To gain insight into the role of H7, an inducible mutant was constructed and shown to have a conditional lethal phenotype: H7 expression and infectious virus formation were dependent on isopropyl-beta-d-thiogalactopyranoside. In the absence of inducer, viral late proteins were made, but membrane and core proteins were not processed by the I7 protease. A block in morphogenesis was demonstrated by transmission electron microscopy: neither typical crescents nor immature virions were detected in the absence of inducer. Instead, factory areas of the cytoplasm contained large, electron-dense inclusions, some of which had partially coated membrane segments at their surfaces. Separate, lower-density inclusions containing the D13 scaffold protein and endoplasmic reticulum membranes were also present. These features are most similar to those previously seen when expression of A11, another conserved nonvirion protein, is repressed.The vertebrate poxviruses, of which vaccinia virus (VACV) is the prototype, encode about 200 proteins, of which almost half are conserved in all species (40). The conserved proteins include those that execute basic functions, which allow poxviruses to replicate and express their double-stranded DNA genomes and assemble infectious particles in the cytoplasm (25). Due to their large number, some of the conserved open reading frames (ORFs) have yet to be characterized. In the present study, we show that the product of the VACV H7R ORF contributes to the formation of the crescent membrane precursors of immature virions (IVs).Crescents are uniformly curved membranes that form within specialized regions of the cytoplasm known as factories (8, 10). The crescents envelop electron-dense granular material containing core precursor proteins to form ∼300-nm spherical IVs, which subsequently undergo internal and external architectural changes to become infectious brick-shaped mature virions (MVs) (6). Several models have been proposed for the structure and mode of formation of crescents and IVs. Transmission electron micrography revealed a single membrane bilayer covered with an external “spicule” coat (8, 24). Although evidence for two closely apposed membranes has been presented (29, 34), other studies support the original single membrane structure (5, 17-19). The outer coat was revealed by deep-etch immunogold electron microscopy to be a lattice comprised of trimers of the D13 protein (18, 36) rather than a layer of discontinuous spikes.The crescent and IV membranes are not fully characterized with regard to their composition or organization, and two main theories regarding their origin have been proposed. One idea, inspired by the spatial separation of crescent and cellular membranes in virus factories, was the de novo origin of poxvirus membranes from lipids and viral proteins (9). An alternative model, positing the derivation of crescents from cellular membranes, was based partly on the lack of precedence of de novo membrane formation in other biological systems, the finding of some viral proteins associated with membranes of the intermediate compartment between the endoplasmic reticulum (ER) and the Golgi apparatus, and the proximity of tubular structures and viral membranes (29, 33). Other studies, however, provided evidence for trafficking of proteins to the viral membrane through the ER rather than the intermediate compartment although the initial membrane nucleation event was not investigated (20, 21).Understanding the mechanism of viral membrane formation depends on the identification of the viral and cellular proteins involved. A role for the cellular coatomer and KDEL receptor in early VACV biogenesis has been suggested (43). Studies of conditional lethal VACV mutants pointed to the involvement of several viral proteins in the formation of crescent membranes. Repression of synthesis of the D13 scaffold protein mimics the effect of the drug rifampin and results in floppy-appearing membranes bordering electron-dense granular material (44). Such membranes seem otherwise normal as they can acquire the scaffold and concomitant rigid curvature within minutes after removal of rifampin and develop into IVs (26). Repression of synthesis of the integral membrane proteins A14 and A17 results in aberrant vesicular or tubular structures that differ from each other in appearance (30, 31, 39, 41). Both A14 and A17 are phosphorylated by the F10 kinase (3, 13, 39), and viral membranes are not detected when cells are infected with conditional lethal F10 mutants under nonpermissive conditions (37, 38). Viral membranes are also not observed under nonpermissive conditions when cells are infected with conditional lethal H5 (12), G5 (7), and A11 (28) mutants though their roles in this process are not yet understood. Here, we characterize the product of the H7R ORF and demonstrate that it is also involved in viral membrane formation and morphogenesis.     

Effect of artificial light on activity in frugivorous bats (Pteropodidae)

Journal of Ethology - Artificial lighting at nights (ALAN) affects behaviour in many animals, especially nocturnal species. However, its effect on frugivorous bats remains less explored, especially...     

Effect of Mycobacterium tuberculosis infection on adipocyte physiology

Tuberculosis (TB) remains as a major threat to human health worldwide despite of the availability of standardized antibiotic therapy. One of the characteristic of pathogenic Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis is its ability to persist in the host in a dormant state and develop latent infection without clinical signs of active disease. However, the mechanisms involved in bacterial persistence and the establishment of latency is not well understood. Adipose tissue is emerging as an important niche that favors actively replicating as well as dormant Mtb during acute and latent infection. This also suggests that Mtb can disseminate from the lungs to adipose tissue during aerosol infection and/or from adipose tissue to lungs during reactivation of latent infection. In this study, we report the interplay between key adipokine levels and the dynamics of Mtb pathogenesis in the lungs and adipose tissue using a rabbit model of pulmonary infection with two clinical isolates that produce divergent outcome in disease progression. Results show that markers of adipocyte physiology and function were significantly altered during Mtb infection and distinct patterns of adipokine expression were noted between adipose tissue and the lungs. Moreover, these markers were differentially expressed between active disease and latent infection. Thus, this study highlights the importance of targeting adipocyte function as potential target for developing better TB intervention strategies.     

The role of arginine-rich motif and beta-annulus in the assembly and stability of Sesbania mosaic virus capsids

Sesbania mosaic virus (SeMV) capsids are stabilized by protein-protein, protein-RNA and calcium-mediated protein-protein interactions. The N-terminal random domain of SeMV coat protein (CP) controls RNA encapsidation and size of the capsids and has two important motifs, the arginine-rich motif (ARM) and the beta-annulus structure. Here, mutational analysis of the arginine residues present in the ARM to glutamic acid was carried out. Mutation of all the arginine residues in the ARM almost completely abolished RNA encapsidation, although the assembly of T=3 capsids was not affected. A minimum of three arginine residues was found to be essential for RNA encapsidation. The mutant capsids devoid of RNA were less stable to thermal denaturation when compared to wild-type capsids. The results suggest that capsid assembly is entirely mediated by CP-dependent protein-protein inter-subunit interactions and encapsidation of genomic RNA enhances the stability of the capsids. Because of the unique structural ordering of beta-annulus segment at the icosahedral 3-folds, it has been suggested as the switch that determines the pentameric and hexameric clustering of CP subunits essential for T=3 capsid assembly. Surprisingly, mutation of a conserved proline within the segment that forms the beta-annulus to alanine, or deletion of residues 48-53 involved in hydrogen bonding interactions with residues 54-58 of the 3-fold related subunit or deletion of all the residues (48-59) involved in the formation of beta-annulus did not affect capsid assembly. These results suggest that the switch for assembly into T=3 capsids is not the beta-annulus. The ordered beta-annulus observed in the structures of many viruses could be a consequence of assembly to optimize intersubunit interactions.     

Vaccinia Virus A19 Protein Participates in the Transformation of Spherical Immature Particles to Barrel-Shaped Infectious Virions

The A19L open reading frame of vaccinia virus encodes a 9-kDa protein that is conserved in all sequenced chordopoxviruses, yet until now it has not been specifically characterized in any species. We appended an epitope tag after the start codon of the A19L open reading frame without compromising infectivity. The protein was synthesized after viral DNA replication and was phosphorylated independently of the vaccinia virus F10 kinase. The A19 protein was present in purified virions and was largely resistant to nonionic detergent extraction, suggesting a location within the core. A conditional lethal mutant virus was constructed by placing the A19 open reading frame under the control of the Escherichia coli lac repressor system. A19 synthesis and infectious virus formation were dependent on inducer. In the absence of inducer, virion morphogenesis was interrupted, and spherical dense particles that had greatly reduced amounts of the D13 scaffold accumulated in place of barrel-shaped mature virions. The infectivity of purified A19-deficient particles was more than 2 log units less than that of A19-containing virions. Nevertheless, the A19-deficient particles contained DNA, and except for the absence of A19 and decreased core protein processing, they appeared to have a similar protein composition as A19-containing virions. Thus, the A19 protein participates in the maturation of immature vaccinia virus virions to infectious particles.