ParA of Mycobacterium smegmatis co‐ordinates chromosome segregation with the cell cycle and interacts with the polar growth determinant DivIVA

Mycobacteria are among the clinically most important pathogens, but still not much is known about the mechanisms of their cell cycle control. Previous studies suggested that the genes encoding ParA and ParB (ATPase and DNA binding protein, respectively, required for active chromosome segregation) may be essential in Mycobacterium tuberculosis. Further research has demonstrated that a Mycobacterium smegmatis parB deletion mutant was viable but exhibited a chromosome segregation defect. Here, we address the question if ParA is required for the growth of M. smegmatis, and which cell cycle processes it affects. Our data show that parA may be deleted, but its deletion leads to growth inhibition and severe disturbances of chromosome segregation and septum positioning. Similar defects are also caused by ParA overproduction. EGFP–ParA localizes as pole‐associated complexes connected with a patch of fluorescence accompanying two ParB complexes. Observed aberrations in the number and positioning of ParB complexes in the parA deletion mutant indicate that ParA is required for the proper localization of the ParB complexes. Furthermore, it is shown that ParA colocalizes and interacts with the polar growth determinant Wag31 (DivIVA homologue). Our results demonstrate that mycobacterial ParA mediates chromosome segregation and co‐ordinates it with cell division and elongation.     

ParA encoded on chromosome II of Deinococcus radiodurans binds to nucleoid and inhibits cell division in Escherichia coli

Bacterial genome segregation and cell division has been studied mostly in bacteria harbouring single circular chromosome and low-copy plasmids. Deinococcus radiodurans, a radiation-resistant bacterium, harbours multipartite genome system. Chromosome I encodes majority of the functions required for normal growth while other replicons encode mostly the proteins involved in secondary functions. Here, we report the characterization of putative P-loop ATPase (ParA2) encoded on chromosome II of D. radiodurans. Recombinant ParA2 was found to be a DNA-binding ATPase. E. coli cells expressing ParA2 showed cell division inhibition and mislocalization of FtsZ-YFP and those expressing ParA2-CFP showed multiple CFP foci formation on the nucleoid. Although, in trans expression of ParA2 failed to complement SlmA loss per se, it could induce unequal cell division in slmAminCDE double mutant. These results suggested that ParA2 is a nucleoid-binding protein, which could inhibits cell division in E. coli by affecting the correct localization of FtsZ and thereby cytokinesis. Helping slmAminCDE mutant to produce minicells, a phenotype associated with mutations in the ‘Min’ proteins, further indicated the possibility of ParA2 regulating cell division by bringing nucleoid compaction at the vicinity of septum growth.     

Competition between ribosome and SecA binding promotes Escherichia coli secA translational regulation.

SecA protein, the protein translocation ATPase of Escherichia coli, autogenously regulates its translation during normal protein secretion by binding to a secretion-responsive element located near the 5' end of its gene on geneX-secA mRNA. In order to characterize this autoregulation further, RNA footprinting and primerextension inhibition (toeprinting) studies were carried out with a segment of geneX-secA RNA, 30S ribosomal subunits and tRNAfMet along with purified SecA protein. The results show that ribosome and SecA-binding sites overlap, indicating that a simple competition for binding of geneX-secA mRNA presumably governs the translation initiation step. Further analysis showed that SecA protein was able to specifically dissociate a preformed 30S-tRNAfMet-geneX-secA RNA ternary complex as indicated by the disappearance of its characteristic toeprint after SecA addition. These findings are consistent with secA autoregulation, and they suggest a novel mechanism for the autoregulatory behavior of this complex protein.     

Competition between trans-translation and termination or elongation of translation

The effects of tRNA, RF1 and RRF on trans-translation by tmRNA were examined using a stalled complex of ribosome prepared using a synthetic mRNA and pure Escherichia coli translation factors. No endoribonucleolytic cleavage of mRNA around the A site was found in the stalled ribosome and was required for the tmRNA action. When the A site was occupied by a stop codon, alanyl-tmRNA competed with RF1 with the efficiency of peptidyl-transfer to alanyl-tmRNA for trans-translation inversely correlated to the efficiency of translation termination. The competition was not affected by RF3. A sense codon also serves as a target for alanyl-tmRNA with competition of aminoacyl-tRNA. The extent of inhibition was decreased with the length of the 3′-extension of mRNA. RRF, only at a high concentration, slightly affected peptidyl-transfer for trans-translation, although it did not affect the canonical elongation. These results indicate that alanyl-tmRNA does not absolutely require the truncation of mRNA around the A site but prefers an mRNA of a short 3′-extension from the A site and that it can operate on either a sense or termination codon at the A site, at which alanyl-tmRNA competes with aminoacyl-tRNA, RF and RRF.     

Localized dimerization and nucleoid binding drive gradient formation by the bacterial cell division inhibitor MipZ

Protein gradients play a central role in the spatial organization of cells, but the mechanisms of their formation are incompletely understood. This study analyzes the determinants responsible for establishing bipolar gradients of the ATPase MipZ, a key regulator of division site placement in Caulobacter crescentus. We have solved the crystal structure of MipZ in different nucleotide states, dissected its ATPase cycle, and investigated its interaction with FtsZ, ParB, and the nucleoid. Our results suggest that the polar ParB complexes locally stimulate the formation of ATP-bound MipZ dimers, which are then retained near the cell poles through association with chromosomal DNA. Due to their intrinsic ATPase activity, dimers eventually dissociate into freely diffusible monomers that undergo spontaneous nucleotide exchange and are recaptured by ParB. These findings clarify the molecular function of a conserved gradient-forming system and reveal mechanistic principles that might be commonly used to sustain protein gradients within cells.     

Phase separation between nucleoid and cytoplasm in Escherichia coli as defined by immersive refractometry.

The refractive indices of nucleoid and cytoplasm in Escherichia coli were derived theoretically and experimentally. For the theoretical estimates, we made use of the known macromolecular composition of E. coli B/r (G. Churchward and H. Bremer, J. Theor. Biol. 94:651-670, 1982) and of estimates of cell and nucleoid volumes. These were obtained from micrographs of living bacteria made with a confocal scanning light microscope. The theoretical values were calculated, assuming that all DNA occurred in the nucleoid and that all protein and RNA occurred in the cytoplasm. Comparison with experimental refractive index values directly obtained by immersive refractometry showed that, besides its DNA, the nucleoid must contain an additional amount of solids equivalent to 8.6% (wt/vol) protein. With the nucleoid containing 6.8% (wt/vol) DNA and 8.6% (wt/vol) protein and the cytoplasm containing 21% (wt/vol) protein and 4% (wt/vol) RNA, a mass difference is obtained, which accounts for the phase separation observed between the nucleoid and cytoplasm in living cells by phase-contrast microscopy. The decrease in the refractive index of the nucleoid relative to that of the cytoplasm observed upon, for instance, OsO4 fixation was interpreted as being indicative of the loss of protein content in the nucleoid.     

DRAM-3 modulates autophagy and promotes cell survival in the absence of glucose

Macroautophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. The process operates under basal conditions as a mechanism to turnover damaged or misfolded proteins and organelles. As a result, it has a major role in preserving cellular integrity and viability. In addition to this basal function, macroautophagy can also be modulated in response to various forms of cellular stress, and the rate and cargoes of macroautophagy can be tailored to facilitate appropriate cellular responses in particular situations. The macroautophagy machinery is regulated by a group of evolutionarily conserved autophagy-related (ATG) proteins and by several other autophagy regulators, which either have tissue-restricted expression or operate in specific contexts. We report here the characterization of a novel autophagy regulator that we have termed DRAM-3 due to its significant homology to damage-regulated autophagy modulator (DRAM-1). DRAM-3 is expressed in a broad spectrum of normal tissues and tumor cells, but different from DRAM-1, DRAM-3 is not induced by p53 or DNA-damaging agents. Immunofluorescence studies revealed that DRAM-3 localizes to lysosomes/autolysosomes, endosomes and the plasma membrane, but not the endoplasmic reticulum, phagophores, autophagosomes or Golgi, indicating significant overlap with DRAM-1 localization and with organelles associated with macroautophagy. In this regard, we further proceed to show that DRAM-3 expression causes accumulation of autophagosomes under basal conditions and enhances autophagic flux. Reciprocally, CRISPR/Cas9-mediated disruption of DRAM-3 impairs autophagic flux confirming that DRAM-3 is a modulator of macroautophagy. As macroautophagy can be cytoprotective under starvation conditions, we also tested whether DRAM-3 could promote survival on nutrient deprivation. This revealed that DRAM-3 can repress cell death and promote long-term clonogenic survival of cells grown in the absence of glucose. Interestingly, however, this effect is macroautophagy-independent. In summary, these findings constitute the primary characterization of DRAM-3 as a modulator of both macroautophagy and cell survival under starvation conditions.Macroautophagy (hereafter autophagy) is a cellular process that delivers cytoplasmic constituents to lysosomes for degradation.¹ Autophagy operates at basal levels in virtually all, if not all, cells. At the initiation of autophagy, membranes termed isolation membranes nucleate in the cytoplasm from a variety of sources.^{2, 3, 4, 5} Two ubiquitin-like conjugation mechanisms involving evolutionarily conserved autophagy-related (Atg) genes then function together to expand these membranes to form the characteristic organelles of autophagy, the autophagosome.^{6, 7} During this process, cargoes are recruited to the lumen of the autophagosome via a protein called LC3, which becomes tethered to autophagosome membranes during biogenesis.⁸ Adapter proteins such as p62/SQSTM1, NBR1 and OPTN then act as ‘bridges'' for cargo recruitment by simultaneously binding LC3, and the ubiquitin moieties on proteins and organelles destined for degradation.⁹Following autophagosome formation, a variety of fusion events can occur with other organelles including multi-vesicular bodies and endosomes.¹⁰ Ultimately, however, fusion occurs with lysosomes to form new organelles called autolysosomes in which lysosomal acidic hydrolases invoke cargo degradation.^{10, 11} Under basal conditions, the breakdown products are then recycled into biosynthetic pathways.^{10, 11} As a result, autophagy is a critical mechanism within cells to remove damaged proteins and organelles, thereby preserving cellular fidelity, homeostasis and ultimately viability of the cell and organism.^{1, 12}Autophagy can also be modulated by a variety of internal and external cues.¹³ This can increase the rate of autophagic flux and/or modulate the cargoes that are digested. In this regard, several selective forms of autophagy have been described including mitophagy – the selective digestion of mitochondria.^{14, 15} The best characterized situation in which autophagy is modulated is in response to starvation conditions.^{16, 17, 18, 19} This evolutionarily conserved response utilizes autophagy to provide fuel for catabolic pathways to maintain ATP levels during periods of diminished nutrient availability.To understand the regulation of autophagy, it is important to identify factors that regulate the process in both general and specific situations. For example, we previously identified DRAM-1 (damage-regulated autophagy modulator-1) as an autophagy regulator downstream of the tumor suppressor p53.^{20, 21} Subsequently, we found that DRAM-1 belongs to a previously undescribed, evolutionarily-conserved protein family.²² To date, however, we have only characterized DRAM-1 and the most related protein in terms of amino-acid sequence that we termed DRAM-2.²² We report here initial characterization of another DRAM-1-related protein that is encoded by TMEM150B and that we have named DRAM-3. This protein localizes to endosomes and autolysosomes/lysosomes, but unlike DRAM-1 is not induced by p53. DRAM-3 does, however, regulate autophagic flux and promotes cell survival in response to nutrient deprivation, but DRAM-3''s effect on cell survival is autophagy-independent.     

Competition between zonae pellucidae and a proteinase inhibitor for sperm binding.

Murine sperm bind a proteinase inhibitor of seminal vesicle origin at ejaculation. The inhibitor binds in the acrosomal region of the sperm head and is removed during in utero or in vitro incubation. Adding inhibitor to sperm reduces their ability to bind zonae, while adding the purified inhibitor binding site to cumulus-free, zona-intact oocytes reduces the ability of the oocytes to bind sperm. Immuno-aggregation of the inhibitor binding site results in exocytosis of the acrosome. These observations suggest that the inhibitor binding site may participate in zona binding and the acrosome reaction. If the inhibitor binding site binds both the zona and the seminal inhibitor, then these components should compete with each other for that site on the sperm. We show that purified seminal inhibitor, as well as other proteinase inhibitors, block zona-induced acrosome reactions. Likewise, zona glycopeptides block inhibitor/anti-inhibitor-induced acrosome reactions in a concentration-dependent fashion. The inhibitor/anti-inhibitor-induced acrosome reaction is sensitive to pertussis toxin and proteinase inhibitor and thus is similar to zona-induced reactions. These findings support the suggestion that the trypsin inhibitor binding site on the head of the sperm functions to insure sperm-zona binding and induction of the acrosome reaction.     

Competition between histone H1 and HMGN proteins for chromatin binding sites

The ability of regulatory factors to access their nucleosomal targets is modulated by nuclear proteins such as histone H1 and HMGN (previously named HMG-14/-17 family) that bind to nucleosomes and either stabilize or destabilize the higher-order chromatin structure. We tested whether HMGN proteins affect the interaction of histone H1 with chromatin. Using microinjection into living cells expressing H1–GFP and photobleaching techniques, we found that wild-type HMGN, but not HMGN point mutants that do not bind to nucleosomes, inhibits the binding of H1 to nucleosomes. HMGN proteins compete with H1 for nucleosome sites but do not displace statically bound H1 from chromatin. Our results provide evidence for in vivo competition among chromosomal proteins for binding sites on chromatin and suggest that the local structure of the chromatin fiber is modulated by a dynamic interplay between nucleosomal binding proteins.     

Competition between erythromycin and virginiamycin for in vitro binding to the large ribosomal subunit

When the S component of virginiamycin binds in vitro to the 50 S ribosomal subunit, a change of fluorescence intensity proportional to the amount of complex formed occurs. Erythromycin competes with virginiamycin S for attachment to ribosomes, and removes previously bound virginiamycin S from its target, as revealed by spectrofluorimetric analysis. The 50 S subunits which are incubated with the M component of virginiamycin (50 S*) have an increased affinity for virginiamycin S (the association constants of virginiamycin S with ribosomes are 2.5 x 10(6) M-1 in the absence of virginiamycin M, and 15 x 10(6) M-1 in its presence). Erythromycin does not compete with virginiamycin S for attachment to 50 S* subunits nor is it able to remove virginiamycin S previously bound to the 50 S* subunit. Thus, virginiamycin M produces a change in ribosomes, which results in a tighter complex virginiamycin S-50 S* subunit. Such change does not require the presence of virginiamycin M, however, as shown by the observation that ribosomes to which labeled virginiamycin M is transiently linked bind virginiamycin S in a form that cannot be removed by erythromycin.     

Evidence for the nature of the link between the arabinogalactan and peptidoglycan of mycobacterial cell walls

The long-posed question of the nature of the link between the mycolylarabinogalactan and the underlying peptidoglycan of the cell walls of Mycobacterium sp. has been addressed. The insoluble cell wall matrix of Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium bovis was partially hydrolyzed with acid either before or after per-O-methylation and the resulting oligosaccharides further derivatized and analyzed by gas chromatography/mass spectrometry. The structures of fragments arising from the reducing end of arabinogalactan demonstrated the existence of the terminal sequence----5)-D-Galf-(1----4)-L-Rhap-(1---3)-D-GlcNAc. Other analyses confirmed the presence of muramyl-6-P within the peptidoglycan of these mycobacteria. Based on the acid lability of the 3-linked GlcNAc unit, the presence of about equimolar amounts of Rhap-(1----3)-D-GlcNAc and muramyl-6-P in an isolated cell wall fragment, and 31P NMR analysis, it was concluded that the GlcNAc residue of the terminal triglycosyl unit of arabinogalactan is joined by 1-O-phosphoryl linkage to the 6-position of some muramyl residues within the peptidoglycan. Thus, it is reasoned that the massive mycolylarabinogalactan of mycobacteria, responsible for aspects of disease pathogenesis and much of the antibody response in infections, is attached to the peptidoglycan framework by the actinomycete-specific diglycosylphosphoryl bridge, L-Rhap-(1----3)-D-GlcNAc-(1----P, perhaps thereby providing a unique target for site-directed chemotherapy of mycobacterial infections.     

Cell length, nucleoid separation, and cell division of rod-shaped and spherical cells of Escherichia coli.

By comparing the dimensions and DNA contents of normal rod-shaped Escherichia coli with those of mutants that grow as spheres or ellipsoids, we have determined that two parameters remain unchanged: the DNA/mass ratio and the average cell length (diameter, for spherical cells). In consequence, the average volumes and DNA contents of the spherical mutant cells are about four to six times greater than those of rod-shaped cells growing at a similar rate. In addition, it was found that cells of both rod and sphere forms had approximately the same number of nucleoids (as seen when the DNA was condensed after inhibition of protein synthesis). The nucleoids of the spherical cells therefore consist of four to six completed chromosomes each (polytene nucleoids). We suggest that the attainment of a minimum cell length is necessary for nucleoid separation after chromosome replication and that such a separation is itself a prerequisite for septum formation.     

Competition of solid and fluid liposomes for binding and metabolism with lipids from the cell surface

The competitive behavior of solid vs. fluid liposomes in liposome-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding and transfer experiments have demonstrated that: solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substance; fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; fluid liposomes that escape lysis dissociate from the cell taking away cell lipid molecules. No lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface.     

Coactivator binding promotes the specific interaction between ligand and the pregnane X receptor

The pregnane X receptor (PXR) detects the presence of a wide variety of endogenous and xenobiotic compounds, and is a master regulator of the expression of genes central to drug metabolism and excretion. We present the 2.0A crystal structure of the human PXR ligand-binding domain (LBD) in complex with the cholesterol-lowering compound SR12813 and a 25 amino acid residue fragment of the human steroid receptor coactivator-1 (SRC-1) containing one LXXLL motif. PXR crystallizes as a homodimer in the asymmetric unit in this structure and possesses a novel alpha2 helix adjacent to its ligand-binding cavity. The SRC-1 peptide forms two distinct helices and binds adjacent to the ligand-dependent transactivation AF-2 helix on the surface of PXR. In contrast with previous PXR structures, in which SR12813 bound in multiple orientations, the small SR12813 agonist in this structure binds in a single, unique orientation within the receptor's ligand-binding pocket and contacts the AF-2 helix. Thermal denaturation studies reveal that the SR12813 ligand and SRC-1 coactivator peptide each stabilize the LBD of PXR, and that together they exert an additive effect on the stability of the receptor. These results indicate that the binding of coactivator to the surface of PXR limits the ability of this promiscuous receptor to "breathe" and helps to trap a single, active conformation of SR12813. They further reveal that specificity is required for PXR activation.