Genetic transformation with cell wall-associated deoxyribonucleic acid in Bacillus subtilis

Cell walls from bacillus subtilis 168 were prepared by conventional methods and found to contain deoxyribonucleic acid (DNA). In transformation assays, after autolysis, it was found that two major regions of the chromosome were selectively enriched in the wall preparations. One region clustered around the replication origin and is represented by the markers purA16, ts8132, thiC5, sacA321, and hisA1. The other region included the replication terminus with representative loci metB10, citK5, gltA292, and pyrA1. All other (internal) loci which were examined showed no statistical enrichment. The two areas of enrichment were similar to but more extensive than those reported for membrane-DNA complexes. The wall preparations also contained protein and lipid, indicating a possible membrane involvement. Analyses of the cell walls revealed that the fatty acid composition of the membrane component was not typical of the for B. subtilis protoplast membranes or for lipoteichoic acids. In addition, radioiodination of cell wall autolysates, followed by gel electrophoresis and autoradiography, demonstrated the presence of proteins not readily detectable in bulk protoplast membranes or on the surfaces of intact cells. These data suggest that a unique component of the membrane and regions of the B. subtilis genome involved in DNA replication events are tightly associated with cell walls. The binding of DNA-membrane complexes to the "rigid" cell wall and the replication of the wall could be a mechanism by which the segregation of growing chromosomes occurs.     

Preprotein translocation by a hybrid translocase composed of Escherichia coli and Bacillus subtilis subunits

Bacterial protein translocation is mediated by translocase, a multisubunit membrane protein complex that consists of a peripheral ATPase SecA and a preprotein-conducting channel with SecY, SecE, and SecG as subunits. Like Escherichia coli SecG, the Bacillus subtilis homologue, YvaL, dramatically stimulated the ATP-dependent translocation of precursor PhoB (prePhoB) by the B. subtilis SecA-SecYE complex. To systematically determine the functional exchangeability of translocase subunits, all of the relevant combinations of the E. coli and B. subtilis secY, secE, and secG genes were expressed in E. coli. Hybrid SecYEG complexes were overexpressed at high levels. Since SecY could not be overproduced without SecE, these data indicate a stable interaction between the heterologous SecY and SecE subunits. E. coli SecA, but not B. subtilis SecA, supported efficient ATP-dependent translocation of the E. coli precursor OmpA (proOmpA) into inner membrane vesicles containing the hybrid SecYEG complexes, if E. coli SecY and either E. coli SecE or E. coli SecG were present. Translocation of B. subtilis prePhoB, on the other hand, showed a strict dependence on the translocase subunit composition and occurred efficiently only with the homologous translocase. In contrast to E. coli SecA, B. subtilis SecA binds the SecYEG complexes only with low affinity. These results suggest that each translocase subunit contributes in an exclusive manner to the specificity and functionality of the complex.     

Membrane association of a Staphylococcus aureus plasmid in Bacillus subtilis.

The Staphylococcus aureus plasmid pUB110 was found to be enriched in deoxyribonucleic acid-membrane complexes isolated from Bacillus subtilis containing pUB110.     

Attachment of the chromosomal terminus of Bacillus subtilis to a fast-sedimenting particle

After gently lysed protoplasts of exponential phase cells of Bacillus subtilis were treated with restriction endonuclease BamHI, 99% of the DNA did not sediment with the plasma membrane. This DNA was fractionated on sucrose gradients into (i) a fast-sedimenting fraction highly enriched for genes from the origin and terminus (purA and ilvA), (ii) a 50 to 100S component also enriched for purA and ilvA, and (iii) the bulk of the DNA. The fast-sedimenting fraction was dissociated by Sarkosyl; this fraction contained a substantial amount of protein and is probably a membrane subparticle. The S value of the 50 to 100S component was not greatly affected by Sarkosyl treatment, but these particles were unable to penetrate an agarose gel during electrophoresis and were retained by nitrocellulose filters. The terminus DNA in the fast-sedimenting fraction and the 50 to 100S component contained a large restriction fragment (1.5 x 10(7) to 2.0 x 10(7) daltons) encoding ilvA, thyB, and ilvD. The bulk of the SP beta prophage and metB, which lie to the right and left, respectively, of the ilvA-ilvD cluster, were not part of the complex. citK, which lies to the right of SP beta, appeared to be present in the fast-sedimenting complexes. The neighboring genes kauA and gltA were not part of the fast-sedimenting complexes. The presence of terminus DNA in the fast-sedimenting components was also demonstrated by a radiochemical method.     

Membrane enrichment of genetic markers close to the origin and terminus during the deoxyribonucleic acid replication cycle in Bacillus subtilis.

A temperature-sensitive Bacillus subtilis initiation mutant was used to achieve one cycle of synchronized deoxyribonucleic acid (DNA) replication. Markers near the origin of replication and the terminus were assayed for association with the cell membrane at intervals during the DNA replication cycle. DNA near the origin and terminus was found to be enriched in the membrane fraction throughout the DNA replication cycle. The magnitude of membrane enrichment or origin and terminus markers varied coincidentally, possibly as a consequence of incubating the cells at 45 degrees C.     

Formation of additional contacts of chromosome with membrane in the process of DNA repair synthesis in bacterial cells

An increase in the amount of membrane-bound DNA was found in B. subtilis cells with UV-induced DNA repair synthesis as compared to untreated cells. It was shown that DNA repair synthesis occurred in DNA membrane complexes (DMC) formed during UV-irradiation. UV-induced formation of DMC was observed in cells of wild type strains which were capable of repairing damaged DNA but not in a mutant defective in DNA-polymerase I. It was demonstrated that DNA-polymerase I is located on the membrane of B. subtilis cells. This suggested a participation of DNA-polymerase I in binding of the chromosome to the membrane in UV-irradiated cells. UV-induced DMC did not dissociate when the cells were treated with inhibitors of DNA-gyrase. It, therefore, was qualitatively different from the DMC found during replication. The mechanisms of binding of the damaged DNA to the membrane in UV-irradiated cells of B. subtilis are discussed.     

Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis.

The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase activities of Bacillus subtilis were found to co-purify as a single multienzyme complex. Mutants of B. subtilis with defects in the pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase complex were correspondingly affected in branched-chain 2-oxo acid dehydrogenase complex activity. Selective inhibition of the E1 or lipoate acetyltransferase (E2) components in vitro led to parallel losses in pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex activity. The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes of B. subtilis at the very least share many structural components, and are probably one and the same. The E3 component appeared to be identical for the pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes in this organism and to be the product of a single structural gene. Long-chain branched fatty acids are thought to be essential for maintaining membrane fluidity in B. subtilis, and it was observed that the ace (pyruvate dehydrogenase complex) mutant 61142 was unable rapidly to take up acetoacetate, unlike the wild-type, indicative of a defect in membrane permeability. A single pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex can be seen as an economical means of supplying two different sets of essential metabolites.     

The levanase operon of Bacillus subtilis expressed in Escherichia coli can substitute for the mannose permease in mannose uptake and bacteriophage lambda infection.

Bacteriophage lambda adsorbs to its Escherichia coli K-12 host by interacting with LamB, a maltose- and maltodextrin-specific porin of the outer membrane. LamB also serves as a receptor for several other bacteriophages. Lambda DNA requires, in addition to LamB, the presence of two bacterial cytoplasmic integral membrane proteins for penetration, namely, the IIC(Man) and IID(Man) proteins of the E. coli mannose transporter, a member of the sugar-specific phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS transporters for mannose of E. coli, for fructose of Bacillus subtilis, and for sorbose of Klebsiella pneumoniae were shown to be highly similar to each other but significantly different from other PTS transporters. These three enzyme II complexes are the only ones to possess distinct IIC and IID transmembrane proteins. In the present work, we show that the fructose-specific permease encoded by the levanase operon of B. subtilis is inducible by mannose and allows mannose uptake in B. subtilis as well as in E. coli. Moreover, we show that the B. subtilis permease can substitute for the E. coli mannose permease cytoplasmic membrane components for phage lambda infection. In contrast, a series of other bacteriophages, also using the LamB protein as a cell surface receptor, do not require the mannose transporter for infection.     

Facilitation of bacteriophage lambda DNA injection by inner membrane proteins of the bacterial phosphoenol-pyruvate: carbohydrate phosphotransferase system (PTS)

Infection of Escherichia coli by bacteriophage lambda depends on two membrane protein complexes: (i) maltoporin (LamB) in the outer membrane for adsorption and (ii) the IIC(Man)-IID(Man) complex of the mannose transporter in the inner membrane for DNA penetration. IIC(Man) and IID(Man) are components of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) which together with the IIAB(Man) subunit mediate transport and phosphorylation of sugars. To identify structural determinants important for penetration of lambda DNA, the homologous IIC-IID complexes of E. coli, K. pneumoniae and B. subtilis, and chimeric complexes between the IIC and IID were characterized. All three complexes support sugar transport in E. coli. Only IIC-IID of E. coli and B. subtilis also support bacteriophage lambda infection. The six chimeric complexes had lost transport activity, but three containing IIC of E. coli or B. subtilis continue to support bacteriophage lambda infection. Complexes containing IIC(Man) and fusion proteins between truncated IID(Man) and alkaline phosphatase or beta-galactosidase support penetration of lambda DNA if less than 100 residues are missing from the C-terminus of IID(Man). Truncation of IIC(Man) renders the complex unstable. Taken together, these results suggest, that IIC is the major specificity determinant for lambda infection but that the IIC subunit is stably expressed only in a complex with the IID subunit. Lambda DNA in transit across the periplasmic space, but not transforming plasmid DNA, is inaccessible to the non-specific nuclease NucA of Anabaena sp. targeted to the periplasmic space either in soluble form or as a fusion protein to the C-terminus of IID(Man).     

Both raft- and non-raft proteins associate with CHAPS-insoluble complexes: some APP in large complexes

Components of caveolae and lipid rafts are characterized by their buoyancy after detergent extraction. Using flotations in density gradients, we now show that non-raft membrane molecules are also associated with detergent-insoluble, buoyant assemblies. When Triton X-100 cellular extracts were spun to equilibrium in Nycodenz, only components of classical rafts floated. In contrast, with the zwitterionic detergent CHAPS, non-raft residents such as calnexin and APP also buoyed. When CHAPS extracts were spun in non-equilibrium (velocity) conditions, some raft components rapidly exited the input fractions while other raft markers and non-raft molecules remained relatively immobile. This pointed to size heterogeneities of CHAPS-insoluble complexes. Combined velocity/equilibrium gradients broadly divided CHAPS-insoluble membrane complexes into three size categories, which all contained cholesterol and the glycosphingolipid GM1. Large complexes were enriched in caveolin and ESA. Medium size complexes were enriched in PrP, whereas small complexes contained non-raft proteins, PrP, and some ESA. While Alzheimer's APP was primarily confined to small assemblies, a portion of its glycosylated form did buoy with large complexes. Large CHAPS-insoluble complexes resemble, but are not equal to, classical rafts. These findings extend considerably the range of detergent-insoluble membranal domains.     

Assembly of glycoprotein-80 adhesion complexes in Dictyostelium. Receptor compartmentalization and oligomerization in membrane rafts

The phospholipid-anchored membrane glycoprotein (gp)-80 mediates cell-cell adhesion through a homophilic trans-interaction mechanism during Dictyostelium development and is enriched in a Triton X-100-insoluble floating fraction. To elucidate how gp80 adhesion complexes assemble in the plasma membrane, gp80-gp80 and gp80-raft interactions were investigated. A low density raft-like membrane fraction was isolated using a detergent-free method. It was enriched in sterols, the phospholipid-anchored proteins gp80, gp138, and ponticulin, as well as DdCD36 and actin, corresponding to components found in the Triton X-100-insoluble floating fraction. Chemical cross-linking revealed that gp80 oligomers were enriched in the raft-like membrane fraction, implicating stable oligomer-raft interactions. However, gp80 oligomers resisted sterol sequestration and were partially dissociated with Triton X-100, suggesting that compartmentalization in rafts was not solely responsible for their formation. The trans-dimer known to mediate adhesion was identified, but cis-oligomerization predominated and displayed greater accumulation during development. In fact, oligomerization was dependent on the level of gp80 expression and occurred among isolated gp80 extracellular domains, indicating that it was mediated by direct gp80-gp80 interactions. Rafts existed in gp80-null cells and such pre-existent membrane domains may provide optimal microenvironments for gp80 cis-oligomerization and the assembly of adhesion complexes.     

Phosphatidylethanolamine and phosphatidylglycerol are segregated into different domains in bacterial membrane. A study with pyrene-labelled phospholipids

To detect and characterize membrane domains that have been proposed to exist in bacteria, two kinds of pyrene-labelled phospholipids, 2-pyrene-decanoyl-phosphatidylethanolamine (PY-PE) and 2-pyrene-decanoyl-phosphatidylglycerol (PY-PG) were inserted into Escherichia coli or Bacillus subtilis membrane. The excimerization rate coefficient, calculated from the excimer-to-monomer ratio dependencies on the probe concentration, was two times higher for PY-PE than for PY-PG at 37 degrees C. This was ascribed to different local concentrations rather than to differences in mobility. The extent of mixing between the two fluorescent phospholipids, estimated by formation of their heteroexcimer, was found very low both in E. coli and B. subtilis, in contrast to model membranes. In addition, these two pyrene derivatives exhibited different temperature phase transitions and different detergent extractability, indicating that the surroundings of these phospholipids in bacterial membrane differ in organization and order. Inhibition of protein synthesis, leading to condensation of nucleoid and presumably to dissipation of membrane domains, indeed resulted in increased formation of heteroexcimers, broadening of phase transitions and equal detergent extractability of both probes. It is proposed that in bacterial membranes these phospholipids are segregated into distinct domains that differ in composition, proteo-lipid interaction and degree of order; the proteo-lipid domain being enriched by PE.     

Some properties of a membrane-deoxyribonucleic acid complex isolated from Bacillus subtilis.

Membrane-deoxyribonucleic acid (DNA) complexes were isolated from Bacillus subtilis by affinity for magnesium-Sarkosyl crystals. These complexes (M-bands) contained greater than 80% of the total cellular DNA; little of the remaining portion could be recovered in a secondary isolation. Isotopic labeling of the origin of replication showed this region of the chromosome to be closely associated with the cell membrane. Interruption of protein or DNA synthesis did not result in detachment of the chromosome from the membrane. Interruption of ribonucleic acid synthesis by rifampin resulted in a decreased ability to isolate DNA in the M-band. Analysis of attachment of the chromosome to membrane during the cell and replication cycles indicated that the chromosome is not released from the membrane at any time during the cell cycle.     

Unstable association in vitro between donor and recipient DNA in Bacillus subtilis.

In addition to stable donor-recipient DNA complexes, unstable complexes between donor and recipient DNA were formed in vitro with Bacillus subtilis. Whereas the stable complexes survived CsCl gradient centrifugation at pH 11.2 and phenol plus sodium p-aminosalicylate extraction with 0.17 M NaCl, the unstable complexes dissociated during these manipulations. The donor moiety from the unstable complexes remained associated with the recipient DNA during phenol plus sodium p-aminosalicylate treatment at 0.85 M NaCl. The unstable complexes could be stabilized artificially by cross-linking with 4,5',8-trimethylpsoralen. Dissociation of the complexes during CsCl gradient centrifugation could be prevented by centrifuging at pH 10. Heterologous DNA fragments derived from phage H1 DNA appeared to be unable to form complexes with the recipient B. subtilis DNA. Unstable complexes were also formed with Escherichia coli DNA, although under all conditions tested, more complex was detectable by using homologous B. subtilis DNA.     

Cell wall-DNA association in Bacillus subtilis.

Autolysis of cell walls of Bacillus subtilis 168 resulted in solubilization of wall-associated DNA. Most of the DNA was solubilized only in the later stages of autolysis. Solubilization of up to 70% of the wall by autolysins resulted in only 25 to 30% solubilization of wall-associated DNA. When the wall fragments remaining after 70% autolysis were analyzed by electron microscopy, it was observed that the preparations were highly enriched for completed septa, or poles. Partial autolysis at pH 5.2 or pH 8.6, both of which reflect hydrogen ion levels that permit either N-acetylglucosaminidase or N-acetylmuramyl-L-alanine amidase, but not both, to act, gave rise to enrichment of cell poles. When walls were incubated with subtilisin, DNase, or RNase, release of DNA (or DNA fragments) was accelerated. Density gradient centrifugation patterns of lysates of cells pulse-labeled with N-[3H]acetylglucosamine and then chased revealed that a small, but significant, proportion of the radioactivity sedimented to a density position equivalent to that of DNA-membrane complexes. Because the pulse-chase sequence enriched for radioactivity in cell poles, the results suggest that at least some molecules from polar cell walls have an affinity for DNA-membrane complexes. We suggest that DNA binds strongly, possibly via a DNA-membrane complex, to cell poles of B. subtilis. The results provide support for a view offered previously (Koch et al., FEMS Microbiol. Lett. 12:201-208, 1981) that some special structure in or very near the poles of gram-positive bacilli is involved in the segregation of DNA during cell division.     

Evidence that the middle T antigen of polyomavirus interacts with the membrane skeleton.

The transforming protein of polyomavirus, middle T antigen, is associated with cellular membranes. We have examined the subcellular location of the middle T antigen in two different cell types by fractionation and detergent phase partitioning. Middle T antigen expressed in human cells by a recombinant adenovirus was detected primarily in the membrane skeleton. Sucrose gradient fractionation revealed that the middle T antigen was associated with complexes with molecular weights of 500,000 to 1,000,000. Several markers for cytoskeleton cofractionate with these complexes, including actin, tubulin, and vimentin. Electron micrographs of membrane skeleton prepared from cells expressing middle T antigen demonstrated that this material contained primarily fibrous structures and was clearly devoid of bilayer membranes. These structures were distinct from the filamentous structures observed in fractions enriched for cytoskeleton. Consistent with a role for membrane skeleton localization in transformation, middle T antigen was detected exclusively in fractions enriched for membrane skeleton in middle T antigen-transformed Rat-2 cells. Our results may resolve the apparent difference between middle T antigen localization as determined by immunomicroscopy and that determined by subcellular fractionation.     

The TatAd component of the Bacillus subtilis twin-arginine protein transport system forms homo-multimeric complexes in its cytosolic and membrane embedded localisation

The twin arginine translocation (Tat) system has the capacity to transfer completely folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The most abundant TatA protein of this system has been suggested to form the protein conducting channel. Here, the molecular organisation of soluble and membrane embedded Bacillus subtilis TatAd was analysed using negative contrast and freeze-fractured electron microscopy. In both compartments, the protein showed homo-oligomerisation. In aqueous solution, TatAd formed homo-multimeric micelle-like complexes. Freeze-fracture analysis of proteoliposomes revealed self association of membrane-integrated TatAd independent from TatCd, the second component of this transport system. Immunogold labelling demonstrated that the substrate prePhoD was co-localised with membrane-integrated TatAd complexes.     

Altered accumulation of a membrane protein unique to a membrane-deoxyribonucleic acid complex in a dna initiation mutant of Bacillus subtilis.

Membrane-deoxyribonucleic acid complexes (M-bands) have been isolated from Bacillus subtilis by their affinity for crystals of Mg2+-Sarkosyl. The membrane proteins of these complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the membrane protein composition of M-band and unfractionated membrane revealed three protein components of 125,000 (mac-1), 57,000 (mac-2), and 42,000 (mac-3) daltons unique to M-band membrane. Growth of a temperature-sensitive dna initiation mutant at the restrictive temperature resulted in an accumulation in the membrane of mac-2. This accumulation did not begin, however, until cell growth had nearly ceased, some 3 to 4 h after the cessation of deoxyribonucleic acid synthesis. Upon return of the mutant to the permissive temperature, mac-2 did not begin to return to normal levels until after the first round of deoxyribonucleic acid synthesis. A protein of 30,000 daltons, common to both M-band and whole membrane, was found to disappear from the membrane when the mutant was grown at the restrictive temperature. This disappearance is the result of increased degradation or removal from the membrane followed by a decreased rate of synthesis or insertion.     

Liposomes comprising anionic but not neutral phospholipids cause dissociation of Rac(1 or 2) x RhoGDI complexes and support amphiphile-independent NADPH oxidase activation by such complexes

Activation of the phagocyte NADPH oxidase involves the assembly of a membrane-localized cytochrome b559 with the cytosolic components p47(phox), p67(phox), p40(phox), and the GTPase Rac (1 or 2). In resting phagocytes, Rac is found in the cytosol as a prenylated protein in the GDP-bound form, associated with the Rho GDP dissociation inhibitor (RhoGDI). In the process of NADPH oxidase activation, Rac is dissociated from RhoGDI and translocates to the membrane, in concert with the other cytosolic components. The mechanism responsible for dissociation of Rac from RhoGDI is poorly understood. We generated Rac(1 or 2) x RhoGDI complexes in vitro from recombinant Rac(1 or 2), prenylated enzymatically, and recombinant RhoGDI, and purified these by anion exchange chromatography. Exposing Rac(1 or 2)(GDP) x RhoGDI complexes to liposomes containing four different anionic phospholipids caused the dissociation of Rac(1 or 2)(GDP) from RhoGDI and its binding to the anionic liposomes. Rac2(GDP) x RhoGDI complexes were more resistant to dissociation, reflecting the lesser positive charge of Rac2. Liposomes consisting of neutral phospholipid did not cause dissociation of Rac(1 or 2) x RhoGDI complexes. Rac1 exchanged to the hydrolysis-resistant GTP analogue, GMPPNP, associated with RhoGDI with lower affinity than Rac1(GDP) and Rac1(GMPPNP) x RhoGDI complexes were more readily dissociated by anionic liposomes. Rac1(GMPPNP) x RhoGDI complexes elicited NADPH oxidase activation in native phagocyte membrane liposomes in the presence of p67(phox), without the need for an anionic amphiphile, as activator. Both Rac1(GDP) x RhoGDI and Rac1(GMPPNP) x RhoGDI complexes elicited amphiphile-independent, p67(phox)-dependent NADPH oxidase activation in phagocyte membrane liposomes enriched in anionic phospholipids but not in membrane liposomes enriched in neutral phospholipids.