The effect of spo0 mutations on the expression of spo0A- and spo0F-lacZ fusions

Summary We have constructedspo0A-lacZ andspo0F-lacZ fusions with a temperate phage vector and have investigated howspo0 gene products are involved in the expression of each of these genes. The expression ofspo0A-lacZ andspo0F-lacZ was stimulated at about the time of cessation of vegetative growth in Spo⁺ cells. This stimulation ofspo0A-lacZ was impaired by mutations in thespo0B, D, E, F orH genes but was not affected by mutations in thespo0J orK genes. Similar results were obtained with thespo0F-lacZ fusion. The effect of thespo0A mutation onspo0A-lacZ expression was characteristic: thespo0A-directed β-galactosidase activity found during vegetative growth was significantly enhanced in thespo0A mutant. This result suggests thatspo0A gene expression is autoregulated being repressed by its own gene product. Another remarkable observation was the effect of thesof-1 mutation, which is known to be aspo0A allele; it suppressed the sporulation deficiency ofspo0B, spo0D andspo0F mutants. Thespo0A-lacZ stimulation, which is impaired by any one of thesespo0 mutations, was restored by the additionalsof-1 mutation.     

Defects in the nutrient-dependent methylation of a membrane-associated protein in spo mutants of Bacillus subtilis

Summary Methylation of a membrane-associated protein with an apparent molecular mass of 40000 daltons has been observed in Bacillus subtilis. The methylation was nutrient dependent and occurred with a doubling time of 4 ± 1 min. In wild-type strains, the half-life of turnover of the methyl group(s) was 17 ± 6 min. Several isogenic strains of B. subtilis containing spo0 mutations (spo0A and spo0H) were found to be normal in glutamate-dependent methylation of the protein and turnover of the methyl group(s). In strains containing spo0B and spo0E mutations, the methyl group(s) were incorporated in response to glutamate addition but turnover was not at a normal rate. The half-life of methyl group turnover was extended to 45 ± 3 min in these strains. In a spo0K mutant and in spoILI and spoIIF mutants, the protein was not significantly methylated. The methylation of a 40000 dalton protein was also found to be dependent on phosphate. This methylation was observed in wild-type and spo0A and spo0H strains with a doubling time of 4 ± 1 min and a half-life of turnover of the methyl group(s) of 11 + 3 min. In strains containing spo0B, spo0E, and spo0F mutations, the phosphate-dependent incorporation of the methyl group(s) was normal (5 ± 1 min) but the turnover half-life was extended to 46 ± 8 min. It is not known whether the nitrogen-dependent and phosphate-dependent systems methylated the same protein. The spo0 mutants are defective in the initial stages of sporulation, and it has been proposed that the spo0 gene products may play a role in nutrient sensing. The discovery of defects in the methylation of the 40 kDa protein in some of these spo0 mutants supports the proposal that the protein methylation may be part of a nutrient sensing system for the control of growth and sporulation in Bacillus species.     

Identification of the Product of Sporulation Gene spo0B in Bacillus subtilis

A 1.4-megadalton EcoRI restriction fragment carrying Bacillus subtilis sporulation gene spo0B was cloned from the specialized transducing phage, φ 105spo0B, into a unique EcoRI site of plasmid vector pUB110, and four plasmids having a deletion in the 1.4-megadalton EcoRI fragment were constructed. Analysis of the polypeptides synthesized in B. subtilis minicells harboring these plasmids and the sporulation ability of strain UOT0436 (spo0B136 recE4) harboring these plasmids showed that the spo0B gene product is a polypeptide of 24,000 daltons. Two-dimensional polyacrylamide gel analysis showed that the isoelectric point of this protein is almost neutral.     

Bacillus sporulation gene spo0H codes for sigma 30 (sigma H).

The DNA sequences of the spo0H genes from Bacillus licheniformis and B. subtilis are described, and the predicted open reading frames code for proteins of 26,097 and 25,447 daltons, respectively. The two spo0H gene products are 91% identical to one another and about 25% identical to most of the procaryotic sigma factors. The predicted proteins have a conserved 14-amino-acid sequence at their amino terminal end, typical of sigma factors. Antibodies raised against the spo0H gene product of B. licheniformis specifically react with RNA polymerase sigma factor protein, sigma 30, purified from B. subtilis. We conclude that the spo0H genes of B. licheniformis and B. subtilis code for sigma 30, now known as sigma H.     

Nickel,a component of factor F 430 from Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum, growing on medium supplemented with 2 mol ⁶³NiCl₂/l, was found to take up 1.2 mol ⁶³Ni per g cells (dry weight). More than 70% of the radioisotope was incorporated into a compound, which dissociated from the protein fraction after heat treatment, was soluble in 70% acetone, and could be purified by chromatography on QAE-Sephadex A-25, Sephadex G-25, and DEAE cellulose. The purified ⁶³Ni labelled compound had an absorption spectrum and properties identical to those of factor F ₄₃₀ and is therefore considered to be identical with factor F ₄₃₀.Factor F ₄₃₀, a compound of molecular weight higher than 1000 with an absorbance maximum at 430 nm, has recently been purified from Methanobacterium thermoautotrophicum (Gunsalus and Wolfe, 1978). The structure and function of this compound are not yet known.     

Purification of the NusB gene product of Escherichia coli K12

Summary The nucleotide sequence of the entire nusB gene of Escherichia coli has recently been determined and the amino acid sequence of its product deduced (Ishii et al. 1984; Swindle et al. 1984). The NusB protein was purified by chromatography on Sephadex G-100, phosphocellulose and hydroxylapatite. Purification of the protein was monitored using ¹⁴C-labelled NusB protein, which was synthesized in a maxicell containing an nusB plasmid as a marker. The final product, which was at least 95% pure as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, had a molecular weight of about 16,000 and an isoelectric point of about 7.3. Analytical data on the amino acid composition of the purified protein agreed with that deduced from the DNA sequence and indicated that this protein was indeed the product of the nusB gene.Abbreviations SDS sodium dodecyl sulphate - kDa kilodaltons - bp base pair(s) - kbp kilobase pair(s)     

Genetic Evidence for the Involvement of the S-Layer Protein Gene sap and the Sporulation Genes spo0A,spo0B,and spo0F in Phage AP50c Infection of Bacillus anthracis

In order to better characterize the Bacillus anthracis typing phage AP50c, we designed a genetic screen to identify its bacterial receptor. Insertions of the transposon mariner or targeted deletions of the structural gene for the S-layer protein Sap and the sporulation genes spo0A, spo0B, and spo0F in B. anthracis Sterne resulted in phage resistance with concomitant defects in phage adsorption and infectivity. Electron microscopy of bacteria incubated with AP50c revealed phage particles associated with the surface of bacilli of the Sterne strain but not with the surfaces of Δsap, Δspo0A, Δspo0B, or Δspo0F mutants. The amount of Sap in the S layer of each of the spo0 mutant strains was substantially reduced compared to that of the parent strain, and incubation of AP50c with purified recombinant Sap led to a substantial reduction in phage activity. Phylogenetic analysis based on whole-genome sequences of B. cereus sensu lato strains revealed several closely related B. cereus and B. thuringiensis strains that carry sap genes with very high similarities to the sap gene of B. anthracis. Complementation of the Δsap mutant in trans with the wild-type B. anthracis sap or the sap gene from either of two different B. cereus strains that are sensitive to AP50c infection restored phage sensitivity, and electron microscopy confirmed attachment of phage particles to the surface of each of the complemented strains. Based on these data, we postulate that Sap is involved in AP50c infectivity, most likely acting as the phage receptor, and that the spo0 genes may regulate synthesis of Sap and/or formation of the S layer.     

Isolation,characterization, and reconstitution of a solubilized fraction containing the hydrophobic sector of the mitochondrial proton pump

The hydrophobic sector of the mitochondrial ATPase complex was purified by sequential extraction with cholate and octylglucoside, by further differential solubilization with guanidine and cholate in the presence of phosphatidylcholine, and by fractionation with ammonium sulfate. A polypeptide with a mass of 28,000 dalton was present in the purified hydrophobic section which was cleaved by trypsin, resulting in loss of reconstitution activity. In contrast, dicyclohexylcarbodiimide-binding proteolipid remained unimpaired after exposure to trypsin. The³²P_i-ATP exchange activity of the reconstituted ATPase complex was inhibited byp-hydroxymercuribenzoate, which reacted primarily with the 28,000-dalton protein, as monitored by acrylamide gel electrophoresis with¹⁴C-labeled inhibitor. The function of a 22,000-dalton polypeptide and of some minor components in the region of the proteolipid remains unknown. An examination of the phospholipid requirements for reconstitution of an active complex revealed an unexpected discrepancy. With an excess of phosphatidylethanolamine, optimal reconstitution of³²P_i-ATP exchange and ATP synthesis in the presence of bacteriorhodopsin and light was achieved; at a high phosphatidylcholine:phosphatidylethanolamine ratio, the rate of ATP synthesis remained high, but the rate of³²P_i-ATP exchange dropped precipitously. A new procedure is described for the reconstitution of the ATPase complex with purified phospholipids which is stable for at least 15 days.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - STE-DTT buffer sucrose (250 mM), Tricine-KOH (50 mM), EDTA (5 mM), DTT (5 mM), pH 8.0 - F _o a membranous preparation from mitochondria conferring oligomycin (or rutamycin) sensitivity to F₁ - F₁F₆ coupling factors 1 (ATPase) and 6 - OSCP oligomycin-sensitivity-conferring protein - BSA bovine serum albumin - SDS sodium dodecyl sulfate - DTT dithiothreitol - STE buffer sucrose (250 mM), Tricine-KOH (50 mM), EDTA (5 mM) - TUA particles submitochondrial particles prepared by stepwise exposure of light-layer submitochondrial particles to trypsin and urea, then sonic oscillation in the presence of dilute ammonia (pH 10.4) - OG-cholate buffer glycerol (20%), Tricine (50 mM), MgSO₄ (5 mM), DTT (5mM), cholate (0.5%), octylglucoside (0.5%), pH 8.0 - p-HMB p-hydroxymercuribenzoate     

Characterization of the gene for a protein kinase which phosphorylates the sporulation-regulatory proteins Spo0A and Spo0F of Bacillus subtilis.

The kinA (spoIIJ) locus contains a single gene which codes for a protein of 69,170 daltons showing strong homology to the transmitter kinases of two component regulatory systems. The purified kinase autophosphorylates in the presence of ATP and mediates the transfer of phosphate to the Spo0A and Spo0F sporulation regulatory proteins. Spo0F protein was a much better phosphoreceptor for this kinase than Spo0A protein in vitro. Mutants with deletion mutations in the kinA gene were delayed in their sporulation. They produced about a third as many spores as the wild type in 24 h, but after 72 h on solid medium, the level of spores approximated that found for the wild-type strain. Such mutations had no effect on the regulation of the abrB gene or on the timing of subtilisin expression and therefore did not impair the repression function of the Spo0A protein. Placement of the kinA locus on a multicopy vector suppressed the sporulation-defective phenotype of spo0B, spo0E, and spo0F mutations but not of spo0A mutations. The results suggest that the spo0B-, spo0E-, and spo0F-dependent pathway of activation (phosphorylation) of the Spo0A regulator may be by-passed through the kinA gene product if it is present at sufficiently high intracellular concentration. The results suggest that multiple kinases exist for the Spo0A protein.     

Over-expression and characterization of the recombinant small heat shock protein from Pyrococcus furiosus

The gene encoding a small heat shock protein (sHSP) from Pyrococcus furiosus was redesigned and chemically synthesized by using bacteria-preferred codons. The gene product was over-expressed in Escherichia coli BL21(DE)₃ and purified to homogeneity. In the presence of this protein, the activities of Taq DNA polymerase, DNA restriction endonuclease HindIII and lysozyme were protected at elevated temperature, and also, thermal aggregation of lysozyme was prevented by this purified recombinant sHSP.Huayou Chen, Zhongmei Chu, Contributed equally to this work     

Early-blocked sporulation mutations alter expression of enzymes under carbon control in Bacillus subtilis

Summary The physiological roles of the gene subset defined by early-blocked sporulation mutations (spo0) and their second-site suppressor alleles (rvtA11 and crsA47) remain cryptic for both vegetative and sporulating Bacillus subtilis cells. To test the hypothesis that spo0 gene products affect global regulation, we assayed the levels of carbon- and nitrogen-sensitive enzymes in wild-type and spo0 strains grown in a defined minimal medium containing various carbon and nitrogen sources. All the spo0 mutations (except spo0J) affected both histidase and arabinose isomerase levels in an unexpected way: levels of both carbon-sensitive enzymes were two- to six-fold higher in spo0 strains compared to wild type, when cells were grown on the derepressing carbon sources arabinose or maltose. There was no difference in enzyme levels with glucose-grown cells, nor was there a significant difference in levels of the carbonindependent enzymes glutamine synthetase and glucose-6-phosphate dehydrogenase. This effect was not due to a slower growth rate for the spo0 mutants on the poor carbon and nitrogen sources used. The levels of carbon-sensitive enzymes were not simply correlated with sporulation ability in genetically suppressed spo0 mutants, but the rvtA and crsA suppressors each had such marked effects on wild-type growth and enzyme levels that these results were difficult to interpret. We conclude that directly or indirectly the spo0 mutations, although blocking the sporulation process, increase levels of carbon-sensitive enzymes, possibly at the level of gene expression.     

Membrane reconstitution in chl-r mutants of Escherichia Coli K 12: VIII. Purification and properties of the FA factor,the product of the chl B gene

The isolation and purification of the product of the chl B gene of Escherichia coli K 12 from the chl A mutant have been attempted. The purified protein gives a single band in 10 % sodium dodecylsulfate/polyacrylamide gel electrophoresis. The molecular weight is estimated to be 35 000. This protein, that we have named “F_A factor”, does not contain any lipid, has a strong tendency to lose its activity by polymerizing but can be kept in an active state when stored in buffer containing NaCl. The addition of purified F_A protein to a soluble extract from the chl B mutant strain grown under anaerobiosis in the presence of nitrate initiates the “complementation reaction”, i.e. the reconstitution of the nitrate reductase activity and the formation of particulate material similar to the native membrane with respect to the structure and enzymatic function. F_A protein acts both on the rate of reconstitution and on the total amount of reconstituted enzyme. The complementation leads to the reconstitution of nonsedimentable nitrate reductase and to the formation of three types of particles of different buoyant densities (1.10, 1.18 and 1.23) the two lightest of which contain nitrate reductase. It is shown that F_A factor is incorporated only into the particles of intermediate density. In vivo, this factor is located in the native membranes of chl A, chl C, chl D and wild-type strains, whatever the growth conditions, aerobiosis or anaerobiosis, and in the presence or absence of nitrate. Protein F_A can be released from either of these membranes (native or reconstituted) by removing Mg²⁺ or by subjecting Kaback's vesicles to mechanical treatments; in the case of 1.18-reconstituted particles and wild-type membranes, the release of F_A protein does not exert any effect on the level of the nitrate reductase activity.     

Photosystem II function and herbicide binding sites during photoinhibition of spinach chloroplasts in-vivo and in-vitro

The time courses of some Photosystem II (PS II) parameters have been monitored during in-vivo and in-vitro photoinhibition of spinach chloroplasts, at room temperature and at 10 °C or 0 °C. Exposing leaf discs of low-light grown spinach at 25 °C to high light led to photoinhibition of chloroplasts in-vivo as manifested by a parallel decrease in the number of functional PS II centres, the variable chlorophyll fluorescence at 77K (F _v /F _m ), and the number of atrazine-binding sites. When the photoinhibitory treatment was given at 10 °C, the former two parameters declined in parallel but the loss of atrazine-binding sites occurred more slowly and to a lesser extent. During in-vitro photoinhibition of chloroplast thylakoids at 25 °C, the loss of functional PS II centres proceeded slightly more rapidly than the loss of atrazine-binding sites, and this difference in rate was further increased when the thylakoids were photoinhibited at 0 °C. During the recovery phase of leaf discs (up to 9 h) the increases in F _v /F _m preceded that of the number of functional PS II centres, while only a further decline in the number of atrazine-binding sites was observed. The recovery of variable chlorophyll fluorescence and the concentration of functional PS II centres occurred more rapidly at 25 °C than at 10 °C. These results suggest that the photoinhibition of PS II function is a relatively temperature-independent early photochemical event, whereas the changes in the concentration of herbicide-binding sites appear to be a more complex biochemical process which can occur with a delayed time course.Abbreviations BSA bovine serum albumin - Chl chlorophyll - D1 32kDa herbicide-binding polypeptide in photosystem II and product of the psbA gene - D2 34kDa polypeptide in photosystem II which is the product of the psbD gene - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolin-dophenol - F ₀, F _v , F _m chlorophyll fluorescence with reaction centres open, variable and maximum fluorescence, respectively - LDS lithium dodecyl sulfate - MES 2-(N-morpholino) ethanesulfonic acid - PSII photosystem II - Q_A, Q_B first and second quinone-type PS II acceptor, respectively     

Purification and characteristics of hydrophobic membrane protein(s) required for DCCD sensitivity of ATPase in mycobacterium phlei

The energy-transducing N,N′-dicyclohexylcarbodiimide-sensitive (DCCD-sensitive) ATPase complex consists of two parts, a soluble catalytic protein (F₁), and an intrinsic membrane protein (F₀). The bacterial coupling factor complex, BCF₀-BCF₁, has recently been purified from Mycobacterium phlei, and used to reconstitute oxidative phosphorylation in detergent-extracted membranes. The BCF₀ moiety has been purified by being recovered from the purified BCF₀-BCF₁ complex by affinity chromatography. BCF₀ is a lipoprotein or lipoprotein complex with an approximate molecular weight of 60,000. The preparation contained 0.15 mg of phospholipid per milligram protein. There appear to be three polypeptides, with approximate molecular weights of 24,000, 18,000, and 8,000 as determined by sodium dodecylsulfate a crylamide gel electrophoresis. Purified BCF₀ conferred DCCD sensitivity on a purified BCF₁ preparation. Reconstitution of oxidative phosphorylation was achieved after incubation of detergent-extracted membranes with purified BCF₀ and purified BCF₁.     

Regulatory proteins of F1F0-ATPase: Role of ATPase inhibitor

An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F₁F₀-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F₁F₀-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F₁F₀-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F₁ in a manner similar to the inhibitor. The 15K protein binds to the F₀ part and holds the inhibitor and the 9K protein on F₁F₀-ATPase even when one of them is detached from the F₁ part.