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We have recently reported (M. Petricek, L. Rutberg, I. Schröder, and L. Hederstedt, J. Bacteriol. 172: 2250-2258, 1990) the cloning and sequence of a Bacillus subtilis chromosomal DNA fragment containing hemA proposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encoding hydroxymethylbilane synthase. In the present communication, we report the sequences and identities of three additional hem genes located immediately downstreatm of hemC, namely, hemD encoding uroporphyrinogen III synthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-1-semialdehyde 2,1-aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for the synthesis of uroporphyrinogen III from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shown to be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create a growth requirement for ALA or heme, indicating that formation of ALA from glutamate-1-semialdehyde can occur spontaneously in vivo or that this reaction may also be catalyzed by other enzymes. An analysis of B. subtilis carrying integrated plasmids or deletions-substitutions in or downstream of hemL indicates that no further genes in heme synthesis are part of the proposed hem operon.

The cytoplasmic membrane of Bacillus subtilis 168, prepared from cells in the stationary Analyses of the membrane lipid revealed the presence of phospholipids (75OiO), neutral lipid and a compound identified as a diglucosyl diglyceride. The major phospholipids were diphosphatidyl glycerol and phosphatidyl ethanolamine, with small amounts of phosphatidyl glycerol and lipoamino acids.Branched chain fatty acids comprised over 7501, of the total fatty acids of both whole cells and membranes. Is0 and anteiso acids with 15 and 17 carbon atoms were the major components, together with small amounts of is0 acids containing 14 and 16 carbon atoms and n-acids. No unsaturated acids were present. phase, has been found to contain protein (62O/,), RNA (22O/,) and lipid (160/0). [4,5] take place a t the membrane.Although the cytoplasmic membrane holds a central position in bacterial metabolism, the detailed composition of the membrane has been studied in relatively few species. Whilst protein and lipid are apparently ubiquitous constituents of the membrane, the amount of RNA and carbohydrate present has been found to vary widely. Many of these variations can no doubt be ascribed to differences in species and culture conditions, but the lack of uniformity in the analyses makes it desirable that further information be obtained.In order to provide a basis for a detailed investigation of membrane structure in Bacillus subtilis, the chemical composition of the membrane has been determined. MATERIALS AND METHODS Organism and Growth ConditionsBacillus subtilis strain 168 was used throughout. Cells were grown in a medium containing (per litre), NH4C1, 2 g ; K,HPO,, 14 g ; KH,PO,, 6 g ; Na citrate 2 H,O, 1 g; Difco casamino acids, 10 g; pH, 7.6. After autoclaving, MgSO,, 0.2 g and glucose, 5 g were added together with trace metals. An overnight Extraction of the casamino acids used in the culture medium by refluxing with chloroform showed that the fatty acid content was less than 0.5 mg per 100 g of casamino acids. Preparation of Protoplasts and MembranesThe cells were suspended in 0.2 M phosphate buffer pH 6.6 containing 0.25 M sucrose, a t a concentration of 1-2 x loll cells/ml. 1.3 mg of lysozyme and 2 pg of pancreatic DNAase were added per ml of suspension and the cells incubated at 30" for 30 to 60 min until microscopic examination showed > 9Q0/, protoplast formation. The protoplasts were collected by centrifugation (40,000 x g for 50 min) and ruptured by resuspension in distilled water containing mM Mg++ and DNAase (1 pg/ml). The mixture was incubated at room temperature until the viscosity decreased to a level where the solution could be readily pipetted. The membranes were collected by centrifugation (48,000 x g for 30 min), washed five times with 0.Qo/, (w/v) NaCl and stored frozen a t Reagents Lysozyme was obtained from the Sigma Chemical Company and DNAase from Worthington Biochemical Corporation. Analytical grade solvents were used as purchased, all other solvents were redistilled before use.-15".

Bacteriophage 4105 is a temperate bacteriophage for Bacillus subtilis 168. Temperature-sensitive and plaque mutants of 4105 were isolated. The results of two-and three-factor crosses with these mutants suggest the vegetative map of 4105 to be circular. The location of prophage 4105 between bacterial markers phe-J and ilvAl was shown by means of PBS1 transduction. Five markers in the prophage were linearly ordered with respect to the bacterial markers. Linkage between bacterial and prophage markers was demonstrated in transformation experiments with deoxyribonucleic acid extracted from lysogenic bacteria. The data demonstrate that prophage 4105 is linearly inserted into the bacterial chromosome.

The nucleotide sequence of a 2.7-kilobase segment of DNA containing the sdhA and sdhB genes encoding the flavoprotein (Fp, sdhA) and iron-sulfur protein (Ip, sdhB) subunits of the succinate dehydrogenase of BaciUus subtilis was determined. This sequence extends the previously reported sequence encoding the cytochrome b558 subunit (sdhC) and completes the sequence of the sdh operon, sdhCAB. The The structural genes encoding the subunits of the SDH complex form an operon at 225°on the B. subtilis linkage map (42). For compatibility with Escherichia coli, the B. subtilis genes have been redefined as sdhC (cytb558, formerly sdhA), sdhA (Fp, formerly sdhB), and sdhB (Ip, formerly sdhC), so the operon is transcribed in the order sdhCAB (11,17,23,33,60 (40), and SDHD (32, 60). The nucleotide sequence of the sdh operon (sdhCDAB) of E. coli has been determined previously and compared with that of the frdABCD operon, which encodes the similar but genetically distinct and anaerobically derepressed enzyme fumarate reductase (FRD) (6,7,11,60). The Fp and Ip subunits of the two enzymes exhibit high degrees of mutual sequence homology both for the predicted amino acid sequences and for the corresponding genes. The nucleotide sequence revealed the presence of two hydrophobic subunits encoded by proximal (sdhCD) rather than distal (frdCD) genes, but despite similarities in size and hydropathy profile, there is little homology between the predicted amino acid sequences for the corresponding gene products. The present work is aimed at defining the differences between the 3-and 4-subunit SDH complexes and comparing the analogous complexes of gram-positive and -negative bacteria. Here the nucleotide sequence of the B. subtilis sdhCAB operon is extended by 2.5 kb to include the structural genes encoding the Fp (sdhA) and Ip (sdhB) subunits, and a comparison of the subunit amino acid sequences with those of the analogous subunits of the E. coli SDH and fumarate reductase is presented. MATERIALS AND METHODSBacteria and plasmids. Plasmid pSH1047 sdhC+ sdhA+ sdhB+ gerE+ has been described elsewhere (17). E. coli MV1OCh3/86 (C600 AtrpES carrying hybrid X prophage Ch3/86 with trfA and trfB of plasmid RK2) was used as a transformation host for preparing plasmid DNA (17). Strain

A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.

Bacillus subtilis mutants unable to catabolize glycerol (Glp mutants) were isolated and mapped. The location of the mutations on the chromosome was determined by a density transfer technique and confirmed by PBS1 transduction and transformation. The different mutations were ordered relative to each other. Mutations rendering the cells glycerol auxotrophic were also mapped and found not to be linked to the Glp mutations.

Antibodies specific for the Mr 65,000 (flavoprotein) and the Mr 28,000 subunits of the succinic dehydrogenase (SDH) of Bacillus subtilis were obtained. By using these antibodies it was shown that both subunits accumulated in the cytoplasm during 5-aminolevulinic acid starvation of a 5-aminolevulinic acid auxotroph. In the cytoplasm the subunits were not associated since they precipitated essentially independently of each other with subunit-specific antibody. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the cytoplasmic subunits migrated identically with the corresponding subunits from the purified membrane-bound SDH complex. Cytoplasmic subunits were pulse-labeled with L-[3S]methionine during 5-aminolevulinic acid starvation. The labeled subunits bound to the membrane when heme synthesis was resumed and also when protein synthesis was blocked by chloramphenicol before readdition of 5-aminolevulinic acid. The experiments thus demonstrated a precursor relationship between cytoplasmic subunits and the subunits of the membrane-bound SDH complex. All SDHnegative mutants isolated so far carry mutations in the citF locus. None of the mutants was found to have either the Mr 65,000 or the Mr 28,000 SDH subunits in the membrane. Four citF mutants, however, contained both subunits in the cytoplasm. Three of these mutants lacked spectrally detectable cytochrome b5m,. The respective mutations mapped at one end of the citF locus. These results strongly support our previous suggestion that cytochrome b&,% is (part of) a membrane binding site for SDH in B. subtilis.