The complete primary structure of the purple membrane protein bacteriorhodopsin, which contains 248 amino acid residues, has been determined. Methods used for separation of the hydrophobic fragments included gel permeation and reverse-phase high-pressure liquid chromatography in organic solvents. The amino acid sequence was determined by a combination of automatic Edman degradation and mass spectrometric methods. The total sequence was derived by ordering of the CNBr fragments on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry and by analysis of N-bromosuccinimide fragments containing overlaps between CNBr fragments. The present sequence differs from that recently reported by Ovchinnikov and coworkers with respect to an additional tryptophan (position 138) and several amino acid assignments.The purple membrane of a number of extremely halophilic bacteria-e.g., Halobacterium halobium-functions as a light-driven proton pump (1, 2). It contains a single protein, bacteriorhodopsin (Mr 26,000) with one molecule of retinaldehyde covalently bound to a lysine residue (3, 4). Bacteriorhodopsin forms a continuum of seven a helices, each of which spans the membrane and is largely embedded in it (5). Knowledge of the primary structure of bacteriorhodopsin is a prerequisite to studies of the mechanism of the proton pump and of the biogenesis of this interesting protein. In a recent paper, we reported (6) on the sequence of the first 102 amino acids and of 39 amino acids from the COOH terminus of this membrane protein. The present paper reports on its complete amino acid sequence (Fig. 1). Although no experimental details have appeared, the complete sequence has also been deduced by Ovchinnikov and coworkers (7,8), and partial sequences have been reported by other laboratories (3, 9, 10). The present sequence differs from that reported by Ovchinnikov and coworkers with respect to an additional tryptophan (position 138) and amino acid assignments at positions 105, 111, 117, 146, and 206. Of the total 248 amino acids present in bacteriorhodopsin, 70% are hydrophobic, and there is significant clustering of the hydrophobic as well as of the hydrophilic amino acids. MATERIALS AND METHODSMaterials. N-Bromosuccinimide (NBS), fluorescamine, and isothiocyanatophenylthiocarbamoylaminopropyl (IPTAP) glass were purchased from Pierce, and trypsin treated with L-(tosylamido-2-phenyl)ethyl chloromethyl ketone, clostripain, and elastase were from Worthington Biochemicals. ['4CISuc-cinic anhydride was from New England Nuclear. Tetraethyltetraamino (TETA) and aminoethylaminopropyl (AEAP) glass were gifts from Mark Horn of Sequemat, Watertown, MA. Other materials were as described previously.Preparation of Chymotryptic and CNBr Fragments. Purple membrane was isolated from H. halobium and apomembrane was prepared as described (6). Digestion of the apomembrane with chymotrypsin gave two fragments, C-1 and C-2, which were collected by centrifugation and separated on a Sephadex LH-60 column ...

The responses of Haemophilus influenzae to DNA gyrase inhibitors were analyzed at the transcriptional and the translational level. High-density microarrays based on the genomic sequence were used to monitor the expression levels of >80% of the genes in this bacterium. In parallel the proteins were analyzed by two-dimensional electrophoresis. DNA gyrase inhibitors of two different functional classes were used. Novobiocin, as a representative of one class, inhibits the ATPase activity of the enzyme, thereby indirectly changing the degree of DNA supercoiling. Ciprofloxacin, a representative of the second class, obstructs supercoiling by inhibiting the DNA cleavage-resealing reaction. Our results clearly show that different responses can be observed. Treatment with the ATPase inhibitor Novobiocin changed the expression rates of many genes, reflecting the fact that the initiation of transcription for many genes is sensitive to DNA supercoiling. Ciprofloxacin mainly stimulated the expression of DNA repair systems as a response to the DNA damage caused by the stable ternary complexes. In addition, changed expression levels were also observed for some genes coding for proteins either annotated as "unknown function" or "hypothetical" or for proteins not directly involved in DNA topology or repair.

We have used high-density oligonucleotide probe arrays (chips) for bacterial transcript imaging. We designed a chip containing probes representing 106 Hemophilus influenzae genes and 100 Streptococcus pneumoniae genes. The apparent lack of polyadenylated transcripts excludes enrichment of mRNA by affinity purification and we thus used total, chemically biotinylated RNA as hybridization probe. We show that hybridization of Streptococcus RNA to a chip allows simultaneous quantification of the transcript levels. The sensitivity was found to be in the range of one to five transcripts per cell. The quantitative chip results were in good agreement with conventional Northern blot analysis of selected genes. This technology allows simultaneous and quantitative measurement of the transcriptional activity of entire bacterial genomes on a single oligonucleotide probe array.

The orientation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by proteolytic , and whole cells are all consistent with the conclusion that the carboxyl terminus as well as the additional cleavage site in the apomembrane are on the cytoplasmic side of the purple membrane. A preliminary account of these findings has appeared (10). MATERIALS AND METHODS MaterialsTrypsin from porcine pancreas was purchased from Miles Laboratory and Pronase from Calbiochem; deoxyribonuclease, ovalbumin, cytochrome c, soybean trypsin inhibitor, basic pancreatic trypsin inhibitor, and carboxypeptidase A (di-isopropylfluorophosphate-treated) were all from Sigma Chemical Co.; proteinase K was from Boehringer-Manheim, Germany. Myoglobin was obtained from Beckman. MethodsPreparation of Purple Membrane. Halobacterium halobium cells were grown, and the purple membrane was isolated by sucrose density gradient as described (9,11). The concentration of the purple membrane was determined by using the molar extinction coefficient at 560 nm of 6.3 X 104 (1, 5); this value was confirmed by amino acid analysis.Preparation of Apomembrane. The purple membrane was suspended (2 mg/ml) in 4.0 M NaCl containing 1.0 M NH20H-HCI (pH 7.0) and the stirred suspension was irradiated at 250 with a 500-W quartz halogen lamp with a Schott 530 filter until the purple color had completely disappeared (12). The apomembrane was collected by centrifugation, washed twice with distilled water, and used immediately for proteolysis.High-Voltage Paper Electrophoresis. The supernatant obtained on centrifugation of the trypsin digest of the purple membrane was lyophilized, the residue was redissolved in water, and the solution was applied to Whatman 3MM paper (up to 0.1 gmol/cm). The paper was wetted with pyridine acetate (pH 6.1) (pyridine/acetic acid/water, 100:4:900) and Abbreviations: Mr, molecular weight; NaDodSO4, sodium dodecyl sulfate.

The sequence of 102 amino acid residues from the NH2 terminus and that of 39 amino acid residues from the COOH terminus of bacteriorhodopsin have been determined. These results are in agreement with those recently published by Ovchinnikov and coworkers [Ovchinnikov, Y. A., Abdulaev, N. G., Feigina, M. Y., Kiselev, A. V. & Lobanov, N. A. (1977) FEBS Lett. 84,[1][2][3][4]. Chymotryptic cleavage of bacteriorhodopsin roduced two fragments, C-1 (Mr 19,000) and C-2 (M, 6900), the latter containing the blocked NH2 terminus (pyroglutamic acid). Further fragmentation with CNBr gave mostly hydrophobic fragments, which were separated by gel permeation and reverse-phase high-pressure liquid chromatography in formic acid/ethanol/water mixtures. The fragments were sequenced by a judicious combination of mass spectrometric peptide sequencing and automated Edman degradation. The C-2 fragments were ordered on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry, while C-1 and C-2 were arranged by analysis of an overlapping CNBr fragment.The purple membranes of a number of extremely halophilic bacteria-e.g., Halobacterium halobium-function as lightdriven proton pumps (1, 2). They contain a single protein, bacteriorhodopsin (Mr 26,000) with one molecule of retinaldehyde covalently bound to a lysine residue (3, 4). Bacteriorhodopsin forms a continuum of seven a helixes, each of which spans the membrane and is largely embedded in it (5). Knowledge of the primary structure of bacteriorhodopsin is a prerequisite to an understanding of the mechanism of proton pumping; in this paper we report on the elucidation of the sequence of the first 102 amino acids as well as-of 39 amino acids at the COOH terminus of this integral membrane protein.Partial amino acid sequences of bacteriorhodopsin have been reported (3, 6), and, more recently, extensive sequence work has been reported by Ovchinnikov and coworkers (7), although no experimental details have appeared so far.Sequence work with membrane proteins is still in its infancy, and progress has been reported only in a relatively few cases (8-10). The straightforward application of current methods for sequencing water-soluble proteins to large hydrophobic membrane proteins such as bacteriorhodopsin is not feasible. Therefore, a major aim of the present work has been the development of methods for sequencing integral membrane proteins. This included gel permeation chromatography with nonaqueous solvents, reverse-phase high-pressure liquid chromatography (HPLC) for separation of the fragments, and a well-balanced combination of gas chromatographic mass spectrometry (GCMS) and automated Edman degradation for sequencing. MATERIALS AND METHODSMaterials. The enzymes used were obtained commercially. Sequencer reagents were purchased from Pierce, LiAl2H4 and B22H6 from Alfa-Ventron (Danvers, MA), Sephadex LH-20 and LH-60 from Pharmacia, and ,uBondapak C18 columns from Waters Associates (Milford, MA).Electrophoresis of Polypeptide Fragments. Fragmenta...