Halocins and sulfolobicins: the emerging story of archaeal protein and peptide antibiotics.

Production of antibiotic peptides and proteins is a near-universal feature of living organisms regardless of phylogenetic classification. Bacteriocins (proteinaceous antimicrobials from the domain Bacteria) have been studied for over 75 years, and the eucaryocins (proteinaceous antimicrobials from the domain Eucarya) since the early 1960s. However, one domain of organisms, the Archaea, containing hyperthermophiles, extreme halophiles and the methanogens, is just beginning to be scrutinized for the production of peptide antibiotics. Production of archaeal proteinaceous antimicrobials (archaeocins) from extreme halophiles (halocins) is a nearly universal feature of the rod-shaped haloarchaea. Halocin activity is first detectable in culture supernatants at the beginning of the transition into stationary phase, concomitant with an induction of transcription of the structural gene. Halocins are diverse in size, consisting of proteins as large as 35 kDa and peptide "microhalocins" as small as 3.6 kDa. The 36 amino acids of microhalocin HalS8 are located in the interior of a 311-residue pro-protein from which they are liberated by an unknown mechanism. Microhalocins are hydrophobic and robust, withstanding heat, desalting and exposure to organic solvents. Unlike the peptide bacteriocins and the eucaryocins, microhalocins possess a large number of neutral residues and are not cationic, leaving their mechanism(s) of action mostly a mystery. While microhalocins affect a variety of haloarchaeal genera (kingdom Euryarchaeota), they also exhibit cross-kingdom toxicity, inhibiting or killing Sulfolobus species (kingdom Crenarchaeota). Finally, archaeocins also are produced by the hyperthermophile "Sulfolobus islandicus". These 20-kDa protein antibiotics are not excreted into the environment, but are associated with small particles apparently derived from the cell's S-layer.

Secreted euryarchaeal microhalocins kill hyperthermophilic crenarchaea.

Few antibiotics targeting members of the archaeal domain are currently available for genetic studies. Since bacterial antibiotics are frequently directed against competing and related organisms, archaea by analogy might produce effective antiarchaeal antibiotics. Peptide antibiotic (halocin) preparations from euryarchaeal halophilic strains S8a, GN101, and TuA4 were found to be toxic for members of the hyperthermophilic crenarchaeal genus Sulfolobus. No toxicity was evident against representative bacteria or eukarya. Halocin S8 (strain S8a) and halocin R1 (strain GN101) preparations were cytostatic, while halocin A4 (strain TuA4) preparations were cytocidal. Subsequent studies focused on the use of halocin A4 preparations and Sulfolobus solfataricus. Strain TuA4 cell lysates were not toxic for S. solfataricus, and protease (but not nuclease) treatment of the halocin A4 preparation inactivated toxicity, indicating that the A4 toxic factor must be a secreted protein. Potassium chloride supplementation of the Sulfolobus assay medium potentiated toxicity, implicating use of a salt-dependent mechanism. The utility of halocin A4 preparations for genetic manipulation of S. solfataricus was assessed through the isolation of UV-induced resistant mutants. The mutants exhibited stable phenotypes and were placed into distinct classes based on their levels of resistance.

Halocin S8: a 36-amino-acid microhalocin from the haloarchaeal strain S8a.

Halocin S8 is a hydrophobic microhalocin of 36 amino acids (3,580 Da) and is the first microhalocin to be described. This peptide antibiotic is unique since it is processed from inside a much larger, 33,962-Da pro-protein. Halocin S8 is quite robust, as it can be desalted, boiled, subjected to organic solvents, and stored at 4 degrees C for extended periods without losing activity. The complete amino acid sequence of halocin S8 was obtained first by Edman degradation of the purified protein and verified from the halS8 gene: H(2)N-S-D-C-N-I-N-S-N-T-A-A-D-V-I-L-C-F-N-Q-V-G-S-C-A-L-C-S-P-T-L-V-G -G-P-V-P-COOH. The halS8 gene is encoded on an approximately 200-kbp megaplasmid and contains a 933-bp open reading frame, of which 108 bp are occupied by halocin S8. Both the halS8 promoter and the "leaderless" halS8 transcript are typically haloarchaeal. Northern blot analysis revealed three halS8 transcripts: two abundant and one minor. Inspection of the 3' end of the gene showed only a single, weak termination site (5'-TTTAT-3'), suggesting that some processing of the larger transcripts may be involved. Expression of the halS8 gene is growth stage dependent: basal halS8 transcript levels are present in low concentrations during exponential growth but increase ninefold during the transition to stationary phase. Initially, halocin activity parallels halS8 transcript levels very closely. However, when halocin activity plateaus, transcripts remain abundant, suggesting inhibition of translation at this point. Once the culture enters stationary phase, transcripts rapidly return to basal levels.

Site-specific prebiotic oligomerization reactions of glycine on the surface of hectorite.

Condensation reactions of the amino acid glycine on the surface of Cu(II)-exchanged hectorite are investigated using the technique of scanning force microscopy. Prebiotic conditions are simulated using alternate wetting and heating cycles. Concentration, immobilization, and subsequent polymerization resulting in glycine oligomers are seen to occur primarily at step edges or faults in the topmost layer. Condensation reactions also occur within tiny micropores or defects in the topmost layer. These reactions are facilitated by the availability of intergallery metal cations at the step edges or pores in the surface region.

Isolation, sequence, and expression of the gene encoding halocin H4, a bacteriocin from the halophilic archaeon Haloferax mediterranei R4.

The first gene to encode a haloarchaeal bacteriocin (halocin H4) has been cloned and sequenced from Haloferax mediterranei R4. Both the signal sequence in the halocin H4 preprotein and the monocistronic halH4 gene have some unusual features. The physiology of halH4 expression reveals that although halH4 transcripts are present at low basal levels during exponential growth, halocin H4 activity first appears as the culture enters stationary phase. As halocin activity levels increase, so do transcript levels, but then activity levels decrease precipitously while transcript levels remain elevated.

bop gene cluster expression in bacteriorhodopsin-overproducing mutants of Halobacterium halobium.

mRNA levels from the bop (bacterio-opsin), brp (bacterio-opsin-related protein), and bat (bacterio-opsin activator) genes in wild-type Halobacterium halobium and two bacteriorhodopsin-overproducing mutants (ET1001 and II-7) were quantitated under conditions in which oxygen levels were steadily depleted and then cultures were either kept in the dark or exposed to light. All three strains showed similar responses to depleted oxygen tensions and the lack of light: bop gene cluster transcript levels first increased in response to steadily declining oxygen, and once oxygen was depleted, transcript levels decreased and became undetectable within 20 to 40 h. In contrast, each strain responded differently to conditions of depleted oxygen and the presence of light. In the wild-type strain, bop gene cluster transcript levels increased 2.4- to 9.2-fold above the highest levels obtained in the dark. In mutant ET1001, bop gene cluster transcript levels did not increase above the highest levels obtained in the dark. In mutant II-7, bop and brp transcript levels did not increase above the highest levels obtained in the dark, but bat transcript levels increased approximately 5.7-fold. This differing response to identical physiological conditions indicates that the mutations resulting in the bacteriorhodopsin-overproducing phenotype in these two mutants are different.

Two-dimensional crystallization of Escherichia coli-expressed bacteriorhodopsin and its D96N variant: high resolution structural studies in projection.

Highly ordered two-dimensional (2-D) crystals of Escherichia coli-expressed bacteriorhodopsin analog (e-bR) and its D96N variant (e-D96N) reconstituted in Halobacterium halobium lipids have been obtained by starting with the opsin protein purified in the denaturing detergent sodium dodecyl sulfate. These crystals embedded in glucose show electron diffraction in projection to better than 3.0 A at room temperature. This is the first instance that expressed bR or a variant has been crystallized in 2-D arrays showing such high order. The crystal lattice is homologous to that in wild-type bR (w-bR) in purple membranes (PM) and permit high resolution analyses of the structure of the functionally impaired D96N variant. The e-bR crystal is isomorphous to that in PM with an overall averaged fractional change of 12.7% (26-3.6-A resolution) in the projection structure factors. The projection difference Fourier map e-bR-PM at 3.6-A resolution indicates small conformational changes equivalent to movement of approximately < 7 C-atoms distributed within and in the neighborhood of the protein envelope. This result shows that relative to w-bR there are no global structural rearrangements in e-bR at this 3.6 A resolution level. The e-D96N crystal is isomorphous to the e-bR crystal with a smaller (9.2%) overall averaged fractional change in the structure factors. The significant structural differences between e-D96N and e-bR are concentrated at high resolution (5-3.6 A); however, these changes are small as quantified from the 3.6 A resolution e-D96N-e-bR Fourier difference map. The difference map showed no statistically significant peaks or valleys within 5 A in projection from the site of D96 substitution on helix C. Elsewhere within the protein envelope the integrated measure of peaks or valleys was < approximately 3 C-atom equivalents. Thus, our results show that for the isosteric substitution of Asp96 by Asn, the molecular conformation of bR in its ground state is essentially unaltered. Therefore, the known effect of D96N on the slowed M412 decay is not due to ground-state structural perturbations.

Effects of Asp-96----Asn, Asp-85----Asn, and Arg-82----Gln single-site substitutions on the photocycle of bacteriorhodopsin.

Bacteriorhodopsin (BR) with the single-site substitutions Arg-82----Gln (R82Q), Asp-85----Asn (D85N), and Asp-96----Asn (D96N) is studied with time-resolved absorption spectroscopy in the time regime from nanoseconds to seconds. Time-resolved spectra are analyzed globally by using multiexponential fitting of the data at multiple wavelengths and times. The photocycle kinetics for BR purified from each mutant are determined for micellar solutions in two detergents, nonyl glucoside and CHAPSO, and are compared to results from studies on delipidated BR (d-BR) in the same detergents. D85N has a red-shifted ground-state absorption spectrum, and the formation of an M intermediate is not observed. R82Q undergoes a pH-dependent transition between a purple and a blue form with different pKa values in the two detergents. The blue form has a photocycle resembling that for D85N, while the purple form of R82Q forms an M intermediate that decays more rapidly than in d-BR. The purple form of R82Q does not light-adapt to the same extent as d-BR, and the spectral changes in the photocycle suggest that the light-adapted purple form of R82Q contains all-trans- and 13-cis-retinal in approximately equal proportions. These results are consistent with the suggestions of others for the roles of Arg-82 and Asp-85 in the photocycle of BR, but results for D96N suggest a more complex role for Asp-96 than previously suggested. In nonyl glucoside, the apparent decay of the M-intermediate is slower in D96N than in d-BR, and the M decay shows biphasic kinetics. However, the role of Asp-96 is not limited to the later steps of the photocycle. In D96N, the decay of the KL intermediate is accelerated, and the rise of the M intermediate has an additional slow phase not observed in the kinetics of d-BR. The results suggest that Asp-96 may play a role in regulating the structure of BR and how it changes during the photocycle.

Expression of the bop gene cluster of Halobacterium halobium is induced by low oxygen tension and by light.

The bop gene cluster consists of at least three genes: bop (bacterio-opsin), brp (bacterio-opsin-related protein), and bat (bacterio-opsin activator). We have quantitated transcript levels from these genes in a wild-type and bacterioruberin-deficient mutant of Halobacterium halobium under conditions which affect purple membrane synthesis. In wild-type cultures grown under high oxygen tension in the dark, bop and bat transcript levels were low during steady-state growth and then increased approximately 29- and approximately 45-fold, respectively, upon entry into stationary phase. brp gene transcription remained very low and essentially unchanged under these conditions. In addition, exposure of wild-type cultures growing under high oxygen tension to 30,000 lx of light stimulated expression of all three genes, especially brp. In contrast to the wild-type, transcription from all three genes in the bacterioruberin mutant was very high during steady-state growth under high oxygen tension in the dark. Cultures of the bacterioruberin mutant were shifted at early stationary phase to low oxygen tension to determine whether oxygen concentrations lower than those present in stationary phase would induce transcription of the bop gene cluster in this strain. Indeed, transcription was induced, suggesting that the bop gene cluster is not completely uncoupled from regulation by oxygen tension in the bacterioruberin mutant. From these data, we propose a regulatory model involving two different mechanisms: (i) bat gene expression is induced under conditions of low oxygen tension and the bat gene product activates bop gene expression and (ii) light induces brp transcription, which stimulates or modulates bat transcription.

Wild-type and mutant bacterioopsins D85N, D96N, and R82Q: high-level expression in Escherichia coli.

The integral membrane protein bacterioopsin, found in the extremely halophilic archaebacterium Halobacterium halobium, was expressed in Escherichia coli as a fusion protein containing 13 heterologous amino acids at the amino terminus. The expressed protein was localized primarily to the E. coli cytoplasmic membrane (greater than 80%) and had an in vivo half-life of 26 min. The amount of bacterioopsin in E. coli crude lysates was quantitated immunologically from Western blots and was expressed at 10-20-fold higher levels than seen previously (i.e., 17 mg/L; 5.6% of the total protein). Three distinct forms of the protein were detected immunologically: two of the forms were generated by the removal of either one or four amino acid residues at the amino terminus; the third form remained unaltered.

Resonance Raman spectra of bacteriorhodopsin mutants with substitutions at Asp-85, Asp-96, and Arg-82.

Detergent solubilized bacteriorhodopsin (BR) proteins which contain alterations made by site-directed mutagenesis (Asp-96----Asn, D96N; Asp-85----Asn, D85N; and Arg-82----Gln, R82Q) have been studied with resonance Raman spectroscopy. Raman spectra of the light-adapted (BRLA) and M species in D96N are identical to those of native BR, indicating that this residue is not located near the chromophore. The BRLA states of D85N and especially R82Q contain more of the 13-cis, C = N syn (BR555) species under ambient illumination compared to solubilized native BR. Replacement of Asp-85 with Asn causes a 25 nm red-shift of the absorption maximum and a frequency decrease in both the ethylenic (-7 cm-1) and the Schiff base C = NH+ (-3 cm-1) stretching modes of BRLA. These changes indicate that Asp-85 is located close to the protonated retinal Schiff base. The BRLA spectrum of R82Q exhibits a slight perturbation of the C = NH+ band, but its M spectrum is unperturbed. The Raman spectra and the absorption properties of D85N and R82Q suggest that the protein counterion environment involves the residues Asp-85-, Arg-82+ and presumably Asp-212-. These data are consistent with a model where the strength of the protein-chromophore interaction and hence the absorption maximum depends on the overall charge of the Schiff base counterion environment.

Wild-type and mutant bacteriorhodopsins D85N, D96N, and R82Q: purification to homogeneity, pH dependence of pumping, and electron diffraction.

Bacterioopsin, expressed in Escherichia coli as a fusion protein with 13 heterologous residues at the amino terminus, has been purified in the presence of detergents and retinylated to give bacteriorhodopsin. Further purification yielded pure bacteriorhodopsin, which had an absorbance ratio (A280/A lambda max) of 1.5 in the dark-adapted state in a single-detergent environment. This protein has a folding rate, absorbance spectrum, and light-induced proton pumping activity identical with those of bacteriorhodopsin purified from Halobacterium halobium. Protein expressed from the mutants D85N, D96N, and R82Q and purified similarly yielded pure protein with absorbance ratios of 1.5. Proton pumping rates of bacteriorhodopsins with the wild-type sequence and variants D85N, D96N, and R82Q were determined in phospholipid vesicles as a function of pH. D85N was inactive at all pH values, whereas D96N was inactive from pH 7.0 to pH 8.0, where wild type is most active, but had some activity at low pH. R82Q showed diminished proton pumping with the same pH dependence as for wild type. Bacteriorhodopsin purified from E. coli crystallized in two types of two-dimensional crystal lattices suitable for low-dose electron diffraction, which permit detailed analysis of structural differences in site-directed variants. One lattice was trigonal, as in purple membrane, and showed a high-resolution electron diffraction pattern from glucose-sustained patches. The other lattice was previously uncharacterized with unit cell dimensions a = 127 A, b = 67 A, and symmetry of the orthorhombic plane group pgg.

Mutational analysis of the histidine operon promoter of Salmonella typhimurium.

We isolated a collection of 67 independent, spontaneous Salmonella typhimurium his operon promoter mutants with decreased his expression. The mutants were isolated by selecting for resistance to the toxic lactose analog o-nitrophenyl-beta-D-thiogalactoside in a his-lac fusion strain. The collection included base pair substitutions. small insertions, a deletion, and one large insertion identified as IS30 (IS121), which is resident on the Mu d1 cts(Apr lac) phage used to construct the his-lac fusion. Of the 37 mutations that were sequenced, 14 were unique. Six of the 14 were isolated more than once, with the IS30 insertion occurring 16 times. The mutations were located throughout the his promoter region, with two in the conserved - 35 hexamer sequence, four in the conserved - 10 hexamer sequence (Pribnow box), seven in the spacer between the - 10 and -35 hexamer sequences, and the IS30 insertions just upstream of the -35 hexamer sequence. Four of the five substitution mutations changed a consensus base pair recognized by E sigma 70 RNA polymerase in the -10 or -35 hexamer. Decreased his expression caused by the 14 different his promoter mutations was measured in vivo. Relative to the wild-type promoter, the mutations resulted in as little as a 4-fold decrease to as much as a 357-fold decrease in his expression, with the largest decreases resulting from changes in the most highly conserved features of E sigma 70 promoters.

Correlation between histidine operon expression and guanosine 5'-diphosphate-3'-diphosphate levels during amino acid downshift in stringent and relaxed strains of Salmonella typhimurium.

We have analyzed the correlation of attenuator-independent expression of the Salmonella typhimurium histidine operon in vivo with levels of the "alarmone" guanosine 5'-diphosphate 3'-diphosphate. Amino acid downshift caused by serine hydroxamate addition increased his expression in a relA+ strain and decreased his expression in a relA mutant, whereas levels of guanosine 5'-diphosphate-3'-diphosphate varied in parallel with the changes in his expression in the two strains. In several experiments, overall variations in his expression ranged from 20- to 60-fold after downshift. The mild downshift allowed growth of the cultures to continue at near-preshift rates. Serine hydroxamate addition was also used to analyze the effect of amino acid downshift on induced expression of wild-type and mutant lac promoters. There was a 12-fold difference in lac expression when a relA+-relA1 pair was subjected to mild starvation but only a 3-fold difference when the strains carried the lacZpL8UV5 promoter mutation. These results suggest that guanosine 5'-diphosphate-3'-diphosphate stimulates gene expression in vivo at the level of transcription initiation.

Regulation of the bacterio-opsin gene of a halophilic archaebacterium.

The protein bacterio-opsin, complexed with retinal, functions as a light-driven proton pump in the purple membrane of the halophilic archaebacterium, Halobacterium halobium. Bacterio-opsin deficient mutants have been characterized in attempts to elucidate regulation of the gene encoding bacterio-opsin (bop). Analysis of the mutational defect in Bop mutants has revealed the existence of at least two genes that affect bop gene expression and (or) purple membrane formation: (i) the brp gene, located 526 base pairs upstream of the bop gene, is transcribed in the opposite orientation, and (ii) the bat gene, located 1602 base pairs upstream of the bop gene, is transcribed in the same orientation as the brp gene. The bat gene start codon overlaps the stop codon of the brp gene. The bat gene could encode an acidic protein of 73,000 Da (674 amino acids) with a predicted secondary structure typical of a soluble alpha--beta type protein. This type of secondary structure is in contrast to the hydrophobic structure predicted for the putative brp protein. Transcriptional analyses of the wild type, 11 Bop mutants, and a Bop revertant suggest that the bat gene has a more direct role than the brp gene in bop gene expression and is involved in activating bop and brp gene expression.