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.

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.Halocins are haloarchaeal equivalents of eubacterial bacteriocins (2) and were first discovered in 1982 by F. RodriguezValera (24). Although nearly universal in halobacterial rods (17, 32), only three halocins have been characterized in any detail: halocin H4 from Haloferax mediterranei R4 (15, 16), halocin H6 from Haloferax gibbonsii Ma2.39 (30, 31), and halocin HalR1 from Halobacterium sp. strain GN101 (23). In parallel with antibiotic production in the domain Bacteria (33), halocins H4, S8, and HalR1 are all initially detected as the cultures leave exponential growth and enter stationary phase (21). Consequently, we chose halocins, beginning with halocin H4, as models to study stationary-phase gene expression in the haloarchaea.Isolation of the halH4 gene. Halocin H4 was purified from H. mediterranei R4 (ATCC 33500) culture supernatants essentially as described elsewhere (4, 15). The amino-terminal sequences of the secreted protein and of two tryptic fragments (numbers 9 and 17 [ Fig. 1]) were used to design inosinecontaining degenerate oligodeoxynucleotide primers. Using these primers, we amplified the 5Ј end of the halocin H4 gene (halH4) by PCR and used the larger product (150 bp) as a probe to recover the gene from an enriched HindIII plasmid library.The start site of transcription of the halH4 gene was determined by primer extension (26). Note that the initiator AUG codon is only 4 bases from the 5Ј end of the message (Fig. 1). Similar leaderless transcripts are produced by other haloarchaeal genes, including bop (7); brp (3); hop (5); and arcA, arcB, and arcC (25). Inspection of the DNA sequence upstream of the halH4 transcriptional start site reveals a box A haloarchaeal promoter hexamer (Fig.