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.