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

Abstract

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.

Halocin Production From Haloarcula Hispanica, a Saltern Extremophile

“…In the present investigation the halocin produced behaved like a typical secondary metabolite Cheung et al (1997). Reported that halocin activity reached a maximum during the transition to stationary phase at the end of the exponential growth phase and then decreased to low levels, and reasoned this might be due to the rapid increase in gene expression level in the late stationary phase.…”

Anaerobic Metabolism in Haloferax Genus

“…mediterranei as a molecule to inhibit the growth of other halophilic archaea (Cheung, Danna, O'Connor, Price & Shand, 1997;Naor, Yair & Gophna, 2013). This strategy reports competitive advantages when Hfx.…”

Microbial defense systems in foods and feeds

“…The only characterized member is from the halocin family produced by halobacteria (Cheung et al, 1997;Shand et al, 1998). S8 is the first discovered halocin and is composed of a short hydrophobic peptide with 36 amino acids, this is derived from larger pro-protein of 34 KDa (Price and Shand, 2000).…”