Halobacterium salinarum NRC-1 contains multiple TBP and TFB proteins required for the recruitment of RNA polymerase for transcription initiation. The presence and the expression of genes encoding TFB were investigated in the two Hbt. salinarum strains NRC-1 and PHH1 and the mutant strain PHH4. The plasmid-encoded tfbC and tfbE genes of NRC-1 were lacking in PHH1 and PHH4. The 5'-end of the tfbF transcript was determined and contained a 5'-untranslated region of 39 nucleotides able to form a stem-loop structure. The expression of these tfb genes was studied in cultures growing at 15, 37°C and under heat shock conditions. Cold temperatures reduced growth and except for tfbF also the amounts of all tfb transcripts. However, the formation of gas vesicles increased in PHH1 and NRC-1. Heat shock reduced growth of PHH1 and NRC-1, but PHH4 was not affected. A 100-fold increase in tfbA and tfbB mRNA was observed in PHH1 and PHH4, whereas NRC-1 reduced the amounts of these transcripts and increased the expression of tfbG. All TFB proteins tested were able to interact with the transcription activator GvpE involved in gas vesicle formation that thus is able to recruit TFB to the gvp promoter.

GvpE is the transcriptional activator of the gvp gene cluster involved in gas vesicle formation in Haloabacterium salinarum. A 20-nucleotide sequence is required for the GvpE-mediated activation of the two oppositely oriented gvp promoters, P ( A ) and P ( D ). This sequence is located adjacent to the TATA-box and the transcription factor-B-binding site BRE, suggesting an interaction between GvpE and proteins of the transcription initiation apparatus. Here, we analysed the interaction of GvpE with the five different TATA-box-binding proteins, TBP, of Hbt. salinarum PHH1. The His-tagged TbpA through TbpE proteins were produced in Escherichia coli, bound to Ni-NTA matrices and tested for interaction with GvpE by protein-protein affinity chromatography. All Tbp(His) proteins retained the two different GvpE proteins from lysates of Haloferax volcanii transformants expressing the respective gvpE reading frame in pJAS35. Also, both GvpE(His) proteins bound to Ni-NTA matrices retained TbpB, whereas the 20-kDa soluble gas vesicle protein GvpH(His) neither bound TbpB nor GvpE from the respective lysates of Hfx. volcanii. From these results, it appears that GvpE interacts with any TBP of Hbt. salinarum. This interaction might attract TBP and subsequently TFB and RNAP to the promoter and thus enhance transcription of the gvp gene cluster.

The presence and expression of the multiple tbp genes encoding TATA-box binding proteins (TBPs) was investigated in various strains and mutants of the archaeon Halobacterium salinarum. Six genes, tbpA through tbpF, are present in the genome of Hbt. salinarum NRC-1 and also in the gas vesicle negative mutant strain R1. The only tbp gene located in the chromosome is tbpE, whereas all others are found in the plasmid DNA. Due to the dynamic nature of the plasmids in the Halobacterium strains, the copy numbers of the alternative tbp genes vary significantly. Five tbp genes (tbpA through tbpE) were present in the wild-type strain Hbt. salinarum PHH1. The tbpC gene of Hbt. salinarum PHH1 carried an ISH27-2 insertion element at the start of the reading frame that prevented the expression. All other tbp genes of PHH1 were expressed under aerobic and anaerobic growth conditions and quantitative RT-PCR yielded tbpE as dominant tbp transcript during the exponential growth phase. The plasmid deletion variant Hbt. salinarum PHH4 lacked all of the tbp genes except for tbpE and showed an altered growth behaviour compared to PHH1 wild-type in the stationary growth phase under anaerobic growth conditions.

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