INCENTIVOS NO TRIBUTARIOS Y TRIBUTARIOS DEL ECUADOR

Transducer inventory of H. salinarum

H. salinarum strain R1 contains a total of 18 transducers (Htrs)

At the beginning of this study the annotation of the genome sequence of halobacterial strain R1 was an ongoing project in the lab (Dieter Oesterhelt et al., unpublished; http://www.halolex.mpg.de) and the annotated genome sequence of halobacterial strain NRC-1 (Ng et al., 2000) was not available, yet. To successfully apply the strategy of generating single- htr-deletions in strain OMI1, it was advisable to know the complete set of htr genes in the halobacterial genome. This should exclude that the responsible transducer would escape the investigation.

BLAST searches (Altschul et al., 1990) with published Htr-sequences against the 6-frame translations of the R1 nucleotide sequence revealed a total of 18 transducer genes. The classification criterion for a transducer gene was the presence of the highly conserved signaling region within its corresponding gene product, which was checked by alignment of the amino acid sequences (Fig. 3.30B). After the publication of the annotated NRC-1 sequence a comparison to the annotated R1 sequence revealed that the nucleotide sequence of the chromosome is practically identical in both strains, apart from the positions and numbers of integrated halobacterial insertion elements (ISH).

The nucleotide sequences at all 17 chromosomal transducer loci are identical in both strains. However, the start codons for htrs 3, 16 and 18 were found to be incorrectly assigned for NRC- 1. Alternative start codons 5' to the ones assigned for NRC-1 result in 5'-extensions of the corresponding open reading frames (orfs). These extensions encode amino acid sequences with significant similarity among each other and also to other transducers (data not shown). This clearly indicates that the longer gene versions are correct.

One of the transducer genes (htr11, car) of strain R1 is not encoded on the chromosome (2001 kb) but on plasmid pHS3 (284 kb) and is completely missing in the NRC-1 genome. Transducer genes are not present on any of the other 3 halobacterial plasmids pHS1 (148 kb), pHS2 (195 kb) and pHS4 (41 kb) (see: http://www.halolex.mpg.de).

The chromosomal locations of the 17 htr genes, 10 che genes and 4 retinal-protein genes are depicted in Fig. 3.4. On the basis of amino acid sequence homology and predicted TM helices, the transducers can be divided into 5 groups: Htrs 9, 10, 11, 12, 13 and 15 (group 1) lack TM helices; Htrs 1 and 14 (group 2) contain 2 TM helices connected by a short hairpin loop; Htrs 2, 3, 4, 5, 6, 16 and 18 (group 3) contain 2 TM helices with an extensive extracellular domain in

between; Htrs 7 and 17 (group 4) contain 3 TM helices and lack an extracellular domain; Htr8 (group 5) contains 6 TM helices and also lacks an extracellular domain.

Several of the htr genes form a transcription unit with an adjacent gene. This is indicated by a short distance between or even a short overlap of successive genes with the same orientation. Most common is an overlap of 4 bases (e.g. an ATGA-overlap of start and stop condon). For several of these transcription units it was demonstrated that the products of their genes form a functional unit.

The 3'-end of htr1 overlaps with sopI (encoding the photoreceptor SRI which interacts with Htr1) and the 3'-end of htr2 is separated by only 2 nucleotides from sopII (encoding the photoreceptor SRII which interacts with Htr2). The 5'-ends of htr3 (basT) and htr5 (cosT) are overlapping with basB and cosB, respectively, which encode putative extracellular lipid- anchored ligand-binding proteins for the respective transducers (Kokoeva et al., 2002). The 5' end of htr6 and the 3' end of htr18 overlap with adjacent orfs OE2170R (tmpC) and OE2196F (potD), respectively. The products of the latter show homology to periplasmic substrate-binding proteins and contain a lipid-anchor motif like BasB and CosB (Fig. 3.5). This suggests that Htr6 and Htr18 are chemotaxis transducers of unknown specificity which interact with a coproduced lipid-anchored binding protein.

Genes htr7 and htr17 both encode transducers with 3 TM regions and overlap at their 5'-ends with orfs OE3472F and OE3438R, respectively, which encode small polypeptides consisting solely of 3 TM regions (Fig. 6.7B). This might hint to a physical interaction of these polypeptides with the TM regions of Htr7 and Htr17, respectively, possibly resulting in heterotetrameric complexes with a 12-TM-region topology resembling that expected for Htr8 dimers.

Figure 3.4 Chromosomal locations of halobacterial genes encoding transducers, opsins and Che- proteins.

The upper and the lower horizontal line symbolize the forward (F) and the reverse (R) strand, respectively, of the 2 Mb chromosome of halobacterial strain R1. The positions of transducer genes (black), opsin genes (blue) and Che protein genes (red) are marked on their respective coding strand (http://www.halolex.mpg.de). The numbers of the corresponding transducers are shown in boxes with a color code reflecting the number of their predicted transmembrane helices (TM): 0 TM, yellow; 2 TM but no extracellular domain, pink; 2 TM plus extracellular domain, red; 3 TM, green; 6 TM, brown. The existence of a transcription unit with the orf at the 3' end or at the 5' end of an

htr is indicated by attributing 3' or 5' to the respective box. Exact htr positions and identifiers of these adjacent orfs are listed in Table 6.2.

The 3'-end of htr15 overlaps with flaD, which encodes a flagellar accessory protein and is followed in sequence by an operon of additional fla genes. However, as the htr15 homolog from the closely related halophile Natronomonas pharaonis has a different genetic neighborhood (Michaela Falb et al., unpublished), a conserved functional interaction of Htr15 and FlaD is unlikely.

Contrary to sopI and sopII, neither bop nor hop are located close to transducer-encoding genes. It is noteworthy that 13 of the 17 chromosomal htr-genes, all che genes and all fla-genes are encoded on the same strand of the chromosomal DNA.