MATCHING

Construction of retinal-protein knockout strains

The aim of this study was to identify the transducer that is responsible for bacteriorhodopsin (BR)-mediated phototaxis and to gain further insights into the nature of this phenomenon. The chosen strategy for its identification was the generation of htr-deletion mutants in a halobacterial strain which contains BR as the only retinal protein.

Although strain Pho81-B4, which had previously been used to demonstrate BR-mediated taxis (Bibikov et al., 1991), met this requirement and was available in the lab, two reasons argued against the use of this strain. (i) Its parent strain Pho81 (Sundberg et al., 1985), as well as Flx15 (Spudich and Spudich, 1982) the parent of Pho81 were both obtained by mutagenesis using N- methyl-N'-nitro-N-nitrosoguanidine, followed by mutant screening procedures. It can be expected that in addition to the loss of functional retinal proteins the two rounds of chemical mutagenesis have caused major changes in the genomes of these strains as compared to the wild- type genome. (ii) Pho81-B4 was generated by transformation of Pho81 with a plasmid containing the bop gene and a mevinolin resistance-conferring gene. The thereby acquired mevinolin resistance would have excluded the use of mevinolin as the selective agent for further transformations of Pho81-B4.

Figure 3.1 Overview of the generated strains leading to retinal protein-deficient strain OMI1, and strains MKK101 and MKK102.

The latter were produced from OMI1 by complementation with the bop and hop gene, respectively, and are the strains of origin for the subsequent htr-deletions. The relevant genotypes are indicated (bop: bacterioopsin, hop: haloopsin, sopI: sensory opsin I, sopII: sensory opsin II, htr: halobacterial transducer, car: htr11, ISH2: halobacterial insertion element 2). The genotype of a new strain is identical to that of the attributed strain in brackets, except for the explicitly added differences.

S9

L33

WHOP

TOM

SAM2

OMI1

MKK101

MKK102

∆hop car∆bop

htr...-deletion strains derived from MKK101

MKK120

As an alternative to Pho81, strain OMI1 (Masato Otsuka, unpublished) was chosen as the parent for the strains to be generated in the course of this study. OMI1 was constructed via defined genetic manipulations instead of chemical mutagenesis (Fig. 3.1). Strain L33 served as the parent strain for these manipulations. It was generated from strain S9 (Wagner et al., 1981) and contained an ISH2 insertion element interrupting its bop gene (DasSarma et al., 1983). Strains WHOP and TOM (Besir, 2001), and strains SAM2 and OMI1 (Masato Otsuka, unpublished) were produced from L33 by successive deletion of all 4 retinal-protein genes.

To produce the parent strain for the htr-deletion mutants, MKK101, the wild-type bop allele was restored in OMI1 at its original position using pUS-Mev-derived plasmid pMKK101 (Fig. 3.2). For investigations on HR-dependent photoreactions another strain, MKK102, was generated from OMI1 by restoration of the wild-type hop allele at its original position using pMKK100-derived plasmid pMKK102.

Southern-blot or PCR analysis of the retinal protein gene loci of OMI1, MKK101 and MKK102 confirmed the expected genotypes (Fig. 3.3). OMI1 and all strains derived from it also lack the genes for the transducers Htr1, Htr2 and Car (Htr11). The loss of the car gene probably happened during strain construction via a partial loss of DNA from the 284 kDa halobacterial plasmid pHS3 (http://www.halolex.mpg.de) which encodes Car.

Figure 3.2 Schematic of the construction of plasmids pMKK101, pMKK102, pMKK114 and pMKK119 to exemplify the genetic strategies employed.

To generate pMKK101 the bop locus (Fig. 3.3) was amplified via PCR from genomic DNA, digested with

BamHI and HindIII, and then cloned into plasmid pUS-Mev digested with BamHI and HindIII. To allow for red-blue selection of colonies, the bgaH-locus from

Haloferax alicantei was cloned between SpeI and SacI sites of pUS-Mev. Furthermore, a multiple cloning site (MCS) was introduced between the BamHI and XbaI sites to produce pMKK100. By cloning PCR-generated DNA fragments, flanked by the appropriate restriction sites, into the MCS, additional deletion and complementation plasmids mentioned in the text were generated. This is described in detail in Table 4.2 and exemplified here by deletion plasmid pMKK114 and complementation plasmids pMKK102 and pMKK119. MevR _{and Amp}R _{indicate the markers for mevinolin-}

and ampicillin-resistance, respectively. pUS-Mev pMKK114 pMKK101 + -locusbop HindIII) (BamHI, +∆14-fragment (BamHI, HindIII) + -locusbgaH

SacI) (SpeI,

+ MCS (BamHI, XbaI)

7213 bp 1 BamHI SmaI XmaI PstI NcoI NheI SphI StuI Bpu10I ClaI XbaI 55 XhoI 2041 SacI 4916 SpeI 7208 MevR (H. volcanii) (pBluescript SK - DNA)- AmpR bgaH (H. alicantei) MCS: pMKK100 + hop-locus (BamHI, HindIII)

pMKK102

pMKK119

+htr14-locus (BamHI, HindIII)

Figure 3.3 Genotypic analysis of strains OMI1, MKK101 and MKK102.

(A)Gene loci of the four halobacterial retinal protein genes (hop, bop, sopI and sopII). Genes deleted in strain OMI1 are depicted as thick arrows containing the gene length in base pairs and the gene name, according to http://www.halolex.mpg.de. Cleavage sites of relevant restriction enzymes are shown. All numbers indicate nucleotide positions given relative to the start of the open reading frame (orf) of the corresponding retinal protein gene. PCR primers are depicted as bent arrows with the position of the first matching 5'-nucleotide indicated. Restriction sequences, attached to or contained in the primer sequences, are indicated by a zigzag line. They were used for cloning the generated PCR fragments to produce complementation vectors. Boxes labeled PRE and CORE depict the DIG-11-dUTP-labeled DNA probes used in Southern-blot analysis at their hybridization positions. (B) Southern-blot analysis of the four retinal protein gene loci in strains S9 (wt) and OMI1. Genomic DNA was cleaved with BamHI in case of the PREsopI and COREsopI blots and with PstI in all other cases. Middle and right lanes represent strains S9 and OMI1, respectively. Left lanes show the positions of marker bands of which the sizes (in kilobasepairs) are given in (C). In all depicted blots generated with PRE DNA probes the size difference of the recognized DNA fragments from S9 and OMI1 matches the size of the deleted DNA regions. On the blots produced with CORE DNA probes band positions were identical to the PRE blots for S9, but bands were missing for OMI1, indicating the absence of the respective opsin gene in the latter. (C) Southern-blot analysis of strain MKK102 using DNA probe COREhop. The presence of the 4.8 kb band for MKK102 demonstrates the successful complementation with the hop gene. Lane 1: marker bands, lanes 2 - 4: PstI-digested MKK102-, S9- and OMI1-DNA, respectively. (D) PCR analysis of bop-complemented strain MKK101. Lane 1 shows marker bands with their sizes indicated. PCR was performed with MKK101- and OMI1-DNA (lanes 2 and 3, respectively) using the primers with starting positions -72 and +869, as shown in (A). Vertical black lines on blot (C) and gel (D) indicate that lanes were not directly adjacent.