Exploration of the biological role of PAL

So far, only little is known about the actinobacterium Nakamurella multipartita. The bacterium was first isolated from active sludge in a study by Yoshimi and colleagues [170]. Due to the phylogenetic position and presence of a unique set of 16S rRNA sequence signatures, the new family of Nakamurellaceae (formerly Microsphaeraceae) was established [171], which today includes four published species (N. multipartita, N. panacisegetis, N. flavida (formerly Humicoccus flavidus) and N.

lactea (formerly Saxeibacter lacteus) that form a robust phylogenetic clan [172]. N. multipartita is a coccus-shaped, gram-positive, strictly aerobic, non-motile and nonspore-forming bacterium [170].

The predominant menaquinone MK-8 and meso-diaminopimelic acid within the cell wall were determined as characteristic chemotaxonomic markers. The N. multipartita genome consists of a single replicon comprising 6,060,298 bp and a GC content of 70.92 %. Within the genome 5471 genes were predicted, including 5415 protein-coding genes and 56 RNA genes of which 66.5 % were assigned a putative function. The evaluation of the distribution of genes within their ‘clusters of orthologous group’ (COG) category revealed that the highest number of genes is involved in transcription (400, 9.1 %), followed by genes involved in carbohydrate transport and metabolism (341, 8.3 %), and genes implicated in amino acid transport and metabolism (334, 8.1 %) [172]. The search for additional ANTAR effectors revealed 15 more hits within the genome via BLAST search [173]. The so far characterized members of the ANTAR family are involved in the regulation of bacterial gene expression via transcriptional antitermination. The presence of this large number of ANTAR proteins within N. multipartita (in contrast, EutV host organism E. faecalis contains 5 ANTAR proteins, while BLAST search in AmiR host P. aeruginosa reveals 20 hits of putative ANTAR proteins) indicates that a multitude of processes may be subject to ANTAR-controlled regulatory mechanisms.

Furthermore, the BLAST search revealed three other putative PAS/ PAC-sensor proteins, one of which represents another LOV protein that belongs to the group of short-LOV proteins that lack a covalently bound effector module. The search for further blue light receptor domains (BLUF, bacteriophytochrome, bacteriorhodopsins, cryptochromes) via BLAST search revealed no further hits within the genome. Potential blue light-induced effects could thus be attributed to the interaction of one the two LOV proteins in perspective studies.

So far, the N. multipartita DSM 44233T strain was the only member of the family Nakamurellaceae for which the complete genome sequence was accessible [174], but through the recent sequencing project of the N. lactea DLS-10 type strain genome in 2017 [175], another genome became available.

However, the N. lactea genome does not seem to involve a PAL-like sequence. We were able to grow the N. multipartita cultures in Trypticase Soy Broth medium by DSMZ, as well as in ordinary LB medium in a temperature range of 25 °C - 30 °C. In order to ensure the growth of pure N. multipartia cultures, I also tested various antibiotics to determine possible resistance levels, which I detected for

ampicillin (100 μg / mL), gentamycin (10 μg / mL) and nalidixic acid (30 μg / mL), which is in accordance with the findings of Kim et al. [172]. In previous experiments of a several-week incubation of streaked N. multipartita cultures on agar plates, I could not detect any signs of differing phenotypic characteristics under light and dark cultivation conditions. To my knowledge, so far no experiments of genetic manipulation have been performed, which complicates the conduction of reverse genetic approaches, such as knock-out or RNA-mediated knock-down studies. Hence, we can only hypothesize about the biological role of PAL in its natural host so far. The efforts of the RIP experiments already initiated are an important step towards the elucidation of the natural function of PAL, as we were able to demonstrate that our generated polyclonal antibody is both specific and suitable for the planned pull-down application, and the cultivation of N. multipartita was successfully established in our laboratory. The identification of the natural target sequences would be the first step in the determination of the natural function of PAL, as the analysis of the RIP data will hopefully reveal RNA sequences associated with the genomic location of corresponding operons controlled by PAL. The thereby identified sequences could be further refined through combined bioinformatics approaches. Yet, the detection of candidate genes identified by this technique will require further validation by additional methods, such as quantitative real-time PCR (qPCR), which would permit the quantification of transcript levels of the candidate genes under dark- and blue light conditions. The identification of the natural targets would further permit to assess if the characteristic antitermination mechanism of the so far characterized ANTAR proteins is preserved within N.

multipartita. As our experiments with the SELEX-derived artificial RNA substrates could already demonstrate that light-activation leads to an increase in affinity of PAL for certain RNA substrates, this suggests an involvement of PAL in posttranscriptional regulatory mechanisms.

6.2.3 Photochemical characterization

UV/Vis absorption spectroscopy was applied to follow the dark state recovery of PAL and the isolated PAL LOV domain after blue light activation. This property plays an important role within in vivo applications, since the reversion rate from signaling to dark state importantly defines the effective light-sensitivity of LOV photoreceptors (see Section 3.2.3). For the heterologously purified flPAL, the recovery to the dark-adapted state occurres with a time constant of 1270 ± 100 s at 25° C, the PAL photoreceptor thus falls within the regime of 'intermediate cycling' LOV proteins in terms of the adduct decay [139]. In contrast, the time constant of the isolated PAL LOV domain is significantly accelerated with 470 ± 40 s at 25° C. I further assessed the temperature dependence of the recovery kinetics, which relates to the cleavage of the covalent bond of the photoproduct, for both full-length PAL and PAL-LOV. As most LOV photoreceptors, both constructs show a linear Arrhenius behavior

regarding the temperature dependence of the recovery kinetics, although non-linear Arrhenius behavior for photoreceptors has been reported, e.g. for the photoactive yellow protein or the transcription factor EL222 from E. litoralis [176,177]. For EL222, sequence and mutagenesis studies have shown that this effect is due to a glutamine to alanine mutation also found in related LOV proteins from different marine bacteria [177]. The activation energy determined for the full-length PAL receptoris in a comparable range (EaflPAL

= 60.6 ± 3.3 kJ /mol) to that of EL222, whose Ea is 63 ± 2 kJ molbelow 45 °C [177].

The dark recovery kinetics were modulated by the introduction of the well-documented mutation of residue T250 (or T32 withinthe isolated LOV domain) of the chromophore binding pocket within the PAL LOV domain. For the mutation of the corresponding residue, a strong decelerating effect on the off-kinetics was found for AsLOV2, YtvA and Vivid LOV domains [56,178,179]. The T32 position corresponds to the residue V416 in AsLOV2 (see Figure 44); V416T substitution was shown to significantly accelerate the AsLOV2 recovery kinetics by a factor of more than 20, whereas V416I and V416L substitution led to a deceleration of dark recovery by a factor of more than ten or 50, respectively (Kawano et al., 2013). Initially, three different variants of the T32 position of the PAL LOV domain were designed and expressed (T32V-/ T32I-/ T32L-PAL-LOV), but only the T32V PAL-LOV mutant yielded sufficient amounts of holoprotein. The introduction of the T32I and T32L mutations resulted in poor protein stability. UV-Vis characterization of the T32V PAL-LOV domain revealed an about 2.5 x decelerated time constant compared to wild-type PAL-LOV (τT32V-PAL-LOV

= 1980± 10 s at 20

°C vs. τ PAL-LOL =

765 ± 5 s), and hence a prolonged lifetime of the signaling state of T32V PAL-LOV. The development of further sets of mutations for the generation of optimized PAL variants with different recovery kinetics will be useful for optogenetic applications, since the off-kinetics also affect the effective photosensitivity in the photostationary state under constant illumination (see Section 3.2.3). Figure 44 summarizes some of the previously altered residues for rate-mutating effects within other LOV proteins within an alignment with the PAL LOV domain.

Figure 44: Alignment of different LOV proteins showing previously altered residues resulting in mutating effects of the dark recovery kinetics highlighted in blue [139].