The Lrs14 family of DNA-binding proteins as nucleoid-associated proteins in the Crenarchaeal order Sulfolobales

Abstract

Organization of archaeal chromatin combines bacterial, eukaryotic, and unique characteristics. Many archaeal lineages harbor a wide diversity of small and highly expressed nucleoid-associated proteins, which are involved in DNA structuring. In Sulfolobales, representing model organisms within the Crenarchaeota, Sul7d, Cren7, Sul10a, and Sul12a are well-characterized nucleoid-associated proteins. Here, we combine evidence that the Lrs14 family of DNA binders is part of the repertoire of nucleoid-associated proteins in Sulfolobales. Lrs14-encoding genes are widespread within genomes of different members of the Sulfolobales, typically encoded as four to nine homologs per genome. The Lrs14 proteins harbor a winged helix-turn-helix DNA-binding domain and are typified by a coiled-coil dimerization. They are characterized by distinct sequence- and structure-based features, including redox-sensitive motifs and residues targeted for posttranslational modification, allowing a further classification of the family into five conserved clusters. Lrs14-like proteins have unique DNA-organizing properties. By binding to the DNA nonsequence specifically and in a highly cooperative manner, with a slight preference for AT-rich promoter regions, they introduce DNA kinks and are able to affect transcription of adjacent transcription units either positively or negatively. Genes encoding Lrs14-type proteins display considerable differential expression themselves in response to various stress conditions, with certain homologs being specific to a particular stressor. Taken together, we postulate that members of the Lrs14 family can be considered nucleoid-associated proteins in Sulfolobales, combining a DNA-structuring role with a global gene expression role in response to stress conditions.

Keywords: Crenarchaeota; DNA binding; Lrs14; archaea; chromatin organization; winged helix‐turn‐helix.

FIGURE 1

(Structural) conservation of homologs belonging to the Lrs14 family of DNA‐binding proteins within the Sulfolobales. (a) Overview of Lrs14‐type protein homologs in representative members of the Sulfolobales, identifiable with locus tag or gene name. This list is the result of iterative BLAST searches starting with the amino acid sequence of AbfR1 as a query and each time expanding the group of homologs using both NCBI and the Archaeal genome browser. The total number of homologs per species is noted between brackets on the right. The simplified phylogenetic tree on top represents the evolutionary relationship between the five Lrs14 clusters. All amino acid sequences were aligned, and a phylogenetic tree was constructed with the MEGA software (Kumar et al., 2018) using the ClustalW algorithm and maximum likelihood method (Felsenstein, 1981), respectively. The phylogenetic tree on the left represents the evolutionary relationship of the Sulfolobales species based on 16S rRNA sequences, as determined by Liu et al. (2021). (b) Sequence alignment of all S. acidocaldarius Lrs14 homologs as well as Sto12a from S. tokodaii. Secondary structure elements according to the crystal structure of Saci_1223 (PDB: 6CMV, Vogt et al., 2018) are depicted above. Amino acids are color coded corresponding to their (potential) function. Conserved and semi‐conserved amino acids are highlighted in dark and light gray, respectively. (c) Crystal structure of AbfR2 (Saci_1223) (PDB: 6CMV, Vogt et al., 2018). Conserved amino acids with a role in dimerization, DNA binding, or PTM are depicted as sticks and colored, orange, green, and purple, respectively. (d) Monomeric structures of AbfR2 (Saci_1223) (PDB: 6CMV, Vogt et al., 2018), the candidate NAP from T. volcanium predicted by Hocher et al. (2022) (Q97B33, AlphaFold‐predicted structure, Jumper et al., 2021), the N‐terminal domain of TrmBL2 of P. furiosus (PDB: 5BPI, Ahmad et al., 2015), Sul12a of S. acidocaldarius (Q4JA12, AlphaFold‐predicted structure, Jumper et al., 2021), and Sso10a of S. solfataricus (PDB: 1R7J, Chen et al., 2004). The core HTH motif is colored blue, the wing is colored green and the dimerization helix and additional α‐helices are colored gray.

FIGURE 2

Abundancy and stress‐responsiveness of Lrs14‐type proteins and hypothetical model of Lrs14‐mediated DNA structuring and gene expression regulation. (a) Intracellular abundance of Lrs14 members in exponentially growing S. acidocaldarius in comparison to “classical” NAPs, transcription initiation factors, and transcription factors at protein level (left) and RNA level (right). Data retrieved from proteomic (mass spectrometry) and transcriptomic (RNA‐seq) experiments described in Baes et al. (2023). Numbers refer to Saci_xxxx locus tags. N/C = no coverage. f = family. (b) Transcriptional expression of lrs14‐encoding genes in S. acidocaldarius in diverse stress conditions including heat shock (Baes et al., 2023), solvent stress in biofilm (Benninghoff et al., 2021), overfeeding (Quehenberger, Sedlmayr & De Kock, unpublished), nutrient starvation (Bischof et al., 2019), biofilm formation (Benninghoff et al., 2021), and UV irradiation (Schult et al., 2018). (c) Conceptual overview of Lrs14 functioning. Top panel: schematic representation of Sulfolobales' chromatin with classical NAPs and Lrs14 proteins structuring the DNA. Bottom panel: a hypothetical model of the role of Lrs14 as a member of the chromatin‐organizing machinery, in which phenotypical triggers, in this case, stress could induce (i) posttranslational modifications (PTM), (ii) differential expression, or (iii) a change in the oxidative state of the Lrs14 proteins leading to restructuring of the chromatin by the Lrs14 proteins and a difference in gene expression of adjacent genes.