Cellular differentiation into hyphae and spores in halophilic archaea

Abstract

Several groups of bacteria have complex life cycles involving cellular differentiation and multicellular structures. For example, actinobacteria of the genus Streptomyces form multicellular vegetative hyphae, aerial hyphae, and spores. However, similar life cycles have not yet been described for archaea. Here, we show that several haloarchaea of the family Halobacteriaceae display a life cycle resembling that of Streptomyces bacteria. Strain YIM 93972 (isolated from a salt marsh) undergoes cellular differentiation into mycelia and spores. Other closely related strains are also able to form mycelia, and comparative genomic analyses point to gene signatures (apparent gain or loss of certain genes) that are shared by members of this clade within the Halobacteriaceae. Genomic, transcriptomic and proteomic analyses of non-differentiating mutants suggest that a Cdc48-family ATPase might be involved in cellular differentiation in strain YIM 93972. Additionally, a gene encoding a putative oligopeptide transporter from YIM 93972 can restore the ability to form hyphae in a Streptomyces coelicolor mutant that carries a deletion in a homologous gene cluster (bldKA-bldKE), suggesting functional equivalence. We propose strain YIM 93972 as representative of a new species in a new genus within the family Halobacteriaceae, for which the name Actinoarchaeum halophilum gen. nov., sp. nov. is herewith proposed. Our demonstration of a complex life cycle in a group of haloarchaea adds a new dimension to our understanding of the biological diversity and environmental adaptation of archaea.

Fig. 1. Characteristics of haloarchaeal strain YIM 93972.

a Salt tolerance of strain YIM 93972 grown in ISP 4 medium after 15 and 29 days (n = 3). b Colony morphology (I) and scanning electron micrographs (EM) of strain YIM 93972 cultivated on solid and liquid ISP 4 medium containing 25% NaCl (n = 3). Scanning EM images of substrate mycelia (II; bar, 10 μm), aerial mycelia (III; bar, 10 μm), and long rod-like spore chains (III; spore size, 0.5–0.7 × 1.0–1.2 μm). Scanning EM images of mycelia grown in liquid ISP 4 medium (IV; bar, 5 μm). Transmission EM images (V and VI) of cells collected from ISP 4 plate after sporulation (bar, 200 nm). c Thin-layer chromatographic analysis of whole-organism methanolysates. Lanes: 1, Haloferax volcanii CGMCC 1.2150^T; 2, YIM 93972; 3, Escherichia coli K12. d Analysis of the polar lipid composition of YIM 93972 using one-dimensional thin layer chromatography. Lanes: 1, YIM 93972; 2, Halomarina oriensis JCM 16495^T; 3, Halomarina salina ZS-57-S^T. The origin is at the bottom. Abbreviations: FAMEs, fatty acid methyl esters; GDEMs, glycerol diether moieties; GLs, glycolipids; PGP-Me, phosphatidylglycerol phosphate methyl ester; PGS, phosphatidylglycero-sulfate; TGD-1, galactosyl mannosyl glucosyl diether; TGD-2, glucosyl mannosyl glucosyl diether; S-TeGD, sulfated galactosyl mannosyl galactofuranosyl glucosyl diether; S-TGD-1, sulfated galactosyl mannosyl glucosyl diether.

Fig. 2. Morphology and phylogeny of morphogenetic haloarchaea.

a Colony morphologies (first row) and scanning electron micrographs (second and third rows) of 7 morphogenetic Halobacteria grown in ISP 4 medium after 14 and 28 days (n = 3). b, Phylogenetic placement of morphogenetic isolates (green) within Halobacteria. The Maximum Likelihood phylogenetic tree (IQ-Tree, LG + F + R10 model) is based on 268 orthologous genes that are universal among 130 Halobacteria genomes and have at most 4 additional paralogs in any of these. The tree is rooted at the Halobacterium-Halodesulfurarchaeum clade following. Branch support values were estimated using aBayes. Scale, substitutions per amino acid site.

Fig. 3. Transcriptomic analysis of strain YIM 93972 wild type and non-differentiating mutants.

a Schematic workflow for transcriptomic analysis. We collected cultures representative for aerial hyphae (AH) and substrate hyphae (SH) from wild-type colony, and the morphological mutants. Two transitional (T) and three bald (B) colonies were chosen for the mutants. Each sample included 3 technical repetitions. Total 16 culture plates were randomly pooled for W-SH and W-AH sample, respectively. Total 32 culture plates were randomly pooled for each mutant sample. b Spearman’s correlation coefficients for transcriptomic profiling of 21 samples. The x and y axes represent the log₂-transformed gene intensities in each two-sample comparison. c Heat maps of differentially expressed genes. Red indicates upregulated genes; blue, downregulated genes. Statistical differences between two groups were analyzed using two-tailed unpaired t-tests. P < 0.05 was considered statistically significant. d The expression clusters of total differentially expressed genes. All dysregulated genes were clustered into different expression groups according to the fold change based on the gene expression value at the mRNA level in different samples by Mfuzz (v2.58.0). e Functional classification of dysregulated genes according to COG functional categories. The COG categories are listed as follows. J: Translation, ribosomal structure and biogenesis; K: Transcription; L: Replication, recombination and repair; B: Chromatin structure and dynamics; D: Cell cycle control, cell division, chromosome partitioning; V: Defense mechanisms; T: Signal transduction mechanisms; M: Cell wall/membrane/envelope biogenesis; N: Cell motility; U: Intracellular trafficking, secretion, and vesicular transport; O: Posttranslational modification, protein turnover, chaperones; C: Energy production and conversion; G: Carbohydrate transport and metabolism; E: Amino acid transport and metabolism; F: Nucleotide transport and metabolism; H: Coenzyme transport and metabolism; I: Lipid transport and metabolism; P: Inorganic ion transport and metabolism; Q: Secondary metabolites biosynthesis, transport and catabolism; R: General function prediction only; S: Function unknown.

Fig. 4. Proteomic analysis of strain YIM 93972 wild type and non-differentiating mutants.

a Overview of TMT methodology for multiplexed comparative analysis. Abbreviations: AH, aerial hyphae; SH, substrate hyphae; W, wild type; T, transitional mutant; B, bald mutant. Two transitional and three bald colonies were selected for the mutants. Each group included two technical repetitions, W-SH1 and W-SH2 in wild group, T1-SH1 and T1-SH2 in transitional group, and B2-SH1 and B2-SH2 in bald group, respectively. Total 16 culture plates were randomly pooled for W-SH and W-AH sample, respectively. Total 32 culture plates were randomly pooled for each mutant sample. b Summary of MS identification and quantitation. c Spearman’s correlation coefficients for proteome profiling of ten samples. The x and y axes represent the log₂-transformed protein intensities in each two-sample comparison. d Heat maps of differentially expressed proteins. Red indicates upregulated proteins; blue, downregulated proteins. Statistical differences between two groups were analyzed using two-tailed unpaired Significance A. P < 0.05 was considered statistically significant. e The expression clusters of total differentially expressed proteins. All dysregulated proteins were clustered into different groups according to the fold change based on the protein expression value in different samples by Mfuzz (v2.58.0). f Functional classification of dysregulated genes according to COG functional categories. The annotations of COG categories are listed in the legend of Fig. 3.

Fig. 5. Sporulation-related genes of strain YIM 93972.

a Typical gene expression profiles in varied differentiating conditions at the mRNA and protein levels. Abbreviations: AH, aerial hyphae; SH, substrate hyphae; W, wild type; T, transitional mutant; B, bald mutant. b Gene organization of the peptide ABC transporter in strain YIM 93972. Arrows indicate the size and directions of ORFs. c Expression levels of the peptide ABC transporter encoding genes at the mRNA and protein level. In transcriptomic data, gene expression at the mRNA level between two groups was calculated by TPM value and shown as means±MSD from W-AH/W-SH (n = 3), T-SH/W-SH (n = 6), and B-SH/W-SH (n = 9). In proteomic data, gene expression at the protein level between two groups was calculated by intensity and shown as means ± MSD from W-AH/W-SH (n = 2), T-SH/W-SH (n = 3), and B-SH/W-SH (n = 4). Statistical differences between two groups were calculated using two-tailed unpaired t-tests (inter-group) at the mRNA level and Significance A (intra-group) at the protein level, respectively. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. The fold change and p-Value were shown in Supplementary Data 4 for Fig. 5c. d The morphological mutants are resistant to the toxic peptide bialaphos (n = 3). The strains listed above were grown for 14 days on ISP 4 agar plate containing 0 (left plate) and 0.8 (right plate) μg/mL bialaphos.

Fig. 6. Role of peptide permeases in morphological differentiation: complementation of a bldKA-KE deletion mutant of Streptomyces coelicolor with the homologous operon of YIM 93972.

a Comparison of the bldK operon of S. coelicolor M145 with ORF_2669-ORF_2673 of YIM 93972; arrows indicate the size and directions of genes; percentage of amino acid sequence identity is indicated for each gene. b Comparison of mycelia phenotypes among M145/pIB139, ΔbldKA-KE/pIB139, ΔbldKA-KE/pIB139-bldKA-KE, and ΔbldKA-KE/pIB139-ORF_2669-ORF_2673 using scanning EM (bar, 5 μm). Deletion of bldKA-KE in M145 abrogated the development of aerial mycelia and spores; this phenotype was restored to near wild-type levels by complementation with bldKA-KE or ORF_2669-ORF_2673 (n = 3).