A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus, lacking flagella, has unusual growth physiology

Abstract

A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus DSM3638 exhibited an extended exponential phase and atypical cell aggregation behavior. Genomic DNA from the mutant culture was sequenced and compared to wild-type (WT) DSM3638, revealing 145 genes with one or more insertions, deletions, or substitutions (12 silent, 33 amino acid substitutions, and 100 frame shifts). Approximately, half of the mutated genes were transposases or hypothetical proteins. The WT transcriptome revealed numerous changes in amino acid and pyrimidine biosynthesis pathways coincidental with growth phase transitions, unlike the mutant whose transcriptome reflected the observed prolonged exponential phase. Targeted gene deletions, based on frame-shifted ORFs in the mutant genome, in a genetically tractable strain of P. furiosus (COM1) could not generate the extended exponential phase behavior observed for the mutant. For example, a putative radical SAM family protein (PF2064) was the most highly up-regulated ORF (>25-fold) in the WT between exponential and stationary phase, although this ORF was unresponsive in the mutant; deletion of this gene in P. furiosus COM1 resulted in no apparent phenotype. On the other hand, frame-shifting mutations in the mutant genome negatively impacted transcription of a flagellar biosynthesis operon (PF0329-PF0338).Consequently, cells in the mutant culture lacked flagella and, unlike the WT, showed minimal evidence of exopolysaccharide-based cell aggregation in post-exponential phase. Electron microscopy of PF0331-PF0337 deletions in P. furiosus COM1 showed that absence of flagella impacted normal cell aggregation behavior and, furthermore, indicated that flagella play a key role, beyond motility, in the growth physiology of P. furiosus.

Figure 1

(Top) Growth of P. furiosusDSM 3638 (O) and the P. furiosusmutant (◆) at 80°C. Samples were taken at 10, 14 and 24 hours for transcriptomic analysis. Inset shows mutant cell density decreasing from cell lysis. (Bottom) A and C: Epifluorescence micrographs (acridine orange stain) of mutant (left two panels) and wild-type (right two panels) P. furiosus cultures after 24 hours post-inoculation, showing significant cell aggregation in the mutant compared to wild-type strain, B and D: Epifluorescence micrographs (acridine orange and calcofluorstain) of the mutant (left panel) and WT (right panel) cultures at 14h showing production of EPS surrounding the cells from exponential phase.

Figure 2

Electron micrographs of wild-type (WT) and mutantP. furiosus post-24h following inoculation. A,B. WT (SEM); C,D. mutant (SEM); E,F. WT (TEM); G,H. mutant (TEM); I. PF0337 KO(SEM); J. PF0331 KO (SEM); K. PF0337 KO(TEM); L. PF0331 KO (TEM).

Figure 3

Distribution of ORFs containing mutations in the P. furiosus mutant genome. Mutations in intergenic regions were also distributed throughout the genome (data not shown).

Figure 4

Heat plot showing ORFs responding to growth phase transitions for the wild-type (WT) and mutant P. furiosus strains growing at 80°C. For the heat plot, red indicates higher transcript levels (log₂(fold change)>1), green indicates lower (log₂(fold change)<-1), and black denotes average transcript levels (The color scale for least squares mean from mixed effects ANOVA model shown on the right, where 0 is average transcript level).

Figure 5. PF2064 locus organization and gene response during growth phase transitions in WT and Mutant

(A)The PF2064 locus includes small hypothetical ORFs, bacteriocin synthesis related genes (yellow), radical SAM proteins (dark blue), transporters (light blue), toxins/antitoxins (pink), and a transcriptional regulator containing HTH domain (grey). PF2065 is the last annotated gene in P. furiosusgenome and hence, PF0001-0005 genes are depicted to be a part of this locus. Gene organization was generated using Vector NTI (Invitrogen). (B) The amino acid sequence (46 residues) of a putative small peptide, presumably bacteriocin-like, discovered in the locus, its N and C termini indicated in red. Cysteines are highlighted in black. Amino acids are noted by their one letter code. (C) Transcriptional response of genes in the PF2064 locus. Red indicates higher transcript levels (log₂(fold change)>1), green indicates lower (log₂(fold change)<-1), and black denotes average transcript levels. The color scale used is the same as in Figure 4.