Production of Polyhydroxyalkanoates in Unsterilized Hyper-Saline Medium by Halophiles Using Waste Silkworm Excrement as Carbon Source

The chlorophyll ethanol-extracted silkworm excrement was hardly biologically reused or fermented by most microorganisms. However, partial extremely environmental halophiles were reported to be able to utilize a variety of inexpensive carbon sources to accumulate polyhydroxyalkanoates. In this study, by using the nile red staining and gas chromatography assays, two endogenous haloarchaea strains: Haloarcula hispanica A85 and Natrinema altunense A112 of silkworm excrement were shown to accumulate poly(3-hydroxybutyrate) up to 0.23 g/L and 0.08 g/L, respectively, when using the silkworm excrement as the sole carbon source. The PHA production of two haloarchaea showed no significant decreases in the silkworm excrement medium without being sterilized compared to that of the sterilized medium. Meanwhile, the CFU experiments revealed that there were more than 60% target PHAs producing haloarchaea cells at the time of the highest PHAs production, and the addition of 0.5% glucose into the open fermentation medium can largely increase both the ratio of target haloarchaea cells (to nearly 100%) and the production of PHAs. In conclusion, our study demonstrated the feasibility of using endogenous haloarchaea to utilize waste silkworm excrement, effectively. The introduce of halophiles could provide a potential way for open fermentation to further lower the cost of the production of PHAs.

Genome Sequence of Hardyhisp2, a Gammapleolipovirus Infecting Haloarcula hispanica

ABSTRACT Hardyhisp2 virus infects the halophilic archaeon Haloarcula hispanica DSM 4426T and is closely related to His2 (Pleolipoviridae family). The viral genome is 16,133 bp long, with terminal inverted repeats of 599 bp. The predicted spike protein is only weakly similar (32% amino acid identity) to that of His2.

The Novel Halovirus Hardycor1, and the Presence of Active (Induced) Proviruses in Four Haloarchaea

The virus Hardycor1 was isolated in 1998 and infects the haloarchaeon Halorubrum coriense. DNA from a frozen stock (HC1) was sequenced and the viral genome found to be 45,142 bp of dsDNA, probably having redundant, circularly permuted termini. The genome showed little similarity (BLASTn) to known viruses. Only twenty-two of the 53 (41%) predicted proteins were significantly similar to sequences in the NCBI nr protein database (E-value ≤ 10−15). Six caudovirus-like proteins were encoded, including large subunit terminase (TerL), major capsid protein (Mcp) and tape measure protein (Tmp). Hardycor1 was predicted to be a siphovirus (VIRFAM). No close relationship to other viruses was found using phylogenetic tree reconstructions based on TerL and Mcp. Unexpectedly, the sequenced virus stock HC1 also revealed two induced proviruses of the host: a siphovirus (Humcor1) and a pleolipovirus (Humcor2). A re-examination of other similarly sequenced, archival virus stocks revealed induced proviruses of Haloferax volcanii, Haloferax gibbonsii and Haloarcula hispanica, three of which were pleolipoviruses. One provirus (Halfvol2) of Hfx. volcanii showed little similarity (BLASTn) to known viruses and probably represents a novel virus group. The attP sequences of many pleolipoproviruses were found to be embedded in a newly detected coding sequence, split in the provirus state, that spans between genes for integrase and a downstream CxxC-motif protein. This gene might play an important role in regulation of the temperate state.

Growth Phase Dependent Cell Shape of Haloarcula

Several haloarchaea are reported to be pleomorphic, while others exhibit remarkable shapes, such as squares. Recently, Haloferax volcanii was found to alter its morphology during growth. Cells are motile rods in early exponential phase, and immotile plates in stationary phase. It is unknown if this growth phase dependent cell shape alteration is a specific feature of Hfx. volcanii, or conserved amongst haloarchaea. Here, we studied the cell shape and motility of two haloarchaea species Haloarcula hispanica and Haloarcula californiae. With a combination of light and electron microscopy, we observed that both strains undergo a growth phase dependent morphological development, albeit in a slightly different fashion as Hfx. volcanii. For both Haloarcula strains, the cell size is changing throughout growth. Cell shape seems to be related with motility, as highly motile cells on semi-solid agar plates are predominantly rod-shaped. We conclude that the growth phase dependent cell morphology alteration might be a common feature amongst haloarchaea, and that cell shape is generally linked with a motile life style. The conservation of this phenomenon underscores the importance of studies of the molecular mechanisms regulating cell shape in archaea.

Haloarcula mannanilytica sp. nov., a galactomannan-degrading haloarchaeon isolated from commercial salt

A mannan-degrading halophilic archaeal strain, MD130-1T, was isolated from a commercial salt sample. Cells were motile, rod-shaped, and stained Gram-negative. Colonies were pink pigmented. Strain MD130-1T was able to grow at 1.5–4.6 M NaCl (optimum, 3.6 M) at pH 6.0–8.0 (optimum, pH 7.0) and at 25–50 °C (optimum, 40 °C). The DNA G+C content was 62.1 mol% (genome). The orthologous 16S rRNA gene sequence showed the highest similarity (99.4 %) to those of Haloarcula japonica JCM 7785T and Haloarcula hispanica JCM 8911T. The values of genome relatedness between strain MD130-1T and Haloarcula species were 84.33–85.96 % in ANIb and 30.4–32.9 % using GGDC formula 2. The polar lipids of strain MD130-1T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and triglycosyl diether-2. Based on the results of phenotypic and phylogenetic analyses, the strain represents a new species of the genus Haloarcula , for which the name Haloarcula mannanilytica sp. nov. is proposed. The type strain is MD130-1T (=JCM 33835T=KCTC 4287T) isolated from commercial salt made in Ishikawa prefecture, Japan.

CTP synthase forms cytoophidia in archaea

CTP synthase (CTPS) is an important metabolic enzyme that catalyzes the rate-limiting reaction of de novo synthesis of the nucleotide CTP. Since 2010, a series of studies have demonstrated that CTPS can form filamentous structures termed cytoophidia in bacteria and eukaryotes. However, it remains unknown whether cytoophidia exist in archaea, the third domain of life. Using Haloarcula hispanica as a model system, here we demonstrate that CTPS forms distinct intracellular compartments in archaeal cells. Under stimulated emission depletion (STED) microscopy, we find that some HhCTPS compartments have elongated filamentous structures, resembling cytoophidia in bacteria and eukaryotes. When Haloarcula cells are cultured in low-salt medium, the occurrence of cytoophidia increases dramatically. Moreover, overexpression of CTPS or glutamine analog treatment promotes cytoophidium assembly in H. hispanica. Our study reveals that CTPS forms cytoophidia in all three domains of life, suggesting that this is an ancient property of CTPS.