AUTHOR=Kim Kyung Hyun , Jia Baolei , Jeon Che Ok 

TITLE=Identification of Trans-4-Hydroxy-L-Proline as a Compatible Solute and Its Biosynthesis and Molecular Characterization in Halobacillus halophilus

JOURNAL=Frontiers in Microbiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.02054

DOI=10.3389/fmicb.2017.02054

ISSN=1664-302X

ABSTRACT=<p><italic>Halobacillus halophilus</italic>, a moderately halophilic bacterium, accumulates a variety of compatible solutes including glycine betaine, glutamate, glutamine, proline, and ectoine to cope with osmotic stress. Non-targeted analysis of intracellular organic compounds using <sup>1</sup>H-NMR showed that a large amount of trans-4-hydroxy-L-proline (Hyp), which has not been reported as a compatible solute in <italic>H. halophilus</italic>, was accumulated in response to high NaCl salinity, suggesting that Hyp may be an important compatible solute in <italic>H. halophilus</italic>. Candidate genes encoding proline 4-hydroxylase (PH-4), which hydroxylates L-proline to generate Hyp, were retrieved from the genome of <italic>H. halophilus</italic> through domain searches based on the sequences of known PH-4 proteins. A gene, HBHAL_RS11735, which was annotated as a multidrug DMT transporter permease in GenBank, was identified as the PH-4 gene through protein expression analysis in <italic>Escherichia coli</italic>. The PH-4 gene constituted a transcriptional unit with a promoter and a rho-independent terminator, and it was distantly located from the proline biosynthetic gene cluster (<italic>pro</italic> operon). Transcriptional analysis showed that PH-4 gene expression was NaCl concentration-dependent, and was specifically induced by chloride anion, similar to the <italic>pro</italic> operon. Accumulation of intracellular Hyp was also observed in other bacteria, suggesting that Hyp may be a widespread compatible solute in halophilic and halotolerant bacteria.</p>