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- Haloferax volcanii (2)
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Iron is part of many redox and other enzymes and, thus, it is essential for all living beings. Many oxic environments have extremely low concentrations of free iron. Therefore, many prokaryotic species evolved siderophores, i.e., small organic molecules that complex Fe3+ with very high affinity. Siderophores of bacteria are intensely studied, in contrast to those of archaea. The haloarchaeon Haloferax volcanii contains a gene cluster that putatively encodes siderophore biosynthesis genes, including four iron uptake chelate (iuc) genes. Underscoring this hypothesis, Northern blot analyses revealed that a hexacistronic transcript is generated that is highly induced under iron starvation. A quadruple iuc deletion mutant was generated, which had a growth defect solely at very low concentrations of Fe3+, not Fe2+. Two experimental approaches showed that the wild type produced and exported an Fe3+-specific siderophore under low iron concentrations, in contrast to the iuc deletion mutant. Bioinformatic analyses revealed that haloarchaea obtained the gene cluster by lateral transfer from bacteria and enabled the prediction of enzymatic functions of all six gene products. Notably, a biosynthetic pathway is proposed that starts with aspartic acid, uses several group donors and citrate, and leads to the hydroxamate siderophore Schizokinen.
The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.