Previously, a 2-D gel expression study revealed that the expression of protein SO1377 was down-regulated during aerobic respiration in a S. oneidensis
FUR (ferric uptake regulator) mutant [11
]. We were curious if the protein was involved, directly or indirectly, in iron metabolism of S. oneidensis
MR-1. Results from this study provided evidence for this hypothesis. The deletion of SO1377
gene altered iron metabolism. Compared with the wild-type control, the mutant accumulated 3 times less intracellular 'free' iron, although the change of total intracellular iron was not evident. Siderophore secretion was lower in the mutant. This was supported by the microarray analysis which showed that the expressions of some genes involved in iron metabolism were altered.
Our study showed that the disruption of SO1377
affected other aspects of cellular activities of S. oneidensis
mutant WG3 was highly sensitive to hydrogen peroxide challenge and had a higher spontaneous, mutation rate of other genes. Microarray analysis showed that even without H2
challenge, some genes involved in oxidative damage protection were up-regulated, presumably in response to an increased level of internal H2
, due to deletion of the SO1377
gene. These phenotypic changes were typical and well documented for mutants of genes involved in iron metabolism and/or oxidative damage protection [20
]. For instance, the study carried out by Touati et al
(1995) showed that the permanent derepression of iron assimilation systems in a fur
deletion mutant of E. coli
produced an oxidative stress and DNA damage including lethal and mutagenic lesions [23
Microarray/proteomic analysis also suggests that dysfunction of SO1377 affects the proteins involved in respiratory electron transfer and thus respiration functions. The overall trend shows, genes involved in aerobic respiration down-regulate, whereas those involved in anaerobic respiration up-regulate. This coincides with the phenotype observation showing that the mutant has a growth deficiency under aerobic but not anaerobic conditions. This suggests that SO1377 perhaps plays a role in oxidative damage protection in the wild-type strain. Without protection from SO1377, cells tend to down-regulate their aerobic respiration and are more prone to produce free radicals under aerobic conditions. Probably as compensation, bacterial anaerobic respiration pathways were up-regulated in the mutant cells. With the same reasoning, decreased secretion of siderophore, for iron absorption and less accumulation of loose intracellular iron in the mutant should be viewed as adaptation responses of bacterial cells to the loss of SO1377 function. Nevertheless, without SO1377, the bacterium becomes more susceptible to H2O2 treatment and gives rise to spontaneous mutation of other genes at a level much higher than that of the wild-type strain.
Little is known about the action of SO1377 and how it biochemically interacts with other proteins is still a speculative issue. Since no sequence/structure clues suggest that SO1377 could directly interact with iron or any other metals, it is more likely that the phenotypes derived from disruption of SO1377 is indirect. That is, through interactions with other proteins, SO1377 may indirectly participate in iron metabolism of S. oneidensis MR-1.
We envision that SO1377 is likely to function as an accessory protein participating in intracellular iron trafficking in S. oneidensis
MR-1. Evidence supporting this idea comes from several lines. The N-terminal sequence of SO1377 belongs to the PHB protein family. Computational analysis suggests that SO1377 (referred as 4840) has potential functions in formation of protein complexes at the inner bacterial cell membrane, ATP/GTP binding, nucleotide binding, protein transport and, molecular chaperone [18
]. TC-BLAST searching shows that partial sequences of SO1376 have similarities with ATX2 and BSD2 of S. cerevisiae
, respectively http://tcdb.ucsd.edu/tcdb/blast/canblast.php
. In S. cerevisiae
, ATX2 encodes a manganese-trafficking protein that localizes to Golgi-like vesicles [24
], whereas BSD2 encodes a copper homeostasis factor [25
]. Also, SO1376 has four cysteine residues at positions 19, 68, 121 and 136, as well as two histidine residues at positions 151 and 155, suggesting that this protein, of 212 amino acids, has potential to be a metalloprotein. Still, a GES hydrophopathy analysis infers SO1376 is a membrane protein. These clues suggest that SO1376 could play a role as a metallochaperone, directly participating in intracellular iron trafficking. Working together, a presumed SO1376/1377 complex could receive ferrous iron from an upstream protein, i.e. SO3034 (a putative ferric iron reductase protein whose transcriptional level was down-regulated 2.2-fold in the SO1377
mutant cells, see Table ), and then transfer it to downstream proteins, such as those involved in cytochrome heme biosynthesis or Fe-S center biosynthesis, etc. The dysfunction of SO1377 could impair the function of SO1376 and compromise the effective process of intracellular iron (II) trafficking resulting in the free iron 'leaking' into the cytoplasm of cells. As a highly reactive species, the 'leaking' free iron could generate very toxic radicals under aerobic conditions, resulting in hypersensitivity to oxidative stress, presumably as a result of Fenton chemistry. Consequently, this elicits DNA damage and onset of SOS response. This may explain why the SO1377
mutant has a higher level of gentamicin/kanamycin resistant mutants.