Comprehensive analysis of iron-regulated genome expression inenvironmental bacterium
Masataka TSUDA

Graduate School of Life Sciences, Tohoku University

Research aims
Iron is an essential element for the growth of almost all bacterial species, althoughmost inhabit environments where the solubility of iron is very low. Bacteria havetherefore evolved numerous mechanisms to efficiently acquire iron (and molecules
containing iron) under limiting conditions. However, a high concentration of
intracellular free iron is detrimental to bacteria because of the generation of highly toxic
reactive oxygen and reactive nitrogen species (ROS and RNS). Bacteria have therefore
evolved sophisticated systems to enzymatically remove ROS and RNS, as well assystems to maintain strict intracellular free iron homeostasis. Burkholderia multivorans
ATCC 17616 is a soil-derived bacterial strain that has an extraordinary metabolic
versatility and has been used to produce many industrially important enzymes1). Our
previous characterization of a fur ATCC 17616 deletant strain revealed that that the Furprotein is a global transcription regulator that is pleiotropically involved in free ironhomeostasis. Consequently, it is also involved in the removal of ROS, tolerance to RNS,
catabolism of TCA-cycle and aromatic carbon compounds, resistance to antibiotics, andsurvival in soil (Fig. 1)2,3). To gain a greater insight into the pleiotropic role of the Furprotein of ATCC 17616, spontaneous suppressor mutants of the ATCC 17616 furdeletant that exhibit restored RNS tolerance were analyzed4). RNS suppression led to
the restoration of phenotypes almost identical to that of the wild-type strain (Table 1).
One such suppressor mutant possessed a mutation in the oxyR gene, a transcriptional
regulator that modulates the adaptive response to ROS/RNS stress. The oxyR mutation
led to overproduction of ROS/RNS-removing enzymes in the fur deletant, explaining
the suppression of ROS/RNS-sensitive phenotypes that are normally exhibited by fur1

mutants. These results demonstrate the genetic interactions between Fur and OxyR
function4). Since this previous study indicated that the analysis of suppressor mutationsin the fur mutant is a viable strategy for demonstrating the transduction system of iron,
we performed a study to identify and characterize additional gene(s) whose mutation(s)suppress the pleiotropic phenotypes of the fur deletant.
Methods
Molecular genetic analysis systems developed by our group, in concert with various
well-established molecular biological and bacterial cultivation methods, were employedin this study 2-4). Illumina re-sequencing was used to identify suppressor mutations andto quantitatively determine transcriptional start sites (TSS analysis) of the ATCC 17616∆fur and its derivative genomes. Homology analysis of DNA and protein sequences wasperformed using NCBI databases. More detailed analysis, when required, wasperformed using our custom computer software(http://www.ige.tohoku.ac.jp/joho/labhome/tool.html).
Results
After examination, five suppressor mutants retaining the wild-type oxyR gene allpossessed the same point mutations in a gene encoding a protein with 81 amino-acidresidues. This protein showed 53% identity with a functionally unknown Yersinia
enterocolitica protein, HemP, which is encoded by the first gene in a hemPRSTUV
operon. Although the exact function of HemP is not known, the hemPRSTUV regioncontains genes responsible for the uptake and usage of heme (Fig. 2). Therefore, theidentified ATCC 17616 gene was also designated hemP. We confirmed that theintroduction of a hemP deletion in the ∆fur mutant gave rise to phenotypes
indistinguishable from those of the original suppressor mutants. The ATCC 17616 hemP
gene is located on the first chromosome, separated from the second chromosome-loaded
hemRSTUV operon. This second region is also predicted to contain genes involved inthe uptake and usage of heme. Transcription of hemP in ATCC 17616 was repressed andinduced under free iron-replete and -depleted conditions, respectively. The transcriptionof hemP strain was higher in the ∆fur under both conditions. Genome-wide TSS
analysis showed that hemP is one of the most transcribed 20 genes in the ∆fur genome.
Upstream of the hemP promoter region is a putative Fur box, a consensus sequence thatspecifically binds the Fur protein. Our genetic analysis revealed that this putative box is2

indeed able to specifically bind the Fur protein, demonstrating that hemP transcription is
directly suppressed by the Fur-Fe2+ complex. Transcription of hemP was also inducedby heme present in the culture medium. The hemP deletant of ACTT 17616 grewnormally in Fe3+-containing medium but was unable to grow in medium containing
heme as the only source of iron (heme medium). This revealed the importance of theHemP protein for the use of heme as an iron source. Our biochemical analysis also
indicated that HemP protein can bind heme and that HemP is located in the cytoplasm.
The ATCC 17616 hemR gene that putatively encodes an outer membrane receptor
specific for heme also possesses the Fur box upstream of the promoter. Transcription ofhemR was (i) repressed under iron-replete conditions by the direct binding of Fur-Fe2+complex at the Fur box, and (ii) induced by heme in the culture medium. HemP wasalso required for the heme-dependent transcriptional induction of hemR. The growthrate of the ATCC 17616 hemR deletant was significantly lower in heme medium
compared to Fe3+-containing medium, indicating the importance of ATCC 17616 HemR
for heme usage.
Conclusions
This study showed that (i) B. multivorans ATCC 17616 is capable of utilizing heme
as a sole source of iron, (ii) hemP and hemR gene products are involved in heme usage,(iii) transcription of both genes under iron-replete conditions is directly repressed byFur, and (iv) transcription of hemR requires hemP. Further transcriptional analysis of
the two genes (i) indicated the preferential usage of free iron rather than heme, and (ii)
led to the proposal of a working model in which heme in the culture medium induces
hemP transcription. The resulting gene product either directly or indirectly induces
hemR transcription. It remains unclear whether (i) HemP directly regulates hemRtranscription by binding to specific sequence(s) around hemR, (ii) if HemP indirectly
regulates hemR transcription by interacting with unidentified gene products or by
regulating the transcription of such genes, or (iii) if Fur and HemP (or a further
unidentified gene product) compete for binding to the hemR-containing region. These
unresolved points will be clarified in future studies. For example, genome-wide SELEX
analysis using the HemP protein and RNA-seq analysis with the hemP mutant. These
analyses will also provide important information concerning the molecular mechanisms
for how addition of hemP mutations to the ∆fur mutant leads to phenotypes very close
to the wild-type strain. Clarification of such mechanisms will contribute to3

understanding a wide variety of bacterial iron signal transduction systems.
References
1) Ohtsubo, Y., Nishiyama, E., Ishibashi, Y., Nagata, Y., and Tsuda, M. (2014) Strategies
to reveal genomic function in natural soil systems. In: Nojiri, H., Tsuda, M., Fukuda,M., and Kamagata, Y. (ed), Biodegradative Bacteria: How Bacteria Degrade, Survive,Adapt, and Evolve. pp 279-291. Springer-Verlag, Tokyo.
2) Yuhara, S., Komatsu, H., Goto, H., Ohtsubo, Y., Nagata, Y., and Tsuda, M. (2008)
Pleiotropic roles of iron-responsive transcriptional regulator Fur in Burkholderia
multivorans. Microbiology 154: 1763-1774.
3) Nagata, Y., Senbongi, J., Ishibashi, Y., Sudo, R., Miyakoshi, M., Ohtsubo, Y., andTsuda, M. (2014) Identification of Burkholderia multivorans ATCC 17616 genetic
determinants for fitness in soil by using signature-tagged mutagenesis. Microbiology
160: 883-891.
4) Kimura, A., Yuhara, S., Ohtsubo, Y., Nagata, Y., and Tsuda, M. (2012) Suppression ofpleiotropic phenotypes of a Burkholderia multivorans fur mutant by oxyR mutation.
Microbiology 158: 1284-1293.
5) Stojiljkovic, I., and Hantke, K. (1992) Hemin uptake system of Yersinia
enterocolitica: similarities with other TonB-dependent systems in gram-negative
bacteria. EMBO J. 11: 4359-4367.

4

Table 1. Phenotypes of B. multivorans ATCC 17616 wild-type strain and its derivatives

PhenotypeWild-type∆fur deletantROS sensitivity
RNS sensitivity

NormalNormalFe3+ uptake
Intracellular free ironUtilization of citrate
Utilization of succinate

NormalNormalNormalNormalSuppressor mutants∆fur∆oxyR∆fur + hemP mutation

Increased
Increased

NormalNormalNormalNormalIncreased
Increased
Decreased
Decreased

Increased
NormalNormalNormalIncreased
NormalNormalNormalFig. 1. Functional pleiotropy of the B. multivorans ATCC 17616 Fur protein

Fig. 2. Organization of genes for uptake and usage of heme. Arrow indicates thetranscriptional start site and direction. Numerals show the percent identity of eachgene product with the corresponding product of Y. enterocolitica. Function of Y.
enterocolitica gene: P, unknown; R, outer-membrane heme receptor; S, heme
monooxygenase; and T, U, and V, ABC transporter.

5