# Presentation: 850341

## Functional annotation of a conserved hypothetical protein in Shewanella oneidensis MR-1: 

**possible roles involved in iron homeostasis and oxidative damage protection**

- _Weimin Gao_1, Christal Secrest2, Carol S. Giometti3, Yongqing Liu1, Liyou Wu1, Xueduan Liu1, Tingfen Yan1,
- Dawn stanek1, Alex Beliaev1 Dorothea K. Thompson1, Matthew W. Fields1, Dong Xu2, Ying Xu2 and Jizhong Zhou1

**Here we report the functional annotation of SO1377, a conserved hypothetical protein of 592 amino acids in *****Shewanella oneidensis***** MR-1. To elucidate the functions of this novel protein, a procedure combing both computational and experimental approaches was applied. Computational analysis of secondary and 3-D structure revealed this protein has potential functions in formation of protein complexes at the inner bacterial cell membrane, ATP/GTP binding, nucleotide binding, protein transport and molecular chaperone. In order to assess possible functions, we generated a knockout mutant of the gene encoding this protein and characterized the phenotype of resulting mutant under various physiological conditions. Our results showed that, compared with the wild type strain, the in-frame deletion mutant becomes very sensitive to the hydrogen peroxide challenge, whereas its siderophore production is enhanced in iron-depleted media. The deletion mutant also produces spontaneous gentamycin resistant mutants at the rate of two-order magnitude higher than that of the wild strain. When cultured in an enriched medium, the mutant also has a decreased  growth rate. To find out how the deletion of this gene influences the expression of other genes of *****S. oneidensis *****MR-1 under such conditions, expression profiles of both the mutant and wild type strains were generated. Analysis of mutant cells revealed numerous gene and protein expression changes relative to wild-type expression. Our study suggested that, SO1377, along with another protein (SO1376) in the same operon, are perhaps play roles involved in the iron homeostasis and oxidative damage protection of *****S. oneidensis***** MR-1. More specifically, SO1376 and 1377 are very likely involved in the process of intracellular iron trafficking and delivery to those iron containing proteins, like many other metallochaperons revealed in recent years. **

**ABSTRACT**

- 1Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN,
- 2The University of Tennessee-Oak Ridge National Laboratory, Knoxville, TN, 3Argonne National Laboratory, Argonne, IL

**Microarray hybridization and data analysis. **Microarray fabrication, hybridization, probe labeling, image acquisition and processing were carried out as described in references**. **Gene expression analysis was performed using 3 independent microarray experiments, with each slid containing 2 replicate array of *S. onedensis* MR-1 genome, a collection of 4,608 distinct ORFs. Total cellular RNA from wild type and SO1377 deletion mutant grown at the stage of late log phase in the enriched medium MR2A was isolated and purified using the TRIzol Reagent (Gibco BRL) according to the manufacture’s instruction. The ratios of the mutant samples to the wild type control were normalized using Pooled-Common-Error model provided by the statistical analysis software ArrayStat v. 2.0. In addition, we also used the following principle to assess the microarray data quality.  We checked possible associated expression patterns for those genes within the same putative operon.  Indeed, many co-regulated genes in response to the deletion of SO1377 are located in the same operons (Figure 7). Also, we selected a few genes that had significant changes between wild type and the mutant for real-time

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**A computational study of SO1377**

**ACKNOWLEDGMENTS**

- This research was funded by the Department of Energy (Office of Biological and Environmental Research, Office of Science) grants from the Microbial Cell Program and Genome To Life Program. Oak Ridge National Laboratory is managed by University of Tennessee-Battelle LLC for the Department of Energy under contract DOE-AC05-00OR22725.

*Shewanella oneidensis* MR-1 is a facultative, Gram-negative bacterium capable of coupling the oxidation of organic carbon to a wide range of electron acceptors such as iron oxides, and thus has a potential to be applied in bioremediation of DOE contaminated sites. The complete 5-Mb genome of *S. oneidensis* MR-1 was recently sequenced by The Institute for Genomic Research (TIGR), and the application of standard sequence-comparison methods revealed that approximately 42% of the MR-1 genome encodes proteins of unknown function. The similar situations can be found in other organisms whose genomes were sequenced recently. Elucidating the functions of these novel genes poses a big challenge to the scientific community yet the significance of such efforts cannot be underestimated to our thoroughly understanding of biological activities of individual organism. In this study, we initiated to characterize the functions of one such hypothetical protein in *S. oneidensis* MR-1. This protein is designated SO1377, having 592 amino acids, and appears its homologue also presents in other bacteria, such as ypiK of *Escherichia coli* with 40% sequence identity. Previous studies suggested this protein probably involves in iron metabolism since it was down-regulated during aerobic respiration in a *Shewanella* FUR (ferric uptake regulator) mutant . In this study, we continued our efforts to elucidate the functions of this protein by combing a computational analysis and experimental approaches.

**INTRODUCTION**

- Secondary structure revealed this protein has potential functions in formation of protein complexes at the inner bacterial cell membrane, ATP/GTP binding, nucleotide binding, protein transport and molecular chaperone (Figure 2). 3-D structure was also generated and found it has a highly similarity with 1dg3a, an interferon-induced guanylate-binding protein which is a member of the dynamin family of proteins (Figure 2). 3-D structures of proteins similar to SO1377 were also shown in Figure 2.

**Hydrogen peroxide challenge. **Cells grown in LB medium to an optical density of 0.5 were distributed into 50-ml Relenmeyer flasks (5 ml each), and hydrogen peroxide was added to various concentrations from 2 mM to 30 mM. After 20 min of incubation with shaking at 30°C, treatment was stopped by addition of catalase (Boehringer-Mannheim) at 400 U/ml and chilling, followed by plating on LB plates. Samples were diluted in cold 10-2 MgSO4 containing 400 U/ml catalase per ml. Colonies were counted after 24 h of incubation at 30°C. The results showed that the mutant is highly sensitive to hydrogen peroxide challenge compared with the wild type strain (Figure 4).

- Figure 1. Schematic representation of ORF0781(SO1377) locus

- ORF0783

- ORF0782

- ORF0781

**ORF0780(SO1378): ThiJ/P family protein**
**ORF0781(SO1377): conserved hypothetical protein**
**ORF0782(SO1376): putative oxidoreductase**
**ORF0783(SO1375): carboxypeptidase **

- ORF0780

- 3-D structure of SO1377

- Structures with similar motifs

- Secondary structure of SO1377

- Figure 2. Secondary and three dimensional  structure of  ORF0781(SO1377)

**In-frame deletion of SO1377(ORF0781)**

- ATG

- TAA

- ORF 0781

- PCR1

- PCR2

- A

- B

- C

- D

- Mix PCR1 & PCR2 with primers A & D

- A

- ATG

- TAA

- D

- Transform ligation product into *E.coli* S17-1

- Transfer ligation product into MR-1 by
- conjugative mating approach

- Screening deletion mutants on LB + 5% Sugar

- Confirmation of deletion mutants by PCR and sequencing

- Ligate PCR product with plasmid vector pDS 3.0

- Figure 3. Schematic illustration of in-frame deletion mutagenesis of ORF0781(SO1377).

**Physiological differentiation between **
**wild type and deletion mutant of SO1377 **

- Figure 4. Response of wild type (MR-1)
- and mutant strain (WG3) to H2O2 treatment

**MR-1**

**WG3**

**MR-1**

**WG3**

- Figure 5. Siderophore production of
-  wild type MR-1 and mutant WG3

**Siderophore production. **A blue agar was used to determine the siderophore production of both wild type and deletion mutant strains following the direction of reference (Schwyn B. and Neilands J. B. 1987. Universal chemical assay for the detection and dertermination of siderophore. Analysis Biochemistry 160: 47-56). Both the wild type strain and mutant grown in LB medium to stationary stage were collected and washed with 0.7% NaCl solution twice and then resuspended  in 0.7% NaCl solution again to make  both strains  reach the same concentration. 5 l suspension of each strain was dropped on the same blue agar, followed by incubation for three days at 30°C. The results showed that the mutant WG3 can secrete more siderophores and then grow better than the wild type strain MR-1 on the blue agar (Figure 5).

- The amino acid sequence for the hypothetical protein was used to predict the secondary structure using PHD and PSIPRED. GenTHREADER and PROSPECT were used for protein threading. The threading results can be visualized using RasMol and CHIME and compared with the three dimensional alignment of the template to the query. Once the best threading template is chosen, the three dimensional structural prediction for the query protein can be generated using MODELLER. Many other web-based programs, such as PSI-BLAST, MOTIF, BLOCKS, ProDom, Pfam, SignalP, TMD,PSORT and PRINTS,  were also used in this study.
- The SO1377 locus is shown as Figure 1. SO1376 and SO1377 are likely organized as an operon, which is flanked by SO1378 and SO1375 from both sides.  SO1377 is divided with 23bp from SO1376. On the upstream of SO1376(ORF0782), a ribosome binding site and likely FUR (Ferric Uptake Regulator) box are identified (Figure 1), suggesting this operon is under the regulation of FUR protein. Also, on the downstream of SO1377(ORF0781),  a -independent transcriptional terminater is identified (data not shown here), inferring SO1377 and SO1376 are on a same transcription unit.

- The whole process of generating the in-frame deletion mutant of SO1377 was illustrated in Figure 2.

**Spontaneous mutation rate of gentamicin resistance. **Both the deletion mutant and wild type strain grown in LB liquid to mid-log phase were spread onto LB agar plus 15 ul/ml gentamycin. After incubation for  two days at 30°C, resistance colony was appeared on LB agar and counted. A series dilution of the same liquid cultures of deletion mutant and wild type strain was  also made and spread onto LB agar with out gentamycin for CFU counting. The spontaneous mutation rate of gentamycin resistance for deletion mutant WG3 is 6.0 x 10-7 whereas the wild type strain MR-1 is 2.3 x 10-8

- Growth of MR-1 and WG3 with 0.1 M NaCl

- Growth of MR-1 and WG3 with 0.5 M NaCl

- Growth of MR-1 and WG3 with 0.6 M NaCl

- wild type

- mutant

- Figure 6. Growth difference between the wild type strain MR-1 and SO1377 deletion mutant
-  WG3 in an enriched medium MR2A.

**Transcription profile differentiation between**
** ****wild type and deletion mutant of SO1377**

**Overview of microarray data**

**Transport and binding protein**

**Energy metabolism**

- Figure 7. Microarray  data
- showing the expression alternation of genes in response to the deletion of SO1377.

- ORF0781(SO1377)

**RPS-BLAST search result showed that, in addition **
**to be included in a conserved hypothetical bacterial**
**protein family COG2268,  partial of SO1377 also **
**belongs to SPFH domain/Band7/ family and PHB **
**(prohibitin homologues) family. **

**Growth features. **In enriched media under aerobic condition, the deletion mutant can grow slower than the wild type strain. Salt stress (NaCl) can signify such difference (Figure 6). However, under anaerobic conditions, no growth difference was observed  between the wild type and mutant strain using fumarate or NO-3 as an electron acceptor while lactate as an electron donor. Also, the same is true in terms of the ability of iron (III) dissimilatory reduction.

- quantitative RTPCR analyses. The gene expression patterns of these genes were confirmed by RTPCR.  Based on above results, we were confident that the microarray data is indeed of good quality. We found that around 500 genes of the mutant have significantly changed their expression compared with that of the wild type strain. Among them, many genes are involved in  the process of transport and binding, energy metabolism (Figure 7).

**References**
- Schwyn B. and Neilands J. B. 1987. Universal chemical assay for the detection and dertermination of siderophore. Analysis Biochemistry 160: 47-56.
- Thompson DK et. al. 2002. Transcriptional and proteomic analysis of a ferric uptake regulator (fur) mutant of Shewanella oneidensis: possible involvement of fur in energy metabolism, transcriptional regulation, and oxidative stress.Appl Environ Microbiol. 2002 Feb;68(2):881-92
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