Differential gene content and gene expression for bacterial evolution and speciation of Shewanella in terms of biosynthesis of heme and heme-requiring proteins

doi:10.1186/s12866-019-1549-9

. 2019 Jul 30;19(1):173.

doi: 10.1186/s12866-019-1549-9.

Differential gene content and gene expression for bacterial evolution and speciation of Shewanella in terms of biosynthesis of heme and heme-requiring proteins

Jingcheng Dai^{1

2}, Yaqi Liu^{1

2}, Shuangyuan Liu^{1

2}, Shuyang Li^{1

2}, Na Gao^{1

2}, Jing Wang^{1

2}, Jizhong Zhou^{3

4}, Dongru Qiu^{5

6}

Affiliations

¹ Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, Hubei Province, China.
² University of Chinese Academy of Sciences, Beijing, 100049, China.
³ Institute for Environmental Genomics, and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73019, USA.
⁴ Earth Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94270, USA.
⁵ Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, Hubei Province, China. qiu@ihb.ac.cn.
⁶ University of Chinese Academy of Sciences, Beijing, 100049, China. qiu@ihb.ac.cn.

PMID: 31362704
PMCID: PMC6664582
DOI: 10.1186/s12866-019-1549-9

Differential gene content and gene expression for bacterial evolution and speciation of Shewanella in terms of biosynthesis of heme and heme-requiring proteins

Jingcheng Dai et al. BMC Microbiol. 2019.

. 2019 Jul 30;19(1):173.

doi: 10.1186/s12866-019-1549-9.

Authors

Jingcheng Dai^{1

2}, Yaqi Liu^{1

2}, Shuangyuan Liu^{1

2}, Shuyang Li^{1

2}, Na Gao^{1

2}, Jing Wang^{1

2}, Jizhong Zhou^{3

4}, Dongru Qiu^{5

6}

Affiliations

¹ Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, Hubei Province, China.
² University of Chinese Academy of Sciences, Beijing, 100049, China.
³ Institute for Environmental Genomics, and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73019, USA.
⁴ Earth Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94270, USA.
⁵ Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, Hubei Province, China. qiu@ihb.ac.cn.
⁶ University of Chinese Academy of Sciences, Beijing, 100049, China. qiu@ihb.ac.cn.

PMID: 31362704
PMCID: PMC6664582
DOI: 10.1186/s12866-019-1549-9

Abstract

Background: Most species of Shewanella harbor two ferrochelatase paralogues for the biosynthesis of c-type cytochromes, which are crucial for their respiratory versatility. In our previous study of the Shewanella loihica PV-4 strain, we found that the disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), but it is different in Shewanella oneidensis MR-1. Hence, the function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2, are investigated in S. oneidensis MR-1.

Result: In the present study, deletion of either hemH1 or hemH2 in S. oneidensis MR-1 did not lead to overproduction of extracellular protoporphyrin IX (PPIX) as previously described in the hemH1 mutants of S. loihica PV-4. Moreover, supplement of exogenous hemins made it possible to generate the hemH1 and hemH2 double mutant in MR-1, but not in PV-4. Under aerobic condition, exogenous hemins were required for the growth of MR-1ΔhemH1ΔhemH2, which also overproduced extracellular PPIX. These results suggest that heme is essential for aerobic growth of Shewanella species and MR-1 could also uptake hemin for biosynthesis of essential cytochrome(s) and respiration. Besides, the exogenous hemin mediated CymA cytochrome maturation and the cellular KatB catalase activity. Both hemH paralogues were transcribed in wild-type MR-1, and the hemH2 transcription was remarkably up-regulated in MR-1ΔhemH1 mutant to compensate for the loss of hemH1. The periplasmic glutathione peroxidase gene pgpD, located in the same operon with hemH2, and a large gene cluster coding for iron, heme (hemin) uptake systems are absent in the PV-4 genome.

Conclusion: Our results indicate that the genetic divergence in gene content and gene expression between these Shewanella species, accounting for the phenotypic difference described here, might be due to their speciation and adaptation to the specific habitats (iron-rich deep-sea vent versus iron-poor freshwater) in which they evolved and the generated mutants could potentially be utilized for commercial production of PPIX.

Keywords: Cytochrome; Ferrochelatase; Hemin; Protoporphyrin IX; Shewanella.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

**Fig. 1**
Effects of *hemH1* and *hemH2* double deletions on phenotypes of *S. oneidensis* MR-1. a The heme biosynthesis pathway; b Organization of the ferrochelatase genes *hemH1* and *hemH2* in *S. loihica* PV-4 and *S. oneidensis* MR-1. The operon encodes a periplasmic glutathione peroxidase (SO_3349) and a ferrochelatase paralogue (*hemH2*) in strain MR-1 while the former is absent in strain PV-4; c Inactivation of both ferrochelatase genes (*hemH1* and *hemH2*) resulted in overproduction of protoporphyrin IX in MR-1. Cell colonies grown from a droplet of mid-log-phase culture (OD₆₀₀ of ~ 0.2) for each indicated strain on LB plates (supplemented with 10 μg/ml of hemin), the *hemH1* and *hemH2* double mutant exhibited a red-color phenotype while deletion of either *hemH1* or *hemH2* did not cause the same phenotypic change. In the genetic complementation analyses on the PPIX-overproducing *hemH1* deletion mutant PV-4Δ*hemH1*, the plasmid-borne wild-type *hemH1* or *hemH2* gene restored the phenotype of the mutant to the same pink color of the wild-type strain carrying the empty vector

**Fig. 2**
Effects of supplemented hemin concentrations on the bacterial growth of the *hemH1* and *hemH2* double mutant. a The *hemH1* and *hemH2* double mutant exhibited red-colored phenotype with supplement of hemin (10 μg/ml) while the double mutant could not grow without supplement of hemin. In the genetic complementation analyses, the plasmid borne wild-type *hemH1* or *hemH2* gene restored phenotype of the *hemH1* and *hemH2* double mutant to that of the wild-type strain carrying the empty vector with or without supplement of hemin; b The bacterial strains were grown in the LB broth containing 0, 0.001, 0.01, 0.1, 1, 10, 100 μg/ml of hemin and 15 μg /ml of gentamycin, and incubated at 28 °C for 18 h; c The growth of the MR-1, MR-1Δ*hemH1*, MR-1Δ*hemH2*, MR-1Δ*hemH1*Δ*hemH2* in the LB broth with 0, 0.1, 1 μg/ml of hemin over time was measured. Growth was determined by measuring OD₆₀₀ values

**Fig. 3**
The chemical analyses of the extracellular compound. The samples and a PPIX standard (Sigma-Aldrich, St. Louis, MO) were dissolved in a solution containing 90% acetone and 10% 0.1 M NH₄OH. a Electrospray ionization tandem mass spectrometry (ESI-MS/MS, precursor ion: 563.2-PPIX) analyses showing almost identical structure of the PPIX standard and the bacterial samples; b Ultraviolet-visible spectrograms of the bacterial extract (the *hemH1* and *hemH2* double mutant) and PPIX standard. The absorbance was measured at every 10 nm (nm) with a spectrometer (UV-1800, Mapada) and quartz cuvettes

**Fig. 4**
Transcriptional analyses of *rpoE2* and *hemH* paralogues in the wild-type strain and the *hemH1*-null mutant of *S. oneidensis* MR-1. a Semi-quantitative RT-PCR analyses of *rpoE2* and *hemH2* transcripts in MR-1 and MR-1Δ*hemH1* strains; b The Real-time PCR analyses of *hemH2* transcripts in MR-1 and MR-1Δ*hemH1* strains; c the relative expression ratio of *hemH1* to *hemH2* in PV-4 and MR-1. Cell samples of the strains were collected for RNA extraction at optical density (OD₆₀₀) of 0.6, 1.3, and 2.2. Transcription of the *16S rRNA* genes was analyzed and used as the internal control gene. The assays were performed in triplicates and the error bars represented the standard deviation (SD) of triplicate independent samples

**Fig. 5**
Effects of visible light and hydrogen peroxide (H₂O₂) on the MR-1Δ*hemH1*Δ*hemH2* mutant. a Effects of hydrogen peroxide (H₂O₂) addition on the growth of the MR-1Δ*hemH1*Δ*hemH2* mutant. The MR-1 wild type strain, the *hemH1*, the *hemH2* in-frame deletion mutants and the *hemH1-hemH2* double mutant strain were grown in LB broth containing 0, 0.1, 0.3, 0.5, 0.7 and 1 mM of hydrogen peroxide and incubated at 28 °C for 18 h. Different concentrations (0, 0.1, 1, 10 μg/ml) of hemin didn’t rescue the growth rate of the hemH1-hemH2 double mutant, when these strains were grown in the LB broth containing 0, 0.1, 0.3, 0.5, 0.7 and 1 mM of hydrogen peroxide and incubated at 28 °C for 18 h; b Effects of visible light on the growth of the MR-1Δ*hemH1*Δ*hemH2* mutant. Cell colonies grown from a droplet of mid-log-phase culture (OD₆₀₀ of ~ 0.2) for each indicated strain on LB plates (supplemented with 10 μg/ml of hemin and 15 μg/ml gentamycin). Experiments were conducted under visible light (about 700~1,000 lx) and dark condition on LB plates. The wild type MR-1 carrying the empty vector was used as control, the double deletion mutant MR-1Δ*hemH1*Δ*hemH2* contained the empty vector, pHERD30T-*hemH1* and pHERD30T-*hemH2*, respectively, were compared. Different concentrations of supplement hemin failed to rescue the phenotype and the growth rate of the *hemH1-hemH2* double mutant, when these strains were grown in the LB broth containing 0, 0.1, 1, 10, 100 μg/ml of hemin and incubated at 28 °C for 18 h; c The KatB-dependent peroxidase activity assays of MR-1 and the double mutant MR-1Δ*hemH1*Δ*hemH2*

**Fig. 6**
Effects of *hemH1* and *hemH2* double deletions on c-type cytochrome synthesis and nitrate reduction in *S. oneidensis* MR-1. a Bacteria were cultivated in the modified M1 minimal media supplemented with 2 mM sodium nitrate under microoxic conditions (in tightly capped tubes incubated without shaking). The blank represents the culture media without bacterial inoculation. Error bars represent SD; b Hemin could rescue the nitrate reduction capacity of MR-1Δ*hemH1*Δ*hemH2* under microoxic conditions; c The effect of different hemin concentration(0, 0.1, 1, 10 μg/ml) on nitration reduction in *hemH1-hemH2* double mutant strain MR-1Δ*hemH1*Δ*hemH2* under microoxic conditions; d Effects of *hemH1-hemH2* double deletion on c-type cytochrome synthesis in MR-1. Total protein (left) content and Heme staining (right) analyses of c-type cytochromes in the following stains: MR-1 wild-type, MR-1Δ*hemH1*, MR-1Δ*hemH2*, MR-1Δ*hemH1*Δ*hemH2* (2X means the double quantity of sample). After cell disruption, the supernatants containing the cellular protein fraction were resuspended in the SDS loading buffer and then incubated at 37 °C for 1 h; e Top panel: The CymA protein content of the wild-type strain MR-1 cells is ∼2-fold higher than that of the double mutant MR-1Δ*hemH1*Δ*hemH2* cells, but the heme-staining analyses showed that the CymA protein were not detected. Cells were grown in M1 medium supplemented with 2 mM sodium nitrate and 0.1 μg/ml hemin under microoxic conditions, and CymA protein content was quantified by using western blotting and densitometry. The lanes contained equivalent total proteins. Bottom panel: The Real-time PCR analyses of *napB* and *cymA* transcripts in MR-1 and MR-1Δ*hemH1hemH2* strains

**Fig. 7**
The uptake of exogenous hemin depends on TonB1 energy transduction system in *S. oneidensis* MR-1. a Organization of the putative hemin uptake gene cluster in MR-1. The region corresponds to the open reading frames from SO_3667 to SO_3675 (left to right); b Effects of exogenous hemin supplement on the PPIX production of MR-1Δ*hemH1*Δ*hemH2*. Ultraviolet-visible spectrograms of the bacterial extract (the *hemH1* and *hemH2* double mutant). The absorbance was measured at a wavelength 405 nm with a spectrometer (UV-1800, Mapada) and quartz cuvettes; c Effects of exogenous hemin supplement on the transcription of *hemA*, *hemB*, *hemD*, *hemF*, *hemG* genes in the double mutant MR-1Δ*hemH1*Δ*hemH2*; d Transcriptional analyses of *hmuX, exbB* and *hmuC* in the wild-type strain and the *hemH1-hemH2* double mutants of MR-1. Effect of exogenous hemin supplement (0.1 μg/ml, 5 μg/ml, 40 μg/ml) on the transcription of *hmuX, exbB* and *hmuC* in the *hemH1-hemH2* double mutants of MR-1

**Fig. 8**
Schematic diagram illustrating the uptake pathway of exogenous hemin and cellular function and the access to cytochrome protein assemble in *S. oneidensis* MR-1 strain. Only the supplement of exogenous hemin made it possible to delete both *hemH1* and *hemH2* genes simultaneously in MR-1. The MR-1 strain could uptake exogenous hemin to compensate for loss of endogenous heme synthesis via the hemin uptake system. The hemin was transported into the cells and was mainly utilized to synthesize the respiration-related hemoproteins, especially the aerobic respiration-related proteins cytochrome c oxidase prior to other heme-requiring proteins such as catalase, peroxidase, and nitrate reductase. The upward red arrow indicates the upregulation of gene expression, the downward red arrow indicates the decrease of enzyme activity

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References

1. Hau HH, Gralnick JA. Ecology and biotechnology of the genus Shewanella. Annu Rev Microbiol. 2007;61:237–258. doi: 10.1146/annurev.micro.61.080706.093257. - DOI - PubMed
1. Fredrickson JK, Romine MF, Beliaev AS, Auchtung JM, Driscoll ME, Gardner TS, Nealson KH, Osterman AL, Pinchuk G, Reed JL, et al. Towards environmental systems biology of Shewanella. Nat Rev Microbiol. 2008;6(8):592–603. doi: 10.1038/nrmicro1947. - DOI - PubMed
1. Bouhenni R, Gehrke A, Saffarini D. Identification of genes involved in cytochrome c biogenesis in Shewanella oneidensis, using a modified mariner transposon. Appl Environ Microb. 2005;71(8):4935–4937. doi: 10.1128/AEM.71.8.4935-4937.2005. - DOI - PMC - PubMed
1. Qiu D, Xie M, Dai J, An W, Wei H, Tian C, Kempher ML, Zhou A, He Z, Gu B, et al. Differential regulation of the two Ferrochelatase paralogues in Shewanella loihica PV-4 in response to environmental stresses. Appl Environ Microbiol. 2016;82(17):5077–5088. doi: 10.1128/AEM.00203-16. - DOI - PMC - PubMed
1. Nakahigashi K, Nishimura K, Miyamoto K, Inokuchi H. Photosensitivity of a protoporphyrin-accumulating, light-sensitive mutant (visA) of Escherichia coli K-12. Proc Natl Acad Sci U S A. 1991;88(23):10520–10524. doi: 10.1073/pnas.88.23.10520. - DOI - PMC - PubMed

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[1] Hau HH, Gralnick JA. Ecology and biotechnology of the genus Shewanella. Annu Rev Microbiol. 2007;61:237–258. doi: 10.1146/annurev.micro.61.080706.093257. - DOI - PubMed

[2] Hau HH, Gralnick JA. Ecology and biotechnology of the genus Shewanella. Annu Rev Microbiol. 2007;61:237–258. doi: 10.1146/annurev.micro.61.080706.093257. - DOI - PubMed

[3] Fredrickson JK, Romine MF, Beliaev AS, Auchtung JM, Driscoll ME, Gardner TS, Nealson KH, Osterman AL, Pinchuk G, Reed JL, et al. Towards environmental systems biology of Shewanella. Nat Rev Microbiol. 2008;6(8):592–603. doi: 10.1038/nrmicro1947. - DOI - PubMed

[4] Fredrickson JK, Romine MF, Beliaev AS, Auchtung JM, Driscoll ME, Gardner TS, Nealson KH, Osterman AL, Pinchuk G, Reed JL, et al. Towards environmental systems biology of Shewanella. Nat Rev Microbiol. 2008;6(8):592–603. doi: 10.1038/nrmicro1947. - DOI - PubMed

[5] Bouhenni R, Gehrke A, Saffarini D. Identification of genes involved in cytochrome c biogenesis in Shewanella oneidensis, using a modified mariner transposon. Appl Environ Microb. 2005;71(8):4935–4937. doi: 10.1128/AEM.71.8.4935-4937.2005. - DOI - PMC - PubMed

[6] Bouhenni R, Gehrke A, Saffarini D. Identification of genes involved in cytochrome c biogenesis in Shewanella oneidensis, using a modified mariner transposon. Appl Environ Microb. 2005;71(8):4935–4937. doi: 10.1128/AEM.71.8.4935-4937.2005. - DOI - PMC - PubMed

[7] Qiu D, Xie M, Dai J, An W, Wei H, Tian C, Kempher ML, Zhou A, He Z, Gu B, et al. Differential regulation of the two Ferrochelatase paralogues in Shewanella loihica PV-4 in response to environmental stresses. Appl Environ Microbiol. 2016;82(17):5077–5088. doi: 10.1128/AEM.00203-16. - DOI - PMC - PubMed

[8] Qiu D, Xie M, Dai J, An W, Wei H, Tian C, Kempher ML, Zhou A, He Z, Gu B, et al. Differential regulation of the two Ferrochelatase paralogues in Shewanella loihica PV-4 in response to environmental stresses. Appl Environ Microbiol. 2016;82(17):5077–5088. doi: 10.1128/AEM.00203-16. - DOI - PMC - PubMed

[9] Nakahigashi K, Nishimura K, Miyamoto K, Inokuchi H. Photosensitivity of a protoporphyrin-accumulating, light-sensitive mutant (visA) of Escherichia coli K-12. Proc Natl Acad Sci U S A. 1991;88(23):10520–10524. doi: 10.1073/pnas.88.23.10520. - DOI - PMC - PubMed

[10] Nakahigashi K, Nishimura K, Miyamoto K, Inokuchi H. Photosensitivity of a protoporphyrin-accumulating, light-sensitive mutant (visA) of Escherichia coli K-12. Proc Natl Acad Sci U S A. 1991;88(23):10520–10524. doi: 10.1073/pnas.88.23.10520. - DOI - PMC - PubMed

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Differential gene content and gene expression for bacterial evolution and speciation of Shewanella in terms of biosynthesis of heme and heme-requiring proteins

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Differential gene content and gene expression for bacterial evolution and speciation of Shewanella in terms of biosynthesis of heme and heme-requiring proteins

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Abstract

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