DPS Proteins Impact on Oxidative Stress Response in Nitrogen Fixing Cyanobacterium Nostoc punctiforme ATCC 29133
Ievgen Dzhygyr
Degree project in applied biotechnology, Master of Science (two years), 2013 Examensarbete i tillämpad bioteknologi 45 hp till masterexamen, 2013
Biology Education Center and Department of Chemistry - Ångström Laboratory, Uppsala University
Supervisor: prof. Karin Stensjö
Content
Abstract... 4
Abbreviations... 5
1 Introduction... 6
1.1The aim of the project... 6
1.2 Cyanobacteria and their biotechnological application... 6
1.3 Hydrogen production with use of Nostoc punctiforme ATCC 29133 ... 7
1.4 Oxidative stress in cyanobacteria and defence mechanisms against it... 8
1.5 Structure of DPS proteins... 9
1.6 DPS proteins from Nostoc punctiforme ATCC 29133... 10
1.6.1 Structural features of Npun_F6212... 11
1.6.2 Structural features of Npun_R5799... 12
1.6.3 Role of Npun_F3730 in oxidative stress defence... 13
2 Results... 14
2.1 Design of genetic constructs ... 14
2.2 Analysis of DPS genes expression under control of native promoters... 15
2.3 Light-induced oxidative stress response measurements………... 16
3 Discussion... 20
3.1 Features of the promoters used for genetic constructs design... 20
3.2 Measurement of the oxidative stress response reveals iron dependence of protection mechanisms... 20
3.3 An insight into the Nostoc punctiforme Npun_F6212 possible structure and functions... 22
3.4 Future studies... 23
3.5 Conclusion... 24
4 Materials and Methods... 25
4.1 Instruments... 25
4.2 Web tools used for calculations, alignment of sequences, protein struc- ture visualisation, and primers design... 25
4.3 Strains of E.coli and N. punctiforme... 25
4.4 Cyanobacterial cultures cultivation……….. 25
4.5 Competent cells preparation... 26
4.6 Genomic DNA extraction... 26
4.7 Construction of plasmids for complementation of the deletion mutants... 26
4.8 Genes amplification for cloning... 27
4.9 Restriction digest of DNA for cloning ... 27
4.10 Purification of restriction digest and PCR products... 27
4.11 Ligation... 27
4.12 E. coli transformation………..……… 28
4.13 Colony PCR (E. coli and cyanobacteria)... 28
4.14 Plasmid extraction... 28
4.15 Sequencing... 28
4.16 RNA extraction... 29
4.17 DNA removing from RNA samples... 29
4.18 cDNA synthesis... 29
4.19 Cyanobacteria transformation (triparental mating)... 29
4.20 Chlorophyll a measurement... 30
4.21 Light induced stress experiments... 30
References... 31
Acknowledgements... 35
Appendix A— Media and Buffers... 36
Appendix B—Primers... 38
Appendix C—Genetic constructs ... 39
Abstract
To facilitate a broader biotechnological application of cyanobacteria in hydrogen production the deeper insight into oxidative stress protection mechanisms is required. For this purpose the role of three DPS proteins: Npun_F3730, Npun_F6212, and Npun_R5799 from Nostoc punctiforme ATCC 29133 was investigated in regards to their influence on oxidative stress tolerance.
A set of genetically engineered strains was created to test the impact of these proteins on the light-induced oxidative stress response and to investigate their role in iron homeostasis. They were exposed to high light of 150 μmol m-2 s-1. After six days of cultivation the level of chlo- rophyll a was measured.
It was found out that DPS proteins play an important role in the oxidative stress protection in N. punctiforme. Deletion mutants of Npun_F3730, Npun_F6212, and Npun_R5799 demon- strated a decreased tolerance to the light-induced oxidative stress when compared to wild type N. punctiforme. At the same time the complementation of deletion strains with a plasmid, car- rying the deleted gene, has significantly increased their stress tolerance. However, the content of chlorophyll a in complemented strains did not reach the level of the wild type. This may indicate that the expression of DPS genes from the plasmid was not as efficient as from the chromosome although the endogenous promoters were used. In the strains where DPS pro- teins were overexpressed, the level of chlorophyll a was higher than in the complemented strains although did not reach the level of the wild type.
Experiments with additional iron in the medium showed that the Npun_F3730 and Npun_F6212 play an important role in iron metabolism under stress conditions. The wild type, when grown in the medium with a six-fold excess of iron than in standard medium, demonstrated a decrease in chlorophyll a content. At the same time, the chlorophyll level of overexpressed strains did not change considerably. This may indicate that DPS proteins can serve as a buffer for an excess of iron.
At the same time, the corresponding complemented strains showed a significant increase in the content of chlorophyll a when growing in a medium with extra iron. This may indicate that under stress conditions at standard iron concentration the insufficient amount of the DPS proteins leads to an iron deficiency, which in its turn, impairs oxidative stress resistance. The fact that the lack of iron in the cyanobacterial cells, as well as its high concentration, increases the oxidative stress underscores the importance of iron homeostasis for the oxidative stress resistance.
Abbreviations ON—over night
PCR—polymerase chain reaction
RT PCR—reverse transcriptase polymerase chain reaction qPCR—quantitative polymerase chain reaction
LB—Luria Broth
SOC—Super-optimal broth with catabolite repression ROS—reactive oxygen species
DNA—deoxyribonucleic acid RNA—ribonucleic acid
mRNA—messenger ribonucleic acid
OE-strains —strains with overexpressed DPS proteins DPS proteins—DNA binding proteins from starved cells
1. Introduction 1.1 The aims of the project
The first goal of this master degree project was to study DPS proteins impact to oxidative stress tolerance using genetically engineered strains of the cyanobacterium Nostoc puncti- forme where different DPS proteins have been inactivated. The second goal of this work was to investigate the possibility to increase the cyanobacteria oxidative stress defence via DPS proteins overexpression.
The project is significant for the understanding of the oxidative stress protection mechanisms in cyanobacteria and for development of a technology for efficient biohydrogen production with a use of Nostoc punctiforme.
To fulfil the aims of the project the several steps were taken. First of all, genetic constructs based on a broad host range vector pPMQAK1 with different DPS proteins were designed to complement DPS-deletion mutants. Native upstream regions of the corresponding genes were taken as promoters to mimic the natural genetic milieu. Then N. punctiforme deletion-mutants and the wild type strain were transformed with the obtained constructs. The new strains’ tol- erance to the light-induced oxidative stress was measured with use of chlorophyll a content as a referent index. In addition, the influence of an excess of Fe2+ in the medium to the oxidative stress resistance was tested.
The most promising DPS proteins, which show the biggest impact on reactive oxygen species (ROS) resistance, were selected and overexpressed in hydrogen producing strain of N. puncti- forme. The investigation of their ability to increase the hydrogen production rate will be car- ried out in future studies.
1.2 Cyanobacteria and their biotechnological application
Cyanobacteria are prokaryotic autotrophic aquatic microorganisms able for photosynthesis.
During the photosynthesis they produce oxygen, take up CO2 andconvert it into organic com- pounds. Their ability to use sunlight as a source of energy as well as their modest nutrition requirements allowed them to occupy various niches in different ecological systems (Heidorn et al., 2011).
In comparison to other photosynthetic organisms such as plants and algae cyanobacteria have higher growth rate and a simpler organization of genetic apparatus which make them more attractive for biotechnological applications. At the same time their autotrophic metabolism makes their use in the process of large scale cultivation more cost effective in comparison to heterotrophic microorganisms.
To date, cyanobacteria are found to be of use in different application. For example, in the field of ecology they can be exploited as biosensor to monitor environmental conditions. Some strains were genetically modified to withstand high concentration of heavy metals and can be applied for bioremediation of polluted water ponds. Cyanobacteria expressing toxins are used to control mosquito populations. In fish industry, genetically engineered cyanobacteria ex- pressing salmon growth hormone are used to increase the fish growth rate (Ruffing, 2011).
Cyanobacteria can also be harnessed for the production of many different chemical com- pounds such as sugars, phycobiliproteins, which can be used as fluorescent tags for molecular biology research (Tooley et al., 2001), polymer feedstock (polyhydroxyalkonates), alkanes, alkenes, isoprene, acetone, fatty acids —the substances which can be converted into biodiesel.
Cyanobacteria can also produce biofuels such as butanol and ethanol (Peralta-Yahya et al., 2012; Wang et al., 2012). The technology for the industrial scale ethanol production has been developed by Algenol Biofuels (Lane, 2012). Among other perspective biofuels is hydrogen.
The use of cyanobacteria for hydrogen production attracts scientific interest because hydrogen has being seen as a substitution for fossil fuels for a long time. Except resolving a vital prob- lem of the depletion of fossil fuels the use of hydrogen can decrease the level of CO2 released in the atmosphere and mitigate the problem of the global warming. From this perspective cyanobacteria have an additional advantage, since they do not only produce hydrogen and oxygen but consume CO2 as well. Moreover technologies for hydrogen storage and use as a fuel in car engines have already been developed (Hwang, 2013).
1.3 Hydrogen production with use of Nostoc punctiforme ATCC 29133
N. punctiforme produces hydrogen as a by product of nitrogen fixation reaction (Lindberg, 2003; Tamagnini et al., 2002):
N2+8H++8e –+16ATP →2NH3+H2+16ADP+16Pi (Equation 1)
However, in N. punctiforme the hydrogen formed during the reaction is reoxidized by uptake hydrogenase which converts it back to hydrogen ions (eqn. 2):
H2→2H++2e– (Equation 2) To make the strain produce and evolve hydrogen the gene coding for the uptake hydrogenase large subunit was knocked out by means of genetic engineering and a new strain Nostoc punc- tiforme ATCC 29133 NHM5 was obtained (Lindberg et al., 2002).
The key enzyme of the hydrogen synthesis, the nitrogenase, contains iron-sulfur active centre which is highly sensitive to oxidation (Tamagnini et al., 2002). To prevent the oxidation of the nitrogenase in the course of evolution N. punctiforme has developed heterocysts, special- ized cells where the process of nitrogen fixation occurs.
Among particularities of heterocysts which make them suitable for nitrogen fixation are a thick cell wall which prevents/reduces oxygen diffusion inside the cell, the absence of photo- system II—the main source of oxidative agents, and also presence of enzymes which can in- activate harmful reactive oxygen species (ROS) (Klipp, 2005; Tamagnini et al., 2002). All this allows for creation of microaerobic environment inside heterocysts which is crucial for nitrogen fixation process.
This evolutionary trait allows N.punctiforme to conduct the reaction of nitrogen fixation even during daylight when the photosynthesis takes place and oxygen is evolved as a product of water photolysis. Such feature makes N. punctiforme a convenient strain for biotechnological production of hydrogen, since the altering of light and darkness or anaerobic growth condition are not needed.
However, a broader use of cyanobacteria in biotechnological production of hydrogen is ham- pered by the high sensitivity of the nitrogenase to oxidation. In order to find ways to decrease a damage to nitrogenase caused by ROS the study of oxidative stress defence mechanisms and the role of DPS proteins was done.
1.4 Oxidative stress in cyanobacteria and defence mechanisms against it
The main cause of the oxidative stress in cyanobacteria is reactive oxygen species (ROS) which are produced inside the cell as a result of the normal photosynthesis and respiration (Blot et al., 2011; Latifi et al., 2009). Energy harvested by pigments of Photosystem II which is used to reduce CO2 can be also transferred to O2 especially if the amount of light harvested by the photosystem exceeds the amount needed for CO2 fixation and other downstream proc- esses. This may result in a formation of singlet oxygen 1O2. Though it is a short living mole- cule it is very reactive and can be a threat to proteins and lipids in a close proximity and to the photosystem itself. Photosystem I can also participate in ROS formation by reducing oxygen to superoxide anion O2— which, reacting with water, may lead to hydrogen peroxide (H2O2) formation. Oxygen itself also can react with different reducers, for example NADH, forming superoxide and hydrogen peroxide (Latifi et al., 2009).
Hydrogen peroxide itself is not very reactive. However, in the reaction of decomposition catalyzed by ferrous ions (Fenton reaction) highly reactive hydroxyl radical (OH*) is formed (eqn. 3) which can cause serious damage to cellular components such as DNA, proteins and cellular membranes (Haikarainen & Papageorgiou, 2010).
H2O2+Fe2+=Fe 3++OH– +OH* (Equation 3) To cope with ROS cyanobaceria have developed different mechanisms of defence such as light energy dissipation through CP43 proteins and carotenoids, which can absorb excess of harvested energy. Cyanobacteria can also increase production of antioxidants (ά-tocopherol and carotenoids) and different enzymes which neutralize ROS (Latifi et al., 2009). Among these enzymes are superoxide dismutases which convert superoxide anion into hydrogen per- oxide and O2, catalases, peroxidases and rubredoxins (Zhao et al., 2007) which convert H2O2
into water.
Among proteins which play an important role in oxidative stress protection are DPS proteins (DNA binding proteins from starved cells)(Calhoun & Kwon, 2010). The first was discovered in E. coli. They belong to the Ferritin -like protein superfamily the members of which are pre- sent in all living organisms (Andrews, 2010). These proteins take part in iron storage and transportation. They also can provide protection to genetic apparatus by binding to DNA and, in such a way, preventing its contact with ROS. However, DNA binding is not always an inherent property of the DPS proteins (Haikarainen & Papageorgiou, 2010; Zeth, 2012).
The catalytic function of DPS proteins is to convert iron and hydrogen peroxide into harm- less ferroxihydroxide FeOOH (eqn. 4), thus preventing the Fenton reaction (Haikarainen &
Papageorgiou, 2010; Zeth, 2012).
2 Fe 2+ +H2O2+2H2O =2FeOOH + 4H+ (Equation 4)
1.5 Structure of DPS proteins
DPS proteins have highly conserved structural and active centre organization even between unrelated species. A monomer of a DPS protein consists of four helices which form a bundle.
The second and the third helices are connected with a long loop with a short helix inside (fig- ure 1). Each subunit takes part in the organization of two ferroxidase centres (FOC) where deactivation of hydrogen peroxide takes place. These active sites are located on the interface between two subunits each of which donates four amino acid residues for their formation, two histidines, aspartate and glutamate, (Zeth, 2012).
Twelve subunits of a single DPS protein form a hollow sphere-shaped structure with the ex- ternal diameter about 8-9 nm and the internal diameter about 4-5 nm. The cavity inside can harbour Fe3+ ions in the form of ferroxihydroxide (Calhoun & Kwon, 2010; Zeth, 2012).
B A
Figure 1. The structure of DPS protein from B. anthracis: A—a fully assembled protein, B—a single subunit.
A B
Figure 2. The structure of FOC of DPS protein from E. coli. A—two FOC are formed at the interface between two subunits. B—two histidines from one subunit and glutamate and aspar- tate from the other coordinate a ferrous ion.
1.6 DPS proteins of Nostoc punctiforme ATCC 29133
An interesting feature of the N. punctiforme and some other related cyanobacteria is the pres- ence of five proteins annotated as DPS proteins (Npun_F3730, Npun_F6212, Npun_R5701, Npun_R5799, and Npun_R3258), while most bacteria have one or two different DPS pro- teins (Ekman et al., 2013). This makes N. punctiforme an interesting candidate for the oxida- tive stress protective mechanism studies.
Such a relatively big number of different DPS proteins can be explained by a very complex regulation of oxidative stress defence, the need to react on different type of ROS, and the ability for cell differentiation. It has been recently shown that some DPS proteins from N.
punctiforme demonstrate cell specificity and are expressed only in one type of cyanobacterial cells, either vegetative cells or heterocysts (Ekman et al., 2013). According to Ekman Npun_F3730 is mainly expressed in vegetative cells while Npun_F6212 and Npun_R5701 in heterocysts. Recent experiments of our group (to be published) point to the fact that Npun_R3258 and Npun_5799 also have higher expression level in heterocysts than in vege- tative cells.
For the experiments three out of five DPS proteins were selected considering time limits of the project. This selection was made on the base of previous work of Martin Ekman and re- searches from our group in the Department of Chemistry Ångström laboratory (see Ekman et al., 2013) and bioinformatic study of the five DPS proteins which I performed during my re- search training in the same group.
The bioinformatic investigation was done with the use of web tools for protein structures pre- diction: Phyre 2 (Kelley & Sternberg, 2009) and I-TASSER (Roy, Kucukura, & Zhang, 2010) which gave 3D models of the DPS proteins. This study pointed out several interesting candi- dates for further research. While three DPS proteins Npun_F3730, Npun_R3258, and Npun_R5701 does not show any deviation from classical DPS structure and FOC organiza- tion structures of Npun_F6212 and Npun_R5799 have interesting particularities which are discussed below.
1.6.1 Structural features of Npun_F6212
As can be seen from the model obtained (figure 3), the Npun_F6212 subunit has a structure which does not match the classical one.
Figure 3. The 3D model of Npun_F6212 DPS protein.
The protein subunit is formed as in all other DPS proteins by four helices but does not contain a short helix inside the loop between helix two and three. Moreover, according to an amino acid sequence alignment, its FOC does not have the classical amino residues composition and is not located on the interface between two subunits but inside the four-helix bundle of single subunits.
An alignment with erythrin from Magnetospirillum magnetotacticum, done with the use of Clustal Omega (Sievers et al., 2011), shows identity of the residues which form FOC of erythrin and the DPS (figure 4).
H56/H53 H127/H138
E129/E135 S53/S50
Y27/Y24
E20/E1 E97/E105 Y104/Y112
Npun_F6212 from N.punctiforme Erythrin from M. magnetotacticum
Figure 4. Pairwise alignment of amino acid sequence of Npun_F6212 from N.punctiforme and erythrin from M. magnetotacticum.
Such arrangement of the FOC is acknowledged (Andrews, 2010) for erythrins except the sub- stitution of glutamate 53 for serine (see scheme on figure 5).
Classical erythrin FOC structure: E---6---Y---ExxH---E---6---Y---ExxH
M. magnetotacticum erythrin
and Npun_F6212 FOC structure: E---6---Y---SxxH---E---6---Y---ExxH
Figure 5. Scheme of ferroxidase centre of erythrins and Npun_F6212
The fact that the predicted amino acid sequence of Npun_F6212 forms a structure that matches the erythrin one’s indicates that this protein was probably annotated incorrectly and is not a DPS but an erythrin. This assumption finds indirect confirmation in the work in which DPS protein cell specificity was investigated (Ekman et al., 2013). It was found that Npun_F6212 is mostly expressed in heterocysts, the cells which function is to provide an- aerobic conditions for nitrogenase. This corresponds to the fact that erythrins are mostly pre- sent in the obligatory anaerobic microorganisms (Andrews, 2010) . The influence of substitu- tion of Glu to Ser on proteins functions is not clear yet.
Taking this information into account, Npun_F6212 was selected as a promising candidate which can help to increase the hydrogen evolution rate if overexpressed in heterocysts.
1.6.2 The structural features of Npun_R5799
The structure prediction of Npun_R5799 has revealed that this protein also have some struc- tural particularities. Its structure matches the classical DPS protein structure overall but, ac- cording to the model from Phyre 2, there is a small helix at the C-terminus of the protein (Figure 6). However, since due to a flexibility of the unstructured protein ends, exact predic- tion of the 3D structure is problematic, and the presence of the helix should be taken with cau- tion.
Figure 6. Structure prediction of Npun_R5799 DPS protein.
A more important feature of this protein is a quite high similarity to DPS A protein from an- other cyanobacterium Thermosynecoccocus elongatus (Figure 7). The structure and functions of this protein where studied in detail by the colleagues from the University of Rome (Alaleona et al., 2010).
.
Figure 7. Alignment of amino acid sequences of Npun_R5799 from N.punctiforme, DPS A from T. elongatus, DPS from Listeria. Inocua. Numeration of Listeria in- nocua. Not all columns are shown.
The protein from T. elongatus has 100 times higher efficiency of Fe2+ oxidation by oxygen than other DPS proteins. It is assumed that such ability is conferred by the substitution of Asp 78 for His 78 and also by participation of an additional histidine (His 164) from the third sub- unit in the FOC formation (figure 8) (Alaleona et al., 2010).
His31/51 His43/63 Asp58/His78
Npun_R5799 from N.punctiforme DPS A from T. elongatus DPS from L. inocua
B
Glu62/82
A
Figure 8. DPS A from T.elongatus. A—structure of FOC. Four histidins and one gluta- mate make up the FOC, B—three adjacent subunits which take part in FOC organization.
Due to its similarity to the unusual DPS A protein from T. elongatus Npun_R5799 was also included into experimental work in order to find out what is its role in protection against oxi- dative stress in N. punctiforme.
1.6.3 The role of Npun_F3730 in oxidative stress defence
The third protein, which was chosen for further studies, was vegetative cells specific N- pun_F3730. It did not show any structural particularities. However, as it is shown in (Ekman et al., 2013), the knock out mutant missing this protein had the lowest tolerance to the oxida- tive stress caused by hydrogen peroxide. To get deeper insight in the function of this protein it was decided to investigate its role in the protection against light-induced oxidative stress.
2 Results 2.1 Design of genetic constructs
In order to study the role of DPS proteins in oxidative stress tolerance in N. punctiforme five deletion mutants (here and further in the text named 3730 Del, 6212 Del, 5799 Del, 3258 Del, 5701 Del) were obtained where corresponding DPS genes were replaced by kanamycin resis- tance cassette (Ekman et al., 2013). In this study, as it was mentioned above, 3730 Del, 6212 Del, and 5799 Del were chosen for investigation.
Besides the investigation of an impact of the DPS genes deletion on the oxidative stress resis- tance, it was decided to study an influence of the deletion complementation and the DPS pro- teins overexpression on N. punctiforme under stress conditions. With this purpose genetic constructs, carrying Npun_F3730, Npun_F6212, and Npun_R5799 genes under control of their respective native promoters, were designed. To avoid negative effects of multiple plas- mid copies in the cells and inclusion-bodies formation low-copy number plasmid pPMQAK-1 (Huang et al., 2010) was chosen as a vector. Subsequently, N.punctiforme deletion strains, as well as the wild type, were transformed with the constructs via triparental mating (see Materi- als and Methods) and new strains were obtained (Table 1).
To investigate the effect of overexpression of DPS proteins on the H2 producing efficiency of heterocyst forming cyanobacteria another set of constructs with Npun_F6212, Npun_F3730, Npun_R5799 under control of HupS and TetR promoters were used to transform the hydrogen producing Nostoc punctiforme NHM5 strain (Lindberg et al., 2002). Due to lack of time ex- periments for hydrogen evolution rate measurements were not completed and their results are not discussed in this work.
Table 1. Strains used in experiments
Name of the strain Description Controls WT N. punctiforme wild type
M5 N. punctiforme hydrogen producing mutant NHM5
Deletion strains 3730 Del N. punctiforme with deleted Npun_F3730 6212 Del N. punctiforme with deleted Npun_F6212 5799 Del N. punctiforme with deleted Npun_R5799 Overexpression strains 3730 OE N. punctiforme with overexpressed Npun_F3730 6212 OE N. punctiforme with overexpressed Npun_F6212 5799 OE N. punctiforme with overexpressed Npun_R5799
Complemented strains 3730 Comp N. punctiforme with deleted Npun_F3730 complemented with a plasmid carrying Npun_F3730 under native promoter
6212 Comp N. punctiforme with deleted Npun_F6212 complemented with a plasmid carrying Npun_F6212 under native promoter
5799 Comp N. punctiforme with deleted Npun_R5799 complemented with a plasmid carrying Npun_R5799 under native promoter
2.2 Analysis of DPS genes expression under control of native promoters
The expression of Npun_F6212 and Npun_F3730 genes in pPMQAK-1 vector under control of the native promoters was confirmed by RT PCR. N. punctiforme cultures carrying the plas- mid with corresponding genes were harvested and RNA was extracted. In order to remove possible genomic DNA contamination the RNA samples were treated with DNase. After this the RNA was reverse transcribed into cDNA with use of random hexamer primers. Subse- quently, the ordinary PCR with gene-specific primers was performed to detect the presence of the target genes. In both cases bands pointing to the presence of the genes transcripts can be seen on gels (figures 14 and 15) while no product can be seen on negative control lanes (sam- ples in which H2O was added instead of reverse transcriptase).
1 2 3
236 bp 300 bp 300 bp
200 bp
Figure 14. Confirmation of expression of Npun_F6212 in pPMQAK-1 vector. PCR was performed on cDNA obtained by reverse transcription of RNA from 6212Comp culture. Lane 1—6212 Comp cDNA with Npun_F6212 primers; lane 2—6212 Comp mRNA with Npun_F6212 primers (negative control for genomic DNA contamina- tion); lane 3—100bp ladder.
1 2 1 2 3
300 bp 228 bp 300 bp
200 bp 200 bp
Figure 15. Confirmation of Npun_F3730 expression in pPMQAK-1 vector. PCR was performed on cDNA obtained by reverse transcription of RNA from 3730 Comp. Left:
lane 1—100bp ladder; lane 2—N. punctiforme genomic DNA with Npun_F3730 prim- ers (positive control); lane 3—3730 Comp cDNA with Npun_F3730 primers. Right:
lane 1—100 bp ladder; lane 2—3730 Comp mRNA with Npun_3730 primers (negative control for genomic DNA contamination).
These data demonstrate that both Npun_F3730 and Npun_F6212 are expressed from the pPMQAK-1 plasmid under the control of the upstream regions taken as endogenous promot- ers. However, relative level of the genes expression was not checked and its efficiency is un- known.
2.3 Light-induced oxidative stress response measurements
According to Blot et al., 2011 and Latifi et al., 2009 photosynthetic apparatus is the main source of ROS in cyanobacterial cells. To find out what level of illumination causes an oxida- tive stress in cyanobacteria light of different intensity was tested: low light (20 μmol m-2 s-1, figure 9), medium light (60 μmol m2 s-1, figure 10), and high light (150 μmol m-2 s-1, figure 11). Chlorophyl a level was measured during 6 days of cultivation. Chlorophyll a content was taken as an indicator of the oxidative stress, since its level corresponds to photosynthetic mi- croorganisms metabolism level and growth rate. This index is widely used in different studies of cyanobacteria, for example Shcolnik et al, 2009.
Only under high light conditions all selected deletion strains showed lower chlorophyll a con- tent after 6 days of cultivation in comparison to the wild type. None of them was able to sur- vive under high light for six days of time. An interesting result was that under medium light conditions 3730 Del showed the same chlorophyll a content as the wild type, and under low light conditions 3730 Del and 5799 Del had even higher amount of chlorophyll per ml of the culture than the wild type. The medium light condition (60 μmol m2 s-1) appeared to be the closest to optimal since in this case all strains showed the highest chlorophyll a content.
Figure 9. Chlorophyll a content in DPS protein deletion strains of N. puncti- forme under low light condition (20 μmol m-2 s-1)
Figure 10. Chlorophyll a content in DPS deletion strains of N. punctiforme under medium light condition (60 μmol m-2 s-1)
Figure 11. Chlorophyll a content in DPS deletion strains of N. punctiforme under high light conditions (150 μmol m-2 s-1)
The next step was to investigate the possibility to complement deletion strains with DPS genes expressed from a plasmid in order to return them to the wild type phenotype. In addi- tion, I tested whether N. punctiforme with overexpressed DPS proteins would be more resis- tant to the oxidative stress. For this purpose, selected complemented and strains with overex- pressed DPS proteins were cultivated under high light conditions and the chlorophyll a con- tent was measured after 6 days of cultivation. The results of these experiments are shown in figure 12.
The experiment showed that strains with overexpresed DPS proteins except 5799 OE did not reach the wild type level of chlorophyll a when cultivated under high light. As to comple- mented strains, they showed higher chlorophyll a content than the corresponding deletion strains but demonstrate considerably lower chlorophyll a content in comparison to the wild type.
Figure 12. Chlorophyll a content in N. punctiforme strains under high light conditions (150 μmol m-2 s-1)
Since one of the DPS proteins functions in a bacterial cell is considered to be iron stor- age/transportation (Zeth, 2012, Andrews, 2010) it was decided to check the influence of a high iron concentration on the oxidative stress response. For this extra iron (six-fold of stan- dard concentration) was added to the cultural medium. The concentration of the iron in the medium can vary with the concentration of iron in ammonium ferric citrate used for the me- dium preparation, and was approximately 15 μM for standard BG11 medium (Shcolnick et al., 2009) and 90 μM for the medium with added iron.
It can be a task for another study to find a concentration of iron required for the optimal growth of each strain (deletion, complemented, and with overexpressed DPS proteins). Unfor- tunately, due to the lack of time it was not done in course of this master project. The given concentration of iron was selected to ensure that the excess of iron will not cause its precipita- tion and that the concentration would be sufficient to cause detectable changes.
As it can be seen from figure 13, the excess of iron caused different impact on different strains. The high iron concentration was detrimental for the wild type and caused a drop in a chlorophyll a level which was expected since iron catalyzes ROS generation in Fenton reac- tion. At the same time, the behaviour of the strains with overexpressed DPS proteins did not indicate any significant influence of extra iron on them which points to the a protective role of overexpressed DPS proteins in iron mediated oxidative stress.
The most interesting results were obtained for the complemented strains where the addition of iron was beneficial and caused an almost two times increase in chlorophyll a content (92%
and 86% for 3730 Comp and 6212 Comp respectively). These strains reached the level of the wild type under the same conditions. In-parallel, the presence of the plasmid was tested in all engineered strains. Unfortunately, it was found out that 5799 Comp and 5799 OE lost the plasmid and can not be used in experiments further.
Figure 13. Chlorophyll a content in N. punctiforme complemented strains and strains with overexpressed DPS proteins under “high light” conditions (150 μmol m-2 s-1) in media with standard iron con- centration (solid bars) and six-fold excess of iron (striped bars).
Overall, deletion strains appeared to be more sensitive to the “high light” conditions than the wild type. Overexpression of the DPS proteins did not result in increase of the chlorophyll a content, as compared to the wild type, instead the chlorophyll a content was slightly lower.
The complementation of the deletion mutants, though increased the level of chlorophyll a, did not lead to the return to the wild type phenotype. The level of iron also appeared to be impor- tant for the ability to resist the light-induced oxidative stress.
3 Discussion
3.1 Features of the promoters used for genetic constructs design
To make the constructs for the deletion complementation, which would be as close to the natural ones as possible, the upstream regions of corresponding DPS genes (see Appendix C) were selected as promoters.
Also several additional constructs were made for the overexpression where the DPS genes are under the control of regulated promoters both of cyanobacterial (HupS) as well as of exoge- nous origin (TetR). HupS promoter is the native promoter from N.punctiforme which is ex- pressed only in heterocysts but not in vegetative cells of N. punctiforme or in E.coli (Holmqvist et al., 2009). The strategy to use a heterocyst was based on an assumption that the expression of the DPS genes only in heterocyst should reduce the burden of extra protein syn- thesis for vegetative cells in a filament and promote its growth. The promoter is induced only when the source of nitrogen in the medium is depleted.
Constructs with Npun_R5799, Npun_ F6212, and Npun_F3730 under control of the HupS promoter were created to investigate the ability to increase hydrogen evolution rate in N.
punctiforme NHM5 strain. The DPS expressed from such constructs should influence ROS level only in heterocyst where the hydrogen synthesis takes place. The aim of overexpression of the DPS proteins in heterocysts is to protect the nitrogenase which is the main enzyme in hydrogen production.
TetR promoter is a strong well-regulated promoter. The promoter has not been tested for N. punctiforme yet but works well in E. coli and in the unicellular cyanobacterium Synecho- cystis sp. The use of this promoter in the genetic constructs can allow a fine control of the level of the gene expression. The promoter is induced if anhydrous tetracycline is added to the medium (Huang & Lindblad, 2013). The main aim was to test TetR in N. punctiforme. In case of successful expression of DPS genes under its control our research group would get a useful tool for the future studies of DPS proteins impact on the cyanobacterial metabolism.
Unfortunately, due to the fact that the cyanobacterial cultures had not yet been fully segre- gated from E.coli strains after the triparental mating, the use of RT-PCR to test whether DPS proteins are expressed was not possible.
3.2 Measurement of the oxidative stress response reveals iron dependence of stress pro- tection mechanisms
The experiments conducted on cyanobacteria with use of the high light as an oxidative stress inducer gave several significant results which should be discussed. The most important is that the deletion mutants demonstrated substantial decrease in chlorophyll a content after cultiva- tion under the high light (150 μmol m-2 s-1) in comparison to the wild type which points to a significant contribution of DPS proteins Npun_F3730, Npun_F6212, and Npun_R5799 into surviving ability of cyanobacteria under stress conditions.
However, in the case when cyanobacteria were cultivated under optimal light conditions (60 μmol m-2 s-1) or under the low light (20 μmol m-2 s-1) the negative effect of the DPS pro- teins absence was not considerable. Thus, it can be concluded that the DPS proteins play not the main but rather auxiliary role in the light-induced oxidative stress protection, and their contribution becomes crucial specifically under the high light.
Moreover, under low light conditions 3730 Del and 5799 Del demonstrated higher chloro- phyll a content than the wild type. This can be explained by the fact that deletion strains have more cellular resources which otherwise would be spent on DPS proteins synthesis. The ab- sence of such effect for 6212 Del may be due to the fact that Npun_F6212 is expressed only in heterocysts (Ekman et al., 2013) where even small changes in ROS concentration may lead to nitrogenase inactivation, shortage of nitrogen, and, as a result, to the decrease in cyanobac- terial growth.
The experiments with OE and complemented strains showed that after 6 days of cultivation the OE strains contained less chlorophyll a per millilitre of culture than the wild type. The complemented strains, though showed an increase in chlorophyll a level in comparison to the deletion mutants, were not able to approach the wild type level. It would be logical to assume that under the high light conditions the OE strains should show higher chlorophyll a content than the wild type since OE strains contain additional copies of DPS genes. The comple- mented strains were expected to demonstrate either the return to the wild type phenotype or higher content of chlorophyll a due to the same reason. Nevertheless, the obtained results were different from expected ones.
The reason why OE and complemented strains did not demonstrate the expected results may be an insufficient level of expression of DPS proteins from the plasmid. Though, the expres- sion itself was confirmed both through the RT PCR and through increase of stress tolerance in the deletion complemented strains, its efficiency is unknown. In case if the size of the up- stream regions chosen as promoters is suboptimal for genes expression or if the absence of the native genetic environment impair expression of the genes from the plasmid the level of DPS proteins in complemented strains can be lower than in the wild type. As to the OE strains, the need to produce extra amount of proteins may cause an additional burden for cell metabolism and, thus, can be an explanation for chlorophyll a level decrease.
The lack of DPS proteins in the complemented strains due to low expression of the genes can also explain the results obtained in the experiments with iron. During the oxidative stress the cellular needs for iron increase because cells require it for building up enzymes, such as cata- lases, peroxidases, and ferridoxins (Kranzler et al., 2013; Shcolnick et al., 2007) which are key parts in the oxidative stress protection mechanism (Latifi et al., 2009). Since except per- oxidase function DPS proteins also take a significant part in the iron metabolism (Haikarainen
& Papageorgiou, 2010; Zhao et al., 2002) insufficient synthesis of the enzymes may lead to iron deficiency. This can be clearly seen from the iron experiments (figure 13) where com- plemented strains demonstrated almost two-fold increase of the chlorophyll a content in the medium with the excess of iron. (see Results).
At the same time the wild type showed a decrease of chlorophyll a level if grown with excess of iron. This agrees with the theory that free ferrous ions generate hydroxyl radicals in reac- tion with H2O2 and increase the level of the oxidative stress (see Introduction). The little re- spond of the OE strains to the increase of iron concentration in the medium can be explained by the fact that overexpressed DPS proteins act as a buffer for extra iron.
The fact that a lack of accessible iron leads to a more severe oxidative stress than an excess of iron in the medium is in line with the work of Shcolnick et al., 2009. It also reveals that the role of DPS proteins in oxidative stress protection lies not only in their ability to neutralize hydrogen peroxide but also in the ability to increase the bioavailability of iron inside cyano- bacterial cells which, probably, is their main function.
The absence of an increase in oxidative stress resistance in the OE strains indicates that the balance in iron homeostasis is more important for N. punctiforme than increasing a potential ROS neutralization capacity. However, the ability of overexpressed DPS proteins to increase resistance to the high iron concentration may be used in designing strains able to absorb iron from environment. Since it was also shown that DPS proteins can bind zinc (Alaleona et al., 2010) it may make them suitable for bioremediation of water ponds contaminated with heavy metals.
It also worth mentioning that for the investigation of the impact of the DPS proteins on the oxidative stress resistance the choice of light as a source of oxidative stress has played a very important role. Using light as a source of oxidative stress induction would also involve secon- dary effects such as need of an increased pool of free iron to form the pigments, which are of importance to cope with the changes in light. In the recent study of DPS proteins from N.
punctiforme (Ekman et al., 2013) the use of hydrogen peroxide as the oxidative stress inducer did not allow the important roles of Npun_F6212 and Npun_R5799 to be detected. The re- sponse of deletion mutants to a treatment with 0.5 mM H2O2 did not deviate much from the wild type response. The possible reason for this phenomenon can be that, according to the Ekman’s work, Npun_F6212 is expressed only in heterocysts and Npun_R5799 is expressed mainly in heterocysts. Such morphological particularity of these cells like a thick cell wall can explain why the concentration of hydrogen peroxide used in the experiment was not enough to cause any effect inside the heterocysts. At the same time, it was not possible to increase the H2O2 concentration because this would cause the death of the vegetative cells, which are more sensitive to H2O2 than heterocysts, and, as a result, the death of whole filament.
3.3 An insight into the Nostoc punctiforme Npun_F6212 possible structure and functions Experimental results show that Npun_F6212 takes part in iron storage/transportation in het- erocysts. This may indicate that, despite structural similarity to erythrin, Npun_F6212 may have other than erythrin organization of subunits. Erythrins with similar type of domain structure are present in bacteria as solo proteins or in form of dimers (Andrews, 2010). Noth- ing was reported about their ability to store iron. However, erythrins remain the less studied ferritin-like proteins (Andrews, 2010) and the ongoing crystallographic study of Npun_F6212 in our group may reveal new interesting features.
3.4 Future studies
In course of this work some preparations and preliminary studies were done which due to the time limits were not finished and can be a subject of a future work. First of all, RT-qPCR on obtained strains is required to determine the number of copies of DPS genes and the strength of their native promoters. Knowing this would be possible to correlate gene dose and cell re- sponse. However, this would only be an indication since the level of mRNA and protein activ- ity might not correlate. Then additional studies on the wild type with use of microarrays can be conducted to elucidate which genes are upregulated under oxidative stress conditions under excess of iron.
Another interesting subject for a further investigation is the behaviour of deletion mutants un- der different light conditions in a medium with excess of iron. This information would eluci- date if the main function of DPS proteins is rather iron transportation than direct ROS neu- tralization.
To obtain more information about functions of Npun_F6212 experiments could be conducted where deletion mutant is complemented with proteins performing peroxidase or iron storage functions for the later option Npun_F3730 or a true bacterioferritin can be used. The results would point to the in vivo role of Npun_F6212 and to what extend the function of this specific protein can be substituted.
Another subject for the future studies is the possibility to overexpress of the DPS proteins un- der the HupS and TetR promoters in a hydrogen producing strain in order to investigate if this strategy can lead to an increase in hydrogen production.
Additional questions which should be solved are: what mechanisms of defence against oxida- tive stress are induced when one of DPS proteins are knocked out, whether other DPS pro- teins’ expression is induced, and what processes are involved in the increase of chlorophyll a level in complemented strains under high light and high iron concentration?
3.5 Conclusion
The application of photoautotrophic cyanobacteria able of performing hydrogen synthesis for biotechnological production of hydrogen at the industrial scale is hampered by high sensitiv- ity of the key enzyme, nitrogenase, to the oxidative stress. To facilitate the process of broad implementation of cyanobacterial cultures into biotechnological processes a deeper under- standing of oxidative stress protection mechanisms is required.
During this work the role of three DPS proteins were investigated. The difference in chloro- phyll a content in deletion and complemented strains of the light induced oxidative-stress ex- periments showed that DPS proteins Npun_F3730, Npun_F6212, and Npun_R5799 play an important role in cyanobacteria resistance to the oxidative stress. Their functions appeared to be crucial for survival under constant high light, but were not vital under light conditions close to the optimal ones.
It was also found that overexpression of the Npun_F3730 and Npun_F6212 in the N.punctiforme wild type resulted in higher resistance to a high iron concentration which points to their important role in iron metabolism, though it did not increase the resistance to the high light treatments as compared to WT. The experiments also revealed an important role of iron in keeping cyanobacterial cell homeostasis under stress conditions. It was shown that both excess and a lack of iron cause an oxidative stress.
Furthermore it was found that the expression of DPS proteins from the plasmid is probably not as efficient as from the chromosome. Under stress conditions lack of DPS proteins leads to iron deficiency in the deletion complemented strains.
It is still unclear whether the overexpression of any of the DPS proteins under strong regu- lated promoter in heterocysts will lead to increase of the hydrogen evolution rate in hydrogen producing strain of N. punctiforme. For the investigation of such possibility additional re- search should be conducted.
4 Materials and Methods 4.1 Instruments
Photosynthetic active radiation sensor:
Skye Quantum Sensor SKP 215 Cell homogenizer:
Precellys 24
Spectrometer:
Varian Cary 50 Bio UV-visible Spectrometer
ThermoScientific Nano-drop 2000 UV-visible Spectrometer Thermocycler:
Biorad MJ Mini Gradient Thermal Cycler
4.2 Web tools used for calculations, sequences alignment, protein structure visualisation, and primers design
Gibton ligation calculator (Collins, 2013)
NCBI nucleotide BLAST, Primer-BLAST, Gene, Genome (NCBI, 2013) Oligocalculator 3.26 (Northwestern University Medical School, 2013) Swiss-Pdb Viewer 4.4 (Swiss Institute of Bioinformatics, 2013) Clustal Omega (Sievers et al., 2011)
Phyre 2 (Kelley L.A. & Sternberg, 2009) I-TASSER (Roy et al., 2010)
4.3 Strains of the E.coli and N. punctiforme
Nostoc punctiforme wild type (Rippka et al., 1979) Nostoc punctiforme NHM5 (Lindberg et al., 2002) E. coli HB101(Elhai et al. 1998; Pansegrau et al. 1994), E. coli DH5ά (Invitrogen, 2013)
4.4 Cyanobacterial cultures cultivation On plates
Cyanobacterial cultures were cultivated on plates with BG11 agar at 30 oC with appropriate antibiotics: neomycin 25 μg/ ml, ampicillin 10 μg/ ml.
In liquid
Cyanobacterial cultures were cultivated in with BG11 medium in 300 ml flasks with aeration or in 100 ml on shaker (120 rpm) at 30 oC, 20 μmol m-2 s-1 with appropriate antibiotics: neo- mycin 12.5 μg/ ml, ampicillin 10 μg/ ml.
4.5 Competent cells preparation
Twenty millilitres of SOC medium was inoculated with 1 ml of stock culture of DH5ά and cultivated ON at 30 oC with shaking 120 rpm. After 18 h of cultivation 12 ml of the culture (OD600=1.0) was transferred into 200 ml of a fresh SOC medium and cultivated at 30 oC with shaking (120 rpm) to OD600=0.22. After these cells were harvested by centrifugation at 3000 g, +4 oC for 10 min. Supernatant was discarded and the pellet was resuspended in 40 ml of ice-cold CCMB 80 buffer. After 40 min of incubation of ice the suspension was spun down (3000 g, +4 oC, 10 min). The supernatant was poured off and the cells were resuspended in 10 ml of CCMB 80 buffer (see Appendix A). The cultured was transferred into 1.5 ml eppendorf tubes, 200 μl in each. This step was done on ice. The tubes with competent cells were frozen at -80 oC.
To check the transformation ability of the cells 40 μl of the culture was transformed with 1 μl of pSCR plasmid (120 ng/μl) and plated on plates with ampicillin. After cultivation ON con- tinuous growth was observed on the plate, the negative control plate (competent cells without the plasmid) was clean.
4.6 Genomic DNA extraction
Five millilitres of a dense cyanobacterial culture was spun down (5min, 3000 g). The pellet was washed twice with 5 ml sodium chloride (5M) to get rid of polysaccharides and trans- ferred to 2 ml screw-cup tube, where 0,5 g of glass beads (0.5 mm diameter), 25 μl of SDS (10%), and 500 μl mixture of phenol:chlorophorm:isopropanol (25:24:1) were added. The cells were disrupted in a bead beater at 6800 rpm for 30 seconds and then spun down at 14000 g for 1 min. Upper aqueous phase was transferred into a new eppendorf tube (1.5 ml) and an equal volume of chloroform was added to get rid of phenol and isopropanol, mixed and spun down at 14000 g for 1 min, the upper aqueous phase was transferred to a new tube. Chloro- phorm extraction was done twice. To precipitate DNA 0.1 volume of sodium acetate (3M) and 2.5 volumes of cold (-20oC) ethanol (100 %) were added to the tube which then was placed in -80 oC freezer for 10 min. After this the tube was spun down at 14000 g for 10 min (+4 oC). The supernatant was discarded and the pellet was washed twice with ice cold 70 % ethanol (14000 g, 10 min). The washed pellet was dried on the bench for 10 min and resus- pended in 50 μl deionised water. The concentration of the extracted DNA was measured with use of NanoDrop 2000 (Thermoscientific).
4.7 Construction of plasmids for complementation of deletion mutants See appendices
4.8 Gene Amplification for Cloning
Thermoscientific Phusion HS II polymerase was used to obtain necessary DNA parts.
Reaction mixture:
5x Phusion HS buffer—10 μl dNTP(10mM)—1 μl
Primers (20 μM)—1 μl each Template (100 ng/ μl )—1 μl
Phusion HS II polymerase (2 u)—0.5 μl Deionised H2O—5.5 μl
Total volume—50 μl
Table 2. Thermocycler programme for gene amplification
Step Temperature, oC Time Number of cycles
Initial denaturation 98 30 sec 1
Denaturation 98 10 sec
Annealing 60 10 sec
Extension 72 20 sec/kb
30
Final extension 72 7 min 1
4.9 Restriction Digest
Restriction digest was performed with the use of Thermoscientific Fast Digest enzymes, ac- cording to the manufacturer instructions. The time of the reaction was prolonged to 45 min.
For the vector restriction shrimp alkaline phosphatase was added to prevent vector self- ligation.
4.10 Restriction digest and PCR product purification
The purification of the PCR products and restriction digest products was done with use of GeneJET PCR purification kit (Thermoscientific) according to the instruction of the manufac- turer. For the elution step deionised water was used.
4.11 Ligation
The amount of DNA parts was calculated with the use of Gibton Calculator.The ratio between vector and DNA parts used was 1:3 for the constructs with native promoters or 1:3:3 (vec- tor:promoter:gene) for the constructs with TetR and HupS promoters. Ligation was performed with the use of Biolabs QuickLigase, according to manufacturer instruction. The time of the reaction was prolonged to 1 hour.
