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The Transport Behavior of a Biflagellated Microswimmer before and after Cargo Loading

Teng XJ, Ng WM, Chong WH, Chan DJC, Mohamud R, Ooi BS, et al.

Langmuir, 2021 08 03;37(30):9192-9201.
PMID: 34255525 DOI: 10.1021/acs.langmuir.1c01345

The changes in the transport behavior of a microswimmer before and after cargo loading are crucial to understanding and control of the motion of a biohybrid microbot. In this work, we show the change in swimming behavior of biflagellated microalgae Chlamydomonas reinhardtii picking up a 4.5 μm polystyrene microbead upon collision. The microswimmer changed from linear forward motion into helical motion upon the attachment of the cargo and swam with a decreased swimming velocity. We revealed the helical motion of the microswimmer upon cargo loading due to suppression of flagella by image analysis of magnified time-lapse images of C. reinhardtii with one microbead attached at the anterior end (between the flagella). Furthered suppression on the flagellum imposed by the loading of the second cargo has led to increased oscillation per displacement traveled and decreased swimming velocity. Moreover, the microswimmer with a microbead attached at the posterior end swam with swimming velocity close to free swimming microalgae and did not exhibit helical swimming behavior. The experimental results and analysis showed that the loading location of the cargo has a great influence over the swimming behavior of the microswimmer. Furthermore, the work balance calculation and mathematical analysis based on Lighthill's model are well consistent with our experimental findings.

Matched MeSH terms: Chlamydomonas reinhardtii*
Dissection of synechococcus rubisco large subunit sections involved in holoenzyme formation in escherichia coli by combinatorial section swapping and sequence analyses

Yee Hung Yeap, Teng Wei Koay, Boon Hoe Lim

Sains Malaysiana, 2018;47:2269-2289.

Engineering the CO2
-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to improve photosynthesis
has long been sought. Rubisco large subunits (RbcL) are highly-conserved but because of certain undefined sequence
differences, plant Rubisco research cannot fully utilise the robust heterologous Escherichia coli expression system and its
GroEL folding machinery. Previously, a series of chimeric cyanobacteria Synechococcus elongatus Rubisco, incorporated
with sequences from the green alga Chlamydomonas reinhardtii, were expressed in E. coli; differences in RbcL sections
essential for holoenzyme formation were pinpointed. In this study, the remaining sections, presumably not crucial for
holoenzyme formation and also the small subunit (RbcS), are substituted to further ascertain the possible destabilising
effects of multiple section mutations. To that end, combinations of Synechococcus RbcL Sections 1 (residues 1-47), 2
(residues 48-97), 5 (residues 198-247) and 10 (residues 448-472), and RbcS, were swapped with collinear Chlamydomonas
sections and expressed in E. coli. Interestingly, only the chimera with Sections 1 and 2 together produces holoenzyme and
an interaction network of complementing amino acid changes is delineated by crystal structure analysis. Furthermore,
sequence-based analysis also highlighted possible GroEL binding site differences between the two RbcLs.

Matched MeSH terms: Chlamydomonas reinhardtii
Characterization of Chlamydomonas Ribulose-1,5-bisphosphate carboxylase/oxygenase variants mutated at residues that are post-translationally modified

Rasineni GK, Loh PC, Lim BH

Biochim Biophys Acta Gen Subj, 2017 Feb;1861(2):79-85.
PMID: 27816753 DOI: 10.1016/j.bbagen.2016.10.027

BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the chloroplast enzyme that fixes CO2 in photosynthesis, but the enzyme also fixes O2, which leads to the wasteful photorespiratory pathway. If we better understand the structure-function relationship of the enzyme, we might be able to engineer improvements. When the crystal structure of Chlamydomonas Rubisco was solved, four new posttranslational modifications were observed which are not present in other species. The modifications were 4-hydroxylation of the conserved Pro-104 and 151 residues, and S-methylation of the variable Cys-256 and 369 residues, which are Phe-256 and Val-369 in land plants. Because the modifications were only observed in Chlamydomonas Rubisco, they might account for the differences in kinetic properties between the algal and plant enzymes.
METHODS: Site-directed mutagenesis and chloroplast transformation have been used to test the essentiality of these modifications by replacing each of the residues with alanine (Ala). Biochemical analyses were done to determine the specificity factors and kinetic constants.
RESULTS: Replacing the modified-residues in Chlamydomonas Rubisco affected the enzyme's catalytic activity. Substituting hydroxy-Pro-104 and methyl-Cys-256 with alanine influenced Rubisco catalysis.
CONCLUSION: This is the first study on these posttranslationally-modified residues in Rubisco by genetic engineering. As these forms of modifications/regulation are not available in plants, the modified residues could be a means to modulate Rubisco activity.
GENERAL SIGNIFICANCE: With a better understanding of Rubisco structure-function, we can define targets for improving the enzyme.

Matched MeSH terms: Chlamydomonas reinhardtii/genetics*

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