Pinpointing Synechococcus Rubisco large subunit sections involved in heterologous holoenzyme formation in Escherichia coli

Aims@#Heterologous holoenzyme formation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has been a challenge due to a limited understanding of its biogenesis. Unlike bacterial Rubiscos, eukaryotic Rubiscos are incompatible with the Escherichia coli (E. coli) chaperone system to fold and assemble into the functional hexadecameric conformation (L8S8), which comprises eight large subunits (RbcL) and eight small subunits (RbcS). Our previous study reported three sections (residues 248-297, 348-397 and 398-447) within the RbcL of Synechococcus elongatus PCC6301, which may be important for the formation of L8S8 in E. coli. The present study further examined these three sections separately, dividing them into six sections of 25 residues (i.e., residues 248-272, 273-297, 348-372, 373-397, 398-422 and 423-447).@*Methodology and results@#Six chimeric Rubiscos with each section within the RbcL from Synechococcus replaced by their respective counterpart sequence from Chlamydomonas reinhardtii were constructed and checked for their effect on holoenzyme formation in E. coli. The present study shows that Section 1 (residues 248-272; section of Synechococcus RbcL replaced by corresponding Chlamydomonas sequence), Section 2 (residues 273-297), Section 3 (residues 348-372) and Section 6 (residues 423-447) chimeras failed to fold and assemble despite successful expression of both RbcL and RbcS. Only Section 4 (residues 373-397) and 5 (residues 398-422) chimeras could form L8S8 in E. coli.@*Conclusion, significance and impact of study@#GroEL chaperonin mediates the folding of bacterial RbcL in E. coli. Therefore, residues 248-297, 348-372 and 423-447 of Synechococcus RbcL may be important for interacting with the GroEL chaperonin for successful holoenzyme formation in E. coli.

Isolation of bacteria with plant growth-promoting activities from a foliar biofertilizer

Aims@#Plant growth-promoting bacteria are the key components of a biofertilizer. This study was aimed to isolate and identify the predominant bacteria found in a foliar biofertilizer and characterizes the potential of the bacterial isolates as plant growth promoters.@*Methodology and results@#Potential bacteria with plant growth-promoting activities were isolated from a foliar biofertilizer on HiCrome™ Bacillus agar and Nutrient agar. Bacteria with unique colonial morphology were selected and categorized by Gram’s differential staining. Subsequently, the bacterial isolates were being further characterized for plant growth-promoting potentials, such as the production of indole acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase and siderophore; as well as the ability of nitrogen fixation and phosphate/potassium solubilization. Based on the characterized traits, three bacterial isolates, namely M17, M22 and M52 showed great potential for being a plant growth promoter. Based on their 16S rRNA gene sequence analysis, M17, M22 and M52 were identified as Leclercia adecarboxylata, Margalitia shackletonii and Lysinibacillus pakistanensis, respectively.@*Conclusion, significance and impact of study@#Bacterial isolates exhibiting plant growth-promoting activities were successfully isolated from a biofertilizer and identified in this study. This finding provides an insight into the potential bacteria of a foliar fertilizer that may promote plant growth. Identification of these plant-growth promoters may help the scientists and agrochemical manufacturers to determine and disclose the key microorganisms of their biofertilizers, thereby contributing to the improvement of biofertilizers and promoting them as reliable alternatives to chemical fertilizers.

Construction of a broad host range expression plasmid vector by Golden Gate cloning

Aims@#Expression of recombinant proteins across a range of different host organisms has profound contribution to the advancement in biotechnology. In this study, we aimed to construct a highly versatile broad host range (BHR) expression vector, designated as pYL101C.@*Methodology and results@#The Golden Gate cloning approach was used to construct pYL101C. Key features of pYL101C include a strong integron promoter (PINTc), a BHR pBBR1 origin of replication (ori), gentamycin resistance gene (GmR) as a selectable marker and a multiple cloning site (MCS) downstream of the promoter for easy-cloning purpose. To verify the functionality of pYL101C, we cloned the superfolder green fluorescent protein (sfGFP) reporter gene into pYL101C and transferred the resultant recombinant plasmid pYL101C::sfGFP into various Gram-negative bacteria. Transformants obtained stably expressed strong green fluorescence under blue light excitation even without selection after four passages. @*Conclusion, significance and impact of study@#The constructed BHR expression vector, pYL101C and recombinant pYL101C::sfGFP are stable and can be used to monitor the presence of Gram-negative bacteria, such as endophytes and pathogens in their hosts and environment.