Pleiotropic effects of PipX, PipY, or RelQ overexpression on growth, cell size, photosynthesis, and polyphosphate accumulation in the cyanobacterium Synechococcus elongatus PCC7942

Abstract

The cyanobacterial protein PipY belongs to the Pyridoxal-phosphate (PLP)-binding proteins (PLPBP/COG0325) family of pyridoxal-phosphate-binding proteins, which are represented in all three domains of life. These proteins share a high degree of sequence conservation, appear to have purely regulatory functions, and are involved in the homeostasis of vitamin B6 vitamers and amino/keto acids. Intriguingly, the genomic context of the pipY gene in cyanobacteria connects PipY with PipX, a protein involved in signaling the intracellular energy status and carbon-to-nitrogen balance. PipX regulates its cellular targets via protein–protein interactions. These targets include the PII signaling protein, the ribosome assembly GTPase EngA, and the transcriptional regulators NtcA and PlmA. PipX is thus involved in the transmission of multiple signals that are relevant for metabolic homeostasis and stress responses in cyanobacteria, but the exact function of PipY is still elusive. Preliminary data indicated that PipY might also be involved in signaling pathways related to the stringent stress response, a pathway that can be induced in the unicellular cyanobacterium Synechococcus elongatus PCC7942 by overexpression of the (p)ppGpp synthase, RelQ. To get insights into the cellular functions of PipY, we performed a comparative study of PipX, PipY, or RelQ overexpression in S. elongatus PCC7942. Overexpression of PipY or RelQ caused similar phenotypic responses, such as growth arrest, loss of photosynthetic activity and viability, increased cell size, and accumulation of large polyphosphate granules. In contrast, PipX overexpression decreased cell length, indicating that PipX and PipY play antagonistic roles on cell elongation or cell division. Since ppGpp levels were not induced by overexpression of PipY or PipX, it is apparent that the production of polyphosphate in cyanobacteria does not require induction of the stringent response.

ppGpp accumulation reduces the expression of the global nitrogen homeostasis-modulating NtcA regulon by affecting 2-oxoglutarate levels

“…1b ). Phenotypic responses, including increased cell size and chlorosis due to ppGpp accumulation, were similar to those observed in the RelQ-ox cells supplied with air under constant illumination (50 µmol photons m −2 s −1 ) in a previous study 21 . Glycogen accumulation, another nitrogen-starved phenotype, was also observed in the RelQ-ox cells, with the glycogen content reaching 37% (total glycogen/dry cell weight) at 120 h (Supplementary Fig.…”

“…These results indicated that (p)ppGpp is important for adaptation under both light and dark conditions. On the other hand, the overexpression of (p)ppGpp synthetase from Bacillus subtilis YjbM/SAS1/RelQ 20 causes apparent phenotypic responses, such as growth arrest, loss of photosynthetic activity and viability, increased cell size, and accumulation of large polyphosphate granules 18 , 21 . In addition, (p)ppGpp accumulation upregulates ribosomal hibernation-promoting factor, which causes ribosomes to dimerize to decrease translation rates of protein synthesis machinery 18 .…”

The ribosome assembly GTPase EngA is involved in redox signaling in cyanobacteria

“…Therefore, acclimatization to strong light is a complex process requiring the implementation of a variety of protective mechanisms to reduce photoinhibition ( Bailey and Grossman, 2008 ; Keren and Krieger-Liszkay, 2011 ; Muramatsu and Hihara, 2012 ). S. elongatus cultures that are acclimated to high light often show the yellowish appearance typical of stressed cultures undergoing chlorosis ( Llop et al, 2023 ) but can nevertheless grow as fast or even faster than at the moderate light intensities used as standard conditions.…”

“…Targets of PII regulation include transcriptional regulators, enzymes, and transporters involved in nitrogen and carbon assimilation ( Burillo et al, 2004 ; Llácer et al, 2007 , 2008 ; Forcada-Nadal et al, 2018 ; Labella et al, 2020b ; Forchhammer et al, 2022 ). Cyanobacterial genomes always contain at least as many copies of glnB as of pipX ( Laichoubi et al, 2011 ) and in S. elongatus , a high PipX/PII ratio prevents growth ( Espinosa et al, 2009 , 2010 ; Laichoubi et al, 2012 ; Chang et al, 2013 ; Jerez et al, 2021 ; Llop et al, 2023 ) suggesting that a relatively high ratio of PII over PipX is required to counteract unwanted interactions with less abundant and/or low affinity PipX partners. So far, EngA is the best candidate for this predicted PipX binding protein.…”

Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions

“…To provide independent evidence of the link between PipX self-interaction and binding to GlnB and/or GlnK proteins, we next analysed the impact of selected pipX mutations on PipX levels in E. coli BTH101, the host for BACTH assays. To strengthen the analysis with additional mutations while testing the effect of co-expression of the downstream gene pipY [54,61] in PipX levels in E. coli we used previously generated shuttle plasmids (Table 1) encoding pipX* (Y6A, Y32A or L65Q in CKX constructs) and constructed additional ones (H9A, F12A or F38A in CKXY constructs).…”