The Histone H1-like protein AlgP facilitates even spacing of polyphosphate granules in Pseudomonas aeruginosa

Abstract

Synthesis of polyphosphate (polyP) is an ancient and universal stress and starvation response in bacteria. In many bacteria, polyP chains come together to form granular superstructures within cells. Some species appear to regulate polyP granule subcellular organization. Despite the critical role of polyP in starvation fitness, the composition of these structures, mechanism(s) underpinning their organization, and functional significance of such organization are poorly understood. We previously determined that granules become transiently evenly spaced on the cell’s long axis during nitrogen starvation in the opportunistic human pathogen Pseudomonas aeruginosa>. Here, we developed a granule-enrichment protocol to screen for polyP granule-localizing proteins. We identified AlgP as a protein that associates with polyP granules. We further discovered that AlgP is required for the even spacing of polyP granules. AlgP is a DNA-binding protein with a 154 amino acid C-terminal domain enriched in ‘KPAA’ repeats and variants of this repeat, with an overall sequence composition similar to the C-terminal tail of eukaryotic histone H1. Granule size, number, and spacing are significantly perturbed in the absence of AlgP, or when AlgP is truncated to remove the C-terminus. The ΔalgP and algPΔCTD mutants having fewer, larger granules. We speculate that AlgP may contribute to spacing by tethering polyP granules to the chromosome, thereby inhibiting fusion with neighboring granules. Our discovery that AlgP facilitates granule spacing allows us for the first time to directly uncouple granule biogenesis from even spacing, and will inform future efforts to explore the functional significance of granule organization on fitness during starvation.

Migration of Polyphosphate Granules in <b><i>Agrobacterium tumefaciens</i></b>

“…Furthermore, the determination of the polyP proteome of A. tumefaciens is necessary to identify other, so far unknown, polyP-associated proteins that might be involved in migration of polyP granules. Interestingly, a similar approach in P. aeruginosa very recently led to the identification of AlgP as a novel polyP-associated protein that is responsible for equidistant spacing of multiple pol-yP granules in P. aeruginosa [Chawla et al, 2021].…”

Polyphosphate affects cytoplasmic and chromosomal dynamics in nitrogen-starved Pseudomonas aeruginosa

“…The unmarked deletion strain referred to as ∆polyP in this study was constructed previously (16), and is a quadruple knockout of P. aeruginosa's four polyP kinases, Ppk1 (PA14_69230), Ppk2A (PA14_01730), Ppk2B (PA14_33240), and Ppk2C (PA14_19410). Strains LR231 and LR238 were used both for chromosomal origin counting and tracking, and were constructed previously (37). Reporter strains with insertions at the attTn7 site were generated by tetraparental conjugation, and exconjugants were selected on VBMM medium with 100mg/mL gentamycin, as described previously (38), and verified by PCR.…”

“…Wt UCBPP-PA14 with ParS pMT1 motif (parB pMT1 protein binding site) and both ssb-mCherry and gfp-ParB pMT1 chimeras expressed under control of the ssb promoter, all integrated at the attTn7 site on the chromosome Ref. (37)…”

“…aeruginosa UCBPP-PA14 ∆ppk1 ∆ppk2A ∆ppk2B ∆ppk2C attTn7::mini-Tn7T-Gm R ParS pMT1 P ssb :ssb-mCherry gfp-parB pMT1 ∆polyP UCBPP-PA14 with ParS pMT1 motif (parB pMT1 protein binding site) and both ssb-mCherry and gfp-ParB pMT1 chimeras expressed under control of the ssb promoter, all integrated at the attTn7 site on the chromosome Ref. (37)…”