Crosstalk between the tricarboxylic acid cycle and peptidoglycan synthesis in <i>Caulobacter crescentus</i> through the homeostatic control of α-ketoglutarate

<div><p>To achieve robust replication, bacteria must integrate cellular metabolism and cell wall growth. While these two processes have been well characterized, the nature and extent of cross-regulation between them is not well understood. Here, using classical genetics, CRISPRi, metabolomics, transcriptomics and chemical complementation approaches, we show that a loss of the master regulator Hfq in <i>Caulobacter crescentus</i> alters central metabolism and results in cell shape defects in a nutrient-dependent manner. We demonstrate that the cell morphology phenotype in the <i>hfq</i> deletion mutant is attributable to a disruption of α-ketoglutarate (KG) homeostasis. In addition to serving as a key intermediate of the tricarboxylic acid (TCA) cycle, KG is a by-product of an enzymatic reaction required for the synthesis of peptidoglycan, a major component of the bacterial cell wall. Accumulation of KG in the <i>hfq</i> deletion mutant interferes with peptidoglycan synthesis, resulting in cell morphology defects and increased susceptibility to peptidoglycan-targeting antibiotics. This work thus reveals a direct crosstalk between the TCA cycle and cell wall morphogenesis. This crosstalk highlights the importance of metabolic homeostasis in not only ensuring adequate availability of biosynthetic precursors, but also in preventing interference with cellular processes in which these intermediates arise as by-products.</p></div

Deletion of <i>hfq</i> alters cell sensitivity to PG-targeting antibiotics.

<p>(A) Representative images from disk diffusion assays evaluating the sensitivity of Δ<i>hfq</i> and control strains toward fosfomycin, cephalexin, and vancomycin. Cells from exponentially growing cultures were mixed with PYE soft agar (0.75%) and poured on top of PYE agar plates in the absence or presence of 100 μM DAP. Plates were incubated at 30°C for 70 h with antibiotic-loaded filter disks. (B) Quantification of antibiotic sensitivity for Δ<i>hfq</i> and control strains, as determined by the diameter of the clear zone of inhibition around the disk relative to the zone of inhibition for the WT strain. The error bars represent the standard deviations from 3 independent experiments. “n.s.”, not significant (<i>p</i>-value > 0.05); *, <i>p</i>-value < 0.01 by two-tailed <i>t</i>-test.</p

Link between the Δ<i>hfq</i> phenotypes and the biosynthesis of pantothenate and CoA.

<p>(A) Schematic of the pantothenate and CoA biosynthesis pathway in <i>C</i>. <i>crescentus</i>. The solid arrows represent a single enzymatic step, while the dashed arrows denote multiple enzymatic steps. PanD and VOR are the only two enzymes in this pathway whose mRNA levels were affected by the <i>hfq</i> deletion. (B) <i>panD</i> and <i>vor</i> mRNA levels in the WT and Δ<i>hfq</i> strains measured in RNA-Seq experiments. The error bars represent the standard deviations from 3 biological replicates. (C) Proposed mechanism underlying KG accumulation in Δ<i>hfq</i> cells. Downregulation of <i>panD</i> expression and upregulation of <i>vor</i> expression lead to reduction in CoA abundance. In turn, lower CoA level reduces KGDH activity leading to KG accumulation. Inactivation of <i>vor</i> in the Δ<i>hfq vor</i>::Tn<i>5</i> suppressor strain partially restores CoA synthesis and improves KGDH activity, resulting in lower KG levels. (D) Phase contrast images of Δ<i>hfq</i> cells grown in PYE with or without 1 mM pantothenate (Pan) for 20 h at 30°C. (E) Scatter plots of cell lengths and widths of populations described in (D). (F) Growth curves of WT and Δ<i>hfq</i> strains cultured at 30°C in PYE with and without 1 mM Pan. Each curve represents the average of 3 replicates with the standard deviation shown in grey.</p

Deletion of <i>hfq</i> impacts central metabolism.

<p>(A) Heatmap showing the changes in the level of various metabolites among the WT, Δ<i>hfq</i>, Δ<i>hfq vor</i>::Tn<i>5</i>, and <i>vor</i>::Tn<i>5</i> strains, as measured by LC-MS. Cells were grown on membrane filters on top of PYE agar. Only the metabolites that were significantly increased or decreased in Δ<i>hfq</i> compared to WT (<i>p</i>-value ≤ 0.05, one-way <i>t</i>-test) are shown. Fold changes were calculated based on the mean of normalized ion counts from 3 biological replicates. (B) Perturbation in the abundance of TCA cycle metabolites between WT and Δ<i>hfq</i> cells based on data shown in (A). (C) Quantification of KG levels in various strains from cells grown in PYE liquid cultures using an enzymatic assay. KG amounts were normalized to the protein content in the metabolite extracts. Error bars denote the standard deviation from 3 biological replicates.</p

The loss of Hfq results in cell morphology defects.

<p>(A) Phase contrast images of the parental (WT) and Δ<i>hfq</i> cells taken from liquid PYE cultures grown at 30°C. The image shown for the Δ<i>hfq</i> strain highlights cells with morphology defects. Arrows denote the presence of granules in Δ<i>hfq</i> cells. Higher magnification of a representative cell containing a granule is shown in the inset (scale bar, 2 μm). (B) Scatter plots of cell lengths and widths of WT and Δ<i>hfq</i> cells grown as in (A).</p

Peptidoglycan (PG) precursor synthesis is limited in the Δ<i>hfq</i> strain.

<p>(A) Schematic of the PG biosynthesis pathway in <i>C</i>. <i>crescentus</i>. The solid arrows represent a single enzymatic step, while the dashed arrows represent multiple enzymatic steps. (B) Proposed mechanism for the reduction of PG precursor synthesis in the absence of Hfq. In the Δ<i>hfq</i> strain, KG accumulation leads to the inhibition of DAP-AT activity resulting in reduced m-DAP and PG synthesis. (C) Representative LC-MS chromatograms for UDP-MurNAc-dipeptide from WT, Δ<i>hfq</i>, Δ<i>hfq vor</i>::Tn<i>5</i>, and <i>vor</i>::Tn<i>5</i> cells grown on membrane filters on top of PYE agar at 30°C. (D) Quantification of UDP-MurNAC-dipeptide level from samples described in (C) using the integrated peak intensity normalized by the protein concentration in the metabolite extracts. The error bars represent the standard deviations from 3 biological replicates. (E) Phase contrast images for Δ<i>hfq</i> cells grown in PYE in the absence and presence of 100 μM DAP for 8 h at 30°C. (F) Scatter plots of cell lengths and widths of populations described in (E).</p

Depletion of KGDH using CRISPRi causes growth and morphology defects.

<p>(A) Schematic representation of the CRISPRi method in <i>C</i>. <i>crescentus</i>. The gene encoding dCas9 was inserted at the chromosomal <i>vanA</i> locus. sgRNA was expressed under a constitutive promoter (Pcon) from a multi-copy plasmid. To achieve KGDH depletion, the vanillic-acid-inducible dCas9 protein was directed to the <i>sucAB</i> locus by sgRNA targeting a 20-bp sequence near the <i>sucA</i> start codon resulting in transcriptional interference. RNAP stands for RNA polymerase. (B) Growth curves of the KGDH depletion strain grown at 30°C in PYE with and without 0.05 mM vanillic acid. Each curve represents the average of three replicates with the standard deviation shown in grey. (C) Phase contrast images of cells grown in the presence (KGDH depletion) or absence (no depletion) of 0.05 mM vanillic acid for 20 h in PYE at 30°C. (D) Scatter plots of cell lengths and widths of populations described in (C).</p

Identification of Tn<i>5</i> suppressors of the Δ<i>hfq</i> growth and cell shape phenotypes.

<p>(A) Schematic of the Tn<i>5</i> screen. The suppressors were identified based on their ability to form bigger colonies on PYE plates. (B) Growth rate measurements for Δ<i>hfq</i> suppressors grown at 30°C in 96-well plates of PYE liquid cultures. The suppressor strains are ordered from fastest to slowest growth rate. Each circle represents an average of 3 replicates with the standard deviation plotted in grey. The growth rates for the WT and Δ<i>hfq</i> strains are shown for comparison. The red shaded box represents the top 30 fastest growing suppressors. (C) Tn<i>5</i> insertion sites for the top 30 suppressors (shaded region in panel B). (D) Phase contrast image of the most common suppressor, Δ<i>hfq vor</i>::Tn<i>5</i>, grown in PYE liquid culture at 30°C. (E) Scatter plot of cell lengths and widths of the population described in (D).</p

Proposed model for the crosstalk between central metabolism and PG precursor synthesis.

<p>(A) In wild-type <i>C</i>. <i>crescentus</i> grown in PYE medium, Hfq is involved in maintaining the homeostasis of central metabolites, such as CoA and KG, presumably by affecting the mRNA levels of metabolic genes <i>panD</i> and <i>vor</i>. KG homeostasis is important to prevent interference with the m-DAP and PG biosynthesis pathways and to maintain normal cell morphogenesis. The solid arrows represent a single enzymatic step, while the dashed arrows denote multiple enzymatic steps. (B) In the absence of Hfq, the mRNA levels of <i>panD</i> and <i>vor</i> are altered, leading to reduced CoA levels and accumulation of KG. High level of KG inhibits m-DAP synthesis, which, in turn, reduces the production of PG precursors and causes cell shape defects.</p