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.2014 Apr 14;9(4):e95189.
doi: 10.1371/journal.pone.0095189. eCollection 2014.

Phase variation in Myxococcus xanthus yields cells specialized for iron sequestration

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Phase variation in Myxococcus xanthus yields cells specialized for iron sequestration

Katarzyna Dziewanowska et al. PLoS One..

Abstract

Myxococcus xanthus undergoes phase variation during growth to produce predominantly two colony phenotypes. The majority are yellow colonies containing swarm-proficient cells and a minority are tan colonies containing swarm-deficient cells. Comparison of the transcriptomes of a yellow variant, a tan variant, and three tan mutants led to the identification of differentially-regulated genes that define key segments of the phase variation pathway. For example, expression of genes for the yellow pigment DKxanthene and the antibiotic myxovirescin was increased significantly in yellow variants. In contrast, expression of the siderophore myxochelin, hemin binding proteins, and iron transport proteins was increased specifically in tan strains. Thus, a consequence of phase variation is that yellow cells shift from producing antibiotic and pigment to producing components involved in acquisition of iron, which may increase fitness during periods of iron limitation. Multiple protein kinases and HTH-Xre DNA-binding proteins identified in this study may be involved in the regulatory hierarchy that governs phase variation.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Tan strains activate pathways dedicated to acquisition of iron.
Many of the genes whose expression is increased significantly in the tan strains comprise the siderophore production and transport, hemin transport, and proteins that provide energy for transport of iron-binding compounds. A partial list of components produced in tan cells is shown in this cartoon. The blue/purple circles represent the siderophore myxochelin pathway (MXAN_3639-3647) , . Myxochelin (mxc) is shown as a yellow star that binds Fe3+ when exported. Mxc•Fe3+ may be imported by a FepA-like protein (candidate is MXAN_6911; dark green shape), located in the outer membrane . Light green shapes represent the ferric siderophore ABC transporter genes (permease and MXAN_0684-0687 in membrane); brown cylinders represent periplasmic energy transduction TonB proteins (MXAN_0276, MXAN0820, and 6485) that may energize the outer membrane proteins (dashed lines). Red/orange icons represent hemin transporters (MXAN_1318-1321). Not shown are RhtX/FptX siderophore transporter (MXAN_5357), iron ABC transporters (MXAN_0770-0772), iron compound ABC transporter, and a periplasmic iron compound binding protein, Fe•ABC (MXAN_6000). Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) software predicts MXAN_3639 (putative iron-chelator utilization protein in the myxochelin operon) interacts with the ferric siderophore ABC transporter permease (MXAN_0685, 0686).
Figure 2
Figure 2. Tan mutants produce and secrete more siderophore.
Chrome azurol S assays were performed as described in Methods. Yellow bars signify the yellow strain; tan bars signify tan strains. (A) The amount of siderophore in 2×108 whole cells after lysis is reported as siderophore units relative to the WT-Y strain (100%). (B) Culture supernate (spent medium) was taken when the density was at ≈5×108 cells ml−1 and was used to quantify the amount of released siderophore. Samples represent the average of three independent assays.
Figure 3
Figure 3. Effect of iron on production of PV molecules.
Parallel cultures of WT-Y and WT-T strains were grown with and without supplements as described in Methods; siderophore and Abs410 were assayed when cells reached 6×108 cells ml−1. Data represent the average of duplicate readings obtained after performing three independent tests comparing the endpoint against the cells used as inoculate. (A) change in siderophore WT-Y, (B) change in siderophore WT-T, (C) change in Abs410 WT-Y, and (D) change in Abs410 WT-T.
Figure 4
Figure 4. Regulation of gene expression by iron.
RNA harvested from WT-Y cells was used to prepare cDNA for qRT-PCR analysis of representative genes whose expression is affected during phase variation;MXAN_0228 (Xre228),MXAN_7370 (ST_kinase),MXAN_3641 (myxochelin),MXAN_6911 (FepA homolog),MXAN_3639 (myxovirescin) andMXAN_4305 (DKX). The results represent three biological and technical replicates; data are from cells grown in CTPM+FeCl3 and CTPM+dipyridyl medium relative to levels from cells grown in CTPM alone (value  = 1) are presented.
Figure 5
Figure 5. WT-T strains do not produce myxovirescin.
Production of myxovirescin, also known as antibiotic TA, was monitored using a zone of inhibition assay withE. coli DH5α. Myxovirescin was produced by WT-Y variants (panel A), but not WT-T variant (B). (C) Like WT-T, myxovirescin was absent inasgB, but present indkxG andxre228 mutants. Production was delayed inxre228.
Figure 6
Figure 6. Expression of regulators and signal transduction proteins show phase-specific behavior.
(A1-40 Four genes encoding nearly identical protein kinase-like proteins are down-regulated in WT-T variants. In each case, the PKc-like gene is downstream of a gene annotated as a serine-threonine protein kinase (S_T Pk). The PKc-like genes that share significant similarity with serine-threonine protein kinases. (A5) A putativekapC gene (KapC =  protein kinase associated protein) is down-regulated in WT-T. (A6–10) Expression of multiple S_T Pk-encoding genes and HTH-Xre-encoding genes (helix-turn-helix xenobiotic response element family of DNA-binding proteins) is increased in WT-T relative to WT-Y strains. Four colored boxes indicate genes whose products share sufficient identity to be considered a protein cluster (ci =  cluster index; NCBI protein cluster database PCLA designation). (B) TheasgB mutant behaves similar to the WT-T with regard to genes in A, but differs from WT-T in that it also shows significant increases in four additional genes predicted to encode a S_T Pk, a hypothetical and two HTH-Xre proteins.
Figure 7
Figure 7. Potential biomarkers of phase variation.
A side-by side comparison illustrates the magnitude of changes between the tan variant and the mutants and highlights some of the outliers. Eight of the ten markers (genes or operons) show significant changes in the WT-T, while the remaining two genes represent changes that are unique to theasgB mutant. The fold change for gene operons represents the average of all genes in the pathway. DKX represents the average of the 14 DKxanthene genes (MXAN_4289-4305), MXV represents the average of 21 myxovirescin genes (MXAN_3930-3950), pKc represents the average of four protein kinase-like genes (MXAN_1234, 2177, 3183, 4841), hemin represents the average of nine myxochelin genes (MXAN_3639-3647), efflux represents the average of four macrolide efflux genes (MXAN_4198-4201), HTH-Xre represents the average of four xenobiotic response element genes (MXAN_4372, 4480, 4481, 6998), and ST_kinase represents the average of seven protein kinases (MXAN_2399 2840, 4371, 4373, 4479, 7370, 7371). BreC isMXAN_0383 and BIg-5 is the average ofMXAN_7217 and7219, which at fold increase of 1764, was off the scale of this graph.
Figure 8
Figure 8. Phase variation yields two cell types.
(A) The data show that the concentration of iron influences the yellow to tan phase switch; in this model, we propose that AsgB or AsgB-dependent products play a key role in the reverse direction switch. The yellow variants produce myxovirescin, the antibiotic needed for predation . Tan variants are mediocre swarmers but are more adept at sequestering iron. (B) The division of labor between yellow and tan variants may be critical for survival ofM xanthus in nature. The robust yellow swarmers are enriched at the periphery of a WT colony whereas slow swarming tan variants would be more enriched at the colony center. The ramifications for development are clear: tan cells, which are predisposed to become spores are well positioned to end up in the interior of the fruiting body and are surrounded by cells that produce DKX, the pigment needed to for production of viable spores .
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