Cell Contact–Dependent Outer Membrane Exchange in Myxobacteria: Genetic Determinants and Mechanism

Biofilms are dense microbial communities. Although widely distributed and medically important, how biofilm cells interact with one another is poorly understood. Recently, we described a novel process whereby myxobacterial biofilm cells exchange their outer membrane (OM) lipoproteins. For the first time we report here the identification of two host proteins, TraAB, required for transfer. These proteins are predicted to localize in the cell envelope; and TraA encodes a distant PA14 lectin-like domain, a cysteine-rich tandem repeat region, and a putative C-terminal protein sorting tag named MYXO-CTERM, while TraB encodes an OmpA-like domain. Importantly, TraAB are required in donors and recipients, suggesting bidirectional transfer. By use of a lipophilic fluorescent dye, we also discovered that OM lipids are exchanged. Similar to lipoproteins, dye transfer requires TraAB function, gliding motility and a structured biofilm. Importantly, OM exchange was found to regulate swarming and development behaviors, suggesting a new role in cell–cell communication. A working model proposes TraA is a cell surface receptor that mediates cell–cell adhesion for OM fusion, in which lipoproteins/lipids are transferred by lateral diffusion. We further hypothesize that cell contact–dependent exchange helps myxobacteria to coordinate their social behaviors

Myxobacterial tools for social interactions

Abstract Myxobacteria exhibit complex social traits during which large populations of cells coordinate their behaviors. An iconic example is their response to starvation: thousands of cells move by gliding motility to build a fruiting body in which vegetative cells differentiate into spores. Here we review mechanisms that the model species Myxococcus xanthus uses for cellecell interactions, with a focus on developmental signaling and social gliding motility. We also discuss a newly discovered cellecell interaction whereby myxobacteria exchange their outer membrane (OM) proteins and lipids. The mechanism of OM transfer requires physical contact between aligned cells on a hard surface and is apparently mediated by OM fusion. The TraA and TraB proteins are required in both donor and recipient cells for transfer, suggesting bidirectional exchange, and TraA is thought to serve as a cell surface adhesin. OM exchange results in phenotypic changes that can alter gliding motility and development and is proposed to represent a novel microbial interacting platform to coordinate multicellular activities. Ó 2012 Published by Elsevier Masson SAS on behalf of Institut Pasteur

Molecular Recognition by a Polymorphic Cell Surface Receptor Governs Cooperative Behaviors in Bacteria

<div><p>Cell-cell recognition is a fundamental process that allows cells to coordinate multicellular behaviors. Some microbes, such as myxobacteria, build multicellular fruiting bodies from free-living cells. However, how bacterial cells recognize each other by contact is poorly understood. Here we show that myxobacteria engage in recognition through interactions between TraA cell surface receptors, which leads to the fusion and exchange of outer membrane (OM) components. OM exchange is shown to be selective among 17 environmental isolates, as exchange partners parsed into five major recognition groups. TraA is the determinant of molecular specificity because: (i) exchange partners correlated with sequence conservation within its polymorphic PA14-like domain and (ii) <i>traA</i> allele replacements predictably changed partner specificity. Swapping <i>traA</i> alleles also reprogrammed social interactions among strains, including the regulation of motility and conferred immunity from inter-strain killing. We suggest that TraA helps guide the transition of single cells into a coherent bacterial community, by a proposed mechanism that is analogous to mitochondrial fusion and fission cycling that mixes contents to establish a homogenous population. In evolutionary terms, <i>traA</i> functions as a rare greenbeard gene that recognizes others that bear the same allele to confer beneficial treatment.</p></div

TraA is a cell surface receptor.

<p>A) Western blot with TraA-PA14 antibodies against whole-cell lysates from <i>traA</i>⁺ (DW1463) and Δ<i>traA</i> (DW1467) strains. Molecular weight markers (kDa) are shown at the left, and the arrow indicates the TraA-specific band at ∼100 kDa. B) TraA immunofluorescence micrographs of live non-permeabilized cells. The same strains and primary antibodies were used as in A. White bar represents 2 µm.</p

TraA-dependent OM exchange confers protection from inter-strain killing.

<p>A) Labeled <i>M. fulvus</i> (stained red with DiD lipid dye) was mixed at a 1∶1 cell ratio with isogenic labeled DK1622 derivative strains (stained green with CFDA SE) that contain either TraA^DK1622 (DK8601*) or TraA<i>^{M. fulvus}</i> (DW1470*). After incubation on an agar surface for the indicated times, cells were collected for microscopic examination to determine the ratio of red to green or yellow cells. Between 300 and >1,000 cells were scored for each time point. B) The experiment was carried out as in A, except DK801 was mixed with isogenic DK1622 derivative strains that contained either TraA^DK1622 (DK8601*) or ΔTraA (DW1467*). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003891#s2" target="_blank">Results</a> are representative from multiple experiments.</p

TraA is the molecular determinant for specificity.

<p>Schematic representations of cell-cell interactions are shown on the left, in which variant TraA receptors are color coded. On the right are merged micrographs from red and green fluorescence images after mixed cells were collected from an agar surface. The laboratory <i>M. xanthus</i> strain was labeled with a red lipophilic DiD membrane dye, which does not transfer to the <i>M. fulvus</i> cells, which were labeled with the green fluorescent tracer dye. In contrast, an isogenic <i>M. xanthus traA</i> allele replacement strain (DW1470), which encodes the <i>traA^{M. fulvus}</i> allele, enables recognition and transfer with <i>M. fulvus</i> (yellow/orange cells).</p

The TraA PA14-like domain is polymorphic and correlates to recognition groupings.

<p>A) TraA amino acid (AA) variation derived from a sequence alignment from 16 <i>M. xanthus</i> strains is plotted. <i>M. fulvus</i> represents a distinct species and was excluded. Black rectangles in the hyper-variable region represent indels that range from one to seven codons in length. The signal sequence (SS) and a putative protein sorting tag (MYXO-CTERM) are also labeled. B) Phylogenetic tree derived from the PA14 polymorphic region, unrooted. Node support values are given as posterior probabilities. The multiple-sequence alignment used to generate the tree is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003891#pgen.1003891.s001" target="_blank">Figure S1</a>. Recognition groups are boxed and labeled. A dashed border indicates the heterogeneous recognition group. C) Domain similarity between three TraA sequences is graphically depicted and color coded. Gray and blue regions contain divergent sequences. Transfer compatibility of TraA variants is shown by green arrows (transfer) or red bars (no transfer). Specificity was determined by PA14 domain relatedness. The apparent chimeric domain architecture, depicting sequence relatedness, suggests that DNA rearrangements occurred between ancestral <i>traA</i> alleles. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003891#pgen.1003891.s002" target="_blank">Figure S2</a> for alignments.</p

<i>traA</i> allele–specific regulation of swarming.

<p>Indicated motile strains were mixed with isogenic engineered nonmotile laboratory strains that encoded the indicated <i>traA</i> alleles. Mixtures in which both strains encoded identical <i>traA</i> alleles or belonged to the same recognition group are highlighted with a black border, and they all exhibited swarm inhibition. A nonmotile Δ<i>traA</i> strain (DW1467) was used as a negative control (full swarming). Stereomicrographs were taken after 2 days of incubation. Assay was done as described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003891#pgen.1003891-Pathak2" target="_blank">[7]</a>.</p

Schematic overview for how TraA-mediated cell-cell interactions can contribute toward myxobacterial social behaviors.

<p>Cell genotypes and TraA receptors are color coded to indicate genetic relatedness. Related TraA receptors bind through proposed homophilic interactions. Populations of low genetic diversity would likely result in only sibling interactions, whereas diverse populations could result in non-kin interactions and could contribute toward group selection dynamics <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003891#pgen.1003891-Traulsen1" target="_blank">[45]</a>. Subsequent OM fusion and component exchange results in the indicated social outcomes. The ability of non-kin cells to interact could result in positive fitness outcomes. For example, if two distinct <i>M. xanthus</i> populations are of insufficient size to build a fruiting body, their combined populations, as mediated by TraA interactions, may be able to surmount this barrier.</p

<i>traA</i> allele–specific interactions in extracellular complementation of gliding motility.

<p>Protein transfer was assayed by the ability of nonmotile recipient mutants (Δ<i>cglC</i> Δ<i>tgl</i>) to be complemented extracellularly by a nonmotile, nonstimulatable donor that encodes the wild-type CglC and Tgl proteins <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003891#pgen.1003891-Pathak2" target="_blank">[7]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003891#pgen.1003891-Nudleman1" target="_blank">[9]</a>. The four engineered donor strains encoded the indicated <i>traA</i> allele replacements. The recipient strains were merodiploid with the indicated <i>traA</i> alleles and the original <i>traA</i>^DK1622 allele. Strains were mixed at a 1∶1 cell ratio, and micrographs were taken after 1 day. Images with black borders show <i>traA</i> allele combinations that restore motility, which occurs by protein transfer <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003891#pgen.1003891-Nudleman1" target="_blank">[9]</a>. Strains are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003891#pgen.1003891.s004" target="_blank">Table S1</a>.</p