Conditional requirement of SglT for type IV pili function and S-motility in Myxococcus xanthus.

Myxobacteria exhibit complex social behaviors such as predation, outer membrane exchange and fruiting body formation. These behaviors depend on coordinated movements of cells on solid surfaces that involve social (S) motility. S-motility is powered by extension-retraction cycles of type 4 pili (Tfp) and exopolysaccharides (EPS) that provide a matrix for group cellular movement. Here, we characterized a new class of S-motility mutants in Myxococcus xanthus. These mutants have a distinctive phenotype: they lack S-motility even though they produce pili and EPS and the phenotype is temperature-sensitive. The point mutations were mapped to a single locus, MXAN_3284, named sglT. Similar to pilT mutants, sglT mutants are hyperpiliated and, strikingly, the temperature-sensitive phenotype is caused by null mutations. Our results indicate that SglT plays a critical role in Tfp function associated with pilus retraction and that the block in pili retraction is caused by a Tfp assembly defect in the absence of SglT at high-temperature growth.

Sibling Rivalry in Myxococcus xanthus Is Mediated by Kin Recognition and a Polyploid Prophage.

UNLABELLED: Myxobacteria form complex social communities that elicit multicellular behaviors. One such behavior is kin recognition, in which cells identify siblings via their polymorphic TraA cell surface receptor, to transiently fuse outer membranes and exchange their contents. In addition, outer membrane exchange (OME) regulates behaviors, such as inhibition of wild-type Myxococcus xanthus (DK1622) from swarming. Here we monitored the fate of motile cells and surprisingly found they were killed by nonmotile siblings. The kill phenotype required OME (i.e., was TraA dependent). The genetic basis of killing was traced to ancestral strains used to construct DK1622. Specifically, the kill phenotype mapped to a large "polyploid prophage," Mx alpha. Sensitive strains contained a 200-kb deletion that removed two of three Mx alpha units. To explain these results, we suggest that Mx alpha expresses a toxin-antitoxin cassette that uses the OME machinery of M. xanthus to transfer a toxin that makes the population "addicted" to Mx alpha. Thus, siblings that lost Mx alpha units (no immunity) are killed by cells that harbor the element. To test this, an Mx alpha-harboring laboratory strain was engineered (by traA allele swap) to recognize a closely related species, Myxococcus fulvus. As a result, M. fulvus, which lacks Mx alpha, was killed. These TraA-mediated antagonisms provide an explanation for how kin recognition specificity might have evolved in myxobacteria. That is, recognition specificity is determined by polymorphisms in traA, which we hypothesize were selected for because OME with non-kin leads to lethal outcomes. IMPORTANCE: The transition from single cell to multicellular life is considered a major evolutionary event. Myxobacteria have successfully made this transition. For example, in response to starvation, individual cells aggregate into multicellular fruiting bodies wherein cells differentiate into spores. To build fruits, cells need to recognize their siblings, and in part, this is mediated by the TraA cell surface receptor. Surprisingly, we report that TraA recognition can also involve sibling killing. We show that killing originates from a prophage-like element that has apparently hijacked the TraA system to deliver a toxin to kin. We hypothesize that this killing system has imposed selective pressures on kin recognition, which in turn has resulted in TraA polymorphisms and hence many different recognition groups.

Cell rejuvenation and social behaviors promoted by LPS exchange in myxobacteria.

Bacterial cells in their native environments must cope with factors that compromise the integrity of the cell. The mechanisms of coping with damage in a social or multicellular context are poorly understood. Here we investigated how a model social bacterium, Myxococcus xanthus, approaches this problem. We focused on the social behavior of outer membrane exchange (OME), in which cells transiently fuse and exchange their outer membrane (OM) contents. This behavior requires TraA, a homophilic cell surface receptor that identifies kin based on similarities in a polymorphic region, and the TraB cohort protein. As observed by electron microscopy, TraAB overexpression catalyzed a prefusion OM junction between cells. We then showed that damage sustained by the OM of one population was repaired by OME with a healthy population. Specifically, LPS mutants that were defective in motility and sporulation were rescued by OME with healthy donors. In addition, a mutant with a conditional lethal mutation in lpxC, an essential gene required for lipid A biosynthesis, was rescued by Tra-dependent interactions with a healthy population. Furthermore, lpxC cells with damaged OMs, which were more susceptible to antibiotics, had resistance conferred to them by OME with healthy donors. We also show that OME has beneficial fitness consequences to all cells. Here, in merged populations of damaged and healthy cells, OME catalyzed a dilution of OM damage, increasing developmental sporulation outcomes of the combined population by allowing it to reach a threshold density. We propose that OME is a mechanism that myxobacteria use to overcome cell damage and to transition to a multicellular organism.

Myxobacteria produce outer membrane-enclosed tubes in unstructured environments.

Myxobacteria are social microbes that exhibit complex multicellular behaviors. By use of fluorescent reporters, we show that Myxococcus xanthus isolates produce long narrow filaments that are enclosed by the outer membrane (OM) and contain proteins. We show that these OM tube (OMT) structures are produced at surprisingly high levels when cells are placed in liquid medium or buffer without agitation. OMTs can be long and easily exceed multiple cell lengths. When viewed by transmission electron microscopy, their morphology varies between tubes and chain-like structures. Intermediate-like structures are also found, suggesting that OMTs may transition between these two morphotypes. In support of this, video epifluorescence microscopy found that OMTs in solution dynamically twist and jiggle. On hard surfaces, myxobacteria glide, and upon cell-cell contact, they can efficiently exchange their OM proteins and lipids by a TraAB-dependent mechanism. Although the structure of OMTs hints at a possible role as conduits for exchange, evidence is presented to the contrary. For example, abundant OMT production occurs in traA or traB mutants and when cells are grown in liquid medium, yet transfer cannot occur under these conditions. Thus, genetic and environmental conditions that promote OMT production are incongruent with OM exchange.

Molecular recognition by a polymorphic cell surface receptor governs cooperative behaviors in bacteria.

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) traA allele replacements predictably changed partner specificity. Swapping traA 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, traA functions as a rare greenbeard gene that recognizes others that bear the same allele to confer beneficial treatment.

Myxobacterial tools for social interactions.

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 cell-cell interactions, with a focus on developmental signaling and social gliding motility. We also discuss a newly discovered cell-cell 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.

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.

Identification of the cglC, cglD, cglE, and cglF genes and their role in cell contact-dependent gliding motility in Myxococcus xanthus.

Within Myxococcus xanthus biofilms, cells actively move and exchange their outer membrane (OM) lipoproteins and lipids. Between genetically distinct strains, OM exchange can regulate recipient cell behaviors, including gliding motility and development. Although many different proteins are thought to be exchanged, to date, only two endogenous OM lipoproteins, CglB and Tgl, are known to be transferred. Protein exchange requires the TraAB proteins in recipient and donor cells, where they are hypothesized to facilitate OM fusion for transfer. To better understand the types of proteins exchanged, we identified the genes for the remaining set of cgl gliding motility mutants. These mutants are unique because their motility defect can be transiently restored by physical contact with donor cells that encode the corresponding wild-type protein, a process called stimulation. Similar to CglB and Tgl, the cglC and cglD genes encode type II signal sequences, suggesting that they are also lipoproteins. Surprisingly, the cglE and cglF genes instead encode type I signal sequences, suggesting that nonlipoproteins are also exchanged. Consistent with this idea, the addition of exogenous synthetic CglF protein (71 amino acids) to a cglF mutant rescued its motility defect. In contrast to a live donor cell, stimulation with purified CglF protein occurred independently of TraA. These results also indicate that CglF may localize to the cell surface. The implications of our findings on OM exchange are discussed.

Heterologous protein transfer within structured myxobacteria biofilms.

Microbial biofilms represent heterogeneous populations of cells that form intimate contacts. Within these populations cells communicate, cooperate and compete. Myxobacteria are noted for their complex social interactions, including gliding motility and lipoprotein exchange. Here, we investigated cis protein sequence and cellular behaviour requirements for lipoprotein transfer between Myxococcus xanthus cells. Specifically, an outer membrane (OM) type II signal sequence (SS) fused to the heterologous mCherry fluorescent reporter resulted in OM localization. When donor cells harbouring SS(OM)-mCherry were mixed with GFP-labelled recipient cells they developed red fluorescence. Our results surprisingly showed that a type II SS for OM localization, but not inner membrane localization, was necessary and sufficient for rapid and efficient heterologous protein transfer. Importantly, transfer did not occur in liquid or on surfaces where cells were poorly aligned. We conclude that cell-cell contact and alignment is a critical step for lipoprotein exchange. We hypothesize that protein transfer facilitates cooperative myxobacteria behaviours.