Two reasons to kill: predation and kin discrimination in myxobacteria.

Myxobacteria are social microbial predators that use cell-cell contacts to identify bacterial or fungal prey and to differentiate kin relatives to initiate cellular responses. For prey killing, they assemble Tad-like and type III-like secretion systems at contact sites. For kin discrimination (KD), they assemble outer membrane exchange complexes composed of the TraA and TraB receptors at contacts sites. A type VI secretion system and Rhs proteins also mediate KD. Following cellular recognition, these systems deliver appropriate effectors into target cells. For prey, this leads to cell death and lysis for nutrient consumption by myxobacteria. In KD, a panel of effectors are delivered, and if adjacent cells are clonal cells, resistance ensues because they express a cognate panel of immunity factors; while nonkin lack complete immunity and are intoxicated. This review compares and contrasts recent findings from these systems in myxobacteria.

The predatory soil bacterium Myxococcus xanthus combines a Tad- and an atypical type 3-like protein secretion system to kill bacterial cells.

Predatory Myxobacteria employ a multilayered predation strategy to kill and lyse soil microorganisms. Aiming to dissect the mechanism of contact-dependent killing of bacteria, we analyze four protein secretion systems in Myxococcus xanthus and investigate the predation of mutant strains on different timescales. We find that a Tad-like and a type 3-like secretion system (Tad and T3SS∗) fulfill distinct functions during contact-dependent prey killing: the Tad-like system is necessary to induce prey cell death, while the needle-less T3SS∗ initiates prey lysis. Fluorescence microscopy reveals that components of both systems interdependently localize to the predator-prey contact site prior to killing. Swarm expansion assays show that both Tad and T3SS∗ are required to handle live prey and that nutrient extraction from prey bacteria is sufficient to power M. xanthus motility. In conclusion, our observations indicate the functional interplay of two types of secretion systems for killing and lysis of bacterial cells.

Myxococcus xanthus predation of Gram-positive or Gram-negative bacteria is mediated by different bacteriolytic mechanisms.

Myxococcus xanthus kills other species to use their biomass as energy source. Its predation mechanisms allow feeding on a broad spectrum of bacteria, but the identity of predation effectors and their mode of action remains largely unknown. We initially focused on the role of hydrolytic enzymes for prey killing and compared the activity of secreted M. xanthus proteins against four prey strains. 72 secreted proteins were identified by mass spectrometry, and among them a family 19 glycoside hydrolase that displayed bacteriolytic activity in vivo and in vitro This enzyme, which we name LlpM (lectin/lysozyme-like protein of M. xanthus), was not essential for predation, indicating that additional secreted components are required to disintegrate prey. Furthermore, secreted proteins lysed only Gram-positive, but not Gram-negative species. We thus compared the killing of different preys by cell-associated mechanisms: Individual M. xanthus cells killed all four test strains in a cell-contact dependent manner, but were only able to disintegrate Gram-negative, not Gram-positive cell envelopes. Thus, our data indicate that M. xanthus uses different, multifactorial mechanisms for killing and degrading different preys. Besides secreted enzymes, cell-associated mechanisms that have not been characterized so far, appear to play a major role for prey killing.IMPORTANCEPredation is an important survival strategy of the widespread myxobacteria, but it remains poorly understood on the mechanistic level. Without a basic understanding of how prey cell killing and consumption is achieved, it also remains difficult to investigate the role of predation for the complex myxobacterial lifestyle, reciprocal predator-prey relationships or the impact of predation on complex bacterial soil communities.We study predation in the established model organism Myxococcus xanthus, aiming to dissect the molecular mechanisms of prey cell lysis. In this study, we addressed the role of secreted bacteriolytic proteins, as well as potential mechanistic differences in the predation of Gram-positive and Gram-negative bacteria. Our observation shows that secreted enzymes are sufficient for killing and degrading Gram-positive species, but that cell-associated mechanisms may play a major role for killing Gram-negative and Gram-positive prey on fast timescales.

The Predation Strategy of Myxococcus xanthus.

Myxobacteria are ubiquitous in soil environments. They display a complex life cycle: vegetatively growing cells coordinate their motility to form multicellular swarms, which upon starvation aggregate into large fruiting bodies where cells differentiate into spores. In addition to growing as saprophytes, Myxobacteria are predators that actively kill bacteria of other species to consume their biomass. In this review, we summarize research on the predation behavior of the model myxobacterium Myxococcus xanthus, which can access nutrients from a broad spectrum of microorganisms. M. xanthus displays an epibiotic predation strategy, i.e., it induces prey lysis from the outside and feeds on the released biomass. This predatory behavior encompasses various processes: Gliding motility and induced cell reversals allow M. xanthus to encounter prey and to remain within the area to sweep up its biomass, which causes the characteristic "rippling" of preying populations. Antibiotics and secreted bacteriolytic enzymes appear to be important predation factors, which are possibly targeted to prey cells with the aid of outer membrane vesicles. However, certain bacteria protect themselves from M. xanthus predation by forming mechanical barriers, such as biofilms and mucoid colonies, or by secreting antibiotics. Further understanding the molecular mechanisms that mediate myxobacterial predation will offer fascinating insight into the reciprocal relationships of bacteria in complex communities, and might spur application-oriented research on the development of novel antibacterial strategies.

Single-Molecule Tracking of DNA Translocases in Bacillus subtilis Reveals Strikingly Different Dynamics of SftA, SpoIIIE, and FtsA.

Like many bacteria, Bacillus subtilis possesses two DNA translocases that affect chromosome segregation at different steps. Prior to septum closure, nonsegregated DNA is moved into opposite cell halves by SftA, while septum-entrapped DNA is rescued by SpoIIIE. We have used single-molecule fluorescence microscopy and tracking (SMT) experiments to describe the dynamics of the two different DNA translocases, the cell division protein FtsA and the glycolytic enzyme phosphofructokinase (PfkA), in real time. SMT revealed that about 30% of SftA molecules move through the cytosol, while a fraction of 70% is septum bound and static. In contrast, only 35% of FtsA molecules are static at midcell, while SpoIIIE molecules diffuse within the membrane and show no enrichment at the septum. Several lines of evidence suggest that FtsA plays a role in septal recruitment of SftA: an ftsA deletion results in a significant reduction in septal SftA recruitment and a decrease in the average dwell time of SftA molecules. FtsA can recruit SftA to the membrane in a heterologous eukaryotic system, suggesting that SftA may be partially recruited via FtsA. Therefore, SftA is a component of the division machinery, while SpoIIIE is not, and it is otherwise a freely diffusive cytosolic enzyme in vivo Our developed SMT script is a powerful technique to determine if low-abundance proteins are membrane bound or cytosolic, to detect differences in populations of complex-bound and unbound/diffusive proteins, and to visualize the subcellular localization of slow- and fast-moving molecules in live cells.IMPORTANCE DNA translocases couple the late events of chromosome segregation to cell division and thereby play an important role in the bacterial cell cycle. The proteins fall into one of two categories, integral membrane translocases or nonintegral translocases. We show that the membrane-bound translocase SpoIIIE moves slowly throughout the cell membrane in B. subtilis and does not show a clear association with the division septum, in agreement with the idea that it binds membrane-bound DNA, which can occur through cell division across nonsegregated chromosomes. In contrast, SftA behaves like a soluble protein and is recruited to the division septum as a component of the division machinery. We show that FtsA contributes to the recruitment of SftA, revealing a dual role of FtsA at the division machinery, but it is not the only factor that binds SftA. Our work represents a detailed in vivo study of DNA translocases at the single-molecule level.

Requirements for Septal Localization and Chromosome Segregation Activity of the DNA Translocase SftA from Bacillus subtilis.

Bacillus subtilis possesses 2 DNA translocases that affect late stages of chromosome segregation: SftA separates nonsegregated DNA prior to septum closure, while SpoIIIE rescues septum-entrapped DNA. We provide evidence that SftA is associated with the division machinery via a stretch of 47 amino acids within its N-terminus, suggesting that SftA is recruited by protein-protein interactions with a component of the division machinery. SftA was also recruited to mid-cell in the absence of its first 20 amino acids, which are proposed to contain a membrane-binding motif. Cell fractionation experiments showed that SftA can be found in the cytosolic fraction, and to a minor degree in the membrane fraction, showing that it is a soluble protein in vivo. The expression of truncated SftA constructs led to a dominant sftA deletion phenotype, even at very low induction rates of the truncated proteins, indicating that the incorporation of nonfunctional monomers into SftA hexamers abolishes functionality. Mobility shift experiments and surface plasmon binding studies showed that SftA binds to DNA in a cooperative manner, and demonstrated low ATPase activity when binding to short nucleotides rather than to long stretches of DNA.

Regulation of cell reversal frequency in Myxococcus xanthus requires the balanced activity of CheY-like domains in FrzE and FrzZ.

The Frz pathway of Myxococcus xanthus controls cell reversal frequency to support directional motility during swarming and fruiting body formation. Previously, we showed that phosphorylation of the response regulator FrzZ correlates with reversal frequencies, suggesting that this activity represents the output of the Frz pathway. Here, we tested the effect of different expression levels of FrzZ and its cognate kinase FrzE on M. xanthus motility. FrzZ overexpression caused a slight increase in phosphorylation and reversals. By contrast, FrzE overexpression abolished phosphorylation of FrzZ; this inhibition required the response regulator domain of FrzE. FrzZ phosphorylation was restored when both FrzE and FrzZ were overexpressed together. Our results show that the response regulator domain of FrzE is a negative regulator of FrzE kinase activity. This inhibition can be modulated by FrzZ, which acts as a positive regulator. Interestingly, fluorescence microscopy revealed that FrzZ and FrzE localize differently: FrzE colocalizes with the FrzCD receptor and the nucleoid, while FrzZ shows dispersed and polar localization. However, FrzZ binds tightly to the truncated variant FrzEΔ(CheY) . This indicates that the response regulator domain of FrzE is required for the interaction between FrzE and FrzZ to be transient, providing an unexpected regulatory output to the Frz pathway.

Phosphorylation-dependent localization of the response regulator FrzZ signals cell reversals in Myxococcus xanthus.

The life cycle of Myxococcus xanthus includes co-ordinated group movement and fruiting body formation, and requires directed motility and controlled cell reversals. Reversals are achieved by inverting cell polarity and re-organizing many motility proteins. The Frz chemosensory pathway regulates the frequency of cell reversals. While it has been established that phosphotransfer from the kinase FrzE to the response regulator FrzZ is required, it is unknown how phosphorylated FrzZ, the putative output of the pathway, targets the cell polarity axis. In this study, we used Phos-tag SDS-PAGE to determine the cellular level of phospho-FrzZ under different growth conditions and in Frz signalling mutants. We detected consistent FrzZ phosphorylation, albeit with a short half-life, in cells grown on plates, but not from liquid culture. The available pool of phospho-FrzZ correlated with reversal frequencies, with higher levels found in hyper-reversing mutants. Phosphorylation was not detected in hypo-reversing mutants. Fluorescence microscopy revealed that FrzZ is recruited to the leading cell pole upon phosphorylation and switches to the opposite pole during reversals. These results are consistent with the hypothesis that the Frz pathway modulates reversal frequency through a localized response regulator that targets cell polarity regulators at the leading cell pole.

Chemosensory signaling controls motility and subcellular polarity in Myxococcus xanthus.

Myxococcus xanthus is a model system for the study of dynamic protein localization and cell polarity in bacteria. M. xanthus cells are motile on solid surfaces enabled by two forms of motility. Motility is controlled by the Che-like Frz pathway, which is essential for fruiting body formation and differentiation. The Frz signal is mediated by a GTPase/GAP protein pair that establishes cell polarity and directs the motility systems. Pilus driven motility at the leading pole of the cell requires dynamic localization of two ATPases and the coordinated production of EPS synthesis. Gliding motility requires dynamic movement of large protein complexes, but the mechanism by which this system generates propulsive force is still an active area of investigation.

Players between the worlds: multifunctional DNA translocases.

DNA translocases play important roles during the bacterial cell cycle and in cell differentiation. Escherichia coli cells contain a multifunctional translocase, FtsK, which is involved in cell division, late steps of chromosome segregation and dimer resolution. In Gram-positive bacteria, the latter two processes are achieved by two translocases, SftA and SpoIIIE. These two translocases operate in a two step fashion, before and after closure of the division septum. DNA translocases have the remarkable ability to translocate DNA in a vectorial manner, orienting themselves according to polar sequences present in bacterial genomes, and perform various additional roles during the cell cycle. DNA translocases genetically interact with Structural Maintenance of Chromosomes (SMC) proteins in a flexible manner in different species, underlining the high versatility of this class of proteins.

Two DNA translocases synergistically affect chromosome dimer resolution in Bacillus subtilis.

In Bacillus subtilis, chromosome dimers that block complete segregation of sister chromosomes arise in about 15% of exponentially growing cells. Two dedicated recombinases, RipX and CodV, catalyze the resolution of dimers by site-specific recombination at the dif site, which is located close to the terminus region on the chromosome. We show that the two DNA translocases in B. subtilis, SftA and SpoIIIE, synergistically affect dimer resolution, presumably by positioning the dif sites in close proximity, before or after completion of cell division, respectively. Furthermore, we observed that both recombinases, RipX and CodV, assemble on the chromosome at the dif site throughout the cell cycle. The preassembly of recombinases probably ensures that dimer resolution can occur rapidly within a short time window around cell division.

Bacillus subtilis CinA is a stationary phase-induced protein that localizes to the nucleoid and plays a minor role in competent cells.

CinA is a conserved bacterial protein that has been reported to play an important role during competence in Streptococcus pneumoniae by recruiting the RecA protein to the cell membrane. Here, we provide information on the homologous CinA in Bacillus subtilis. We found that the synthesis of CinA is upregulated during stationary phase in all cells. The loss of CinA has a mild effect during competence, but it has no influence on the localization of RecA. CinA was observed to be associated with the nucleoid in the cell, and not with the cell membrane, as shown for S. pneumoniae. Purified CinA is a soluble protein, probably forming trimers, like other homologues, which share a domain with CinA that has been reported to be involved in molybdopterine biosynthesis. Our results suggest that CinA plays a nucleoid-associated general role in cells entering stationary phase that is not specific to competence in B. subtilis and possibly in many other bacteria.

SpoIIIE and a novel type of DNA translocase, SftA, couple chromosome segregation with cell division in Bacillus subtilis.

Cell division must only occur once daughter chromosomes have been fully separated. However, the initiating event of bacterial cell division, assembly of the FtsZ ring, occurs while chromosome segregation is still ongoing. We show that a two-step DNA translocase system exists in Bacillus subtilis that couples chromosome segregation and cell division. The membrane-bound DNA translocase SpoIIIE assembled very late at the division septum, and only upon entrapment of DNA, while its orthologue, SftA (YtpST), assembled at each septum in B. subtilis soon after FtsZ. Lack of SftA resulted in a moderate segregation defect at a late stage in the cell cycle. Like the loss of SpoIIIE, the absence of SftA was deleterious for the cells during conditions of defective chromosome segregation, or after induction of DNA damage. Lack of both proteins exacerbated all phenotypes. SftA forms soluble hexamers in solution, binds to DNA and has DNA-dependent ATPase activity, which is essential for its function in vivo. Our data suggest that SftA aids in moving DNA away from the closing septum, while SpoIIIE translocates septum-entrapped DNA only when septum closure precedes complete segregation of chromosomes.