James E. Berleman

Characterization of Four Outer Membrane Proteins Involved in Binding Starch to the Cell Surface of Bacteroides thetaiotaomicron

Bacteroides thetaiotaomicron, a gram-negative obligate anaerobe, utilizes polysaccharides by binding them to its cell surface and allowing cell-associated enzymes to hydrolyze them into digestible fragments. We use the starch utilization system as a model to analyze the initial steps involved in polysaccharide binding and breakdown. In a recent paper, we reported that one of the outer membrane proteins involved, SusG, had starch-degrading activity but was not sufficient for growth on starch. Moreover, SusG alone did not have detectable starch binding activity. Previous studies have shown that starch binding is essential for starch utilization. In this paper, we report that four other outer membrane proteins, SusC through SusF, are responsible for starch binding. Results of (14)C-starch binding assays show that SusC and SusD both contribute a significant amount of starch binding. SusE also appears to contribute substantially to starch binding. Using affinity chromatography, we show in vitro that these Sus proteins interact to bind starch. Moreover, protease accessibility of either SusC or SusD greatly increased when one was expressed without the other. This finding supports the hypothesis that SusC and SusD interact in the outer membrane. Evidence from additional protease accessibility studies suggests that SusC, SusE, and SusF are exposed on the cell surface. Our results demonstrate that SusC and SusD act as the major starch binding proteins on the cell surface, with SusE enhancing binding. SusFs role in starch utilization has yet to be determined, although the fact that starch protected it from proteolytic attack suggests that it does bind starch.

Deciphering the hunting strategy of a bacterial wolfpack

Myxococcus xanthus is a common soil bacterium with an intricate multicellular lifestyle that continues to challenge the way in which we conceptualize the capabilities of prokaryotic organisms. Myxococcus xanthus is the preferred laboratory representative from the Myxobacteria, a family of organisms distinguished by their ability to form highly structured biofilms that include tentacle-like packs of surface-gliding cell groups, synchronized rippling waves of oscillating cells and massive spore-filled aggregates that protrude upwards from the substratum to form fruiting bodies. But most of the Myxobacteria are also predators that thrive on the degradation of macromolecules released through the lysis of other microbial cells. The aim of this review is to examine our understanding of the predatory life cycle of M. xanthus. We will examine the multicellular structures formed during contact with prey, and the molecular mechanisms utilized by M. xanthus to detect and destroy prey cells. We will also examine our understanding of microbial predator-prey relationships and the prospects for how bacterial predation mechanisms can be exploited to generate new antimicrobial technologies.

The role of bacterial outer membrane vesicles for intra- and interspecies delivery.

An increasing number of Gram-negative bacteria have been observed to secrete outer membrane vesicles (OMVs). Many mysteries remain with respect to OMV formation, the regulation of OMV content and mode of targeting and fusion. Bacterial OMVs appear to serve a variety of purposes in intra- and interspecies microbial extracellular activities. OMVs have been shown to mediate cell-to-cell exchange of DNA, protein and small signalling molecules. The impact of such material exchanges on microbial communities and pathogenic processes, including the delivery of toxins at high concentration through OMVs, is discussed. This rather recent aspect of microbial ecology is likely to remain an important area of research as an in-depth understanding of OMVs may allow new approaches for combating bacterial infections and provide new routes for selective drug delivery.

Predataxis behavior in Myxococcus xanthus

Spatial organization of cells is important for both multicellular development and tactic responses to a changing environment. We find that the social bacterium, Myxococcus xanthus utilizes a chemotaxis (Che)-like pathway to regulate multicellular rippling during predation of other microbial species. Tracking of GFP-labeled cells indicates directed movement of M. xanthus cells during the formation of rippling wave structures. Quantitative analysis of rippling indicates that ripple wavelength is adaptable and dependent on prey cell availability. Methylation of the receptor, FrzCD is required for this adaptation: a frzF methyltransferase mutant is unable to construct ripples, whereas a frzG methylesterase mutant forms numerous, tightly packed ripples. Both the frzF and frzG mutant strains are defective in directing cell movement through prey colonies. These data indicate that the transition to an organized multicellular state during predation in M. xanthus relies on the tactic behavior of individual cells, mediated by a Che-like signal transduction pathway.

Bacterial social networks: structure and composition of Myxococcus xanthus outer membrane vesicle chains

The social soil bacterium, Myxococcus xanthus, displays a variety of complex and highly coordinated behaviours, including social motility, predatory rippling and fruiting body formation. Here we show that M. xanthus cells produce a network of outer membrane extensions in the form of outer membrane vesicle chains and membrane tubes that interconnect cells. We observed peritrichous display of vesicles and vesicle chains, and increased abundance in biofilms compared with planktonic cultures. By applying a range of imaging techniques, including three-dimensional (3D) focused ion beam scanning electron microscopy, we determined these structures to range between 30 and 60 nm in width and up to 5 μm in length. Purified vesicle chains consist of typical M. xanthus lipids, fucose, mannose, N-acetylglucosamine and N-acetylgalactoseamine carbohydrates and a small set of cargo protein. The protein content includes CglB and Tgl outer membrane proteins known to be transferable between cells in a contact-dependent manner. Most significantly, the 3D organization of cells within biofilms indicates that cells are connected via an extensive network of membrane extensions that may connect cells at the level of the periplasmic space. Such a network would allow the transfer of membrane proteins and other molecules between cells, and therefore could provide a mechanism for the coordination of social activities.

Multicellular Development in Myxococcus xanthus Is Stimulated by Predator-Prey Interactions

Myxococcus xanthus is a predatory bacterium that exhibits complex social behavior. The most pronounced behavior is the aggregation of cells into raised fruiting body structures in which cells differentiate into stress-resistant spores. In the laboratory, monocultures of M. xanthus at a very high density will reproducibly induce hundreds of randomly localized fruiting bodies when exposed to low nutrient availability and a solid surface. In this report, we analyze how M. xanthus fruiting body development proceeds in a coculture with suitable prey. Our analysis indicates that when prey bacteria are provided as a nutrient source, fruiting body aggregation is more organized, such that fruiting bodies form specifically after a step-down or loss of prey availability, whereas a step-up in prey availability inhibits fruiting body formation. This localization of aggregates occurs independently of the basal nutrient levels tested, indicating that starvation is not required for this process. Analysis of early developmental signaling relA and asgD mutants indicates that they are capable of forming fruiting body aggregates in the presence of prey, demonstrating that the stringent response and A-signal production are surprisingly not required for the initiation of fruiting behavior. However, these strains are still defective in differentiating to spores. We conclude that fruiting body formation does not occur exclusively in response to starvation and propose an alternative model in which multicellular development is driven by the interactions between M. xanthus cells and their cognate prey.

Exopolysaccharide microchannels direct bacterial motility and organize multicellular behavior

The myxobacteria are a family of soil bacteria that form biofilms of complex architecture, aligned multilayered swarms or fruiting body structures that are simple or branched aggregates containing myxospores. Here, we examined the structural role of matrix exopolysaccharide (EPS) in the organization of these surface-dwelling bacterial cells. Using time-lapse light and fluorescence microscopy, as well as transmission electron microscopy and focused ion beam/scanning electron microscopy (FIB/SEM) electron microscopy, we found that Myxococcus xanthus cell organization in biofilms is dependent on the formation of EPS microchannels. Cells are highly organized within the three-dimensional structure of EPS microchannels that are required for cell alignment and advancement on surfaces. Mutants lacking EPS showed a lack of cell orientation and poor colony migration. Purified, cell-free EPS retains a channel-like structure, and can complement EPS− mutant motility defects. In addition, EPS provides the cooperative structure for fruiting body formation in both the simple mounds of M. xanthus and the complex, tree-like structures of Chondromyces crocatus. We furthermore investigated the possibility that EPS impacts community structure as a shared resource facilitating cooperative migration among closely related isolates of M. xanthus.

The predatory life cycle of Myxococcus xanthus.

Myxococcus xanthus is a predatory bacterium and a model system for social behaviour in bacteria. Myx. xanthus forms thin biofilms, where cells work together to colonize new territory, invade prey colonies and lyse prey cells. Prey-cell lysis occurs at close proximity, and utilizes antibiotics such as myxovirescin, hydrolytic enzymes such as the protease MepA and extracellular outer-membrane vesicles that may facilitate delivery. Many questions about the mechanism of prey lysis remain, as well as a complete understanding of the vast hydrolytic and secondary metabolite potential present in the Myx. xanthus genome. However, it is clear that predation presents unique challenges for this bacterium, which are solved, in part, through the social behaviours at the disposal of Myx. xanthus. Here, we discuss the life cycle of Myx. xanthus, and the hypothesis that multicellular behaviour in this organism is critical to, and derives from, the challenges of growth as a bacterial predator.

Outer Membrane Vesicles of Bacteria: Structure, Biogenesis, and Function

Extracellular membrane vesicles (EMVs), a characteristic present across each domain of life, are subcellular shuttles of biologically active cargo that have a variety of functions ranging from cell-to-cell communication to predatory behavior. Mechanism(s) governing EMV biogenesis remain elusive; however, several initiators have been determined such as stress stimuli, sensing a potential prey or intruder, and signaling molecules. Regardless of function, increased membrane curvature and bulging is a key characteristic that leads to budding and release. This chapter highlights the differences between biogenesis processes of the bacteria, archaea and eukarya. We then focus on the outer membrane vesicles (OMVs) specific to Gram-negative bacteria, including several mechanism(s) that potentially explain how the loss of crucial OM-peptidoglycan (PGN) and OMPGN-inner membrane (IM) interactions can destabilize the OM to result in OMV biogenesis. Despite gaps present in the current understanding of these novel organelles, OMVs are one mechanism that allow microbial cells to function as multicellular organisms, as pathogens, and act as key predators in their environment. We discuss the importance in better understanding OMV biogenesis for greater insight into how this form of membrane architecture can be utilized for vaccines and targeted/specific treatments for infections. A. Nasarabadi (*) • M. Auer Lawrence Berkeley National Laboratory, Berkeley, CA, USA e-mail: anasarabadi@lbl.gov; mauer@lbl.gov J.E. Berleman Lawrence Berkeley National Laboratory, Berkeley, CA, USA Saint Mary’s College of California, Moraga, CA, USA e-mail: jeb8@stmarys-ca.edu # Springer International Publishing AG (outside the USA) 2017 O. Geiger (ed.), Biogenesis of Fatty Acids, Lipids and Membranes, Handbook of Hydrocarbon and Lipid Microbiology, DOI 10.1007/978-3-319-43676-0_44-1 1

Cyclic GMP controls Rhodospirillum centenum cyst development: cGMP control of encystment

Adenylyl cyclases are widely distributed across all kingdoms whereas guanylyl cyclases are generally thought to be restricted to eukaryotes. Here we report that the α‐proteobacterium Rhodospirillum centenum secretes cGMP when developing cysts and that a guanylyl cyclase deletion strain fails to synthesize cGMP and is defective in cyst formation. The R. centenum cyclase was purified and shown to effectively synthesize cGMP from GTP in vitro, demonstrating that it is a functional guanylyl cyclase. A homologue of the Escherichia coli cAMP receptor protein (CRP) is linked to the guanylyl cyclase and when deleted is deficient in cyst development. Isothermal calorimetry (ITC) and differential scanning fluorimetry (DSF) analyses demonstrate that the recombinant CRP homologue preferentially binds to, and is stabilized by cGMP, but not cAMP. This study thus provides evidence that cGMP has a crucial role in regulating prokaryotic development. The involvement of cGMP in regulating bacterial development has broader implications as several plant‐interacting bacteria contain a similar cyclase coupled by the observation that Azospirillum brasilense also synthesizes cGMP when inducing cysts.