An extracellular matrix-associated zinc metalloprotease is required for dilauroyl phosphatidylethanolamine chemotactic excitation in Myxococcus xanthus

An extracellular matrix connects bacteria that live in organized assemblages called biofilms. While the role of the matrix in the regulation of cell behavior has not been extensively examined in bacteria, we suggest that, like mammalian cells, the matrix facilitates cell-cell interactions involved with regulation of cohesion, motility, and sensory transduction. The extracellular matrix of the soil bacterium Myxococcus xanthus is essential for biofilm formation and fruiting body development. The matrix material is extruded as long, thin fibrils that mediate adhesion to surfaces, cohesion to other cells, and excitation by the chemoattractant dilauroyl phosphatidylethanolamine. We report the identification of a putative matrix-associated zinc metalloprotease called FibA (fibril protein A). Western blotting with FibA-specific monoclonal antibody 2105 suggests extensive proteolytic processing of FibA during assembly into fibrils, consistent with the autoprocessing observed with other members of the M4 metalloprotease family. Disruption of fibA had no obvious effect on the structure of the fibrils and did not inhibit cell cohesion, excitation by dioleoyl phosphatidylethanolamine, or activity of the A- or S-motility motors. However, the cells lost the ability to respond to dilauroyl phosphatidylethanolamine and to form well-spaced fruiting bodies, though substantial aggregation was observed. Chemotactic excitation of the fibA mutant was restored by incubation with purified wild-type fibrils. The results suggest that this metalloprotease is involved in sensory transduction.     

FibA and PilA act cooperatively during fruiting body formation of Myxococcus xanthus

The extracellular matrix (ECM) of Myxococcus xanthus is essential for social (S-) motility and fruiting body formation. An ECM-bound protein, FibA, is homologous to M4 zinc metalloproteases and is important for stimulation by a phosphatidylethanolamine (PE) chemoattractant and for formation of discrete aggregation foci. In this work, we demonstrate that a correlation exists between a reduced ability to respond to PE and the observed defects in fruiting body morphogenesis. Furthermore, the fibA aggregation defect is accentuated by the absence of either PilA, the structural subunit of type IV pili, or DifD, a chemosensory response regulator. The inability to form fruiting bodies is not due to a loss of S-motility, but rather the loss of PilA and pili as pilT fibA mutants form fruiting bodies. The FibA active site residue E342 is important for fruiting body morphogenesis in the absence of PilA. Mutants exhibiting defects in fruiting body morphogenesis also produce fewer viable spores. It is proposed that FibA and PilA act as extracellular sensors for developmental signals.     

Myxococcus xanthus protein C is a major spore surface protein.

Fruiting body formation in Myxococcus xanthus involves the aggregation of cells to form mounds and the differentiation of rod-shaped cells into spherical myxospores. The surface of the myxospore is composed of several sodium dodecyl sulfate (SDS)-soluble proteins, the best characterized of which is protein S (Mr, 19,000). We have identified a new major spore surface protein called protein C (Mr, 30,000). Protein C is not present in extracts of vegetative cells but appears in extracts of developing cells by 6 h. Protein C, like protein S, is produced during starvation in liquid medium but is not made during glycerol-induced sporulation. Its synthesis is blocked in certain developmental mutants but not others. When examined by SDS-polyacrylamide gel electrophoresis, two forms of protein C are observed. Protein C is quantitatively released from spores by treatment with 0.1 N NaOH or by boiling in 1% SDS. It is slowly washed from the spore surface in water but is stabilized by the presence of magnesium. Protein C binds to the surface of spores depleted of protein C and protein S. Protein C is a useful new marker for development in M. xanthus because it is developmentally regulated, spore associated, abundant, and easily purified.     

Myxococcus xanthus developmental cell fate production: heterogeneous accumulation of developmental regulatory proteins and reexamination of the role of MazF in developmental lysis

Myxococcus xanthus undergoes a starvation-induced multicellular developmental program during which cells partition into three known fates: (i) aggregation into fruiting bodies followed by differentiation into spores, (ii) lysis, or (iii) differentiation into nonaggregating persister-like cells, termed peripheral rods. As a first step to characterize cell fate segregation, we enumerated total, aggregating, and nonaggregating cells throughout the developmental program. We demonstrate that both cell lysis and cell aggregation begin with similar timing at approximately 24 h after induction of development. Examination of several known regulatory proteins in the separated aggregated and nonaggregated cell fractions revealed previously unknown heterogeneity in the accumulation patterns of proteins involved in type IV pilus (T4P)-mediated motility (PilC and PilA) and regulation of development (MrpC, FruA, and C-signal). As part of our characterization of the cell lysis fate, we set out to investigate the unorthodox MazF-MrpC toxin-antitoxin system which was previously proposed to induce programmed cell death (PCD). We demonstrate that deletion of mazF in two different wild-type M. xanthus laboratory strains does not significantly reduce developmental cell lysis, suggesting that MazF's role in promoting PCD is an adaption to the mutant background strain used previously.     

Cloning of fibA, encoding an immunogenic subunit of the fibril-like surface structure of Peptostreptococcus micros

Although we are currently unaware of its biological function, the fibril-like surface structure is a prominent characteristic of the rough (Rg) genotype of the gram-positive periodontal pathogen Peptostreptococcus micros. The smooth (Sm) type of this species as well as the smooth variant of the Rg type (RgSm) lack these structures on their surface. A fibril-specific serum, as determined by immunogold electron microscopy, was obtained through adsorption of a rabbit anti-Rg type serum with excess bacteria of the RgSm type. This serum recognized a 42-kDa protein, which was subjected to N-terminal sequencing. Both clones of a lambdaTriplEx expression library that were selected by immunoscreening with the fibril-specific serum contained an open reading frame, designated fibA, encoding a 393-amino-acid protein (FibA). The 15-residue N-terminal amino acid sequence of the 42-kDa antigen was present at positions 39 to 53 in FibA; from this we conclude that the mature FibA protein contains 355 amino acids, resulting in a predicted molecular mass of 41,368 Da. The putative 38-residue signal sequence of FibA strongly resembles other gram-positive secretion signal sequences. The C termini of FibA and two open reading frames directly upstream and downstream of fibA exhibited significant sequence homology to the C termini of a group of secreted and surface-located proteins of other gram-positive cocci that are all presumably involved in anchoring of the protein to carbohydrate structures. We conclude that FibA is a secreted and surface-located protein and as such is part of the fibril-like structures.     

A Dynamic Response Regulator Protein Modulates G-Protein-Dependent Polarity in the Bacterium Myxococcus xanthus

Migrating cells employ sophisticated signal transduction systems to respond to their environment and polarize towards attractant sources. Bacterial cells also regulate their polarity dynamically to reverse their direction of movement. In Myxococcus xanthus, a GTP-bound Ras-like G-protein, MglA, activates the motility machineries at the leading cell pole. Reversals are provoked by pole-to-pole switching of MglA, which is under the control of a chemosensory-like signal transduction cascade (Frz). It was previously known that the asymmetric localization of MglA at one cell pole is regulated by MglB, a GTPase Activating Protein (GAP). In this process, MglB specifically localizes at the opposite lagging cell pole and blocks MglA localization at that pole. However, how MglA is targeted to the leading pole and how Frz activity switches the localizations of MglA and MglB synchronously remained unknown. Here, we show that MglA requires RomR, a previously known response regulator protein, to localize to the leading cell pole efficiently. Specifically, RomR-MglA and RomR-MglB complexes are formed and act complementarily to establish the polarity axis, segregating MglA and MglB to opposite cell poles. Finally, we present evidence that Frz signaling may regulate MglA localization through RomR, suggesting that RomR constitutes a link between the Frz-signaling and MglAB polarity modules. Thus, in Myxococcus xanthus, a response regulator protein governs the localization of a small G-protein, adding further insight to the polarization mechanism and suggesting that motility regulation evolved by recruiting and combining existing signaling modules of diverse origins.     

Behavior of peripheral rods and their role in the life cycle of Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium with a complex life cycle including a developmental phase in which cells aggregate and sporulate in response to starvation. In previous papers, we have described a heretofore unsuspected layer of complexity in the development of M. xanthus: vegetatively growing cells differentiate into two cell types during development. In addition to the differentiation of spores within fruiting bodies, a second cell type, peripheral rods, arises outside fruiting bodies. The pattern of expression of proteins in peripheral rods is different from that of either vegetatively growing cells or spores, and peripheral rods express a number of recognized developmental markers. In this report, we examine four aspects of the biology of peripheral rods: (i) the influence of nutrients on the proportion of peripheral rods in a population of developing cells, (ii) the capacity of peripheral rods to recapitulate development, (iii) the development of peripheral rods on conditioned medium, and (iv) the ability of peripheral rods to resume growth on low amounts of exogenously added nutrients. The results of these studies suggest that peripheral rods play a significant role in the life cycle of M. xanthus by allowing the exploitation of low amounts or transient influxes of nutrients without the investment of energy in spore germination. The differentiation of vegetatively growing cells into two cell types that differ significantly in biology, shape, and localization within the population has been incorporated into a model of the life cycle of M. xanthus.     

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.     

Identification and localization of Myxococcus xanthus porins and lipoproteins

Myxococcus xanthus DK1622 contains inner (IM) and outer membranes (OM) separated by a peptidoglycan layer. Integral membrane, β-barrel proteins are found exclusively in the OM where they form pores allowing the passage of nutrients, waste products and signals. One porin, Oar, is required for intercellular communication of the C-signal. An oar mutant produces CsgA but is unable to ripple or stimulate csgA mutants to develop suggesting that it is the channel for C-signaling. Six prediction programs were evaluated for their ability to identify β-barrel proteins. No program was reliable unless the predicted proteins were first parsed using Signal P, Lipo P and TMHMM, after which TMBETA-SVM and TMBETADISC-RBF identified β-barrel proteins most accurately. 228 β-barrel proteins were predicted from among 7331 protein coding regions, representing 3.1% of total genes. Sucrose density gradients were used to separate vegetative cell IM and OM fractions, and LC-MS/MS of OM proteins identified 54 β-barrel proteins. Another class of membrane proteins, the lipoproteins, are anchored in the membrane via a lipid moiety at the N-terminus. 44 OM proteins identified by LC-MS/MS were predicted lipoproteins. Lipoproteins are distributed between the IM, OM and ECM according to an N-terminal sorting sequence that varies among species. Sequence analysis revealed conservation of alanine at the +7 position of mature ECM lipoproteins, lysine at the +2 position of IM lipoproteins, and no noticable conservation within the OM lipoproteins. Site directed mutagenesis and immuno transmission electron microscopy showed that alanine at the +7 position is essential for sorting of the lipoprotein FibA into the ECM. FibA appears at normal levels in the ECM even when a +2 lysine is added to the signal sequence. These results suggest that ECM proteins have a unique method of secretion. It is now possible to target lipoproteins to specific IM, OM and ECM locations by manipulating the amino acid sequence near the +1 cysteine processing site.     

Gene expression during development of Myxococcus xanthus. Analysis of the genes for protein S

Protein S is an abundant spore coat protein produced during fruiting body formation (development) of the bacterium Myxococcus xanthus. We have cloned the DNA which codes for protein S and have found that this DNA hybridizes to three protein S RNA species from developmental cells but does not hybridize to RNA from vegetative cells. The half-life of protein S RNA was found to be unusually long, about 38 minutes, which, at least in part, accounts for the high level of protein S synthesis observed during development. Hybridization of restriction fragments from cloned M. xanthus DNA to the developmental RNAs enabled us to show that M. xanthus has two directly repeated genes for protein S (gene 1 and gene 2) which are separated by about 10(3) base-pairs on the bacterial chromosome. To study the expression of the protein S genes in M. xanthus, eight M. xanthus strains were isolated with Tn5 insertions at various positions in the DNA which codes for protein S. The strains which contained insertions in gene 1 or between gene 1 and gene 2 synthesized all three protein S RNA species and exhibited normal levels of protein S on spores. In contrast, M. xanthus strains exhibited normal levels of protein S on spores. In contrast, M. xanthus strains with insertions in gene 2 had no detectable protein S on spores and lacked protein S RNA. Thus, gene 2 is responsible for most if not all of the production of protein S during M. xanthus development. M. xanthus strains containing insertions in gene 1, gene 2 or both genes, were found to aggregate and sporulate normally even though strains bearing insertions in gene 2 contained no detectable protein S. We examined the expression of gene 1 in more detail by constructing a fusion between the lacZ gene of Escherichia coli and the N-terminal portion of protein S gene 1 of M. xanthus. The expression of beta-galactosidase activity in an M. xanthus strain containing the gene fusion was shown to be under developmental control. This result suggests that gene 1 is also expressed during development although apparently at a much lower level than gene 2.     

Genetic circuitry controlling motility behaviors of Myxococcus xanthus

M. xanthus has a complex multicellular lifestyle including swarming, predation and development. These behaviors depend on the ability of the cells to achieve directed motility across solid surfaces. M. xanthus cells have evolved two motility systems including Type-IV pili that act as grappling hooks and a controversial engine involving mucus secretion and fixed focal adhesion sites. The necessity for cells to coordinate the motility systems and to respond rapidly to environmental cues is reflected by a complex genetic network involving at least three complete sets of chemosensory systems and eukaryotic-like signaling proteins. In this review, we discuss recent advances suggesting that motor synchronization results from spatial oscillations of motility proteins. We further propose that these dynamics are modulated by the action of multiple upstream complementary signaling systems. M. xanthus is thus an exciting emerging model system to study the intricate processes of directed cell migration.     

Heat shock proteins of vegetative and fruiting Myxococcus xanthus cells.

The heat shock response of Myxococcus xanthus was investigated and characterized. When shifted from 28 to 40 degrees C, log-phase cells rapidly ceased growth, exhibited a 50% reduction in CFU, and initiated the synthesis of heat shock proteins (HTPs). Heat-shocked log-phase M. xanthus cells labeled with [35S]methionine were found to produce 18 major HTPs. The HTPs, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, were characterized with regard to molecular mass, subcellular location (periplasm, membrane, or cytoplasm), and temperature required for expression. Most HTPs were expressed at 36 degrees C, the optimum growth temperature of M. xanthus. Cells preincubated at 36 degrees C for 1 h before being shifted to 40 degrees C demonstrated increased thermotolerance compared with cells shifted directly from 28 to 40 degrees C. The HTPs produced by heat-shocked starvation-induced fruiting cells and glycerol-induced sporulating cells were also analyzed and characterized. Thirteen HTPs were detected in fruiting cells shifted from 28 to 40 degrees C. Six of these HTPs were not seen in vegetative M. xanthus cells. Log-phase cells induced to sporulate by the addition of glycerol produced 17 HTPs after being shifted to 40 degrees C. These HTPs were found to be a mixture of HTPs detected in heat-shocked log-phase cells and heat-shocked fruiting cells.     

Aggregation during fruiting body formation in Myxococcus xanthus is driven by reducing cell movement

When starved, Myxococcus xanthus cells assemble themselves into aggregates of about 10(5) cells that grow into complex structures called fruiting bodies, where they later sporulate. Here we present new observations on the velocities of the cells, their orientations, and reversal rates during the early stages of fruiting body formation. Most strikingly, we find that during aggregation, cell velocities slow dramatically and cells orient themselves in parallel inside the aggregates, while later cell orientations are circumferential to the periphery. The slowing of cell velocity, rather than changes in reversal frequency, can account for the accumulation of cells into aggregates. These observations are mimicked by a continuous agent-based computational model that reproduces the early stages of fruiting body formation. We also show, both experimentally and computationally, how changes in reversal frequency controlled by the Frz system mutants affect the shape of these early fruiting bodies.     

Sensory adaptation during negative chemotaxis in Myxococcus xanthus.

Myxococcus xanthus exhibits many tactic movements that require the frz signal transduction system, such as colony swarming and cellular aggregation during fruiting body formation. Previously we demonstrated that the Frz proteins control the chemotactic movements of M. xanthus (W. Shi, T. Köhler, and D. R. Zusman, Mol. Microbiol. 9:601-611, 1993). However it was unclear from that study how chemotaxis might be achieved at the cellular level. In this study, we showed that M. xanthus cells not only modulate the reversal frequency of cell movement in response to repellent stimuli but also exhibit sensory adaptation in response to the continuous presence of nonsaturating repellent stimuli. The sensory adaptation behavior requires FrzF (a putative methyltransferase) and is correlated with the methylation-demethylation of FrzCD, a methyl-accepting chemotaxis protein. These results indicate that negative chemotaxis in M. xanthus is achieved by chemokinesis plus sensory adaptation in a manner analogous to that of the free-swimming enteric bacteria.     

Localization of neonatal secretory proteins in different cell types of the rat submandibular gland from embryogenesis to adulthood

In the neonatal rat submandibular gland, Type III cells contain a group of related proteins that we call the B1-immunoreactive proteins (B1-IP; 23.5, 26, and 27.5 kDa). Type I cells lack these, but synthesize a different protein, Protein C (89 kDa). With maturation of the gland, these neonatal cell types are no longer seen in the seromucous acini, which are no longer reactive for the B1-IP. Here, we report the ultrastructural immunocytochemical localization of the B1-IP and Protein C over the course of development. From their first appearance in the embryo, the B1-IP and Protein C are present in different cells which become morphologically typical Type I and III cells prior to birth. At all stages, Type I cells have strong Protein C labeling and no B1 labeling. By 3 days postpartum, ultrastructurally atypical Type III cells are seen (Type IIIP); these label for the B1-IP, but also show labeling with antibody to Protein C. In the next week, as mucous cells appear in the acini, these show both B1-IP and C labeling; the B1 marker is lost by 30 days postpartum, but adult mucous acinar cells continue to show Protein C reactivity. In view of the appearance of Protein C reactivity in neonatal Type IIIP and then in mucous cells, and the presence of B1 reactivity in early but not mature mucous cells, we suggest that Type III cells differentiate into mucous cells and that Type IIIP cells are intermediates in this transformation. We see no evidence for the differentiation of either Type III or mucous cells from Type I cells, although our data cannot rule out this possibility. In adult glands, cells with B1 labeling are seen in intercalated ducts. Cells that appear to be Type I cells are also present in these ducts and label for Protein C. Double labeling for B1-IP and Protein C demonstrated that the two markers were exclusively present in different cells within intercalated ducts. This is of considerable interest, as intercalated ducts have been reported to be the stem cell population for normal and trauma-induced cellular replacement.