Stepwise metamorphosis of the tubeworm Hydroides elegans is mediated by a bacterial inducer and MAPK signaling

Diverse animal taxa metamorphose between larval and juvenile phases in response to bacteria. Although bacteria-induced metamorphosis is widespread among metazoans, little is known about the molecular changes that occur in the animal upon stimulation by bacteria. Larvae of the tubeworm Hydroides elegans metamorphose in response to surface-bound Pseudoalteromonas luteoviolacea bacteria, producing ordered arrays of phage tail-like metamorphosis-associated contractile structures (MACs). Sequencing the Hydroides genome and transcripts during five developmental stages revealed that MACs induce the regulation of groups of genes important for tissue remodeling, innate immunity, and mitogen-activated protein kinase (MAPK) signaling. Using two MAC mutations that block P. luteoviolacea from inducing settlement or metamorphosis and three MAPK inhibitors, we established a sequence of bacteria-induced metamorphic events: MACs induce larval settlement; then, particular properties of MACs encoded by a specific locus in P. luteoviolacea initiate cilia loss and activate metamorphosis-associated transcription; finally, signaling through p38 and c-Jun N-terminal kinase (JNK) MAPK pathways alters gene expression and leads to morphological changes upon initiation of metamorphosis. Our results reveal that the intricate interaction between Hydroides and P. luteoviolacea can be dissected using genomic, genetic, and pharmacological tools. Hydroides' dependency on bacteria for metamorphosis highlights the importance of external stimuli to orchestrate animal development. The conservation of Hydroides genome content with distantly related deuterostomes (urchins, sea squirts, and humans) suggests that mechanisms of bacteria-induced metamorphosis in Hydroides may have conserved features in diverse animals. As a major biofouling agent, insight into the triggers of Hydroides metamorphosis might lead to practical strategies for fouling control

Characterization of bacterial operons consisting of two tubulins and a kinesin-like gene by the novel Two-Step Gene Walking method

Tubulins are still considered as typical proteins of Eukaryotes. However, more recently they have been found in the unusual bacteria Prosthecobacter (btubAB). In this study, the genomic organization of the btub-genes and their genomic environment were characterized by using the newly developed Two-Step Gene Walking method. In all investigated Prosthecobacters, btubAB are organized in a typical bacterial operon. Strikingly, all btub-operons comprise a third gene with similarities to kinesin light chain sequences. The genomic environments of the characterized btub-operons are always different. This supports the hypothesis that this group of genes represents an independent functional unit, which was acquired by Prosthecobacter via horizontal gene transfer. The newly developed Two-Step Gene Walking method is based on randomly primed polymerase chain reaction (PCR). It presents a simple workflow, which comprises only two major steps—a Walking-PCR with a single specific outward pointing primer (step 1) and the direct sequencing of its product using a nested specific primer (step 2). Two-Step Gene Walking proved to be highly efficient and was successfully used to characterize over 20 kb of sequence not only in pure culture but even in complex non-pure culture samples

Microtubules in Bacteria: Ancient Tubulins Build a Five-Protofilament Homolog of the Eukaryotic Cytoskeleton

Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as “bacterial microtubules” (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening

Characterization and evolution of cell division and cell wall synthesis genes in the bacterial phyla Verrucomicrobia, Lentisphaerae, Chlamydiae and Planctomycetes and phylogenetic comparison with rRNA genes

In the past, studies on the relationships of the bacterial phyla Planctomycetes, Chlamydiae, Lentisphaerae, and Verrucomicrobia using different phylogenetic markers have been controversial. Investigations based on 16S rRNA sequence analyses suggested a relationship of the four phyla, showing the branching order Planctomycetes, Chlamydiae, Verrucomicrobia/Lentisphaerae. Phylogenetic analyses of 23S rRNA genes in this study also support a monophyletic grouping and their branching order—this grouping is significant for understanding cell division, since the major bacterial cell division protein FtsZ is absent from members of two of the phyla Chlamydiae and Planctomycetes. In Verrucomicrobia, knowledge about cell division is mainly restricted to the recent report of ftsZ in the closely related genera Prosthecobacter and Verrucomicrobium. In this study, genes of the conserved division and cell wall (dcw) cluster (ddl, ftsQ, ftsA, and ftsZ) were characterized in all verrucomicrobial subdivisions (1 to 4) with cultivable representatives (1 to 4). Sequence analyses and transcriptional analyses in Verrucomicrobia and genome data analyses in Lentisphaerae suggested that cell division is based on FtsZ in all verrucomicrobial subdivisions and possibly also in the sister phylum Lentisphaerae. Comprehensive sequence analyses of available genome data for representatives of Verrucomicrobia, Lentisphaerae, Chlamydiae, and Planctomycetes strongly indicate that their last common ancestor possessed a conserved, ancestral type of dcw gene cluster and an FtsZ-based cell division mechanism. This implies that Planctomycetes and Chlamydiae may have shifted independently to a non-FtsZ-based cell division mechanism after their separate branchings from their last common ancestor with Verrucomicrobia

A contractile injection system stimulates tubeworm metamorphosis by translocating a proteinaceous effector

The swimming larvae of many marine animals identify a location on the sea floor to undergo metamorphosis based on the presence of specific bacteria. Although this microbe–animal interaction is critical for the life cycles of diverse marine animals, what types of biochemical cues from bacteria that induce metamorphosis has been a mystery. Metamorphosis of larvae of the tubeworm Hydroides elegans is induced by arrays of phage tail-like contractile injection systems, which are released by the bacterium Pseudoalteromonas luteoviolacea. Here we identify the novel effector protein Mif1. By cryo-electron tomography imaging and functional assays, we observe Mif1 as cargo inside the tube lumen of the contractile injection system and show that the mif1 gene is required for inducing metamorphosis. Purified Mif1 is sufficient for triggering metamorphosis when electroporated into tubeworm larvae. Our results indicate that the delivery of protein effectors by contractile injection systems may orchestrate microbe–animal interactions in diverse contexts

BtubA-BtubB Heterodimer Is an Essential Intermediate in Protofilament Assembly

BACKGROUND:BtubA and BtubB are two tubulin-like genes found in the bacterium Prosthecobacter. Our work and a previous crystal structure suggest that BtubB corresponds to alpha-tubulin and BtubA to beta-tubulin. A 1:1 mixture of the two proteins assembles into tubulin-like protofilaments, which further aggregate into pairs and bundles. The proteins also form a BtubA/B heterodimer, which appears to be a repeating subunit in the protofilament. METHODOLOGY/PRINCIPAL FINDINGS:We have designed point mutations to disrupt the longitudinal interfaces bonding subunits into protofilaments. The mutants are in two classes, within dimers and between dimers. We have characterized one mutant of each class for BtubA and BtubB. When mixed 1:1 with a wild type partner, none of the mutants were capable of assembly. An excess of between-dimer mutants could depolymerize preformed wild type polymers, while within-dimer mutants had no activity. CONCLUSIONS:An essential first step in assembly of BtubA + BtubB is formation of a heterodimer. An excess of between-dimer mutants depolymerize wild type BtubA/B by sequestering the partner wild type subunit into inactive dimers. Within-dimer mutants cannot form dimers and have no activity

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy

The cell cycle of the planctomycete Gemmata obscuriglobus with respect to cell compartmentalization

Background: Gemmata obscuriglobus is a distinctive member of the divergent phylum Planctomycetes, all known members of which are peptidoglycan-less bacteria with a shared compartmentalized cell structure and divide by a budding process. G. obscuriglobus in addition shares the unique feature that its nucleoid DNA is surrounded by an envelope consisting of two membranes forming an analogous structure to the membrane-bounded nucleoid of eukaryotes and therefore G. obscuriglobus forms a special model for cell biology. Draft genome data for G. obscuriglobus as well as complete genome sequences available so far for other planctomycetes indicate that the key bacterial cell division protein FtsZ is not present in these planctomycetes, so the cell division process in planctomycetes is of special comparative interest. The membrane-bounded nature of the nucleoid in G. obscuriglobus also suggests that special mechanisms for the distribution of this nuclear body to the bud and for distribution of chromosomal DNA might exist during division. It was therefore of interest to examine the cell division cycle in G. obscuriglobus and the process of nucleoid distribution and nuclear body formation during division in this planctomycete bacterium via light and electron microscopy. Results: Using phase contrast and fluorescence light microscopy, and transmission electron microscopy, the cell division cycle of G. obscuriglobus was determined. During the budding process, the bud was formed and developed in size from one point of the mother cell perimeter until separation. The matured daughter cell acted as a new mother cell and started its own budding cycle while the mother cell can itself initiate budding repeatedly. Fluorescence microscopy of DAPI-stained cells of G. obscuriglobus suggested that translocation of the nucleoid and formation of the bud did not occur at the same time. Confocal laser scanning light microscopy applied to cells stained for membranes as well as DNA confirmed the behaviour of the nucleoid and nucleoid envelope during cell division. Electron microscopy of cryosubstituted cells confirmed deductions from light microscopy concerning nucleoid presence in relation to the stage of budding, and showed that the nucleoid was observed to occur in both mother and bud cells only at later budding stages. It further suggested that nucleoid envelope formed only after the nucleoid was translocated into the bud, since envelopes only appeared in more mature buds, while naked nucleoids occurred in smaller buds. Nucleoid envelope appeared to originate from the intracytoplasmic membranes (ICM) of both mother cell and bud. There was always a connecting passage between mother cell and bud during the budding process until separation of the two cells. The division cycle of the nucleated planctomycete G. obscuriglobus appears to be a complex process in which chromosomal DNA is transported to the daughter cell bud after initial formation of the bud, and this can be performed repeatedly by a single mother cell. Conclusion: The division cycle of the nucleated planctomycete G. obscuriglobus is a complex process in which chromosomal nucleoid DNA is transported to the daughter cell bud after initial formation of a bud without nucleoid. The new bud nucleoid is initially naked and not surrounded by membrane, but eventually acquires a complete nucleoid envelope consisting of two closely apposed membranes as occurs in the mother cell. The membranes of the new nucleoid envelope surrounding the bud nucleoid are derived from intracytoplasmic membranes of both the mother cell and the bud. The cell division of G. obscuriglobus displays some unique features not known in cells of either prokaryotes or eukaryotes

Uncharacterized bacterial structures revealed by electron cryotomography

Electron cryotomography (ECT) can reveal the native structure and arrangement of macromolecular complexes inside intact cells. This technique has greatly advanced our understanding of the ultrastructure of bacterial cells. We now view bacteria as structurally complex assemblies of macromolecular machines rather than as undifferentiated bags of enzymes. To date, our group has applied ECT to nearly 90 different bacterial species, collecting more than 15,000 cryotomograms. In addition to known structures, we have observed, to our knowledge, several uncharacterized features in these tomograms. Some are completely novel structures; others expand the features or species range of known structure types. Here, we present a survey of these uncharacterized bacterial structures in the hopes of accelerating their identification and study, and furthering our understanding of the structural complexity of bacterial cells

A contractile injection system stimulates tubeworm metamorphosis by translocating a proteinaceous effector

The swimming larvae of many marine animals identify a location on the sea floor to undergo metamorphosis based on the presence of specific bacteria. Although this microbe–animal interaction is critical for the life cycles of diverse marine animals, what types of biochemical cues from bacteria that induce metamorphosis has been a mystery. Metamorphosis of larvae of the tubeworm Hydroides elegans is induced by arrays of phage tail-like contractile injection systems, which are released by the bacterium Pseudoalteromonas luteoviolacea. Here we identify the novel effector protein Mif1. By cryo-electron tomography imaging and functional assays, we observe Mif1 as cargo inside the tube lumen of the contractile injection system and show that the mif1 gene is required for inducing metamorphosis. Purified Mif1 is sufficient for triggering metamorphosis when electroporated into tubeworm larvae. Our results indicate that the delivery of protein effectors by contractile injection systems may orchestrate microbe–animal interactions in diverse contexts