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1.  Two new families of the FtsZ-tubulin protein superfamily implicated in membrane remodeling in diverse bacteria and archaea 
Biology Direct  2010;5:33.
Several recent discoveries reveal unexpected versatility of the bacterial and archaeal cytoskeleton systems that are involved in cell division and other processes based on membrane remodeling. Here we apply methods for distant protein sequence similarity detection, phylogenetic approaches, and genome context analysis to described two previously unnoticed families of the FtsZ-tubulin superfamily. One of these families is limited in its spread to Proteobacteria whereas the other is represented in diverse bacteria and archaea, and might be the key component of a novel, multicomponent membrane remodeling system that also includes a Von Willebrand A domain-containing protein, a distinct GTPase and membrane transport proteins of the OmpA family.
This article was reviewed by Purificación López-García and Gáspár Jékely; for complete reviews, see the Reviewers Reports section.
doi:10.1186/1745-6150-5-33
PMCID: PMC2875224  PMID: 20459678
2.  A canonical FtsZ protein in Verrucomicrobium spinosum, a member of the Bacterial phylum Verrucomicrobia that also includes tubulin-producing Prosthecobacter species 
Background
The origin and evolution of the homologous GTP-binding cytoskeletal proteins FtsZ typical of Bacteria and tubulin characteristic of eukaryotes is a major question in molecular evolutionary biology. Both FtsZ and tubulin are central to key cell biology processes – bacterial septation and cell division in the case of FtsZ and in the case of tubulins the function of microtubules necessary for mitosis and other key cytoskeleton-dependent processes in eukaryotes. The origin of tubulin in particular is of significance to models for eukaryote origins. Most members of domain Bacteria possess FtsZ, but bacteria in genus Prosthecobacter of the phylum Verrucomicrobia form a key exception, possessing tubulin homologs BtubA and BtubB. It is therefore of interest to know whether other members of phylum Verrucomicrobia possess FtsZ or tubulin as their FtsZ-tubulin gene family representative.
Results
Verrucomicrobium spinosum, a member of Phylum Verrucomicrobia of domain Bacteria, has been found to possess a gene for a protein homologous to the cytoskeletal protein FtsZ. The deduced amino acid sequence has sequence signatures and predicted secondary structure characteristic for FtsZ rather than tubulin, but phylogenetic trees and sequence analysis indicate that it is divergent from all other known FtsZ sequences in members of domain Bacteria. The FtsZ gene of V. spinosum is located within a dcw gene cluster exhibiting gene order conservation known to contribute to the divisome in other Bacteria and comparable to these clusters in other Bacteria, suggesting a similar functional role.
Conclusion
Verrucomicrobium spinosum has been found to possess a gene for a protein homologous to the cytoskeletal protein FtsZ. The results suggest the functional as well as structural homology of the V. spinosum FtsZ to the FtsZs of other Bacteria implying its involvement in cell septum formation during division. Thus, both bacteria-like FtsZ and eukaryote-like tubulin cytoskeletal homologs occur in different species of the phylum Verrucomicrobia of domain Bacteria, a result with potential major implications for understanding evolution of tubulin-like cytoskeletal proteins and the origin of eukaryote tubulins.
doi:10.1186/1471-2148-7-37
PMCID: PMC1845146  PMID: 17349062
3.  Split decision: A thaumarchaeon encoding both FtsZ and Cdv cell division proteins chooses Cdv for cytokinesis 
Molecular Microbiology  2011;82(3):535-538.
Cytoskeletal proteins play a pivotal role in cytokinesis in prokaryotes and eukaryotes. Most bacteria and a major branch of the archaea called the Euryarchaeota harbor a tubulin homolog, FtsZ, which assembles into a dynamic polymeric ring structure required for cytokinesis. However, Crenarchaeota, another branch of the archaea, lack FtsZ and instead use Cdv proteins, which are homologs of the ESCRT-III-like system involved in vesicular sorting and cytokinesis in eukaryotes, for cell division. Recently, a group of Crenarchaeota that grow in non-extreme environments was found to be sufficiently divergent to warrant its own branch of the archaea called the Thaumarchaeota. Notably, Thaumarchaeota have both Cdv and FtsZ homologs, which begs the question of which system is used for cell division. In this issue of Molecular Microbiology, Pelve and colleagues tackle this question. They found that cells of the thaumarchaeon Nitrosopumilus maritimus likely divide using the Cdv system and not FtsZ, based on localization of Cdv proteins but not FtsZ to division sites. The authors also provide evidence that the cell cycle during growth of N. maritimus differs significantly from those of other archaea.
doi:10.1111/j.1365-2958.2011.07833.x
PMCID: PMC3298680  PMID: 21895799
4.  Microtubules in Bacteria: Ancient Tubulins Build a Five-Protofilament Homolog of the Eukaryotic Cytoskeleton 
PLoS Biology  2011;9(12):e1001213.
The unequivocal identification of microtubules in bacteria throws light on the evolution of modern eukaryotic microtubules from a primordial structure.
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.
Author Summary
Bacteria are generally distinguished from the cells of fungi, plants, and animals (eukaryotes) not only by their much smaller size but also by the absence of certain subcellular structures such as nuclei, internal organelles, and microtubules. Using state-of-the-art microscopy, we demonstrate here that microtubules do exist in some bacteria. These bacterial microtubules are built from proteins that are closely related to the microtubule proteins in eukaryotes. Bacterial microtubules are smaller in diameter than their counterparts in eukaryotic cells but have the same basic architecture. We propose that bacterial microtubules represent primordial structures that preceded eukaryotic microtubules evolutionarily. Because bacterial microtubules can be produced and handled in the lab more easily than their eukaryotic counterparts, they may become useful tools for microtubule research and anti-cancer drug screening.
doi:10.1371/journal.pbio.1001213
PMCID: PMC3232192  PMID: 22162949
5.  The Nitrosopumilus maritimus CdvB, but Not FtsZ, Assembles into Polymers 
Archaea  2013;2013:104147.
Euryarchaeota and Crenarchaeota are two major phyla of archaea which use distinct molecular apparatuses for cell division. Euryarchaea make use of the tubulin-related protein FtsZ, while Crenarchaea, which appear to lack functional FtsZ, employ the Cdv (cell division) components to divide. Ammonia oxidizing archaeon (AOA) Nitrosopumilus maritimus belongs to another archaeal phylum, the Thaumarchaeota, which has both FtsZ and Cdv genes in the genome. Here, we used a heterologous expression system to characterize FtsZ and Cdv proteins from N. maritimus by investigating the ability of these proteins to form polymers. We show that one of the Cdv proteins in N. maritimus, the CdvB (Nmar_0816), is capable of forming stable polymers when expressed in fission yeast. The N. maritimus CdvB is also capable of assembling into filaments in mammalian cells. However, N. maritimus FtsZ does not assemble into polymers in our system. The ability of CdvB, but not FtsZ, to polymerize is consistent with a recent finding showing that several Cdv proteins, but not FtsZ, localize to the mid-cell site in the dividing N. maritimus. Thus, we propose that it is Cdv proteins, rather than FtsZ, that function as the cell division apparatus in N. maritimus.
doi:10.1155/2013/104147
PMCID: PMC3684127  PMID: 23818813
6.  Assembly of an FtsZ Mutant Deficient in GTPase Activity Has Implications for FtsZ Assembly and the Role of the Z Ring in Cell Division 
Journal of Bacteriology  2001;183(24):7190-7197.
FtsZ, the ancestral homologue of eukaryotic tubulins, assembles into the Z ring, which is required for cytokinesis in prokaryotic cells. Both FtsZ and tubulin have a GTPase activity associated with polymerization. Interestingly, the ftsZ2 mutant is viable, although the FtsZ2 mutant protein has dramatically reduced GTPase activity due to a glycine-for-aspartic acid substitution within the synergy loop. In this study, we have examined the properties of FtsZ2 and found that the reduced GTPase activity is not enhanced by DEAE-dextran-induced assembly, indicating it has a defective catalytic site. In the absence of DEAE-dextran, FtsZ2 fails to assemble unless supplemented with wild-type FtsZ. FtsZ has to be at or above the critical concentration for copolymerization to occur, indicating that FtsZ is nucleating the copolymers. The copolymers formed are relatively stable and appear to be stabilized by a GTP-cap. These results indicate that FtsZ2 cannot nucleate assembly in vitro, although it must in vivo. Furthermore, the stability of FtsZ-FtsZ2 copolymers argues that FtsZ2 polymers would be stable, suggesting that stable FtsZ polymers are able to support cell division.
doi:10.1128/JB.183.24.7190-7197.2001
PMCID: PMC95568  PMID: 11717278
7.  Comparison of Small Molecule Inhibitors of the Bacterial Cell Division Protein FtsZ and Identification of a Reliable Cross-Species Inhibitor 
ACS chemical biology  2012;7(11):1918-1928.
FtsZ is a guanosine triphosphatase (GTPase) that mediates cytokinesis in bacteria. FtsZ is homologous in structure to eukaryotic tubulin and polymerizes in a similar head-to-tail fashion. The study of tubulin’s function in eukaryotic cells has benefited greatly from specific and potent small molecule inhibitors, including colchicine and taxol. Although many small molecule inhibitors of FtsZ have been reported, none has emerged as a generally useful probe for modulating bacterial cell division. With the goal of establishing a useful and reliable small molecule inhibitor of FtsZ, a broad biochemical cross-comparison of reported FtsZ inhibitors was undertaken. Several of these molecules, including phenolic natural products, are unselective inhibitors that seem to derive their activity from the formation of microscopic colloids or aggregates. Other compounds, including the natural product viriditoxin and the drug development candidate PC190723, exhibit no inhibition of GTPase activity using protocols in this work or under published conditions. Of the compounds studied, only zantrin Z3 exhibits good levels of inhibition, maintains activity under conditions that disrupt small molecule aggregates, and provides a platform for exploration of structure-activity relationships (SAR). Preliminary SAR studies have identified slight modifications to the two sidechains of this structure that modulate the inhibitory activity of zantrin Z3. Collectively these studies will help focus future investigations toward the establishment of probes for FtsZ that fill the roles of colchicine and taxol in studies of tubulin.
doi:10.1021/cb300340j
PMCID: PMC3514448  PMID: 22958099
8.  Evolution of the cytoskeleton 
Summary
The eukaryotic cytoskeleton appears to have evolved from ancestral precursors related to prokaryotic FtsZ and MreB. FtsZ and MreB show 40−50% sequence identity across different bacterial and archaeal species. Here I suggest that this represents the limit of divergence that is consistent with maintaining their functions for cytokinesis and cell shape. Previous analyses have noted that tubulin and actin are highly conserved across eukaryotic species, but so divergent from their prokaryotic relatives as to be hardly recognizable from sequence comparisons. One suggestion for this extreme divergence of tubulin and actin is that it occurred as they evolved very different functions from FtsZ and MreB. I will present new arguments favoring this suggestion, and speculate on pathways. Moreover, the extreme conservation of tubulin and actin across eukaryotic species is not due to an intrinsic lack of variability, but is attributed to their acquisition of elaborate mechanisms for assembly dynamics and their interactions with multiple motor and binding proteins. A new structure-based sequence alignment identifies amino acids that are conserved from FtsZ to tubulins. The highly conserved amino acids are not those forming the subunit core or protofilament interface, but those involved in binding and hydrolysis of GTP.
doi:10.1002/bies.20601
PMCID: PMC2630885  PMID: 17563102
9.  Did DNA replication evolve twice independently? 
Nucleic Acids Research  1999;27(17):3389-3401.
DNA replication is central to all extant cellular organisms. There are substantial functional similarities between the bacterial and the archaeal/eukaryotic replication machineries, including but not limited to defined origins, replication bidirectionality, RNA primers and leading and lagging strand synthesis. However, several core components of the bacterial replication machinery are unrelated or only distantly related to the functionally equivalent components of the archaeal/eukaryotic replication apparatus. This is in sharp contrast to the principal proteins involved in transcription and translation, which are highly conserved in all divisions of life. We performed detailed sequence comparisons of the proteins that fulfill indispensable functions in DNA replication and classified them into four main categories with respect to the conservation in bacteria and archaea/eukaryotes: (i) non-homologous, such as replicative polymerases and primases; (ii) containing homologous domains but apparently non-orthologous and conceivably independently recruited to function in replication, such as the principal replicative helicases or proofreading exonucleases; (iii) apparently orthologous but poorly conserved, such as the sliding clamp proteins or DNA ligases; (iv) orthologous and highly conserved, such as clamp-loader ATPases or 5'-->3' exonucleases (FLAP nucleases). The universal conservation of some components of the DNA replication machinery and enzymes for DNA precursor biosynthesis but not the principal DNA polymerases suggests that the last common ancestor (LCA) of all modern cellular life forms possessed DNA but did not replicate it the way extant cells do. We propose that the LCA had a genetic system that contained both RNA and DNA, with the latter being produced by reverse transcription. Consequently, the modern-type system for double-stranded DNA replication likely evolved independently in the bacterial and archaeal/eukaryotic lineages.
PMCID: PMC148579  PMID: 10446225
10.  Rationalization of paclitaxel insensitivity of yeast β-tubulin and human βIII-tubulin isotype using principal component analysis 
BMC Research Notes  2012;5:395.
Background
The chemotherapeutic agent paclitaxel arrests cell division by binding to the hetero-dimeric protein tubulin. Subtle differences in tubulin sequences, across eukaryotes and among β-tubulin isotypes, can have profound impact on paclitaxel-tubulin binding. To capture the experimentally observed paclitaxel-resistance of human βIII tubulin isotype and yeast β-tubulin, within a common theoretical framework, we have performed structural principal component analyses of β-tubulin sequences across eukaryotes.
Results
The paclitaxel-resistance of human βIII tubulin isotype and yeast β-tubulin uniquely mapped on to the lowest two principal components, defining the paclitaxel-binding site residues of β-tubulin. The molecular mechanisms behind paclitaxel-resistance, mediated through key residues, were identified from structural consequences of characteristic mutations that confer paclitaxel-resistance. Specifically, Ala277 in βIII isotype was shown to be crucial for paclitaxel-resistance.
Conclusions
The present analysis captures the origin of two apparently unrelated events, paclitaxel-insensitivity of yeast tubulin and human βIII tubulin isotype, through two common collective sequence vectors.
doi:10.1186/1756-0500-5-395
PMCID: PMC3477043  PMID: 22849332
11.  Phylum Verrucomicrobia representatives share a compartmentalized cell plan with members of bacterial phylum Planctomycetes 
BMC Microbiology  2009;9:5.
Background
The phylum Verrucomicrobia is a divergent phylum within domain Bacteria including members of the microbial communities of soil and fresh and marine waters; recently extremely acidophilic members from hot springs have been found to oxidize methane. At least one genus, Prosthecobacter, includes species with genes homologous to those encoding eukaryotic tubulins. A significant superphylum relationship of Verrucomicrobia with members of phylum Planctomycetes possessing a unique compartmentalized cell plan, and members of the phylum Chlamydiae including human pathogens with a complex intracellular life cycle, has been proposed. Based on the postulated superphylum relationship, we hypothesized that members of the two separate phyla Planctomycetes and Verrucomicrobia might share a similar ultrastructure plan differing from classical prokaryote organization.
Results
The ultrastructure of cells of four members of phylum Verrucomicrobia – Verrucomicrobium spinosum, Prosthecobacter dejongeii, Chthoniobacter flavus, and strain Ellin514 – was examined using electron microscopy incorporating high-pressure freezing and cryosubstitution. These four members of phylum Verrucomicrobia, representing 3 class-level subdivisions within the phylum, were found to possess a compartmentalized cell plan analogous to that found in phylum Planctomycetes. Like all planctomycetes investigated, they possess a major pirellulosome compartment containing a condensed nucleoid and ribosomes surrounded by an intracytoplasmic membrane (ICM), as well as a ribosome-free paryphoplasm compartment between the ICM and cytoplasmic membrane.
Conclusion
A unique compartmentalized cell plan so far found among Domain Bacteria only within phylum Planctomycetes, and challenging our concept of prokaryote cell plans, has now been found in a second phylum of the Domain Bacteria, in members of phylum Verrucomicrobia. The planctomycete cell plan thus occurs in at least two distinct phyla of the Bacteria, phyla which have been suggested from other evidence to be related phylogenetically in the proposed PVC (Planctomycetes-Verrucomicrobia-Chlamydiae) superphylum. This planctomycete cell plan is present in at least 3 of 6 subdivisions of Verrucomicrobia, suggesting that the common ancestor of the verrucomicrobial phylum was also compartmentalized and possessed such a plan. The presence of this compartmentalized cell plan in both phylum Planctomycetes and phylum Verrucomicrobia suggest that the last common ancestor of these phyla was also compartmentalized.
doi:10.1186/1471-2180-9-5
PMCID: PMC2647929  PMID: 19133117
12.  The Deep Archaeal Roots of Eukaryotes 
Molecular Biology and Evolution  2008;25(8):1619-1630.
The set of conserved eukaryotic protein-coding genes includes distinct subsets one of which appears to be most closely related to and, by inference, derived from archaea, whereas another one appears to be of bacterial, possibly, endosymbiotic origin. The “archaeal” genes of eukaryotes, primarily, encode components of information-processing systems, whereas the “bacterial” genes are predominantly operational. The precise nature of the archaeo–eukaryotic relationship remains uncertain, and it has been variously argued that eukaryotic informational genes evolved from the homologous genes of Euryarchaeota or Crenarchaeota (the major branches of extant archaea) or that the origin of eukaryotes lies outside the known diversity of archaea. We describe a comprehensive set of 355 eukaryotic genes of apparent archaeal origin identified through ortholog detection and phylogenetic analysis. Phylogenetic hypothesis testing using constrained trees, combined with a systematic search for shared derived characters in the form of homologous inserts in conserved proteins, indicate that, for the majority of these genes, the preferred tree topology is one with the eukaryotic branch placed outside the extant diversity of archaea although small subsets of genes show crenarchaeal and euryarchaeal affinities. Thus, the archaeal genes in eukaryotes appear to descend from a distinct, ancient, and otherwise uncharacterized archaeal lineage that acquired some euryarchaeal and crenarchaeal genes via early horizontal gene transfer.
doi:10.1093/molbev/msn108
PMCID: PMC2464739  PMID: 18463089
archaea; eukaryotes; Euryarchaeota; Crenarchaeota; phylogenetic analysis
13.  Evolution of diverse cell division and vesicle formation systems in Archaea 
Nature Reviews. Microbiology  2010;8(10):731-741.
Recently a novel cell division system comprised of homologues of eukaryotic ESCRT-III (endosomal sorting complex required for transport III) proteins was discovered in the hyperthermophilic crenarchaeote Sulfolobus acidocaldarius. On the basis of this discovery, we undertook a comparative genomic analysis of the machineries for cell division and vesicle formation in Archaea. Archaea possess at least three distinct membrane remodelling systems: the FtsZ-based bacterial-type system, the ESCRT-III-based eukaryote-like system and a putative novel system that uses an archaeal actin-related protein. Many archaeal genomes encode assortments of components from different systems. Evolutionary reconstruction from these findings suggests that the last common ancestor of the extant Archaea possessed a complex membrane remodelling apparatus, different components of which were lost during subsequent evolution of archaeal lineages. By contrast, eukaryotes seem to have inherited all three ancestral systems.
doi:10.1038/nrmicro2406
PMCID: PMC3293450  PMID: 20818414
14.  Targeted Gene Knockouts Reveal Overlapping Functions of the Five Physcomitrella patens FtsZ Isoforms in Chloroplast Division, Chloroplast Shaping, Cell Patterning, Plant Development, and Gravity Sensing 
Molecular Plant  2009;2(6):1359-1372.
Chloroplasts and bacterial cells divide by binary fission. The key protein in this constriction division is FtsZ, a self-assembling GTPase similar to eukaryotic tubulin. In prokaryotes, FtsZ is almost always encoded by a single gene, whereas plants harbor several nuclear-encoded FtsZ homologs. In seed plants, these proteins group in two families and all are exclusively imported into plastids. In contrast, the basal land plant Physcomitrella patens, a moss, encodes a third FtsZ family with one member. This protein is dually targeted to the plastids and to the cytosol. Here, we report on the targeted gene disruption of all ftsZ genes in P. patens. Subsequent analysis of single and double knockout mutants revealed a complex interaction of the different FtsZ isoforms not only in plastid division, but also in chloroplast shaping, cell patterning, plant development, and gravity sensing. These results support the concept of a plastoskeleton and its functional integration into the cytoskeleton, at least in the moss P. patens.
doi:10.1093/mp/ssp076
PMCID: PMC2782794  PMID: 19946616
Bryophyte; cell wall; gravitropism; GTPase; chloroplast; plastoskeleton; P. patens; moss
15.  Evaluating the microtubule cytoskeleton and its interacting proteins in monocots by mining the rice genome 
Annals of Botany  2008;103(3):387-402.
Background
Microtubules (MTs) are assembled by heterodimers of α- and β-tubulins, which provide tracks for directional transport and frameworks for the spindle apparatus and the phragmoplast. MT nucleation and dynamics are regulated by components such as the γ-tubulin complex which are conserved among eukaryotes, and other components which are unique to plants. Following remarkable progress made in the model plant Arabidopsis thaliana toward revealing key components regulating MT activities, the completed rice (Oryza sativa) genome has prompted a survey of the MT cytoskeleton in this important crop as a model for monocots.
Scope
The rice genome contains three α-tubulin genes, eight β-tubulin genes and a single γ-tubulin gene. A functional γ-tubulin ring complex is expected to form in rice as genes encoding all components of the complex are present. Among proteins that interact with MTs, compared with A. thaliana, rice has more genes encoding some members such as the MAP65/Ase1p/PRC1 family, but fewer for the motor kinesins, the end-binding protein EB1 and the mitotic kinase Aurora. Although most known MT-interacting factors have apparent orthologues in rice, no orthologues of arabidopsis RIC1 and MAP18 have been identified in rice. Among all proteins surveyed here, only a few have had their functions characterized by genetic means in rice. Elucidating functions of proteins of the rice MT cytoskeleton, aided by recent technical advances made in this model monocot, will greatly advance our knowledge of how monocots employ their MTs to regulate their growth and form.
doi:10.1093/aob/mcn248
PMCID: PMC2707338  PMID: 19106179
Cytoskeleton; kinesins; microtubules (MTs); microtubule-associated proteins (MAPs); motors; rice; Oryza sativa
16.  Phylogenomic analysis of the cystatin superfamily in eukaryotes and prokaryotes 
Background
The cystatin superfamily comprises cysteine protease inhibitors that play key regulatory roles in protein degradation processes. Although they have been the subject of many studies, little is known about their genesis, evolution and functional diversification. Our aim has been to obtain a comprehensive insight into their origin, distribution, diversity, evolution and classification in Eukaryota, Bacteria and Archaea.
Results
We have identified in silico the full complement of the cystatin superfamily in more than 2100 prokaryotic and eukaryotic genomes. The analysis of numerous eukaryotic genomes has provided strong evidence for the emergence of this superfamily in the ancestor of eukaryotes. The progenitor of this superfamily was most probably intracellular and lacked a signal peptide and disulfide bridges, much like the extant Giardia cystatin. A primordial gene duplication produced two ancestral eukaryotic lineages, cystatins and stefins. While stefins remain encoded by a single or a small number of genes throughout the eukaryotes, the cystatins have undergone a more complex and dynamic evolution through numerous gene and domain duplications. In the cystatin superfamily we discovered twenty vertebrate-specific and three angiosperm-specific orthologous families, indicating that functional diversification has occurred only in multicellular eukaryotes. In vertebrate orthologous families, the prevailing trends were loss of the ancestral inhibitory activity and acquisition of novel functions in innate immunity. Bacterial cystatins and stefins may be emergency inhibitors that enable survival of bacteria in the host, defending them from the host's proteolytic activity.
Conclusion
This study challenges the current view on the classification, origin and evolution of the cystatin superfamily and provides valuable insights into their functional diversification. The findings of this comprehensive study provide guides for future structural and evolutionary studies of the cystatin superfamily as well as of other protease inhibitors and proteases.
doi:10.1186/1471-2148-9-266
PMCID: PMC2784779  PMID: 19919722
17.  Glutamate 83 and arginine 85 of helix H3 bend are key residues for FtsZ polymerization, GTPase activity and cellular viability of Escherichia coli: lateral mutations affect FtsZ polymerization and E. coli viability 
BMC Microbiology  2013;13:26.
Background
FtsZ is an essential cell division protein, which localizes at the middle of the bacterial cell to mediate cytokinesis. In vitro, FtsZ polymerizes and induces GTPase activity through longitudinal interactions to form the protofilaments, whilst lateral interactions result within formation of bundles. The interactions that participate in the protofilaments are similar to its eukaryotic homologue tubulin and are well characterized; however, lateral interactions between the inter protofilaments are less defined. FtsZ forms double protofilaments in vitro, though the key elements on the interface of the inter-protofilaments remain unclear as well as the structures involved in the lateral interactions in vivo and in vitro. In this study, we demonstrate that the highly conserved negative charge of glutamate 83 and the positive charge of arginine 85 located in the helix H3 bend of FtsZ are required for in vitro FtsZ lateral and longitudinal interactions, respectively and for in vivo cell division.
Results
The effect of mutation on the widely conserved glutamate-83 and arginine-85 residues located in the helix H3 (present in most of the tubulin family) was evaluated by in vitro and in situ experiments. The morphology of the cells expressing Escherichia coli FtsZ (E83Q) mutant at 42°C formed filamented cells while those expressing FtsZ(R85Q) formed shorter filamented cells. In situ immunofluorescence experiments showed that the FtsZ(E83Q) mutant formed rings within the filamented cells whereas those formed by the FtsZ(R85Q) mutant were less defined. The expression of the mutant proteins diminished cell viability as follows: wild type > E83Q > R85Q. In vitro, both, R85Q and E83Q reduced the rate of FtsZ polymerization (WT > E83Q >> R85Q) and GTPase activity (WT > E83Q >> R85Q). R85Q protein polymerized into shorter filaments compared to WT and E83Q, with a GTPase lag period that was inversely proportional to the protein concentration. In the presence of ZipA, R85Q GTPase activity increased two fold, but no bundles were formed suggesting that lateral interactions were affected.
Conclusions
We found that glutamate 83 and arginine 85 located in the bend of helix H3 at the lateral face are required for the protofilament lateral interaction and also affects the inter-protofilament lateral interactions that ultimately play a role in the functional localization of the FtsZ ring at the cell division site.
doi:10.1186/1471-2180-13-26
PMCID: PMC3626584  PMID: 23384248
Bacterial division; FtsZ polymerization; Longitudinal/lateral interactions; ZipA
18.  Characterization of bacterial operons consisting of two tubulins and a kinesin-like gene by the novel Two-Step Gene Walking method 
Nucleic Acids Research  2007;35(20):e135.
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.
doi:10.1093/nar/gkm836
PMCID: PMC2175320  PMID: 17942428
AMO41149; AMO41150; EF650087; AM743196
19.  Guanine nucleotide-dependent assembly of FtsZ into filaments. 
Journal of Bacteriology  1994;176(9):2754-2758.
FtsZ is an essential cell division protein that is localized to the leading edge of the bacterial septum in a cytokinetic ring. It contains the tubulin signature motif and is a GTP binding protein with a GTPase activity. Further comparison of FtsZ with eukaryotic tubulins revealed some additional sequence similarities, perhaps indicating a similar GTP binding site. Examination of FtsZ incubated in vitro by electron microscopy revealed a guanine nucleotide-dependent assembly into protein filaments, supporting the hypothesis that the FtsZ ring is formed through self-assembly. FtsZ3, which is unable to bind GTP, does not polymerize, whereas FtsZ2, which binds GTP but is deficient in GTP hydrolysis, is capable of polymerization.
Images
PMCID: PMC205420  PMID: 8169229
20.  Common evolutionary origins of mechanosensitive ion channels in Archaea, Bacteria and cell-walled Eukarya 
Archaea  2001;1(1):35-44.
The ubiquity of mechanosensitive (MS) channels triggered a search for their functional homologs in Archaea. Archaeal MS channels were found to share a common ancestral origin with bacterial MS channels of large and small conductance, and sequence homology with several proteins that most likely function as MS ion channels in prokaryotic and eukaryotic cell-walled organisms. Although bacterial and archaeal MS channels differ in conductive and mechanosensitive properties, they share similar gating mechanisms triggered by mechanical force transmitted via the lipid bilayer. In this review, we suggest that MS channels of Archaea can bridge the evolutionary gap between bacterial and eukaryotic MS channels, and that MS channels of Bacteria, Archaea and cell-walled Eukarya may serve similar physiological functions and may have evolved to protect the fragile cellular membranes in these organisms from excessive dilation and rupture upon osmotic challenge.
PMCID: PMC2685541  PMID: 15803657
Arabidopsis; mechanosensitivity; phylogeny; yeast
21.  Cell division and the ESCRT complex 
The Archaea constitute the third domain of life, a separate evolutionary lineage together with the Bacteria and the Eukarya.1 Species belonging to the Archaea contain a surprising mix of bacterial (metabolism, life style, genomic organization) and eukaryotic (replication, transcription, translation) features.2 The archaeal kingdom comprises two main phyla, the Crenarchaeota and the Euryarchaeota. Regarding the cell division process in archaeal species (reviewed in ref. 3), members of the Euryarchaeota rely on an FtsZ-based cell division mechanism4 whereas, previously, no division genes had been detected in the crenarchaea. However, we recently reported the discovery of the elusive cell division machinery in crenarchaea from the genus Sulfolobus.5 The minimal machinery consists of three genes, which we designated cdvA, B and C (for cell division), organized into an operon that is widely conserved among crenarchaea. The gene products polymerize between segregating nucleoids at the early mitotic stage, forming a complex that remains associated with the leading edge of constriction throughout cytokinesis. Interestingly, CdvB and CdvC were shown to be related to the eukaryotic ESCRT-III protein sorting machinery (reviewed in ref. 6), indicating shared common ancestry and mechanistic similarities to endosomal vesicle formation and viral (HIV) budding in eukaryotes. We also demonstrated that the cdv operon is subject to checkpoint-like regulation, and that the genes display a complementary phylogenetic distribution within the Archaea domain relative to FtsZ-dependent division systems.5 Here, the findings are further explored and discussed, and topics for further investigation are suggested.
PMCID: PMC2686351  PMID: 19704896
Archaea; cell cycle; cell division; cytokinesis; ESCRT; HIV; Sulfolobus; vesicle formation; virion release
22.  Multidimensional View of the Bacterial Cytoskeleton 
Journal of Bacteriology  2013;195(8):1627-1636.
The perspective of the cytoskeleton as a feature unique to eukaryotic organisms was overturned when homologs of the eukaryotic cytoskeletal elements were identified in prokaryotes and implicated in major cell functions, including growth, morphogenesis, cell division, DNA partitioning, and cell motility. FtsZ and MreB were the first identified homologs of tubulin and actin, respectively, followed by the discovery of crescentin as an intermediate filament-like protein. In addition, new elements were identified which have no apparent eukaryotic counterparts, such as the deviant Walker A-type ATPases, bactofilins, and several novel elements recently identified in streptomycetes, highlighting the unsuspected complexity of cytostructural components in bacteria. In vivo multidimensional fluorescence microscopy has demonstrated the dynamics of the bacterial intracellular world, and yet we are only starting to understand the role of cytoskeletal elements. Elucidating structure-function relationships remains challenging, because core cytoskeletal protein motifs show remarkable plasticity, with one element often performing various functions and one function being performed by several types of elements. Structural imaging techniques, such as cryo-electron tomography in combination with advanced light microscopy, are providing the missing links and enabling scientists to answer many outstanding questions regarding prokaryotic cellular architecture. Here we review the recent advances made toward understanding the different roles of cytoskeletal proteins in bacteria, with particular emphasis on modern imaging approaches.
doi:10.1128/JB.02194-12
PMCID: PMC3624557  PMID: 23417493
23.  Evolution of Tubulin Gene Arrays in Trypanosomatid parasites: genomic restructuring in Leishmania 
BMC Genomics  2006;7:261.
Background
α- and β-tubulin are fundamental components of the eukaryotic cytoskeleton and cell division machinery. While overall tubulin expression is carefully controlled, most eukaryotes express multiple tubulin genes in specific regulatory or developmental contexts. The genomes of the human parasites Trypanosoma brucei and Leishmania major reveal that these unicellular kinetoplastids possess arrays of tandem-duplicated tubulin genes, but with differences in organisation. While L. major possesses monotypic α and β arrays in trans, an array of alternating α- and β tubulin genes occurs in T. brucei. Polycistronic transcription in these organisms makes the chromosomal arrangement of tubulin genes important with respect to gene expression.
Results
We investigated the genomic architecture of tubulin tandem arrays among these parasites, establishing which character state is derived, and the timing of character transition. Tubulin loci in T. brucei and L. major were compared to examine the relationship between the two character states. Intergenic regions between tubulin genes were sequenced from several trypanosomatids and related, non-parasitic bodonids to identify the ancestral state. Evidence of alternating arrays was found among non-parasitic kinetoplastids and all Trypanosoma spp.; monotypic arrays were confirmed in all Leishmania spp. and close relatives.
Conclusion
Alternating and monotypic tubulin arrays were found to be mutually exclusive through comparison of genome sequences. The presence of alternating gene arrays in non-parasitic kinetoplastids confirmed that separate, monotypic arrays are the derived state and evolved through genomic restructuring in the lineage leading to Leishmania. This fundamental reorganisation accounted for the dissimilar genomic architectures of T. brucei and L. major tubulin repertoires.
doi:10.1186/1471-2164-7-261
PMCID: PMC1621084  PMID: 17044946
24.  The Common Ancestor of Archaea and Eukarya Was Not an Archaeon 
Archaea  2013;2013:372396.
It is often assumed that eukarya originated from archaea. This view has been recently supported by phylogenetic analyses in which eukarya are nested within archaea. Here, I argue that these analyses are not reliable, and I critically discuss archaeal ancestor scenarios, as well as fusion scenarios for the origin of eukaryotes. Based on recognized evolutionary trends toward reduction in archaea and toward complexity in eukarya, I suggest that their last common ancestor was more complex than modern archaea but simpler than modern eukaryotes (the bug in-between scenario). I propose that the ancestors of archaea (and bacteria) escaped protoeukaryotic predators by invading high temperature biotopes, triggering their reductive evolution toward the “prokaryotic” phenotype (the thermoreduction hypothesis). Intriguingly, whereas archaea and eukarya share many basic features at the molecular level, the archaeal mobilome resembles more the bacterial than the eukaryotic one. I suggest that selection of different parts of the ancestral virosphere at the onset of the three domains played a critical role in shaping their respective biology. Eukarya probably evolved toward complexity with the help of retroviruses and large DNA viruses, whereas similar selection pressure (thermoreduction) could explain why the archaeal and bacterial mobilomes somehow resemble each other.
doi:10.1155/2013/372396
PMCID: PMC3855935  PMID: 24348094
25.  An archaeal origin for the actin cytoskeleton 
A hallmark of the eukaryotic cell is the actin cytoskeleton, involved in a wide array of processes ranging from shape determination and phagocytosis to intracellular transport and cytokinesis. Recently, we reported the discovery of an actin-based cytoskeleton also in Archaea. The archaeal actin ortholog, Crenactin, was shown to belong to a conserved operon, Arcade (actin-related cytoskeleton in Archaea involved in shape determination), encoding an additional set of cytoskeleton-associated proteins. Here, we elaborate on the implications of these findings for the evolutionary relation between archaea and eukaryotes, with particular focus on the possibility that eukaryotic actin and actin-related proteins have evolved from an ancestral archaeal actin gene. Archaeal actin could thus have played an important role in cellular processes essential for the origin and early evolution of the eukaryotic lineage. Further exploration of uncharacterized archaeal lineages is necessary to find additional missing pieces in the evolutionary trajectory that ultimately gave rise to present-day organisms.
PMCID: PMC3306326  PMID: 22446522
actin; arcade; archaea; ARPs; crenactin; cell shape; cytoskeleton; eukaryogenesis; MreB; phagocytosis

Results 1-25 (218137)