Architecture of the Bacterial Flagellar Distal Rod and Hook of Salmonella

The bacterial flagellum is a large molecular complex composed of thousands of protein subunits for motility. The filamentous part of the flagellum, which is called the axial structure, consists of the filament, the hook, and the rods, with other minor components—the cap protein and the hook associated proteins. They share a common basic architecture of subunit arrangement, but each part shows quite distinct mechanical properties to achieve its specific function. The distal rod and the hook are helical assemblies of a single protein, FlgG and FlgE, respectively. They show a significant sequence similarity but have distinct mechanical characteristics. The rod is a rigid, straight cylinder, whereas the hook is a curved tube with high bending flexibility. Here, we report a structural model of the rod constructed by using the crystal structure of a core fragment of FlgG with a density map obtained previously by electron cryomicroscopy. Our structural model suggests that a segment called L-stretch plays a key role in achieving the distinct mechanical properties of the rod using a structurally similar component protein to that of the hook.

Download Full-text

Independent pseudogenizations and losses of sox15 during amniote diversification following asymmetric ohnolog evolution

BMC Ecology and Evolution ◽

10.1186/s12862-021-01864-z ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yusaku Ogita ◽

Kei Tamura ◽

Shuuji Mawaribuchi ◽

Nobuhiko Takamatsu ◽

Michihiko Ito

Keyword(s):

Sequence Similarity ◽

The Other ◽

Vertebrate Evolution ◽

Skeletal Muscle Regeneration ◽

Cns Development ◽

Placental Development ◽

Relaxed Selection ◽

Significant Sequence Similarity ◽

Anuran Amphibians ◽

Divergent Gene

Abstract Background Four ohnologous genes (sox1, sox2, sox3, and sox15) were generated by two rounds of whole-genome duplication in a vertebrate ancestor. In eutherian mammals, Sox1, Sox2, and Sox3 participate in central nervous system (CNS) development. Sox15 has a function in skeletal muscle regeneration and has little functional overlap with the other three ohnologs. In contrast, the frog Xenopus laevis and zebrafish orthologs of sox15 as well as sox1-3 function in CNS development. We previously reported that Sox15 is involved in mouse placental development as neofunctionalization, but is pseudogenized in the marsupial opossum. These findings suggest that sox15 might have evolved with divergent gene fates during vertebrate evolution. However, knowledge concerning sox15 in other vertebrate lineages than therian mammals, anuran amphibians, and teleost fish is scarce. Our purpose in this study was to clarify the fate and molecular evolution of sox15 during vertebrate evolution. Results We searched for sox15 orthologs in all vertebrate classes from agnathans to mammals by significant sequence similarity and synteny analyses using vertebrate genome databases. Interestingly, sox15 was independently pseudogenized at least twice during diversification of the marsupial mammals. Moreover, we observed independent gene loss of sox15 at least twice during reptile evolution in squamates and crocodile-bird diversification. Codon-based phylogenetic tree and selective analyses revealed an increased dN/dS ratio for sox15 compared to the other three ohnologs during jawed vertebrate evolution. Conclusions The findings revealed an asymmetric evolution of sox15 among the four ohnologs during vertebrate evolution, which was supported by the increased dN/dS values in cartilaginous fishes, anuran amphibians, and amniotes. The increased dN/dS value of sox15 may have been caused mainly by relaxed selection. Notably, independent pseudogenizations and losses of sox15 were observed during marsupial and reptile evolution, respectively. Both might have been caused by strong relaxed selection. The drastic gene fates of sox15, including neofunctionalization and pseudogenizations/losses during amniote diversification, might be caused by a release from evolutionary constraints.

Download Full-text

Discriminating between JCPyV and BKPyV in Urinary Virome Data Sets

Viruses ◽

10.3390/v13061041 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1041

Author(s):

Rita Mormando ◽

Alan J. Wolfe ◽

Catherine Putonti

Keyword(s):

Jc Virus ◽

Sequence Similarity ◽

Bk Virus ◽

Data Sets ◽

Metagenomic Sequencing ◽

Significant Sequence Similarity ◽

Sequence Identity ◽

Shotgun Metagenomic Sequencing ◽

Urinary Microbiome ◽

Six Genes

Polyomaviruses are abundant in the human body. The polyomaviruses JC virus (JCPyV) and BK virus (BKPyV) are common viruses in the human urinary tract. Prior studies have estimated that JCPyV infects between 20 and 80% of adults and that BKPyV infects between 65 and 90% of individuals by age 10. However, these two viruses encode for the same six genes and share 75% nucleotide sequence identity across their genomes. While prior urinary virome studies have repeatedly reported the presence of JCPyV, we were interested in seeing how JCPyV prevalence compares to BKPyV. We retrieved all publicly available shotgun metagenomic sequencing reads from urinary microbiome and virome studies (n = 165). While one third of the data sets produced hits to JCPyV, upon further investigation were we able to determine that the majority of these were in fact BKPyV. This distinction was made by specifically mining for JCPyV and BKPyV and considering uniform coverage across the genome. This approach provides confidence in taxon calls, even between closely related viruses with significant sequence similarity.

Download Full-text

An intergenic G-rich region in Leishmania tarentolae kinetoplast maxicircle DNA is a pan-edited cryptogene encoding ribosomal protein S12

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.56-67.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 56-67

Author(s):

D A Maslov ◽

N R Sturm ◽

B M Niner ◽

E S Gruszynski ◽

M Peris ◽

...

Keyword(s):

Amino Acid ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Sequence Similarity ◽

Rich Region ◽

Leishmania Tarentolae ◽

Significant Sequence Similarity ◽

The Family ◽

Intergenic Regions ◽

Ribosomal Protein S12

Six short G-rich intergenic regions in the maxicircle of Leishmania tarentolae are conserved in location and polarity in two other kinetoplastid species. We show here that G-rich region 6 (G6) represents a pan-edited cryptogene which contains at least two domains edited independently in a 3'-to-5' manner connected by short unedited regions. In the completely edited RNA, 117 uridines are added at 49 sites and 32 uridines are deleted at 13 sites, creating a translated 85-amino-acid polypeptide. Similar polypeptides are probably encoded by pan-edited G6 transcripts in two other species. The G6 polypeptide has significant sequence similarity to the family of S12 ribosomal proteins. A minicircle-encoded gRNA overlaps 12 editing sites in G6 mRNA, and chimeric gRNA/mRNA molecules were shown to exist, in agreement with the transesterification model for editing.

Download Full-text

Nanosurgical Manipulation of Titin and Its M-Complex

Nanomaterials ◽

10.3390/nano12020178 ◽

2022 ◽

Vol 12 (2) ◽

pp. 178

Author(s):

Dominik Sziklai ◽

Judit Sallai ◽

Zsombor Papp ◽

Dalma Kellermayer ◽

Zsolt Mártonfalvi ◽

...

Keyword(s):

Mechanical Properties ◽

Thick Filament ◽

Plastic Properties ◽

Force Microscopy ◽

Atomic Force ◽

Thick Filaments ◽

Volume Increment ◽

Associated Proteins ◽

Cross Links ◽

Globular Head

Titin is a multifunctional filamentous protein anchored in the M-band, a hexagonally organized supramolecular lattice in the middle of the muscle sarcomere. Functionally, the M-band is a framework that cross-links myosin thick filaments, organizes associated proteins, and maintains sarcomeric symmetry via its structural and putative mechanical properties. Part of the M-band appears at the C-terminal end of isolated titin molecules in the form of a globular head, named here the “M-complex”, which also serves as the point of head-to-head attachment of titin. We used high-resolution atomic force microscopy and nanosurgical manipulation to investigate the topographical and internal structure and local mechanical properties of the M-complex and its associated titin molecules. We find that the M-complex is a stable structure that corresponds to the transverse unit of the M-band organized around the myosin thick filament. M-complexes may be interlinked into an M-complex array that reflects the local structural and mechanical status of the transversal M-band lattice. Local segments of titin and the M-complex could be nanosurgically manipulated to achieve extension and domain unfolding. Long threads could be pulled out of the M-complex, suggesting that it is a compact supramolecular reservoir of extensible filaments. Nanosurgery evoked an unexpected volume increment in the M-complex, which may be related to its function as a mechanical spacer. The M-complex thus displays both elastic and plastic properties which support the idea that the M-band may be involved in mechanical functions within the muscle sarcomere.

Download Full-text

Legionella pneumophila CRISPR-Cas suggests recurrent encounters with one or more phages in the family Microviridae .

Applied and Environmental Microbiology ◽

10.1128/aem.00467-21 ◽

2021 ◽

Author(s):

Shayna R. Deecker ◽

Malene L. Urbanus ◽

Beth Nicholson ◽

Alexander W. Ensminger

Keyword(s):

Legionella Pneumophila ◽

Sequence Similarity ◽

Mobile Element ◽

Current Data ◽

Significant Sequence Similarity ◽

Remediation Strategies ◽

The Family ◽

Legionnaires Disease ◽

Outstanding Question

Legionella pneumophila is a ubiquitous freshwater pathogen and the causative agent of Legionnaires’ disease. L. pneumophila growth within protists provides a refuge from desiccation, disinfection, and other remediation strategies. One outstanding question has been whether this protection extends to phages. L. pneumophila isolates are remarkably devoid of prophages and to date no Legionella phages have been identified. Nevertheless, many L. pneumophila isolates maintain active CRISPR-Cas defenses. So far, the only known target of these systems is an episomal element that we previously named Legionella Mobile Element-1 (LME-1). The continued expansion of publicly available genomic data promises to further our understanding of the role of these systems. We now describe over 150 CRISPR-Cas systems across 600 isolates to establish the clearest picture yet of L. pneumophila ’s adaptive defenses. By searching for targets of 1,500 unique CRISPR-Cas spacers, LME-1 remains the only identified CRISPR-Cas targeted integrative element. We identified 3 additional LME-1 variants - all targeted by previously and newly identified CRISPR-Cas spacers - but no other similar elements. Notably, we also identified several spacers with significant sequence similarity to microviruses, specifically those within the subfamily Gokushovirinae . These spacers are found across several different CRISPR-Cas arrays isolated from geographically diverse isolates, indicating recurrent encounters with these phages. Our analysis of the extended Legionella CRISPR-Cas spacer catalog leads to two main conclusions: current data argue against CRISPR-Cas targeted integrative elements beyond LME-1, and the heretofore unknown L. pneumophila phages are most likely lytic gokushoviruses. IMPORTANCE Legionnaires’ disease is an often-fatal pneumonia caused by Legionella pneumophila , which normally grows inside amoebae and other freshwater protists. L. pneumophila trades diminished access to nutrients for the protection and isolation provided by the host. One outstanding question is whether L. pneumophila is susceptible to phages, given the protection provided by its intracellular lifestyle. In this work, we use Legionella CRISPR spacer sequences as a record of phage infection to predict that the “missing” L. pneumophila phages belong to the microvirus subfamily Gokushovirinae . Gokushoviruses are known to infect another intracellular pathogen, Chlamydia . How do gokushoviruses access L. pneumophila (and Chlamydia ) inside their “cozy niches”? Does exposure to phages happen during a transient extracellular period (during cell-to-cell spread) or is it indicative of a more complicated environmental lifestyle? One thing is clear, 100 years after their discovery, phages continue to hold important secrets about the bacteria upon which they prey.

Download Full-text

Genomic comparison of archaeal conjugative plasmids fromSulfolobus

Archaea ◽

10.1155/2004/151926 ◽

2004 ◽

Vol 1 (4) ◽

pp. 231-239 ◽

Cited By ~ 66

Author(s):

Bo Greve ◽

Susanne Jensen ◽

Kim Brügger ◽

Wolfram Zillig ◽

Roger A. Garrett

Keyword(s):

Sequence Similarity ◽

Comparative Sequence Analysis ◽

Genomic Comparison ◽

Variable Regions ◽

Integrase Gene ◽

Significant Sequence Similarity ◽

Genes Encoding ◽

Putative Origin ◽

Main Components ◽

Intercellular Exchange

All of the known self-transmissable plasmids of the Archaea have been found in the genusSulfolobus. To gain more insight into archaeal conjugative processes, four newly isolated self-transmissable plasmids, pKEF9, pHVE14, pARN3 and pARN4, were sequenced and subjected to a comparative sequence analysis with two earlier sequenced plasmids, pNOB8 and pING1. The analyses revealed three conserved and functionally distinct sections in the genomes. Section A is considered to encode the main components of the conjugative apparatus, where two genes show low but significant sequence similarity to sections of genes encoding bacterial conjugative proteins. A putative origin of replication is located in section B, which is highly conserved in sequence and contains several perfect and imperfect direct and inverted repeats. Further downstream, in section C, an operon encoding six to nine smaller proteins is implicated in the initiation and regulation of replication. Each plasmid carries an integrase gene of the type that does not partition on integration, and there is strong evidence for their integration into host chromosomes, where they may facilitate intercellular exchange of chromosomal genes. Two plasmids contain hexameric short regularly spaced repeats (SRSR), which have been implicated in plasmid maintenance, and each plasmid carries multiple recombination motifs, concentrated in the variable regions, which likely provide sites for genomic rearrangements.

Download Full-text

Dexamethasone enhances ras-recision gene expression in cultured murine fetal lungs: role in development

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2000.279.2.l312 ◽

2000 ◽

Vol 279 (2) ◽

pp. L312-L318 ◽

Cited By ~ 8

Author(s):

Mala R. Chinoy ◽

Steven E. Zgleszewski ◽

Robert E. Cilley ◽

Thomas M. Krummel

Keyword(s):

Lysyl Oxidase ◽

Sequence Similarity ◽

Data Bank ◽

Arbitrary Primers ◽

Significant Sequence Similarity ◽

Anchor Primer ◽

Enhanced Expression ◽

Fetal Lungs ◽

Cytoskeletal Elements ◽

Existing Data

We have shown that dexamethasone (Dex) accelerates maturation and differentiation of cultured fetal murine lungs (Cilley RE, Zgleszewski SE, Krummel TM, and Chinoy MR. Surg Forum 47: 692–695, 1996). We now demonstrate that although Dex inhibits thinning of acinar walls and secondary septa formation, it does, however, promote lung growth. CD-1 murine fetal lungs were cultured for 7 days in the presence and absence of 10 nM Dex. Dex-modulated genes were investigated and identified by differential display of mRNAs performed with specific anchor primer H-T11G and 24 arbitrary primers. Thirty-five differentially expressed cDNAs were isolated, subcloned, sequenced, and identified through BLAST searches. One of these cDNAs, termed Dex2, with enhanced expression in Dex-treated lungs, had 100% similarity with ras-recision gene ( rrg), also known as the lysyl oxidase ( LOX) gene that encodes lysyl oxidase. LOX gene is very highly conserved, with significant sequence similarity among mouse, rat, and human. Two other cDNAs, termed Dex1 and Dex4, were also identified as rrg, with 92 and 97% sequence similarity with the existing data bank sequence of rrg. LOX enzyme is known to downregulate p21 ras protein and play a central role in the maturation of collagen and elastin in the extracellular matrix as well as modulate the cytoskeletal elements. Thus LOX may be important in lung developmental processes involving epithelial-mesenchymal interactions.

Download Full-text

Analysis of structural MtrC models based on homology with the crystal structure of MtrF

Biochemical Society Transactions ◽

10.1042/bst20120132 ◽

2012 ◽

Vol 40 (6) ◽

pp. 1181-1185 ◽

Cited By ~ 18

Author(s):

Marcus J. Edwards ◽

James K. Fredrickson ◽

John M. Zachara ◽

David J. Richardson ◽

Thomas A. Clarke

Keyword(s):

Crystal Structure ◽

Outer Membrane ◽

Domain Structure ◽

Spectroscopic Data ◽

Sequence Similarity ◽

Shewanella Oneidensis ◽

Significant Sequence Similarity ◽

X Ray ◽

X Ray Crystallography ◽

Homology Models

The outer-membrane decahaem cytochrome MtrC is part of the transmembrane MtrCAB complex required for mineral respiration by Shewanella oneidensis. MtrC has significant sequence similarity to the paralogous decahaem cytochrome MtrF, which has been structurally solved through X-ray crystallography. This now allows for homology-based models of MtrC to be generated. The structure of these MtrC homology models contain ten bis-histidine-co-ordinated c-type haems arranged in a staggered cross through a four-domain structure. This model is consistent with current spectroscopic data and shows that the areas around haem 5 and haem 10, at the termini of an octahaem chain, are likely to have functions similar to those of the corresponding haems in MtrF. The electrostatic surfaces around haem 7, close to the β-barrels, are different in MtrF and MtrC, indicating that these haems may have different potentials and interact with substrates differently.

Download Full-text

Complete Nucleotide Sequence of Plasmid Rts1: Implications for Evolution of Large Plasmid Genomes

Journal of Bacteriology ◽

10.1128/jb.184.12.3194-3202.2002 ◽

2002 ◽

Vol 184 (12) ◽

pp. 3194-3202 ◽

Cited By ~ 37

Author(s):

Takahiro Murata ◽

Makoto Ohnishi ◽

Takeshi Ara ◽

Jun Kaneko ◽

Chang-Gyun Han ◽

...

Keyword(s):

Nucleotide Sequence ◽

Complete Nucleotide Sequence ◽

Sequence Similarity ◽

Open Reading Frames ◽

Conjugative Plasmid ◽

Proteus Vulgaris ◽

Significant Sequence Similarity ◽

Modular Evolution ◽

Broad Array ◽

Reading Frames

ABSTRACT Rts1, a large conjugative plasmid originally isolated from Proteus vulgaris, is a prototype for the IncT plasmids and exhibits pleiotropic thermosensitive phenotypes. Here we report the complete nucleotide sequence of Rts1. The genome is 217,182 bp in length and contains 300 potential open reading frames (ORFs). Among these, the products of 141 ORFs, including 9 previously identified genes, displayed significant sequence similarity to known proteins. The set of genes responsible for the conjugation function of Rts1 has been identified. A broad array of genes related to diverse processes of DNA metabolism were also identified. Of particular interest was the presence of tus-like genes that could be involved in replication termination. Inspection of the overall genome organization revealed that the Rts1 genome is composed of four large modules, providing an example of modular evolution of plasmid genomes.

Download Full-text

Evaluation of a Structural Model ofPseudomonas aeruginosa Outer Membrane Protein OprM, an Efflux Component Involved in Intrinsic Antibiotic Resistance

Journal of Bacteriology ◽

10.1128/jb.183.1.367-374.2001 ◽

2001 ◽

Vol 183 (1) ◽

pp. 367-374 ◽

Cited By ~ 46

Author(s):

Kendy K. Y. Wong ◽

Fiona S. L. Brinkman ◽

Roland S. Benz ◽

Robert E. W. Hancock

Keyword(s):

Crystal Structure ◽

Antibiotic Resistance ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Structural Model ◽

Sequence Similarity ◽

Homologous Protein ◽

Related Family ◽

Resistance Nodulation Division

ABSTRACT The outer membrane protein OprM of Pseudomonas aeruginosa is involved in intrinsic and mutational multiple-antibiotic resistance as part of two resistance-nodulation-division efflux systems. The crystal structure of TolC, a homologous protein in Escherichia coli, was recently published (V. Koronakis, A. Sharff, E. Koronakis, B. Luisl, and C. Hughes, Nature 405:914–919, 2000), demonstrating a distinctive architecture comprising outer membrane β-barrel and periplasmic helical-barrel structures, which assemble differently from the common β-barrel-only conformation of porins. Based on their sequence similarity, a similar content of α-helical and β-sheet structure determined by circular dichroism spectroscopy, and our observation that OprM, like TolC, reconstitutes channels in planar bilayer membranes, OprM and TolC were considered to be structurally homologous, and a model of OprM was constructed by threading its sequence to the TolC crystal structure. Residues thought to be important for the TolC structure were conserved in space in this OprM model. Analyses of deletion mutants and previously isolated insertion mutants of OprM in the context of this model allowed us to propose roles for different protein domains. Our data indicate that the helical barrel of the protein is critical for both the function and the integrity of the protein, while a C-terminal domain localized around the equatorial plane of this helical barrel is dispensable. Extracellular loops appear to play a lesser role in substrate specificity for this efflux protein compared to classical porins, and there appears to be a correlation between the change in antimicrobial activity for OprM mutants and the pore size. Our model and channel formation studies support the “iris” mechanism of action for TolC and permit us now to form more focused hypotheses about the functional domains of OprM and its related family of efflux proteins.

Download Full-text