ParA proteins of secondary genome elements cross-talk and regulate radioresistance through genome copy number reduction in Deinococcus radiodurans

Abstract Deinococcus radiodurans, an extremely radioresistant bacterium has a multipartite genome system and ploidy. Mechanisms underlying such types of bacterial genome maintenance and its role in extraordinary radioresistance are not known in this bacterium. Chromosome I (Chr I), chromosome II (Chr II) and megaplasmid (Mp) encode its own set of genome partitioning proteins. Here, we have characterized P-loop ATPases of Chr II (ParA2) and Mp (ParA3) and their roles in the maintenance of genome copies and extraordinary radioresistance. Purified ParA2 and ParA3 showed nearly similar polymerization kinetics and interaction patterns with DNA. Electron microscopic examination of purified proteins incubated with DNA showed polymerization on nicked circular dsDNA. ParA2 and ParA3 showed both homotypic and heterotypic interactions to each other, but not with ParA1 (ParA of Chr I). Similarly, ParA2 and ParA3 interacted with ParB2 and ParB3 but not with ParB1 in vivo. ParB2 and ParB3 interaction with cis-elements located upstream to the corresponding parAB operon was found to be sequence-specific. Unlike single mutant of parA2 and parA3, their double mutant (ΔparA2ΔParA3) affected copy number of cognate genome elements and resistance to γ-radiation as well as hydrogen peroxide in this bacterium. These results suggested that ParA2 and ParA3 are DNA-binding ATPases producing higher order polymers on DNA and are functionally redundant in the maintenance of secondary genome elements in D. radiodurans. The findings also suggest the involvement of secondary genome elements such as Chr II and Mp in the extraordinary radioresistance of D. radiodurans.

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PprA interacts with replication proteins and affects their physicochemical properties required for replication initiation in Deinococcus radiodurans

10.1101/2020.03.25.007906 ◽

2020 ◽

Cited By ~ 2

Author(s):

Ganesh K. Maurya ◽

Hari S. Misra

Keyword(s):

Atpase Activity ◽

Physicochemical Properties ◽

Deinococcus Radiodurans ◽

Specific Interaction ◽

Replication Initiation ◽

Gene Encoding ◽

Heterotypic Interactions ◽

Pleiotropic Functions ◽

Chromosome Ii ◽

Chromosome I

AbstractThe deletion mutant of pprA, a gene encoding pleiotropic functions in radioresistant bacterium Deinococcus radiodurans, showed an increased genomic content and ploidy in chromosome I and chromosome II. We identified oriC in chromosome I (oriCI) and demonstrated the sequence specific interaction of deinococcal DnaA (drDnaA) with oriCI. drDnaA and drDnaB showed ATPase activity while drDnaB catalyzed 5′→3′ dsDNA helicase activity. These proteins showed both homotypic and heterotypic interactions. The roles of C-terminal domain of drDnaA in oriCI binding and its stimulation of ATPase activity were demonstrated. Notably, PprA showed ~2 times higher affinity to drDnaA as compared to drDnaB and attenuated both homotypic and heterotypic interactions of these proteins. Interestingly, the ATPase activity of drDnaA but not drDnaB was inhibited in presence of PprA. These results suggested that PprA influences the physicochemical properties of drDnaA and drDnaB that are required for initiation of DNA replication at oriCI site in this bacterium.

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Scanning electron microscopic examination of enamel surface after fixed orthodontic treatment: In-vivo study

Srpski arhiv za celokupno lekarstvo ◽

10.2298/sarh1202022s ◽

2012 ◽

Vol 140 (1-2) ◽

pp. 22-28 ◽

Cited By ~ 9

Author(s):

Tijana Sessa ◽

Jelena Civovic ◽

Tina Pajevic ◽

Jovana Juloski ◽

Milos Beloica ◽

...

Keyword(s):

Electron Microscopic Examination ◽

Surface Damage ◽

Multivariate Regression Analysis ◽

Enamel Surface ◽

Orthodontic Appliances ◽

Electron Microscopic ◽

Fixed Orthodontic Appliances ◽

Tooth Surfaces ◽

Scanning Electron

Introduction. Therapy with fixed orthodontic appliances starts with bracket bonding and ends with debonding of brackets, leaving enamel surface varied. Objective. The aim of this pilot study was to examine enamel surface before and after debonding of orthodontic brackets by the use of scanning electron microscopy (SEM). Methods. Epoxy replicas of four patients? premolars indicated for therapy with fixed orthodontic appliances were made and brackets were bonded to their teeth with a different adhesives (Enlight, No-mix, Fuji Ortho LC and Heliosit Orthodontic) (n=4). Two months later, brackets on premolars were debonded and amounts of adhesive left on the tooth surfaces and the bracket bases were evaluated with the adhesive remnant index (ARI). After resin removal, epoxy replicas were made and the surface of premolars was evaluated with the enamel surface index (ESI). All replicas of premolars (n=32) were prepared for SEM examination and compared under different magnifications. Tooth damage was estimated based on correlation between ARItooth and ESI. Results. Pearson?s ?2 test showed no significant differences between ARItooth and ARIbracket of four materials used. Nonparametric correlations showed significant differences between ARItooth and ARIbracket, ESI and ARItooth, and between ESI and ARIbracket. Increasing of ARItooth is followed with the descent of ARIbracket and the ascent of ESI. Multivariate regression analysis showed a significant correlation between ESI and ARItooth. Conclusion. Most bond failures took place at enamel-adhesive interface. ARItooth was a predictor to enamel surface damage. The type of material did not affect enamel surface damage.

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Cell interactions of Listeria monocytogenes L forms and peritoneal exudative cells in rats

Canadian Journal of Microbiology ◽

10.1139/m93-154 ◽

1993 ◽

Vol 39 (11) ◽

pp. 1014-1021 ◽

Cited By ~ 5

Author(s):

L. Mihailova ◽

N. Markova ◽

T. Radoucheva ◽

D. Veljanov ◽

S. Radoevska

Keyword(s):

Listeria Monocytogenes ◽

Peritoneal Cavity ◽

Electron Microscopic Examination ◽

Lavage Fluid ◽

Host Cells ◽

Electron Microscopic ◽

Cellular Defense ◽

Scanning Electron Microscopic ◽

Scanning Electron

Listeria monocytogenes 4b and its forms without cell walls (L forms of a protoplastic type) were used to study in vivo interactions with host cells. Samples of peritoneal lavage fluid were obtained from rats intraperitoneally inoculated at intervals between 1 and 15 days after challenge, for scanning electron microscopic, bacteriological, biochemical, and cytometrical investigations. Scanning electron microscopic examination revealed continuous adhesion of L forms on the macrophage surface up to 15 days after inoculation. The persistence of the L forms within the peritoneal cavity was also shown bacteriologically at all sample times, while the parental bacterial forms were isolated from the peritoneal cavity up to 7 days after challenge. The total count of peritoneal exudative cells determined by automated flow peroxidase cytometry peaked on the 15th day in animals infected with parental forms, while in animals infected with L forms the peak was lower and the macrophage population was predominant. The glycolytic and acid phosphatase activity of peritoneal exudative cells was two times higher in rats infected with L forms as compared with rats infected with the L. monocytogenes parental forms on the 3rd day after challenge. An understanding of the nature of the interactions between L forms of L. monocytogenes and peritoneal exudative cells found in vivo could be used to establish the influence of L forms on host cellular defense mechanisms.Key words: Listeria monocytogenes, L forms, peritoneal exudative cells, electron microscopy.

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The binding of Pseudomonas aeruginosa outer membrane ghosts to human buccal epithelial cells

Canadian Journal of Microbiology ◽

10.1139/m86-032 ◽

1986 ◽

Vol 32 (2) ◽

pp. 160-166 ◽

Cited By ~ 4

Author(s):

P. Doig ◽

A. L. Franklin ◽

R. T. Irvin

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Outer Membrane ◽

Copy Number ◽

Electron Microscopic Examination ◽

Equilibrium Analysis ◽

Metabolic Effects ◽

Electron Microscopic ◽

Buccal Epithelial Cells ◽

Number Class

The binding of outer membrane (OM) ghosts derived from Pseudomonas aeruginosa strain 492c to human buccal epithelial cells (BECs) was examined. Electron microscopic examination of the binding of OM ghosts to BECs revealed direct OM ghost–BEC interaction. Equilibrium analysis of the binding of OM ghosts to trypsinized BECs employing the Langmuir adsorption isotherm indicated the number of binding sites (iV) to be 1.3 × 10−1 μg protein per BEC with an apparent association constant (Ka) of 3.4 × 10−2 mL/μg protein. The Langmuir analysis of binding of OM ghosts to untrypsinized BECs was complex, suggesting two possible classes of receptors, a high affinity–low copy number class (Ka, 1.8 × 10−2 mL/μg protein; N, 8.6 × 10−5 μg protein per BEC) and a low affinity – high copy number class(Afa, 3.7 × 10−3 mL/μg protein; N, 9.2 × 10−4 μg protein per BEC). Sugar inhibition studies incorporating D-galactose enhanced binding to each BEC type. N-Acetylneuraminic acid and N-acetyl-glucosamine both enhanced binding of OM ghosts to untrypsinized BECs, while inhibiting binding to trypsinized BECs. D-Arabinose inhibited binding to both BEC types. Binding of OM ghosts to both BEC types was greatly inhibited by D-fucose, while L-fucose only greatly inhibited binding to untrypsinized BECs. These sugar inhibition data demonstrated a difference in the binding of OM ghosts to trypsinized and untrypsinized BECs and possibly reveal the nature of the receptor(s), free of possible bacterial metabolic effects. These data indicated that OM ghosts from 492c appear to bind to BECs in a similar manner to the intact bacteria and represent a simple model system to study the adhesion of P. aeruginosa to BECs.

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The effects of intravitreally injected bevacizumab on the retina and retina pigment epithelium: experimental in-vivo electron microscopic study in intact versus vitrectomized eyes

Open Medicine ◽

10.2478/s11536-009-0120-8 ◽

2010 ◽

Vol 5 (6) ◽

pp. 745-751 ◽

Cited By ~ 1

Author(s):

Nilufer Kocak ◽

Candan Ozogul ◽

Suleyman Kaynak ◽

Ulker Sonmez ◽

Mehmet Zengin ◽

...

Keyword(s):

Pigment Epithelium ◽

Retinal Pigment ◽

Intravitreal Bevacizumab ◽

Electron Microscopic Examination ◽

Photoreceptor Cells ◽

Control Group ◽

Electron Microscopic ◽

Tissue Samples ◽

Electron Microscopes

AbstractTo analyze the retinal toxicity of bevacizumab at various doses both in vitrectomized and non-vitrectomized rabbit models. Twenty- eight rabbits were included in the study. Twenty- four rabbits were assigned to six groups, with 4 of the rabbits in the control group. The animals in Groups 1, 2 and 3 received bevacizumab at a dose of 0.3 mg, 0.5 mg and 1.5 mg /eye, respectively. The rabbits in Groups 4, 5 and 6 received intravitreal bevacizumab of 0.3 mg, 0.5 mg and 1.5mg/eye, respectively, after gas compression vitrectomy. Two weeks after the procedure, the rabbits were euthanized. Retina tissue samples were then obtained and examined with both light and electron microscopes. In Groups 1, 2 and 3 after bevacizumab injection, toxic degeneration in the photoreceptor and retinal pigment epithelium cells was observed via electron microscopic examination. The findings in Groups 4 and 5 were normal as compared to the control group. In Group 6, toxicity in the bipolar neurons and photoreceptor cells was noticed. Increased toxicity and retinal penetration were noticed in all administered doses of bevacizumab in the presence of vitreous. In addition, ocular toxicity occurred through the injection of the highest dose of bevacizumab after vitrectomy. It is possible that the bevacizumab dose and the, vitreous are as important as the drug half-life in the vitreous.

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Studies on Schistocephalus solidus

Parasitology ◽

10.1017/s0031182000068748 ◽

1965 ◽

Vol 55 (2) ◽

pp. 257-268 ◽

Cited By ~ 17

Author(s):

Morag L. O. McCaig ◽

C. Adrian Hopkins

Keyword(s):

Growth Rate ◽

Water Content ◽

Gasterosteus Aculeatus ◽

Technical Assistance ◽

Horse Serum ◽

Electron Microscopic Examination ◽

Dry Weight ◽

Electron Microscopic ◽

Schistocephalus Solidus

Schistocephalus plerocercoids in the weight range 2–200mg F.W. recovered from the perivisceral cavity of Gasterosteus aculeatus were cultured in various media. In a medium composed of 25% horse serum, 0·5% yeast extract, 0·65% glucose and Hanks's saline at pH 7·1, 21°C, 95% air +5% CO2, dry weight increases of up to 500% were recorded in 8 days. The specific growth rate of large plerocercoids was only one-tenth of the rate observed in small plerocercoids. A plerocercoid of double the weight of another had approximately half the specific growth rate.Worms after 8 days cultivation were found to have only slightly higher than normal glycogen and water content, and to be able to mature when heated to 40°C. However, the rate of growth slowed to zero by the 24th day in culture at 21°C. Electron microscopic examination showed a ‘deposit’ formed over the microvilli, thin at 8 days but dense after 21 days.The in vivo glycogen and water content of plerocercoids from 3–300 mg F.W. was determined. Glycogen rose from 24% in plerocercoids of 10mg F. W. to 50–55% in plerocercoids over 80mg F. W. The water content was found to mimic precisely this change, falling from 82% to a plateau of 67–69%.We wish to thank Professor Gareth Owen for permission to use the photograph shown in the Plate and for his help while using the electron microscope. It is also a pleasure to thank Miss Patricia Grant for her technical assistance.

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Overlapping oriC and centromere-like functions in secondary genome replicons determine their maintenance independent of chromosome I in Deinococcus radiodurans

10.1101/2020.03.28.013953 ◽

2020 ◽

Author(s):

Ganesh K Maurya ◽

Hari S. Misra

Keyword(s):

Radiation Resistance ◽

Copy Number ◽

Deinococcus Radiodurans ◽

Specific Interactions ◽

Wild Type ◽

Γ Radiation ◽

E Coli ◽

Copy Numbers ◽

Type Pattern ◽

Chromosome I

AbstractThe Deinococcus radiodurans multipartite genome system (MGS) consists of chromosome I (ChrI) and secondary genome elements; Chr II and megaplasmid (MP). The sequences upstream to parAB operons in Chr II (cisII) and MP (cisMP) helped an E. coli plasmid maintenance in D. radiodurans and showed sequence specific interactions with DnaA and ParBs. The cells devoid of cisII (ΔcisII) or cisMP (ΔcisMP) showed reduced γ radiation resistance and copy number of Chr II and MP. Fluorescent Reporter-Operator System (FROS) developed for ChrI, ChrII and MP in ΔcisII or ΔcisMP mutants showed no change in wild type pattern of Chr I localization. However, the relative copy numbers of Chr II and MP had reduced while anucleate cells had increased in mutants. These results suggested that cisII and cisMP elements contain both ori and centromere-like functions, and like other MGS bacteria, the Chr I and secondary genome are maintained independently in D. radiodurans.

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Yes-associated protein impacts adherens junction assembly through regulating actin cytoskeleton organization

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00027.2016 ◽

2016 ◽

Vol 311 (3) ◽

pp. G396-G411 ◽

Cited By ~ 21

Author(s):

Haibo Bai ◽

Qingfeng Zhu ◽

Alexandra Surcel ◽

Tianzhi Luo ◽

Yixin Ren ◽

...

Keyword(s):

Adherens Junction ◽

Electron Microscopic Examination ◽

Blood Stream ◽

In Vivo Studies ◽

Cell Junction ◽

Electron Microscopic ◽

Liver Size ◽

Cortical Tension ◽

E Cadherin

The Hippo pathway effector Yes-associated protein (YAP) regulates liver size by promoting cell proliferation and inhibiting apoptosis. However, recent in vivo studies suggest that YAP has important cellular functions other than controlling proliferation and apoptosis. Transgenic YAP expression in mouse hepatocytes results in severe jaundice. A possible explanation for the jaundice could be defects in adherens junctions that prevent bile from leaking into the blood stream. Indeed, immunostaining of E-cadherin and electron microscopic examination of bile canaliculi of Yap transgenic livers revealed abnormal adherens junction structures. Using primary hepatocytes from Yap transgenic livers and Yap knockout livers, we found that YAP antagonizes E-cadherin-mediated cell-cell junction assembly by regulating the cellular actin architecture, including its mechanical properties (elasticity and cortical tension). Mechanistically, we found that YAP promoted contractile actin structure formation by upregulating nonmuscle myosin light chain expression and cellular ATP generation. Thus, by modulating actomyosin organization, YAP may influence many actomyosin-dependent cellular characteristics, including adhesion, membrane protrusion, spreading, morphology, and cortical tension and elasticity, which in turn determine cell differentiation and tissue morphogenesis.

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Tight Junction–associated MARVEL Proteins MarvelD3, Tricellulin, and Occludin Have Distinct but Overlapping Functions

Molecular Biology of the Cell ◽

10.1091/mbc.e09-08-0734 ◽

2010 ◽

Vol 21 (7) ◽

pp. 1200-1213 ◽

Cited By ~ 188

Author(s):

David R. Raleigh ◽

Amanda M. Marchiando ◽

Yong Zhang ◽

Le Shen ◽

Hiroyuki Sasaki ◽

...

Keyword(s):

Tight Junction ◽

Protein Interactions ◽

Electron Microscopic Examination ◽

Electron Microscopic ◽

Junction Protein ◽

Tight Junction Regulation ◽

Sirna Knockdown ◽

Related Proteins

In vitro studies have demonstrated that occludin and tricellulin are important for tight junction barrier function, but in vivo data suggest that loss of these proteins can be overcome. The presence of a heretofore unknown, yet related, protein could explain these observations. Here, we report marvelD3, a novel tight junction protein that, like occludin and tricellulin, contains a conserved four-transmembrane MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain. Phylogenetic tree reconstruction; analysis of RNA and protein tissue distribution; immunofluorescent and electron microscopic examination of subcellular localization; characterization of intracellular trafficking, protein interactions, dynamic behavior, and siRNA knockdown effects; and description of remodeling after in vivo immune activation show that marvelD3, occludin, and tricellulin have distinct but overlapping functions at the tight junction. Although marvelD3 is able to partially compensate for occludin or tricellulin loss, it cannot fully restore function. We conclude that marvelD3, occludin, and tricellulin define the tight junction–associated MARVEL protein family. The data further suggest that these proteins are best considered as a group with both redundant and unique contributions to epithelial function and tight junction regulation.

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Distinct Centromere-Like parS Sites on the Two Chromosomes of Vibrio spp.

Journal of Bacteriology ◽

10.1128/jb.00416-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5314-5324 ◽

Cited By ~ 41

Author(s):

Yoshiharu Yamaichi ◽

Michael A. Fogel ◽

Sarah M. McLeod ◽

Monica P. Hui ◽

Matthew K. Waldor

Keyword(s):

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Focus Formation ◽

Origin Region ◽

Chromosome Ii ◽

Chromosome I ◽

Functional Partitioning ◽

Circular Chromosomes ◽

Derivatives Of

ABSTRACT Vibrio cholerae, the cause of cholera, has two circular chromosomes. The parAB genes on each V. cholerae chromosome act to control chromosome segregation in a replicon-specific fashion. The chromosome I (ChrI) parAB genes (parAB1) govern the localization of the origin region of ChrI, while the chromosome II (ChrII) parAB genes (parAB2) control the segregation of ChrII. In addition to ParA and ParB proteins, Par systems require ParB binding sites (parS). Here we identified the parS sites on both V. cholerae chromosomes. We found three clustered origin-proximal ParB1 binding parS1 sites on ChrI. Deletion of these three parS1 sites abrogated yellow fluorescent protein (YFP)-ParB1 focus formation in vivo and resulted in mislocalization of the ChrI origin region. However, as observed in a parA1 mutant, mislocalization of the ChrI origin region in the parS1 mutant did not compromise V. cholerae growth, suggesting that additional (non-Par-related) mechanisms may mediate the partitioning of ChrI. We also identified 10 ParB2 binding parS2 sites, which differed in sequence from parS1. Fluorescent derivatives of ParB1 and ParB2 formed foci only with the cognate parS sequence. parABS2 appears to form a functional partitioning system, as we found that parABS2 was sufficient to stabilize an ordinarily unstable plasmid in Escherichia coli. Most parS2 sites were located within 70 kb of the ChrII origin of replication, but one parS2 site was found in the terminus region of ChrI. In contrast, in other sequenced vibrio species, the distribution of parS1 and parS2 sites was entirely chromosome specific.

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