Cell viability in magnetotactic multicellular prokaryotes

A magnetotactic multicellular prokaryote (MMP) is an assembly of bacterial cells organized side by side in a hollow sphere in which each cell faces both the external environment and an internal acellular compartment in the center of the multicellular organism. MMPs swim as a unit propelled by the coordinated beating of the many flagella on the external surface of each cell. At every stage of its life cycle, MMPs are multicellular. Initially, a spherical MMP grows by enlarging the size of each of its cells, which then divide. Later, the cells separate into two identical spheres. Swimming individual cells of MMPs have never been observed. Here we have used fluorescent dyes and electron microscopy to study the viability of individual MMP cells. When separated from the MMP, the cells cease to move and they no longer respond to magnetic fields. Viability tests indicated that, although several cells could separate from a MMP before completely losing their motility and viability, all of the separated cells were dead. Our data show that the high level of cellular organization in MMPs is essential for their motility, magnetotactic behavior, and viability. [Int Microbiol 2006; 9(4):267-272

Salinity dependence of the distribution of multicellular magnetotactic prokaryotes in a hypersaline lagoon

Candidatus Magnetoglobus multicellularis is an unusual magnetotactic multicellular microorganism composed of a highly organized assemblage of gram-negative bacterial cells. In this work, the salinity dependence of Ca. M. multicellularis and its abundance in the hypersaline Araruama Lagoon, Brazil were studied. Viability experiments showed that Ca. M. multicellularis died in salinities >55‰ and < 40‰. Low salinities were also observed to modify the cellular assemblage. In microcosms prepared with different salinities, the microorganism grew better at intermediate salinities whereas in high or low salinities, the size of the population did not increase over time. The concentrations of Ca. M. multicellularis in the lagoon were related to salinity; sites with lower and higher salinities than the lagoon average contained less Ca. M. multicellularis. These results demonstrate the influence of salinity on the survival and distribution of Ca. M. multicellularis in the environment. In sediments, the abundance of Ca. M. multicellularis ranged from 0 to 103 microorganisms/ml, which represented 0.001% of the counts of total bacteria. The ability of Ca. M. multicellularis to accumulate iron and sulfur in high numbers of magnetosomes (up to 905 per microorganism) suggests that its impact on the sequestration of these elements (0.1% for biogenic bacterial iron) is not proportional to its abundance in the lagoon. [Int Microbiol 2009; 12(3):193-201

Greigite magnetosome membrane ultrastructure in ‘Candidatus Magnetoglobus multicellularis’

The ultrastructure of the greigite magnetosome membrane in the multicellular magnetotactic bacteria ‘Candidatus Magnetoglobus multicellularis’ was studied. Each cell contains 80 membrane-enclosed iron-sulfide magnetosomes. Cytochemistry methods showed that the magnetosomes are enveloped by a structure whose staining pattern and dimensions are similar to those of the cytoplasmic membrane, indicating that the magnetosome membrane likely originates from the cytoplasmic membrane. Freeze-fracture showed intramembrane particles in the vesicles surrounding each magnetosome. Observations of cell membrane invaginations, the trilaminar membrane structure of immature magnetosomes, and empty vesicles together suggested that greigite magnetosome formation begins by invagination of the cell membrane, as has been proposed for magnetite magnetosomes

The oxygen sensor MgFnr controls magnetite biomineralization by regulation of denitrification in Magnetospirillum gryphiswaldense

Background: Magnetotactic bacteria are capable of synthesizing magnetosomes only under oxygen-limited conditions. However, the mechanism of the aerobic repression on magnetite biomineralization has remained unknown. In Escherichia coli and other bacteria, Fnr (fumarate and nitrate reduction regulator) proteins are known to be involved in controlling the switch between microaerobic and aerobic metabolism. Here, we report on an Fnr-like protein (MgFnr) and its role in growth metabolism and magnetite biomineralization in the alphaproteobacterium Magnetospirillum gryphiswaldense. Results: Deletion of Mgfnr not only resulted in decreased N-2 production due to reduced N2O reductase activity, but also impaired magnetite biomineralization under microaerobic conditions in the presence of nitrate. Overexpression of MgFnr in the WT also caused the synthesis of smaller magnetite particles under anaerobic and microaerobic conditions in the presence of nitrate. These data suggest that proper expression of MgFnr is required for WT-like magnetosome synthesis, which is regulated by oxygen. Analyses of transcriptional gusA reporter fusions revealed that besides showing similar properties to Fnr proteins reported in other bacteria, MgFnr is involved in the repression of the expression of denitrification genes nor and nosZ under aerobic conditions, possibly owing to several unique amino acid residues specific to MTB-Fnr. Conclusions: We have identified and thoroughly characterized the first regulatory protein mediating denitrification growth and magnetite biomineralization in response to different oxygen conditions in a magnetotactic bacterium. Our findings reveal that the global oxygen regulator MgFnr is a genuine O-2 sensor. It is involved in controlling expression of denitrification genes and thereby plays an indirect role in maintaining proper redox conditions required for magnetite biomineralization

Differential cellular FGF-2 upregulation in the rat facial nucleus following axotomy, functional electrical stimulation and corticosterone: a possible therapeutic target to Bell's palsy

<p>Abstract</p> <p>Background</p> <p>The etiology of Bell's palsy can vary but anterograde axonal degeneration may delay spontaneous functional recovery leading the necessity of therapeutic interventions. Corticotherapy and/or complementary rehabilitation interventions have been employed. Thus the natural history of the disease reports to a neurotrophic resistance of adult facial motoneurons leading a favorable evolution however the related molecular mechanisms that might be therapeutically addressed in the resistant cases are not known. Fibroblast growth factor-2 (FGF-2) pathway signaling is a potential candidate for therapeutic development because its role on wound repair and autocrine/paracrine trophic mechanisms in the lesioned nervous system.</p> <p>Methods</p> <p>Adult rats received unilateral facial nerve crush, transection with amputation of nerve branches, or sham operation. Other group of unlesioned rats received a daily functional electrical stimulation in the levator labii superioris muscle (1 mA, 30 Hz, square wave) or systemic corticosterone (10 mgkg^-1). Animals were sacrificed seven days later.</p> <p>Results</p> <p>Crush and transection lesions promoted no changes in the number of neurons but increased the neurofilament in the neuronal neuropil of axotomized facial nuclei. Axotomy also elevated the number of GFAP astrocytes (143% after crush; 277% after transection) and nuclear FGF-2 (57% after transection) in astrocytes (confirmed by two-color immunoperoxidase) in the ipsilateral facial nucleus. Image analysis reveled that a seven days functional electrical stimulation or corticosterone led to elevations of FGF-2 in the cytoplasm of neurons and in the nucleus of reactive astrocytes, respectively, without astrocytic reaction.</p> <p>Conclusion</p> <p>FGF-2 may exert paracrine/autocrine trophic actions in the facial nucleus and may be relevant as a therapeutic target to Bell's palsy.</p

Genome-Wide Identification of Essential and Auxiliary Gene Sets for Magnetosome Biosynthesis in Magnetospirillum gryphiswaldense

Magnetotactic bacteria (MTB) stand out by their ability to manufacture membrane-enclosed magnetic organelles, so-called magnetosomes. Previously, it has been assumed that a genomic region of approximately 100 kbp, the magnetosome island (MAI), harbors all genetic determinants required for this intricate biosynthesis process. Recent evidence, however, argues for the involvement of additional auxiliary genes that have not been identified yet. In the present study, we set out to delineate the full gene complement required for magnetosome production in the alphaproteobacterium Magnetospirillum gryphiswaldense using a systematic genome-wide transposon mutagenesis approach. By an optimized procedure, a Tn5 insertion library of 80,000 clones was generated and screened, yielding close to 200 insertants with mild to severe impairment of magnetosome biosynthesis. Approximately 50% of all Tn5 insertion sites mapped within the MAI, mostly leading to a nonmagnetic phenotype. In contrast, in the majority of weakly magnetic Tn5 insertion mutants, genes outside the MAI were affected, which typically caused lower numbers of magnetite crystals with partly aberrant morphology, occasionally combined with deviant intracellular localization. While some of the Tn5-struck genes outside the MAI belong to pathways that have been linked to magnetosome formation before (e.g., aerobic and anaerobic respiration), the majority of affected genes are involved in so far unsuspected cellular processes, such as sulfate assimilation, oxidative protein folding, and cytochrome c maturation, or are altogether of unknown function. We also found that signal transduction and redox functions are enriched in the set of Tn5 hits outside the MAI, suggesting that such processes are particularly important in support of magnetosome biosynthesis. IMPORTANCE Magnetospirillum gryphiswaldense is one of the few tractable model magnetotactic bacteria (MTB) for studying magnetosome biomineralization. So far, knowledge on the genetic determinants of this complex process has been mainly gathered using reverse genetics and candidate approaches. In contrast, nontargeted forward genetics studies are lacking, since application of such techniques in MTB has been complicated for a number of technical reasons. Here, we report on the first comprehensive transposon mutagenesis study in MTB, aiming at systematic identification of auxiliary genes necessary to support magnetosome formation in addition to key genes harbored in the magnetosome island (MAI). Our work considerably extends the candidate set of novel subsidiary determinants and shows that the full gene complement underlying magnetosome biosynthesis is larger than assumed. In particular, we were able to define certain cellular pathways as specifically important for magnetosome formation that have not been implicated in this process so far

Unusual source of tachycardia in an adolescent

Mahaim fiber tachycardia is an uncommon cause of palpitations among the pediatric population. This case report describes an adolescent female who presented with recurrent episodes of tachycardia with chest pain and dizziness. Her ECG showed tachycardia with wide QRS complexes and left bundle branch block pattern. Repeat ECG after adenosine treatment revealed sinus rhythm with persistence of the left bundle branch block pattern. Metoprolol was started however she continued to have episodes of sustained tachycardia

Multidrug-Resistant Nontuberculous Mycobacteria Isolated from Cystic Fibrosis Patients

Worldwide, nontuberculous mycobacteria (NTM) have become emergent pathogens of pulmonary infections in cystic fibrosis (CF) patients, with an estimated prevalence ranging from 5 to 20%. This work investigated the presence of NTM in sputum samples of 129 CF patients (2 to 18 years old) submitted to longitudinal clinical supervision at a regional reference center in Rio de Janeiro, Brazil. From June 2009 to March 2012, 36 NTM isolates recovered from 10 (7.75%) out of 129 children were obtained. Molecular identification of NTM was performed by using PCR restriction analysis targeting the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene, and susceptibility tests were performed that followed Clinical and Laboratory Standards Institute recommendations. for evaluating the genotypic diversity, pulsed-field gel electrophoresis (PFGE) and/or enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) was performed. the species identified were Mycobacterium abscessus subsp. bolletii (n = 24), M. abscessus subsp. abscessus (n = 6), Mycobacterium fortuitum (n = 3), Mycobacterium marseillense (n = 2), and Mycobacterium timonense (n = 1). Most of the isolates presented resistance to five or more of the antimicrobials tested. Typing profiles were mainly patient specific. the PFGE profiles indicated the presence of two clonal groups for M. abscessus subsp. abscessus and five clonal groups for M. abscesssus subsp. bolletii, with just one clone detected in two patients. Given the observed multidrug resistance patterns and the possibility of transmission between patients, we suggest the implementation of continuous and routine investigation of NTM infection or colonization in CF patients, including countries with a high burden of tuberculosis disease.Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)PDTIS-FIOCRUZUniv Fed Rio de Janeiro, Programa Posgrad Clin Med, Hosp Univ Clementino Fraga Filho, Rio de Janeiro, BrazilUniv Fed Rio Grande do Sul, Programa Posgrad Ciencias Med, Porto Alegre, RS, BrazilUniv Fed Rio de Janeiro, Fac Ciencias Med, Dept Microbiol Imunol & Parasitol, Rio de Janeiro, BrazilInst Fernandes Figueira Fiocruz, Rio de Janeiro, BrazilUniv Estado Rio de Janeiro, Hosp Univ Pedro Ernesto, Rio de Janeiro, BrazilUniv Fed Rio de Janeiro, Inst Microbiol, BR-21941 Rio de Janeiro, BrazilFundacao Oswaldo Cruz, Inst Pesquisa Evandro Chagas, Rio de Janeiro, BrazilInst Doencas Torax, Rio de Janeiro, BrazilJohns Hopkins Univ, Baltimore, MD USAUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv Fed Fluminense, Inst Biomed, Niteroi, RJ, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFAPERJ: 103.225/2011FAPERJ: 103.287/2011FAPERJ: 110.272/2010FAPERJ: 110.761/2010FAPERJ: 111.497/2008CNPq: 476536/2012-0CNPq: 473444/2010-0CNPq: 567037/2008-8Web of Scienc

Intracellular mGluR5 plays a critical role in neuropathic pain

Spinal mGluR5 is a key mediator of neuroplasticity underlying persistent pain. Although brain mGluR5 is localized on cell surface and intracellular membranes, neither the presence nor physiological role of spinal intracellular mGluR5 is established. Here we show that in spinal dorsal horn neurons >80% of mGluR5 is intracellular, of which ∼60% is located on nuclear membranes, where activation leads to sustained Ca(2+) responses. Nerve injury inducing nociceptive hypersensitivity also increases the expression of nuclear mGluR5 and receptor-mediated phosphorylated-ERK1/2, Arc/Arg3.1 and c-fos. Spinal blockade of intracellular mGluR5 reduces neuropathic pain behaviours and signalling molecules, whereas blockade of cell-surface mGluR5 has little effect. Decreasing intracellular glutamate via blocking EAAT-3, mimics the effects of intracellular mGluR5 antagonism. These findings show a direct link between an intracellular GPCR and behavioural expression in vivo. Blockade of intracellular mGluR5 represents a new strategy for the development of effective therapies for persistent pain