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DNA-Binding HU proteins are essential for the maintenance of genomic DNA supercoiling and compaction in prokaryotic cells and are promising pharmacological targets for the design of new antibacterial agents. The virtual screening for low-molecular-weight compounds capable of specifically interacting with the DNA-recognition loop of the HU protein from the mycoplasma Spiroplasma melliferum was performed. The ability of the initially selected ligands to form stable complexes with the protein target was assessed by molecular dynamics simulation. One compound, which forms an unstable complex, was eliminated by means of a combination of computational methods, resulting in a decrease in the number of compounds that will pass to the experimental test phase. This approach can be used to solve a wide range of problems related to the search for and validation of low-molecular-weight inhibitors specific for a particular protein target.

We report here the draft genome sequence of a Dyella -like bacterium (DLB) isolated from Hyalesthes obsoletus , the insect vector of the uncultivable mollicute bacterium “ Candidatus Phytoplasma.” This isolate inhibits Spiroplasma melliferum , a cultivable mollicute. The draft genome of DLB consists of 4,196,214 bp, with a 68.6% G+C content, and 3,757 genes were predicted.

In a post on May 12, 2008, Elio asked where ribosomes are located in bacterial cells. According to a 2006 paper by Ortiz et al. (1.usa.gov/1NzRno7), “the ribosomes are evenly distributed throughout the cytosol” and “no distinct nucleoid territory was observed.” This prompts one to also ask, “Where art thou, O nucleoid?” Do their observations indeed indicate that, in the small cells of Spiroplasma melliferum ( Figure E ; volume ~0.02 µm3), the ribosomes (number ~1000) and DNA (genome size 1.46 Mbp) co-mingle and that a phase separation between cytoplasm and nucleoid is absent? In larger, fast-growing E. coli cells ( Figure B ; volume 1–3 µm3), the nucleoid is clearly visible in living cells as a low-density compartment, as was already documented by Mason and Powelson in 1956. However, distinct nucleoids are difficult to see in smaller, slow-growing E. coli cells. In the small Caulobacter, nucleoids have even been reported to be absent (R.B. Jensen, 2006) (1.usa.gov/1jQ96ht). The existence of a discrete DNA phase was calculated by Odijk (1998) (1.usa.gov/1RdvvzJ), taking into account the excluded volume interactions between DNA and the soluble proteins as present in small E. coli cells ( Figure A ; volume 0.46 µm3; genome size 4.6 Mbp). To me it seems unlikely that the physical laws that predict the visible phase separation in the larger cells would not hold for the smaller cells, as well.

HU proteins belong to the nucleoid-associated proteins (NAPs) that are involved in vital processes such as DNA compaction and reparation, gene transcriptionetc.No data are available on the structures of HU proteins from mycoplasmas. To this end, the HU protein from the parasitic mycoplasmaSpiroplasma melliferumKC3 was cloned, overexpressed inEscherichia coliand purified to homogeneity. Prismatic crystals of the protein were obtained by the vapour-diffusion technique at 4°C. The crystals diffracted to 1.36 Å resolution (the best resolution ever obtained for a HU protein). The diffraction data were indexed in space groupC2 and the structure of the protein was solved by the molecular-replacement method with one monomer per asymmetric unit.

Two species of Spiroplasma (Mollicutes) bacteria were isolated from and described as pathogens of the European honey bee, Apis mellifera, ~30 years ago but recent information on them is lacking despite global concern to understand bee population declines. Here we provide a comprehensive survey for the prevalence of these two Spiroplasma species in current populations of honey bees using improved molecular diagnostic techniques to assay multiyear colony samples from North America (U.S.A.) and South America (Brazil). Significant annual and seasonal fluctuations of Spiroplasma apis and Spiroplasma melliferum prevalence in colonies from the U.S.A. (n = 616) and Brazil (n = 139) occurred during surveys from 2011 through 2013. Overall, 33% of U.S.A. colonies and 54% of Brazil colonies were infected by Spiroplasma spp., where S. melliferum predominated over S. apis in both countries (25% vs. 14% and 44% vs. 38% frequency, respectively). Colonies were co-infected by both species more frequently than expected in both countries and at a much higher rate in Brazil (52%) compared to the U.S.A. (16.5%). U.S.A. samples showed that both species were prevalent not only during spring, as expected from prior research, but also during other seasons. These findings demonstrate that the model of honey bee spiroplasmas as springtime-restricted pathogens needs to be broadened and their role as occasional pathogens considered in current contexts.

Anti-bacterial activities were compared for two series of voltage-dependent pore-formers: (i) alamethicin (Alm) and its synthetic analogs (Alm-dUL) where α-amino-isobutyric acid residues (Aibs) were replaced by leucines and selected key residues substituted and (ii) homologous voltage sensors of the electric eel sodium channel (repeats S4L45 (III) and S4L45 (IV)). Spiroplasma melliferum, a bacterium related to the mycoplasmas, was used as a target cell. The data show that with respect to growth inhibition, cell deformation and plasma membrane depolarization, the highest efficient peptide remained natural Alm although the minimal inhibitory concentrations of its Leu analogs were within the same range as the parent molecule, except for Alm-dUL P14A. Thus, as for the pore-forming activity observed in artificial membranes and for the toxicity towards mammalian cells, proline-14 proved to be a critical residue for the anti-bacterial activity of alamethicin. Regarding the sodium voltage sensors, their anti-bacterial efficiency was at least 10 times lower although they promoted spiroplasma cell agglutination. The anti-bacterial activities of the peptides were correlated with their pore-forming properties, especially with the apparent and mean number of monomers per conducting aggregate (〈N〉) when both peptide families were considered and, secondly, with mean open times (τo) within each family. This suggests that although they may form ‘raft-like’ structures, the mechanism underlying anti-bacterial activity of Alm and its active analogs, as well as the S4L45 voltage sensors with the S. melliferum plasma membrane, is predominantly through pore-formation according to the ‘barrel-stave’ mechanism.

Spiroplasma infections in honey bees have been reported in Europe and Asia quite recently, due to intensive studies on the epidemiology of honey bee diseases. The situation in the US is less well analyzed. Here, we examined the honey bee colonies in Beltsville, MD, where Spiroplasmamelliferum was originally reported and found S. melliferum infection in honey bees. Our data showed high variation of S. melliferum infection in honey bees with a peak prevalence in May during the course of one-year study period. The colony prevalence increased from 5% in February to 68% in May and then decreased to 25% in June and 22% in July. Despite that pathogenicity of spiroplasmas in honey bee colonies remains to be determined, our results indicated that spiroplasma infections need to be included for the consideration of the impacts on honey bee health.

Spiroplasma apis and Spiroplasma melliferum are known as honey bee pathogens and are detected by unspecific methodologies like culturing or dark field microscopy. We developed a multiplex PCR being able to differentiate between both species and detect the genus Spiroplasma. This PCR can directly be used on culture samples or on DNA extracted bees. By PCR on cultured samples we were able to identify S. apis in Bombus pratorum and S. melliferum in Bombus pascuorum.

Diese Arbeit dokumentiert die Ergebnisse der Untersuchungen des bakteriellen Cytoskeletts von Spiroplasma melliferum, die durch Anwendung der Kryo-Elektronentomographie und biochemischer Methoden gewonnen wurden. Die Elektronentomographie ist eine Abbildungstechnik, die eine drei-dimensionale (3-D) Abbildung pleomorpher, biologischer Objekte, wie z.B. Zellen, Organellen, Viren oder Proteine, mit einer Auflösung von derzeit 4-6 Nanometern ermöglicht. Durch Kombination mit Kryo-Präparationsmethoden, d.h. der Einbettung der Probe in einen amorphen Eisfilm, kann das Objekt in einem quasi-nativen Zustand untersucht werden. Diese Technik eignet sich daher bestens, um die Struktur und räumliche Anordnung abbildbarer makromolekularer Aggregate eines Organismus aufzuklären. Ein limitierender Faktor für die Elektronentomographie ist die Größe des zu untersuchenden Objekts, welches eine Dicke von weniger als einem Mikrometer aufweisen sollte. Aus diesem Grund wurde für diese Arbeit das helikale Bakterium S. melliferum (Klasse Mollicutes) ausgewählt, welches einen Durchmesser von etwa 200 nm hat und nach bisherigen Untersuchungen ein Cytoskelett aufweisen soll. Um die Existenz dieses Cytoskeletts zu überprüfen und einen detaillierten Einblick in dessen Struktur, Funktionsweise und Zusammensetzung zu erhalten, wurden in dieser Arbeit Elektronentomogramme aufgenommen, computergestützte Bewegungsmodelle aufgestellt und biochemische Untersuchungen der Cytoskelett-Filamente durchgeführt. Die elektronentomographische Datenaufzeichnung ganzer Zellen erfolgte mit Hilfe eines 300 kV Transmissions-Elektronenmikroskops, welches mit einer Feldemissionskathode und einem Energiefilter ausgestattet ist. Der Energiefilter ermöglicht die Abtrennung inelastisch gestreuter Elektronen, die sich durch Kontrasterniedrigung negativ auf die Bildaufnahmen auswirken würden. Dennoch weisen die Tomogramme aufgrund der niedrigen Elektronendosis, die für vitrifizierte, biologische Proben unabdingbar ist, ein geringes Signal-zu-Rausch-Verhältnis auf. In den aufgenommenen Tomogrammen konnte ein Cytoskelett nachgewiesen werden, welches aus drei Bändern aus parallel angeordneten Filamenten unterschiedlicher Dicke besteht. Zwischen zwei Bändern aus je fünf dicken (11 nm) Filamenten liegt ein Band aus neun dünnen (4 nm) Filamenten. Diese Filamentbänder erstrecken sich helikal, dicht unterhalb der Zellmembran, über die ganze Länge der Zelle. Durch Western-Blot-Analyse und Sequenzierung konnten desweiteren zwei Filament-bildende Proteine nachgewiesen werden, das sogenannte „Fibril“-Protein und das Aktin-ähnliche Protein MreB. Isolierungs- und Aufreinigungsversuche gelangen nur für das „Fibril“-Protein, für das elektronenmikroskopisch gezeigt werden konnte, daß es etwa 10 nm dicke Filamente bildet. Es wird angenommen, daß das Protein MreB die dünnen Filamente bildet, welche jedoch sehr instabil sind und daher bei der Isolierung zerfallen. Die aufgestellten Bewegungsmodelle ließen zudem eine Erklärung verschiedener Bewegungsmechanismen dieser schwimmenden Bakterien zu. Demnach ermöglichen Längen- oder Abstandsänderungen der beiden äuβeren Filamentbänder mittels des dazwischen liegenden Bandes eine Fortbewegung dieser Bakterien. This work documents the results of the investigations of the bacterial cytoskeleton of Spiroplasma melliferum, which have been obtained using cryo-electron tomography and biochemical methods.Electron tomography is an imaging technique that is presently capable of providing three-dimensional (3-D) images of pleomorphic, biological objects such as cells, organelles, viruses or proteins at a resolution of 4-6 nanometers. In combination with cryo-preparation techniques, which means embedding of the specimen in amorphous ice, the object can be investigated in a close-to-native state. Therefore, this technique is most suitable to reveal the structure and spatial arrangement of illustratable macromolecular aggregates of an organism. A limiting factor for electron tomography is the size of the investigated object, which should have a thickness of less than one micrometer. For this reason the helical bacterium S. melliferum (class Mollicutes), which has a diameter of about 200 nm and has a cytoskeleton according to previous investigations, was chosen for this work. Apart from the recording of electron tomograms, computer-based motility models were built up and biochemical investigations of the cytoskeletal filaments were performed in this work in order to examine the existence of this cytoskeleton and to get a detailed insight into its structure, function and composition. The electron tomographic data acquisition of whole cells was performed using a 300 kV transmission electron microscope, equipped with a field emission gun and an energy filter. The energy filter enables the separation of inelastically scattered electrons, which would have a negative effect on image acquisition due to contrast reduction. Nevertheless, the tomograms show a low signal-to-noise ratio due to the low electron dose, which is necessary for vitrified, biological specimen. In the acquired tomograms a cytoskeleton could be identified which comprises three ribbons of parallel filaments of different thickness. A ribbon of nine thin (4 nm) filaments is sandwiched between two ribbons of five thick (11 nm) filaments each. These ribbons run helically through the whole cell just underneath the cell membrane. Additionally, two filament-forming proteins, the so-called fibril protein and the actin-like protein MreB, could be identified using western blot and sequence analysis. Isolation and purification experiments only succeeded for the fibril protein, which was shown to build 10 nm wide filaments by electron microscopy. It is assumed that MreB forms the thin filaments, which, however, are very instable and therefore depolymerize during the isolation procedure. The proposed motility models explained different motility modes of these swimming bacteria. Thus, motility of these bacteria can be achieved by length or distance changes of the two outer ribbons via the ribbon lying in between.

A chemically defined medium (CC-494M) was used to study the effect of inorganic salts on the growth of three spiroplasmas representing three distinct serogroups: Spiroplasma melliferum (AS 576), Spiroplasma floricola (23–6), and SR 3 spiroplasma. KH2PO4 or NaH2PO4∙H2O was required. KH2PO4 supported faster growth and higher yield for spiroplasmas than NaH2PO4∙H2O. The optimal concentration of KH2PO4 for S. melliferum, S. floricola, and SR 3 spiroplasma was 0.5, 0.5, and 0.25 mM, respectively, whereas that of NaH2PO4∙H2O was 0.5, 0.05, and 2.5 mM. When supplemented with NaH2PO4∙H2O, KCl promoted growth comparable with that obtained from KH2PO4-supplemented medium and RbCl supported limited growth, whereas NaCl, LiCl, CsCl, CaCl2, and MgSO4∙7H2O were inhibitory. Spiroplasma growth decreased as the concentration of LiCl and CsCl increased. At 200 mM LiCl totally inhibited the growth of S. melliferum and SR 3 spiroplasma, whereas limited growth was obtained for S. floricola. CsCl at 150 mM totally inhibited the growth of S. floricola, while limited growth was observed for S. melliferum and SR 3 spiroplasma. RbCl supported limited growth when supplemented with NaH2PO4∙H2O, whereas it was inhibitory when added in KH2PO4-supplemented medium. All the 10 potassium salts (KCH2COOH, KBr, K2CO3, KHSO4, KCl, KOH, Kl, KNO3, KH2PO4, and K2HPO4) at concentrations of 20 mM promoted growth when supplemented with NaH2PO4∙H2O.