Highly Dynamic Genomic Loci Drive the Synthesis of Two Types of Capsular or Secreted Polysaccharides within the Mycoplasma mycoides Cluster

Mycoplasmas of the Mycoplasma mycoides cluster are all ruminant pathogens. Mycoplasma mycoides subsp. mycoides is responsible for contagious bovine pleuropneumonia and is known to produce capsular polysaccharide (CPS) and exopolysaccharide (EPS). Previous studies have strongly suggested a role for Mycoplasma mycoides subsp. mycoides polysaccharides in pathogenicity. Mycoplasma mycoides subsp. mycoides-secreted EPS was recently characterized as a β(1→6)-galactofuranose homopolymer (galactan) identical to the capsular product. Here, we extended the characterization of secreted polysaccharides to all other members of the M. mycoides cluster: M. capricolum subsp. capripneumoniae, M. capricolum subsp. capricolum, M. leachii, and M. mycoides subsp. capri (including the LC and Capri serovars). Extracted EPS was characterized by nuclear magnetic resonance, resulting in the identification of a homopolymer of β(1→2)-glucopyranose (glucan) in M. capricolum subsp. capripneumoniae and M. leachii. Monoclonal antibodies specific for this glucan and for the Mycoplasma mycoides subsp. mycoides-secreted galactan were used to detect the two polysaccharides. While M. mycoides subsp. capri strains of serovar LC produced only capsular galactan, no polysaccharide could be detected in strains of serovar Capri. All strains of M. capricolum subsp. capripneumoniae and M. leachii produced glucan CPS and EPS, whereas glucan production and localization varied among M. capricolum subsp. capricolum strains. Genes associated with polysaccharide synthesis and forming a biosynthetic pathway were predicted in all cluster members. These genes were organized in clusters within two loci representing genetic variability hot spots. Phylogenetic analysis showed that some of these genes, notably galE and glf, were acquired via horizontal gene transfer. These findings call for a reassessment of the specificity of the serological tests based on mycoplasma polysaccharides.     

Genetic Structure and Diversity in Wild and Cultivated Populations of the Mangrove Oyster Crassostrea gasar from Southern Brazil

Marine Biotechnology - The mangrove oyster (Crassostrea gasar) is Brazil’s second most cultured species and presents a high potential for aquaculture. However, artificial selection in a...     

Biotin biosynthetic pathway in recombinant strains of Escherichia coli overexpressing bio genes: evidence for a limiting step upstream from KAPA

The effect of the overexpression of the bioABFCD operon on the biotin biosynthetic pathway was investigated in an Escherichia coli K12 bioR mutant with a chromosomal deletion for the biotin operon. When transformed with a multicopy number plasmid containing bioABFCD, this strain synthetized 10,000 times more biotin than a wild-type E. coli strain. In order to further increase biotin production, the bioA and bioB operons were subcloned into plasmids with stronger promoters and in some cases optimal ribosome binding sites. The new constructions led to the accumulation of large amounts of soluble Bio proteins (although not BioC) but did not improve biotin production. In all the constructed strains, BioA, BioD, and BioB activities were greatly amplified but these activitie did not correlate with the level of protein syntthesis. These strains accumulated only low levels of vitamers, auggesting that the major limiting step for higher biotin production occurs upstream from the first intermediate of the Bio pathway we assayed (7,keto-8-aminopelargonic acid). As BioC overproduction was shown to impair cell growth, we could not determine if this early step of pathway was limiting. Correspondence to: S. Lévy-Schil     

Isolation and analysis of a gene from the marine microalga Isochrysis galbana that encodes a lipase-like protein

Microalgae constitute a novel study area for characterising new lipolytic enzymes of biotechnological interest. A new gene from the microalga Isochrysis galbana has been isolated and a preliminary characterisation performed. The full-length cDNA contains 2,060 base pairs with an open reading frame of 1,374 nucleotides encoding a polypeptide of 457 amino acids with a predicted molecular mass of 49 kDa, and a theoretical pI of 5.65. The deduced protein includes highly conserved motifs found in α/β fold hydrolases, and shares some similarities with putative or known lipases. Sequence comparison indicated that the catalytic triad corresponds to residues Ser¹⁸⁸, Asp³⁰⁶ and His³⁹¹, with the nucleophilic residue Ser¹⁸⁸ positioned within the consensus G-X-S¹⁸⁸-X-G pentapeptide. Phylogenetic analyses established close relationships with fungal lipases and microalgal sequences.     

Determination of the major zinc fractions in human serum by ultrafiltration

In this paper we report a method for measuring ultrafiltrable zinc in human serum by electrothermal atomic absorption spectrophtometry. We show also that ultrafiltration permits to determine alpha-2 macroglobulin bound zinc and losely bound zinc if a strong zinc ligand (EDTA) is added to serum before ultrafiltration. This last fraction, after deduction of ultrafiltrable zinc, represents roughly all albumin bound zinc. In 20 controls we found that ultrafiltrable zinc amounted 0.311 μmol/L (S.D.=0.117 μmol/L), alpha-2 macroglobulin bound zinc 3.08 μmol/L (S.D.=0.221 μmol/L), and albumin bound zinc 12.11 μmol/L (S.D.=1.95 μmol/L). Our method needs only a small volume of serum, it is simple and rapid but also very accurate and reliable. The losely bound fraction is very dynamic and, representing the physiologically active part of serum zinc, it could be a good marker of zinc deficiency.     

In vitro regulation of the innervation pattern of quail muscle fibres by quail and mouse neurons

Myoblasts from rudiments of slow and fast muscle, anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) respectively, of 9-day-old quail embryos were cultured in vitro for a period of up to 60 days in order to give rise to well-differentiated muscle fibres. These fibres were innervated by neurons from either quail or mouse embryo spinal cord and their innervation pattern was examined by the visualization of acetylcholine receptors (ACh-R) and of acetylcholinesterase (ACh-E) activity at the neuromuscular contacts. In the culture system used, quail neurons always innervated muscle fibres at several sites and only when a fast-type activity was imposed on these neurons did a reduction in the number of the previously established neuromuscular contacts take place. In contrast, in the muscle fibres innervated by mouse neurons, a spontaneous reduction in the number of the previously established neuromuscular contacts occurred but this spontaneous reduction depended upon the level of differentiation reached by the muscle fibres in vitro. In the cultures of muscle fibres previously innervated by mouse neurons, the addition of quail neurons did not provoke any modification in the initial innervation pattern, and no quail ACh-R cluster was observed. In contrast, in the muscle fibres previously innervated by quail neurons, the mouse neurons contacted these fibres, resulting in a decrease in the number of quail ACh-R clusters. These results emphasize the part played by neurons in the establishment of the innervation pattern when muscle fibres have reached a high level of differentiation. In vitro, the slow and fast characteristics of the muscle fibres do not influence this pattern.