- Volume 150, Issue 9, 2004

An epidemic of tremor disease has been a serious problem in Chinese mitten crabs, Eriocheir sinensis, in China in recent years. The disease-causing agent was previously considered to be a rickettsia-like organism. Here, analysis of the 16S rRNA gene sequence, light and electron microscopy and cultivation in vitro were used to identify the agent. Sequence analysis of the 16S rRNA gene found it to have 98 % identity with that of Spiroplasma mirum. The agent was able to be passed through membrane filters with pores 220 nm in diameter and could be cultivated by inoculating the yolk sac of embryonated chicken eggs and M1D medium. Rotary motion and flexional movement were seen by light microscopy, and electron microscopy showed that the organism had a helical morphology and lacked a cell wall. The organism produced small colonies with a diameter of 40–50 μm after 17–25 days of incubation on solid M1D medium. The agent was found in blood cells, muscles, nerves and connective tissues of crabs inoculated with a filtrate of yolk sacs or with cultures grown in M1D medium, and it was similar in structure to those grown in eggs and cultivation broth. Disease was reproduced by experimental infection with the cultivated organisms. This study has demonstrated that the causative agent of tremor disease in the Chinese mitten crab is a member of the genus Spiroplasma. This is believed to be the first time a spiroplasma has been found in a crustacean. These findings are not only significant for studies on pathogenic spiroplasmas, but also have implications for studies of freshwater ecology.

Yeast cells of Candida albicans are induced by serum at 37 °C to produce germ tubes, the first step in a transition from yeast to hyphal growth. Previously, it has been shown that the active component is not serum albumin but is present in the dialysable fraction of serum. In this study, serum induction of germ-tube formation is shown to occur even in the presence of added exogenous nitrogen sources and is therefore not signalled by nitrogen derepression. The active component in serum was purified by ion-exchange, reverse-phase and size-exclusion chromatography from the dialysable fraction of serum and was identified by NMR to be d-glucose. Enzymic destruction of glucose, using glucose oxidase, demonstrated that d-glucose was the only active component in these fractions. Induction of germ-tube formation by d-glucose required a temperature of 37 °C and the pH optimum was between pH 7·0 and 8·0. d-Glucose induced germ-tube formation in a panel of clinical isolates of C. albicans. Although d-glucose is the major inducer in serum, a second non-dialysable, trichloroacetic acid precipitable inducer is also present. However, whereas either 1·4 % (v/v) serum or an equivalent concentration of d-glucose induced 50 % germ-tube formation, the non-dialysable component required a 10-fold higher concentration to induce 50 % germ-tube formation. Serum is, therefore, the most effective induction medium for germ-tube formation because it is buffered at about pH 8·5 and contains two distinct inducers (glucose and a non-dialysable component), both active at this pH.

Eubacterium pyruvativorans I-6T is a non-saccharolytic, amino-acid-fermenting anaerobe from the rumen, isolated by its ability to grow on pancreatic casein hydrolysate (PCH) as sole C source. This study investigated its metabolic properties and its likely ecological niche. Additional growth was supported by pyruvate, vinyl acetate, and, to a lesser extent, lactate and crotonate, and also by a mixture of amino acids (alanine, glycine, serine and threonine) predicted to be catabolized to pyruvate. No single amino acid supported growth, and peptides were required for growth on amino acids. Alanine, followed by leucine, serine and proline, were used most extensively during growth, but only alanine and asparate were extensively modified before incorporation. Growth on PCH, but not on pyruvate, was increased by the addition of acetate, propionate and butyrate. l-Lactate was fermented incompletely, mainly to acetate, but no lactate-C was incorporated. Propionate and butyrate were utilized during growth, forming valerate and caproate, respectively. Labelling experiments suggested a metabolic pattern where two C atoms of butyrate, valerate and caproate were derived from amino acids, with the others being formed from acetate, propionate and butyrate. The metabolic strategy of E. pyruvativorans therefore resembles that of Clostridium kluyveri, which ferments ethanol only when the reaction is coupled to acetate, propionate or butyrate utilization. The fermentative niche of E. pyruvativorans appears to be to scavenge amino acids, lactate and possibly other metabolites in order to generate ATP via acetate formation, using volatile fatty acid elongation with C2 units derived from other substrates to dispose of reducing equivalents.

Cells of the magnetotactic marine vibrio, strain MV-1, produce magnetite-containing magnetosomes when grown anaerobically or microaerobically. Stable, spontaneous, non-magnetotactic mutants were regularly observed when cells of MV-1 were cultured on solid media incubated under anaerobic or microaerobic conditions. Randomly amplified polymorphic DNA analysis showed that these mutants are not all genetically identical. Cellular iron content of one non-magnetotactic mutant strain, designated MV-1nm1, grown anaerobically, was ∼20- to 80-fold less than the iron content of wild-type (wt) MV-1 for the same iron concentrations, indicating that MV-1nm1 is deficient in some form of iron uptake. Comparative protein profiles of the two strains showed that MV-1nm1 did not produce several proteins produced by wt MV-1. To understand the potential roles of these proteins in iron transport better, one of these proteins was purified and characterized. This protein, a homodimer with an apparent subunit mass of about 19 kDa, was an iron-regulated, periplasmic protein (p19). Two potential ‘copper-handling’ motifs (MXM/MX2M) are present in the amino acid sequence of p19, and the native protein binds copper in a 1 : 1 ratio. The structural gene for p19, chpA (copper handling protein) and two other putative genes upstream of chpA were cloned and sequenced. These putative genes encode a protein similar to the iron permease, Ftr1, from the yeast Saccharomyces cerevisiae, and a ferredoxin-like protein of unknown function. A periplasmic, copper-containing, iron(II) oxidase was also purified from wt MV-1 and MV-1nm1. This enzyme, like p19, was regulated by media iron concentration and contained four copper atoms per molecule of enzyme. It is hypothesized that ChpA, the iron permease and the iron(II) oxidase might have analogous functions for the three components of the S. cerevisiae copper-dependent high-affinity iron uptake system (Ctr1, Ftr1 and Fet3, respectively), and that strain MV-1 may have a similar iron uptake system. However, iron(II) oxidase purified from both wt MV-1 and MV-1nm1 displayed comparable iron oxidase activities using O2 as the electron acceptor, indicating that ChpA does not supply the multi-copper iron(II) oxidase with copper.

Application of a promoter-trapping strategy to identify plant-inducible genes carried on an indigenous Pseudomonas plasmid, pQBR103, revealed the presence of a putative oligoribonuclease (orn) gene that encodes a highly conserved 3′ to 5′ exoribonuclease specific for small oligoribonucleotides. The deduced amino acid sequence of the plasmid-derived orn (orn pl) showed three conserved motifs characteristic of Orn from both prokaryotes and eukaryotes. Deletion of orn pl generated no observable phenotype, but inactivation of the chromosomal copy caused slow growth in Pseudomonas putida KT2440. This defect was fully restored by complementation with orn from Escherichia coli (orn E.coli). Plasmid-derived orn pl was capable of partially complementing the P. putida orn mutant, demonstrating functionality of orn pl. Phylogenetic analysis showed that plasmid-encoded Orn was distinct from Orn encoded by the chromosome of proteobacteria. A survey of orn pl from related Pseudomonas plasmids showed a sporadic distribution but no sequence diversity. These data suggest that the orn pl was acquired by pQBR103 in a single gene-transfer event: the donor is unknown, but is unlikely to be a member of the Proteobacteria.