Stenotrophomonas chelatiphaga sp. nov., a new aerobic EDTA-degrading bacterium

A new ethylenediaminetetraacetic acid (EDTA)-utilizing gammaproteobacterial strain LPM-5^T was isolated from municipal sewage sludge. Aerobic, gram-negative, motile rods multiply by binary fission. Neutrophilic and mesophilic, these are unable to grow in the presence of 3% NaCl (w/v), and unable to reduce nitrate to nitrite, and are oxidase and catalase positive, but lipase negative. The major cellular fatty acids are C_i15:0, C_a15:0 and C_16:1w7c. The dominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol (cardiolipin). The DNA G+C content is 68.3 mol% (T_m). The 16S rRNA gene sequence analysis showed a high similarity of strain LPM-5^T to the species members of genus Stenotrophomonas: S. maltophilia LMG 958^T (98.6%), S. rhizophila CCUG 47042^T (98.3%), S. koreensis TR6-01^T (97.6%) and S. acidaminiphila CIP 106456^T (97.0%). Based on these results and modest DNA–DNA hybridization levels with S. maltophilia VKM B-591^T (=LMG 958^T) (51%) and S. rhizophila CCUG 47042^T (52%), the isolate was classified as a novel species, Stenotrophomonas chelatiphaga sp. nov. (type strain LPM-5^T=VKM B-2486=DSM-21508=CCUG 57178).     

The cone-specific calcium sensor guanylate cyclase activating protein 4 from the zebrafish retina

Guanylate cyclase activating proteins (GCAPs) serve as neuronal Ca²⁺-sensor proteins in vertebrate rod and cone photoreceptor cells. Zebrafish express in their retina a variety of six different GCAPs, of which four are specific for cone cells. One isoform, zGCAP4, is mainly expressed in double cones and long single cones. We cloned the zGCAP4 gene, purified non-myristoylated and myristoylated forms of the protein after heterologous expression in Escherichia coli and studied its properties: zGCAP4 was a strong activator of membrane-bound guanylate cyclases from bovine and zebrafish retina, showing half-maximal activation at 520–570 nM free Ca²⁺ concentration. Furthermore, the Ca²⁺-sensitive activation properties of non-myristoylated and myristoylated zGCAP4 were similar, indicating no influence of the myristoyl moiety on Ca²⁺-sensor function. Myristoylated zGCAP4 showed low affinity for membranes and did not exhibit a Ca²⁺–myristoyl switch, a feature typical of some but not all neuronal Ca²⁺-sensor proteins. However, tryptophan fluorescence studies and Ca²⁺-dependent differences in protease accessibility revealed Ca²⁺-induced conformational changes in myristoylated and non-myristoylated zGCAP4, indicating the operation as a Ca²⁺ sensor. Thus, expression and biochemical properties of zGCAP4 are in agreement with its function as an efficient Ca²⁺-sensitive regulator of guanylate cyclase activity in cone vision.     

Substitution of methionine 63 or 83 in S100A9 and cysteine 42 in S100A8 abrogate the antifungal activities of S100A8/A9: potential role for oxidative regulation

S100A8 and S100A9 and their heterocomplex calprotectin (S100A8/A9) are abundant cytosolic constituents in human neutrophils previously shown to possess antifungal activity. This study was designed to investigate mechanisms involved in the modulation of the antifungal properties of S100A8/A9. S100A8, S100A9 and site-directed mutants of both proteins were tested for their antifungal effect against Candida albicans in microplate dilution assays. Whereas S100A8 alone did not inhibit fungal growth, S100A9 by itself had a moderate antifungal effect. Combining both proteins had the strongest effect. Supporting a potential role for oxidation in S100A8/A9, substitution of methionine 63 or 83 of S100A9 resulted in the loss of antifungal activity. Additionally, the substitution to alanine of cysteine 42 of S100A8 also caused a loss of S100A8's ability to enhance S100A9's antifungal effect. Overall, our data indicate that both S100A8 and S100A9 are required for their fully active antifungal effect and that oxidation regulates S100A8/A9 antifungal activity through mechanisms that remain to be elucidated and evaluated. Finally, together with our previous work describing the oxidation-sensitive anti-inflammatory effects of S100A8/A9, we propose that S100A8/A9 exerts an anti-inflammatory activity in healthy state and that conditions associated with oxidative stress activate the antifungal activity of S100A8/A9.     

Protein unfolding is an essential requirement for transport across the parasitophorous vacuolar membrane of Plasmodium falciparum

Plasmodium falciparum traffics a large number of proteins to its host cell, the mature human erythrocyte. How exactly these proteins gain access to the red blood cell is poorly understood. Here we have investigated the effect of protein folding on the transport of model substrate proteins to the host cell. We find that proteins must pass into the erythrocyte cytoplasm in an unfolded state. Our data strongly support the presence of a protein-conducing channel in the parasitophorous vacoular membrane, and additionally imply an important role for molecular chaperones in keeping parasite proteins in a 'translocation competent' state prior to membrane passage.     

N-(1-Carboxyethyl)alanine (alanopine), a new non-sugar component of lipopolysaccharides of Providencia and Proteus

O-Polysaccharides were released by mild acid degradation of lipopolysaccharides of Providencia alcalifaciens O35 and Proteus vulgaris O76 and were studied by 1D and 2D ¹H and ¹³C NMR spectroscopies, including HMBC and NOESY (ROESY) experiments. Both polysaccharides were found to contain N-(1-carboxyethyl)alanine (alanopine) that is N-linked to 4-amino-4,6-dideoxyglucose. Analysis of published data [Vinogradov, E.; Perry, M. B. Eur. J. Biochem.2000, 267, 2439-2446] shows that alanopine is present also on the same sugar in the lipopolysaccharide core of Proteus mirabilis O6 and O57.     

The Plasmid-Encoded Regulator Activates Factors Conferring Lysozyme Resistance on Enteropathogenic Escherichia coli Strains

We demonstrate that enhanced lysozyme resistance of enteropathogenic Escherichia coli requires the plasmid-encoded regulator, Per, and is mediated by factors outside the locus for enterocyte effacement. EspC, a Per-activated serine protease autotransporter protein, conferred enhanced resistance on nonpathogenic E. coli, and a second Per-regulated, espC-independent lysozyme resistance mechanism was identified.