Discovery and characterization of a new class of O-linking oligosaccharyltransferases from the Moraxellaceae family.

Bacterial protein glycosylation is commonly mediated by oligosaccharyltransferases (OTases) that transfer oligosaccharides en bloc from preassembled lipid-linked precursors to acceptor proteins. Natively, O-linking OTases usually transfer a single repeat unit of the O-antigen or capsular polysaccharide to the side chains of serine or threonine on acceptor proteins. Three major families of bacterial O-linking OTases have been described: PglL, PglS, and TfpO. TfpO is limited to transferring short oligosaccharides both in its native context and when heterologously expressed in glycoengineered Escherichia coli. On the other hand, PglL and PglS can transfer long-chain polysaccharides when expressed in glycoengineered E. coli. Herein, we describe the discovery and functional characterization of a novel family of bacterial O-linking OTases termed TfpM from Moraxellaceae bacteria. TfpM proteins are similar in size and sequence to TfpO enzymes but can transfer long-chain polysaccharides to acceptor proteins. Phylogenetic analyses demonstrate that TfpM proteins cluster in distinct clades from known bacterial OTases. Using a representative TfpM enzyme from Moraxella osloensis, we determined that TfpM glycosylates a C-terminal threonine of its cognate pilin-like protein and identified the minimal sequon required for glycosylation. We further demonstrated that TfpM has broad substrate tolerance and can transfer diverse glycans including those with glucose, galactose, or 2-N-acetyl sugars at the reducing end. Last, we find that a TfpM-derived bioconjugate is immunogenic and elicits serotype-specific polysaccharide IgG responses in mice. The glycan substrate promiscuity of TfpM and identification of the minimal TfpM sequon renders this enzyme a valuable additional tool for expanding the glycoengineering toolbox.

Analysis of bacterial FAMEs using gas chromatography - vacuum ultraviolet spectroscopy for the identification and discrimination of bacteria.

The identification of microorganisms is very important in different fields and alternative methods are necessary for a rapid and simple identification. The use of fatty acids for bacterial identification is gaining attention as phenotypic characteristics are reflective of the genotype and are more easily analyzed. In this work, gas chromatography-vacuum ultraviolet spectroscopy (GC-VUV) was used to determine bacteria fatty acid methyl esters (FAMEs), to identify and discriminate different environmental bacteria based on their fatty acid profile. Microorganisms were grown in agar and their fatty acids extracted, saponified, and esterified before analysis. Unique FAME profiles were obtained for each microorganism mainly composed of branched, cyclopropane, hydroxy, saturated, and unsaturated fatty acid methyl esters. S. maltophilia showed a higher diversity of fatty acids while Bacillus species showed higher complexity in terms of branched-chain FAMEs, with several iso and anteiso forms. 12 different bacteria genera and 15 species were successfully differentiated based on their fatty acid profiles after performing PCA and cluster analysis. Some difficult to differentiate species, such as Bacillus sp., which are genetically very similar, were differentiated with the developed method.

Ultrastructural analysis of the rugose cell envelope of a member of the Pasteurellaceae family.

Bacterial membranes serve as selective environmental barriers and contain determinants required for bacterial colonization and survival. Cell envelopes of Gram-negative bacteria consist of an outer and an inner membrane separated by a periplasmic space. Most Gram-negative bacteria display a smooth outer surface (e.g., Enterobacteriaceae), whereas members of the Pasteurellaceae and Moraxellaceae families show convoluted surfaces. Aggregatibacter actinomycetemcomitans, an oral pathogen representative of the Pasteurellaceae family, displays a convoluted membrane morphology. This phenotype is associated with the presence of morphogenesis protein C (MorC). Inactivation of the morC gene results in a smooth membrane appearance when visualized by two-dimensional (2D) electron microscopy. In this study, 3D electron microscopy and atomic force microscopy of whole-mount bacterial preparations as well as 3D electron microscopy of ultrathin sections of high-pressure frozen and freeze-substituted specimens were used to characterize the membranes of both wild-type and morC mutant strains of A. actinomycetemcomitans. Our results show that the mutant strain contains fewer convolutions than the wild-type bacterium, which exhibits a higher curvature of the outer membrane and a periplasmic space with 2-fold larger volume/area ratio than the mutant bacterium. The inner membrane of both strains has a smooth appearance and shows connections with the outer membrane, as revealed by visualization and segmentation of 3D tomograms. The present studies and the availability of genetically modified organisms with altered outer membrane morphology make A. actinomycetemcomitans a model organism for examining membrane remodeling and its implications in antibiotic resistance and virulence in the Pasteurellaceae and Moraxellaceae bacterial families.

[Taxonomical status of the psychrotolerant Antarctic microorganisms].

The aerobic chemoorganotrophic bacteria, dominating in soils and phytocenosis of the Antarctic Region, on combination of morphological and biochemical properties belong to several taxons of Bacteria domain. Gram-negative strains 3189, 3415 (fam. Halomonadaceae, Halomonas sp.) and 3088, 3468, 3469 (fam. Moraxellaceae, Psychrobacter sp.) belong to phylum Proteobacteria, to class Gammaproteobacteria. Gram-negative strains 3294 3392 (Rhizobiales, fam. Methylobacteriaceae, Methylobacterium sp.) relate to class Alphaproteobacteria of this phylum. Gram-positive strains 3179, 3275, 3470, 3471 (fam. Microbacteriaceae, Cryobacterium sp.), 3054, 3058, 3411 (fam. Corynebacteriaceae, Corynebacterium sp.) and 3194, 3398 (fam. Micrococcaceae, Micrococcus sp.) relate to phylum Actinobacteria, class Actinobacteria. Thus, the psychrophilic and psychrotolerant Antarctic bacteria (aerobic chemoorganotrophic) isolated from phytocenosis and soils of polar region are characterized by wide taxonomic variety.

Genus Enhydrobacter Staley et al. 1987 should be recognized as a member of the family Rhodospirillaceae within the class Alphaproteobacteria.

The genus Enhydrobacter, first reported as a member of the family Vibrionaceae, has been placed in the family Moraxellaceae, but as a genus incertae sedis in Bergey's Manual of Systematic Bacteriology 2nd edition. During our taxonomic investigation of Enhydrobacter-like organisms, we observed that the 16S rRNA sequences of E. aerosaccus-type strain versions NCIMB 12535(T) , ATCC 27094( T) and CCUG 58314(T) were very different from the accessible data (accession no. AJ550856). Phylogenetic analysis of our 16S rRNA sequence data revealed that these organisms were located within the family Rhodospirillaceae. The genera Inquilinus, Oceanibaculum, Skermanella and Nisaea were closely related (sequence similarities were 88.3~87.0%), but Enhydrobacter could be distinguished from these genera by growth characteristics, fatty acid profiles (C(19:0) cyclo ω8c; 38.4% C(18:1) ω7c; 32.2%, and C(16:0) ; 8.9% were major components), in being non-flagellated, and differing in enzymatic activities, including trypsin and ß-glucosidase. From these data, we conclude that the genus Enhydrobacter should be recognized as an independent genus of the family Rhodospirillaceae within the class Alphaproteobacteria.

[Novel halotolerant bacterium from cryopeg in permafrost: description of Psychrobacter muriicola sp. nov].

A novel halotolerant psychrotrophic gram-negative bacterium, strain 2pS, was isolated from lenses of water brine in Arctic permafrost (cryopeg). The optimal growth of the new strain was observed at 16-18 degrees C; the maximal and minimal growth temperatures were 37 degrees C and -2 degrees C, respectively. The pH growth range was 5.8 to 8.5 (optimum 6.5-7.5) and the range of medium salinity was 0 to 100 g/l (optimum 3-8 g/l NaCl). The strain 2pS did not produce acid from carbohydrates and utilized acetate, yeast extract, pyruvate, glutarate, fumarate, caproate, heptanoate, butyrate, malate, DL-lactate, citrate, L-proline, L-tyrosine, butanol, and dulcitol as the sole carbon and energy sources. The major fatty acids of the cell wall at optimal growth temperature were C18:1(omega 7) and C18:1(omega 9). The G + C DNA content was 46.0 mol.%. Phylogenetic analysis of the 16S rRNA gene sequences showed that the studied strain was the closest (97% similarity) to Psychrobacter nivimaris DSM 16093T, a halotolerant psychrotrophic bacterium isolated from the Arctic sea's ice. Genotypic and phenotypic differences of the new bacterium from closely related species lead to the conclusion that strain 2pS belongs to a novel species of the genus Psychrobacter: Psychrobacter muriicola sp. nov.

Alkanindiges illinoisensis gen. nov., sp. nov., an obligately hydrocarbonoclastic, aerobic squalane-degrading bacterium isolated from oilfield soils.

An alkane-degrading bacterium, designated GTI MVAB Hex1(T), was isolated from chronically crude oil-contaminated soil from an oilfield in southern Illinois. The isolate grew very weakly or not at all in minimal or rich media without hydrocarbons. Straight-chain aliphatic hydrocarbons, such as hexadecane and heptadecane, greatly stimulated growth; shorter-chain (