Alvin Fox

Monomeric and Polymeric Gram-Negative Peptidoglycan but Not Purified LPS Stimulate the Drosophila IMD Pathway

Insects depend solely upon innate immune responses to survive infection. These responses include the activation of extracellular protease cascades, leading to melanization and clotting, and intracellular signal transduction pathways inducing antimicrobial peptide gene expression. In Drosophila, the IMD pathway is required for antimicrobial gene expression in response to gram-negative bacteria. The exact molecular component(s) from these bacteria that activate the IMD pathway remain controversial. We found that highly purified LPS did not stimulate the IMD pathway. However, lipid A, the active portion of LPS in mammals, activated melanization in the silkworm Bombyx morii. On the other hand, the IMD pathway was remarkably sensitive to polymeric and monomeric gram-negative peptidoglycan. Recognition of peptidoglycan required the stem-peptide sequence specific to gram-negative peptidoglycan and the receptor PGRP-LC. Recognition of monomeric and polymeric peptidoglycan required different PGRP-LC splice isoforms, while lipid A recognition required an unidentified soluble factor in the hemolymph of Bombyx morii.

Utility of 16S–23S rRNA spacer region methodology: how similar are interspace regions within a genome and between strains for closely related organisms?

Abstract Use of 16S–23S interspace region sequence variability, as a relatively new method, is becoming an important supplement to 16S rRNA sequencing as the standard for differentiating bacterial species. If interspace regions are as variable within a genome as between strains for closely related organisms, this limits the utility of the technique. Strains W23 and 168 represent two distinct genetic clusters within the species Bacillus subtilis . B. atrophaeus var. niger was selected as a member of a group of species closely related to B. subtilis . Comparison of the 10 rDNA operons, available from Genbank, for B. subtilis 168 shows three distinct types of interspace (ISR) regions. Two of the ten 16S–23S ISRs contain the sequences for isoleucine and alanine tRNA and are identical in sequence. The remaining eight ISRs lack tRNA sequences and have two distinct sizes. Variability among non-tRNA operons ranged from 97–100%. Counting the tRNA insert as one change, variability between tRNA and non-tRNA containing sequences ranged from 95.3–97%. The sequences of equivalent 16S–23S ribosomal operon interspace regions (ISRs) are highly conserved between W23 and 168 (99.9–100%). Thus the sequence differences between strains 168 and W23 are less than between multiple operons within 168. However, the sequence of an ISR from B. atrophaeus var. niger is quite distinct from any of the ISRs found in B. subtilis (range 88.2–91.6%). These observations are consistent with the previous suggestion that B . atrophaeus is distinct genetically from the B . subtilis sub-groups represented by W23 and 168 respectively. This is the first study to make sequence comparisons at the genome, strain and species level for the rRNA interspace region. Considerations of this issue will be important in using ISR methodology to differentiate other closely related bacterial species.

Modifications in the alditol acetate method for analysis of muramic acid and other neutral and amino sugars by capillary gas chromatography-mass spectrometry with selected ion monitoring.

Two alditol acetate methods for the gas chromatographic (GC) analysis of neutral and amino sugars were compared. Following sodium borohydride reduction, one method uses methylimidazole as an acetylation catalyst without prior removal of water or borate salts and the other method uses sodium acetate after removal of borate and water. Depending on the acetylation conditions, muramic acid produced different derivatives. With methylimidazole, reliable derivatization of muramic acid was not possible, although other sugars derivatized reliably. With sodium acetate, all sugars tested were reproducibly derivatized. The utility of the sodium acetate method is shown by the trace GC-mass spectrometric analysis of muramic acid and rhamnose derived from bacterial peptidoglycan-polysaccharide complexes in mammalian tissue.

Discrimination among the B. Cereus group, in comparison to B. Subtilis, by structural carbohydrate profiles and ribosomal RNA spacer region PCR

Summary The B. cereus group (B. anthracis, B. thuringiensis and B. cereus) and B. subtilis were physiologically, molecularly and chemically characterized. Within the B. cereus group, strains grew anaerobically and were not lysozyme susceptible. B. anthracis strains were non-hemolytic unlike B. cereus or B. thuringiensis. Only B. thuringiensis produced parasporal bodies. The 16S / 23S rRNA spacer region was amplified giving 3 major products. The PCR products found in strains of B. subtilis were approximately 270, 400, and 430 nucleotides, with the corresponding bands in the B. cereus group at 250, 430, and 480. These patterns allowed B. subtilis to be differentiated from the other 3 species. Using gas chromatography-mass spectrometry, sugar profiles of vegetative cells were indistinguishable for B. cereus and B. thuringiensis. B. anthracis contained high levels of galactose which generally distinguished it from B. cereus/B. thuringiensis, while B. subtilis was distinguished from the B. cereus group by low mannosamine levels. Spore profiles differed from vegetative profiles in all 4 species. Like vegetative profiles, spore profiles were distinctive for B. cereus/B. thuringiensis, B. anthracis, and B. subtilis. B. cereus and B. thuringiensis spores both contained rhamnose, fucose, 2-O-methyl rhamnose and 3-O-methyl rhamnose, unlike B. anthracis spores which contained only rhamnose and 3-O-methyl rhamnose. B. subtilis strains were heterogeneous with some resembling B. anthracis and others B. cereus/B. thuringiensis, although B. subtilis strains typically contained quinovose. The B. cereus group can be easily distinguished from B. subtilis, however, differentiation within this group has always been problematic. Using carbohydrate profiling, B. anthracis is readily distinguished from B. cereus(B. thuringiensis. Additionally, changes in carbohydrate composition between vegetative cells and spores occurs in the B. cereus group and B. subtilis.

Mass Spectrometry for Species or Strain Identification after Culture or without Culture: Past, Present, and Future

This minireview discusses the use of mass spectrometry in biomarker discovery, the current utility of these markers for bacterial identification after culture, and the potential for non-culture-based diagnosis of infectious diseases. The bases of these thoughts are the independent revolutions that

Restriction fragment length polymorphism of rRNA operons for discrimination and intergenic spacer sequences for cataloging of Bacillus subtilis sub-groups

Restriction fragment length polymorphism of rRNA operons (RFLP) and 16S-23S rRNA intergenic region (ISR) sequences of Bacillus subtilis subsp. subtilis, B. subtilis subsp. spizizenii, and B. atrophaeus were compared. ISR sequences of the B. subtilis subspecies were extremely similar (W23 versus 168 rrn H, J, G,W; 96.8%; rrn D, E; 98.4%; rrnB; 97.9%) and, therefore, not useful for their differentiation. However, RFLP of rRNA operons of the B. subtilis subspecies were distinct in terms of numbers and organization within the genome (e.g. the 168 sub-group generally contained 8.3- and 8.0-kb fragments absent in the W23 sub-group). The more distantly related B. atrophaeus was distinct from both B. subtilis subspecies in terms of ISR sequence and rRNA operon number and organization. RFLP of rRNA operons discriminates the two sub-groups of Bacillus subtilis that are indistinguishable by ISR sequence. However, ISR sequence defines the relatedness of B. subtilis to other species (e.g. B. atrophaeus) within the genus Bacillus.

Carbohydrates and glycoproteins of Bacillus anthracis and related bacilli: targets for biodetection.

The spore is the form released in a bioterrorism attack. There is a real need for definition of new targets for Bacillus anthracis that might be incorporated into emerging biodetection technologies. Particularly of interest are macromolecules found in B. anthracis that are (1) spore-specific, (2) readily accessible on the spore surface and (3) distinct from those present in related organisms. One of the few biochemical methods to identify the spores of B. anthracis is based on the presence of rhamnose and 3-O-methyl rhamnose as determined by gas chromatography-mass spectrometry. Related organisms additionally contain 2-O-methyl rhamnose and fucose. Carbohydrates and glycoproteins of the B. cereus group of organisms and the related B. subilis group are reviewed here. It is hypothesized that the spore-specific carbohydrate is a component of the newly described glycoprotein of the exosporium of B. anthracis. Further work to define the protein and carbohydrate components of the glycoprotein of B. anthracis could be highly useful in developing new technologies for rapid biodetection.

Investigation of the Concentration of Bacteria and Their Cell Envelope Components in Indoor Air in Two Elementary Schools

ABSTRACT Bacterial cell envelope components are widely distributed in airborne dust, where they act as inflammatory agents causing respiratory symptoms. Measurements of these agents and other environmental factors are assessed in two elementary schools in a southeastern city in the United States. Muramic acid (MA) was used as a marker for bacterial peptidoglycan (PG), and 3-hydroxy fatty acids (3-OH FAs) were used as markers for Gram-negative bacterial lipopolysaccharide (LPS). Culturable bacteria were collected using an Andersen sampler with three different culture

Increased levels of bacterial markers and CO2 in occupied school rooms

Our group previously demonstrated that carbon dioxide (CO2) levels in heavily occupied schools correlate with the levels of airborne bacterial markers. Since CO2 is derived from the room occupants, it was hypothesized that in schools, bacterial markers may be primarily increased in indoor air because of the presence of children; directly from skin microflora or indirectly, by stirring up dust from carpets and other sources. The purpose of this project was to test the hypothesis. Muramic acid (Mur) is found in almost all bacteria whereas 3-hydroxy fatty acids (3-OH FAs) are found only in Gram-negative bacteria. Thus Mur and 3-OH FA serve as markers to assess bacterial levels in indoor air (pmol m(-3)). In our previous school studies, airborne dust was collected only from occupied rooms. However, in the present study, additional dust samples were collected from the same rooms each weekend when unoccupied. Samples were also collected from outside air. The levels of dust, Mur and C10:0, C12:0, C14:0, and C16:0 3-OH FAs were each much higher (range 5-50 fold) in occupied rooms than in unoccupied school rooms. Levels in outdoor air were much lower than that of indoor air from occupied classrooms and higher than the levels in the same rooms when unoccupied. The mean CO2 concentrations were around 420 parts per million (ppm) in unoccupied rooms and outside air; and they ranged from 1017 to 1736 ppm in occupied rooms, regularly exceeding 800-1000 ppm, which are the maximum levels indicative of adequate indoor ventilation. This indicates that the children were responsible for the increased levels of bacterial markers. However, the concentration of Mur in dust was also 6 fold higher in occupied rooms (115.5 versus 18.2 pmole mg(-1)). This further suggests that airborne dust present in occupied and unoccupied rooms is quite distinct. In conclusion in unoccupied rooms, the dust was of environmental origin but the children were the primary source in occupied rooms.

Capillary gas chromatographic analysis of alditol acetates of neutral and amino sugars in bacterial cell walls

Several improvements in the preparation of alditol acetates of neutral and amino sugars and in the preparation of glass capillary columns for their separation are described. Modifications in sample preparation permitted the simultaneous processing of multiple samples and eliminated extraneous background peaks. Efficient and inert columns were tailor-made for the separation of alditol acetates of neutral and amino sugars by leaching glass capillaries with aqueous hydrochloric acid and dynamically coating with SP-2330.