Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria

Regulated covalent modifications of lipid A are implicated in virulence of pathogenic Gram-negative bacteria. The Salmonella typhimurium PhoP/PhoQ-activated gene pagP is required both for biosynthesis of hepta-acylated lipid A species containing palmitate and for resistance to cationic anti-microbial peptides. Palmitoylated lipid A can also function as an endotoxin antagonist. We now show that pagP and its Escherichia coli homolog (crcA) encode an unusual enzyme of lipid A biosynthesis localized in the outer membrane. PagP transfers a palmitate residue from the sn-1 position of a phospholipid to the N-linked hydroxymyristate on the proximal unit of lipid A (or its precursors). PagP bearing a C-terminal His(6)-tag accumulated in outer membranes during overproduction, was purified with full activity and was shown by cross-linking to behave as a homodimer. PagP is the first example of an outer membrane enzyme involved in lipid A biosynthesis. Additional pagP homologs are encoded in the genomes of YERSINIA: and BORDETELLA: species. PagP may provide an adaptive response toward both Mg(2+) limitation and host innate immune defenses.     

Acyl-acyl carrier protein specificity of UDP-GlcNAc acyltransferases from gram-negative bacteria: relationship to lipid A structure.

Lipid A, the component of lipopolysaccharide that provides the membrane anchor of the core and O-antigen sugars, is known to contain characteristic R-3-hydroxy fatty acids bound to the 2,2' (N-linked) and 3,3' (O-linked) positions of the glucosamine disaccharide in different gram-negative bacteria. The studies reported here show that it is the acyl-acyl carrier protein specificities of the enzymes UDP-GlcNAc-O-acyltransferase and UDP-3-O-[(R)-3-hydroxyacyl]-GlcN-N-acyltransferase that determine the nature of these fatty acids.     

A novel glycosphingolipid from gram-negative aquatic bacteria.

The chloroform-methanol extractable lipids of the Gram-negative fresh-water bacteria Arcocella aquatica NO-502 and Flectobacillus major FM were found to contain an unusual ninhydrin-positive glycolipid. It was purified by two-stage silica gel-column chromatography. By the use of IR and (1)H-NMR spectroscopy, mass spectrometry and chemical-degradation experiment, the lipid was established to be 1-O-monoglycosyl ceramide, the carbohydrate moiety of which was the alpha-pyranose-ring form of 7-desoxy-7-amino-D-manno-heptulosonic acid, or 1-hydroxycarbonyl-6-deoxy-6-amino-alpha-D-mannopyranose. The ceramide portion consisted mainly (by 95% in the A. aquatica glycolipid and 80% in the F. major glycolipid) of 2-N-(2'-D-hydroxy-13'-methyltetradecanoyl)-15-methyl-4(E)-hexad ecasph ingenine. The minor molecular species differed from the major one only in fatty acid structure. The glycolipid accounted for 8 and 11% of the total lipids extracted from A. aquatica NO-502 and F. major FM cells, respectively.     

Different lipid A types in lipopolysaccharides of phototrophic and related non-phototrophic bacteria

Lipid A analyses confirm not only the present taxa of the purple nonsulfur bacteria (formerly Rhodospirillaceae), but also phylogenetical relatedness of distinct phototrophic to distinct non-phototrophic bacteria, as was suggested by cataloguing 16S rRNA. For example, lipid A with ester-bound 3-OH-10:0 and the rare amide-linked 3-oxo-14:0 is common to the phototrophic Rhodobacter capsulatus and Rhodobacter sphaeroides and also to Paracoccus denitrificans and Thiobacillus versutus. 'Lipid ADAG' (lipid A with 2,3-diamino-D-glucose (DAG)) occurs in the phototrophic Rhodopseudomonas viridis and Rhodopseudomonas palustris and also in the related non-phototrophic species, e.g., Nitrobacter winogradskyi, Pseudomonas diminuta, or Thiobacillus ferrooxidans. The phylogenetically more coherent purple sulfur bacteria (Chromatiaceae) uniformly contain D-mannose in their phosphate-free lipid A. Among the green bacteria, only the Chlorobiaceae but not the likewise chlorosome-containing Chloroflexaceae contain lipopolysaccharide. Lipid ADAG from R. viridis is a structural analogue of a biosynthetic precursor (lipid X) of enterobacterial lipid A. Lipid A synthase from Salmonella accepts not only lipid X but also the synthetic di-N-acyl-2,3-diamino-D-glucose analogue as substrate (Raetz, C.R.H., unpublished results). More and more naturally occurring lipid A's with both, 2,3-diaminoglucose and glucosamine ('mixed' lipid A, with 2,3-diaminoglucose or glucosamine dominating) are being found. Newly recognized lipid A and lipid ADAG types might offer the possibility of differentially stimulating desired biological activities in animals without also having the undesired endotoxic activities. The non-toxic lipid A from Rhodopseudomonas viridis for example is able to stimulate prostaglandin secretion in peritoneal macrophages and can be used as an antagonist to the endotoxic shock caused by Salmonella lipopolysaccharide.     

A numerical taxonomic study of non-motile non-fermentative gram-negative bacteria from foods

A numerical taxonomic study using 155 unit characters was performed on 63 strains of Gram-negative non-motile non-fermentative bacteria isolated from proteinaceous foods. Similar bacteria from other sources and several Pseudomonas strains from meat were included for reference purposes. Three clusters were observed at 76% S which contained all the food strains. Cluster 1 was composed entirely of Acinetobacter strains including 17 isolated from foods that were provisionally identified with Acinetobacter johnsonii. Cluster 2 contained 22 strains identified as Psychrobacter immobilis, of which 20 were from food. Cluster 3 contained all the Pseudomonas reference strains and 26 non-motile strains isolated from meat. These were shown to be non-motile variants of Pseudomonas fragi. A simple identification scheme, based on five tests, is presented for the distinction of the three types of bacteria.     

Improved method of lipopolysaccharide isolation from gram-negative bacteria

A modification of the traditional method for lipoplysaccharide isolation from the cells of grammnegative bacteria was elaborated on the basis of extraction by the hot water solution of phenol (the method of Westfahl). To make the method simpler and to raise the yield of the product it was proposed to use the water-phenol extract without its division for plases. The nucleic acids are eliminated by precipitation from dialyzed extract at pH 3,2-3,4 achieved by addition of acetic acid. The comparative isolation of lipopolysaccharides by the classic and modified methods has confirmed the advantages of a new technique.     

Dissemination of lipid A deacylases (pagL) among gram-negative bacteria: identification of active-site histidine and serine residues

Lipopolysaccharide (LPS) is one of the main constituents of the Gram-negative bacterial outer membrane. It usually consists of a highly variable O-antigen, a less variable core oligosaccharide, and a highly conserved lipid moiety, designated lipid A. Several bacteria are capable of modifying their lipid A architecture in response to external stimuli. The outer membrane-localized lipid A 3-O-deacylase, encoded by the pagL gene of Salmonella enterica serovar Typhimurium, removes the fatty acyl chain from the 3 position of lipid A. Although a similar activity was reported in some other Gram-negative bacteria, the corresponding genes could not be identified. Here, we describe the presence of pagL homologs in a variety of Gram-negative bacteria. Although the overall sequence similarity is rather low, a conserved domain could be distinguished in the C-terminal region. The activity of the Pseudomonas aeruginosa and Bordetella bronchiseptica pagL homologs was confirmed upon expression in Escherichia coli, which resulted in the removal of an R-3-hydroxymyristoyl group from lipid A. Upon deacylation by PagL, E. coli lipid A underwent another modification, which was the result of the activity of the endogenous palmitoyl transferase PagP. Furthermore, we identified a conserved histidine-serine couple as active site residues, suggesting a catalytic mechanism similar to serine hydrolases. The biological function of PagL remains unclear. However, because PagL homologs were found in both pathogenic and nonpathogenic species, PagL-mediated deacylation of lipid A probably does not have a dedicated role in pathogenicity.     

Sensitivity of nonfermentative gram-negative bacteria to essential oils of different origin

The sensitivity of MDR (multi drug resistant) strains of Stenotrophomonas maltophilia, Acinetobacter baumannii, and Pseudomonas aeruginosa to essential oils and their individual components was studied; bacteriostatic and bactericidal concentrations of 16 substances were determined. Crimean rose oil exhibited the highest activity, with the minimum inhibitory concentration of 1.95 μL/ mL. Growth of bacterial batch cultures in the presence of subinhibitory concentrations of essential oils or their individual components was studied. Kinetic models analysis revealed positive correlations of growth characteristics of the studied bacteria the effects of essential oils (p-level < 0.05). Correlations between lag phase duration and the death of bacterial cultures and correlations between the concentration of linalool (a component of essential oils) and the degree of growth suppression for S. maltophilia and A. baumannii were revealed.     

Nature and location of amide-bound (R)-3-acyloxyacyl groups in lipid A of lipopolysaccharides from various gram-negative bacteria

It has previously been demonstrated [Eur. J. Biochem. 124, 191-198 (1982) and 137, 15-22 (1983)] that the lipid A component of Salmonella and Proteus lipopolysaccharides contains amide-linked (R)-3-acyloxyacyl residues. In the present study lipid A of other gram-negative bacteria was analysed for the presence of amide-bound 3-acyloxyacyl residues. It was found that such residues are constituents of all lipid A tested (Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeruginosa, Xanthomonas sinensis, Bacteroides fragilis, Vibrio cholerae, Fusobacterium nucleatum, Rhodospirillum tenue, Acinetobacter calcoaceticus, and Escherichia coli). Amide-linked (R)-3-acyloxyacyl groups, therefore, represent common and ubiquitous structural elements of bacterial lipid A. The composition of 3-acyloxyacyl groups differed considerably among different bacteria. As amide-bound (R)-3-hydroxy fatty acids straight chain and isobranched acyl groups with 10-17 carbon atoms were identified. The most frequently encountered fatty acids, substituting the 3-hydroxyl group of 3-hydroxy fatty acids, were nonhydroxylated straight chain and isobranched acyl residues with 10-17 carbon atoms as well as (S)-2-hydroxy fatty acids with 12 carbon atoms. In some cases, using laser desorption mass spectrometry, the distribution of 3-acyloxyacyl residues over the two available glucosamine amino groups of the lipid A backbone was investigated.     

Metabolism of o-phthalic acid by different gram-negative and gram-positive soil bacteria

Different bacteria, isolated from soil by the enrichment method, were able to grow on phthalic acid as carbon source. Protocatechuate was identified as intermediate in phthalate metabolism. All phthalategrown bacteria oxidized phthalate and protocatechuate rapidly without having a lag-period. Benzoic acid, terephthalic acid, protocatechuic acid, salicylic acid, di- and mono-butyl phthalate were also metabolized by some of the organisms, benzoic acid being degraded via catechol and terephthalic acid via protocatechuate as intermediate. All organisms tested cleaved protocatechuate or catechol, respectively, by the ortho fission, when grown on phthalate, terephthalate, or benzoate as carbon source. A characterization and tentative identification of the organisms is given.     

Rapid method for distinction of gram-negative from gram-positive bacteria

Summary A rapid method for distinction between gram-negative and grampositive bacteria by means of a 3% solution of potassium hydroxide is tested on 71 gram-positive and 55 gram-negative bacterial strains. The method proved reliable with one exception only, a Bacillus macerans strain. That strain was definately gram-negative on staining. Other Bacillus strains were proved gram-positive by the test, even those being gram-negative on staining.     

Directed isolation of gram-negative asporogenous bacteria from natural substrates

A method for selective isolation of gramnegative nonsporulating bacteria from soil substrates was developed. The method consists of plating out the substrates on a glucose-yeast medium with addition of benzylpenicillin and nalidixic acid. The isolation frequency of gramnegative nonsporulating bacteria increased under such conditions from 9-16 (control) to 80-100 per cent. At the same time the isolation frequency of gram-positive bacteria decreased from 88.5 to 9.6 per cent.     

Heterogeneity from increasing crop types: effect on carabid beetles

We investigated the effect of crop type heterogeneity on carabid beetles in intensively managed agricultural landscapes. We compared different crops and habitat type configurations and compositions. In addition we assessed the effect of artificial fragmentation by paved roads and water channels in an agricultural landscape. Crop type heterogeneity negatively affected carabid assemblages. Spatial dissection by artificial structures had an even more negative effect on carabid diversity. The nested structure of the carabid assemblages among crop types was caused by a negative response to heterogeneity in the agricultural landscape. Even though crop types in an intensive agricultural landscape increased, the landscape may tend to fragment habitat. Although artificially fragmented habitats had more unstable carabid assemblages, heterogeneity from different crop types has also been shown to be a characteristic of fragmented landscapes. To produce a positive relationship between biodiversity and heterogeneity in an agricultural landscape, farming intensity should be reduced to allow for the conversion of highly productive lands into more natural habitats.     

Protein secretion in gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria.

The xcp genes are required for protein secretion by Pseudomonas aeruginosa. They are involved in the second step of the process, i.e. the translocation across the outer membrane, after the exoproteins have reached the periplasm in a signal peptide dependent fashion. The nucleotide sequence of a 2.5 kb DNA fragment containing xcp genes showed at least two complete open reading frames, potentially encoding proteins with molecular weights of 41 and 19 kd. Products with these apparent molecular weights were identified after expression of the DNA fragment in vitro and in vivo. Subcloning and complementation experiments showed that both proteins are required for secretion. The two products are located in the inner membrane and share highly significant homologies with the PulL and PulM proteins which are required for the specific secretion of pullulanase in Klebsiella pneumoniae. These homologies reveal the existence of a common mechanism for protein secretion in Pseudomonas aeruginosa and Klebsiella pneumoniae.