Increased uptake of putrescine in the rhizosphere inhibits competitive root colonization by Pseudomonas fluorescens strain WCS365.

Sequence analysis of the chromosomal Tn5lacZ flanking regions of the Pseudomonas fluorescens WCS365 competitive root colonization mutant PCL1206 showed that the Tn5lacZ is inserted between genes homologous to bioA and potF. The latter gene is the first gene of the potF1F2GHI operon, which codes for a putrescine transport system in Escherichia coli. The position of the Tn5lacZ suggests an effect on the expression of the pot operon. A mutation in the potF1 gene as constructed in PCL1270, however, had no effect on competitive root colonization. The rate of uptake of [1,4-14C]putrescine by cells of mutant PCL1206 appeared to be increased, whereas cells of strain PCL1270 were strongly impaired in the uptake of putrescine. Dansylation of tomato root exudate and subsequent thin-layer chromatography showed the presence of a component with the same Rf value as dansyl-putrescine, which was identified as dansyl-putrescine by mass spectrometric analyses. Other polyamines such as spermine and spermidine were not detected in the root exudate. Growth of mutant strains, either alone or in competition with the wild type, was tested in media containing putrescine, spermine, or spermidine as the sole nitrogen source. The results show that mutant PCL1206 is strongly impaired in growth on putrescine and slightly impaired on spermine and spermidine. The presence of the polyamines had a similar effect on the growth rate of strain PCL1270 in the presence of putrescine but a less severe effect in the presence of spermine and spermidine. We conclude that an increased rate of putrescine uptake has a bacteriostatic effect on Pseudomonas spp. cells. We have shown that putrescine is an important tomato root exudate component and that root-colonizing pseudomonads must carefully regulate their rate of uptake because increased uptake causes a decreased growth rate and, therefore, a decreased competitive colonization ability.

Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations.

Quantitative analyses of fast- and slow-growing soybean rhizobia populations in soils of four different provinces of China (Hubei, Shan Dong, Henan, and Xinjiang) have been carried out using the most probable number technique (MPN). All soils contained fast- (FSR) and slow-growing (SSR) soybean rhizobia. Asiatic and American soybean cultivars grown at acid, neutral and alkaline pH were used as trapping hosts for FSR and SSR strains. The estimated total indigenous soybean-rhizobia populations of the Xinjiang and Shan Dong soil samples greatly varied with the different soybean cultivars used. The soybean cultivar and the pH at which plants were grown also showed clear effects on the FSR/SSR rations isolated from nodules. Results of competition experiments between FSR and SSR strains supported the importance of the soybean cultivar and the pH on the outcome of competition for nodulation between FSR and SSR strains. In general, nodule occupancy by FSRs significantly increased at alkaline pH. Bacterial isolates from soybean cultivar Jing Dou 19 inoculated with Xinjiang soil nodulate cultivars Heinong 33 and Williams very poorly. Plasmid and lipopolysaccharide (LPS) profiles and PCR-RAPD analyses showed that cultivar Jing Dou 19 had trapped a diversity of FSR strains. Most of the isolates from soybean cultivar Heinong 33 inoculated with Xinjiang soil were able to nodulate Heinong 33 and Williams showed very similar, or identical, plasmid, LPS and PCR-RAPD profiles. All the strains isolated from Xinjiang province, regardless of the soybean cultivar used for trapping, showed similar nodulation factor (LCO) profiles as judged by thin layer chromatographic analyses. These results indicate that the existence of soybean rhizobia sub-populations showing marked cultivar specificity, can affect the estimation of total soybean rhizobia populations indigenous to the soil, and can also affect the diversity of soybean rhizobial strains isolated from soybean nodules.

Introduction of the phzH gene of Pseudomonas chlororaphis PCL1391 extends the range of biocontrol ability of phenazine-1-carboxylic acid-producing Pseudomonas spp. strains.

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. Its biocontrol activity is mediated by the production of phenazine-1-carboxamide (PCN). In contrast, the take-all biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84, which produce phenazine-1-carboxylic acid (PCA), do not control this disease. To determine the role of the amide group in biocontrol, the PCN biosynthetic genes of strain PCL1391 were identified and characterized. Downstream of phzA through phzG, the novel phenazine biosynthetic gene phzH was identified and shown to be required for the presence of the 1-carboxamide group of PCN because a phzH mutant of strain PCL1391 accumulated PCA. The deduced PhzH protein shows homology with asparagine synthetases that belong to the class II glutamine amidotransferases, indicating that the conversion of PCA to PCN occurs via a transamidase reaction catalyzed by PhzH. Mutation of phzH caused loss of biocontrol activity, showing that the 1-carboxamide group of PCN is crucial for control of tomato foot and root rot. PCN production and biocontrol activity of the mutant were restored by complementing the phzH gene in trans. Moreover, transfer of phzH under control of the tac promoter to the PCA-producing biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84 enabled these strains to produce PCN instead of PCA and suppress tomato foot and root rot. Thus, we have shown, for what we believe is the first time, that the introduction of a single gene can efficiently extend the range of the biocontrol ability of bacterial strains.

Phenazine-1-carboxamide production in the biocontrol strain Pseudomonas chlororaphis PCL1391 is regulated by multiple factors secreted into the growth medium.

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. The production of phenazine-1-carboxamide (PCN) is crucial for this biocontrol activity. In vitro production of PCN is observed only at high-population densities, suggesting that production is under the regulation of quorum sensing. The main autoinducer molecule produced by PCL1391 was identified structurally as N-hexanoyl-L-homoserine lactone (C6-HSL). The two other autoinducers that were produced comigrate with N-butanoyl-L-homoserine lactone (C4-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL). Two PCL1391 mutants lacking production of PCN were defective in the genes phzI and phzR, respectively, the nucleotide sequences of which were determined completely. Production of PCN by the phzI mutant could be complemented by the addition of exogenous synthetic C6-HSL, but not by C4-HSL, C8-HSL, or any other HSL tested. Expression analyses of Tn5luxAB reporter strains of phzI, phzR, and the phz biosynthetic operon clearly showed that phzI expression and PCN production is regulated by C6-HSL in a population density-dependent manner. The introduction of multiple copies of the regulatory genes phzI and phzR on various plasmids resulted in an increase of the production of HSLs, expression of the PCN biosynthetic operon, and consequently, PCN production, up to a sixfold increase in a copy-dependent manner. Surprisingly, our expression studies show that an additional, yet unidentified factor(s), which are neither PCN nor C4-HSL or C8-HSL, secreted into the growth medium of the overnight cultures, is involved in the positive regulation of phzI, and is able to induce PCN biosynthesis at low cell densities in a growing culture, resulting in an increase of PCN production.

Effect of genetically modified Pseudomonas putida WCS358r on the fungal rhizosphere microflora of field-grown wheat.

We released genetically modified Pseudomonas putida WCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the phz biosynthetic gene locus of strain P. fluorescens 2-79 and constitutively produced the antifungal compound phenazine-1-carboxylic acid (PCA). In the springs of 1997 and 1998 we sowed wheat seeds treated with either GMM 2, GMM 8, or WCS358r (approximately 10(7) CFU per seed), and measured the numbers, composition, and activities of the rhizosphere microbial populations. During both growing seasons, all three bacterial strains decreased from 10(7) CFU per g of rhizosphere sample to below the limit of detection (10(2) CFU per g) 1 month after harvest of the wheat plants. The phz genes were stably maintained, and PCA was detected in rhizosphere extracts of GMM-treated plants. In 1997, but not in 1998, fungal numbers in the rhizosphere, quantified on 2% malt extract agar (total filamentous fungi) and on Komada's medium (mainly Fusarium spp.), were transiently suppressed in GMM 8-treated plants. We also analyzed the effects of the GMMs on the rhizosphere fungi by using amplified ribosomal DNA restriction analysis. Introduction of any of the three bacterial strains transiently changed the composition of the rhizosphere fungal microflora. However, in both 1997 and 1998, GMM-induced effects were distinct from those of WCS358r and lasted for 40 days in 1997 and for 89 days after sowing in 1998, whereas effects induced by WCS358r were detectable for 12 (1997) or 40 (1998) days. None of the strains affected the metabolic activity of the soil microbial population (substrate-induced respiration), soil nitrification potential, cellulose decomposition, plant height, or plant yield. The results indicate that application of GMMs engineered to have improved antifungal activity can exert nontarget effects on the natural fungal microflora.

Rhizobial NodL O-acetyl transferase and NodS N-methyl transferase functionally interfere in production of modified Nod factors.

The products of the rhizobial nodulation genes are involved in the biosynthesis of lipochitin oligosaccharides (LCOs), which are host-specific signal molecules required for nodule formation. The presence of an O-acetyl group on C-6 of the nonreducing N-acetylglucosamine residue of LCOs is due to the enzymatic activity of NodL. Here we show that transfer of the nodL gene into four rhizobial species that all normally produce LCOs that are not modified on C-6 of the nonreducing terminal residue results in production of LCOs, the majority of which have an acetyl residue substituted on C-6. Surprisingly, in transconjugant strains of Mesorhizobium loti, Rhizobium etli, and Rhizobium tropici carrying nodL, such acetylation of LCOs prevents the endogenous nodS-dependent transfer of the N-methyl group that is found as a substituent of the acylated nitrogen atom. To study this interference between nodL and nodS, we have cloned the nodS gene of M. loti and used its product in in vitro experiments in combination with purified NodL protein. It has previously been shown that a chitooligosaccharide N deacetylated on the nonreducing terminus (the so-called NodBC metabolite) is the preferred substrate for NodS as well as for NodL. Here we show that the NodBC metabolite, acetylated by NodL, is not used by the NodS protein as a substrate while the NodL protein can acetylate the NodBC metabolite that has been methylated by NodS.

The occurrence of internal (1 --> 5)-linked arabinofuranose and arabinopyranose residues in arabinogalactan side chains from soybean pectic substances.

CDTA-extractable soybean pectic substances were subjected to enzymatic digestion with arabinogalactan degrading enzymes yielding a resistant polymeric pectic backbone and arabino-, galacto-, and arabinogalacto-oligomers. The complex digest was fractionated using size-exclusion chromatography. Monosaccharide composition analysis, HPAEC fractionation and MALDI-TOF MS analysis of the resulting fractions showed that each contained a mixture of oligosaccharides of essentially the same degree of polymerisation, composed of only arabinose and galactose. MALDI-TOF MS analysis was used for molecular mass screening of oligosaccharides in underivatised HPAEC fractions. The monosaccharide sequence and the branching pattern of oligosaccharides (degree of polymerisation from 4 to 8) were determined using linkage analysis and ES-CID tandem MS analysis of the per-O-methylated oligosaccharides in each of the HPAEC fractions. These analyses indicated the presence of common linear (1 --> 4)-linked galacto-oligosaccharides, and both linear and branched arabino-oligosaccharides. In addition, the results unambiguously showed the presence of oligosaccharides containing (1 --> 4)-linked galactose residues bearing an arabinopyranose residue as the non-reducing terminal residue, and a mixture of linear oligosaccharides constructed of (1 --> 4)-linked galactose residues interspersed with an internal (1 --> 5)-linked arabinofuranose residue. The consequences of these two new structural features of pectic arabinogalactan side chains are discussed.

Analysis of the pmsCEAB gene cluster involved in biosynthesis of salicylic acid and the siderophore pseudomonine in the biocontrol strain Pseudomonas fluorescens WCS374.

Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb EcoRI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of P. putida. Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (pmsC and pmsB) showed homologies with chorismate-utilizing enzymes; a third ORF (pmsE) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene (pmsA). A putative promoter region and two predicted iron boxes were localized upstream of pmsC. We determined by reverse transcriptase-mediated PCR that the pmsCEAB genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in P. putida and Escherichia coli cells. In E. coli, deletions affecting the first ORF (pmsC) diminished SA production, whereas deletion of pmsB abolished it completely. The pmsB gene induced low levels of SA production in E. coli when expressed under control of the lacZ promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn5 mutant (374-05) that is simultaneously impaired in SA and pseudomonine production.

Growth temperature regulation of host-specific modifications of rhizobial lipo-chitin oligosaccharides: the function of nodX is temperature regulated.

Lipo-chitin oligosaccharides (LCOs) are usually produced and isolated for structural analysis from bacteria cultured under laboratory rather than field conditions. We have studied the influence of bacterial growth temperature on the LCO structures produced by different Rhizobium leguminosarum strains, using thin-layer chromatographic, high-performance liquid chromatographic, and mass spectrometric analyses. Wild-type R. leguminosarum bv. viciae A1 was shown to produce larger relative amounts of nodX-mediated, acetylated LCOs at 12 degrees C than at 28 degrees C, indicating that the activity of nodX (a gene encoding an LCO O-acetyl transferase) is temperature dependent. Interestingly, symbiotic resistance genes sym1 and sym2 found in primitive pea cultivars are also temperature sensitive, only being active at low temperatures, at which they block nodulation by R. leguminosarum bv. viciae strains lacking nodX. We therefore propose that the gene-for-gene relationship between plant and bacterium has a temperature-sensitive mechanism as an adaptation to environmental conditions. An R. leguminosarum bv. trifolii strain was also shown to produce larger relative amounts of nodX-mediated, acetylated LCOs at 12 degrees C than at 28 degrees C. The major components synthesized by the two strains are produced at both temperatures but in different relative amounts, while some minor components are only produced at one of the two temperatures.

A Lotus japonicus nodulation system based on heterologous expression of the fucosyl transferase NodZ and the acetyl transferase NoIL in Rhizobium leguminosarum.

Heterologous expression of NodZ and NolL proteins in Rhizobium leguminosarum bv. viciae led to the production of acetyl fucosylated lipo-chitin oligosaccharides (LCOs), indicating that the NolL protein obtained from Mesorhizobium loti functions as an acetyl transferase. We show that the NolL-dependent acetylation is specific for the fucosyl penta-N-acetylglucosamine species. In addition, the NolL protein caused elevated production of LCOs. Efficient nodulation of Lotus japonicus by the NodZ/NolL-producing strain was demonstrated. Nodulation efficiency was further improved by the addition of the ethylene inhibitor L-alpha-(2-aminoethoxyvinyl) glycine (AVG).

The structure of the linkage between the O-specific polysaccharide and the core region of the lipopolysaccharide from Salmonella enterica serovar Typhimurium revisited.

Salmonella enterica sv. Typhimurium strain 1135 possesses smooth(S)-form lipopolysaccharide (LPS). Although the structures of the core region and the O-specific polysaccharide were investigated intensively between the 1960s and the 1980s, the structure of the linkage region between the O-chain and the core was not elucidated unequivocally. By using modern MS and high-field NMR spectroscopy for analysis of the isolated carbohydrate backbone of the LPS, it has been shown that it is a beta-D-Galp residue that links the first repeating unit of the O-specific polysaccharide to O-4 of the last D-Glcp residue of the core region. Interestingly, this particular D-Galp residue is alpha-linked in all following repeating units. The data are discussed with regard to the ligation of O-specific polysaccharide and core region during LPS biosynthesis.

Identification of arsenosugars at the picogram level using nanoelectrospray quadrupole time-of-flight mass spectrometry.

State-of-the-art analytical methods for arsenic speciation rely typically on the availability of standards of defined structure, limiting the applicability of such methods to the determination of known compounds. Our previous high-energy tandem mass spectrometric studies demonstrated the strength of mass spectrometry for generating structurally diagnostic ions that allow for the identification of arsenic-containing ribofuranosides (arsenosugars) without the use of standards. We now report a more widely applicable and more sensitive approach, using negative-ion nano-electrospray low-energy tandem mass spectrometry for the generation of structurally useful product ions that allow for identification of arsenosugars at the picogram level. In the negative-ion mode, numerous product ions, suitable for characterizing naturally occurring dimethylated arsenosugars, were generated in high abundance. Application of the method to an algal extract unequivocally demonstrated the presence of a single dimethylated arsenosugar. In the positive-ion mode, characteristic tandem mass spectra were obtained for four trimethylarsonioribosides, allowing their identification without the need for standards. Overall it was demonstrated that nano-ES-MS/MS techniques can be used for characterizing arsenosugars on a routine basis, a necessary requirement for assessing potential health risks associated with consuming foods containing elevated levels of arsenosugars and for improving our understanding of arsenic biochemistry.

Structural characterisation of lipo-chitin oligosaccharides isolated from Bradyrhizobium aspalati, microsymbionts of commercially important South African legumes.

The shoots of the South African legume Aspalathus linearis spp. linearis (A. linearis) are used in the manufacture of an increasingly popular beverage that has acclaimed beneficial effects on health; this important export product is known as Rooibos (or Redbush) tea. Three strains of Bradyrhizobium aspalati, which are the nitrogen-fixing symbionts of Aspalathus carnosa, A. hispida and A. linearis, were tested for the production of lipo-chitin oligosaccharide signal molecules using thin-layer chromatographic analysis after induction with different inducers, including Rooibos tea extract, and radioactive labelling. Large-scale separation, using high-performance liquid chromatography, of lipo-chitin oligosaccharides from B. aspalati isolated from A. carnosa was performed for structural characterisation using fast-atom bombardment mass spectrometry and chemical modifications followed by gas chromatography-mass spectrometric analysis. The strain was shown to secrete a family of unusual lipo-chitin oligosaccharides that are highly substituted on the nonreducing-terminal residue but unsubstituted on the reducing-terminal residue. They have a backbone of three to five beta-(1-->4)-linked N-acetyl-D-glucosamine residues substituted on the nonreducing terminus with a C16:0, C16:1, C18:0, C18:1, C19:1cy, or C20:1 fatty acyl chain, and are both N-methylated and 4,6-dicarbamoylated.

Structural studies of a heteroxylan from Plantago major L. seeds by partial hydrolysis, HPAEC-PAD, methylation and GC-MS, ESMS and ESMS/MS.

The seed mucilage from Plantago major L. contains acidic heteroxylan polysaccharides. For further structural analysis, oligosaccharides were generated by partial acid hydrolysis and then isolated by high-pH anion-exchange chromatography (HPAEC). Each HPAEC fraction was shown by ESMS to contain one major oligosaccharide and several minor components. Partial structures of the oligosaccharides were determined using GC-MS, ESMS and ES tandem mass spectrometry (ESMS/MS). A (1-->4)-linked xylan trisaccharide and (1-->3)-linked xylan oligosaccharides with DP 6-11 suggested that the backbone of the heteroxylan polysaccharide consisted of blocks of (1-->4)-linked and (1-->3)-linked Xylp residues. A (1-->2)-linked Xylp disaccharide and a branched tetrasaccharide were also found, revealing that single Xylp residues are linked to the O-2 of some of the (1-->4)-linked Xylp residues in the backbone. In addition, our results confirm the presence of side chains consisting of the disaccharide GlcpA-(1-->3)-Araf.

Mass spectrometric analysis of Klebsiella pneumoniae ssp. pneumoniae rough strain R20 (O1-: K20-) lipopolysaccharide preparations: identification of novel core oligosaccharide components and three 3-deoxy-D-manno-oct-2-ulopyranosonic artifacts.

In an attempt to find the best approach for the mass spectrometric analysis of the whole range of lipopolysaccharide (LPS) structures from Klebsiella pneumoniae ssp. pneumoniae rough strain R20 (O1-:K20-), various methods of LPS preparation were applied and the products were analyzed using a range of mass spectrometric techniques. The most productive approach proved to be the removal of lipid A by mild acid hydrolysis and the study of the core oligosaccharide structures using nanoelectrospray time-of-flight mass spectrometry (TOF-MS) in combination with collision-induced dissociation tandem mass spectrometry. This procedure is very sensitive, but results in the generation of a reducing 3-deoxy-D-manno-oct-2-ulopyranosonic acid residue (Kdo) that is susceptible to the formation of artifacts, which give rise to pseudomolecular ions 18, 46, and 88 Da below the pseudomolecular ion for the unmodified species. Alternatively, matrix-assisted laser desorption/ionization TOF-MS combined with post-source decay can be used to study the de-O-acylated LPS preparation and especially to identify those residues bearing phosphate groups and the residues involved in the linkage between the core and lipid A. In addition to the five LPS core structures defined using NMR spectroscopy by Süsskind et al., several extra related LPS structure were identified. Larger LPS species were observed, which surprisingly do not represent species containing longer versions of the novel Klebsiella heptoglycan, but instead are species having the defined core and heptoglycan extended with up to three extra hexuronic acid and one or two extra hexose residues.

The structures of the carbohydrate backbones of the lipopolysaccharides from Escherichia coli rough mutants F470 (R1 core type) and F576 (R2 core type).

The lipopolysaccharides (LPS) from Escherichia coli rough mutant strains F470 (R1 core type) and F576 (R2 core type) were deacylated yielding in each case a mixture of oligosaccharides with one predominant product which was isolated using high-performance anion-exchange chromatography. In addition, one oligosaccharide present in minor quantities was isolated from LPS of E. coli strain F576 (R2 core type). The structures of the oligosaccharides were determined by chemical analyses and NMR spectroscopic experiments. Furthermore, de-O-acylated and dephosphorylated LPS preparations were investigated by fast-atom bombardment and collision induced dissociation tandem mass spectrometry. The combined data allow us to deduce the following carbohydrate backbones of the E. coli R1 and R2 core types which share the following structure (Scheme 1): but differ in the substituents R1 and R2 which for the R1 core type are predominantly: and to a minor extent: and for the R2 core type predominantly: and to a minor extent: in which all sugars are d-pyranoses (l,d-Hep, lglycerodmanno-heptopyranose; P, phosphate).

The regulator gene phoB mediates phosphate stress-controlled synthesis of the membrane lipid diacylglyceryl-N,N,N-trimethylhomoserine in Rhizobium (Sinorhizobium) meliloti.

Bacteria react to phosphate starvation by activating genes involved in the transport and assimilation of phosphate as well as other phosphorous compounds. Some soil bacteria have evolved an additional mechanism for saving phosphorous. Under phosphate-limiting conditions, they replace their membrane phospholipids by lipids not containing phosphorus. Here, we show that the membrane lipid pattern of the free-living microsymbiotic bacterium Rhizobium (Sinorhizobium) meliloti is altered at low phosphate concentrations. When phosphate is growth limiting, an increase in sulpholipids, ornithine lipids and the de novo synthesis of diacylglyceryl trimethylhomoserine (DGTS) lipids is observed. Rhizobium meliloti phoCDET mutants, deficient in phosphate uptake, synthesize DGTS constitutively at low or high medium phosphate concentrations, suggesting that reduced transport of phosphorus sources to the cytoplasm causes induction of DGTS biosynthesis. Rhizobium meliloti phoU or phoB mutants are unable to form DGTS at low or high phosphate concentrations. However, the functional complementation of phoU or phoB mutants with the phoB gene demonstrates that, of the two genes, only intact phoB is required for the biosynthesis of the membrane lipid DGTS.

Comparison of characteristics of the nodX genes from various Rhizobium leguminosarum strains.

We have analyzed the nucleotide sequences of the nodX genes from two strains of Rhizobium leguminosarum bv. viciae able to nodulate Afghan peas (strains A1 and Himalaya) and from two strains of R. leguminosarum bv. trifolii (ANU843 and CSF). The nodX genes of strains A1 and ANU843 were shown to be functional for the induction of nodules on Afghan peas. To analyze the cause of phenotypic differences of strain A1 and strain TOM we have studied the composition of the lipochitin-oligosaccharides (LCOs) produced by strain A1 after induction by the flavonoid naringenin or various pea root exudates. The structural analysis of the LCOs by mass spectrometry revealed that strain A1 synthesizes a family of at least 23 different LCOs. The use of exudates instead of naringenin resulted only in quantitative differences in the ratios of various LCOs produced.

Characterization of a novel branched tetrasaccharide of 3-deoxy-D-manno-oct-2-ulopyranosonic acid. The structure of the carbohydrate backbone of the lipopolysaccharide from Acinetobacter baumannii strain nctc 10303 (atcc 17904).

For the first time, the tetrasaccharide Kdoalpha2-->5Kdoalpha2-->5(Kdoalpha2-->4)Kdo (Kdo is 3-deoxy-D-manno-oct-2-ulopyranosonic acid) has been identified in a bacterial lipopolysaccharide (LPS), i.e. in the core region of LPS from Acinetobacter baumannii NCTC 10303. The LPS was analyzed using compositional analysis, mass spectrometry, and NMR spectroscopy. The disaccharide D-GlcpNbeta1-->6D-GlcpN, phosphorylated at O-1 and O-4', was identified as the carbohydrate backbone of the lipid A. The Kdo tetrasaccharide is attached to O-6' of this disaccharide and is further substituted by short L-rhamnoglycans of varying length and by the disaccharide D-GlcpNAcalpha1-->4D-GlcpNA (GlcpNA, 2-amino-2-deoxy-glucopyranosuronic acid). The core region is not substituted by phosphate residues and represents a novel core type of bacterial LPS. The complete carbohydrate backbone of the LPS is shown in Structure I as follows: where Rha is rhamnose. Except were indicated, monosaccharides possess the D-configuration. Sugars marked with an asterisk are present in non-stoichiometric amounts.

Sodium-cationized oligosaccharides do not appear to undergo 'internal residue loss' rearrangement processes on tandem mass spectrometry.

The phenomenon of 'internal residue loss' of protonated native- and per-O-methylated oligosaccharides has recently been described as occurring on high-energy collision conditions. Awareness of this phenomenon in the mass spectrometric analysis of oligosaccharides is of great importance since the rearrangement ions produced by this process may complicate monosaccharide sequence assignment. In this research, oligosaccharides having N-acetyl-glucosamine residues as the reducing or non-reducing terminal residue have been included in our MS/MS analyses in order to try to better understand the factors that influence 'internal residue loss'. Native and per-O-methylated compounds were submitted to positive and negative MS/MS, selecting protonated, sodium-cationized, or de-protonated pseudomolecular ions as precursors. High- and low-energy collision induced dissociation tandem mass spectrometry experiments were performed using a four sector instrument and a hybrid quadrupole time-of-flight mass spectrometer respectively. The phenomenon of 'internal residue loss' was not observed on either high- or low-energy CID-MS/MS when sodium-cationized precursor ions of either native or per-O-methylated oligosaccharides were examined. Similarly, MS/MS analysis performed in the negative ionization mode also failed to generate ions resulting from 'internal residue loss'. This combination of experiments therefore offers a way to be sure whether ions observed in the tandem mass spectra of protonated native or per-O-methylated oligosaccharides originate from 'internal residue loss' or from direct glycosidic linkage fragmentation.