Fundamental differences in physiology of Bordetella pertussis dependent on the two-component system Bvg revealed by gene essentiality studies.

The identification of genes essential for a bacterium's growth reveals much about its basic physiology under different conditions. Bordetella pertussis, the causative agent of whooping cough, adopts both virulent and avirulent states through the activity of the two-component system, Bvg. The genes essential for B. pertussis growth in vitro were defined using transposon sequencing, for different Bvg-determined growth states. In addition, comparison of the insertion indices of each gene between Bvg phases identified those genes whose mutation exerted a significantly different fitness cost between phases. As expected, many of the genes identified as essential for growth in other bacteria were also essential for B. pertussis. However, the essentiality of some genes was dependent on Bvg. In particular, a number of key cell wall biosynthesis genes, including the entire mre/mrd locus, were essential for growth of the avirulent (Bvg minus) phase but not the virulent (Bvg plus) phase. In addition, cell wall biosynthesis was identified as a fundamental process that when disrupted produced greater fitness costs for the Bvg minus phase compared to the Bvg plus phase. Bvg minus phase growth was more susceptible than Bvg plus phase growth to the cell wall-disrupting antibiotic ampicillin, demonstrating the increased susceptibility of the Bvg minus phase to disruption of cell wall synthesis. This Bvg-dependent conditional essentiality was not due to Bvg-regulation of expression of cell wall biosynthesis genes; suggesting that this fundamental process differs between the Bvg phases in B. pertussis and is more susceptible to disruption in the Bvg minus phase. The ability of a bacterium to modify its cell wall synthesis is important when considering the action of antibiotics, particularly if developing novel drugs targeting cell wall synthesis.

The evolution of Bordetella pertussis has selected for mutations of acr that lead to sensitivity to hydrophobic molecules and fatty acids.

Whooping cough, or pertussis, is resurgent in numerous countries worldwide. This has renewed interest in Bordetella pertussis biology and vaccinology. The in vitro growth of B. pertussis has been a source of difficulty, both for the study of the organism and the production of pertussis vaccines. It is inhibited by fatty acids and other hydrophobic molecules. The AcrAB efflux system is present in many different bacteria and in combination with an outer membrane factor exports acriflavine and other small hydrophobic molecules from the cell. Here, we identify that the speciation of B. pertussis has selected for an Acr system that is naturally mutated and displays reduced activity compared to B. bronchiseptica, in which the system appears intact. Replacement of the B. pertussis locus with that of B. bronchiseptica conferred higher levels of resistance to growth inhibition by acriflavine and fatty acids. In addition, we identified that the transcription of the locus is repressed by a LysR-type transcriptional regulator. Palmitate de-represses the expression of the acr locus, dependent on the LysR regulator, strongly suggesting that it is a transcriptional repressor that is regulated by palmitate. It is intriguing that the speciation of B. pertussis has selected for a reduction in activity of the Acr efflux system that typically is regarded as protective to bacteria.

A curated genome-scale metabolic model of Bordetella pertussis metabolism.

The Gram-negative bacterium Bordetella pertussis is the causative agent of whooping cough, a serious respiratory infection causing hundreds of thousands of deaths annually worldwide. There are effective vaccines, but their production requires growing large quantities of B. pertussis. Unfortunately, B. pertussis has relatively slow growth in culture, with low biomass yields and variable growth characteristics. B. pertussis also requires a relatively expensive growth medium. We present a new, curated flux balance analysis-based model of B. pertussis metabolism. We enhance the model with an experimentally-determined biomass objective function, and we perform extensive manual curation. We test the model's predictions with a genome-wide screen for essential genes using a transposon-directed insertional sequencing (TraDIS) approach. We test its predictions of growth for different carbon sources in the medium. The model predicts essentiality with an accuracy of 83% and correctly predicts improvements in growth under increased glutamate:fumarate ratios. We provide the model in SBML format, along with gene essentiality predictions.

Drivers and Barriers for Adopting Accreditation at Local Health Departments for Their Performance Improvement Effort.

CONTEXT: A national system of voluntary public health accreditation for state, local, and tribal health departments (local health departments [LHDs]) is part of a movement that aims to improve public health performance with ultimate impact on population health outcomes. Indiana is a good setting for the study of LHD accreditation adoption because several LHDs reported de-adopting accreditation in a recent statewide survey and because 71% of Indiana counties serve populations of 50 000 or less. DESIGN: A systematic method of analyzing qualitative data based on the Performance Improvement Model framework to expand our understanding of de-adoption of public health accreditation. SETTING/PARTICIPANTS: In 2015, we conducted a key informant interview study of the 3 LHDs that decided to delay their engagement in the accreditation based on findings from an Indiana survey on LHD accreditation adoption. The study is an exploration of LHD accreditation de-adoption and of the contributions made to its understanding by the Performance Improvement Model. RESULT: The study found that top management team members are those who champion accreditation adoption, and that organizational structure and culture facilitate the staff's embracing of the change. The Performance Improvement Model was found to enhance the elucidation of the inner domain elements of Consolidated Framework for Implementation Research in the context of de-adoption of public health accreditation. CONCLUSION: Governing entities' policies and priorities appear to mediate whether the LHDs are able to continue accreditation pursuit. Lacking any of these driving forces appears to be associated with decisions to de-adoption of accreditation. Further work is necessary to discern specific elements mediating decisions to pursue accreditation. This study demonstrates the added knowledge of Performance Improvement Model (PIM) to the CFIR framework. A large scale study is called to further clarify and discern supports of specific to the needs of individual LHDs for their performance improvement effort.

It's Not Just a Yes or No Answer: Expressions of Local Health Department Accreditation.

UNLABELLED: The identification and exploration of moderators of health department accreditation remain limited by current dichotomous conceptualizations of pursuit. METHODS: A 2015 survey measured Indiana local health department (LHD) accreditation pursuit and progress, classifying respondents by progress evidence. Covariates included attitudes about the future impact of accreditation on funding and performance, health department size, geography, health outcome ranking, and quality improvement (QI) programing. RESULTS: Four classifications of accreditation pursuit emerged and were found to have greater association with covariates than standard dichotomous measures. "Active Pursuit" was associated with formal QI programing and a belief that accreditation will impact future funding and performance. "Intent Only" was associated with no QI programing and no completion of accreditation prerequisites. "Discontinued" was associated with the belief that accreditation will not impact future performance. "Not Pursuing" was associated with no interest or plan to complete prerequisites and reported belief that accreditation will not impact future health department funding or performance. CONCLUSION: More granular characterizations of accreditation pursuit may improve understanding of influential factors. This measurement framework should be validated in studies of LHDs in other states.

Bacteriophage-mediated Glucosylation Can Modify Lipopolysaccharide O-Antigens Synthesized by an ATP-binding Cassette (ABC) Transporter-dependent Assembly Mechanism.

Lysogenic bacteriophages may encode enzymes that modify the structures of lipopolysaccharide O-antigen glycans, altering the structure of the bacteriophage receptor and resulting in serotype conversion. This can enhance virulence and has implications for antigenic diversity and vaccine development. Side chain glucosylation is a common modification strategy found in a number of bacterial species. To date, glucosylation has only been observed in O-antigens synthesized by Wzy-dependent pathways, one of the two most prevalent O-antigen synthesis systems. Here we exploited a heterologous system to study the glucosylation potential of a model O-antigen produced in an ATP-binding cassette (ABC) transporter-dependent system. Although O-antigen production is cryptic in Escherichia coli K-12, because of a mutation in the synthesis genes, it possesses a prophage glucosylation cluster, which modifies the GlcNAc residue in an α-l-Rha-(1→3)-d-GlcNAc motif found in the original O16 antigen. Raoultella terrigena ATCC 33257 produces an O-antigen possessing the same disaccharide motif, but its assembly uses an ABC transporter-dependent system. E. coli harboring the R. terrigena O-antigen biosynthesis genes produced an O-antigen displaying reduced reactivity toward antisera raised against the native R. terrigena repeat structure, indicative of an altered chemical structure. Structural determination using NMR revealed the addition of glucose side chains to the repeat units. O-antigen modification was dependent on a functional ABC transporter, consistent with modification in the periplasm, and was eliminated by deletion of the glucosylation genes from the E. coli chromosome, restoring native level antisera sensitivity and structure. There are therefore no intrinsic mechanistic barriers for bacteriophage-mediated O-antigen glucosylation in ABC transporter-dependent pathways.

Measuring Accreditation Activity and Progress: Findings from a Survey of Indiana Local Health Departments, 2013.

A 2013 survey of Indiana local health departments (LHDs) measured accreditation activity and progress. Reported activities were categorized using the Public Health Accreditation Board's (PHAB's) accreditation steps as a guiding framework and matched with selected sociodemographic, organizational, and technical assistance variables. Findings indicated that 42 (59.2%) of responding Indiana LHDs reported pursuing accreditation. Of LHDs pursuing accreditation, 21 were at the initial introductory step, 18 were at the prerequisite step, one reported submitting an application to PHAB, and two reported no activity, yet intent to pursue accreditation. Reported receipt of technical assistance was associated with accreditation progress (p=0.01) and, specifically, with being at the prerequisite step. Facilitating the pursuit of LHD accreditation in states with low public health investment is possible with targeted accreditation resources. Finding meaningful measures of accreditation progress will help advance the study of factors associated with LHD accreditation on a broad scale and for the long term.

Lipopolysaccharide O antigen size distribution is determined by a chain extension complex of variable stoichiometry in Escherichia coli O9a.

The lengths of bacterial polysaccharides can be critical for their biological function. Unlike DNA or protein synthesis, where polymer length is implicit in the nucleic acid template, the molecular mechanisms for regulating polysaccharide length are poorly understood. Two models are commonly cited: a "molecular clock" regulates length by controlling the duration of the polymer extension process, whereas a "molecular ruler" determines length by measurement against a physical structure in the biosynthetic complex. Escherichia coli O9a is a prototype for the biosynthesis of O polysaccharides by ATP-binding cassette transporter-dependent processes. The length of the O9a polysaccharide is determined by two proteins: an extension enzyme, WbdA, and a termination enzyme, WbdD. WbdD is known to self-oligomerize and also to interact with WbdA. Changing either enzyme's concentration can alter the polysaccharide length. We quantified the O9a polysaccharide length distribution and the enzyme concentration dependence in vivo, then made mathematical models to predict the polymer length distributions resulting from hypothetical length-regulation mechanisms. Our data show qualitative features that cannot be explained by either a molecular clock or a molecular ruler model. Therefore, we propose a "variable geometry" model, in which a postulated biosynthetic WbdA-WbdD complex assembles with variable stoichiometry dependent on relative enzyme concentration. Each stoichiometry produces polymers with a distinct, geometrically determined, modal length. This model reproduces the enzyme concentration dependence and modality of the observed polysaccharide length distributions. Our work highlights limitations of previous models and provides new insight into the mechanisms of length control in polysaccharide biosynthesis.

Modern supercritical fluid technology for food applications.

This review provides an update on the use of supercritical fluid (SCF) technology as applied to food-based materials. It advocates the use of the solubility parameter theory (SPT) for rationalizing the results obtained when employing sub- and supercritical media to food and nutrient-bearing materials and for optimizing processing conditions. Total extraction and fractionation of foodstuffs employing SCFs are compared and are illustrated by using multiple fluids and unit processes to obtain the desired food product. Some of the additional prophylactic benefits of using carbon dioxide as the processing fluid are explained and illustrated with multiple examples of commercial products produced using SCF media. I emphasize the role of SCF technology in the context of environmentally benign and sustainable processing, as well as its integration into an overall biorefinery concept. Conclusions are drawn in terms of current trends in the field and future research that is needed to secure new applications of the SCF platform as applied in food science and technology.

Five new genes are important for common polysaccharide antigen biosynthesis in Pseudomonas aeruginosa.

UNLABELLED: Common polysaccharide antigen (CPA) is a conserved cell surface polysaccharide produced by Pseudomonas aeruginosa. It contains a rhamnan homopolymer and is one of the two forms of O polysaccharide attached to P. aeruginosa lipopolysaccharide (LPS). Our laboratory has previously characterized an eight-gene cluster (pa5447-pa5454 in P. aeruginosa PAO1) required for biosynthesis of CPA. Here we demonstrate that an adjacent five-gene cluster pa5455-pa5459 is also involved. Using reverse transcriptase PCR (RT-PCR), we showed that the original eight-gene cluster and the new five-gene cluster are both organized as operons. We have analyzed the LPS phenotypes of in-frame deletion mutants made in each of the five genes, and the results verified that these five genes are indeed required for CPA biosynthesis, extending the CPA biosynthesis locus to contain 13 contiguous genes. By performing overexpression experiments of different sets of these biosynthesis genes, we were able to obtain information about their possible functions in CPA biosynthesis. IMPORTANCE: Lipopolysaccharide (LPS) is an important cell surface structure of Gram-negative bacteria. The human opportunistic pathogen Pseudomonas aeruginosa simultaneously produces an O-antigen-specific (OSA) form and a common polysaccharide antigen (CPA) form of LPS. CPA, the focus of this study, is composed of α-1-2, α1-3-linked d-rhamnose sugars and has been shown to be important for attachment of the bacteria to human airway epithelial cells. Genome sequencing of this species revealed a new five-gene cluster that we predicted to be involved in CPA biosynthesis and modification. In this study, we have generated chromosomal knockouts by performing in-frame deletions and allelic replacements. Characterizing the function of each of the five genes is important for us to better understand CPA biosynthesis and the mechanisms of chain length termination and regulation of this unique D-rhamnan polysaccharide.

Design and optimization of a semicontinuous hot-cold extraction of polyphenols from grape pomace.

Grape pomace contains appreciable amounts of polyphenolic compounds such as anthocyanins and procyanidins which can be recovered for use as food supplements. The extraction of these polyphenols from the pomace is usually accomplished at slightly elevated temperatures, frequently employing hydroethanolic solvents. Due to governmental regulations and the cost involved in using ethanol as a solvent, as well as the loss in polyphenolics due to thermal degradation, improved extraction techniques are required. In this study, a semicontinuous extraction apparatus employing only water was developed to maximize the recovery of anthocyanins and procyanidins from red grape pomace (Vitis vinifera). Water is preheated prior to its entry to the extraction cell containing the grape pomace sample, where it is allowed to then flow continuously through the unheated extraction vessel prior to its collection at ambient conditions. Extraction variables that impacted the polyphenolic recovery included pomace moisture content (crude or dried), sample mass, water flow rate, and extraction temperature. A response surface method was used to analyze the results from the extraction, and the optimal conditions were found to be 140 °C and 9 mL/min water flow rate. These conditions can produce an extract containing 130 mg/100 g DW of anthocyanins and 2077 mg/100 g DW of procyanidins. Higher yields of polyphenolics were observed using crude (wet) rather than dried pomace, hence avoiding the need to dry the pomace prior to extraction. The described semicontinuous extraction method using only water as the extraction solvent under subcritical conditions allowed the efficient extraction of polyphenols from red grape pomace without the attendant loss of polyphenolic content due to having to heat the extraction vessel prior to commencement of extraction.

Oligosaccharide conjugates of Bordetella pertussis and bronchiseptica induce bactericidal antibodies, an addition to pertussis vaccine.

Pertussis is a highly contagious respiratory disease that is especially dangerous for infants and children. Despite mass vaccination, reported pertussis cases have increased in the United States and other parts of the world, probably because of increased awareness, improved diagnostic means, and waning vaccine-induced immunity among adolescents and adults. Licensed vaccines do not kill the organism directly; the addition of a component inducing bactericidal antibodies would improve vaccine efficacy. We investigated Bordetella pertussis and Bordetella bronchiseptica LPS-derived core oligosaccharide (OS) protein conjugates for their immunogenicity in mice. B. pertussis and B. bronchiseptica core OS were bound to aminooxylated BSA via their terminal Kdo residues. The two conjugates induced similar anti-B. pertussis LPS IgG levels in mice. B. bronchiseptica was investigated because it is easier to grow than B. pertussis. Using B. bronchiseptica genetically modified strains deficient in the O-specific polysaccharide, we isolated fractions of core OS with one to five repeats of the terminal trisaccharide, having at the nonreducing end a GlcNAc or GalNAc, and bound them to BSA at different densities. The highest antibody levels in mice were elicited by conjugates containing an average of 8-17 OS chains per protein and with one repeat of the terminal trisaccharide. Conjugate-induced antisera were bactericidal against B. pertussis, and the titers correlated with ELISA-measured antibody levels (r = 0.74). Such conjugates are easy to prepare and standardize; added to a recombinant pertussis toxoid, they may induce antibacterial and antitoxin immunity.

Physicochemical characterization of dilute n-alcohol/biodiesel mixtures by inverse gas chromatography.

Inverse gas chromatography (IGC) has been used to determine the physicochemical parameters that characterize solution thermodynamic interactions in biodiesel-n-alcohol solute systems. Such data is of value to chemical engineers and separation scientists in optimizing separation processes to separate alcoholic solutes at low concentrations in soybean oil methyl ester mixtures (biodiesel). The derived activity and Henry's Law coefficient data can be used to rationalize the interaction of four members of an n-alcoholic homologous series and the soya-based methyl ester solvent in terms of such esters as "green" renewable solvents. Sorption isotherm data confirm linear behavior in most cases between the solute (alcohol) vapor state concentrations and their uptake into the biodiesel phase. Overall, the experimentally determined activity coefficients agree well with those predicted by solution thermodynamic theories as well as correlative chemical engineering equations.

Antigenic Variation among Bordetella: Bordetella bronchiseptica strain MO149 expresses a novel o chain that is poorly immunogenic.

The O chain polysaccharide (O PS) of Bordetella bronchiseptica and Bordetella parapertussis lipopolysaccharide is a homopolymer of 2,3-diacetamido-2,3-dideoxygalacturonic acid (GalNAc3NAcA) in which some of the sugars are present as uronamides. The terminal residue contains several unusual modifications. To date, two types of modification have been characterized, and a survey of numerous strains demonstrated that each contained one of these two modification types. Host antibody responses against the O PS are directed against the terminal residue modifications, and there is little cross-reactivity between the two types. This suggests that Bordetella O PS modifications represent a means of antigenic variation. Here we report the characterization of the O PS of B. bronchiseptica strain MO149. It consists of a novel two-sugar repeating unit and a novel terminal residue modification, with the structure Me-4-alpha-L-GalNAc3NAcA-(4-beta-D-GlcNAc3NAcA-4-alpha-L-GalNAc3NAcA-)(5-6)-, which we propose be defined as the B. bronchiseptica O3 PS. We show that the O3 PS is very poorly immunogenic and that the MO149 strain contains a novel wbm (O PS biosynthesis) locus. Thus, there is greater diversity among Bordetella O PSs than previously recognized, which is likely to be a result of selection pressure from host immunity. We also determine experimentally, for the first time, the absolute configuration of the diacetimido-uronic acid sugars in Bordetella O PS.

Biosynthesis of uronamide sugars in Pseudomonas aeruginosa O6 and Escherichia coli O121 O antigens.

The major component of the outer leaflet of the outer membrane of Gram-negative bacteria is lipopolysaccharide (LPS). The outermost domain of LPS is a polysaccharide called O antigen. Pseudomonas aeruginosa establishes biofilms on wet surfaces in a wide range of habitats and mutations in O-antigen biosynthesis genes affect bacterial adhesion and the structure of these biofilms. The P. aeruginosa O6 O antigen contains a 2-acetamido-2-deoxy-d-galacturonamide (d-GalNAcAN) residue. O-antigen biosynthesis in this serotype requires the wbpS gene, which encodes a protein with conserved domains of the glutamine-dependent amidotransferase family. Replacement of conserved amino acids in the N-terminal glutaminase conserved domain of WbpS inhibited O-antigen biosynthesis under restricted-ammonia conditions, but not in rich media; suggesting that this domain functions to provide ammonia for O-antigen biosynthesis under restricted-ammonia conditions, by hydrolysis of glutamine. Escherichia coli O121 also produces a d-GalNAcAN-containing O antigen, and possesses a homologue of wbpS called wbqG. An E. coli O121 wbqG mutant was cross-complemented by providing wbpS in trans, and vice versa, showing that these two genes are functionally interchangeable. The E. coli O121 wbqG mutant O antigen contains 2-acetamido-2-deoxy-d-galacturonate (d-GalNAcA), instead of d-GalNAcAN, demonstrating that wbqG is specifically required for biosynthesis of the carboxamide in this sugar.

The role of cytotoxic therapy with hematopoietic stem cell transplantation in the treatment of follicular lymphoma: an evidence-based review.

Clinical research examining the role of hematopoietic stem cell transplantation (SCT) in the therapy of follicular non-Hodgkin lymphoma in adults is presented and critically evaluated in this systematic evidence-based review. Specific criteria were used for searching the published literature and for grading the quality and strength of the evidence and the strength of the treatment recommendations. Treatment recommendations reached unanimously by a panel of follicular lymphoma experts are: (1) autologous SCT is recommended as salvage therapy based on pre-rituximab data, with a significant improvement in overall survival (OS) and progression-free (PFS) survival; (2) autologous SCT is not recommended as first-line treatment for most patients because of no significant improvement in OS; (3) autologous SCT is recommended for transformed follicular lymphoma patients; (4) reduced intensity conditioning before allogeneic SCT appears to be an acceptable alternative to myeloablative regimens; (5) an HLA-matched unrelated donor appears to be as effective an HLA-matched related donor for reduced intensity conditioning allogeneic SCT. There are insufficient data to make a recommendation on the use of autologous SCT after rituximab-based salvage therapy. Eleven areas of needed research in the treatment of follicular lymphoma with SCT were identified and are presented in the review.

Subcritical solvent extraction of anthocyanins from dried red grape pomace.

Accelerated solvent extraction (ASE) was used to optimize and determine the effectiveness of an alternative, environmentally friendly extraction procedure using subcritical solvents to recover anthocyanins from freeze-dried, ground Sunbelt red grape pomace. Anthocyanins were extracted from pomace using the following ASE variables: pressure (6.8 MPa), one extraction cycle, and temperature (40, 60, 80, 100, 120, and 140 degrees C). Conventional solvent extraction with methanol/water/formic acid (60:37:3 v/v/v) was compared to four hydroethanolic solvents (10, 30, 50, and 70% ethanol in water, v/v). Anthocyanins in the extracts were identified and quantified by HPLC-MS and HPLC. There was an insignificant interaction between solvent and temperature (p = 0.0663). Solvents containing 70 and 50% ethanol in water extracted more total anthocyanins (463 and 455 mg/100 g of DW, respectively) than other solvents. The total amounts of anthocyanins extracted at 100 degrees C (450 mg/100 g of DW), 80 degrees C (436 mg/100 g of DW), and 120 degrees C (411 mg/100 g of DW) were higher than at the other temperatures. Solvents containing 70 and 50% ethanol in water extracted similar amounts of anthocyanins as conventional extraction solvent.

Subcritical solvent extraction of procyanidins from dried red grape pomace.

Procyanidins in dried Sunbelt ( Vitis labrusca L.) red grape pomace were extracted using accelerated solvent extraction (ASE) with pressure (6.8 MPa), one extraction cycle, and temperature (40, 60, 80, 100, 120, and 140 degrees C). Six ethanol/water solvents (0, 10, 30, 50, 70, and 90%, v/v) were compared to conventional extraction with acetone/water/acetic acid (70:29.5:0.5, v/v/v). Procyanidins in the extracts were identified by HPLC-ESI-MS/MS and contained degrees of polymerization (DP) of 1-5 (monomers through pentamers) and polymers (DP > 10). Generally, 50% ethanol/water (v/v) extracted more total procyanidins than other ethanol/water compositions, and contained up to 115% of total procyanidins extracted by the acetone-based conventional solvent. Additionally, 50% ethanol/water (v/v) extracted 205, 221, and 113% more epicatechin, catechin, and dimers, respectively, than conventional extraction. Results indicated greater extraction of low oligomeric procyanidins using 50% ethanol/water (v/v) solvent between 80 and 140 degrees C.

Review: Lipopolysaccharide biosynthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa causes serious nosocomial infections, and an important virulence factor produced by this organism is lipopolysaccharide (LPS). This review summarizes knowledge about biosynthesis of all three structural domains of LPS - lipid A, core oligosaccharide, and O polysaccharides. In addition, based on similarities with other bacterial species, this review proposes new hypothetical pathways for unstudied steps in the biosynthesis of P. aeruginosa LPS. Lipid A biosynthesis is discussed in relation to Escherichia coli and Salmonella, and the biosyntheses of core sugar precursors and core oligosaccharide are summarised. Pseudomonas aeruginosa attaches a Common Polysaccharide Antigen and O-Specific Antigen polysaccharides to lipid A-core. Both forms of O polysaccharide are discussed with respect to their independent synthesis mechanisms. Recent advances in understanding O-polysaccharide biosynthesis since the last major review on this subject, published nearly a decade ago, are highlighted. Since P. aeruginosa O polysaccharides contain unusual sugars, sugar-nucleotide biosynthesis pathways are reviewed in detail. Knowledge derived from detailed studies in the O5, O6 and O11 serotypes is applied to predict biosynthesis pathways of sugars in poorly-studied serotypes, especially O1, O4, and O13/O14. Although further work is required, a full understanding of LPS biosynthesis in P. aeruginosa is almost within reach.

The structural basis for catalytic function of GMD and RMD, two closely related enzymes from the GDP-D-rhamnose biosynthesis pathway.

The rare 6-deoxysugar D-rhamnose is a component of bacterial cell surface glycans, including the D-rhamnose homopolymer produced by Pseudomonas aeruginosa, called A-band O polysaccharide. GDP-D-rhamnose synthesis from GDP-D-mannose is catalyzed by two enzymes. The first is a GDP-D-mannose-4,6-dehydratase (GMD). The second enzyme, RMD, reduces the GMD product (GDP-6-deoxy-D-lyxo-hexos-4-ulose) to GDP-d-rhamnose. Genes encoding GMD and RMD are present in P. aeruginosa, and genetic evidence indicates they act in A-band O-polysaccharide biosynthesis. Details of their enzyme functions have not, however, been previously elucidated. We aimed to characterize these enzymes biochemically, and to determine the structure of RMD to better understand what determines substrate specificity and catalytic activity in these enzymes. We used capillary electrophoresis and NMR analysis of reaction products to precisely define P. aeruginosa GMD and RMD functions. P. aeruginosa GMD is bifunctional, and can catalyze both GDP-d-mannose 4,6-dehydration and the subsequent reduction reaction to produce GDP-D-rhamnose. RMD catalyzes the stereospecific reduction of GDP-6-deoxy-D-lyxo-hexos-4-ulose, as predicted. Reconstitution of GDP-D-rhamnose biosynthesis in vitro revealed that the P. aeruginosa pathway may be regulated by feedback inhibition in the cell. We determined the structure of RMD from Aneurinibacillus thermoaerophilus at 1.8 A resolution. The structure of A. thermoaerophilus RMD is remarkably similar to that of P. aeruginosa GMD, which explains why P. aeruginosa GMD is also able to catalyze the RMD reaction. Comparison of the active sites and amino acid sequences suggests that a conserved amino acid side chain (Arg185 in P. aeruginosa GMD) may be crucial for orienting substrate and cofactor in GMD enzymes.