Structural elucidation of the lipopolysaccharide core regions of the wild-type strain PAO1 and O-chain-deficient mutant strains AK1401 and AK1012 from Pseudomonas aeruginosa serotype O5

Lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype O5 wild-type strain PAO1 and derived rough-type mutant strains AK1401 and AK1012 was isolated by a modified phenol/chloroform/petroleum-ether extraction method. Deoxycholate/PAGE of the LPS from the rough mutant AK1401 indicated two bands near the dye front with mobilities similar to those of the parent strain, indicating that both LPS contain a complete core and a species comprising a core and one repeating unit. Composition analysis of the LPS from strains PAO1 and AK1401 indicated that the complete core oligosaccharide was composed of D-glucose (four units), L-rhamnose (one unit), 2-amino-2-deoxy-D-galactose (one unit), L-glycero-D-manno-heptose (Hep; two units), 3-deoxy-D-manno-octulosonic acid (Kdo; two units), L-alanine (one unit) and phosphate (three units). The glycan structure of the LPS was determined by one-dimensional and two-dimensional (2D) NMR techniques in combination with MS-based methods on oligosaccharide samples obtained from the LPS by delipidation procedures. The locations of three phosphomonoester groups on the first heptose residue were established by a two-dimensional 31P (omega1)-half-filtered COSY experiment on the reduced core oligosaccharide sample of the LPS from the wild-type strain. The presence of a 7-O-carbamoyl substituent was observed on the second heptose. The structure of the core region of the O-chain-deficient LPS from P. aeruginosa serotype 05 is as follows: [structure: see text] where R1 is beta-D-Glcp-(1-->2)-alpha-L-Rhap-(1-->6)-alpha-D-Glcp-(1--> and R2 is alpha-D-Glcp-(1-->6)-beta-D-Glcp-(1->. A structural model is presented that is also representative of that for P. aeruginosa serotype O6 LPS. A revised structure for the serotype O6 mutant strain A28 is presented.     

Structural roles of the spore coat proteins in Dictyostelium discoideum

The integrity of spores formed by mutant strains of Dictyostelium discoideum lacking the major spore coat proteins, SP96, SP70, or SP60, was compared to that of wild-type strains. Single, double, and triple knock-out strains developed normally and produced spores which were indistinguishable from wild-type spores by light or electron microscopy. However, the mutant strains were susceptable to staining with the lectin, ricin A, which recognizes a galactose-rich polysaccharide that is normally hidden by overlying spore coat proteins. The intensity of staining with fluorescently labeled ricinA increased as the spore coat proteins were incrementally lost. While these results indicate that the major outer spore coat proteins are not essential for the construction of a multi-layered spore coat in Dictyostelium, they show that the spores are more porous which might make them at risk to predators before germination.     

Neuromuscular blocking agents in the emergency department

The pbp4 gene of a Staphylococcus aureus strain selected stepwise in vitro for growth on increasing concentrations of penicillin and of its susceptible parent strain showed overall identity except in the promoter region. In the mutant a deletion upstream of the pbp4 structural gene removed 90 nucleotides (nt) that were framed by a 12 nt inverted repeat. This deletion occurred in step 4 of the in vitro selection procedure and was paralleled by a significant increase in the penicillin-binding protein 4 (PBP4) production. The in vitro step selected mutant showed a remarkable increase in the cross-linking of the peptidoglycan compared to its parent. This was linked to morphological changes in the appearance of the cells, which were surrounded by a very thick and fuzzy cell wall.     

Inhibition of cell wall turnover and autolysis by vancomycin in a highly vancomycin-resistant mutant of Staphylococcus aureus

A highly vancomycin-resistant mutant (MIC = 100 microg/ml) of Staphylococcus aureus, mutant VM, which was isolated in the laboratory by a step-pressure procedure, continued to grow and synthesize peptidoglycan in the presence of vancomycin (50 microg/ml) in the medium, but the antibiotic completely inhibited cell wall turnover and autolysis, resulting in the accumulation of cell wall material at the cell surface and inhibition of daughter cell separation. Cultures of mutant VM removed vancomycin from the growth medium through binding the antibiotic to the cell walls, from which the antibiotic could be quantitatively recovered in biologically active form. Vancomycin blocked the in vitro hydrolysis of cell walls by autolytic enzyme extracts, lysostaphin and mutanolysin. Analysis of UDP-linked peptidoglycan precursors showed no evidence for the presence of D-lactate-terminating muropeptides. While there was no significant difference in the composition of muropeptide units of mutant and parental cell walls, the peptidoglycan of VM had a significantly lower degree of cross-linkage. These observations and the results of vancomycin-binding studies suggest alterations in the structural organization of the mutant cell walls such that access of the vancomycin molecules to the sites of wall biosynthesis is blocked.     

Cloning and characterization of the fmt gene which affects the methicillin resistance level and autolysis in the presence of triton X-100 in methicillin-resistant Staphylococcus aureus

In methicillin-resistant Staphylococcus aureus (MRSA) strains, Triton X-100 reduced the oxacillin resistance level, although the degree of reduction varied from strain to strain. To study the responses of MRSA strains to Triton X-100, we isolated a Tn551 insertion mutant of the COL strain that became more susceptible to oxacillin in the presence of 0.02% Triton X-100. The Tn551 insertion of the mutant was transduced back to the parent strain, other MRSA strains (strains KSA8 and NCTC 10443), and methicillin-susceptible strain RN450. All transductants of MRSA strains had reduced levels of resistance to oxacillin in the presence of 0.02% Triton X-100, while those of RN450 did not. Tn551 mutants of KSA8 and NCTC 10443 also had reduced levels of resistance in the absence of 0.02% Triton X-100. The autolysis rates of the transductants in the presence of 0.02% Triton X-100 were significantly increased. Amino acid analysis of peptidoglycan and testing of heat-inactivated cells for their susceptibilities to several bacteriolytic enzymes showed that there were no significant differences between the parents and the respective Tn551 mutants. The Tn551 insertion site mapped at a location different from the previously identified fem and llm sites. Cloning and sequencing showed that Tn551 had inserted at the C-terminal region of a novel gene designated fmt. The putative Fmt protein showed a hydropathy pattern similar to that of S. aureus penicillin-binding proteins and contained two of the three conserved motifs shared by penicillin-binding proteins and beta-lactamases, suggesting that fmt may be involved in cell wall synthesis.     

Human pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1 alpha. Characterization and kinetic analysis of wild-type and mutant enzymes

Pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor-1 alpha is a protein with two different functions. We have overexpressed and purified the human wild-type protein, and its Cys81Ser and Cys81Arg mutants. The Cys81Arg mutant has been proposed to be causative in a hyperphenylalaninaemic patient [Citron, B. A., Kaufman, S., Milstien, S., Naylor, E. W., Greene, C. L. & Davis, M. D. (1993) Am. J. Hum. Genet. 53, 768-774]. The dehydratase behaves as a tetramer on gel filtration, while cross-linking experiments showed mono-, di-, tri-, and tetrameric forms, irrespective of the presence of the single Cys81. Sulfhydryl-modifying reagents did not affect the activity, but rather showed that Cys81 is exposed. Various pterins bind and quench the tryptophan fluorescence suggesting the presence of a specific binding site. The fluorescence is destroyed upon light irradiation. Wild-type and the Cys81Ser protein enhance the rate of the phenylalanine hydroxylase assay approximately 10-fold, a value similar to that of native dehydratase from rat liver; the Cys81Arg mutant, in contrast, has significantly lower activity. This is compatible with the hypothesis that the dehydratase is a rate-limiting factor for the in vivo phenylalanine hydroxylase reaction. The three proteins enhance the spontaneous dehydration of the synthetic substrate 6,6-dimethyl-7,8-dihydropterin-4a-carbinolamine approximately 50-70-fold at 4 degrees C and pH 8.5. The results are discussed in view of the recently solved three-dimensional structure of the enzyme [Ficner, R., Sauer, U. W., Stier, G. & Suck, D. (1995) EMBO J. 14, 2032-2042].     

Site-directed mutagenesis of yeast eEF1A. Viable mutants with altered nucleotide specificity

Site-directed mutants of eEF1A (formerly eEF-1alpha) were generated using a modification of a highly versatile yeast shuttle vector (Cavallius, J., Popkie, A. P., and Merrick, W. C. (1997) Biochim. Biophys. Acta 1350, 345-358). The nucleotide specificity sequence NKMD (residues number 153-156) was targeted for mutagenesis, and the following mutants were obtained: N153D (DKMD), N153T (TKMD), D156N (NKMN), D156W (NKMW), and the double mutant N153T,D156E (TKNE). All of the yeast strains containing the mutant eEF1As as the sole source of eEF1A were viable except for the N153D mutant. Most of the purified mutant eEF1As had specific activities in the poly(U)-directed synthesis of polyphenylalanine similar to wild type, although with a Km for GTP increased by 1-2 orders of magnitude. The mutants showed a reduced rate of GTP hydrolysis, and most displayed misincorporation rates greater than wild type. The mutant NKMW eEF1A showed unusual properties. The yeast strain was temperature sensitive for growth, although the purified protein was not. Second, this form of eEF1A was 10-fold more accurate in protein synthesis, and its rate of GTP hydrolysis was about 20% of wild type. In total, the wild-type protein contains the most optimal nucleotide specificity sequence, NKMD, and even subtle changes in this sequence have drastic consequences on eEF1A function in vitro or yeast viability.     

Structural studies on the short-chain lipopolysaccharide of Vibrio cholerae O139 Bengal

A Vibrio cholerae O139 strain, MO10-T4, lacking capsular polysaccharide, produces a short-chain lipopolysaccharide (LPS), similar to enterobacterial SR strains. It was studied by acidic and alkaline degradation, dephosphorylation, sugar and methylation analysis, high-performance anion-exchange chromatography, one- and two-dimensional 1H-, 13C-, and 31P-NMR spectroscopy, and electrospray ionization mass spectrometry. The following structure was proposed for the core region of the LPS: [structure: see text] where PEtn stands for 2-aminoethyl phosphate, Fru for fructose, Hep for L-glycero-D-manno-heptose, and Kdo for 3-deoxy-D-manno-octulosonic acid; unless otherwise stated, the monosaccharide residues are D and present in the pyranose form. An O-acetyl group is present on a secondary position, tentatively O4 of the alpha-linked glucosyl group. Some LPS species contain an additional putative fructose residue whose location remains unknown. An O139-negative mutant strain, Bengal-2R, derived from V. cholerae O139, has also been investigated and shown to produce an O-antigen-lacking LPS similar to those from enterobacterial R strains, some of the LPS species containing the same core region as the strain MO10-T4 LPS and the other lacking the lateral heptose residue. The carbohydrate backbone core structure is the same for the V. cholerae O139 and V. cholerae O1 LPS, thus confirming the close relation between these bacteria; however, the 2-aminoethyl phosphate, the O-acetyl group, and the second fructose residue have not been reported for the O1 LPS. In the V. cholerae O139 strain MO10-T4 LPS, a short O-side chain is attached at position 3 of the 7-substituted heptose residue and has the same structure as one repeating unit of the V. cholerae O139 capsular polysaccharide. Some details of the structure proposed are at variance with those recently published for another V. cholerae O139 strain [Cox, A. D., Brisson, J.-R., Varma, V. & Perry, M. B. (1996) Carbohydr. Res. 290, 43-58; Cox, A. D. & Perry, M. B. (1996) Carbohydr. Res. 290, 59-65.]     

Identification of histidine 118 in the D1 polypeptide of photosystem II as the axial ligand to chlorophyll Z

Chlorophyll Z (ChlZ) is a redox-active chlorophyll (Chl) which is photooxidized by low-temperature (<100 K) illumination of photosystem II (PSII) to form a cation radical, ChlZ+. This cofactor has been proposed to be an "accessory" Chl in the PSII reaction center and is expected to be buried in the transmembrane region of the PSII complex, but the location of ChlZ is unknown. A series of single-replacement site-directed mutants of PSII were made in which each of two potentially Chl-ligating histidines, D1-H118 or D2-H117, was substituted with amino acids which varied in their ability to coordinate Chl. Assays of the wild-type and mutant strains showed parallel phenotypes for the D1-118 and D2-117 mutants: noncoordinating or poorly coordinating residues at either position decreased photosynthetic competence and impaired assembly of PSII complexes. Only the mutants substituted with glutamine (D1-H118Q and D2-H117Q) had phenotypes comparable to the wild-type strain. The ChlZ+ cation was characterized by low-temperature electron paramagnetic resonance (EPR), near-infrared (IR) absorbance, and resonance Raman (RR) spectroscopies in wild-type, H118Q, and H117Q PSII core complexes. The quantum yield of ChlZ+ formation is the same (approximately 2.5% per saturating flash at 77 K) for wild-type, H118Q, and H117Q, indicating that its efficiency of photooxidation is unchanged by the mutations. Similarly, the EPR and near-IR absorbance spectra of ChlZ+ are insensitive to the mutations made at D1-118 and D2-117. In contrast, the RR signature of ChlZ+ in H118Q PSII, obtained by selective near-IR excitation into the ChlZ+ cation absorbance band, is significantly altered relative to wild-type PSII while the RR spectrum of ChlZ+ in the H117Q mutant remains identical to wild-type. Shifts in the RR spectrum of ChlZ+ in H118Q reflect a change in the structure of the Chl ring, most likely due to a perturbation of the core size and/or extent of doming caused by a change in the axial ligand to Mg(II). Thus, we conclude that the axial ligand to ChlZ is H118 of the D1 polypeptide. Furthermore, we propose that H117 of the D2 polypeptide is the ligand to a homologous redox-inactive accessory Chl which we term ChlD. The Chl Z and D terminology reflects the 2-fold structural symmetry of PSII which is apparent in the redox-active tyrosines, YZ and YD, and the active/inactive branch homology of the D1/D2 polypeptides with the L/M polypeptides of the bacterial reaction center.     

Peptidoglycan structural dynamics during germination of Bacillus subtilis 168 endospores

Peptidoglycan structural dynamics during endospore germination of Bacillus subtilis 168 have been examined by muropeptide analysis. The first germination-associated peptidoglycan structural changes are detected within 3 min after the addition of the specific germinant L-alanine. We detected in the spore-associated material new muropeptides which, although they have slightly longer retention times by reversed-phase (RP)-high-pressure liquid chromatography (HPLC) than related ones in dormant spores, show the same amino acid composition and molecular mass. Two-dimensional nuclear magnetic resonance (NMR) analysis shows that the chemical changes to the muropeptides on germination are minor and are probably limited to stereochemical inversion. These new muropeptides account for almost 26% of the total muropeptides in spore-associated material after 2 h of germination. The exudate of germinated spores of B. subtilis 168 contains novel muropeptides in addition to those present in spore-associated material. Exudate-specific muropeptides have longer retention times, have no reducing termini, and exhibit a molecular mass 20 Da lower than those of related reduced muropeptides. These new products are anhydro-muropeptides which are generated by a lytic transglycosylase, the first to be identified in a gram-positive bacterium. There is also evidence for the activity of a glucosaminidase during the germination process. Quantification of muropeptides in spore-associated material indicates that there is a heterogeneous distribution of muropeptides in spore peptidoglycan. The spore-specific residue, muramic delta-lactam, is proposed to be a major substrate specificity determinant of germination-specific lytic enzymes, allowing cortex hydrolysis without any effect on the primordial cell wall.     

The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity

The murI gene of Escherichia coli was recently identified on the basis of its ability to complement the only mutant requiring D-glutamic acid for growth that had been described to date: strain WM335 of E. coli B/r (P. Doublet, J. van Heijenoort, and D. Mengin-Lecreulx, J. Bacteriol. 174:5772-5779, 1992). We report experiments of insertional mutagenesis of the murI gene which demonstrate that this gene is essential for the biosynthesis of D-glutamic acid, one of the specific components of cell wall peptidoglycan. A special strategy was used for the construction of strains with a disrupted copy of murI, because of a limited capability of E. coli strains grown in rich medium to internalize D-glutamic acid. The murI gene product was overproduced and identified as a glutamate racemase activity. UDP-N-acetylmuramoyl-L-alanine (UDP-MurNAc-L-Ala), which is the nucleotide substrate of the D-glutamic-acid-adding enzyme (the murD gene product) catalyzing the subsequent step in the pathway for peptidoglycan synthesis, appears to be an effector of the racemase activity.     

Glu-320 and Asp-323 are determinants of the CYP4A1 hydroxylation regiospecificity and resistance to inactivation by 1-aminobenzotriazole

Little information is available on the active site structure of the CYP4A family of enzymes or the mechanism by which their omega-hydroxylation regiospecificity is enforced. We report here that the E320A, D323E, and E320/D323E mutations decrease the catalytic rate of CYP4A1 approximately 5-fold and cause up to a 10-fold shift from omega- to (omega-1)-hydroxylation. The decreased catalytic rate is due to an increase in the uncoupled reduction of molecular oxygen. Tighter binding of 1- and 4-substituted imidazoles to the double mutant than to the other proteins suggests that its active site is less constrained. The reaction of these proteins with phenyldiazene causes heme degradation without the detectable formation of a phenyl-iron complex. CYP4A1 and its E320A mutant are not inactivated by 1-aminobenzotriazole (1-ABT), but the D323E and E320A/D323E mutants are inactivated. The resistance of purified CYP4A1 to inactivation by 1-ABT is surprising in view of the fact that 1-ABT causes the loss of the omega-hydroxylase activity both in microsomal preparations and in vivo. Collectively, the results establish that Glu-320, and particularly Asp-323, help to define the active site dimensions, the degree of coupled versus uncoupled versus uncoupled turnover, the omega-versus (omega-1)-hydroxylation regiospecificity, and the susceptibility to inactivation by mechanism-based inhibitors. Furthermore, they provide experimental evidence for a structural analogy between the CYP4A1 and P450BM-3 active sites.     

The assembly system for the outer core portion of R1- and R4-type lipopolysaccharides of Escherichia coli. The R1 core-specific beta-glucosyltransferase provides a novel attachment site for O-polysaccharides

The major core oligosaccharide biosynthesis operons from prototype Escherichia coli strains displaying R1 and R4 lipopolysaccharide core types were polymerase chain reaction-amplified and analyzed. Comparison of deduced products of the open reading frames between the two regions indicate that all but two share total similarities of 94% or greater. Core oligosaccharide structures resulting from nonpolar insertion mutations in each gene of the core OS biosynthesis operon in the R1 strain allowed assignment of all of the glycosyltransferase enzymes required for outer core assembly. The difference between the R1 and R4 core oligosaccharides results from the specificity of the WaaV protein (a beta1, 3-glucosyltransferase) in R1 and WaaX (a beta1, 4-galactosyltransferase) in R4. Complementation of the waaV mutant of the R1 prototype strain with the waaX gene of the R4 strain converted the core oligosaccharide from an R1- to an R4-type lipopolysaccharide core molecule. Aside from generating core oligosaccharide specificity, the unique beta-linked glucopyranosyl residue of the R1 core plays a crucial role in organization of the lipopolysaccharide. This residue provides a novel attachment site for lipid A-core-linked polysaccharides and distinguishes the R1-type LPS from existing models for enterobacterial lipopolysaccharides.     

A higher control center of locomotor behavior in the Drosophila brain

In order to elucidate the behavioral significance of the central complex (CC), we have examined walking in 15 Drosophila mutant strains belonging to eight independent X-linked genes that affect the structure of the CC. Compared to four different wild-type strains, all are impaired either in a general or in a paradigm-dependent manner. Behavioral deficits concern walking activity, walking speed, or "straightness of walking" as measured in an object fixation task, in fast phototaxis, and in negative geotaxis. Behavioral deficits of three strains with mutations in different genes were studied in detail using mosaic analysis and high-speed cinematography. In all cases the focus for declining walking activity is located in the brain and is fully correlated with the respective defect of the CC. A high correlation between the degree of the behavioral impairment and the severity of the structural defect in two strains further adds to the evidence. Declining walking activity is not an unspecific side effect of structural brain defects, as steady walking is observed in structural mutants of the visual system and mushroom bodies. In mutant flies no-bridgeKS49 (nob), step size as a function of the stepping period is reduced. The focus of the resulting reduced average and maximum walking speeds resides in the brain and, again, the behavioral impairment fully correlates with the structural defects of the CC. While no indication is found for a role of the CC in setting up the basic stepping rhythm in straight walking (a respective phenotype in mutant central-complexKS181 flies resides in the ventral ganglion), a role in turning and start/stop maneuvers is suggested by aberrations in the stepping pattern of nob flies during such episodes.     

U1 small nuclear ribonucleoprotein particle-protein interactions are revealed in Saccharomyces cerevisiae by in vivo competition assays

Two highly conserved regions of the 586-nucleotide yeast (Saccharomyces cerevisiae) U1 small nuclear RNA (snRNA) can be mutated or deleted with little or no effect on growth rate: the universally conserved loop II (corresponding to the metazoan A loop) and the yeast core region (X. Liao, L. Kretzner, B. Séraphin, and M. Rosbash, Genes Dev. 4:1766-1774, 1990). To examine the contribution of these regions to U1 small nuclear ribonucleoprotein particle (snRNP) activity, a competitor U1 gene, encoding a nonfunctional U1 snRNA molecule, was introduced into a number of strains carrying a U1 snRNA gene with loop II or yeast core mutations. The presence of the nonfunctional U1 gene lowered the growth rate of these mutant strains but not wild-type strains, consistent with the notion that mutant U1 RNAs are less active than wild-type U1 snRNAs. A detailed analysis of the U1 snRNA levels and half-lives in a number of merodiploid strains suggests that these mutant U1 snRNAs interact with U1 snRNP proteins less well than do their wild-type counterparts. Competition for protein factors during snRNP assembly could account for a number of previous observations in both yeast and mammalian cells.     

A Serum Response Factor homolog is required for spore differentiation in Dictyostelium

A homolog of the Serum Response Factor (SRF) has been isolated from Dictyostelium discoideum and its function studied by analyzing the consequences of its gene disruption. The MADS-box region of Dictyostelium SRF (DdSRF) is highly conserved with those of the human, Drosophila and yeast homologs. srfA is a developmentally regulated gene expressed in prespore and spore cells. This gene plays an essential role in sporulation as its disruption leads to abnormal spore morphology and loss of viability. The mutant spores were round and cellulose deposition seemed to be partially affected. Initial prestalk and prespore cell differentiation did not seem to be compromised in the mutant since the expression of several cell-type-specific markers were found to be unaffected. However, the mRNA level of the spore marker spiA was greatly reduced. Activation of the cAMP-dependent protein kinase (PKA) by 8-Br-cAMP was not able to fully bypass the morphological defects of srfA- mutant spores, although this treatment induced spiA mRNA expression. Our results suggest that DdSRF is required for full maturation of spores and participates in the regulation of the expression of the spore-coat marker spiA and probably other maturation genes necessary for proper spore cell differentiation.