Galacto-oligosaccharide production from lactose by Sirobasidium magnum CBS6803

N. ONISHI, I. KIRA AND K. YOKOZEKI. 1996. Galacto-oligosaccharide (Gal-OS) was produced from lactose by a yeast, Sirobasidium magnum CBS6803. With toluene-treated resting cells, 136 mg ml⁻¹ of Gal-OS was produced from 360 mg ml⁻¹ of lactose at 50°C for 42 h. Then, the yield of Gal-OS was increased by a culture method in which cell growth followed the enzymatic reaction : 224 mg ml⁻¹ of Gal-OS was produced at 30°C for 60 h. Finally, combination of the toluene-treated resting cells and glucose oxidase plus catalase was applied to improve productivity by the removal of a by-product, glucose, which inhibits the Gal-OS production, from the reaction mixture. In this case, 242 mg ml⁻¹ 4-galactosyl-lactose. of Gal-OS was produced at 50°C for 42 h without cell growth. The structure of the major product ws identified as 4-galactosyl-laetos.     

Galacto-oligosaccharide production using a recycling cell culture of Sterigmatomyces elviae CBS8119

Addition of small amounts of Fe²⁺, Zn²⁺, Cu²⁺ and thiamine-HCl to the culture medium was required for promoting the galacto-oligosaccharide (Gal-OS)-producing activity of Sterigmatomyces elviae CBS8119, when the concentration of yeast extract in the medium was lowered to 0·1 g l⁻¹. Galacto-oligosaccharide production using a recycling cell culture was performed in a medium containing 360 mg ml⁻¹ of lactose supplemented with optimal concentrations of Fe²⁺ (1·5 mg l⁻¹ of FeSO₄.7H₂O), Zn²⁺ (15 mg l⁻¹ of ZnSO₄.7H₂O), Cu²⁺ (0·5 mg l⁻¹ of CuSO₄.5H₂O) and thiamine-HCl (1 mg l⁻¹ ) . Galacto-oligosaccharide production was maintained at high levels during six cycles of production, with the amount of Gal-OS produced in each cycle being more than 216 mg ml⁻¹ (weight yield of more than 60%).     

Purification and properties of a novel thermostable galacto-oligosaccharide-producing beta-galactosidase from Sterigmatomyces elviae CBS8119.

A thermostable beta-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was solubilized from a cell wall preparation of Sterigmatomyces elviae CBS8119. The enzyme was purified to homogeneity by means of chromatography on DEAE-Toyopearl, Butyl-Toyopearl, Chromatofocusing, and p-aminobenzyl 1-thio-beta-D-galactopyranoside agarose columns. The molecular weight of the purified enzyme was estimated to be about 170,000 by gel filtration with a Highload-Superdex 200pg column and 86,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its isoelectric point, determined by polyacrylamide gel electrofocusing, was 4.1. The optimal temperature for enzyme activity was 85 degrees C. It was stable at temperatures up to 80 degrees C for 1 h. The optimal pH range for the enzyme was 4.5 to 5.0, it was stable at pH 2.5 to 7.0, and its activity was inhibited by Hg2+. The Km values for o-nitrophenyl-beta-D-galactopyranoside and lactose were 9.5 and 2.4 mM, respectively, and the maximum velocities for these substrates were 96 and 240 mumol/min per mg of protein, respectively. In addition, this enzyme possessed a high level of transgalactosylation activity. Galacto-oligosaccharides, including tri- and tetrasaccharides, were produced with a yield, by weight, of 39% from 200-mg/ml lactose.     

Suppressor analysis of mutations in the loop 2-3 motif of lactose permease: evidence that glycine-64 is an important residue for conformational changes.

A superfamily of transport proteins, which includes the lactose permease of Escherichia coli, contains a highly conserved motif, G-X-X-X-D/E-R/K-X-G-R/K-R/K, in the loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9. Previous analysis of this motif in the lactose permease (A. E. Jessen-Marshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995) has shown that the conserved glycine residue found at the first position in the motif (i.e., Gly-64) is important for transport function. Every substitution at this site, with the exception of alanine, greatly diminished lactose transport activity. In this study, three mutants in which glycine-64 was changed to cysteine, serine, and valine were used as parental strains to isolate 64 independent suppressor mutations that restored transport function. Of these 64 isolates, 39 were first-site revertants to glycine or alanine, while 25 were second-site mutations that restored transport activity yet retained a cysteine, serine, or valine at position 64. The second-site mutations were found to be located at several sites within the lactose permease (Pro-28 --> Ser, Leu, or Thr; Phe-29 --> Ser; Ala-50 --> Thr, Cys-154 --> Gly; Cys-234 --> Phe; Gln-241 --> Leu; Phe-261 --> Val; Thr-266 --> Iso; Val-367 --> Glu; and Ala-369 --> Pro). A kinetic analysis was conducted which compared lactose uptake in the three parental strains and several suppressor strains. The apparent Km values of the Cys-64, Ser-64, and Val-64 parental strains were 0.8 mM, 0.7 mM, and 4.6 mM, respectively, which was similar to the apparent Km of the wild-type permease (1.4 mM). In contrast, the Vmax values of the Cys-64, Ser-64, and Val-64 strains were sharply reduced (3.9, 10.1, and 13.2 nmol of lactose/min x mg of protein, respectively) compared with the wild-type strain (676 nmol of lactose/min x mg of protein). The primary effect of the second-site suppressor mutations was to restore the maximal rate of lactose transport to levels that were similar to the wild-type strains. Taken together, these results support the notion that Gly-64 in the wild-type permease is at a site in the protein which is important in facilitating conformational changes that are necessary for lactose translocation across the membrane. According to our tertiary model, this site is at an interface between the two halves of the protein.     

Lactose carrier mutants of Escherichia coli with changes in sugar recognition (lactose versus melibiose).

The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli. The lactose carrier transports the alpha-galactoside sugar melibiose as well as the beta-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside. Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport. Accumulation (uphill) of melibiose was completely defective in all of the mutants. Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.     

The role of helix VIII in the lactose permease of Escherichia coli: I. Cys-scanning mutagenesis.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VIII and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262-->Cys, Gly 268-->Cys, Asn 272-->Cys, Pro 280-->Cys, Asn 284-->Cys, Gly 287-->Cys, and Gly 288-->Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As expected (Ujwal ML, Sahin-Tóth M, Persson B, Kaback HR, 1994, Mol Membr Biol 1:9-16), Cys replacement for Glu 269 abolishes lactose transport. Immunoblot analysis reveals that the mutants are inserted into the membrane at concentrations comparable to C-less permease, with the exceptions of mutants Pro 280-->Cys, Gly 287-->Cys, and Lys 289-->Cys, which are expressed at reduced levels. The transport activity of the mutants is inhibited by N-ethylmaleimide (NEM) in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to inactivation by NEM at positions that cluster in manner indicating that they are on one face of an alpha-helix (Gly 262-->Cys, Val 264-->Cys, Thr 265-->Cys, Gly 268-->Cys. Asn 272-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, and Ala 279-->Cys). The results indicate that transmembrane domain VIII is in alpha-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S. Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ.     

The role of helix VIII in the lactose permease of Escherichia coli: II. Site-directed sulfhydryl modification.

Cys-scanning mutagenesis of putative transmembrane helix VIII in the lactose permease of Escherichia coli (Frillingos S. Ujwal ML, Sun J, Kaback HR, 1997, Protein Sci 6:431-437) indicates that, although helix VIII contains only one irreplaceable residue (Glu 269), one face is important for active lactose transport. In this study, the rate of inactivation of each N-ethylmaleimide (NEM)-sensitive mutant is examined in the absence or presence of beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). Remarkably, the analogue affords protection against inactivation with mutants Val 264-->Cys, Gly 268-->Cys, and Asn 272-->Cys, and alkylation of these single-Cys mutants in right-side-out membrane vesicles with [14C]NEM is attenuated by TDG. In contrast, alkylation of Thr 265-->Cys, which borders the three residues that are protected by TDG, is enhanced markedly by the analogue. Furthermore, NEM-labeling in the presence of the impermeant thiol reagent methanethiosulfonate ethylsulfonate demonstrates that ligand enhances the accessibility of position 265 to solvent. Finally, no significant alteration in NEM reactivity is observed for mutant Gly 262-->Cys, Glu 269-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, or Ala 279-->Cys. The findings indicate that a portion of one face of helix VIII (Val 264, Gly 268, and Asn 272), which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligand-induced conformational change.     

Measurement of beta(1-->3)-glucans in occupational and home environments with an inhibition enzyme immunoassay.

beta (1-->3)-Glucans are known for their potent ability to induce nonspecific inflammatory reactions and are believed to play a role in bioaerosol-induced respiratory symptoms. An inhibition enzyme immunoassay (EIA) was developed for the quantitation of beta (1-->3)-glucans in dust samples from occupational and residential environments. Immunospecific rabbit antibodies were produced by immunization with bovine serum albumin-conjugated laminarin [beta (1-->3)-glucan] and affinity chromatography on epoxy-Sepharose-coupled beta (1-->3)-glucans. The laminarin-based calibration curve in the inhibition EIA ranged from approximately 40 to 3,000 ng/ml (15 to 85% inhibition). Another beta (1-->3)-glucan (curdlan) showed a similar inhibition curve but was three to five times less reactive on a weight basis. Pustulan, presumed to be a beta (1-->6)-glucan, showed a parallel dose-response curve at concentrations 10 times higher than that of laminarin. Control experiments with NaIO4 and beta (1-->3)-glucanase treatment to destroy beta (1-->6)- and beta (1-->3)-glucan structures, respectively, indicate that the immunoreactivity of pustulan in the assay was due to beta (1-->3)-glucan and not to beta (1-->6)-glucan structures. Other polysaccharides, such as mannan and alpha (1-->6)-glucan, did not react in the inhibition EIA. Beta (1-->3)-Glucan extraction of dust samples in water (with mild detergent) was performed by heat treatment (120 degrees C) because aqueous extracts obtained at room temperature did not contain detectable beta (1-->3)-glucan levels. The assay was shown to detect heat-extractable beta (1-->3)-glucan in dust samples collected in a variety of occupational and environmental settings. On the basis of duplicate analyses of dust samples, a coefficient of variation of approximately 25% was calculated. It was concluded that the new inhibition EIA offers a useful method for indoor beta (1-->3)-glucan exposure assessment.     

The role of transmembrane domain III in the lactose permease of Escherichia coli.

Deletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. Similarly, replacement of this region en bloc with 23 contiguous Ala, Leu, or Phe residues abolishes active lactose transport. The observations suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less permease) each residue from Tyr 75 to Leu 99 was individually replaced with Cys. Twenty-one of the 25 mutants accumulate lactose to > 70% of the steady-state exhibited by C-less permease, and an additional 3 mutants transport to lower, but significant levels (40-60% of C-less). Cys replacement for Leu 76 results in low transport activity (18% of C-less). However, when placed in the wild-type background, mutant Leu 76-->Cys exhibits highly significant rates of transport (55% of wild type) and steady-state levels of lactose accumulation (65% of wild type). Immunoblots reveal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N-ethylmaleimide show that mutant Gly 96-->Cys is rapidly inactivated, whereas the other single-Cys mutants are not altered significantly by the alkylating agent. Moreover, the rate of inactivation of Gly 96-->Cys permease is enhanced at least 2-fold in the presence of beta-galactopyranosyl 1-thio-beta, D-galactopyranoside. The observations demonstrate that although no residue per se appears to be essential, structural properties of helix III are important for active lactose transport.     

Continuous production of lactosucrose by immobilized Sterigmatomyces elviae mutant

In this study, in order to develop a continuous production process of lactosucrose in a packed-bed reactor, Sterigmatomyces elviae ATCC 18894 was selected and mutated. The mutant strain of S. elviae showed 54.3% higher lactosucrose production than the wild type. Reaction conditions such as temperature, pH, substrate concentration and flow rate were also optimized. Under optimized reaction conditions (50 degrees C, pH 6.0, 25% sucrose and 25% lactose as substrate, flow rate 1.2 ml/min), the maximum concentration of lactosucrose (192 g/l) was obtained. In a packed-bed reactor, continuous production of lactosucrose was performed using S. elviae mutant immobilized in calcium alginate, and about 180 g/l of lactosucrose production was achieved for 48 days.     

Ligand-induced conformational changes in the lactose permease of Escherichia coli: evidence for two binding sites.

By using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right-side-out membrane vesicles containing Val 331-->Cys permease, lactose transport is inactivated by either N-ethylmaleimide (NEM) or 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Remarkably, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) enhances the rate of inactivation by CPM, a hydrophobic sulfhydryl reagent, whereas NEM inactivation is attenuated by the ligand. Val 331-->Cys permease was then purified and studied in dodecyl-beta,D-maltoside by site-directed fluorescence spectroscopy. The reactivity of Val 331-->Cys permease with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS) is not changed over a low range of TDG concentrations (< 0.8 mM), but the fluorescence of the MIANS-labeled protein is quenched in a saturable manner (apparent Kd approximately equal to 0.12 mM) without a change in emission maximum. In contrast, over a higher range of TDG concentrations (1-10 mM), the reactivity of Val 331-->Cys permease with MIANS is enhanced and the emission maximum of MIANS-labeled permease is blue shifted by 3-7 nm. Furthermore, the fluorescence of MIANS-labeled Val 331 -->Cys permease is quenched by both acrylamide and iodide, but the former is considerably more effective. A low concentration of TDG (0.2 mM) does not alter quenching by either compound, whereas a higher concentration of ligand (10 mM) decreases the quenching constant for iodide by about 50% and for acrylamide by about 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and some properties of alpha-L-arabinofuranosidase from Bacillus subtilis 3-6.

alpha-L-Arabinofuranosidase (EC 3.2.1.55) was purified from culture supernatant of Bacillus subtilis 3-6. The enzyme had a molecular weight of 61,000 and displayed maximum activity at pH 7.0 and 60 degrees C. It released arabinose from O-alpha-L-arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-x ylopyranos e (A1X2), O-beta-D-xylopyranosyl-(1-->4)-[O-alpha-L-arabinofuranosyl-(1-->3)]- O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (A1X3), and arabinan, but not from O-beta-D-xylopyranosyl-(1-->2)-O-alpha-L- arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-O-beta-D-xylopyr anosyl- (1-->4)-D-xylopyranose (A1X4), arabinoxylan, gum arabic, or arabinogalactan.     

Structural studies of lipooligosaccharides from Haemophilus ducreyi ITM 5535, ITM 3147, and a fresh clinical isolate, ACY1: evidence for intrastrain heterogeneity with the production of mutually exclusive sialylated or elongated glycoforms.

The structures of the lipooligosaccharides (LOSs) from Haemophilus ducreyi ITM 5535 and ITM 3147 and a fresh clinical isolate, ACY1, have been investigated. Oligosaccharides were obtained from phenol-water-extracted LOS by mild acid hydrolysis and were studied by methylation analysis, fast atom bombardment and electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy. The major oligosaccharide obtained from all strains was a nonasaccharide with the structure beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-D-Galp-(1-->4)-D-a lpha-D-Hepp- (1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp - (1-->3)]4)-L-alpha-D-Hepp-Kdo (Kdo stands for 3-deoxy-D-manno-octulosonic acid) and is thus identical to that identified as the major oligosaccharide in H. ducreyi ITM 2665 (E. K. H. Schweda, A. C. Sundström, L. M. Eriksson, J.A. Jonasson, and A. A. Lindberg, J. Biol. Chem. 269:12040-12048, 1994). Electrospray ionization mass spectrometry on O-deacylated LOS from H. ducreyi ITM 5535 obtained after treatment with anhydrous hydrazine gave evidence for the presence of a sialylated major compound, Neu5Ac alpha(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-D-Gal p- (1-->4)-D-alpha-D-Hepp-(1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp -(1-->2)-L- alpha-D-Hepp-(1-->3)]4)-L-alpha-D-Hepp-Kdo(P)-O-deacylated lipid A (Neu5Ac stands for N-acetylneuraminic acid). However, an even larger oligosaccharide could be isolated from all strains as a minor component, viz., the undecasaccharide beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-d-Galp-(1-->4)-beta-D-glcNAcp-(1-->3)-beta-D-Galp-(1-->4)-D-alpha-D-Hepp-(1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)]4-L-alpha-D-Hepp-Kdo, which represents an N-acetyl lactosamine disaccharide unit elongation of the LOS outer core. No Sialylation of this latter minor component undecasaccharide was detected.     

Stereochemistry during aflatoxin biosynthesis: conversion of norsolorinic acid to averufin.

A reaction sequence, norsolorinic acid (NA)-->averantin (AVN)-->5'-hydroxyaverantin (HAVN)-->averufin (AVR), is the early part of a biosynthetic pathway for aflatoxins. In this study, we determined the stereochemical relationship among these metabolites by using chiral high-performance liquid chromatography. In cell-free experiments using the cytosol fraction of Aspergillus parasiticus NIAH-26, (1'S)-AVN was exclusively produced from NA in the presence of NADPH. Also, only (1'S)-AVN, and not (1'R)-AVN, served as a substrate for the reverse reaction from AVN to NA. When the microsome fraction of NIAH-26 was incubated with (1'S)-AVN in the presence of NADPH, two HAVN diastereomers and one AVR enantiomer were formed, whereas these substances were never produced from (1'R)-AVN. Moreover, (1'S,5'R)-AVR was exclusively formed from both HAVN diastereomers by the cytosol fraction in the presence of NAD. The feeding experiments using this mutant showed that aflatoxins were produced from (1'S,5'R)-AVR but not from (1'R,5'S)-AVR. These results indicate that the enzymes involved in this pathway show strict stereospecificity to their substrates and that the configuration of (1'S,5'R)-AVR leading to the formation of aflatoxins is due to the stereospecificity of NA dehydrogenase which catalyzes the reaction between (1'S)-AVN and NA.     

Purification and characterization of a novel enzyme, alpha-neoagarooligosaccharide hydrolase (alpha-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107.

A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose], agarotriose [O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose], agarobiose [O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose], 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp. strain JT0107 and characterized. This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography. The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric. Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases. The optimum temperature and pH were 30 degrees C and 7.7, respectively. The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.     

Cysteine scanning mutagenesis of putative transmembrane helices IX and X in the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino-acid residue in putative transmembrane helices IX and X and the short intervening loop was systematically replaced with Cys (from Asn-290 to Lys-335). Thirty-four of 46 mutants accumulate lactose to high levels (70-100% or more of C-less), and an additional 7 mutants exhibit lower but highly significant lactose accumulation. As expected (see Kaback, H.R., 1992, Int. Rev. Cytol. 137A, 97-125), Cys substitution for Arg-302, His-322, or Glu-325 results in inactive permease molecules. Although Cys replacement for Lys-319 or Phe-334 also inactivates lactose accumulation, Lys-319 is not essential for active lactose transport (Sahin-Tóth, M., Dunten, R.L., Gonzalez, A., & Kaback, H.R., 1992, Proc. Natl. Acad. Sci. USA 89, 10547-10551), and replacement of Phe-334 with leucine yields permease with considerable activity. All single-Cys mutants except Gly-296 --> Cys are present in the membrane in amounts comparable to C-less permease, as judged by immunological techniques. In contrast, mutant Gly-296 --> Cys is hardly detectable when expressed at a relatively low rate from the lac promoter/operator but present in the membrane in stable form when expressed at a high rate from T7 promoter. Finally, studies with N-ethylmaleimide (NEM) show that only a few mutants are inactivated significantly. Remarkably, the rate of inactivation of Val-315 --> Cys permease is enhanced at least 10-fold in the presence of beta-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or an H+ electrochemical gradient (delta mu-H+). The results demonstrate that only three residues in this region of the permease -Arg-302, His-322, and Glu-325-are essential for active lactose transport. Furthermore, the enhanced reactivity of the Val-315 --> Cys mutant toward NEM in the presence of TDG or delta mu-H+ probably reflects a conformational alteration induced by either substrate binding or delta mu-H+.     

Characterization of a transposon Tn916-generated mutant of Haemophilus ducreyi 35000 defective in lipooligosaccharide biosynthesis.

To define the role of the surface lipooligosaccharide (LOS) of Haemophilus ducreyi in the pathogenesis of chancroid, Tn916 mutants of H. ducreyi 35000 defective in expression of the murine monoclonal antibody (MAb) 3F11 epitope on H. ducreyi LOS were identified by immunologic screening. One mutant, designated 1381, has an LOS which lacks the MAb 3F11 epitope and migrates with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene disrupted by the Tn916 element in strain 1381 was identified by cloning the sequences flanking the Tn916 element. The sequences were then used to probe a lambda DASHII genomic library. In strain 1381, Tn916 interrupts a gene which encodes an open reading frame (ORF) with an Mr of 40,246. This ORF has homology to the product of the rfaK gene of Escherichia coli. The major LOS glycoform produced by strain 1381 was analyzed by using a combination of mass spectrometry, linkage and composition analysis, and 1H nuclear magnetic resonance spectroscopy. The major LOS species was found to terminate in a single glucose attached to the heptose (L-glycero-D-manno-heptose, or Hep) trisaccharide core. In the wild-type strain 35000, glucose serves as the acceptor for the addition of the D-glycero-D-manno-heptose (or DDHep), which extends to form the mature branch of the H. ducreyi LOS. This mature oligosaccharide is in turn partially capped by the addition of sialic acid (NeuAc), i.e., NeuAc2 alpha-->3Gal beta1-->4GlcNAc beta1-->3Gal beta1-->4DDHep alpha1-->6Glc beta1 (W. Melaugh et al., Biochemistry 33:13070-13078, 1994). Since this LOS terminates prior to the addition of the branch DD-heptose, this gene is likely to encode the D-glycero-D-manno-heptosyltransferase. Strain 1381 exhibits a significant reduction in adherence to and invasion of primary human keratinocytes. This defect was complemented by the cloned heptosyltransferase gene, indicating that the terminal portion of the LOS oligosaccharide plays an important role in adherence to human keratinocytes.     

A virulent isolate of Salmonella enteritidis produces a Salmonella typhi-like lipopolysaccharide.

The lipopolysaccharide (LPS) of Salmonella enteritidis has been implicated as a virulence factor of this organism. Therefore, the LPS from a stable virulent isolate, SE6-E21, was compared with that from an avirulent isolate, SE6-E5. The LPSs were extracted, and the high-molecular-weight (HMW) LPS was separated from the low-molecular-weight (LMW) LPS for both isolates. Both the HMW and LMW LPSs were characterized by glycosyl composition and linkage analyses. Immunochemical characterization was performed by Western blotting using factor 9 antiserum and using S. typhimurium antiserum which contains factors 1, 4, 5, and 12(2). In addition, the polysaccharides released by mild acid hydrolysis were isolated and subjected to hydrolysis by bacteriophage P22, which contains endorhamnosidase activity. The resulting oligosaccharides were purified by using Bio-Gel P4 gel permeation chromatography and characterized by nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry (FAB-MS), tandem MS-MS, and matrix-assisted laser desorption time of flight MS. The results show that the HMW LPS O-antigen polysaccharides from both isolates are comprised of two different repeating units, -[-->2)-[alpha-Tyvp-(1-->3)]beta-D-Manp-(1-->4)-alpha-L-R hap-(1-->3)-alpha-D-Galp-(1-->]- (structure I) and [-->2)-[alpha-Tyvp-(1-->3)]beta-D-Manp-(1-->4)-alpha--L-R hap-(1-->3)-[alpha-D-Glcp-(1-->4)]alpha-D-Galp-(1-->]- (structure II). The LMW LPSs from both isolates contains truncated O-antigen polysaccharide which is comprised of only structure I. In the virulent SE6-E21 isolate, the HMW LPS has a structure I/II ratio of 1:1, while in the avirulent SE6-E5 isolate, this ratio is 7:1. While the 7:1 ratio represents the published level of glucosylation for S. enteritidis LPS as well as for S. enteritidis LPS purchased from Sigma Chemical Co., the 1:1 ratio found for the virulent SE6-E21 is identical to the high level of glucosylation reported for S. typhi LPS. Thus, the LPS from the virulent SE6-E21 isolate produces an S. typhi-like LPS. Furthermore, the amount of O-antigen polysaccharide in SE6-E21 was twice that in SE6-E5.     

Autoprocessing of the protease that degrades small, acid-soluble proteins of spores of Bacillus species is triggered by low pH, dehydration, and dipicolinic acid.

The sequence-specific protease (termed GPR) that degrades small, acid-soluble proteins (SASP) during germination of spores of Bacillus species is synthesized during sporulation as an inactive precursor termed P46. Approximately 2 h later in sporulation, P46 is converted proteolytically to a smaller form, termed P41, which is active in vitro, but which does not act significantly on SASP in vivo until spore germination is initiated. While it appears likely that P46-->P41 conversion is an autoprocessing event, the mechanisms regulating P46-->P41 conversion in vivo are not clear. In this work we found that P46-->P41 conversion in vitro was stimulated tremendously in an allosteric manner by pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) plus Ca2+ but not by Ca2+ in combination with a variety of DPA analogs. The processing reaction stimulated by Ca(2+)-DPA was seen at pH 5.1 but not at pH 6.2 or 7, and under these conditions P46-->P41 conversion exhibited a linear time course and was a first-order reaction, indicative of an intramolecular autoprocessing reaction. At pH 5.1, P46-->P41 conversion was stimulated markedly by very high ionic strength. At pHs from 5.1 to 6.6, P46-->P41 conversion also occurred when P46 was dehydrated to approximately 54% relative humidity. This processing was stimulated markedly when dehydration was carried out in the presence of DPA and NaCl but was greatly decreased when dehydration was to 10, 33, or 75% relative humidity. Since previous work has shown that P(46)-->P(41) processing in vivo takes place (i) after a fall in forespore pH to 6.3 to 6.9 and approximately in parallel with (ii) DPA accumulation by the forespore and (iii) dehydration of the forespore, out new finings in vitro suggest that these three changes may synergistically trigger P(46)-->P(41) autoprocessing in the developing forespore. Presumably the conditions in vivo during this authoprocessing preclude significant attack of the P(41) generated on its SASP substrates.     

Cysteine scanning mutagenesis of the N-terminal 32 amino acid residues in the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in the hydrophilic N-terminus and the first putative transmembrane helix was systematically replaced with Cys (from Tyr-2 to Trp-33). Twenty-three of 32 mutants exhibit high lactose accumulation (70-100% or more of C-less), and an additional 8 mutants accumulate to lower but highly significant levels. Surprisingly, Cys replacement for Gly-24 or Tyr-26 yields fully active permease molecules, and permease with Cys in place of Pro-28 also exhibits significant transport activity, although previous mutagenesis studies on these residues suggested that they may be required for lactose transport. As expected, Cys replacement for Pro-31 completely inactivates, in agreement with previous findings indicating that "helix-breaking" propensity at this position is necessary for full activity (Consler TG, Tsolas O, Kaback HR, 1991, Biochemistry 30:1291-1297). Twenty-nine mutants are present in the membrane in amounts comparable to C-less permease, whereas membrane levels of mutants Tyr-3-->Cys and Phe-12-->Cys are slightly reduced, as judged by immunological techniques. Dramatically, mutant Phe-9-->Cys is hardly detectable when expressed from the lac promoter/operator at a relatively low rate, but is present in the membrane in a stable form when expressed at a high rate from the T7 promoter. Finally, studies with N-ethylmalemide show that 6 Cys-replacement mutants that cluster at the C-terminal end of putative helix I are inactivated significantly.(ABSTRACT TRUNCATED AT 250 WORDS)