) describe two unique ␤-N-acetylglucosaminidases from Vibrio furnissii. A third, ExoII, is reported here. The gene, exoII, was cloned into Escherichia coli, sequenced, and ExoII purified to apparent homogeneity (36 kDa). The molecular weight and N-terminal 16 amino acids of the protein conform to the predicted sequence. ExoII exhibited unique substrate specificity. It rapidly cleaved p-nitrophenyl and 4-methylumbelliferyl ␤-GlcNAc, was slightly active with p-nitrophenyl-␤-GalNAc, and was inactive with all other GlcNAc derivatives tested, including N,N-diacetylchitobiose and (GlcNAc) n , n ‫؍‬ 3-6. Unlike GlcNAc (K i , 210 M), (GlcNAc) n are poor inhibitors of ExoII.The predicted protein sequence is unique among ␤-Nacetylglucosaminidases excepting Cht60, recently cloned from a marine Alteromonas (Tsujibo, H., Fujimoto, K., Tanno, H., Miyamoto, K., Imada, C., Okami, Y., and Inamori, Y. (1994) Gene (Amst.) 146, 111-115). Cht60, a chitobiase, is 26.9% identical to ExoII in a 182-amino acid overlap, but the two enzymes differ in substrate specificity and other properties. ExoII shares similarity with five bacterial and yeast ␤-glucosidases, up to 44% identity in the 25-amino acid catalytic domain. By analogy, ExoII may play a role in signal transduction between invertebrate hosts and V. furnissii.The chitin catabolic cascade of the marine bacterium Vibrio furnissii comprises a complex array of genes and gene products required for the utilization of chitin. The accompanying papers describe the molecular cloning and characterization of two hexosaminidases that are part of this cascade (1, 2).In this report we describe the molecular cloning of a thirdThis novel enzyme differs from all reported ␤-GlcNAcidases, possibly excepting one, in its substrate specificity and amino acid sequence. The enzyme is designated ExoII to differentiate it from the periplasmic exohexosaminidase, ExoI (2); the corresponding structural genes are exoI and exoII, respectively. EXPERIMENTAL PROCEDURES MaterialsThe following chemicals were purchased or were gifts from the indicated sources. Chitin, GlcNAc, 2 p-nitrophenyl (PNP) glycosides, and GlcNAc-6-P were from Sigma; chitin oligosaccharides, (GlcNAc) n (n ϭ 2-6), were from Seikagaku America, Inc. (Rockville, MD); MUF-␤-GlcNAc and MUF-␤-(GlcNAc) 2 were from Calbiochem; phenyl-␤-GlcNAc, benzyl-␤-GlcNAc, p-methylphenyl-␤-GlcNAc, 6-aminohexyl-␤-GlcNAc, ␤-GlcNAc-Asn, and Thr/Ser-Asn-GlcNAc-GlcNAc-(Man) 9 were kindly provided by Dr. Y. C. Lee (Department of Biology, The Johns Hopkins University, Baltimore, MD). . Reagents for molecular biology were obtained from New England Biolabs, U. S. Biochemical Corp., Life Technologies, Inc., Stratagene, and Boehringer Mannheim. Vector pVEX-11 was a kind gift from Dr. Chaudhary (Laboratory of Molecular Biology, National Institutes of Health, Bethesda, MD). Primers T7, T3 were purchased from Stratagene. Additional primers were synthesized in the laboratory of Dr. Robert Schleif (Department of Biology, The Johns Hopkins University, Baltimore, MD). Radioisotopes...

Changes in phosphometabolites, following osmotic shock, were analyzed by two-dimensional thin layer chromatography, in extracts of the halotolerant alga DunalielIa salina in order to clarify the regulation of glycerol synthesis from starch. The experiments were carried out in wild-type and in osmotically defective mutant cells. It is demonstrated that hyperosmotic shock induces a decrease in fructose 6-phosphate and an increase in fructose-1,6-bisphosphate indicating the activation of phosphofructokinase. Two mutants, which are specifically defective in their response to hyperosmotic shock, accumulate glucose 6-phosphate or phosphogluconate following shock, and have remarkably reduced activities of glucose-6-phosphate dehydrogenase and of phosphogluconate dehydrogenase, respectively. These results indicate that the pentose-phosphate oxidative pathway has a major role in glycerol synthesis. Hyperosmotic shock leads to a transient accumulation of phosphoryicholine and to a decrease of inositolbisphosphate in D. salina extracts. Accumulation of phosphorylcholine is not detected in osmotically defective mutants. Hypoosmotic shock induces an increase in inositolbisphosphate but not in phosphorylcholine. These results are consistent with previous indications for differential activations of phospholipases by hyper or hypoosmotic shock in DunalielIa. Based on these results we suggest that (a) phosphofructokinase is an important checkpoint enzyme in the regulation of glycerol production, and (b) that the pentose-phosphate pathway has a major role in keeping oxidation-reduction balance during glycerol synthesis. The possible role of lipid breakdown products as second messengers in regulating glycerol production in DunalielIa is discussed.Dunaliella is a unicellular halotolerant green alga which adapts to a large range of NaCl concentrations, from 0.1 to 5 M, by the synthesis of intracellular glycerol. Previous studies have shown that glycerol is made primarily from starch reserves in the chloroplast (3, 4, 6, 9), but the exact metabolic pathway leading to glycerol synthesis is not known. Four enzymes which apparently are involved in glycerol metabolism have been described, three of them unique to Dunaliella. It has been proposed that glycerol is made from DHAP (Table I contains complete list of abbreviations) via a GP-DH and a GP-ase, or reconverted to DHAP via a G-DH and a DHA-K (reviewed in ref. 4). The exact enzymatic pathway leading to DHAP formation from starch is still unclear and the energetic requirements in ATP and reduced pyridine nucleotides are not known. The observation that glycerol production proceeds either in the light or in the dark suggests that there is no obligatory requirement for photosynthesis neither as an energy source nor as a carbon source. It is also unclear how is glycerol synthesis and its reconversion to starch being regulated. It has been clearly established that protein synthesis is not involved in the induction of glycerol production since inhibitors of protein synthesis do not ...

A technique for selection of Dunaliella mutants defective in their capacity to recover from osmotic shocks has been developed. The selection is based on physical separation of mutants on density gradients. This technique takes advantage of the fact that DunalielIa cells, when exposed to osmotic shocks, initially change volume and density due to water gain or loss and subsequently recover their volume and density by readjusting their intracellular glycerol. Eight mutants that do not recover their original density following hyperosmotic shocks have been isolated. The mutants grow similar to wild type cells in I molar NaCI, and recover like the wild type from hypotonic shocks but are defective in recovering from hypertonic shocks. A partial characterization of one of the mutants is described.The unicellular green alga Dunaliella has the remarkable capacity to grow and adapt to media ranging in salinity from 50 mM to 5 M NaCl. The major means of osmoregulation of this wall-less alga are by production of intracellular glycerol at concentrations that are proportional to the external NaCl concentration.The response of Dunaliella to changes in the extracellular osmotic pressure occurs in two distinct phases. In the first phase the cells rapidly shrink or swell under hypertonic or hypotonic conditions, respectively. The second phase of adaptation is slower (2-3 h) and involves synthesis or elimination of glycerol. By the end of this period the cells recover their original volume.Although it is clear that recovery of Dunaliella cells from hypertonic shocks involves glycerol production, and a few novel enzymes that seem to be involved in glycerol metabolism have been identified (2, 5), it is still uncertain what triggers glycerol production or elimination in response to osmotic shocks. Changes in Na+ content (6, 13) pH level (9), phosphate (8), inositol phospholipids (7), and in ultrastructure (1 1) have been observed following osmotic shocks and were suggested to be involved in triggering glycerol production or elimination.A valuable approach to study the mechanism of osmoregulation could be characterization of mutants defective in osmoregulation. Towards

Different microorganisms, including yeast and algae, accumulate large amounts of polyphosphates. However, the physiological role of polyphosphates is largely unknown. In vivo a~ p NMR studies, carried out in the unicellular alga, Dunaliella salina, demonstrate that cytoplasmic alkalization induces massive hydrolysis of polyphosphates, which is correlated kinetically with the recovery of cytoplasmic pH. Analysis of acid extracts of the cells indicates that long-chain polyphosphates are hydrolysed mainly to tripolyphosphate. It is suggested that the hydrolysis of polyphosphates provides a pH-stat mechanism to counterbalance alkaline stress.

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