Molecular and physiological characterisation of a 3-phytase from soil bacterium<Emphasis Type="Italic"> Klebsiella</Emphasis> sp. ASR1

Klebsiella sp. strain ASR1 isolated from an Indonesian rice field is able to hydrolyse myo-inositol hexakis phosphate (phytate). The phytase protein was purified and characterised as a 42 kDa protein accepting phytate, NADP and sugar phosphates as substrates. The corresponding gene (phyK) was cloned from chromosomal DNA using a combined approach of protein and genome analysis, and expressed in Escherichia coli. The recombinant enzyme was identified as a 3-phytase yielding myo-inositol monophosphate, Ins(2)P, as the final product of enzymatic phytate hydrolysis. Based on its amino acid sequence, PhyK appears to be a member of a hitherto unknown subfamily of histidine acid phytate-degrading enzymes with the active site RHGXRXP and HD sequence motifs, and is different from other general phosphatases and phytases. Due to its ability to degrade sodium phytate to the mono phosphate ester, the phyK gene product is an interesting candidate for industrial and agricultural applications to make phytate phosphorous available for plant and animal nutrition.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Biochemical properties and substrate specificities of alkaline and histidine acid phytases

Phytases are a special class of phosphatase that catalyze the sequential hydrolysis of phytate to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are added to animal feedstuff to reduce phosphate pollution in the environment, since monogastric animals such as pigs, poultry, and fish are unable to metabolize phytate. Based on biochemical properties and amino acid sequence alignment, phytases can be categorized into two major classes, the histidine acid phytases and the alkaline phytases. The histidine acid phosphatase class shows broad substrate specificity and hydrolyzes metal-free phytate at the acidic pH range and produces myo-inositol monophosphate as the final product. In contrast, the alkaline phytase class exhibits strict substrate specificity for the calcium–phytate complex and produces myo-inositol trisphosphate as the final product. This review describes recent findings that present novel viewpoints concerning the molecular basis of phytase classification.     

Novel Glucose-1-Phosphatase with High Phytase Activity and Unusual Metal Ion Activation from Soil Bacterium Pantoea sp. Strain 3.5.1

Phosphorus is an important macronutrient, but its availability in soil is limited. Many soil microorganisms improve the bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes, and urban landscapes. Bacterial isolate Pantoea sp. strain 3.5.1 with the highest level of phytase activity was isolated from forest soil and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy, and biochemically characterized. The AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest K_m values toward phytate, glucose-6-phosphate, and glucose-1-phosphate. Unexpectedly, stimulation of enzymatic activity by several divalent metal ions was observed for the AgpP enzyme. High-performance liquid chromatography (HPLC) and high-performance ion chromatography (HPIC) analyses of phytate hydrolysis products identify dl-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction, indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features.     

A Novel Phytase appA from <Emphasis Type="Italic">Citrobacter amalonaticus</Emphasis> CGMCC 1696: Gene Cloning and Overexpression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A novel phytase gene appA, with upstream and downstream sequences from Citrobacter amalonaticus CGMCC 1696, was cloned by degenerate polymerase chain reaction (PCR), and thermal asymmetric interlaced (TAIL) PCR and was overexpressed in Pichia pastoris. Sequence analysis revealed one open reading frame that consisted of 1311 bp encoding a 436–amino-acid protein, which had a deduced molecular mass of 46.3 kDa. The phytase appA belongs to the histidine acid phosphatase family and exhibits the highest identity (70.1%) with C. braakii phytase. The gene was overexpressed in P. pastoris. The secretion yield of recombinant appA protein was accumulated to approximately 4.2 mg·mL⁻¹, and the enzyme activity level reached 15,000 U·mL⁻¹, which is higher than any previous reports. r-appA was glycosylated, as shown by Endo H treatment. r-appA was purified and characterized. The specific activity of r-appA for sodium phytate was 3548 U·mg⁻¹. The optimum pH and temperature for enzyme activity were 4.5 and 55°C, respectively. r-appA was highly resistant to pepsin or trypsin treatment. This enzyme could be an economic and efficient alternative to the phytases currently used in the feed industry.     

Catalytic efficiency of HAP phytases is determined by a key residue in close proximity to the active site

The maximum activity of Yersinia enterocolitica phytase (YeAPPA) occurs at pH 5.0 and 45 °C, and notably, its specific activity (3.28 ± 0.24 U mg⁻¹) is 800-fold less than that of its Yersinia kristeensenii homolog (YkAPPA; 88% amino acid sequence identity). Sequence alignment and molecular modeling show that the arginine at position 79 (Arg79) in YeAPPA corresponding to Gly in YkAPPA as well as other histidine acid phosphatase (HAP) phytases is the only non-conserved residue near the catalytic site. To characterize the effects of the corresponding residue on the specific activities of HAP phytases, Escherichia coli EcAPPA, a well-characterized phytase with a known crystal structure, was selected for mutagenesis—its Gly73 was replaced with Arg, Asp, Glu, Ser, Thr, Leu, or Tyr. The results show that the specific activities of all of the corresponding EcAPPA mutants (17–2,400 U mg⁻¹) were less than that of the wild-type phytase (3,524 U mg⁻¹), and the activity levels were approximately proportional to the molecular volumes of the substituted residues’ side chains. Site-directed replacement of Arg79 in YeAPPA (corresponding to Gly73 of EcAPPA) with Ser, Leu, and Gly largely increased the specific activity, which further verified the key role of the residue at position 79 for determining phytase activity. Thus, a new determinant that influences the catalytic efficiency of HAP phytases has been identified.     

Impact of oxygen supply on rtPA expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 (DE3): ammonia effects

A phytase with high activity at neutral pH and typical water temperatures (∼25°C) could effectively hydrolyze phytate in aquaculture. In this study, a phytase-producing strain, Pedobacter nyackensis MJ11 CGMCC 2503, was isolated from glacier soil, and the relevant gene, PhyP, was cloned using degenerate PCR and thermal asymmetric interlaced PCR. To our knowledge, this is the first report of detection of phytase activity and cloning of phytase gene from Pedobacter. PhyP belongs to beta-propeller phytase family and shares very low identity (∼28.5%) with Bacillus subtilis phytase. The purified recombinant enzyme (r-PhyP) from Escherichia coli displayed high specific activity for sodium phytate of 24.4 U mg⁻¹. The optimum pH was 7.0, and the optimum temperature was 45°C. The K _m, V _max, and k _cat values were 1.28 mM, 71.9 μmol min⁻¹ mg⁻¹, and 45.1 s⁻¹, respectively. Compared with Bacillus phytases, r-PhyP had higher relative activity at 25°C (r-PhyP (>50%), B. subtilis phytase (<8%)) and hydrolyzed phytate from soybean with greater efficacy at neutral pH. These characteristics suggest that r-PhyP might be a good candidate for an aquatic feed additive in the aquaculture industry.     

Cloning, biochemical characterisation, tissue localisation and possible post-translational regulatory mechanism of the cytosolic phosphoglucose isomerase from developing sunflower seeds

Lipid biosynthesis in developing sunflower (Helianthus annuus L.) seeds requires reducing power. One of the main sources of cellular NADPH is the oxidative pentose phosphate pathway (OPPP), generated from the oxidation of glucose-6-phosphate. This glycolytic intermediate, which can be imported to the plastid and enter in the OPPP, is the substrate and product of cytosolic phosphoglucose isomerase (cPGI, EC 5.3.1.9). In this report, we describe the cloning of a full-length cDNA encoding cPGI from developing sunflower seeds. The sequence was predicted to code for a protein of 566 residues characterised by the presence of two sugar isomerase domains. This cDNA was heterologously expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified using immobilised metal ion affinity chromatography and biochemically characterised. The enzyme had a specific activity of 1,436 μmol min⁻¹ mg⁻¹ and 1,011 μmol min⁻¹ mg⁻¹ protein when the reaction was initiated with glucose-6-phosphate and fructose-6-phosphate, respectively. Activity was not affected by erythrose-4-phosphate, but was inhibited by 6-P gluconate and glyceraldehyde-3-phosphate. A polyclonal immune serum was raised against the purified enzyme, allowing the study of protein levels during the period of active lipid synthesis in seeds. These results were compared with PGI activity profiles and mRNA expression levels obtained from Q-PCR studies. Our results point to the existence of a possible post-translational regulatory mechanism during seed development. Immunolocalisation of the protein in seed tissues further indicated that cPGI is highly expressed in the procambial ring.     

Effects of Pyrocatechol on Some Metabolic Processes of Mature Sugar Beet (Beta vulgaris) Plants

Pyrocatechol (PC), 10^-2M, was applied to the foliage of mature plants of sugar beet (Beta vulgaris L.). Its effect on the activity of nitrate reductase, transaminase, invertase, phosphatases, sucrose synthetase, sucrose phosphate synthetase, and UDPG-pyrophosphorylase were determined 7, 14, and 21 days after treatment. Significant reductions in the activity of nitrate reductase, transaminase, invertase, and phosphatases (including phenyl phosphatase, glucose-1-, glucose-6-, fructose-6-phosphatase, and adenosine triphosphatase) in the treated plants occurred. On the other hand, activities of the enzymes of sucrose biosynthesis, uridine, diphosphate glucose pyrophosphorylase (UDPG-pyrophosphorylase), sucrose synthetase, and sucrose phosphate synthetase were significantly stimulated by the application of pyrocatechol. The results suggest that the growth inhibition following the application of PC to sugar beet plants may stem in part from an amino acid stress resulting from a PC-induced decrement in nitrate reductase and transaminase activity. Its application also creates an enzymatic condition favorable for sucrose biosynthesis and storage.     

Biochemical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Catalytic Properties

Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined. The specific activities of the fungal phytases at 37°C ranged from 23 to 196 U · (mg of protein)⁻¹, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which left myo-inositol 2-monophosphate as the end product. A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the A. niger, Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, the Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, α- and β-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the myo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumigatus phytase. In contrast, phosphate liberation by A. niger or A. terreus phytase decreased with incubation time, and the myo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either A. fumigatus phytase or A. niger phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the A. fumigatus phytase than by the A. niger phytase at later stages of incubation.     

Molecular characterization,physicochemical properties,known and potential applications of phytases: An overview

Phytases (myo-inositol hexakisphosphate phosphohydrolases) hydrolyze the phosphate ester bonds of phytate-releasing phosphate and lower myo-inositol phosphates and/or myo-inositol. Phytases, in general, are known to enhance phosphate and mineral uptake in monogastric animals such as poultry, swine, and fish, which cannot metabolize phytate besides reducing environmental pollution significantly. In this study, the molecular, biophysical, and biochemical properties of phytases are reviewed in detail. Alterations in the molecular and catalytic properties of phytases, upon expression in heterologous hosts, are discussed. Diverse applications of phytases as feed additives, as soil amendment, in aquaculture, development of transgenic organisms, and as nutraceuticals in the human diet also are dealt with. Furthermore, phytases are envisaged to serve as potential enzymes that can produce versatile lower myo-inositol phosphates of pharmaceutical importance. Development of phytases with improved attributes is an important area being explored through genetic and protein engineering approaches, as no known phytase can fulfill all the properties of an ideal feed additive.     

Purification and Properties of a Phytate-degrading Enzyme from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

A periplasmatic phytate-degrading enzyme from Pantoea agglomerans isolated from soil was purified about 470-fold to apparent homogeneity with a recovery of 16% referred to the phytate-degrading activity in the crude extract. It behaved as a monomeric protein with a molecular mass of about 42 kDa. The purified enzyme exhibited a single pH optimum at 4.5. Optimum temperature for the degradation of phytate was 60°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K_M = 0.34 mmol/l and k_cat = 21 s^-1 at pH 4.5 and 37°C. The enzyme exhibited a narrow substrate selectivity. Only phytate and glucose-1-phosphate were identified as good substrates. Since this Pantoea enzyme has a strong preference for glucose-1-phosphate over phytate, under physiological conditions glucose-1-phosphate is its most likely substrate. The maximum amount of phosphate released from phytate by the purified enzyme suggests myo-inositol pentakisphosphate as the final product of enzymatic phytate degradation.     

Improved sensitivity,accuracy and prediction provided by a high-performance liquid chromatography screen for the isolation of phytase-harbouring organisms from environmental samples

HPLC methods are shown to be of predictive value for classification of phytase activity of aggregate microbial communities and pure cultures. Applied in initial screens, they obviate the problems of ‘false-positive’ detection arising from impurity of substrate and imprecision of methodologies that rely on phytate-specific media. In doing so, they simplify selection of candidates for biotechnological applications. Combined with 16S sequencing and simple bioinformatics, they reveal diversity of the histidine phosphatase class of phytases most commonly exploited for biotechnological use. They reveal contribution of multiple inositol-polyphosphate phosphatase (MINPP) activity to aggregate soil phytase activity, and they identity Acinetobacter spp. as harbouring this prevalent soil phytase activity. Previously, among bacteria MINPP was described exclusively as an activity of gut commensals. HPLC methods have also identified, in a facile manner, a known commercially successful histidine (acid) phosphatase enzyme. The methods described afford opportunity for isolation of phytases for biotechnological use from other environments. They reveal the position of attack on phytate by diverse histidine phosphatases, something that other methods lack.     

Functional insights revealed by the crystal structures of Escherichia coli glucose-1-phosphatase

The Escherichia coli periplasmic glucose-1-phosphatase is a member of the histidine acid phosphatase family and acts primarily as a glucose scavenger. Previous substrate profiling studies have demonstrated some of the intriguing properties of the enzyme, including its unique and highly selective inositol phosphatase activity. The enzyme is also potentially involved in pathogenic inositol phosphate signal transduction pathways via type III secretion into the host cell. We have determined the crystal structure of E. coli glucose-1-phosphatase in an effort to unveil the structural mechanism underlying such unique substrate specificity. The structure was determined by the method of multiwavelength anomalous dispersion using a tungstate derivative together with the H18A inactive mutant complex structure with glucose 1-phosphate at 2.4-A resolution. In the active site of glucose-1-phosphatase, there are two unique gating residues, Glu-196 and Leu-24, in addition to the conserved features of histidine acid phosphatases. Together they create steric and electrostatic constraints responsible for the unique selectivity of the enzyme toward phytate and glucose-1-phosphate as well as its unusually high pH optimum for the latter. Based on the structural characterization, we were able to derive simple structural principles that not only precisely explains the substrate specificity of glucose-1-phosphatase and the hydrolysis products of various inositol phosphate substrates but also rationalizes similar general characteristics across the histidine acid phosphatase family.     

<Emphasis Type="Italic">APHO1</Emphasis> from the yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis> encodes an acid phosphatase of broad substrate specificity

The extracellular acid phosphatase-encoding Arxula adeninivorans APHO1 gene was isolated using degenerated specific oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1449 bp encoding a protein of 483 amino acids with a calculated molecular mass of 52.4 kDa. The sequence includes an N-terminal secretion sequence of 17 amino acids. The deduced amino acid sequence exhibits 54% identity to phytases from Aspergillus awamori, Asp. niger and Asp. ficuum and a more distant relationship to phytases of the yeasts Candida albicans and Debaryomyces hansenii (36–39% identity). The sequence contains the phosphohistidine signature and the conserved active site sequence of acid phosphatases. APHO1 expression is induced under conditions of phosphate limitation. Enzyme isolates from wild and recombinant strains with the APHO1 gene expressed under control of the strong A. adeninivorans-derived TEF1 promoter were characterized. For both proteins, a molecular mass of approx. 350 kDa, corresponding to a hexameric structure, a pH optimum of pH 4.8 and a temperature optimum of 60°C were determined. The preferred substrates include p-nitrophenyl-phosphate, pyridoxal-5-phosphate, 3-indoxyl-phosphate, 1-naphthylphosphate, ADP, glucose-6-phosphate, sodium-pyrophosphate, and phytic acid. Thus the enzyme is a secretory acid phosphatase with phytase activity and not a phytase as suggested by strong homology to such enzymes.     

Construction,expression and characterization of fusion enzyme from <Emphasis Type="Italic">Arthrobacter oxydans</Emphasis> dextranase and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> amylase

An artificial fusion protein of Arthrobacter oxydans dextranase and Klebsiella pneumoniae α-amylase was constructed and expressed in Escherichia coli. Most of the expressed protein existed as an insoluble fraction, which was solubilized with urea. The purified fusion enzyme electrophoretically migrated as a single protein band; M = 137 kDa, and exhibited activities of both dextranase (10.8 U mg⁻¹) and amylase (7.1 U mg⁻¹), which were lower than that of reference dextranase (13.3 U mg⁻¹) and α-amylase (103 U mg⁻¹). The fusion enzyme displayed bifunctional enzyme activity at pH 5–7 at 37°C. These attributes potentially make the fusion enzyme more convenient for use in sugar processing than a two-enzyme system.     

Partial purification and characterization of phytases from pollen of lily (Lilium longiflorum Thunb.)

Summary Two phytases from lily pollen (Lilium longiflorum Thunb.) were partially purified and characterized. The first (pH optimum 5.0) was purified 40-fold from ungerminated pollen. The second (pH optimum 6.5) appeared during germination and was purified 68-fold from pollen germinated 2 h. Molecular weight of the first was 72 kD, and the second was 36 kD as determined by gel filtration. Both were active against phosphate esters other than phytate, although purification of the first reduced its activity against AMP and myo-inositol 2-P to 10% of activity against phytate. Phytase from germinated pollen (but not ungerminated) was inhibited by the sulfhydryl agent parahydroxy mercuribenzoate; P _i inhibited phytase from ungerminated but not germinated pollen. Such different catalytic and physical properties may reflect different biochemical functions.Abbreviations HPLC High performance liquid chromatography - DEAE diethyl aminoethyl - P _i orthophosphate - PP _i pyrophosphate - p-NPP para-nitrophenyl phosphate - pNP para-nitrophenol - MI myo-inositol - MI 2-P myo-inositol 2-P - MI penta P myo-inositol pentakisphosphate - PHMB para-hydroxy mercuribenzoate - PMSF phenyl methyl sulfonyl fluoride - AMP adenosine monophosphate - GMP guanosine monophosphate - EGTA ethylene glycol-bis (-aminoethyl ether) N, N, N, N-tetraacetic acid     

Improvement of Yersinia frederiksenii phytase performance by a single amino acid substitution

A new phytase (APPA) with optimum pH 2.5—substantially lower than that of most of microbial phytases (pH 4.5–6.0)—was cloned from Yersinia frederiksenii and heterologously expressed in Escherichia coli. Containing the highly conserved motifs typical of histidine acid phosphatases, APPA has the highest identity (84%) to the Yersinia intermedia phytase (optimal pH 4.5), a member of histidine acid phosphatase family. Based on sequence alignment and molecular modeling of APPA and related phytases, APPA has only one divergent residue, Ser51, in close proximity to the catalytic site. To understand the acidic adaptation of APPA, five mutants (S51A, S51T, S51D, S51K, and S51I) were constructed by site‐directed mutagenesis, expressed in E. coli, purified, and characterized. Mutants S51T and S51I exhibited a shift in the optimal pH from 2.5 to 4.5 and 5.0, respectively, confirming the role of Ser51 in defining the optimal pH. Thus, a previously unrecognized factor other than electrostatics—presumably the side‐chain structure near the active site—contributes to the optimal pH for APPA activity. Compared with wild‐type APPA, mutant S51T showed higher specific activity, greater activity over pH 2.0–5.5, and increased thermal and acid stability. These properties make S51T a better candidate than the wild‐type APPA for use in animal feed. Biotechnol. Bioeng. 2009;103: 857–864. © 2009 Wiley Periodicals, Inc.     

The glucose-6-phosphatase enzyme in developing human trachea and oesophagus

Summary Glucose-6-phosphatase is an endoplasmic reticulum system which is found primarily in liver and kidney. Recently, it has become clear that it is also present in lower amounts in a variety of other tissues. Previous histochemical studies of glucose-6-phosphate hydrolysis in trachea have given equivocal results and only one study on adult oesophagus has shown glucose-6-phosphatase, enzymatic activity but without cellular localization. We have now shown, using microassay techniques, that microsomes isolated from human foetal trachea and oesophagus both contain low levels of specific glucose-6-phosphatase activity (mean= 0.9 and 1.5 nmol min⁻¹ mg⁻¹ microsomal protein, respectively) which are less than 10% of the levels in microsomes of human foetal liver of similar age. In the developing trachea, glucose-6-phosphatase immunoreactivity has been found, using a monospecific antibody to the catalytic subunit of the glucose-6-phosphatase enzyme, to be first present at 10–11 weeks' gestation, and thereafter in foetal life, predominantly present in ciliated cells, with smaller amounts in non-ciliated secretory cells, duct lining cells, and occasional basal cells. The foetal oesophageal epithelium is transiently ciliated from 10 to 11 weeks' gestation, but ciliated cells are gradually replaced by squamous cells from 14 to 16 weeks onwards. Glucose-6-phosphatase immunoreactivity in human foetal oesophagus is predominantly confined to ciliated cells, but non-ciliated luminal cells are also reactive, as are occasional basal cells. Mucus secretory cells in foetal trachea and oesophagus are immunonegative, as is the entire epithelium of both organs in the embryo (up to 56 postovulatory days).     

Mapping of the Escherichia coli acid glucose-1-phosphatase gene agp and analysis of its expression in vivo by use of an agp-phoA protein fusion.

The agp gene of Escherichia coli encodes an acid glucose-1-phosphatase, one of the numerous phosphatases optimally active between pH 4 and 6 found in the periplasmic space of this bacterium. An agp-phoA protein fusion linked to a gene conferring kanamycin resistance was inserted into the chromosome in place of agp by homologous recombination and was mapped to minute 22.6. Because the activity of glucose-1-phosphatase cannot be measured accurately in whole cells, the alkaline phosphatase activity of the agp-phoA hybrid protein was used to monitor the expression of the chromosomal agp gene. The expression of agp was subject to catabolite repression but was unaffected by the concentration of inorganic phosphate in the growth medium. The product of the agp gene was required for growth on glucose-1-phosphate as the sole carbon source, a function for which alkaline phosphatase or other acid phosphatases cannot substitute.