A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 ␤-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18°C and 50% activity remaining at 0°C) and heat labile (inactivated within 10 min at 37°C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both ␤-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-␤-D-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20°C than the comparable LacZ ␤-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4°C but dissociated into inactive monomers at 25°C. Comparisons of family 2 ␤-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.Glycosyl hydrolases (EC 3.2.1 to 3.2.3) cleave the glycosidic bond between two or more carbohydrates or between a carbohydrate and another moiety. Traditionally, glycosyl hydrolases were classified based on functional similarity. More recently, however, Henrissat and his coworkers have organized these enzymes into 90 glycosyl hydrolase families characterized by hydrophobicity plots, amino acid sequence similarities, and reaction mechanisms (10-12). This system also identifies possible common structural domains, thereby defining evolutionary connections and suggesting reaction mechanisms for the glycosyl hydrolases. Based on these criteria, the once-unified group of enzymes that exhibit ␤-galactosidase activity (EC 3.2.1.23) are now subdivided into four different families: 1, 2, 35, and 42.Of these, the best studied is family 2, which includes the well-characterized ␤-galactosidase from Escherichia coli that is encoded by the lacZ gene. Although there is considerable information about the regulation (1), biochemistry (18,23,35,47), reaction mechanism (17, 45), and structure (16) of this LacZ ␤-galactosidase, few other ␤-galactosidases within this family have been characterized biochemically (4, 7, 13-15, 26, 27, 43), while most examples exist only as a published sequence. Because of the emphasis on the E. coli LacZ enzyme, opportunities to lea...

During our work on psychrophilic microorganisms we obtained a large collection of new isolates. In order to identify six of these, we examined their growth properties, cell wall compositions, and their 16S rRNA gene sequences. The results showed that all of the isolates are gram-positive, aerobic, contain lysine in their cell walls, and belong to the high mol% G+C Arthrobacter subgroup. Phylogenetic analysis of the 16S rRNA genes grouped five isolates obtained from a small geographical region into a monophyletic clade. Isolate B7 had a 16S rRNA sequence that was 94.3% similar to that of Arthrobacter polychromogenes and 94.4% similar to that of Arthrobacter oxydans. Primary characteristics that distinguish isolate B7 from the Arthrobacter type strain (Arthrobacter globiformis) and A. polychromogenes include lack of growth at 37 degrees C, growth at 0-5 degrees C, the ability to use lactose as a sole carbon source, and the absence of blue pigments. Because of these differences, isolate B7 was chosen as a type strain representing a new Arthrobacter species, Arthrobacter psychrolactophilus. The sixth isolate, LV7, differed from the other five because it did not have the rod/ coccus morphological cycle and was most closely related to Arthrobacter agilis.

We have characterized a new psychrotrophic Arthrobacter isolate which produces ␤-galactosidase isozymes. When DNA from this isolate was transformed into an Escherichia coli host, we obtained three different fragments, designated 12, 14, and 15, each encoding a different ␤-galactosidase isozyme. The ␤-galactosidase produced from fragment 12 was of special interest because the protein subunit was smaller (about 71 versus 116 kDa) than those typically encoded by the lacZ family. The isozyme encoded by fragment 12 was purified, and its activity and thermostability were examined. Although the enzyme is highly specific towards ␤-Dgalactoside substrates, its levels in the isolate do not increase in cells grown with lactose. Nucleotide sequence determination showed that the gene encoding isozyme 12 is not similar to the other members of the lacZ family but has regions similar to ␤-galactosidase isozymes from Bacillus stearothermophilus and B. circulans. Addition of the isozyme 12 sequence to the database made it possible to examine these enzymes as possible members of a new, separate family. Our analysis of this new family showed some conserved amino acids corresponding to the lacZ acid-base catalytic region but no homology with the nucleophilic region. On the basis of these comparisons, we designated this a new lacG family.The intricate genetic and biochemical studies with the lacZencoded ␤-galactosidase (EC 3.2.1.23) of Escherichia coli provide an excellent foundation for examining and comparing the structures of other ␤-galactosidases. Because the lacZ enzyme is used so frequently, we often overlook other interesting and unusual ␤-galactosidases. The study of these new enzymes, however, can help explain the roles of various amino acids in enzyme structure, provide insight into the evolution of genes encoding ␤-galactosidases, suggest structural relationships to other glycosidases, and yield enzymes with valuable properties that differ from those of the E. coli enzyme.We had isolated psychrotrophic Arthrobacter strains which produced cold-active ␤-galactosidases and demonstrated that cells of one strain, B7, contained at least two isozymes when grown with lactose as a carbon source (22). When we used DNA from isolate B7 to transform an E. coli host, we obtained three transformants, each containing a different DNA segment encoding a ␤-galactosidase activity. One gene, designated 15, encoded the predominant isozyme found in lactose-grown cells. This enzyme has a temperature optimum about 20ЊC below that of the E. coli ␤-galactosidase and has been purified and characterized, and the gene that encodes it has been sequenced (33). This enzyme has the conserved acid-base and nucleophilic sites involved in catalysis that are typically found with the lacZ family of ␤-galactosidases. The presence of the other isozymes in this isolate raised questions about their possible functions in enabling these psychrotrophic strains to survive rapid temperature changes or in using other glycoside substrates. Our initial work with the secon...

Investigations of psychrotrophic microorganisms have been limited even though the dominant environment of the Earth is cold and enzymes with high activities at low temperatures could have commercial uses. We have isolated and characterized three psychrotrophic strains with 13-galactosidase activities. The isolates, B7, D2, and D5, were gram-positive, catalase-positive, obligate aerobes. Cells observed with a scanning electron microscope appeared as rods during the early stages of growth but became coccoid during the stationary phase. An analysis of the amino acid composition of the cell walls demonstrated the presence of lysine as the predominant diamino acid in all three isolates. The cell cycle morphology and cell wall composition suggest that the three isolates are members of the genus Arthrobacter. The 13-galactosidase activities in whole cells were labile when incubated at 40°C and had temperature optima about 20°C below that of the enzyme encoded by the lacZ gene ofEscherichia coli. Electrophoresis of extracts from the isolates in nondenaturing polyacrylamide gels detected at least two protein bands that hydrolyzed 5-bromo-4-chloro-3-indolyl-13-D-galactopyranoside (X-Gal), suggesting the presence of 13-galactosidase isozymes.

An Arthrobacter ␤-galactosidase has homology with the lysosomal acid ␤-galactosidases from humans and mice and with a Xanthomonas manihotis enzyme. Phylogenetic analysis of the deduced amino acid sequence showed an unusual pattern, with this procaryotic enzyme clustering within the animal clade. The gene encodes a subunit of 52 kDa, and the enzyme appears to be active as a dimer. The enzyme hydrolyzed substrates with either a ␤-1,4 or a ␤-1,3 linkage.We are interested in the function of ␤-galactosidases from psychrophilic microorganisms capable of growth at or below 5°C (9). Strains hydrolyzing the chromogen X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) were isolated and one, isolate B7, was identified as an Arthrobacter species with a growth range from 0 to 30°C (7). Chromosomal DNA from strain B7 was recombined into a plasmid and transformed into Escherichia coli. Surprisingly, three transformants with different genes encoding ␤-galactosidase activities were found. Previous work demonstrated that one gene encodes a ␤-galactosidase with a subunit size of 111 kDa and the conserved acid-base and nucleophilic regions found for lacZ enzymes (13). The second gene, however, encodes a ␤-galactosidase with a smaller subunit (71 kDa) which lacks significant homology with the lacZ nucleophilic site and has homology with an isozyme from the thermophile Bacillus stearothermophilus. Studies of this gene helped define a new ␤-galactosidase family, which we designated lacG (2). In this report, we characterized the most unusual gene and isozyme of the three. This gene encoded a small subunit (52 kDa), designated 14.2. The enzyme was active as a dimer and hydrolyzed substrates with either a ␤-1,4 or -1,3 glycoside linkage. The amino acid sequence derived from the nucleotide sequence was homologous with that of a newly discovered enzyme from a Xanthomonas species and those of the lysosomal acid ␤-galactosidases of humans and mice. These results raise several interesting questions regarding the presence and function of a gene with homology to eucaryotic DNA in a psychrophilic soil bacterium.Expression of the Arthrobacter gene in Salmonella typhimurium. The Arthrobacter genes were originally cloned into strain JM109 (13), where the ␤-galactosidase level was sufficient to detect colonies hydrolyzing X-Gal. However, transformants carrying the gene 14.2 had low specific activities (0.02 to 0.03 U/mg of protein), as measured by the release of o-nitrophenyl from o-nitrophenyl-␤-D-galactopyranoside (ONPG) (8), making it difficult to purify the enzyme. Thus, the gene was transformed into a variety of strains. There was no change in the activity with any of the E. coli strains; however, a 100-fold increase to 2.25 U/mg of protein was obtained when the construct pUC18/14.2 was transformed into S. typhimurium CH3 FЈ cells containing an FЈ (⌬lacZ138 and Tn10::proAB81) carrying the lactose permease gene. These levels permitted the detection of a protein migrating at 52 kDa following electrophoresis on a sodium dodecyl sulfate-7.5% ...