Evaluation of cyclohexenoesculetin-beta-D-galactoside and 8-hydroxyquinoline-beta-D-galactoside as substrates for the detection of beta-galactosidase.

We describe the synthesis of two new substrates for the detection of beta-galactosidase and evaluate their performance in comparison with that of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). Of 171 Enterobacteriaceae strains that were able to hydrolyze X-Gal, 166 (97.1%) also hydrolyzed cyclohexenoesculetin-beta-D-galactoside whereas only 96 (56.1%) showed evidence of hydrolysis of 8-hydroxyquinoline-beta-D-galactoside. No false-positive results were observed with either substrate.     

Rapid screening of quorum-sensing signal N-acyl homoserine lactones by an in vitro cell-free assay

A simple, sensitive, and rapid cell-free assay system was developed for detection of N-acyl homoserine lactone (AHL) autoinducers involved in bacterial quorum sensing (QS). The present approach improves upon previous whole-cell biosensor-based approaches in its utilization of a cell-free assay approach to conduct bioassays. The cell-free assay was derived from the AHL biosensor bacterium Agrobacterium tumefaciens NTL4(pCF218)(pCF372), allowing the expression of beta-galactosidase upon addition of exogenous AHLs. We have shown that beta-galactosidase expression is possible in cell-free solution [lysate from Agrobacterium tumefaciens NTL4(pCF218)(pCF372) culture]. Assay detection limits with the use of chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) ranged from approximately 100 nM to 300 nM depending on the specific AHL. Replacement (of X-Gal) with the luminescent substrate Beta-Glo increased sensitivity to AHLs by 10-fold. A major advantage of the cell-free assay system is elimination of time-consuming steps for biosensor cell culture conditioning, which are required prior to whole-cell bioassays. This significantly reduced assay times from greater than 24 h to less than 3 h, while maintaining high sensitivity. Assay lysate may be prepared in bulk and stored (-80 degrees C) over 6 months for future use. Finally, the present protocol may be adapted for use with other biosensor strains and be used in high-throughput AHL screening of bacteria or metagenomic libraries.     

Characterization of an unusual cold-active beta-glucosidase belonging to family 3 of the glycoside hydrolases from the psychrophilic isolate Paenibacillus sp. strain C7

We selected for spore-forming psychrophilic bacteria able to use lactose as a carbon source and one isolate, designated Paenibacillus sp. strain C7, that was phylogenetically related to, but distinct from both Paenibacillus macquariensis and Paenibacillus antarcticus. Some Escherichia coli transformants obtained with genomic DNA from this isolate hydrolyzed X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside) only below 30 degrees C, an indication of cold-active beta-galactosidase activity. Sequencing of the cloned insert revealed an open reading frame encoding a 756-amino acid protein that, rather than belonging to a family typically known for beta-galactosidase activity, belonged to glycoside hydrolase family 3, a family of beta-glucosidases. Because of this unusual placement, the recombinant enzyme (BglY) was purified and characterized. Consistent with its classification, the enzyme had seven times greater activity with the glucoside substrate ONPGlu (o-nitrophenyl-beta-D-glucopyranoside) than with the galactoside substrate ONPGal (o-nitrophenyl-beta-D-galactopyranoside). In addition, the enzyme had, with ONPGlu, a thermal optimum around 30 to 35 degrees C, activity over a broad pH range (5.5 to 10.9), and an especially low Km (<0.003 mM). Further examination of substrate preference showed that the BglY enzyme also hydrolyzed other aryl-beta-glucosides such as helicin, MUG (4-methylumbelliferyl-beta-D-glucopyranoside), esculin, indoxyl-beta-D-glucoside (a natural indigo precursor), and salicin, but had no activity with glucosidic disaccharides or lactose. These characteristics and substrate preferences make the BglY enzyme unique among the family 3 beta-glucosidases. The hydrolysis of a variety of aryl-beta-glucosides suggests that the enzyme may allow the organism to use these substrates in the environment and that its low Km on indoxyl-beta-D-glucoside may make it useful for producing indigo.     

A fluorimetric method using fluorescein di-beta-D-galactopyranoside for quantifying the senescence-associated beta-galactosidase activity in human foreskin fibroblast Hs68 cells

The senescence-associated beta-galactosidase (SA-betaG) assay is one of the few accepted markers of cell aging. However, the cytochemical method using 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal) as substrate is limited in sensitivity and is only semiquantitative. Here, we modified the X-Gal method by replacing X-Gal with fluorescein di-beta-D-galactopyranoside (FDG) as substrate for SA-betaG, and the activity was measured fluorimetrically. We showed in Hs68 cells that the FDG fluorescein fluorescence increased with increasing passages of the cells in parallel with the X-Gal method. A major advantage of the FDG method is that it is a quantitative method for the SA-betaG activity. For example, we showed that the FDG fluorescein in p30(+1) of Hs68 cells was generally stronger than that in p26(+1) cells, whereas the X-Gal method gave similar results (95 and 100%) for p26(+1) and p30(+1) cells. The FDG method was precise with a relative standard deviation lower than 10%. We further demonstrated that FDG and X-Gal could be added simultaneously for SA-betaG assay because the FDG fluorescein diffused readily through formaldehyde-fixed cell membrane and could be detected in the suspension buffer. Thus, a double-substrate method, i.e., X-Gal for rapid qualitative assay and FDG for quantitative assay, can be conducted simultaneously to provide a simple and reliable assay of SA-betaG activity as a marker of cell aging.     

Expression of a mouse metallothionein-Escherichia coli beta-galactosidase fusion gene (MT-beta gal) in early mouse embryos

We have microinjected DNA containing the inducible mouse metallothionein-I (MT-I) promoter, coupled to the structural gene for Escherichia coli beta-galactosidase (lacZ), into the pronuclei of one-cell mouse embryos. A qualitative histochemical assay, with 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal) as a substrate, was used to detect expression of lacZ at several preimplantation stages. We observed staining indicative of exogenous beta-galactosidase activity in 5-17% of DNA-injected embryos assayed at preimplantation stages after 16-24 h treatment with ZnSO4. Thus, lacZ can be used as an indicator gene for promoter function during early mouse embryogenesis, and the incorporation of the MT-I promoter into fusion genes can be a useful means of controlling the expression of exogenous genes in preimplantation mouse embryos.     

Use of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside for the isolation of beta-galactosidase-positive bacteria from municipal water supplies

A new medium, mX-Gal, has been developed for the membrane filter enumeration of beta-galactosidase-positive bacteria in municipal water supplies. mX-Gal medium contains the chromogenic beta-galactosidase substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). All Aeromonas, Citrobacter, and Enterobacter strains isolated from raw water on mX-Gal medium were beta-galactosidase positive. In contrast, only 10 to 20% of these strains produced a red colony with a metallic sheen on m-Endo agar LES medium. Of 674 chlorinated water samples analyzed for total coliforms on m-Endo agar LES medium and for beta-galactosidase-positive bacteria on mX-Gal medium, 18 that were negative for coliforms on m-Endo agar LES showed beta-galactosidase-positive bacteria on mX-Gal. Of a total of 50 beta-galactosidase-positive bacteria isolated from these samples, 76% were identified as Aeromonas hydrophila.     

Alizarin-beta-D-galactoside: a new substrate for the detection of bacterial beta-galactosidase

We describe the synthesis of a new substrate for the detection of bacterial beta-galactosidase. This substrate, alizarin-beta-D-galactoside, is readily hydrolysed to release alizarin which complexes with various metal ions to form brightly coloured chelates. A total of 367 strains of Gram-negative bacteria were examined for their ability to hydrolyse three chromogenic substrates: alizarin-beta-D-galactoside (Aliz-gal), cyclohexenoesculetin-beta-D-galactoside (CHE-gal) and 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). A total of 182 strains (49.6%) were found to hydrolyse at least one of the three substrates. All of these 182 strains (100%) hydrolysed Aliz-gal whereas only 170 (93.4%) and 173 (95.1%) hydrolysed CHE-gal and X-gal, respectively. We conclude that alizarin-beta-D-galactoside is a highly sensitive substrate for the demonstration of beta-galactosidase.     

Characterization of psychrotrophic microorganisms producing beta-galactosidase activities.

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 beta-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 beta-galactosidase activities in whole cells were labile when incubated at 40 degrees C and had temperature optima about 20 degrees C below that of the enzyme encoded by the lacZ gene of Escherichia 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-beta-D-galactopyranoside (X-Gal), suggesting the presence of beta-galactosidase isozymes.     

表达β-半乳糖苷酶的gal B16肿瘤模型的建立及其在肿瘤DNA疫苗研究中的应用

本文工作的目的是建立以β-半乳糖苷酶为标志性抗原的小鼠黑色素瘤模型,并进行肿瘤免疫的研究。我们首先在pcDNA3质粒中引入一个β-半乳糖苷酶编码基因从而建立转染质粒p3gal。p3gal转染小鼠黑色素瘤细胞B16后,再通过G418筛选及X-Gal细胞染色得到表达β-半乳糖苷酶的gal B16细胞株。接着用该细胞株成功地在C57小鼠上建立了表达β-半乳糖苷酶的gal B16肿瘤模型。并在此模型上观察了β-半乳糖苷酶编码基因作为DNA疫苗抑制gal B16肿瘤生长的作用。     

Signal strains that can detect certain DNA replication and membrane mutants of Escherichia coli: isolation of a new ssb allele, ssb-3.

Mutations in several dna genes of Escherichia coli, when introduced into a strain with a lac fusion in the SOS gene sulA, resulted in formation of blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal). Unexpectedly, several lines of evidence indicated that the blue colony color was not primarily due to induction of the SOS system but rather was due to a membrane defect, along with the replication defect, making the cell X-Gal extrasensitive (phenotypically Xgx), possibly because of enhanced permeability to X-Gal or leakage of beta-galactosidase. (i) In most cases, beta-galactosidase specific activity increased only two- to threefold. (ii) Mutations conferring tolerance to colicin E1 resulted in blue colony color with no increase in beta-galactosidase specific activity. (iii) Mutations in either the dnaA, dnaB, dnaC, dnaE, dnaG, or ssb gene, when introduced into a strain containing a bioA::lac fusion, produced a blue colony color without an increase in beta-galactosidase synthesis. These lac fusion strains can serve as signal strains to detect dna mutations as well as membrane mutations. By localized mutagenesis of the 92-min region of the chromosome of the sulA::lac signal strain and picking blue colonies, we isolated a novel ssb allele that confers the same extreme UV sensitivity as a delta recA allele, which is a considerably greater sensitivity than that conferred by the two well-studied ssb alleles, ssb-1 and ssb-113. The technique also yielded dnaB mutants; fortuitously, uvrA mutants were also found.     

trans-Sialidase catalyzed sialylation of beta-galactosyldisaccharide with an introduction of beta-galactosidase

Introduction of beta-galactosidase into a trans-sialidase reaction, i.e. sialic acid transfer reaction from a donor substrate (alpha2,3-sialyllactose) to an acceptor substrate (beta-galactosyldisaccharide), could improve the yield of desired sialylated trisaccharide by hydrolyzing lactose, a byproduct from the donor. When trans-sialidase reaction was performed with stoichiometric amounts (2 mM) of alpha2,3-sialyllactose and Galbeta(1,3)GlcNAc, the yield of NeuAcalpha(2,3)Galbeta(1,3)GlcNAc increased from 45% to 75% by the coupling of Escherichia coli beta-galactosidase. Furthermore, by changing the substrate ratio in the coupled reaction, i.e. two-fold excess of alpha2,3-sialyllactose to Galbeta(1,3)GlcNAc, above 95% of yield was achieved based on the amount of Galbeta(1,3)GlcNAc. However, two-fold excess of Galbeta(1,3)GlcNAc to alpha2,3-sialyllactose in this reaction was more desirable for the purification of NeuAcalpha(2,3)Galbeta(1,3)GlcNAc, since complete consumption of alpha2,3-sialyllactose was achieved. Efficiency of the coupled reaction was affected by the specificity of beta-galactosidase for acceptor substrate. When Galbeta(1,6)GlcNAc was used as the acceptor, E. coli beta-galactosidase hydrolyzed Galbeta(1,6)GlcNAc as well as lactose in the coupled reaction, resulting in a significant decrease in the yield of desired sialylated trisaccharide. The conversion yield of the sialylation of Galbeta(1,6)GlcNAc could be improved by employing Bacillus circulans beta-galactosidase.     

A bifunctional chimeric protein consisting of MutS and beta-galactosidase

A bifunctional protein consisting of MutS, a mismatch binding protein and a beta-galactosidase reporter domain has been constructed. The fusion of beta-galactosidase to the MutS C-terminus was obtained by cloning the Escherichia coli lacZ gene encoding beta-galactosidase into a plasmid vector carrying the Thermus thermophilus mutS gene. Milligram amounts of this huge chimeric protein (217 kDa monomer) were purified from 1l of overexpressing E. coli cells using metal-chelate affinity chromatography. The mismatch binding properties of the fusion protein were confirmed by DNA mobility shift assay in polyacrylamide gels. Binding to biotinylated mismatched DNA immobilized on streptavidin microplates followed by colorimetric reaction with X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), demonstrated both mismatch recognition and beta-galactosidase activity of the chimeric protein. The activity of beta-galactosidase domain of the fusion was similar to that of the native enzyme. A colorimetric assay for beta-galactosidase activity using X-Gal supplemented with NBT (nitro blue tetrazolium) allowed detection of 50 and 500 fmol of the chimeric protein with naked eye in 45 microl volumes after 120 and 15 min incubation, respectively.     

New approach to cell lineage analysis in mammals using the Cre-loxP system

The Cre-loxP site-specific recombination system was used for cell lineage analysis in mammals. We constructed an expression plasmid, pCETZ-17, which consists of cytomegalovirus enhancer/chicken beta-actin promoter (CAG), a portion of the rabbit beta-globin gene, loxP-flanked DNA sequence (containing enhanced green fluorescent protein (EGFP) cDNA), and lacZ gene encoding E. coli beta-galactosidase (beta-gal). When circular pCETZ-17 plasmid DNA was microinjected into the pronuclei of fertilized eggs and these eggs were allowed to develop to two-cell stage, 62.8% (59/94) of the two-cell embryos exhibited distinct fluorescence in one or both blastomeres, but never expressed lacZ protein, as evaluated by histochemical staining with X-Gal, a substrate for beta-gal. When both circular plasmids, pCETZ-17 and pCAG/NCre (containing CAG and DNA sequences encoding nuclear location signal and Cre), were co-injected into fertilized eggs, almost all (87.0%, 47/54) embryos exhibited low or no fluorescence, but 51.9% (27/52) exhibited positive staining for beta-gal activity. This indicates that transient expression of the Cre recombinase gene removed the loxP-flanked DNA sequence in pCETZ-17 and then caused expression of the downstream lacZ sequence. We next microinjected pCETZ-17 into the pronuclei of fertilized eggs, cultured these injected eggs for 1 day, and collected only two-cell embryos expressing EGFP in both blastomeres. One blastomere of the EGFP-expressing two-cell embryos was microinjected with pCAG/NCre, and these treated embryos were cultured for 1 day up to four-cell stage. When the developing four-cell embryos were subjected to staining with X-Gal, cell lineage-related staining pattern for beta-gal activity was observed in most (77.8%, 7/9) embryos. These findings were further confirmed using two-cell embryos derived from a transgenic mouse line carrying CETZ-17 transgene. Thus, our system, which is based on transient expression of the Cre recombinase gene directly introduced into nuclei of embryonic cells by microinjection, is a powerful means for cell lineage analysis in mammals.     

Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the experimentally injured rabbit cornea and tear fluid using the sensitive substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC)

Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the rabbit eye after various experimental injuries were performed using the same sensitive fluorogenic substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC). The aim of the study was to examine whether the severity of corneal damage corresponds with the level of the enzyme activity in the tear fluid. As until recently the substrate beta-galactoside-4-HFC had not been used for the histochemical detection of acid beta-galactosidase in the cornea, results obtained with this substrate in a fluorescent method were compared in parallel cryostat sections with results obtained using the substrate 5-bromo-4-chloro-3-indoxyl beta-galactoside in the indigogenic method (previously shown to be very sensitive for the detection of acid beta-galactosidase activity in the cornea). Both methods revealed similar localization and changes in enzyme activity; using beta-galactoside-4-HFC an acceptable cellular localization was achieved. For the measurement of acid beta-galactosidase activity in the tear fluid a semiquantitative biochemical method was elaborated using filter paper punches with the substrate (beta-galactoside-4-HFC) soaked with tears and incubated at 37 degrees C. The time of the first appearance of a greenish-yellow fluorescence (enzyme positivity) was recorded by UV lamp and compared with the appearance of fluorescence in calibrated punches containing known acid beta-galactosidase activities. The results show that beta-galactoside-4-HFC is useful for the biochemical assessment of acid beta-galactosidase activity in the tear fluid. Comparing histochemical and biochemical results, it can be concluded that increased enzymatic activity in tears parallels the severity of corneal damage. Further studies are necessary to evaluate whether the detection of acid beta-galactosidase activity in tears might be useful for diagnostic purposes in humans.     

Active-site-directed inactivation of Aspergillus oryzae beta-galactosidase with beta-D-galactopyranosylmethyl-p-nitrophenyltriazene

beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene (beta-GalMNT), a specific inhibitor of beta-galactosidase, was isolated as crystals by HPLC and its chemical and physicochemical characteristics were examined. Aspergillus oryzae beta-galactosidase was inactivated by the compound. We studied the inhibition mechanism in detail. The inhibitor was hydrolyzed by the enzyme to p-nitroaniline and an active intermediate (beta-galactopyranosylmethyl carbonium or beta-galactopyranosylmethyldiazonium), which inactivated the enzyme. The efficiency of inactivation of the enzyme (the ratio of moles of inactivated enzyme to moles of beta-GalMNT hydrolyzed by the enzyme) was 3%; the efficiency of Escherichia coli beta-galactosidase was 49%. In spite of the low efficiency, the rate of inactivation of A. oryzae enzyme was not very different from that of the E. coli enzyme, because the former hydrolyzed beta-GalMNT faster than the latter did. A. oryzae beta-galactosidase was also inactivated by p-chlorophenyl, p-tolyl, and m-nitrophenyl derivatives of beta-galactopyranosylmethyltriazene. However, E. coli beta-galactosidase was not inactivated by these triazene derivatives. The results showed that the inactivation of A. oryzae and E. coli beta-galactosidases by beta-GalMNT was an enzyme-activated and active-site-directed irreversible inactivation. The possibility of inactivation by intermediates produced nonenzymatically was ruled out for E. coli, but not for the A. oryzae enzyme.     

A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures

beta-Galactosidase (beta-Gal) activity is a widely accepted biomarker to detect senescence both in situ and in vitro. A cytochemical assay based on production of a blue-dyed precipitate that results from the cleavage of the chromogenic substrate X-Gal is commonly used. Blue and nonblue cells are counted under the microscope and a semiquantitative percentage of senescent cells can be obtained. Here, we present a quantitative, fast, and easy to use chemiluminescent assay to detect senescence. The Galacton chemiluminescent method used to detect the prokaryotic beta-Gal reporter enzyme in transfection studies was adapted to assay mammalian beta-Gal. The assay showed linear production of luminescence in a time- and cell-number-dependent manner. The chemiluminescent assay showed significant correlation with the cytochemical assay in detecting replicative senescence (Pearson r=0.8486, p<0.005). Moreover, the chemiluminescent method (Galacton) also detected stress-induced senescence in cells treated with H2O2 similar to the cytochemical assay (X-Gal) (Galacton: control 25,207.3+/-6548.6, H2O2 52,487.4+/-16,284.9, p<0.05; X-Gal: control 41.31+/-7.0%, H2O2 92.97+/-2.8%, p<0.01). Thus, our method is well suited to the detection of replicative and stress-induced senescence in cell culture.     

Characterization of a psychrotrophic Arthrobacter gene and its cold-active beta-galactosidase.

Enzymes with high specific activities at low temperatures have potential uses for chemical conversions when low temperatures are required, as in the food industry. Psychrotrophic microorganisms which grow at low temperatures may be a valuable source of cold-active enzymes that have higher activities at low temperatures than enzymes found for mesophilic microorganisms. To find cold-active beta-galactosidases, we isolated and characterized several psychrotrophic microorganisms. One isolate, B7, is an Arthrobacter strain which produces beta-galactosidase when grown in lactose minimal media. Extracts have a specific activity at 30 degrees C of 2 U/mg with o-nitrophenyl-beta-D-galactopyranoside as a substrate. Two isozymes were detected when extracts were subjected to electrophoresis in a nondenaturing polyacrylamide gel and stained for activity with 5-bromo-4-chloro-indolyl-beta-D-galactopyranoside (X-Gal). When chromosomal DNA was prepared and transformed into Escherichia coli, three different genes encoding beta-galactosidase activity were obtained. We have subcloned and sequenced one of these beta-galactosidase genes from the Arthrobacter isolate B7. On the basis of amino acid sequence alignment, the gene was found to have probable catalytic sites homologous to those from the E. coli lacZ gene. The gene encoded a protein of 1,016 amino acids with a predicted molecular mass of 111 kDa. The enzyme was purified and characterized. The beta-galactosidase from isolate B7 has kinetic properties similar to those of the E. coli lacZ beta-galactosidase but has a temperature optimum 20 degrees C lower than that of the E. coli enzyme.     

Development of a rapid positive/absent test for coliforms using sensitive bioluminescence assay

We have developed a sensitive bioluminescence assay for beta-galactosidase using a luminescent substrate, D-luciferin-O-beta-galactopyranoside (LuGal). The detection limit for beta-galactosidase was 3 x 10-20 mol per assay, which was approximately 50-fold more sensitive than the test using a fluorescent substrate. This assay was applied to a positive/absent (P/A) test for coliforms. Observations made after 7 h of culture followed by a 10-min enzyme assay using LuGal were comparable to those made after a 22-24-h culture by the current method. Therefore, the LuGal method allows a rapid P/A test for coliforms.     

Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli.

A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the beta-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichia coli. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the beta-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium beta-galactosidase gene in E. coli was not subject to glucose repression. By using transposon Tn5 mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for beta-galactosidase expression in E. coli. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and Klebsiella pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus II, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but Tn5 insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of beta-galactosidase in E. coli.