β-Galactosidase activity assay using far-red-shifted fluorescent substrate DDAOG

β-Galactosidase (β-gal) is commonly used as a reporter gene in biological research, and a wide variety of substrates have been developed to assay its activity. One substrate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) β-d-galactopyranoside (DDAOG), can be cleaved by β-gal to produce 7-hydroxy-9H(I,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). On excitation, DDAO generates a far-red-shifted fluorescent signal. Using this substrate, we developed a β-gal activity assay method. The DDAO signal was stable for at least 18 h. The signal intensity was linearly related to both the enzyme amount and substrate concentration. An optimized buffer for the β-gal/DDAOG assay was also formulated. When compared with the colorimetric substrate o-nitrophenyl-β-d-galactopyranoside (ONPG), the signal-to-background ratio of the DDAOG method was approximately 12-fold higher. The β-gal/DDAOG assay method was also tested in transiently transfected cells employing both pharmacologically and genetically inducible gene expression systems. The ability to detect signal induction is comparable to a similar assay using luciferase as the signal generating moiety. The β-gal/DDAOG assay method should provide a fluorescent reporter assay system for the wide variety of β-gal systems currently in use.     

2-Arylbenzothiazole, benzoxazole and benzimidazole derivatives as fluorogenic substrates for the detection of nitroreductase and aminopeptidase activity in clinically important bacteria

A series of 2-(2-nitrophenyl)benzothiazole 7, 2-(2-nitrophenyl)benzoxazole 10 and 2-(2-nitrophenyl)benzimidazole 13 derivatives have been synthesised and assessed as indicators of nitroreductase activity across a range of clinically important Gram negative and Gram positive bacteria. The majority of Gram negative bacteria produced strongly fluorescent colonies with substrates 7 and 10 whereas fluorescence production in Gram positive bacteria was less widespread. The l-alanine 16 and 19 and β-alanine 21 and 23 derivatives have been prepared from 2-(2-aminophenyl)benzothiazole 14 and 2-(2-aminophenyl)benzoxazole 17. These four compounds have been evaluated as indicators of aminopeptidase activity. The growth of Gram positive bacteria was generally inhibited by these substrates but fluorescent colonies were produced with the majority of Gram negative bacteria tested.     

Escherichia coli β-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii

We have developed several protocols for the use of β-galactosidase (βGal) from Escherichia coli as a reporter enzyme in transfection studies of Toxoplasma gondii (Tg) and as a readily screenable marker for stable transformation. Three Tg expression vectors with different promoters driving lacZ were constructed and shown in transient transfections to differ in their relative expression levels. Using a fluorescent βGal substrate, it was possible to detect enzymatic activity with as little as 50 ng of transfected lacZ-containing plasmid DNA. When stably transformed intracellular parasites were cultivated in microtiter plates in the presence of the color substrate, chlorophenol red-β-D-galactopyranoside (CPRG), the signal from as few as 400 Tg could be readily detected by eye. Using serial dilutions of transfected parasite cultures in the presence of CPRG, we were able to clone stably expressing βGal-positive Tg without the need for another selectable marker. Such lacZ transgenics could also be visualized histochemically in the tissue of infected mice. Thus, the application of βGal to studies on Tg provides not only a much needed second reporter for transient transfection, it also comprises a safe and sensitive marker for the generation and analysis of stably transfected parasites     

A Fluorescent Gram Stain for Flow Cytometry and Epifluorescence Microscopy

The fluorescent nucleic acid binding dyes hexidium iodide (HI) and SYTO 13 were used in combination as a Gram stain for unfixed organisms in suspension. HI penetrated gram-positive but not gram-negative organisms, whereas SYTO 13 penetrated both. When the dyes were used together, gram-negative organisms were rendered green fluorescent by SYTO 13; conversely, gram-positive organisms were rendered red-orange fluorescent by HI, which simultaneously quenched SYTO 13 green fluorescence. The technique correctly predicted the Gram status of 45 strains of clinically relevant organisms, including several known to be gram variable. In addition, representative strains of gram-positive anaerobic organisms, normally decolorized during the traditional Gram stain procedure, were classified correctly by this method.Gram’s staining method is considered fundamental in bacterial taxonomy. The outcome of the Gram reaction reflects major differences in the chemical composition and ultrastructure of bacterial cell walls. The Gram stain involves staining a heat-fixed smear of cells with a rosaniline dye such as crystal or methyl violet in the presence of iodine, with subsequent exposure to alcohol or acetone. Organisms that are decolorized by the alcohol or acetone are designated gram negative.Alternative Gram staining techniques have recently been proposed. Sizemore et al. (19) reported on the use of fluorescently labeled wheat germ agglutinin. This lectin binds specifically to N-acetylglucosamine in the peptidoglycan layer of gram-positive bacteria, whereas gram-negative organisms contain an outer membrane that prevents lectin binding. Although simpler and faster than the traditional Gram stain, this method requires heat fixation of organisms.Other Gram stain techniques suitable for live bacteria in suspension have been described. Allman et al. (1) demonstrated that rhodamine 123 (a lipophilic cationic dye) rendered gram-positive bacteria fluorescent, but its uptake by gram-negative organisms was poor. This reduced uptake by gram-negative bacteria was attributed to their outer membranes. The outer membrane can be made more permeable to lipophilic cations by exposure to the chelator EDTA (4). Shapiro (18) took advantage of this fact to form the basis of another Gram stain, one which involved comparing the uptake of a carbocyanine dye before and after permeabilizing organisms with EDTA. All of these methods, however, rely on one-color fluorescence, making analysis of mixed bacterial populations difficult.An alternative to the use of stains is the potassium hydroxide (KOH) test. The method categorizes organisms on the basis of differences in KOH solubility. After exposure to KOH, gram-negative bacteria are more easily disrupted than gram-positive organisms. This technique has been used to classify both aerobic and facultatively anaerobic bacteria, including gram-variable organisms (8). In a study by Halebian et al. (9), however, this technique incorrectly classified several anaerobic strains, giving rise to the recommendation that the method should only be used in conjunction with the traditional Gram stain.In this study we demonstrate a Gram staining technique for unfixed organisms in suspension, by using clinically relevant bacterial strains and organisms notorious for their gram variability. The method uses two fluorescent nucleic acid binding dyes, hexidium iodide (HI) and SYTO 13. Sales literature (11) published by the manufacturers of HI (Molecular Probes, Inc., Eugene, Oreg.), which displays a red fluorescence, suggests that the dye selectively stains gram-positive bacteria. SYTO 13 is one of a group of cell-permeating nucleic acid stains and fluoresces green (11). These dyes have been found to stain DNA and RNA in live or dead eukaryotic cells (16). Both dyes are excited at 490 nm, permitting their use in fluorescence instruments equipped with the most commonly available light sources. We reasoned that a combination of these two dyes applied to mixed bacterial populations would result in all bacteria being labeled, with differential labeling of gram-positive bacteria (HI and SYTO 13) and gram-negative bacteria (SYTO 13 only). The different fluorescence emission wavelengths of the two dyes would ensure differentiation of gram-positive from gram-negative bacteria by either epifluorescence microscopy or flow cytometry when equipped with the appropriate excitation and emission filters. While a commercial Gram stain kit produced by Molecular Probes includes HI and an alternative SYTO dye, SYTO 9, we are unaware of any peer-reviewed publications regarding either its use or its effectiveness with traditionally gram-variable organisms.     

Discovery of human Golgi β-galactosidase with no identified glycosidase using a QMC substrate design platform for exo-glycosidase

Post-translational modifications (PTMs) of proteins play important roles in the physiology of eukaryotes. In the PTMs, non-reversible glycosylations are classified as N-glycosylations and O-glycosylations, and are catalyzed by various glycosidases and glycosyltransferases. However, β-glycosidases are not known to play a role in N- and O-glycan processing, although both glycans provide partial structures as substrates for β-galactosidase and β-N-acetylglucosaminidase in the Golgi apparatus of human cells. We explored human Golgi β-galactosidase using fluorescent substrates based on a quinone methide cleavage (QMC) substrate design platform that was previously developed to image exo-type glycosidases in living cells. As a result, we discovered a novel Golgi β-galactosidase in human cells. It is possible to predict a novel and important function in glycan processing of this β-galactosidase, because various β-galactosyl linkages in N- and O-glycans exist in Golgi apparatus. In addition, these results show that the QMC platform is excellent for imaging exo-type glycosidases.     

Synthesis of 2-arylbenzothiazole derivatives and their application in bacterial detection

A series of 2-arylbenzothiazole derivatives have been prepared as fluorogenic enzyme substrates in order to detect aminopeptidase, esterase, phosphatase and β-galactosidase activity in clinically important Gram-negative and Gram-positive bacteria. Substrates were incorporated into an agar-based culture medium and this allowed growth of intensely fluorescent bacterial colonies based on hydrolysis by specific enzymes. Substrate 20 targeted l-alanine aminopeptidase activity and was hydrolysed exclusively by a range of Gram-negative bacteria and inhibited the growth of a range of Gram-positive bacteria. Substrate 19a targeted β-alanyl aminopeptidase activity and generated fluorescent colonies of selected Gram-negative species including Pseudomonas aeruginosa. Substrate 21b targeted C8-esterase activity and resulted in strongly fluorescent colonies of selected species known to harbour such enzyme activity (e.g., Salmonella and Pseudomonas). Most Gram-negative species produced colonies with an intense blue fluorescence due to hydrolysis of phosphatase substrates 24a–c and substrate 24c was also hydrolysed by strains of Staphylococcus aureus. Compounds 26b and 26c targeted β-galactosidase activity and generated strongly fluorescent colonies with coliform bacteria that produced this enzyme (e.g., Escherichia coli).     

A sensitive assay of galactocerebroside beta-galactosidase by high-performance liquid chromatography using galactosyl-N-dansyl-sphingosine as substrate

A rapid and sensitive method was devised for determining β-galactosidase activity specific for galactocerebroside. A fluorescent derivative of galactocerebroside, 1-O-galactosyl-2-N-1-dimethylaminonaphthalene-5-sulfonyl-sphingosine, was used as substrate, and the product, 2-N-1-dimethylaminonaphthalene-5-sulfonyl-sphingosine, was taken into organic solvent phase. Quantitative analysis of 2-N-dimethylaminonaphthalene-5-sulfonyl-sphingosine was carried out fluorometrically by use of high-performance liquid chromatography on silica gel column.     

Divergent genetic control of protein solubility and conformational quality in Escherichia coli

In bacteria, protein overproduction results in the formation of inclusion bodies, sized protein aggregates showing amyloid-like properties such as seeding-driven formation, amyloid-tropic dye binding, intermolecular β-sheet architecture and cytotoxicity on mammalian cells. During protein deposition, exposed hydrophobic patches force intermolecular clustering and aggregation but these aggregation determinants coexist with properly folded stretches, exhibiting native-like secondary structure. Several reports indicate that inclusion bodies formed by different enzymes or fluorescent proteins show detectable biological activity. By using an engineered green fluorescent protein as reporter we have examined how the cell quality control distributes such active but misfolded protein species between the soluble and insoluble cell fractions and how aggregation determinants act in cells deficient in quality control functions. Most of the tested genetic deficiencies in different cytosolic chaperones and proteases (affecting DnaK, GroEL, GroES, ClpB, ClpP and Lon at different extents) resulted in much less soluble but unexpectedly more fluorescent polypeptides. The enrichment of aggregates with fluorescent species results from a dramatic inhibition of ClpP and Lon-mediated, DnaK-surveyed green fluorescent protein degradation, and it does not perturb the amyloid-like architecture of inclusion bodies. Therefore, the Escherichia coli quality control system promotes protein solubility instead of conformational quality through an overcommitted proteolysis of aggregation-prone polypeptides, irrespective of their global conformational status and biological properties.     

Purification of β-glucosidase from the salivary glands of the green rice leafhopper, Nephotettix cincticeps (Uhler) (Hemiptera: Cicadellidae), and its detection in the salivary sheath

The green rice leafhopper, Nephotettix cincticeps (Uhler), is an insect pest of rice and discharges β-glucosidase (EC 3.2.1.21) from its salivary glands during feeding. To investigate the biological function of this enzyme, we purified it from the heads of 18,000 adult females by acetone precipitation and a series of chromatography steps: gel filtration, cation-exchange chromatography, metal-affinity chromatography and hydrophobic interaction chromatography. During cation-exchange chromatography, β-glucosidases were eluted in three peaks (isozymes). These β-glucosidases were monomeric proteins of 58 kDa as estimated by SDS-PAGE and 62 kDa based on gel filtration. All of the purified β-glucosidase isozymes exhibited maximum activity for p-nitrophenyl β-glucoside (NPGlc) and p-nitrophenyl β-galactopyranoside (NPGal) at pH 5.5 and 5.0, respectively. There was no significant difference in substrate specificity among the three isozymes. The Km values were estimated to be 0.13 μM for NPGlc and 0.9 μM for NPGal. Among the oligosaccharide substrates examined, laminaribiose (Glc β1-3 Glc) was the most extensively hydrolyzed, sophorose (Glc β1-2 Glc) and cellobiose (Glc β1-4 Glc) were comparatively well hydrolyzed, and gentiobiose (Glc β1-6 Glc), lactose (Gal β1-4 Glc), laminaritriose, cellotriose and cellotetraose were poorly hydrolyzed. Among the glycoside substrates examined, salicin was considerably well hydrolyzed. β-Glucosidase was detected in the salivary sheaths by activity staining with a fluorescent substrate. The salivary β-glucosidase of N. cincticeps may be involved in the hydrolysis of a phenol glucoside present in the saliva, which is a step in the solidification of gelling saliva to form salivary sheaths.     

Simultaneous enrichment of cereals with polyunsaturated fatty acids and pigments by fungal solid state fermentations

Four Mucor strains were tested for their ability to grow on four cereal substrates and enriched them with gamma-linolenic acid (GLA) and β-carotene. M. circinelloides CCF-2617 as the best producer accumulated of both GLA and β-carotene in high amounts during utilization of rye bran/spent malt grains (3:1). The first growth phase was characterized by rapid GLA biosynthesis, while distinct β-carotene formation was found in the stationary fungal growth. Therefore various cultivation conditions were tested in order to optimize the yield of either GLA or β-carotene. The fungus grown on cereal substrate supplemented with glucose produced maximal 8.5 mg β-carotene and 12.1 g GLA in 1 kg fermented substrate, respectively. On the other hand, the highest amount of GLA in the fermented substrate (24.2 g/kg) was achieved when 30% of sunflower oil was employed to the substrate. Interestingly, β-carotene biosynthesis was completely inhibited when either whey or linseed oil were added to the substrate.     

Novel fluorescent protein from Hydnophora rigida possesses green emission

Fluorescent proteins are a family of proteins capable of producing fluorescence at various specific wavelengths of ultra violet light. We have previously reported the identification and characterization of a novel cyan fluorescent protein (HriCFP) from a reef coral species, Hydnophora rigida. In search of new members of the diverse family of fluorescent proteins, here we report a new green fluorescent protein (HriGFP) from H. rigida. HriGFP was identified, cloned, expressed in Escherichia coli and purified to homogeneity by metal affinity and size exclusion chromatography. The dynamic light scattering and gel filtration experiments suggested the presence of monomers in solution. The peptide mass fingerprint on the purified protein established the identity of HriGFP. HriGFP had excitation peak at 507 nm and emission peak at 527 nm. HriGFP was similar to HriCFP except the last 16 amino acid sequence at the C-terminal; however, they have shown least similarity with other known fluorescent proteins. Moreover the computational model suggests that HriGFP is a globular protein which consists of 6 α-helices and 3 β-sheets. Taken together our results suggested that HriGFP is a novel naturally occurring fluorescent protein that exists as a monomer in solution.     

An efflux pump is involved in secretion of newly synthesized siderophore by Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes the fluorescent siderophore, pyoverdine (PVD), to enable iron acquisition. Epifluorescence microscopy and cellular fractionation were used to investigate the role of an efflux pump, PvdRT-OpmQ, in PVD secretion. Bacteria lacking this efflux pump accumulated PVD, or a fluorescent precursor, in the periplasm, due to their inability to efficiently secrete into the media newly synthesized PVD. PvdRT-OpmQ is only the second system identified for secretion of newly synthesized siderophores by Gram negative bacteria.     

Transient and permanent gene transfer into the brain of the teleost fish medaka (Oryzias latipes) using human adenovirus and the Cre-loxP system

In this study, we demonstrated that human type-5 adenovirus infected the brain of the teleost fish, medaka (Oryzias latipes), in vivo. Injection of adenoviral vector into the mesencephalic ventricle of medaka larvae induced the expression of reporter genes in some parts of the telencephalon, the periventricular area of the mesencephalon and diencephalon, and the cerebellum. Additionally, the Cre-loxP system works in medaka brains using transgenic medaka carrying a vector containing DsRed2, flanked by loxP sites under control of the β-actin promoter and downstream promoterless enhanced green fluorescent protein (EGFP). We demonstrated that the presence of green fluorescence depended on injection of adenoviral vector expressing the Cre gene and confirmed that EGFP mRNA was transcribed in the virus-injected larvae.     

Synthesis of nucleotide-activated disaccharides with recombinant β3-galactosidase C from Bacillus circulans

Nucleotide-activated di- and oligosaccharides represent a novel class of glycoconjugates. They are components of human milk with still unknown biological function. Synthetic access to a wide range of nucleotide di- and oligosaccharides would also facilitate their utilization as donor substrates or inhibitors of Leloir-glycosyltransferases. We here present for the first time the synthesis of β1-3-linked nucleotide activated disaccharides by recombinant β3-galactosidase C from Bacillus circulans. UDP-Glc, UDP-GlcNAc, and UDP-GalNAc reacted as acceptor substrates in the transglycosylation reaction with p-nitrophenyl-β-galactoside as donor substrate. In an attempt to optimise the transglycosylation reaction, focused microwave irradiation was investigated. In comparison to conventional thermal heating product compositions and product yields were affected by microwave irradiation and depended on the used acceptor substrate. Microwave irradiation was advantageous for syntheses with UDP-GlcNAc as preferred acceptor substrate of β3-galactosidase C. The β1,3 linked UDP-disaccharide was the main product with minor fractions of UDP-tri- and UDP-tetrasaccharide. In summary, access to important UDP-disaccharides such as UDP-LacNAc type 1 and UDP-Thomsen-Friedenreich(T)-antigen was accomplished for further studies of their role as donor substrates or inhibitors of glycosyltransferases.     

Expression of recombinant actin 5C from Drosophila in the methylotrophyc yeast Pichia pastoris

A system of expression for the foreign actin gene in yeast cells Pichia pastoris has been developed. As a target protein, the Drosophila cytoplasmic actin 5C, which has 90% homology to the β-actin of higher eukaryotes, was used. In the present work, in order to develop conditions for biosynthesis of the target protein in yeast cells and a purification procedure for the recombinant protein, a GFP-actin fusion protein containing green fluorescent protein (GFP) as a fusion tag was expressed and purified. The size and survival of P. pastoris cells producing recombinant protein were characterized and shown to depend on the accumulation of recombinant actin. The purified fusion protein was used to obtain a polyclonal antibody necessary for testing for recombinant actin.     

Improved plasmid vectors for the production of multiple fluorescent protein fusions in Bacillus subtilis

The intrinsically fluorescent green fluorescent protein has been used in many laboratories as a cytological marker to monitor protein localisation in live cells. Multiple spectrally modified mutant versions and novel fluorescent proteins from other species have subsequently been reported and used for labelling cells with multiple fluorescent protein fusions. In this work we report the design and use of vectors containing some of these spectral variants of GFP for use in the Gram positive bacterium Bacillus subtilis. These vectors complement those previously described (Lewis and Marston, 1999. Gene 227, 101-109) to provide a large suite of plasmid vectors for use in this and other related Gram positive organisms. Using these vectors we have been able to directly demonstrate the sequential assembly/disassembly of proteins involved in the generation of cellular asymmetry during development.     

Fluorescent Method for Monitoring Cheese Starter Permeabilization and Lysis

A fluorescence method to monitor lysis of cheese starter bacteria using dual staining with the LIVE/DEAD BacLight bacterial viability kit is described. This kit combines membrane-permeant green fluorescent nucleic acid dye SYTO 9 and membrane-impermeant red fluorescent nucleic acid dye propidium iodide (PI), staining damaged membrane cells fluorescent red and intact cells fluorescent green. For evaluation of the fluorescence method, cells of Lactococcus lactis MG1363 were incubated under different conditions and subsequently labeled with SYTO 9 and PI and analyzed by flow cytometry and epifluorescence microscopy. Lysis was induced by treatment with cell wall-hydrolyzing enzyme mutanolysin. Cheese conditions were mimicked by incubating cells in a buffer with high protein, potassium, and magnesium, which stabilizes the cells. Under nonstabilizing conditions a high concentration of mutanolysin caused complete disruption of the cells. This resulted in a decrease in the total number of cells and release of cytoplasmic enzyme lactate dehydrogenase. In the stabilizing buffer, mutanolysin caused membrane damage as well but the cells disintegrated at a much lower rate. Stabilizing buffer supported permeabilized cells, as indicated by a high number of PI-labeled cells. In addition, permeable cells did not release intracellular aminopeptidase N, but increased enzyme activity was observed with the externally added and nonpermeable peptide substrate lysyl-p-nitroanilide. Finally, with these stains and confocal scanning laser microscopy the permeabilization of starter cells in cheese could be analyzed.     

Green fluorescent protein marks skeletal muscle in murine cell lines and zebrafish

The green fluorescent protein (GFP) acts as a vital dye upon the absorption of blue light. When the gfp gene is expressed in bacteria, flies or nematodes, green fluorescence can be directly observed in the living organism. We inserted the cDNA encoding this 238-amino-acid (aa) jellyfish protein into an expression vector containing the rat myosin light-chain enhancer (MLC-GFP) to evaluate its ability to serve as a muscle-specific marker. Transiently, as well as stably, transfected C2C12 cell lines produced high levels of GFP distributed homogeneously throughout the cytoplasm and was not toxic through several cell passages. Expression of MLC-GFP was strictly muscle-specific, since Cos 7 fibroblasts transfected with MLC-GFP did not fluoresce. When GFP and βGal markers were compared, the GFP signal was visible in the cytoplasm of the living cell, whereas visualization of βGal required fixation and resulted in deformation of the cells. When the MLC-GFP construct was injected into zebrafish embryos, muscle-specific gfp expression was apparent within 24 h of development, gfp expression was never observed in non-muscle tissues using the MLC-GFP construct. Transgenic fish continued to express high levels of gfp in skeletal muscle at 1.5 months, demonstrating that GFP is an effective marker of muscle cells in vivo.     

Effects of phospholipids on substrate specificity of β-galactosidase purified from Aspergillus oryzae

When entrapped into liposomes composed of phosphatidylcholine and other lipids, β-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) purified from Aspergillus oryzae could cleave the β-galactosidic bond of the terminal galactose of galactocerebroside and GM₁-ganglioside (II³NeuAc-GgOse₄Cer, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminosyl)-galactosylglucosylceramide), while the free enzyme could not. The products of the hydrolysis of galactocerebroside were found to be β-galactose and ceramide, which was confirmed by using a fluorescent analog of galactocerebroside, 1-O-galactosyl-2-N-(1-dimethylaminonaphthalene-5-sulfonyl)-sphingosine, as substrate. The formation of GM₂-ganglioside (II³NeuAc-GgOse₃Cer, N-acetylgalactosaminyl-(N-acetylneuraminosyl)-galactosylglucosylceramide) by the hydrolysis of GM₁-ganglioside was also demonstrated. The lipid composition of the liposomes influenced the amount of the enzyme entrapped and the activity of the trapped enzyme. A large amount of the enzyme was entrapped into the liposomes composed of phosphatidylcholine-cholesterol-stearoylamine (molar ratio, 7:2:1). The enzyme trapped in the liposomes and that in those of phosphatidylcholine-cholesterol-sulfatide (molar ratio, 7:2:1) had higher activity on galactocerebroside and GM₁-ganglioside than that in other liposomes. The activity of β-galactosidase trapped in liposomes was increased in the presence of detergent, while that of the free enzyme was not changed.By a similar procedure to introduce enzymes into hydrophobic environments, enzymes other than β-galactosidase might come to possess different substrate specificities.     

Functional analysis of an α-helical antimicrobial peptide derived from a novel mouse defensin-like gene

Gene-encoded antimicrobial peptides (AMPs) are an essential component of the innate immune system in many species. Analysis of β-defensin gene expression in mouse tissue using primers that were specific for conserved sequences located outside of the β-defensin translated region identified a novel small gene. The novel gene had an open reading frame of 114 bp and encoded a predicted protein of 37 amino acid residues. A search of the genome database revealed that the gene locus and the sequence of exon 1 of this novel gene were similar to subgroup 1 mouse β-defensins. A small peptide, K17 (FSPQMLQDIIEKKTKIL), derived from the amino acid sequence of this novel gene was synthesized. Circular dichroism (CD) spectroscopic analysis of chemically synthesized peptide demonstrated that the peptide exhibited random coil conformation in aqueous solution, but the peptide adopted helical conformation in the presence of trifluoroethanol or sodium dodecyl sulfate, a membrane-mimicking environment. The peptide exhibited bactericidal activity against Salmonella enterica serovar Typhimurium (Gram negative) and Staphylococcus aureus (Gram positive); it was not cytotoxic in cultures of mammalian cells or hemolytic in cultures of erythrocytes. These results suggested that K17 may be a candidate therapeutic for the treatment of bacterial infection.