Grape antioxidant dietary fibre prevents mitochondrial apoptotic pathways by enhancing Bcl-2 and Bcl-xL expression and minimising oxidative stress in rat distal colonic mucosa.

Grape antioxidant dietary fibre (GADF) is a grape product rich in dietary fibre and natural antioxidants. We reported previously that GADF intake reduced apoptosis and induced a pro-reducing shift in the glutathione (GSH) redox status of the rat proximal colonic mucosa. The aim of the study was to elucidate the molecular mechanisms responsible for the anti-apoptotic effect of GADF and their association with the oxidative environment of the distal colonic mucosa. The ability of GADF to modify colonic crypt cell proliferation was also investigated. Male Wistar rats (n 20) were fed with diets containing either cellulose (control group) or GADF (GADF group) as fibre for 4 weeks. GADF did not modify cell proliferation but induced a significant reduction of colonic apoptosis. The anti-apoptotic proteins Bcl-2 (B-cell lymphoma-2) and Bcl-xL (B-cell lymphoma extra large) were up-regulated in the mitochondria and down-regulated in the cytosol of the GADF mucosa, whereas the opposite was found for the pro-apoptotic protein Bax (Bcl-2-associated X protein), leading to an anti-apoptotic shift in the pattern of expression of the Bcl-2 family. Cytosolic cytochrome c and cleaved caspase-3 levels and caspase-3 activity were reduced by GADF. The modulation of the antioxidant enzyme system and the increase of the cytosolic GSH:glutathione disulfide (GSSG) ratio elicited by GADF helped to reduce oxidative damage. The cytosolic GSH:GSSG ratio was negatively related to apoptosis. These results indicate that GADF acts on the expression of the pro- and anti- apoptotic Bcl-2 proteins, attenuating the mitochondrial apoptotic pathway in the distal colonic mucosa. This effect appears to be associated with the antioxidant properties of GADF.

Grape antioxidant dietary fiber stimulates Lactobacillus growth in rat cecum.

UNLABELLED: The digesta is a highly active biological system where epithelial cells, microbiota, nondigestible dietary components, and a large number of metabolic products interact. The gut microbiota can be modulated by both endogenous and exogenous substrates. Undigested dietary residues are substrates for colonic microbiota and may influence gut microbial ecology. The objective of this work was to study the capacity of grape antioxidant dietary fiber (GADF), which is rich in polyphenols, to modify the bacterial profile in the cecum of rats. Male adult Wistar rats were fed for 4 wk with diets containing either cellulose or GADF as dietary fiber. The effect of GADF on bacterial growth was evaluated in vitro and on the cecal microbiota of rats using quantitative real time polymerase chain reaction (RT-PCR). The results showed that GADF intake stimulates proliferation of Lactobacillus and slightly affects the composition of Bifidobacterium species. GADF was also found to have a stimulative effect on Lactobacillus reuteri and Lactobacillus acidophilus in vitro. These findings suggest that the consumption of a diet rich in plant foods with high dietary fiber and polyphenol content may enhance the gastrointestinal health of the host through microbiota modulation. PRACTICAL APPLICATION: Grape antioxidant fiber combines nutritional and physiological properties of dietary fiber and natural antioxidants from grapes. Grape antioxidant fiber could be used as an ingredient for functional foods and as a dietary supplement to increase the intake of dietary fiber and bioactive compounds.

Interaction of the Salmonella Typhimurium effector protein SopB with host cell Cdc42 is involved in intracellular replication.

The phosphoinositide phosphatase SopB/SigD is a type III secretion system effector that plays multiple roles in Salmonella internalization and intracellular survival. We previously reported that SopB complexed with and inhibited the small GTPase Cdc42 when expressed in a yeast model system, independently of its phosphatase activity. Here we show that human Cdc42, but not Rac1, interacts with catalytically inactive SopB when coexpressed in Saccharomyces cerevisiae. This interaction occurs with both constitutively active and non-activatable Cdc42, suggesting that SopB binds Cdc42 independently of its activation state. By mutational analysis we have narrowed the Cdc42-interacting region of SopB to the first 142 amino acids, and isolated a collection of point mutations in this region, mainly affecting leucine residues conserved in the related Shigella IpgD protein. Such mutations yielded SopB unable to interact with Cdc42 but maintained phosphatase activity. SopB mutant proteins defective for binding Cdc42 were ubiquitinated upon translocation in mammalian cells, but their localization to the Salmonella-containing vacuole was reduced compared with wild-type SopB. Whereas invasion of mammalian cells by Salmonella bearing these sopB mutations was not affected, intracellular replication was less efficient, suggesting that SopB-Cdc42 interaction contributes to the adaptation of Salmonella to the intracellular environment.

[Polymorphism of mucA and fpvA genes in Pseudomonas aeruginosa isolates from cystic fibrosis patients: co-existence of genetically different variants]. / Polimorfismo de los genes mucA y fpvA en Pseudomonas aeruginosa de pacientes con fibrosis quística: coexistencia de variantes genéticas en el mismo paciente.

INTRODUCTION: Pseudomonas aeruginosa is able to colonize the lungs of cystic fibrosis patients (CF) in an adaptive process that results in the selection of a dominant strain through a process of genetic variation. METHODS: One hundred and twenty tree isolates of P. aeruginosa were sequentially recovered from 6 CF patients during the routine follow-up or exacerbations over periods of 2 to 12 years in the Ramon y Cajal University Hospital (Madrid, Spain). Another 13 isolates were obtained from a single CF patient in a short-term study. They were analysed by restriction fragment length polymorphism (RFLP) and sequencing of mucA and fpvA genes, which code for the alginate biosynthesis regulator and a pyoverdin receptor, respectively, and their antibiotic susceptibility was studied by microdilution. RESULTS: A dominant colonising strain was found in each patient based on the RFLP profile. The polymorphisms of mucA and fpvA genes correlated well with these profiles, but suggested a relationship between strains isolated from two brothers, not inferred by RFLP. Stop codon mutations in mucA were unique to each dominant strain, indicating the adaptive process suffered. The alternate detection of the same mucA and/or fpvA genotypic variants suggested the coexistence of several subpopulations. This hypothesis was confirmed in a prospective study in which 6 variants were isolated in 7 days from the same patient. CONCLUSIONS: Genotypic variants of the P. aeruginosa dominant strains can coexist in the chronic colonization in CF patients. These variants can be undetected by RFLP and they might present variable antibiotic susceptibility.

Polimorfismo de los genes mucA y fpvA en Pseudomonas aeruginosa de pacientes con fibrosis quística: coexistencia de variantes genéticas en el mismo paciente / Polymorphism of mucA and fpvA genes in Pseudomonas aeruginosa isolates from cystic fibrosis patients: co-existence of genetically different variants

Introducción Pseudomonas aeruginosa coloniza la mucosa respiratoria del paciente con fibrosis quística (FQ), seleccionándose en general una única cepa dominante en un proceso de variación genética. Métodos Se obtuvieron secuencialmente 123 aislados de P. aeruginosa en 6 pacientes con FQ atendidos durante su seguimiento habitual o en las exacerbaciones a lo largo de 2-12 años en el Hospital Universitario Ramón y Cajal (Madrid). Otros 13 aislados se obtuvieron de un sólo paciente en un estudio a corto plazo (7 días). Se estudió su sensibilidad antimicrobiana por microdilución y caracterizaron mediante polimorfismo de fragmentos de restricción (RFLP) y secuenciación de los genes mucA y fpvA, que codifican respectivamente el regulador de la síntesis de alginato y un receptor de pioverdina. Resultados Se identificó en cada paciente una cepa dominante según el perfil de RFLP. Los polimorfismos de los genes mucA y fpvA se correlacionaron bien con dicho perfil, pero en dos pacientes hermanos se observó una relación entre cepas no evidente por RFLP. Las mutaciones sin sentido en mucA fueron exclusivas de la cepa dominante de cada paciente, reflejando el proceso adaptativo. La detección alternativa de los mismos polimorfismos en mucA o fpvA mostró la coexistencia de subpoblaciones en cada paciente. Esta hipótesis se confirmó en el estudio prospectivo de 7 días al aislar 6 variantes en un único paciente. Conclusiones En la colonización crónica por P. aeruginosa en pacientes con FQ coexisten variantes genotípicas no siempre detectadas por RFLP y con diferentes perfiles de sensibilidad (AU)

Introduction Pseudomonas aeruginosa is able to colonize the lungs of cystic fibrosis patients (CF) in an adaptive process that results in the selection of a dominant strain through a process of genetic variation. Methods One hundred and twenty tree isolates of P. aeruginosa were sequentially recovered from 6 CF patients during the routine follow-up or exacerbations over periods of 2 to 12 years in the Ramon y Cajal University Hospital (Madrid, Spain). Another 13 isolates were obtained from a single CF patient in a short-term study. They were analysed by restriction fragment length polymorphism (RFLP) and sequencing of mucA and fpvA genes, which code for the alginate biosynthesis regulator and a pyoverdin receptor, respectively, and their antibiotic susceptibility was studied by microdilution. Results A dominant colonising strain was found in each patient based on the RFLP profile. The polymorphisms of mucA and fpvA genes correlated well with these profiles, but suggested a relationship between strains isolated from two brothers, not inferred by RFLP. Stop codon mutations in mucA were unique to each dominant strain, indicating the adaptive process suffered. The alternate detection of the same mucA and/or fpvA genotypic variants suggested the coexistence of several subpopulations. This hypothesis was confirmed in a prospective study in which 6 variants were isolated in 7 days from the same patient. Conclusions Genotypic variants of the P. aeruginosa dominant strains can coexist in the chronic colonization in CF patients. These variants can be undetected by RFLP and they might present variable antibiotic susceptibility (AU)

Chronic colonization by Pseudomonas aeruginosa of patients with obstructive lung diseases: cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary disease.

Pseudomonas aeruginosa is isolated in sputum cultures from cystic fibrosis (CF) patients and adults with bronchiectasis (BS) and chronic obstructive pulmonary disease, but it is not well known if the characteristics of colonization in these latter patients are similar to those with CF. We examined 125 P. aeruginosa isolates obtained from 31 patients suffering from these diseases by pulsed field gel electrophoresis and genotyping of mucA and fpvA genes. The pattern of colonization, with dominance of a clonal strain and incidence of mucoid phenotypes, was similar in every group of patients; however, in some CF and BS patients, we detected the replacement or coexistence of 2 main clones. The main differences were found in the nucleotide position of less common mucA mutations, other than mucA22, and in the predominance of the different types of the pyoverdine receptor. Our results support a similar colonization pattern by P. aeruginosa in the different obstructive pulmonary diseases.

Microbiological quality of saffron from the main producer countries.

A microbiological study of saffron spice was undertaken in the context of a European research project (Methodologies for Implementing International Standards for Saffron Purity and Quality, the acronym for which is SAFFIC), analyzing 79 samples obtained from the main producer countries, namely Greece, Iran, Italy, Morocco, and Spain. Current microbiological quality criteria are the same as for other spices, but saffron is added in minute quantities during the cooking process, so the health risk associated with microbial contamination might be lower. We did not detect Salmonella either by culture or by PCR methods in any sample, and Escherichia coli was only found in five samples. Enterobacteriaceae were frequently found (70.9% of the samples), but most of them belonged to species of probable environmental origin. Aerobic sporulated bacteria were also common, but only three samples contained Bacillus cereus at low levels (<200 CFU g(-1)). Clostridium perfringens counts were also very low, with only one sample reaching >100 CFU g(-1), an acceptable value. Overall, microbial contamination in saffron was markedly lower than it was in other spices.

Stimulatory role of grape pomace polyphenols on Lactobacillus acidophilus growth.

Grape pomace is a natural product rich in dietary fiber and polyphenols. A major part of dietary polyphenols is not absorbed in the small intestine and can interact with colonic microbiota. The influence of grape polyphenols on Lactobacillus acidophilus CECT 903 growth was investigated through agar diffusion assays and cultures in liquid media. Grape phenolic extracts and some standards of phenolic compounds (caffeic acid, gallic acid, tannic acid, catechin, epicatechin, and quercetin) were assayed. All phenolic compounds tested did not exert an inhibitory effect on L. acidophilus growth at a maximum concentration of 5000 microg/disk in agar diffusion assays. In addition, a stimulatory trend in bacterial growth was observed in both grape phenolic extracts and tannic acid. The major finding was that grape pomace phenolic extract (1mg/mL) induced a significant biomass increase of L. acidophilus grown in liquid culture media. Further research into the interaction between phenolic compounds and other intestinal bacteria, as well as healthy consequences, is required.

Addressing the effects of Salmonella internalization in host cell signaling on a reverse-phase protein array.

Through acute enteric infection, Salmonella invades host enterocytes and reproduces intracellularly into specialized vacuolae. This involves changes in host cell signaling elicited by bacterial proteins delivered via type III secretion systems (TTSS). One of the two TTSSs of Salmonella enterica serovar Typhimurium encoded by the Salmonella pathogenicity island-1, triggers bacterial internalization. Among the effector proteins translocated by this TTSS, the GTPase modulator SopE/E2 and the phosphoinositide phosphatase SigD are known to play key roles in these processes. To better understand their contribution to re-programming host cell pathways, we used ZeptoMARK reverse-phase protein array technology, which allows printing 32-sample lysate arrays that can be analyzed with phospho-specific antibodies to evaluate the phosphorylation of signaling proteins. Lysates were obtained at different times after infection of HeLa cells with WT, TTSS-deficient, sopE/E2 and sigD single and double deletants, as well as different sigD Salmonella mutants. Our analysis detected activation of p38, JNK and ERK mitogen-activated protein kinases, mainly dependent on SopE/E2, as well as SigD-dependent phosphorylation of PKB/Akt and its targets GSK-3beta and FKHR/FoxO. This is the first time that reverse-phase protein array technology is used in the cellular microbiology field, demonstrating its value to screen for host signaling events through bacterial infection.

A yeast-based genetic screen for identification of pathogenic Salmonella proteins.

Salmonella uses type III secretion systems (TTSS) to deliver pathogenic proteins into the host cells. These translocated effectors induce bacterial internalization and intracellular proliferation by targeting important cellular processes that are conserved among eukaryotes. Here, we assessed the feasibility of performing a genetic screen in yeast to identify novel Salmonella effectors, by searching for genes that produce toxicity when expressed in this model system. We identified several known TTSS-translocated effectors and found that two of them, SteC and SseF, from Salmonella enterica serovar Typhimurium, interfere with cytoskeletal dynamics as they do in mammalian cells. We also identified 11 genes of unknown function (seven from S. Typhi and four from S. Typhimurium) that display features commonly showed by effector proteins, such as a (G+C) content lower than the average for the chromosome, suggesting their acquisition by horizontal transfer processes. Five of these proteins are highly conserved only among Salmonella serovars, whereas the other six are also conserved in other pathogenic or opportunistic enterobacteria. Moreover, we identified other proteins that share specific activity domains with either translocated or bacterial-confined proteins known to be involved in pathogenesis, which might also act as virulence proteins.

Inhibition of Cdc42-dependent signalling in Saccharomyces cerevisiae by phosphatase-dead SigD/SopB from Salmonella typhimurium.

Heterologous expression of bacterial virulence factors in Saccharomyces cerevisiae is a feasible approach to study their molecular function. The authors have previously reported that the Salmonella typhimurium SigD protein, a phosphatidylinositol phosphatase involved in invasion of the host cell, inhibits yeast growth, presumably by depleting an essential pool of phosphatidylinositol 4,5-bisphosphate, and also that a catalytically inactive version, SigD(R468A), was able to arrest growth by a different mechanism that involved disruption of the actin cytoskeleton. This paper describes marked differences between the phenotypes elicited by expression of SigD and SigD(R468A) in yeast. First, expression of SigD(R468A) caused accumulation of large unbudded cells and loss of septin organization, while SigD expression caused none of these effects. Second, growth inhibition by SigD(R468A) was mediated by a cell cycle arrest in G2 dependent on the Swe1 morphogenetic checkpoint, but SigD-induced growth inhibition was cell cycle independent. And third, SigD caused strong activation of the yeast MAP kinase Slt2, whereas SigD(R468A) rather inactivated another MAP kinase, Kss1. In a screen for suppressors of SigD(R468A)-induced growth arrest by overexpression of a yeast cDNA library, the Cdc42 GTPase was isolated. Furthermore, SigD(R468A) was co-purified with Cdc42 from yeast lysates. It is concluded that the Salmonella SigD protein deprived of its phosphatase activity is able to disrupt yeast morphogenesis by interfering with Cdc42 function, opening the possibility that the SigD N-terminal region might directly modulate small GTPases from the host during infection.

The amino-terminal non-catalytic region of Salmonella typhimurium SigD affects actin organization in yeast and mammalian cells.

The internalization of Salmonella into epithelial cells relies on the function of bacterial proteins which are injected into the cell by a specialized type III secretion system. Such bacterial effectors interfere with host cell signalling and induce local cytoskeletal rearrangements. One of such effectors is SigD/SopB, which shares homology with mammalian inositol phosphatases. We made use of the Saccharomyces cerevisiae model for elucidating new aspects of SigD function. Endogenous expression of SigD in yeast caused severe growth inhibition. Surprisingly, sigD alleles mutated in the catalytic site or even deleted for the whole C-terminal phosphatase domain still inhibited yeast growth by inducing loss of actin polarization and precluding the budding process. Accordingly, when expressed in HeLa cells, the same sigD alleles lost the ability of depleting phosphatidylinositol 4,5-bisphosphate from the plasma membrane, but still caused disappearance of actin fibres and loss of adherence. We delineate a region of 25 amino acids (residues 118-142) that is necessary for the effect of SigD on actin in HeLa cells. Our data indicate that SigD exerts a toxic effect linked to its N-terminal region and independent of its phosphatase activity.

Molecular and microbiological analysis of caecal microbiota in rats fed with diets supplemented either with prebiotics or probiotics.

The potential health-improving effects of both a prebiotic and a probiotic infant formula have been evaluated in a rat model. Two groups of 10 rats were fed with either prebiotics containing fructo-oligosaccharides or probiotics containing viable Bifidobacterium lactis and Streptococcus thermophilus. The composition of their caecal microbiota was analyzed both by classical plate count of the main bacterial groups and by PCR amplification of a V3 fragment of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE). Both diets induced a significant reduction of clostridia and Bacteroides spp. compared to a control diet, whereas prebiotics were also able to reduce the number of coliforms and to increase the presence of bifidobacteria. DGGE analysis showed a significant increase of 16S rRNA gene fragments in rats fed with either probotics or prebiotics. Nineteen bands were sequenced and most of them showed similarity to cultured bacteria. Detection of Bifidobacterium spp. by this technique using genus-specific primers only permitted these bacteria to be detected in prebiotics-fed rats, whereas the use of Lactobacillus group-specific primers gave similar results in rats fed with any diet, in agreement with the plate count results.

Detection of integrons and antibiotic-resistance genes in Salmonella enterica serovar Typhimurium isolates with resistance to ampicillin and variable susceptibility to amoxicillin-clavulanate.

We characterized 29 antimicrobial-resistant Salmonella enterica serovar Typhimurium strains, including four belonging to the monophasic variant 4,5,12:i:-, mostly isolated from infants. They were selected from 3230 strains isolated in the years 1990-2001 on the basis of resistance to ampicillin and variable susceptibility to the amoxicillin-clavulanate combination. Twenty-three strains were resistant to more than four antibiotics. All the strains carried the bla(TEM) gene and most were able to transfer this gene by conjugation. Sequencing of the gene from one of the amoxicillin-clavulanate-resistant strains allowed identification of the encoded beta-lactamase as TEM-1; all of these strains carried a second gene encoding beta-lactamase production, either pse-1 or oxa1. However, the association of bla(TEM) plus pse-1 genes did not always confer resistance to amoxicillin-clavulanate. The pse-1 gene, found in 17 strains, was located in the Salmonella Genomic Island-1 (SGI1), which carries two integrons and encodes multiple drug-resistance. None of the oxa1-bearing strains had the SGI1, yet this gene was found as part of an integron that also carried the aadA1 gene and was not plasmid-associated. Thirteen of the strains harbouring SGI1 belonged to the definitive phage type (DT) 104, and most of those remaining to DT104b and U302; particularly, strains carrying the oxa1-aadA1 integron belonged to the last two phage types. Pulsed field electrophoresis confirmed the clonal organization of DT104 strains, whereas U302 strains fell into different groups, depending on their resistance determinants.

A proteomic approach to study Salmonella typhi periplasmic proteins altered by a lack of the DsbA thiol: disulfide isomerase.

Two-dimensional electrophoresis (2-DE) was used to analyze the pleiotropic effects of a deficiency in DsbA, a periplasmic disulfide-bond oxidoreductase, in Salmonella typhi. With this aim, the dsbA gene was cloned and assayed for activity in a dsbA-null mutant of Escherichia coli. A dsbA/chloramphenicol acetylase construct was then used to disrupt the wild-type gene of S. typhi. The resultant dsbA-null mutant of S. typhi, like the E. coli mutant, exhibited a lack of flagellation and of glucose-1-phosphatase activity. Periplasmic extracts from the parental and mutant strains were analyzed by 2-DE using standard denaturing and nondenaturing conditions. Differences in protein expression were more marked in nondenaturing conditions. Ninety-nine protein spots were analyzed by peptide mass fingerprinting, and 65 spots were identified by searching a S. typhi database. Twenty-five spots were exclusively detected in the wild-type strain, 10 were found only in the mutant strain, and 21 were common to both strains. We observed a lack of DsbA, glucose-1-phosphatase and flagellin in the dsbA-null mutant, which explains two of the observed phenotypes. The AI-2 autoinducer-producing protein LuxS, which is involved in quorum-sensing signalling was also absent.

A novel connection between the yeast Cdc42 GTPase and the Slt2-mediated cell integrity pathway identified through the effect of secreted Salmonella GTPase modulators.

Modulation of host cellular GTPases through the injection of the effector proteins SopE2 and SptP is essential for Salmonella typhimurium to enter into non-phagocytic cells. Here we show that expression of the guanine nucleotide exchange factor for Cdc42 SopE2 in Saccharomyces cerevisiae leads to the activation of Fus3 and Kss1 MAPKs, which operate in the mating and filamentation pathways, causing filamentous growth in haploid yeast cells. Furthermore, it promotes the activation of the cell integrity MAPK Slt2. Cdc42 activation by removal of its putative intrinsic GTPase-activating proteins (GAPs), Rga1, Rga2, and Bem3, also results in the phosphorylation of Kss1, Fus3, and Slt2 MAPKs. These data support the role of these GAP proteins as negative regulators of Cdc42, confirm the modulating effect of this GTPase on the filamentation and mating pathways and point to a novel connection between Cdc42 and the cell integrity pathway. Cdc42-induced activation of Slt2 occurs in a mating and filamentation pathway-dependent manner, but it does not require the function of Rho1, which is the GTPase that operates in the cell integrity pathway. Moreover, we report that Salmonella SptP can act as a GAP for Cdc42 in S. cerevisiae, down-regulating MAPK-mediated signaling. Thus, yeast provides a useful system to study the interaction of bacterial pathogenic proteins with eukaryotic signaling pathways. Furthermore, these proteins can be used as a tool to gain insight into the mechanisms that regulate MAPK-mediated signaling in eukaryotes.

Homologous regions of the Salmonella enteritidis virulence plasmid and the chromosome of Salmonella typhi encode thiol: disulphide oxidoreductases belonging to the DsbA thioredoxin family.

The nucleotide sequence relatedness between the chromosome of Salmonella typhi and the virulence plasmid of Salmonella enteritidis was investigated using short DNA probes of < 2 kb covering the whole virulence plasmid sequence. Only one homologous region was detected. This region was subsequently cloned and partially sequenced. Sequences closely related to the pefl gene and the ORFs orf7, orf8 and orf9, which are located downstream of the fimbrial pef operon of the Salmonella typhimurium virulence plasmid, were detected. Sequencing of the cloned S. typhi DNA fragment also revealed identity with genes of the fimbrial sef operon characterized in the chromosome of S. enteritidis. These nucleotide sequences mapped upstream of the S. typhi chromosomal region homologous to the S. enteritidis virulence plasmid. The general organization of the cloned S. typhi chromosomal fragment was similar to the fimbriae-encoding region of the S. typhimurium virulence plasmid. The deduced product of orf8 in the S. typhimurium virulence plasmid, as well as those of the corresponding ORFs in the homologous region of the S. typhi chromosome and in the S. enteritidis virulence plasmid (designated dlt and dlp, respectively), appeared to be related to the thioredoxin family of thiol: disulphide oxidoreductases. The dlp gene was able to complement the DTT-sensitive phenotype, the inability to metabolize glucose 1-phosphate and the low alkaline phosphatase activity of a dsbA mutant of Escherichia coli. The dlt gene partially complemented the lack of alkaline phosphatase activity, but not the other mutant phenotypes. The products of both genes could be detected using the T7 RNA polymerase promoter expression system. The estimated molecular masses of the products of the dlt and dlp genes by SDS-PAGE were 26 and 23 kDa, respectively, the first being in agreement with the deduced amino acid sequence and the latter, somewhat smaller. The processing of a possible leader peptide in the Dlp protein, but not in the Dlt protein, could be responsible for this difference. The Dlp protein appeared as a doublet band on SDS-PAGE, which is characteristic of the oxidized and reduced states of this kind of protein.