Breast-fed infants often have intestinal microbiota dominated by bifidobacteria in contrast to formula-fed infants. We found that several bifidobacterial strains produce a lacto-N-biosidase that liberates lacto-N-biose I (Gal␤1,3GlcNAc; type 1 chain) from lacto-N-tetraose (Gal␤1,3GlcNAc␤1,3Gal␤1,4Glc), which is a major component of human milk oligosaccharides, and subsequently isolated the gene from Bifidobacterium bifidum JCM1254. The gene, designated lnbB, was predicted to encode a protein of 1,112 amino acid residues containing a signal peptide and a membrane anchor at the N and C termini, respectively, and to possess the domain of glycoside hydrolase family 20, carbohydrate binding module 32, and bacterial immunoglobulin-like domain 2, in that order, from the N terminus. The recombinant enzyme showed substrate preference for the unmodified ␤-linked lacto-Nbiose I structure. Lacto-N-biosidase activity was found in several bifidobacterial strains, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli, under the tested conditions. These results, together with our recent finding of a novel metabolic pathway specific for lacto-N-biose I in bifidobacterial cells, suggest that some of the bifidobacterial strains are highly adapted for utilizing human milk oligosaccharides with a type 1 chain.

The iolABCDEFGHIJ operon of Bacillus subtilis is responsible for myo-inositol catabolism involving multiple and stepwise reactions. Previous studies demonstrated that IolG and IolE are the enzymes for the first and second reactions, namely dehydrogenation of myo-inositol to give 2-keto-myo-inositol and the subsequent dehydration to 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione. In the present studies the third reaction was shown to be the hydrolysis of 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione catalyzed by IolD to yield 5-deoxy-D-glucuronic acid. The fourth reaction was the isomerization of 5-deoxy-D-glucuronic acid by IolB to produce 2-deoxy-5-keto-D-gluconic acid. Next, in the fifth reaction 2-deoxy-5-keto-D-gluconic acid was phosphorylated by IolC kinase to yield 2-deoxy-5-keto-D-gluconic acid 6-phosphate. IolR is known as the repressor controlling transcription of the iol operon. In this reaction 2-deoxy-5-keto-Dgluconic acid 6-phosphate appeared to be the intermediate acting as inducer by antagonizing DNA binding of IolR. Finally, IolJ turned out to be the specific aldolase for the sixth reaction, the cleavage of 2-deoxy-5-keto-D-gluconic acid 6-phosphate into dihydroxyacetone phosphate and malonic semialdehyde. The former is a known glycolytic intermediate, and the latter was previously shown to be converted to acetyl-CoA and CO 2 by a reaction catalyzed by IolA. The net result of the inositol catabolic pathway in B. subtilis is, thus, the conversion of myo-inositol to an equimolar mixture of dihydroxyacetone phosphate, acetyl-CoA, and CO 2 . myo-Inositol (MI)2 is abundant in soil and also common and essential in plants and animals. A number of microorganisms, including Bacillus subtilis (1), Cryptococcus melibiose (2), Aerobacter aerogenes (reclassified as Enterobacter aerogenes/Klebsiella mobilis) (3), Rhizobium leguminosarum bv. viciae (4), Sinorhizobium meliloti (5), Sinorhizobium fredii (6), Corynebacterium glutamicum (7), and Lactobacillus casei (8) can grow on MI as the sole carbon source. MI catabolism in A. aerogenes was studied biochemically, and a pathway of the catabolism finally yielding acetyl-CoA and dihydroxyacetone phosphate (DHAP) was proposed (9). However, our knowledge of the molecular genetics of bacterial MI catabolism has been restricted to B. subtilis (1, 10 -12). In B. subtilis, the iol divergon, comprising the operons iolABCDEFGHIJ and iolRS (1), and the iolT gene (12) were shown to be required for inositol catabolism (Fig. 1). Nowadays, a large number of bacteria have genes annotated iol in their genome sequence, but the annotation is only based on sequence similarity to B. subtilis iol genes, as relatively few studies have been done to demonstrate the participation of the deduced iol genes in MI catabolism.In B. subtilis, a repressor encoded by iolR is responsible for the regulation of all the iol genes (11, 12). In the absence of MI in the growth medium, the IolR repressor binds to the operator site within the promoter regions to repress the transcription. In its presence, howev...

The PCR primers for O, H, and Vi antigen genes, tyv (rfbE), prt (rfbS), fliC-d, fliC-a, and viaB, were designed and used for the rapid identification of Salmonella enterica serovars Typhi and Paratyphi A with multiplex PCR. The results showed that all the clinical isolates examined of Salmonella serovars Typhi and Paratyphi A were accurately identified by this assay.Enteric fever remains an important public health problem in many countries of the world. Typhoid fever and paratyphoid fever are still serious public health problems in many geographic areas and are endemic in most countries, especially those of Southeast Asia and Africa. Recently, multiple-drugor fluoroquinolone-resistant strains of Salmonella enterica serovars Typhi and Paratyphi A have been emerging on the Indian subcontinent, spreading to, and becoming major problems, throughout the world (1, 4, 6). Typhoid fever and paratyphoid fever are sometimes-fatal infections of adults and children that cause bacteremia and inflammatory destruction of the intestine and other organs and that require urgent treatment by the administration of appropriate antibiotics. The diagnosis of typhoid fever or paratyphoid fever is made by ordinary culture methods and biochemical tests. The classic diagnosis method for typhoid fever or paratyphoid fever requires at least 4 or 5 days for positive results. A rapid, alternative method is needed for the diagnosis of typhoid fever or paratyphoid fever. Some researchers have already reported serovar Typhi detection methods with PCR that use the fliC-d gene (7), the Vi capsular antigen gene (3), and the 16S rRNA gene (9). As only one gene was targeted for the identification of serovar Typhi in these methods, strains of Salmonella serovars other than serovar Typhi were detected in some cases. In this study, we developed a more specific diagnosis method for both typhoid fever and paratyphoid fever based on a multiplex PCR technique that detected the Vi antigen gene (viaB), H antigen genes (fliC-d and fliC-a), and O antigen synthesis genes (tyv and prt). This system enabled us to identify and differentiate serovars Typhi and Paratyphi A, which are clinically important human pathogens, by only a single PCR, when we isolated the bacteria from blood or stool cultures from clinical patients.The bacterial strains used in this study were collected from the regional public health office in Japan, and all isolates were identified by biochemical and serological tests. A suspension of bacteria was heated at 100°C for 10 min. The samples were then used for the PCRs. We designed the primers tyv-s and tyv-as for detection of the tyvelose epimerase gene (tyv, previously called rfbE) and the primers fliCcom-s and fliCd-as for detection of the fliC-d gene (phase-1 flagellin gene for d antigen [H:d]) of Salmonella serovar Typhi. The primers parat-s and parat-as were designed for detection of a paratose synthase gene (prt, previously called rfbS), and the primers fliCcom-s and fliCa-as were designed for detection of a fliC-a gene (phase-1 flagellin...

Background:Phenotypically lacto-N-biosidase-positive Bifidobacterium longum JCM1217 does not possess a gene homologous to previously identified lacto-N-biosidase. Results: Hypothetical proteins BLLJ_1505 and BLLJ_1506 encode lacto-N-biosidase and its designated chaperone, respectively. Conclusion:The enzyme showed unique and unexpected substrate specificity. Significance: The enzyme is important for understanding how B. longum consumes human milk oligosaccharides and also may serve as a new tool in glycobiology.

Modification of the surface structure of the commercial ion exchange membrane Nafion®117, by low dose electron beam ͑EB͒ exposure, to produce an improved polymer electrolyte membrane for direct methanol fuel cells ͑DMFC͒ is described. Nafion 117 film was exposed to low dose EB irradiation, at an accelerating voltage of 35 kV. Subsequently the properties of the film itself, in terms of conductance, methanol permeability, percentage water uptake and shrinkage, together with the performance of its membrane electrode assembly in the DMFC were analyzed and contrasted with the untreated material. Low-dose EB treatment is shown to be effective in the reduction of methanol crossover, 600 C/cm 2 exposure reducing crossover to 7% of that of the parent material. In terms of overall DMFC performance ͑maximum power output͒, improvements of up to 51% are reported in comparison to the use of untreated Nafion 117. A simple analytical protocol, allowing film properties to be directly related to subsequent DMFC performance, is also reported. IR reflectance ͑attenuated total reflectance͒ spectroscopy was used to study film surface composition and determine the effect of low-dose EB exposure on Nafion 117 structure. These observations are contrasted with previous findings using traditional EB systems.International interest in both renewable power sources and alternatives to curb current fuel emission levels makes the development of a new fuel cell system an attractive proposition. On consideration of the main issues, such as safety, device fabrication, market, costs, and potential applications, the direct methanol fuel cell ͑DMFC͒ appears to be the system of choice. This view is supported by the vast number of research groups, including academic, industrial, and governmental laboratories, currently targeting the development of DMFCs for a wide range of potential uses, from portable power sources for small electronic devices to vehicular applications. Although major steps forward have been achieved in terms of DMFC design since the onset of research in this area, further developments allowing widespread commercialization of this technology are more likely to come from improvements in the central DMFC components.Although a number of other factors directly influence performance, the operational part of the DMFC can be thought of in terms of having two main features, the electrodes and the polymerelectrolyte membrane. Looking at the basic DMFC design and mode of operation, improving catalytic activity at the cathode and reducing the extent of methanol crossover through the polymer-electrolyte membrane can be identified as two ways of improving overall cell performance. During operation methanol is fed into the system at the anode, where it is oxidized generating carbon dioxide ͓CH 3 OH ϩ H 2 O → CO 2 ϩ 6H ϩ ϩ 6e Ϫ ͔, while the corresponding reduction process takes place at the cathode ͓4H ϩ ϩ O 2 ϩ 4e Ϫ → 2H 2 O͔. However, due to the similar composition of the two electrodes, if methanol crossover through the polymer-electrolyte membran...

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D-chiro-Inositol (DCI) is a drug candidate for the treatment of type 2 diabetes and polycystic ovary syndrome, since it improves the efficiency with which the body uses insulin and also promotes ovulation. Here, we report genetic modification of Bacillus subtilis for production of DCI from myo-inositol (MI). The B. subtilis iolABC-DEFGHIJ operon encodes enzymes for the multiple steps of the MI catabolic pathway. In the first and second steps, MI is converted to 2-keto-MI (2KMI) by IolG and then to 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione by IolE. In this study, we identified iolI encoding inosose isomerase, which converts 2KMI to 1-keto-D-chiroinositol (1KDCI), and found that IolG reduces 1KDCI to DCI. Inactivation of iolE in a mutant constitutively expressing the iol operon blocked the MI catabolic pathway to accumulate 2KMI, which was converted to DCI via the activity of IolI and IolG. The mutant was able to convert at least 6% of input MI in the culture medium to DCI.

The myo-inositol catabolism pathway of Bacillus subtilis has not been fully characterized but was proposed to involve step-wise multiple reactions that finally yielded acetyl-CoA and dihydroxyacetone phosphate. It is known that the iolABCDEFGHIJ operon is responsible for the catabolism of inositol. IolG catalyses the first step of myo-inositol catabolism, the dehydrogenation of myo-inositol, producing 2-keto-myo-inositol (inosose). The second step was thought to be the dehydration of inosose. Genetic and biochemical analyses of the iol genes led to the identification of iolE, encoding the enzyme for the second step of inositol catabolism, inosose dehydratase. The reaction product of inosose dehydratase was identified as D-2,3-diketo-4-deoxy-epi-inositol.