Housing data-based socioeconomic index and risk of invasive pneumococcal disease: an exploratory study.

We previously developed and validated an index of socioeconomic status (SES) termed HOUSES (housing-based index of socioeconomic status) based on real property data. In this study, we assessed whether HOUSES overcomes the absence of SES measures in medical records and is associated with risk of invasive pneumococcal disease (IPD) in children. We conducted a population-based case-control study of children in Olmsted County, MN, diagnosed with IPD (1995-2005). Each case was age- and gender-matched to two controls. HOUSES was derived using a previously reported algorithm from publicly available housing attributes (the higher HOUSES, the higher the SES). HOUSES was available for 92·3% (n = 97) and maternal education level for 43% (n = 45). HOUSES was inversely associated with risk of IPD in unmatched analysis [odds ratio (OR) 0·22, 95% confidence interval (CI) 0·05-0·89, P = 0·034], whereas maternal education was not (OR 0·77, 95% CI 0·50-1·19, P = 0·24). HOUSES may be useful for overcoming a paucity of conventional SES measures in commonly used datasets in epidemiological research.

First Report of Pantoea sp. Induced Soft Rot Disease of Pleurotus eryngii in Korea.

The king oyster mushroom, Pleurotus eryngii, has become a popular crop because of its unique flavor and texture and is cultivated in many areas in Korea. In 2003, symptoms of water-soaked lesions and soft rot in the stipes and pileus of cultivated P. eryngii was observed in Jinju, Korea. Diseased tissue was plated on nutrient media. Dominate colonies were yellow, convex, circular with smooth margins, and had a shiny texture. Computer analysis of the data gathered, using the API kit (50CHE, bioMérieux, Marcy-l'Etoile, France), showed that the strain belongs to the Enterobacteriaceae. Although the API system did not give an exact identification, the metabolic profile of the bacterial strain closely resembled the database profile of Pantoea sp. (positive for acid production from the fermentation of d-fructose, d-galactose, d-glucose, d-trehalose, and d-ribose and negative for oxidase, urease, pectate, and thiosulfate). The 16S rDNA sequence of the bacterium was determined (GenBank Accession No. AY530796). When compared with those in GenBank, the bacterium was determined to belong to the Enterobacteriaceae family of the Gammaproteobacteria, and the highest degree of sequence similarity was found to be with Pantoea ananatis strain BD 588 (97.4%) and Pantoea ananatis strain Pna 97-1 (97.3%). In the phylogenetic tree, the bacterium clearly related to the Pantoea lineage, as evidenced by the high bootstrap value. A BLAST search with 16S rDNA sequence of the bacterium supported the API results that the isolate belongs to a species of Pantoea. Pathogenicity tests of this new Pantoea isolate were carried out with bacterial suspensions (approximately 1 × 106 CFU/ml) that were grown for 24 h in Luria-Bertani broth cultures. These were used to inoculate directly on the mycelia of P. eryngii that had been cultivated for 35 days in a plastic bottle. The water and broth were also inoculated to another set of bottles as a control experiment. Inoculated bottles were incubated in a cultivation room at 16 to 17°C with relative humidity between 80 and 95%. Early symptoms of the disease included a dark brown water drop that developed on hypha and primordium of the mushrooms after 5 to 7 days. After 13 days, water-soaked lesions developed on the stipes and pileus, and the normal growth of the mushrooms was inhibited. An offensive odor then developed along with a severe soft rot that was similar to the disease symptoms observed under natural conditions. Mushrooms in control bottles did not develop symptoms. Koch's postulates were fulfilled by isolating bacteria from typical lesions from inoculated mushrooms that were identical to the inoculated strain in colony morphology and biochemical characteristics. Pantoea ananatis was first reported as a pathogen of pineapple fruit causing brown rot (3). Several bacterial diseases, such as brown blotch on cultivated mushrooms by Pseudomonas tolaasii (2) and bacterial soft rot on winter mushroom by Erwinia carotovora subsp. Carotovora, causing severe damage to mushrooms are known (1). However, no Pantoea sp. induced disease of edible mushroom has been previously reported. To our knowledge, this is the first report of soft rot disease on P. eryngii caused by Pantoea sp. References: (1) H. Okamoto et al. Ann. Phytopathol. Soc. Jpn. 65:460. 1999. (2) S. G. Paine. Ann. Appl. Biol. 5:206. 1919. (3) F. B. Serrano. Philipp. J. Sci. 36:271, 1928.

Phylogenetic analysis of protozoa in the rumen contents of cow based on the 18S rDNA sequences.

AIMS: To examine the diversity of protozoa in the rumen contents of cow. METHODS AND RESULTS: Protozoa that inhabit the rumen were detected by PCR using protozoan-specific primers. Libraries of protozoan rDNA sequences were constructed from rumen fluid, solid tissues and epithelium. Twenty-three clones isolated from rumen fluid fell into two genera identified as Entodinium (69.6% of clones) and Epidinium (31.4% of clones). Of the clones isolated from rumen fluid, a moderate number were unidentifiable (30.4%). CONCLUSIONS: The predominant protozoan genus identified in the whole rumen belonged to the Entodinium group (81.1%). Protozoa were not detected in the rumen epithelium. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that rumen fluid and solid tissues contain different protozoan populations that may play specific roles in rumen function. Quantitative PCR techniques and a more specific set of phylogenetic probes that distinguish between protozoan species are needed to determine the significance of newly identified groups and to determine the distribution of identified protozoan clusters in rumen microbial communities.

Cloning and characterization of ce/8A gene from Rhizobium leguminosarum bv. trifolii 1536.

AIMS: To isolate the cellulase gene from Rhizobium leguminosarum bv. trifolii 1536. METHODS AND RESULTS: By the shot-gun method a clone (cel8A) harbouring 3.1 kb genomic DNA fragment from R. leguminosarum bv. trifolii 1536 was obtained. The cel8A gene coded 348 amino acids and it belongs to the glycosyl hydrolase family 8. The molecular mass of Cel8A protein induced from Escherichia coli DH5alpha, appeared to be 35 kDa. The optimum pH and optimum temperature was 7.0, and about 30 degrees C for its enzymatic activity respectively. CONCLUSIONS: R. leguminosarum bv. trifolii 1536 had cel8A gene having an open reading frame of 1047 bp coded for the activity of hydrolyzation of carboxymethyl cellulose. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of celluloytic enzyme by R. leguminosarum bv. trifolii was confirmed, which would play specific roles in rhizobia. Future study should focus on its role in the infection and nodulation phenomena.

The effect of a disrupted yhjQ gene on cellular morphology and cell growth in Escherichia coli.

The 5' upstream region of the cellulose synthase operon ( bcs operon) has been isolated by cloning from Escherichia coli. A gene encoding YhjQ is located 1.0 kb upstream of the bcs operon in E. coli. The function of YhjQ remains unknown. Insertional inactivation of the yhjQ gene causes abnormal cell division, resulting in incomplete partition of the chromosome and filamentous cells of various sizes. These results suggest that the product of yhjQ may affect normal doubling and cellular morphology.

Cloning of the cel9A gene and characterization of its gene product from marine bacterium Pseudomonas sp. SK38.

The yellow-pigmented bacterial strain causing green spot rot and death of layer was isolated from Porphyra dentata. This strain has been identified as Pseudomonas sp., harboring agarase, xylanase, and protease activity, as well as carboxymethyl-cellulase (CMCase). Using genomic DNA from the Pseudomonas sp. SK38 digested with Sau3AI and ligated into pBluescript II KS+, we isolated a cel gene encoding a CMCase in Pseudomonas sp. SK38. A 4.5-kb fragment was subcloned into pKR400. The structure of the cel9A gene consists of an open reading frame of 1,521 bp starting with a GTG start codon and ending with a TAG stop codon. It thus encodes 506 amino acid residues of a protein with a calculated molecular weight of 52,636 daltons plus a signal peptide of 22 amino acids. The deduced amino acid sequence of the cel9A protein is similar to the same protein of Clostridium thermocellum. It contains, in particular, the two conserved regions of the glycoside hydrolase family 9. The apparent molecular mass of the Cel9A protein is 52 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is most active at pH 6-7 and an optimal temperature of around 30 degrees C.

Cloning and characterization of an intracellular isoamylase gene from Pectobacterium chrysanthemi PY35.

The gene encoding an intracellular isoamylase from the Pectobacterium chrysanthemi PY35 was cloned in Escherichia coli DH5alpha and sequenced. The isoamylase gene (amyX) had an open reading frame of 1974 bp encoding 657 amino acid residues with a calculated molecular weight of 74,151 Da. The molecular weight of the enzyme was also estimated to be 74 kDa by activity staining of a SDS-PA gel. Isoamylase from P. chrysanthemi PY35 had 59% pairwise amino acid identity with glycogen debranching enzyme from E. coli and contained the four regions conserved among all amylolytic enzymes. The isoamylase was optimally active at pH 7 and 40 degrees C. AmyX hydrolyzed alpha-1,6-glycosidic linkages of amylopectin, while did not hydrolyze alpha-1,4-glycosidic linkages of amylose.

Cloning and characterization of thermostable endoglucanase (Cel8Y) from the hyperthermophilic Aquifex aeolicus VF5.

Aquifex aeolicus is the hyperthermophilic bacterium known, with growth-temperature maxima near 95 degrees C. The cel8Y gene, encoding a thermostable endoglucanase (Cel8Y) from Aquifex aeolicus VF5, was cloned into a vector for expression and expressed in Escherichia coli XL1-Blue. A clone of 1.7 kb fragment containing endoglucanase activity, designated pKYCY100, was sequenced and found to contain an ORF of 978 bp encoding a protein of 325 amino acid residues, with a calculated molecular mass of 38,831 Da. This endoglucanase was designated cel8Y gene. The endoglucanase has an 18-amino-acid signal peptide but not cellulose-binding domain. The endoglucanase of A. aeolicus VF5 had significant amino acid sequence similarities with endoglucanases from glycosyl hydrolase family 8. The predicted amino acid sequence of the Cel8Y protein was similar to that of CMCase of Cellulomonas uda, BcsC of Escherichia coli, CelY of Erwinia chrysanthemi, and CMCase of Acetobacter xylinum. The molecular mass of Cel8Y was calculated to be 36,750 Da, which is consistent with the value obtained from result of CMC-SDS-PAGE of the purified enzyme. Cel8Y was thermostable, exhibiting maximal activity at 80 degrees C and pH optima of 7.0 and with half-lives of 2 h at 100 degrees C, 4 h at 90 degrees C.

Cloning and sequencing of cel5Z gene from Erwinia chrysanthemi PY35.

The phytopathogenic bacterium Erwinia chrysanthemi (Ech) secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanases. We cloned genomic DNA from Ech PY35 digested with Sau3AI and ligated into pBluescript II SK+. One of the E. coli XL1-blue clones had the ability to hydrolyze carboxymethyl cellulose and polygalacturonic acid. By subsequent subcloning from this 2.9 kb fragment, we obtained a 2.0 kb (pPY401), designated cel5Z, which had the activity of hydrolyzation of carboxymethyl cellulose. The cel5Z gene had an open reading frame (ORF) of 1,281 bp starting with an ATG start codon and followed by a TAA stop codon, encoding 426 amino acids with a signal peptide of 41 amino acids. Since the deduced amino acid sequence of this protein was very similar to that of CelE of Pseudomonas fluorescens, and had the conserved region, VIYEIYNEPL, it belonged to the glycoside hydrolase family 5 of EC 3.2.1.4. The molecular mass of Cel5Z protein from E. coli XL1-blue, as analyzed by CMC-SDS-PAGE, appeared to be 42 kDa. The optimum pH was 6, and the optimum temperature was about 40 degrees C for its enzymatic activity.

Cloning and sequencing of pel gene responsible for CMCase activity from Erwinia chrysanthemi PY35.

The phytopathogenic bacterium Erwinia chrysanthemi secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanase. We cloned genomic DNA from Erwinia chrysanthemi PY35. One of the E. coli XL1-Blue clones contained a 5.1-kb BamHI fragment and hydrolyzed carboxymethyl cellulose and polygalacturonic acid. By subsequent subcloning, we obtained a 2.9-kb fragment (pPY100) that contained the pel gene responsible for CMCase and pectate lyase activities. The pel gene had an open reading frame (ORF) of 1,278 bp encoding 425 amino acids with a signal peptide of 25 amino acids. Since the deduced amino acid sequence of this protein was very similar to that of PelL of E. chrysanthemi EC16, we concluded that it belonged to the pectate lyase family EC 4.2.2.2, and we designated it PelL1. Sequencing showed that the PeIL1 protein contains 400 amino acids and has a calculated pI of 7.15 and a molecular mass of 42,925 Da. The molecular mass of PelL1 protein expressed in E. coli XL1-Blue, as analyzed by SDS-PAGE, appeared to be 43 kDa. The optimum pH for its enzymatic activity was 9, and the optimum temperature was about 40 decreased C.

Characterization of Erwinia chrysanthemi PY35 cel and pel gene existing in tandem and rapid identification of their gene products.

Genomic DNA of the phytopathogenic Erwinia chrysanthemi PY35 was partially digested with Sau3AI, ligated into the BamHI site of pBluescript II SK+, and introduced into E. coli. One clone that was able to hydrolyse carboxymethylcellulose and polygalacturonic acid was selected. A 2.9 kb fragment containing the pelL1 gene (pPY300) and cel5Z gene (pPY401) in tandem was subcloned and sequenced. The pelL1 and cel5Z genes had open reading frames of 1,278 bp and 1,281 bp encoding 425 and 426 amino acid residues with calculated molecular weights of 45,649 Da and 46,473 Da, respectively. pelL1 and cel5Z carried a typical prokaryotic signal peptide of 24 and 41 amino acid residues, respectively. The apparent molecular masses of the proteins when expressed in E. coli cells were approximately 43 kDa (PelL1) and 42 kDa (Cel5Z) as assessed by PGA-SDS-PAGE and CMC-SDS-PAGE.

Cloning of the Escherichia coli endo-1,4-D-glucanase gene and identification of its product.

A plasmid (pYP17) containing a genomic DNA insert from Escherichia coli K-12 that confers the ability to hydrolyze carboxymethylcellulose (CMC) was isolated from a genomic library constructed in the cosmid vector pLAFR3 in E. coli DH5alpha. A small 1.65-kb fragment, designated bcsC (pYP300), was sequenced and found to contain an ORF of 1,104 bp encoding a protein of 368 amino acid residues, with a calculated molecular weight of 41,700 Da. BcsC carries a typical prokaryotic signal peptide of 21 amino acid residues. The predicted amino acid sequence of the BcsC protein is similar to that of CelY of Erwinia chrysanthemi, CMCase of Cellulomonas uda, EngX of Acetobacter xylinum, and CelC of Agrobacterium tumefaciens. Based on these sequence similarities, we propose that the bcsC gene is a member of glycosyl hydrolase family 8. The apparent molecular mass of the protein, when expressed in E. coli, is approximately 40 kDa, and the CMCase activity is found mainly in the extracellular space. The enzyme is optimally active at pH 7 and a temperature of 40 degrees C.

Cloning and sequencing of the celA gene encoding CMCase of Erwinia carotovora subsp. carotovora LY34.

The phytopathogenic Erwinia carotovora subsp. carotovora LY34 secretes multiple isozymes of the plant cell wall-disintegrating enzyme, endoglucanases. Genomic DNA from Ecc LY34 was digested with Sau3AI and ligated into the BamHI site of pBluescript II SK+. One of the E. coli clones containing a Sau3AI fragment of Ecc genomic DNA hydrolyzed carboxymethyl cellulose and was shown to contain the 2.2 kb BamHI restriction fragment, which was subcloned to generate pLYCA100 named as celA. The structural organization of a celA gene encoding 387 amino acids consists of an open reading frame (ORF) of 1161 bp starting with an ATG start codon and followed by a TAA stop codon. CelA protein contained a typical catalytic domain, interdomain, cellulose binding domain, and prokaryotic signal peptide of 32 amino acids. Since the deduced amino acid sequences of CelA protein was very similar to those of CelV of Erwinia carotovora subsp. carotovora SCC3193 enzyme and to those of CelN of Erwinia atroceptica enzyme, it belongs to the cellulase family 5. The apparent molecular mass of CelA protein was calculated to be 39 kDa by carboxymethylcellulose-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (CMC-SDS-PAGE). Activity staining of carboxymethyl cellulase (CMCase) in sodium dodecyl sulfate polyacrylamide gel containing 0.1% CMC revealed that the cloned isozyme comigrated with a corresponding isozyme produced by Ecc LY34. The CelA had a calculated pI of 5.42. The optimum pH was 7 and the optimum temperature was about 45 degrees C.

Characterization of Erwinia carotovora subsp. carotovora LY34 endo-1,4-beta-glucanase genes and rapid identification of their gene products.

Genomic DNA of the phytopathogenic Erwinia carotovora subsp. carotovora LY34 was partially digested with Sau3AI, ligated into the BamHI site of pBlue-script II SK+, and introduced into E. coli. Two clones that were able to hydrolyse carboxymethylcellulose were selected. 1.5 kb and 1.2 kb fragments containing the celA and celB genes, respectively, were subcloned and sequenced. The celA and celB genes had open reading frames of 1,161 bp and 792 bp encoding 487 and 264 amino acid residues with calculated molecular weights of 42,003 Da and 29,890 Da, respectively. Each, CelA and CelB, carried a typical prokaryotic signal peptide of 32 and 36 amino acid residues, respectively. The apparent molecular masses of the proteins when expressed in E. coli cells were approximately 39 kDa (CelA) and 26 kDa (CelB) as assessed by CMC-SDS-PAGE. Activity staining of CMCase in an SDS-PAGE gel containing 0.1% CMC revealed that the cloned endoglucanase isozymes comigrated with the corresponding ones present in Erwinia carotovora subsp. carotovora LY34.