Molecular cloning and functional characterization of a sucrose isomerase (isomaltulose synthase) gene from Enterobacter sp. FMB-1

Aims:  Isomaltulose (palatinose) is a slowly digestible sucrose isomer that can reduce both the glycemic and insulinemic response to foods. The aim of this study was to clone and express a sucrose isomerase (SIase) gene and characterize the protein that is responsible for the production of isomaltulose in the micro-organism Enterobacter sp. FMB-1.
Methods and Results:  A cosmid clone containing c. 6 kbp region encoding an SIase gene was identified. The 5969-bp chromosomal DNA fragment covering the SIase ( esi ) gene in Enterobacter sp. FMB-1 was sequenced. Although this DNA fragment contained several open reading frames other than esi , only the presence of esi was sufficient to produce isomaltulose in recombinant Escherichia coli . The esi gene was expressed in E. coli , leading to the characterization of its SIase activity.
Conclusions:  The Enterobacter sp. FMB-1 esi gene was successfully cloned and expressed in E. coli . This gene encoded a functional SIase that produced isomaltulose from sucrose.
Significance and Impact of the Study:  This is the first molecular analysis of an SIase gene in an Enterobacter strain. The functional expression of the Enterobacter sp. FMB-1 esi gene in E. coli offers an alternative choice for the industrial production of isomaltulose.     

Molecular characterization of Irish E. coli O157:H7 isolates of human, bovine, ovine and porcine origin

Aims:  To determine the degree of relatedness between isolates of Escherichia coli O157:H7 of human, bovine, ovine and porcine origin.
Methods and Results:  Escherichia coli O157:H7 isolates were compared using (i) PFGE Xba I patterns, (ii) PCR profiles of virulence genes and (iii) the DNA sequences of genes reported to play a role in pathogenicity. The 77 E. coli O157:H7 isolates demonstrated 49 different PFGE patterns of which, eight were common to multiple isolates, and the remaining 41 were distinct. Isolates of different origin did not correlate, except for one cluster consisting of two human and two beef isolates. The majority of animal isolates had the same PCR profiles of virulence genes as those isolated from clinical patients. Single nucleotide polymorphisms (SNPs) were identified in the sequence of a 255-bp region of the vtx2 subunit A gene.
Conclusions:  Six SNPs were detected in the vtx2 A gene, defining four different haplotypes. One nonsynonymous substitution encoded for an amino acid change from glutamic to aspartic acid.
Significance and Impact of the Study:  Results indicate that although E. coli O157:H7 isolates of differing origin were distinct by PFGE, the DNA sequences of the main virulence genes associated with human clinical illness were conserved.     

Assessment of anti-oxidant activity of plant extracts using microbial test systems

Aims:  To evaluate the anti-oxidant properties of extracts from 20 medicinal herbs growing in western Siberia using microbial test systems and different in vitro methods.
Methods and results:  In vivo anti-oxidant activity of extracts was evaluated for their capacity to protect bacteria, Escherichia coli , against bacteriostatic and bactericidal effects of H₂O₂ and menadione, and action on anti-oxidant gene expression. In vitro anti-oxidant activity has been examined by a number of methods including: the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH^•)-scavenging assay, chelating activity and capacity to protect plasmid DNA against oxidative damage. In addition, total polyphenol content was determined. The extracts of Fragaria vesca , Rosa majalis , Pentaphylloides fruticosa , Alchemilla vulgaris and Pulmonaria mollis possessed the highest levels of anti-oxidant activity in vivo and in vitro . The protective properties were more closely related to the DPPH^• radical-scavenging activity, tannin content and action on anti-oxidant gene expression than to other parameters.
Conclusion:  The extracts of medicinal plants may have anti-oxidant effects on bacteria simultaneously through several different pathways, including direct inhibition of reactive oxygen species, iron chelation and anti-oxidant genes induction.
Significance and Impact of the Study:  Using microbial test systems, we revealed herbs that may be used as potential sources of natural anti-oxidants.     

Detection of heat-stable and heat-labile enterotoxin genes of Escherichia coli in diarrhoeic faecal samples of mithun (Bos frontalis) calves by polymerase chain reaction

Aims:  To find out the prevalence of different serogroups of Escherichia coli ( E. coli ) and to detect heat-stable (ST) and heat-labile (LT) enterotoxin genes of enterotoxigenic E. coli (ETEC) from the faeces of mithun calves with diarrhoea.
Methods and Results:  Faecal samples obtained from 65 diarrhoeic mithun calves of under 2 months of age were examined for E. coli using polymerase chain reaction (PCR). Fifty-four E. coli isolates were obtained from those samples, which belonged to 38 different serogroups. Out of 54 isolates tested by PCR, two isolates (3·70%) belonging to serogroups O26 and O55 were found to possess gene that code for ST enterotoxin and one isolate (1·85%) belonging to serogroup O125 was found to carry LT enterotoxin gene.
Conclusions:  Escherichia coli isolates from diarrhoeic mithun calves were found to possess ST and LT enterotoxin genes, which are designated as ETEC, and these isolates can be detected through PCR using specific primers.
Significance and Impact of the Study:  This study reports the isolation of ETEC possessing ST and LT enterotoxin genes for the first time and ETEC could be a cause of diarrhoea in mithun calves leading to calf mortality.     

Identification, cloning and sequencing of Escherichia coli strain χ1378 (O78:K80) iss gene isolated from poultry colibacillosis in Iran

Aims:  To identify, clone and sequence the iss (increased serum survival) gene from E. coli strain χ1378 isolated from Iranian poultry and to predict its protein product, Iss.
Methods and Results:  The iss gene from E. coli strain χ1378 was amplified and cloned into the pTZ57R/T vector and sequenced. From the DNA sequence, the Iss predictive protein was evaluated using bioinformatics. Iss from strain χ1378 had 100% identity with other E. coli serotypes and isolates from different origins and also 98% identity with E. coli O157:H7 Iss protein. Phylogenetic analysis showed no significant different phylogenic groups among E. coli strains.
Conclusions:  The strong association of predicted Iss protein among different E. coli strains suggests that it could be a good antigen to control and detect avian pathogenic E. coli (APEC).
Significance and Impact of the study:  Because the exact pathogenesis and the role of virulence factors are unknown, the Iss protein could be used as a target for vaccination in the future, but further research is required.     

Quantification of the expression of reference and alcohol dehydrogenase genes of some acetic acid bacteria in different growth conditions

Aims:  The aim of this study was to develop a reliable system to analyse the expression of the pyrroloquinoline quinone (PQQ)–alcohol dehydrogenase (ADH) and test its ability to predict the growth and oxidative activity of some acetic acid bacteria (AAB).
Methods and Results:  Specific primers were designed for use in RT-PCR to quantify ADH expression and several housekeeping genes in four species of AAB. 16S rRNA gene was selected as an internal control. The relative expression of adh A was measured in Acetobacter aceti , Acetobacter pasteurianus , Gluconacetobacter hansenii and Gluconobacter oxydans grown in two media that had glucose or ethanol as the carbon source. AAB adh A expression was shown to be related to the two Acetobacter species' ability to oxidise and grow on ethanol, whereas G. oxydans were unable to grow on ethanol and the growth of Ga. hansenii was not related to adh A expression.
Conclusions:  The differential expression of ADH could be a marker to analyse both growth and oxidation ability in some AAB, especially those of the genus Acetobacter .
Significance and Impact of the Study:  Several housekeeping genes were tested in AAB and after growth in different media and it was evident that only the ribosomal coding genes were adequate as reference genes for RT-PCR.     

Over 1000 genes are involved in the DNA damage response of Escherichia coli

Changes in gene expression after treatment of Escherichia coli cultures with mitomycin C were assessed using gene array technology. Unexpectedly, a large number of genes (nearly 30% of all genes) displayed significant changes in their expression level. Analysis and classification of expression profiles of the corresponding genes allowed us to assign this large number of genes into one or two dozen small clusters of genes with similar expression profiles. This assignment allowed us to describe systematically the changes in the level of gene expression in response to DNA damage. Among the damage-induced genes, more than 100 are novel. From those genes involved in DNA metabolism that have not previously been shown to be induced by DNA damage, the mutS gene involved in mismatch repair is especially noteworthy. In addition to the SOS response, we observed the induction of other stress response pathways, such as those of oxidative stress and osmotic protection. Among the genes that are downregulated in response to DNA damage are numerous protein biosynthesis genes. Analysis of the gene expression data highlighted the essential involvement of sigma(s)-regulated genes and the general stress response network in the response to DNA damage.     

Cellular responses and expression profiling of human bone marrow stromal cells stimulated with enamel matrix proteins in vitro

Objectives:  The aim of this study was to investigate biological effects and gene expression profiles of enamel matrix proteins (EMPs), on human bone marrow stromal cells (HBMSCs), for preliminary understanding of mechanisms involved in promoting periodontal regeneration by EMPs.
Materials and methods:  EMPs were extracted using the acetic acid method, and HBMSCs from human bone marrow aspirates were cultured. Attachment levels, level of cells morphologically attenuated, cell proliferation, alkaline phosphatase (ALP) activity and staining of HBMSCs were measured in the absence and in the presence of EMPs. Microarray analysis was performed to detect gene profiles of HBMSCs by treatment with 200 μg/ml EMPs, for 5 days. Four differential genes were selected for validation of the microarray data using real-time PCR.
Results:  EMPs promoted proliferation and ALP activity of HBMSCs in a time- and dose-dependent manner, and at a concentration of 200 μg/ml significantly enhanced proliferation and ALP expression. However, there were no significant changes between EMP-treated groups and the control group in cell attachment and cell process attenuation levels. Twenty-seven genes were differentially expressed by HBMSCs in the presence of EMPs. Expressions of 18 genes were upregulated and expressions of nine genes were found to be downregulated. There was good consistency between data obtained from the validation group and microarray results.
Conclusions:  EMPs promoted cell proliferation and differentiation and gene expression profiles of HBMSCs were affected. This may help elucidation of mechanisms involved in promoting regeneration of periodontal tissues by EMPs.