Physiological function of the maltose operon regulator, MalR, in Lactococcus lactis

BACKGROUND: Maltose metabolism is initiated by an ATP-dependent permease system in Lactococcus lactis. The subsequent degradation of intracellular maltose is performed by the concerted action of P(i)-dependent maltose phosphorylase and β-phosphoglucomutase. In some Gram-positive bacteria, maltose metabolism is regulated by a maltose operon regulator (MalR), belonging to the LacI-GalR family of transcriptional regulators. A gene presumed to encode MalR has been found directly downstream the maltose phosphorylase-encoding gene, malP in L. lactis. The purpose of this study was to investigate the physiological role of the MalR protein in maltose metabolism in L. lactis. RESULTS: A L. lactis ssp. lactis mutant, TMB5004, deficient in the putative MalR protein, was physiologically characterised. The mutant was not able to ferment maltose, while its capability to grow on glucose as well as trehalose was not affected. The activity of maltose phosphorylase and β-phosphoglucomutase was not affected in the mutant. However, the specific maltose uptake rate in the wild type was, at its lowest, five times higher than in the mutant. This difference in maltose uptake increased as the maltose concentration in the assay was increased. CONCLUSION: According to amino acid sequence similarities, the presumed MalR is a member of the LacI-GalR family of transcriptional regulators. Due to the suggested activating effect on maltose transport and absence of effect on the activities of maltose phosphorylase and β-phosphoglucomutase, MalR of L. lactis is considered rather as an activator than a repressor

Randomly amplified polymorphic DNA (RAPD) typing of Salmonella Senftenberg in animal feed production.

Randomly Amplified Polymorphic DNA (RAPD) was studied as genotyping method typing for a strain collection of Salmonella, belonging to the serotype Senftenberg. This collection consisted of 48 strains/isolates that were sampled in Sweden during 1995-96. In this study the aim was evaluate the usefulness of RAPD in terms of discriminatory power, reproducibility and typeability to compare it with other genotyping methods. By using Taq and Tth DNA polymerase in separate reactions the ability to increase discriminatory power and reproducibility was studied. When Tth DNA polymerase was used in the RAPD reaction brighter bands were obtained and the reproducibility was increased. The results suggest that RAPD can be used for rapid screening of a strain material and in combination with other more discriminating genotyping methods to more truly reflect the genetic diversity

Comparison of heterologous xylose transporters in recombinant Saccharomyces cerevisiae

Abstract Background Baker's yeast (Saccharomyces cerevisiae) has been engineered for xylose utilization to enable production of fuel ethanol from lignocellulose raw material. One unresolved challenge is that S. cerevisiae lacks a dedicated transport system for pentose sugars, which means that xylose is transported by non-specific Hxt transporters with comparatively low transport rate and affinity for xylose. Results In this study, we compared three heterologous xylose transporters that have recently been shown to improve xylose uptake under different experimental conditions. The transporters Gxf1, Sut1 and At5g59250 from Candida intermedia, Pichia stipitis and Arabidopsis thaliana, respectively, were expressed in isogenic strains of S. cerevisiae and the transport kinetics and utilization of xylose was evaluated. Expression of the Gxf1 and Sut1 transporters led to significantly increased affinity and transport rates of xylose. In batch cultivation at 4 g/L xylose concentration, improved transport kinetics led to a corresponding increase in xylose utilization, whereas no correlation could be demonstrated at xylose concentrations greater than 15 g/L. The relative contribution of native sugar transporters to the overall xylose transport capacity was also estimated during growth on glucose and xylose. Conclusions Kinetic characterization and aerobic batch cultivation of strains expressing the Gxf1, Sut1 and At5g59250 transporters showed a direct relationship between transport kinetics and xylose growth. The Gxf1 transporter had the highest transport capacity and the highest xylose growth rate, followed by the Sut1 transporter. The range in which transport controlled the growth rate was determined to between 0 and 15 g/L xylose. The role of catabolite repression in regulation of native transporters was also confirmed by the observation that xylose transport by native S. cerevisiae transporters increased significantly during cultivation in xylose and at low glucose concentration.</p

Development of an in vitro method for detection of Clostridium botulinum types A and E using real-time PCR

Specific primers for C. botulinum types A and E neurotoxin genes were evaluated both from the literature and of own construction. Furthermore, a real-time system with specific hybridisation probes was designed for C. botulinum type E neurotoxin gene, and is under construction for type A. Sequencing of part of the neurotoxin gene for type E showed some differences with sequences reported in GenBank. The PCR methods were optimised regarding amplification efficiency, linear range and specificity. The detection limit for type E using real-time PCR is at least 0,1 ng/ml, corresponding to 0,4 pg of total DNA in the tube, and at least 0,5 ng/ml (2,5 pg of total DNA) for type A using conventional PCR. Quantitative reverse transcription PCR was used to study the relative expression of the neurotoxin gene in different growth phases

Evaluation of false-positive PCR signals due to free DNA in food samples

To assess the risk of false-positive signals in PCR, caused by DNA originating from dead cells, the natural degradation of DNA in food samples was studied. A sample preparation method was developed in connection with real-time PCR to quantify the degradation of DNA in carcass-rinse from chicken and pork homogenate. Our results indicate that the risk of obtaining false-positive PCR signals is significant in pork homogenate, since DNA degradation is slow, but less pronounced in carcass-rinse from poultry

Detection of Salmonella spp in animal feed by PCR

The aim of this study was to develop a rapid PCR-based detection method to confirm presumptive isolates of Salmonella spp. as well as to directly detect Salmonella in feed samples. The specificity of the assay was confirmed by testing I 04 Salmonella strains and 43 strains of other Enterobacteriaceae species. A simple sample preparation method was developed based on dilution of suspended cells picked from agar plates after selective enrichment. The method was evaluated on 109 food and feed samples and there was 100% agreement between the traditional method based on biochemical tests and the PCR method. The inhibitory effect on PCR for 15 different feed samples was overcome by using Tth DNA polymerase in combination with addition of glycerol, which enabled direct detection of Salmonella cells in feed

Detection of C. botulinum in PCR-positive pig faecal samples by traditional isolation and characterization methods

A recently developed semi-quantitatively PCR procedure, specific in the detection of part of the types B, E and F neurotoxin genes, have been used to establish the prevalence of C. botulinum in faeces from slaughter pigs in Sweden. To verify PCR-positive samples, tests were performed to isolate and to identify C. botulinum by traditional microbiological methods and by mouse bioassay. Of twenty isolates from selective and non-selective media, two isolates were found positive by the preliminary screening e.g. lipase reaction and gelatine liquefication. None of the isolates were confirmed positive by RapiD ANA II, PCR or by the mouse bioassay. This describes the difficulties in isolating C. botulinum from heavily contaminated samples such as faeces, because of the lack of satisfactory selective media for C. botulinum and the presence of toxic compounds other than botulinum toxin in the sample