Biomedical subjects

Lars Sundström

Publications and source records attributed to Lars Sundström.

7 recordsLinked to original sources

PubMed2006

Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data.

The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCyder MS Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data. Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan MDLC, online connected to an LTQTM linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired MS data were subjected to DeCyder MS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared. This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels.

Amino Acid Sequence↗

PubMed2004

Integron integrase binds to bulged hairpin DNA.

Gene cassettes are short, monogenic DNA elements that translocate between integrons through site-specific excision and integration. These events require that an integron-coded tyrosine recombinase forms a reactive complex with two sites, at least one of which belongs to the attC class. An attC site can be divided into two pairs of short repeats flanking a palindromic central region. The nucleotide sequence of attC among different cassettes varies extensively, implying that the site contains a structural recognition determinant with low sequence constraints. Oligonucleotides representing many different sequence modifications in either strand of the site were examined for integrase binding by using an electrophoresis mobility shift assay. The inner repeats, a central triplet and two single-nucleotide asymmetries in the site had the strongest influence on binding strength and strand choice. Our data show that the recombinase binds to a bulged hairpin in attC and that the hairpin distortion due to bulging could define the appropriate orientation of the otherwise symmetrical site. This is consistent with the strong bias for binding of recombinase to the bottom-strand oligonucleotides in vitro. Moreover, it was observed that the mobility-shifted complexes persisted under protein-denaturing assay conditions, indicating that a covalent link is indeed formed between integrase and DNA. Upon substitution of the presumed DNA-attacking residue, Y312, with a phenylalanine, DNA binding remained but there was no covalent linkage.

Base Sequence↗

PubMed2003

Sulphonamide resistance gene sul3 found in Escherichia coli isolates from human sources.

OBJECTIVES: The aim of this study was to investigate the molecular basis of an observed increasing resistance to trimethoprim and sulphonamides despite a simultaneous decline in co-trimoxazole consumption. The distribution of sulphonamide resistance genes sul1, sul2 and the recently discovered sul3 was studied in a collection of clinical isolates of Enterobacteriaceae. METHODS: PCR with primers specific for sul1, sul2 and sul3 was used to detect the three known sulphonamide resistance genes in the isolate collection. Sequence analysis was used for confirmation of results. Restriction endonuclease digestion and conjugational transfer assays were used for plasmid analysis. RESULTS: In 64 sulphonamide-resistant isolates, 39 sul1 genes and 48 sul2 genes were detected. Twenty-five isolates carried both sul1 and sul2 and two were negative for both genes. With PCR and sequence analysis these two were shown to harbour the new sulphonamide resistance gene sul3, which was carried by different plasmids. CONCLUSIONS: Sulphonamide resistance gene sul3, which is widespread among pigs in Switzerland, has now also been identified in two different clinical isolates of Escherichia coli, located in urinary tract infections in patients in Sweden.

Anti-Infective Agents↗

PubMed2003

Purification and partial characterization by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the recombinant transposase, TniA.

A recombinant transposase, TniA, a basic DNA binding protein, was chromatographically purified and characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) methods. Escherichia coli cells, overexpressing native TniA, were ultrasonically disrupted and the clarified supernatant was used as starting material for anion-exchange chromatography on SOURCE1 15Q 4.6/100 PE (Tricorn), at pH 7.5. This initial step was proven to be a fast and simple way of removing acidic proteins like proteases. TniA was collected from the flow-through fraction and applied onto HiTrap heparin HP 5 ml in order to capture the basic TniA. This was followed by cation-exchange chromatography through Mono S 5/50 GL (Tricorn), at pH 6.5 which resulted in a purity of TniA of about 95%. The molecular mass of TniA was determined to 62 869 rel. mol. mass units with MALDI-TOF-MS and the identity of the protein was confirmed by peptide mass fingerprinting of trypsin-digested TniA. Partial amino acid sequencing was achieved after derivatization of tryptic peptides using Ettan CAF MALDI Sequencing Kit and post source decay. The fact that transposases are DNA-binding and that many of them possess basic isoelectric point values suggest that the outlined purification protocol may serve as a general method for the purification of recombinant nontagged transposases and other basic DNA-binding proteins.

Amino Acid Sequence↗

PubMed2003

New dfr2 gene as a single-gene cassette in a class 1 integron from a trimethoprim-resistant Escherichia coli isolate.

Resistance to trimethoprim among Enterobacteriaceae is increasing in spite of a stable or decreasing use of the drug. Integrons are common among these bacteria and many of them contain dfr gene cassettes. A clinical isolate of Escherichia coli from a urine specimen obtained at the Karolinska Hospital in Stockholm was resistant to trimethoprim, ampicillin, sulfonamides, chloramphenicol, streptomycin, and norfloxacin. PCR analysis with primers specific for the 5' and 3' sequences flanking the cassettes in class 1 integrons, detected a very short cassette region of no more than approximately 400 bp. Sequence analysis of the entire integron was performed, revealing a single-gene cassette of 411 bp encoding a new dihydrofolate reductase. The gene cassette comprised all sequences between the flanking conserved sequences and encoded only 78 amino acids. By homology it belongs to the unique group of dfr2 gene cassettes and being the fourth described gene in this group the new gene is called dfr2d. The enzymes encoded by the dfr2 genes are 67% identical and are not related to any other known dfr gene products.

Amino Acid Sequence↗

PubMed2002

IntI2 integron integrase in Tn7.

Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7 and its relatives. We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon. This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies. The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7. However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E.

3' Untranslated Regions↗

PubMed2002

Antibiotic-resistant cell-detaching Escherichia coli strains from Nigerian children.

The properties of 23 cell-detaching Escherichia coli strains that were isolated from stool specimens in Nigeria are described. Common properties of the strains included the presence of genes encoding alpha-hemolysin (100%), pyelonephritis-associated pili (100%), and cytotoxic necrotizing factor 1 (70%) as well as lactose negativity (70%) and multiple antibiotic resistance (74%). Antibiotic resistance was shown in most cases to be transferable and associated with the presence of class 1 integrons. Phenotypic properties and pulsed-field gel electrophoresis analysis demonstrated that the majority of the strains, particularly multiply resistant, lactose-negative O4:H40 strains, were closely related. Multiply-resistant cell-detaching E. coli strains may represent an important reservoir for antibiotic resistance genes.

Bacterial Typing Techniques↗