Evaluation of Petrifilm™ Select <Emphasis Type="Italic">E. coli</Emphasis> Count Plate medium to discriminate antimicrobial resistant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Screening and enumeration of antimicrobial resistant Escherichia coli directly from samples is needed to identify emerging resistant clones and obtain quantitative data for risk assessment. Aim of this study was to evaluate the performance of 3M™ Petrifilm™ Select E. coli Count Plate (SEC plate) supplemented with antimicrobials to discriminate antimicrobial-resistant and non-resistant E. coli.     

A role of <Emphasis Type="Italic">ygfZ</Emphasis> in the <Emphasis Type="Italic">Escherichia coli</Emphasis> response to plumbagin challenge

Plumbagin is found in many herbal plants and inhibits the growth of various bacteria. Escherichia coli strains are relatively resistant to this drug. The mechanism of resistance is not clear. Previous findings showed that plumbagin treatment triggered up-regulation of many genes in E. coli including ahpC, mdaB, nfnB, nfo, sodA, yggX and ygfZ. By analyzing minimal inhibition concentration and inhibition zones of plumbagin in various gene-disruption mutants, ygfZ and sodA were found critical for the bacteria to resist plumbagin toxicity. We also found that the roles of YgfZ and SodA in detoxifying plumbagin are independent of each other. This is because of the fact that ectopically expressed SodA reduced the superoxide stress but not restore the resistance of bacteria when encountering plumbagin at the absence of ygfZ. On the other hand, an ectopically expressed YgfZ was unable to complement and failed to rescue the plumbagin resistance when sodA was perturbed. Furthermore, mutagenesis analysis showed that residue Cys228 within YgfZ fingerprint region was critical for the resistance of E. coli to plumbagin. By solvent extraction and HPLC analysis to follow the fate of the chemical, it was found that plumbagin vanished apparently from the culture of YgfZ-expressing E. coli. A less toxic form, methylated plumbagin, which may represent one of the YgfZ-dependent metabolites, was found in the culture supernatant of the wild type E. coli but not in the ΔygfZ mutant. Our results showed that the presence of ygfZ is not only critical for the E coli resistance to plumbagin but also facilitates the plumbagin degradation.     

Enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EPEC) in Antarctic fur seals <Emphasis Type="Italic">Arctocephalus gazella</Emphasis>

Rectal swabs were collected from Antarctic fur seal pups Arctocephalus gazella at Cape Shirreff, South Shetland Islands, and analyzed for the presence of anthropogenic pathogens. Two of the 33 pups tested positive for enteropathogenic Escherichia coli (EPEC). These samples are the first records of EPEC in Antarctic wildlife and suggest that more needs to be done to protect the Antarctic fauna from exotic anthropogenic pathogens.     

Host Cell Reactivation in Strains of <Emphasis Type="Italic">E. coli</Emphasis> lacking DNA Polymerase Activity <Emphasis Type="Italic">in vitro</Emphasis>

BACTERIAL cells can repair DNA which has been damaged by irradiation with ultraviolet light (UV). A repair process which does not depend on light is known as dark reactivation. Because the cells can repair damaged infecting DNA, as well as their own by the same mechanism the phenomenon has also been called host cell reactivation (HCR). HCR seems to consist of the following reaction steps¹: (1) endonucleolytic incision close to the UV photoproduct which most frequently is a pyrimidine dimer; (2) excision of the photoproduct as an oligonucleotide; (3) resynthesis of the removed nucleotide sequence using the opposite strand as a template; and (4) rejoining of the polynucleotide chains.     

The backbone structure of the thermophilic <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis> ribose binding protein is essentially identical to its mesophilic <Emphasis Type="Italic">E. coli</Emphasis> homolog

Background  

Comparison of experimentally determined mesophilic and thermophilic homologous protein structures is an important tool for understanding the mechanisms that contribute to thermal stability. Of particular interest are pairs of homologous structures that are structurally very similar, but differ significantly in thermal stability.     

Sensitivity of various <Emphasis Type="Italic">Escherichia coli</Emphasis> strains to 2,4,6-trinitrotoluene

The sensitivity of Escherichia coli strains K-12 and 055 to 2,4,6-trinitrotoluene (TNT) was found to correlate with the structural and functional properties of the outer lipoprotein membrane. The protective ability of the membrane of strain 055 is much lower than that of K-12. This is the cause of the greater sensitivity of 055 to the toxic action of TNT. High TNT concentrations (100–200 mg/l) suppressed the growth of 055, whereas K-12 grew at all TNT concentrations studied. Both strains adapted to high TNT concentrations by converting it by either nitroreduction or denitritation depending on concentration. The denitritation system of strain 055 started TNT degradation earlier than that of K-12.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 53–57.Original Russian Text Copyright © 2005 by Kurinenko, Denivarova, Yakovleva.     

Translation initiation region sequence preferences in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

The mRNA translation initiation region (TIR) comprises the initiator codon, Shine-Dalgarno (SD) sequence and translational enhancers. Probably the most abundant class of enhancers contains A/U-rich sequences. We have tested the influence of SD sequence length and the presence of enhancers on the efficiency of translation initiation.     

Enhanced production and characterization of a β-glucosidase from <Emphasis Type="Italic">Bacillus halodurans</Emphasis> expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A putative β-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-β-D-1-thiogalactopyranoside, the enzyme expressed at ∼40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A ₆₀₀ 12 in auto-inducing M9NG medium, β-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the β-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45°C and 8.0, respectively, under the assay conditions. K _m and k _cat against p-nitrophenyl-β-D-glucopyranoside were 4 mM and 0.75 sec⁻¹, respectively. To our knowledge, this is the first report of high-level expression and characterization of a β-glucosidase from B. halodurans.     

Deletion of methylglyoxal synthase gene (<Emphasis Type="Italic">mgsA</Emphasis>) increased sugar co-metabolism in ethanol-producing <Emphasis Type="Italic">Escherichia coli</Emphasis>

The use of lignocellulose as a source of sugars for bioproducts requires the development of biocatalysts that maximize product yields by fermenting mixtures of hexose and pentose sugars to completion. In this study, we implicate mgsA encoding methylglyoxal synthase (and methylglyoxal) in the modulation of sugar metabolism. Deletion of this gene (strain LY168) resulted in the co-metabolism of glucose and xylose, and accelerated the metabolism of a 5-sugar mixture (mannose, glucose, arabinose, xylose and galactose) to ethanol.     

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC α and β subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.     

<Emphasis Type="Italic">Bacillus anthracis</Emphasis>, <Emphasis Type="Italic">Francisella tularensis</Emphasis> and <Emphasis Type="Italic">Yersinia pestis</Emphasis>. The most important bacterial warfare agents — <Emphasis Type="Italic">review</Emphasis>

There are three most important bacterial causative agents of serious infections that could be misused for warfare purposes: Bacillus anthracis (the causative agent of anthrax) is the most frequently mentioned one; however, Fracisella tularensis (causing tularemia) and Yersinia pestis (the causative agent of plague) are further bacterial agents enlisted by Centers for Disease Control and Prevention into the category A of potential biological weapons. This review intends to summarize basic information about these bacterial agents. Military aspects of their pathogenesis and the detection techniques suitable for field use are discussed.     

Formation of glycated recombinant leghemoglobin in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

A nonenzymatic glycation of the recombinant leghemoglobin expressed in Escherichia coli cells was demonstrated for the first time. This process involved the heme pocket and gave low-spin leghemoglobin species. A correlation between the degree of E. coli protein glycation and synthesis of poly-β-hydroxybutyric acid was found, suggesting that the accumulation of reserve carbon sources and nonenzymatic glycation could be alternative processes.     

PriA participates in nascent DNA synthesis in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The question of whether discontinuous DNA replication operates only for the lagging strand or for both strands in E. coli remains unresolved. In this study, the participation of priA, B, C and rep genes in discontinuous DNA replication was examined by analyzing the size distribution of nascent DNA synthesized in wild-type, lig-7 and polA4113 genetic backgrounds. Inactivation of priA, but not priB, priC or rep, resulted in a significant increase of high molecular weight (HMW) DNA in the short pulse-labeled DNA in the wild-type lig ⁺ polA ⁺ strains. Inactivation of priA also produced a significant increase of HMW DNA in the nascent DNA synthesized in lig-7 and polA4113 strains. These results indicate that PriA is involved in the discontinuous synthesis of nascent DNA.     

Probing the role of copper in the biosynthesis of the molybdenum cofactor in <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The crystal structure of Cnx1G, an enzyme involved in the biosynthesis of the molybdenum cofactor (Moco) in Arabidopsis thaliana, revealed the remarkable feature of a copper ion bound to the dithiolene unit of a molybdopterin intermediate (Kuper et al. Nature 430:803-806, 2004). To characterize further the role of copper in Moco biosynthesis, we examined the in vivo and/or in vitro activity of two Moco-dependent enzymes, dimethyl sulfoxide reductase (DMSOR) and nitrate reductase (NR), from cells grown under a variety of copper conditions. We found the activities of DMSOR and NR were not affected when copper was depleted from the media of either Escherichia coli or Rhodobacter sphaeroides. These data suggest that while copper may be utilized during Moco biosynthesis when it is available, copper does not appear to be strictly required for Moco biosynthesis in these two organisms.     

Mutators and hypermutability in bacteria: the <Emphasis Type="Italic">Escherichia coli</Emphasis> paradigm

Mutators (also called hypermutators) are mutants which show higher than normal spontaneous mutation frequencies, ranging from 10–20 fold to 100–1000 fold higher, or sometimes even more, than wild-type cells. Being a mutator is advantageous to the organism when adapting to environmental changes or stressful situations, such as moving from one habitat to another, one host to another, exposure to antibiotics etc. However, this advantage is only a short-term benefit. In the long run, hypermutability leads to a fitness disadvantage due to accumulation of deleterious mutations or antagonistic pleiotropy or both. Contrary to intuitive expectations, hypermutability is commonly encountered in natural bacterial populations, especially among clinical isolates. It is believed to be involved in the emergence of antibiotic resistance and a hindrance to the treatment of infectious diseases. Here, I review the state of knowledge on the common mechanisms of hypermutability such as errors/defects in DNA replication, proof reading, mismatch repair, oxidative DNA damage, mistranslation etc., as well as phenomena associated with these processes, using Escherichia coli as a paradigmatic organism.     

Presence of <Emphasis Type="Italic">C. albidus</Emphasis>, <Emphasis Type="Italic">C. laurentii</Emphasis> and <Emphasis Type="Italic">C. uniguttulatus</Emphasis> in Crop and Droppings of Pigeon Lofts (<Emphasis Type="Italic">Columba livia</Emphasis>)

Columba livia is an important reservoir and carrier of Cryptococcus neoformans, Cryptococcus uniguttulatus, Cryptococcus laurentii and Cryptococcus albidus. Upper digestive tract of this species is also known as a habitat for Cryptococcus neoformans. Given the increasing clinical interest of this microorganism, 331 swabs from crop and 174 dropping samples from pigeon lofts in Grand Canary Island have been studied. The obtained results show an extensive presence samples 81 positive (24.47%) of Cryptococcus spp. in analysed crops: 32 (9.66%) for C. neoformans, 24 (7.2%) for C. uniguttulatus, 23 (6.9%) for C. albidus and 2 (0.6%) for C. laurentii. In the same way, Cryptococcus spp was also isolated in 82 (47.13%), dropping samples: C. neoformans in 59 (33.9%), C. uniguttulatus, in 9 (5.17%), C. laurentii in 8 (4.59%) and C. albidus in 6 (3.44%) of the investigated samples, respectively. The cryptococcosis produced by species of cryptococci other than C. neoformans has become more important during the last decade, supporting the study on the role of pigeon in the epidemiology of this disease.