Influence of the culture conditions on extracellular lyase activity related to K5 polysaccharide

The K5 capsular polysaccharide antigen of some Escherichia coli strains is the non-sulphated precursor in heparin biosynthesis. It is composed by two components, 16000 and 1500 Da, whose ratio depends on the activity of the extracellular form of a lyase synthesized by the same K5 producer strain. The lyase activity on the K5 chain size was greatly influenced by the medium composition employed for the lyase production. The control of lyase activity results in defined ratios of the two components in the K5 polysaccharide that is suitable for the semisynthetic production of heparin-like molecules.     

Purification and properties of swine kidney phosphoprotein phosphatase

Summary Pig intestinal brush borders (BB) were radiolabeled by iodination using the lactoperoxidase-hydrogen peroxide procedure. The BB were then detergent solubilized, centrifuged to remove particulate material, and chromatographed on Sepharose CL-4B. The fractions were incubated with K88+ E. coli using an in vitro binding assay. Binding of the iodinated membranes to K88+ E. coli occurred throughout a wide range of molecular weight components, in excess of 690K daltons to near 25K daltons. The system utilizing intact K88+ E. coli and solubilized BB was shown to be saturable. Prior contact of K88+ E. coli with nonradiolabeled membranes or specific antibodies to K88+ pili inhibited binding of the radiolabeled BB. Simple sugars were tested for their ability to block binding of the labeled BB; partial inhibition occurred with galactose (17.9%), galactosamine (32%), glucose (10.6%), and N-acetylglucosamine (32%). Calcium enhanced binding with as little as 10 M. A 10 × increase in binding occurred with 500 M calcium. Affinity chromatography using K88+ pili coupled on agarose beads avidly bound the labeled BB. The receptor membranes were eluted with high molar concentrations of salt, however considerable degradation occurred. Despite low yields from the affinity system, receptor membranes with higher binding activities were recovered. Protein: glycoprotein ratios were 1:4. Elution with SDS and electrophoresis on 12.5% polyacrylamide gels in the presence of a reducing agent produced two major subunits 35–32K and 23K daltons. These components were recovered from the gels and retained their binding activity. This information suggests that the intestinal receptor responsible for binding of K88+ E. coli is a glycoprotein, that in the native state exists in multimeric forms.     

The influence of pH and external H+ concentration on caesium toxicity and accumulation inEscherichia coli andBacillus subtilis

Summary Toxicity screening ofEscherichia coli NCIB 9484 andBacillus subtilis 007, NCIB 168 and NCIB 1650 has shown Cs⁺ to be the most toxic Group 1 metal cation. However, toxicity and accumulation of Cs⁺ by the bacteria was affected by two main external factors; pH and the presence of other monovalent cations, particularly K⁺. Over the pH range 6–9 bothE. coli andB. subtilis showed increasing sensitivity towards caesium as the pH was raised. The presence of K⁺ and Na⁺ in the laboratory media used lowered caesium toxicity and lowered acumulation of the metal. In order to assess accurately Cs⁺ toxicity towards the bacterial strains it was therefore necessary to define the K⁺:Cs⁺ ratio in the external medium. The minimum inhibitory K⁺:Cs⁺ concentration ratio for theBacillus strains tested was in the range 12–13 whileE. coli had a minimum inhibitory K⁺:Cs⁺ concentration ratio of 16.     

Cloning and expression of the genes for xylose isomerase and xylulokinase from Klebsiella pneumoniae 1033 in Escherichia coli K12

Summary The genes xy1A and xy1B were cloned together with their promoter region from the chromosome of Klehsiella pneumoniae var. aerogenes 1033 and the DNA sequence (3225 bp) was determined. The gene xy1A encodes the enzyme xylose isomerase (XI or XylA) consisting of 440 amino acids (calculated M_r of 49 793). The gene xy1B encodes the enzyme xylulokinase (XK or Xy1B) with a calculated M, of 51 783 (483 amino acids). The two genes successfully complemented xy1 mutants of Escherichia coli K12, but no gene dosage effect was detected. E. coli wild-type cells which harbored plasmids with the intact xylA _Kp 5 upstream region in high copy number (but lacking an active xy1B gene on the plasmids) were phenotypically xylose-negative and xylose isomerase and xylulokinase activities were drastically diminished. Deletion of 5 upstream regions of xy1A on these plasmids and their substitution by a lac promoter resulted in a xylose-positive phenotype. This also resulted in overproduction of plasmid-encoded xylose isomerase and xylulokinase activities in recombinant E. coli cells.     

Intergeneric conjugal transfer of plasmid DNA from<Emphasis Type="Italic"> Escherichia coli</Emphasis> to<Emphasis Type="Italic"> Kitasatospora setae</Emphasis>, a bafilomycin B<Subscript>1</Subscript> producer

An effective transformation procedure for Kitasatospora setae was established based on transconjugation from Escherichia coli ET12567 (pUZ8002) using a C31-derived integration vector, pSET152, containing oriT and attP fragments. While no transconjugation was observed under the standard transconjugation conditions for Streptomyces species, sufficient transconjugation (>1×10^-6) was achieved on ISP4 medium containing 30 mM MgCl₂ using a 25- to 125-fold excess of E. coli donor cells. In addition, the sequence and location of the chromosomal integration site attB of K. setae was identified for the first time in genera of non-Streptomyces actinomycetes. K. setae contains a single C31 attB site. Similar to the case of Streptomyces species, the attB site of K. setae is present within an ORF encoding a pirin-homolog, but the K. setae-attB sequence deviates slightly from the consensus sequence of Streptomyces attB sequences.     

The electrooptical parameters of suspensions of <Emphasis Type="Italic">Escherichia coli</Emphasis> XL-1 cells interacting with helper phage M13K07

The electrophysical properties of Escherichia coli XL-1 cells interacting with helper phage M13K07 were studied as a function of the phage-to-cell ratio and the contact time. The electro-optical signal of bacterial cells changed considerably as soon as 10 min after the onset of their incubation with phage particles, presumably due to phage adsorption on the cell surface. The maximum changes in the orientational spectra of cell suspensions were observed when the phage-to-cell ratio was 20. Selectivity studies showed that E. coli XL-1 cells interacting with the helper phage M13K07 in the presence of foreign microflora, such as E. coli K-12 or Azospirillum brasilense Sp7, can be identified by using their electrophysical properties. Changes in the orientational spectra of cell suspensions are interpreted with the stage of phage-bacterium interaction taken into account. The results obtained can probably be used to devise a new rapid method for identification of microorganisms and to study the particular stages of cell infection by bacteriophages.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 198–203.Original Russian Text Copyright © 2005 by Bunin, Ignatov, Guliy, Zaitseva, ONeil, Ivnitskii     

The isolation and preliminary characterisation of a novel Escherichia coli mutant rorB with enhanced sensitivity to ionising radiation

Summary Escherichia coli K803 cells were mutagenized and screened for the presence of clones sensitive to -rays but not to ultraviolet light. One new mutant of this type, named rorB, was isolated. This mutant is both cross-sensitive to mitomycin C and shows reduced conjugal recombination frequencies, but to a lesser extent than the phenotypically similar mutant recN. Unlike previously reported mutants of E. coli or yeast with an enhanced sensitivity to ionising radiations, rorB appears to be near wild type in ability to rejoin DNA double-strand breaks. The rorB gene maps close to ilvGEDAC at 84.5 min of the E. coli chromosome.     

A newly discovered tRNA(1Asp) gene (aspV) of Escherichia coli K12

Summary We report a new tRNA ₁ ^Asp gene near the dnaQ gene, which is located at 5 min on the Escherichia coli linkage map. We named it aspV. The sequence corresponding to the mature tRNA is identical with that of the two previously identified tRNA ₁ ^Asp genes (aspT and aspU), but there is no homology in the sequences of their 3-and 5-flanking regions.Abbreviations kb kilo base pair(s) - rrn ribosomal RNA     

Transgenic mice expressing a chimaeric anti-E. coli immunoglobulin α heavy chain gene

To induce constitutive immunity against a pathogenic strain ofEscherichia coli (K99), a rearranged immunoglobulin (Ig) heavy chain (HC) gene was constructed. Because the route ofE. coli infection is enteric, an IgA transgene was desirable. A chimaeric gene construct was cloned that coded for a HC that recognized a specificE. coli pilus antigen. The construct comprised a gene promoter, murine VDJ, and bovine -HC constant region. Following microinjection of the HC construct into murine zygotes, of 50 liveborn mice, three were identified as transgenic. In all three transgenic founders, transgene-encoded mRNA expression was detected by northern blot. The transgenic founders were analysed for transgene-encoded RNA expression in splenic tissue before and after challenge with pathogenicE. coli. Founder 4-3 expressed transgene-encoded RNA both before and after challenge; expression was detected in the other two founders only post-challenge. As no differences were found when sera were analysed for bovine IgA in control and transgenic mice, protein expression was assessed by challenge of HC founders with K99E. coli by gavage. Control mice challenged with K99E. coli were moribund within 24 h post-gavage, but there was no observable affect in the three transgenic founders. Unfortunately, after obtaining offspring from all founders, no transgenic offspring were identified (0/108). The low yield of transgenic founders, coupled with the apparent germ-line mosaicism may point to either mechanical or critical developmental anomalies. However, the production of transgenic mice harbouring an Ig HC gene construct confirmed that an Ig transgene coding for an antibody to an animal pathogen could function in a tissue-specific and protective manner in a mammalian system.     

Plasmid mediated silver resistance in Acinetobacter baumannii

Acinetobacter baumannii BL88, an environmental isolate, was resistant to 13 metals and 10 antibiotics. Plumbagin cured resistance to silver, cadmium, antimony, streptomycin and ampicillin at varying frequencies. However, only silver resistance transferred (1 × 10^–6 recepient^–1) to Escherichia coli K12 during conjugation. Correspondingly there was transfer of a 54 kb plasmid (pUPI199) from A. baumannii BL88. The plasmid transformed E. coli DH5 cells at a frequency of 1 × 10^–8 recepient^–1. The growth rate of E. coli DH5; (pUPI199) was slower as compared with E. coli DH5. Plasmid pUPI199 was 76 and 9.6% stable in the host A. baumannii BL88 in the presence and absence of selection pressure, respectively. A. baumannii BL88 was found to accumulate and retain silver whereas E. coli DH5 (pUPI199) effluxed 63% of the accumulated silver ions.     

The Status and the Role of Glutathione under Disturbed Ionic Balance and pH Homeostasis in Escherichia coli

The study of glutathione status in aerobically grown Escherichia coli cultures showed that the total intracellular glutathione (GSH_in + GSSG_in) level falls by 63% in response to a rapid downshift in the extracellular pH from 6.5 to 5.5. The incubation of E. coli cells in the presence of 50 mM acetate or 10 g/ml gramicidin S decreased the total intracellular glutathione level by 50 and 25%, respectively. The fall in the total intracellular glutathione level was accompanied by a significant decrease in the (GSH_in : GSSG_in) ratio. The most profound effect on the extracellular glutathione level was exerted by gramicidin S, which augmented the total glutathione level by 1.8 times and the (GSH_out : GSSG_out) ratio by 2.1 times. The gramicidin S treatment and acetate stress inhibited the growth of mutant E. coli cells defective in glutathione synthesis 5 and 2 times more severely than the growth of the parent cells. The pH downshift and the exposure of E. coli cells to gramicidin S and 50 mM acetate enhanced the expression of the sodA gene coding for superoxide dismutase SodA.     

Analysis of Sequence Periodicity in <Emphasis Type="Italic">E. coli</Emphasis> Proteins: Empirical Investigation of the “Duplication and Divergence” Theory of Protein Evolution

Periodicity was quantified in 4289 Escherichia coli K12 confirmed and putative protein sequences, using a simple chi-square technique previously shown to reveal triplet period periodicity in coding DNA. Periodicities were calculated from period n = 2 to period n = 50 in nine different alphabetic representations of the proteins. By comparison with a randomly generated proteome of the same compositional content, the E. coli proteome does not contain a significant excess of periodic proteins. However, 60 proteins do appear to be significantly periodic in at least one alphabetic representation, after Bonferroni correction, at p < 0.01, and 30 at p < 0.001. These are compared with significantly periodic proteins of solved three-dimensional structure, detected by an identical analysis of the sequences from a protein structure database. It is concluded that there is no evidence for the presence of a proteome-wide quasi-periodicity as predicted by the duplication and divergence model of protein evolution and that the major periodicity detected is a consequence of the repetitive tendencies within -helices. However, it is not possible to explain all sequence periodicities in terms of observable secondary structure, as in cases where sequence periodicity can be compared to solved structure, there is often no structural regularity that would provide an obvious explanation in terms of natural selection on protein function.     

The combined effect of high hydrostatic pressure,heat and bacteriocins on inactivation of foodborne pathogens in milk and orange juice

The objective of this study was to combine pressure (345 MPa) with heat (50 C), and bacteriocins (5000 AU/ml sample) for a short time (5 min) for the inactivation of relatively pressure-resistant strains of four foodborne pathogens: Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella in pasteurized milk and orange juice. Without bacteriocin addition, 5.5 log-cycle reduction was obtained for S. aureus 485 in milk whereas more than 8 log-cycle reduction was achieved for all the other strains studied. After storage of samples for 24 h at 4 C, S. aureus 765 also gave positive results on selective media, where no growth was observed for all the other micro-organisms assayed. Incubation of the same pressurized samples at 37 C for 48 h showed growth of L. monocytogenes strains in addition to S. aureus strains, where still no growth was observed for E. coli O157:H7 and Salmonella strains in their respective selective media. For orange juice samples, more than 8 log-cycle reduction was achieved for all the bacterial species studied. No growth was seen for these species on their respective selective media agar plates after storage at 4 C for 24 h and at 37 C for 48 h. When a bacteriocin-based biopreservative (BP₁) was combined with pressurization, more than 8 log-cycle reduction in cell population of the resistant strains of S. aureus and L. monocytogenes were achieved in milk after pressurization. Milk samples were stored at 25 C up to 30 days to test the effect of treatment and samples showed no growth whereas all the controls were positive.     

The bacteriophage T4 dexA gene: sequence and analysis of a gene conditionally required for DNA replication

Summary We have cloned and sequenced a bacteriophage T4 EcoRI fragment that complements T4 del (39-56) infections of an optA defective Escherichia coli strain. Bacteria containing this recombinant plasmid synthesize two new proteins with molecular weights of 9 and 26 kilodaltons. We have identified the gene encoding the 26 kilodalton protein as essential for T4 infections of optA defective E. coli. Genetic and biochemical results are consistent with the identification of this protein as the product of the dexA gene, which encodes a 3 to 5 exonuclease.     

Primary structure of the Synechococcus PCC 7942 PAPS reductase gene

The structural gene encoding a thioredoxin-dependent 5-phosphoadenylyl sulphate (PAPS) reductase (EC 1.8.4.-) from cyanobacterium Synechococcus PCC 7942 (Anacystis nidulans) was detected by heterologous hybridization with the cysH gene from Escherichia coli K12. The cyanobacterial gene (further called par gene) comprised 696 nt which are 57.8% homologous to the enterobacterial gene. The putative open reading frame encoded a polypeptide consisting of 232 amino acid residues (deduced molecular weight 26635) which showed significant homologies to the polypeptide from E. coli (50.8%) and to the polypeptide from Saccharomyces cerevisiae (30.3%). A single cysteine located at the C-terminus of the polypeptide of E. coli (Cys₂₃₉) was conserved in Synechococcus. Conservation of this cysteinyl residue seems indispensable for catalysis. Complementation of a cysH-deficient mutant of E. coli by the cyanobacterial gene indicated that the cloned DNA is the structural gene of the PAPS reductase.Abbreviations IPTG isopropyl-1-thio--D-galactoside - PAPS 3-phosphoadenosine-5-phosposulphate     

Comparison of the in situ survival and activity ofKlebsiella pneumoniae andEscherichia coli in tropical marine environments

A near-shore coastal mangrove island receiving untreated sewage and a coastal cove receiving rum distillery effluent in Puerto Rico were examined for their ability to support survival and activity ofKlebsiella pneumoniae andEscherichia coli. Pure cultures of both bacteria were monitored for 96 hours in situ at both locations using membrane diffusion chambers.K. pneumoniae survived at all sites as measured by AODC and Coulter Counter direct counts. However, at the mangrove island less than 20% of theK. pneumoniae population was active (AODC) after the first 3 hours and less than 10% of this population was respiring (INT). In contrast, the coastal area which was receiving rum distillery effluent was able to maintain 40% of theK. pneumoniae population in an active state with 90% respiring. TheE. coli population declined by two orders of magnitude at the mangrove island, but remained unchanged at the rum distillery outfall. TheE. coli population had a higher proportion of active cells and respiring cells thanK. pneumoniae at all sites. At the rum distillery site, theE. coli population was remarkable in that 95% remained active and 99% were respiring. This study suggests that, when sufficient organic loading exists,E. coli, a nonsurvivor, can overcome the bactericidal effects of tropical marine waters.K. pneumoniae, a survivor, could survive under all conditions but could not maintain the activity or respiration that theE. coli population could, even when high organic loads were present. Morphological changes related to nutrient stress in the tropical marine environment were apparent inE. coli, but not inK. pneumoniae. Based on physiological activityE. coli is just as much a survivor asK. pneumoniae in tropical marine waters.     

Immunofluorescent localization of phosphoenolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase/oxygenase proteins in leaves of C3, C4 and C3−C4 intermediate Flaveria species

The concentrations of 17 nucleotides and three nucleosides have been determined in a batch suspension culture of Datura innoxia using a new procedure for extraction, purification and high-performance liquid chromatography separation of these compounds. The nucleotide pools change appreciably in the different phases of growth. These changes indicate the preparation for and initiation of cell proliferation, and reflect metabolic events during cell division, cell elongation and starvation. The main components of the nucleotide pool are uracil nucleotides, with uridine 5-diphosphate sugars as the predominant fraction, and the adenine nucleotides. Although their concentrations vary by a factor of more than 6 the ratio of the uracil to adenine nucleotides is kept fairly constant during growth. The energy charge is maintained at a rather high value. The correlation of these events with nutrient uptake and macromolecular synthesis by the batch culture is presented in the following paper.Abbreviations Glc glucose - GlcNAc 2-acetamido-2-deoxy-d-glucose - HPLC high performance liquid chromatography - UDP uridine 5-diphosphate     

Cloning and characterization of the bifunctional alcohol/acetaldehyde dehydrogenase gene (<Emphasis Type="BoldItalic">adhE</Emphasis>) in <Emphasis Type="BoldItalic">Leuconostoc mesenteroides</Emphasis> isolated from kimchi

A bifunctional alcohol/acetaldehyde dehydrogenase (AdhE) gene (adhE) was cloned from Leuconostoc mesenteroides C7 (LMC7), which is the dominant lactic acid bacterium produced during heterofermentation of kimchi. The nucleotide sequence of the DNA fragment containing putative adhE, which is 2685 bp long and encodes an 886 amino acid polypeptide, exhibits 99% homology with Leu. mesenteroides sp. cremoris. The deduced AdhE comprises two conserved domains: alcohol dehydrogenase (Adh) and acetaldehyde dehydrogenase (Aldh). Moreover, two NAD-binding sites were observed, based on the presence of the GXGXXG motif. A pADHE containing the adhE gene expressed AdhE at the translational level in Escherichia coli BL21, which was at a higher level than in E. coli DH5 and E. coli JM109. The AdhE of LMC7 showed Adh and Aldh activities that, when expressed in E. coli. BL21, were 7.5 and 5.7 U mg^-1 , respectively.