Cytosine deaminase that hydrolyzes creatinine to N-methylhydantoin in various cytosine deaminase-forming microorganisms

Creatinine deimination has been newly detected in the following various cytosine deaminase-forming microorganisms: Escherichia coli, Proteus mirabilis, Pseudomonas aureofaciens, Pseudomonas chlororaphis and Pseudomonas cruciviae. All these microorganisms, except for E. coli, formed cytosine deaminase in a constitutive or repressive way. P. putida 77 and E. coli showed highly increased formation of creatinine deiminase in the presence of creatinine and cytosine. Throughout serial DEAE-Sephacel and Sephacryl S-300 column chromatographies, the cytosine deaminases of these microorganisms, except for that of P. ovalis, were found to hydrolyze both creatinine and cytosine at comparable rates. No concrete evidence was obtained for the presence of any other protein that hydrolyzed creatine and/or cytosine than the cytosine deaminases in the three test microorganisms randomly selected for investigation.Different from P. putida 77, none of the test microorganisms degraded N-methylhydantoin; neither N-methylhydantoin amidohydrolase nor N-carbamoylsarcosine amidohydrolase was formed in the presence of creatinine in these microorganisms. As a result, the wide occurrence of cytosine deaminases in microorganisms was found to be related to the wide distribution of those microorganisms which hydrolyze creatinine to N-methylhydantoin without further degradation.     

A naturally occurring large chromosomal inversion in Escherichia coli K12

Summary Strain 1485IN and its derivatives were found to have a large inversion extending to about 35% of the chromosome. Because of this, the question arose as to whether 1485IN had arisen from an Escherichia coli strain other than K12. However, 1485IN had a flagellar antigen and a restriction-modification system indistinguishable from those of W3110, a major line of K12, and had retained an amber suppressor and sensitivity that are characteristics of W1485 from which this strain seems to have arisen. Strain 1485IN had acquired proline auxotrophy, but showed the same growth rate as W1485 in nutrient broth at 37°C. Interrupted matings with Hfr strains of 1485IN revealed a gene arrangement of nalA-gal-trp-his-lac-proA-thrleu-ilv, in which gal, trp, and his were on the inverted segment. The termini of the inversion were inferred to be situated between tsx (9.5 min) and purE (12 min) and between his (44 min) and cdd (46.5 min).     

A DNA replication gene maps near terC in Escherichia coli K12

Summary Temperature-sensitive mutants that filamented at the non-permissive temperature were isolated by specific mutagenesis of the terminus region of the Escherichia coli chromosome. Two of them, mapping at about 35 min, failed to divide due to inhibition of DNA replication. Further characterization indicated that these mutants are temperature-sensitive for DNA chain elongation.     

Methylated cytosine at Dcm (CC T A GG) sites in Escherichia coli: Possible function and evolutionary implications

The frequency and distribution of methylated cytosine (5-MeC) at CC _T ^A GG (Dcm sites) in 49 E. coli DNA loci (207,530 bp) were determined. Principal observations of this analysis were: (1) Dcm frequency was higher than expected from random occurrence but lower than calculated with Markov chain analysis; (2) CCTGG sites were found more frequently in coding than in noncoding regions, while the opposite was true for CCAGG sites; (3) Dcm site distribution does not exhibit any identifiably regular pattern on the chromosome; (4) Dcm sites at oriC are probably not important for accurate initiation of DNA replication; (5) 5-MeC in codons was more frequently found in first than in second and third positions; (6) there are probably few genes in which the mutation rate is determined mainly by DNA methylation. It is proposed that the function of Dcm methylase is to protect chromosomal DNA from restriction-enzyme EcoRII. The Dcm methylation contribution to determine frequency of oligonucleotides, mutation rate, and recombination level, and thus evolution of the E. coli genome, could be interpreted as a consequence of the acquisition of this methylation.Correspondence to: M.C. Gómez-Eichelmann     

Escherichia coli K12 does not colonize the gastrointestinal tract of Fischer-344 rats

Summary The colonizing potential ofEscherichia coli K12 containing a vector coding for somidobove (bovine somatotropin) was determined. Treated male and female Fischer-344 rats were given a single oral gavage inoculum of sucrose with/without tetracycline (15 g/ml). Untreated control animals received similar drinking water regimes. All animals survived until termination. There were no clinical signs of toxicity observed and no treatment-related effect upon body weight, food consumption, or efficiency of food utilization. Fresh fecal samples were collected from each rat every 24 h following inoculation and the population of the marked strain was quantitated until no bacterial colonies were observed for two consecutive days. While all inoculated rats were positive at 24 h, by 72 and 96 h all had become negative for the test (marked) strain, as were the corresponding control group throughout the test. The frozen stock of the marked strain used as the positive control demonstrated that the agar plates were selective for the test strain. Fourteen days following inoculation, all groups of rats were killed and the gastrointestinal tracts removed and treated to recover the marked strain. There was no evidence of the marked strain in the gastrointestinal tract of any rat from any group. Thus, theE. coli K12 host/vector system used in this experiment does not colonize the gastrointestinal tract of Fischer-344 rats.     

Reduction of methylglyoxal in Escherichia coli K12 by an aldehyde reductase and alcohol dehydrogenase

Two enzymes, one NADPH-dependent and another NADH-dependent which catalyze the reduction of methylglyoxal to acetol have been isolated and substantially purified from crude extracts of Escherichia coli K₁₂ cells. Substrate specificity and formation of acetol as the reaction product by both the enzymes, reversibility of NADH-dependent enzyme with alcohols as substrates and inhibitor study with NADPH-dependent enzyme indicate that NADPH-dependent and NADH-dependent enzymes are identical with an aldehyde reductase (EC 1.1.1.2) and alcohol dehydrogenase (EC 1.1.1.1) respectively. The K_m for methylglyoxal have been determined to be 0.77 mM for NADPH-dependent and 3.8 mM for NADH-dependent enzyme. Stoichiometrically equimolar amount of acetol is formed from methylglyoxal by both NADPH- and NADH-dependent enzymes. In phosphate buffer, both the enzymes are active in the pH range of 5.8–6.6 with no sharp pH optimum. Molecular weight of both the enzymes were found to be 100,000 ± 3,000 by gel filtration on a Sephacryl S-200 column. Both NADPH- and NADH-dependent enzymes are sensitive to sulfhydryl group reagents.     

Vitamin B12 transport in Escherichia coli K12 does not require the btuE gene of the btuCED operon

Summary Transport of vitamin B₁₂ across the cytoplamic membrane ofEscherichia coli requires the products ofbtuC andbtuD, two genes in thebtuCED operon. The role ofbtuE, the central gene of this operon, was examined. Deletions withinbtuE were constructed by removal of internal restriction fragments and were crossed onto the chromosome by allelic replacement. In-frame deletions that removed 20% or 82% of thebtuE coding region did not affect expression of the distalbtuD gene. These nonpolar deletions had little effect on vitamin B₁₂ binding (whole cells or periplasmic fraction) and transport. They did not affect the utilization of vitamin B₁₂ or other cobalamins for methionine biosynthesis, even in strains with decreased outer membrane transport of vitamin B₁₂. ThebtuE mutations did not impair adenosyl-cobalamin dependent catabolism of ethanolamine or repression ofbtuB expression. Thus, despite its genetic location in the transport operon, thebtuE product plays no essential role in vitamin B₁₂ transport.     

Characteristics of capsules in enterotoxemic Escherichia coli O139:K12 strains causing swine edema disease

The characteristics of the capsule of the enterotoxemic Escherichia coli (ETEEC) O139:K12 strains that strongly adhere to Hep-2 cells were examined. Electron microscopic studies using the freeze-substitution technique revealed that ETEEC strains had a capsule of approximately 25 nm. These strains show hydrophobic surface properties and strong adherence to human polymorphonuclear leukocytes (PMNs). In contrast, ETEEC strains RK-O139 and ED-1 show weak adherence to HEp-2 cells and fail to express the capsule layer on the cell surface. These ETEEC strains possess hydrophilic surface properties and also adhere to PMNs. The lipopolysaccharide (LPS) analysis by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that ETEEC strains had the same LPS profile and long O-side chains of LPS. Furthermore, all strains were resistant to serum killing activity. These results suggest that the capsule of ETEEC strains does not contribute as an antiphagocytic factor, but as an adherence factor to host cells.     

Effect of recF, recJ, recN, recO and ruv mutations on ultraviolet survival and genetic recombination in a recD strain of Escherichia coli K12

Summary DNA repair and recombination were investigated in a recD mutant of Escherichia coli which lacked the nuclease activity of the RecBCD enzyme. The resistance of this mutant to ultraviolet (UV) light was shown to be a function of recJ. A recD recJ double mutant was found to be more sensitive to UV radiation than a recB mutant, whereas recD and recJ single mutants were resistant. Recombination in conjugational crosses with Hfr donors was also reduced in recD recJ strains, but the effect was modest in comparison with the sensitivity to UV. Within certain limits, mutations in recF, recN, recO, lexA and ruv did not affect sensitivity to UV and recombination in a recD mutant any more than in a recD⁺ strain. The possibility that recD and recJ provide overlapping activities, either of which can promote DNA repair and recombination in the absence of the other, is discussed.     

Identifying Escherichia coli genes involved in intrinsic multidrug resistance

Multidrug resistance is a major cause of clinical failure in treating bacterial infections. Increasing evidence suggests that bacteria can resist multiple antibiotics through intrinsic mechanisms that rely on gene products such as efflux pumps that expel antibiotics and special membrane proteins that block the penetration of drug molecules. In this study, Escherichia coli was used as a model system to explore the genetic basis of intrinsic multidrug resistance. A random mutant library was constructed in E. coli EC100 using transposon mutagenesis. The library was screened by growth measurement to identify the mutants with enhanced or reduced resistance to chloramphenicol (Cm). Out of the 4,000 mutants screened, six mutants were found to be more sensitive to Cm and seven were more resistant compared to the wild-type EC100. Mutations in 12 out of the 13 mutants were identified by inverse polymerase chain reaction. Mutants of the genes rob, garP, bipA, insK, and yhhX were more sensitive to Cm compared to the wild-type EC100, while the mutation of rhaB, yejM, dsdX, nagA, yccE, atpF, or htrB led to higher resistance. Overexpression of rob was found to increase the resistance of E. coli biofilms to tobramycin (Tob) by 2.7-fold, while overexpression of nagA, rhaB, and yccE significantly enhanced the susceptibility of biofilms by 2.2-, 2.5-, and 2.1-fold respectively.     

Expression of genes from the marine bacteriumAlteromonas haloplanktis 214 inEscherichia coli K-12

DNA from the marine bacteriumAlteromonas haloplanktis 214 was partially digested withSau 3A and inserted into theBam HI site of the cloning vector pBR322. The ligation mixture was used to transformEscherichia coli HB101. The gene bank plasmid preparation obtained was used to transformEscherichia coli K-12 strain EO2717, an organism auxotrophic for histidine, arginine, adenine, uracil and thiamin. Prototrophic transformants for each of the five metabolites were isolated using appropriate minimal media for selection. Plasmids isolated from each of the transformants were shown by hybridization to containA. haloplanktis DNA and to be capable of complementing the appropriate mutation inE. coli EO2717. Restriction maps showed that each of the plasmids was different.     

Expression of Anabaena ferredoxin genes in Escherichia coli

The genes for ferredoxin from heterocysts (fdx H) and vegetative cells (pet F) of Anabaena sp. strain 7120 were subcloned into plasmid pUC 18/19. Both genes were expressed in Escherichia coli at high levels (10% of total protein). Pet F could be expressed from its own promoter. The ferredoxins were correctly assembled to the holoprotein. Heterocyst ferredoxin was purified from E. coli extracts on a large scale. Its biochemical and biophysical properties were identical to those of the authentic ferredoxin, isolated from Anabaena heterocysts.This paper is dedicated to Prof. A. Trebst on the occasion of his 60th birthday.     

Identification and DNA sequence of tdcR, a positive regulatory gene of the tdc operon of Escherichia coli

Summary Efficient in vivo expression of the biodegradative threonine dehydratase (tdc) operon of Escherichia coli is dependent on a regulatory gene, tdcR. The tdcR gene is located 198 base pairs upstream of the tdc operon and is transcribed divergently from this operon. The nucleotide sequence of tdcR and two unrelated reading frames has been determined. The deduced amino acid sequence of TdcR indicates that is is a polypeptide of M_r 12000 with 99 amino acid residues and contains a potential helix-turnhelix DNA binding motif. Deletion analysis and minicell expression of the tdcR gene suggest that TdcR may serve as a trans-acting positive activator for the tdc operon.     

Filamentous growth of Escherichia coli K12 elicited by dimeric,mixed-valence complexes of ruthenium

Dimeric, mixed-valence [(Ru(II), Ru(III)] compounds of ruthenium caused filament formation in growing cultures of Escherichia coli K12. Three compounds with the general formula Ru₂(NH₃)₆X₅ · H₂O (where X is a halide) were tested; in order of decreasing effectiveness (and with the concentration giving maximum effect), these were the bromo (10^-5M), chloro (10^-4 to 10^-5M), and iodo (10^-3 to 10^-4M) analogues. Filamentation elicited by the bromo and chloro compounds was spontaneously reversible after 3–4 h, and tentatively attributed to oxidation of the active mixed-valence form to inactive Ru(III) complexes. Several compounds known to accelerate division of filaments formed under other conditions were ineffective in reversing the filamentation, but the presence of 0,43 M-dimethylsulphoxide totally inhibited filamentation caused by the bromo or chloro compounds and by cis-Pt(NH₃)₂Cl₂ (cisplatin), an established filamenting and antitumour agent. The ruthenium complexes bound to mammalian DNA, but were without effect on the UV spectrum or cellular content of DNA in E. coli, despite showing marked mutagenic activity in reverse mutation tests with Salmonella typhimurium. Cells remained sensitive to inhibition of division by the ruthenium complexes until immediately prior to the division event. Possibilities for the (probably complex) mode of action and the potential of related compounds for therapeutic use are discussed.Non-standard abbreviation DMSO dimethylsulphoxide     

Iron-hydroxamate transport into Escherichia coli K12: localization of FhuD in the periplasm and of FhuB in the cytoplasmic membrane

Summary ThefhuB, fhuC andfhuD genes encode proteins which catalyze transport of iron(III)-hydroxamate compounds from the periplasm into the cytoplasm ofEscherichia coli. ThefhuB, C, D genes were cloned downstream of a strong phage T7 promoter and transcribed by T7 RNA polymerase. The overexpressed FhuD protein appeared in two forms of 31 and 28 kDa and was released upon conversion of vegetative cells into spheroplasts, suggesting synthesis of FhuD as a precursor and export into the periplasm. The very hydrophobic FhuB protein was found in the cytoplasmic membrane. These properties, together with the previously found homologies in the FhuC protein to ATP-binding proteins, display the characteristics of a periplasmic binding protein dependent transport system across the cytoplasmic membrane. The molecular weight of FhuB and the sequence offhuC, as previously published by us, was confirmed. FhuB exhibited double the size of most hydrophobic proteins of such systems and showed homology between the amino- and carboxy-terminal halves of the protein, indicating duplication of an original gene and subsequent fusion of the two DNA fragments.