The fadD gene of Escherichia coli K12 is located close to rnd at 39.6 min of the chromosomal -map and is a new member of the AMP-binding protein family

The fadD gene of Escherichia coli K12 was cloned and sequenced. The gene was identified by its ability to complement the corresponding mutant and by measuring the enzymatic activity after its expression in this mutant. The deduced polypeptide sequence exhibits similarity to other long chain acyl-CoA (coenzyme A) synthetases and a variety of other proteins, which together form a family of AMP-binding proteins. This family is extended by several new members and subdivided into four groups. fadD is assigned to a subgroup that does not include long chain acyl-CoA synthetases from eukaryotic organisms.     

Identification of Escherichia coli genes whose expression increases as a function of external pH

Summary Using transposon TnphoA and a plate screening method, we have isolated a set of Escherichia coli strains carrying phoA fusions with genes whose expression is modulated as a function of external pH. Besides fusions with the ompF gene and the malB locus, thirteen independent fusions were analysed whose expression is maximal during growth at pHs ranging from 7.0 to 8.5 and minimal during growth at pH 5.0. Six different genetic loci, called phmA, phmB, phmC, phmD, phmE and phmF (for pH modulated) were characterized and localized on the E. coli chromosome at approx. 12, 18, 41, 45, 75 and 84 min, respectively. Expression of phmA: :phoA fusions is also influenced when internal pH or environmental conditions such as osmolarity or anaerobiosis are modified. EnvZ protein is not involved in the regulation of phm : :phoA fusions.     

Nisin biosynthesis genes are encoded by a novel conjugative transposon

Summary Genes for biosynthesis of the lactococcal peptide antibiotic nisin were shown to be encoded by a novel chromosomally located transposon Tn5301. The element is 70 kb in size and lacks inverted repeats at its termini. Although a copy of the insertion sequence IS904 is located near to one end, this did not appear to be involved in the transposition process. The integrated element is flanked by the directly repeated sequence 5-TTTTTG-3. Analysis of ten independent transconjugants revealed that Tn5301 integration is site-specific; two chromosomal targets were identified and shown to have some sequence homology. The element shares features with the Tn916 family of conjugative transposons and with Tn554 but is also exhibits some unique properties. Tn5301 is thus considered to be the prototype of a novel class of conjugative transposon.     

Isolation and characterization of Paracoccus denitrificans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn) DNA-modifying property

A selection scheme was devised to isolate Paracoccus denitrificans mutants with increased recipient qualities in transfer experiments, using broad host range plasmids. In some of the mutants obtained, a DNA modifying activity that prevents the activity of the restriction endonucleases BamHI and BglII on isolated P. denitrificans DNA had simultaneously been lost. From a detailed analysis of the restriction properties of the enzymes SAU3 AI, MboI and DpnI, it was concluded that a subset of GATC sequences in P. denitrificans DNA may be methylated at an unusual position. It was concluded that P. denitrificans possesses at least one potent host-dependent restriction/modification system which affects conjugation. In addition to the class of restriction-defective mutants, at least one other class of enhanced transfer mutants with unknown defect(s) was isolated. Strains, in which the two mutant classes were combined, exhibited transfer frequencies which were significantly higher than strains containing either mutation alone. Such double mutant strains appeared to be well suited for future experiments like complementation analysis, transposon mutagenesis and gene replacement by homologous recombination.     

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).     

Pyrimidine,purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K12. Involvement of the glnLG and purR genes in the regulation of codA expression

Cytosine deaminase, encoded by the codA gene in Escherichia coli catalyzes the deamination of cytosine to uracil and ammonia. Regulation of codA expression was studied by determining the level of cytosine deaminase in E. coli K12 grown in various defined media. Addition of either pyrimidine or purine nucleobases to the growth medium caused repressed enzyme levels, whereas growth on a poor nitrogen source such as proline resulted in derepression of cytosine deaminase synthesis. Derepression of codA expression was induced by starvation for either uracil or cytosine nucleotides. Nitrogen control was found to be mediated by the glnLG gene products, and purine repression required a functional purR gene product. Studies with strains harbouring multiple mutations affecting both pyrimidine, purine and nitrogen control revealed that the overall regulation of cytosine deaminase synthesis by the different metabolites is cumulative.This paper is dedicated to Professor John Ingraham, Department of Bacteriology, University of California, Davis, on the occasion of his retirement, in recognition of his many contributions in the field of bacterial growth and metabolism     

Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases

Summary We have cloned and sequenced the Escherichia coli K-12 ppsA gene. The ppsA gene codes for PEP synthase, which converts pyruvate into phosphoenolpyruvate (PEP), an essential step in gluconeogenesis when pyruvate or lactate are used as a carbon source. The open reading frame consists of 792 amino acids and shows homology with other phosphohistidine-containing enzymes that catalyze the conversion between pyruvate and PEP. These enzymes include pyruvate, orthophosphate dikinases from plants and Bacteroides symbiosus and Enzyme I of the bacterial PEP:carbohydrate phosphotransferase system.     

Assessing the toxic effect of 2,4,6-trinitrotoluene on cells of <Emphasis Type="Italic">Escherichia coli</Emphasis> K12 by flow cytofluorometry

The magnitude of transmembrane potential Δψ in cells of Escherichia coli K12 was determined by the method of flow cytofluorometry for different phases of growth. It was large in the log phase, whereas in the lag and stationary phases, the population was shown to consist of two subpopulations with low and large values of Δψ in cells. In the presence of 200 mg/l of 2,4,6-trinitrotoluene (TNT), this bimodal distribution of Δψ over the population was observed during the entire growth period until TNT was almost completely eliminated from the cultivation medium (to a concentration of 18–20 mg/l). The mean value of Δψ in cells of the population grown in the presence of TNT was substantially smaller than that in controls due to the larger fraction of the subpopulation with a low value of Δψ. Upon elimination of TNT, the distribution of Δψ in cells of the culture became unimodal and close to that in the control culture in the early log phase of growth. These findings are discussed from the standpoint that considers heterogeneity of the culture of Escherichia coli K12 as a mechanism of its adaptation to the presence of xenobiotics.     

Cell division inhibition gene dicB is regulated by a locus similar to lamboid bacteriophage immunity loci

Summary A mutation (dicA1) of a repressor gene located in the terminus region of the Escherichia coli chromosome has previously been shown to lead to temperature-dependent inhibition of division, and to be complemented by plasmids carrying either dicA or an adjacent gene dicC. In this study, operon fusions in the region coding for the division inhibition gene dicB have been used to show that temperature sensitivity does not result from high temperature inactivation of the dicA repressor. Sequence comparisons indicate that dicA and dicC are similar to genes c2 and cro respectively of bacteriophage P22, and carry similarly organized tandem operators, indicating a common evolutionary origin for dicAC and P22 immC. Nevertheless, the consensus half-operator sequence of dicAC, TGTTAGYYA, differs significantly from that of P22 immC (ATTTAAGAN). an analysis of the in vivo control of promoters dicAp, dicBp and dicCp placed upstream of malQ shows that the dicAC system is functionally similar to that of an immunity region, with the possible exception of an absence of pairwise cooperative binding. Our results also indicate that the dicA1 mutation causes a switch to permanent control by dicC at all temperatures.     

Growth phase-dependent changes in the expression of global regulatory genes and associated metabolic pathways in Escherichia coli

Metabolic regulation in Escherichia coli was studied in terms of the changes in the expression of the global regulatory genes rpoD, rpoS, soxRS, cra, fadR, iclR and arcA at three different growth phases, in batch culture. The expression of rpoS and several rpoS-dependent metabolic pathway genes, such as tktB, talA, fumC, acnA, sucA, acs and sodC, were increased (∼1.5 to 2-fold) as the cells entered the late phase of growth. The changes in the expression of other global regulators and their effects on different metabolic pathway genes were less significant, as compared to rpoS, during the later phases of growth.