Regulation of Raoultella terrigena comb.nov. phytase expression.

Phytases catalyze the release of phosphate from phytate (myo-inositol hexakisphosphate) to inositol polyphosphates. Raoultella terrigena comb.nov.

On the origin of plasmid-borne, extended-spectrum, antibiotic resistance mutations in bacteria.

Many antibiotic resistance mutations arise in pathogenic bacteria that harbor plasmids (R-plasmids). Resistance to third generation cephalosporins, for instance, largely occurs by one or more point mutations in plasmid bla genes that expand the resistance spectrum of beta-lactamases. Here I review relevant evidence underlying the worldwide emergence of extended spectrum beta-lactamases (ESBLs).

An IS4 insertion at the glnA control region of Escherichia coli creates a new promoter by providing the -35 region of its 3'-end.

An insertion element (IS)4 insertion selected as suppressor of the rpoN73::Tn5 alelle was located inside the control region of the glnA gene in Escherichia coli. In the rpoN73::Tn5 background the IS4 insertion promotes glnA transcription at a low constitutive level sufficient to sustain glutamine-independent growth. The IS4 insertion mutation in either rpoN73::Tn5 or wild-type backgrounds promotes glnA transcription from a new start site located two bases downstream of the glnAp2 start site.

Nitrogen regulation in an Escherichia coli strain with a temperature sensitive glutamyl-tRNA synthetase.

Escherichia coli cells carrying the gltX351 allele are unable to grow at 42 degrees C (Ts phenotype) due to an altered glutamyl-tRNA synthetase. We found that gltX351 cells display a new phenotype termed Gsd-, i.e.

Mutations affecting the Shine-Dalgarno sequences of the untranslated region of the Escherichia coli gltBDF operon.

Individual mutations which affected each of the two Shine-Dalgarno sequences at the 5' untranslated region of the gltB gene of Escherichia coli were characterized. They were isolated in plasmids carrying a gltB'-'lacZ protein fusion preceded by the regulatory region of the gltBDF operon. Subcloning and nucleotide sequencing of approximately 1,206 bp of DNA encompassing the gltBDF regulatory region showed that the mutations affected the first base at each of the two identical Shine-Dalgarno sequences, SD1 and SD2, located 40 and 8 bases, respectively, upstream from the putative gltB open reading frame.

gltBDF operon of Escherichia coli.

A 2.0-kilobase DNA fragment carrying antibiotic resistance markers was inserted into the gltB gene of Escherichia coli previously cloned in a multicopy plasmid. Replacement of the chromosomal gltB+ gene by the gltB225::omega mutation led to cells unable to synthesize glutamate synthase, utilize growth rate-limiting nitrogen sources, or derepress their glutamine synthetase.

glnA mutations conferring resistance to methylammonium in Escherichia coli K12.

Cells of Escherichia coli K12 were sensitive to 100 mM-methylammonium when cultured under nitrogen limitation, and resistant when grown with an excess of either NH4Cl or glutamine. Glutamine synthetase activity was required for expression of the methylammonium-sensitive phenotype. Mutants were isolated which were resistant to 100 mM-methylammonium, even when grown under nitrogen limitation.

Glutamine synthetase-constitutive mutation affecting the glnALG upstream promoter of Escherichia coli.

The spontaneous gln-76 mutation of Escherichia coli (Osorio et al., Mol. Gen.

Nitrogen regulation of synthesis of the high affinity methylammonium transport system of Escherichia coli.

Uptake of 14CH3NH+3 (methylammonium) was measured as a probe of NH+4 transport in intact Escherichia coli cells and derivatives impaired in the Ntr regulatory system. The results suggest that expression of the high affinity 14CH3NH+3 transport system (a) requires de novo polypeptide synthesis, (b) is activated by the glnG and glnF regulatory products under nitrogen limitation, and (c) is repressed under nitrogen excess by the glnL product. Cells deficient in glutamate synthase activity by virtue of their harbouring the gltB31 mutation were unable to activate synthesis of 14CH3NH+3 transport.

glnF-lacZ fusions in Escherichia coli: studies on glnF expression and its chromosomal orientation.

The regulatory gene, glnF, of Escherichia coli was fused to the structural genes of the lac operon by use of the hybrid Mu phage derivative Mudl (Ap lac). Analysis of two of these fusions showed that the glnF gene is expressed constitutively, i.e.

cis-Dominant, glutamine synthetase constitutive mutations of Escherichia coli independent of activation by the glnG and glnF products.

Mutants resistant to 80 microM L-methionine-DL-sulfoximine (MS) were isolated on glucose-minimal 15 mM NH4+ medium plates from Escherichia coli cells which were hypersensitive to this concentration of the analogue by virtue of their harboring glnG mutations. MS-resistant mutants derived from strain MX902 carried, in addition to its glnG74 ::Tn5 allele, mutations tightly linked to glnA, as shown by P1-mediated transduction experiments. One particular allele, gln-76, which suppressed the MS-sensitivity conferred by glnG74 ::Tn5 but not its Ntr- phenotype (inability to transport and utilize compounds such as arginine or proline as the only nitrogen sources), was shown to allow constitutive expression of glutamine synthetase in the absence not only of a functional glnG product but also of a functional glnF product.

Nucleotide sequence of the glnA control region of Escherichia coli.

The RNA polymerase binding sites present along a DNA segment encompassing the glnA, glnL, and glnG genes have been identified in a hybrid plasmid carrying this chromosomal region of Escherichia coli. The DNA sequence was determined of an 817 base pair segment that contains the region coding for the first 42 amino acids of the NH2-terminal and of the glnA structural gene, as well as its regulatory region. Analysis of this nucleotide sequence revealed three probable RNA polymerase recognition sites, imperfect palindromes, inverted repeats, and direct repeated sequences.

Cloning and physical mapping of the glnA gene of Escherichia coli K-12.

We have isolated part of the glnA region of Escherichia coli K-12 as a 6.4 Md DNA fragment on the ColE1 hybrid plasmid pACR1. DNA fragments from pACR1 obtained by cleavage with certain restriction endonucleases were subcloned into the pBR322 cloning vehicle.

Progress in the resolution of the cytoplasmic membrane DNA initiation complex of Escherichia coli.

The attachment of the bacterial chromosome to the cytoplasmic membrane in Escherichia coli was studied. The initiator DNA was specifically labeled and the outer and cytoplasmic membranes were separated in a step sucrose gradient. The labeled DNA was localized mainly in the cytoplasmic membrane fraction.

Genetic and physicochemical characterization of Escherichia coli strains carrying fused F' elements derived from KLF1 and F57.

Although the two F' elements, F57 (carrying his(+)) and KLF1 (carrying leu(+)), cannot normally coexist in the same cell, crosses between RecA(-) strains of Escherichia coli carrying these two elements gave rise, at a low frequency, to progeny carrying both the his(+) and leu(+) markers in an extrachromosomal state. Genetic studies showed that the his(+) and leu(+) markers were now linked, and centrifugation studies of F' DNA isolated from these strains confirmed the presence of a new species of DNA molecule of altered size. It is proposed that the his(+) and leu(+) genes in these strains are covalently linked on a single "fused" F' element.

IMMUNOLOGICALLY ACTIVE POLYSACCHARIDES FROM NOCARDIA ASTEROIDES AND NOCARDIA BRASILIENSIS.

Zamora, A. (University of Mexico, Mexico, D.F.