Evolutionary origins and sequence of the Escherichia coli O4 O-antigen gene cluster

Escherichia coli express many types of O antigen, present in the outer membrane of the Gram-negative bacterial cell wall. O-Antigen biosynthesis genes are clustered together and differences seen in O-antigen types are due to genetic variation within this gene cluster. Sequencing of the E. coli O4 O-antigen gene cluster revealed a similar gene order and high levels of similarity to that of E. coli O26; indicating a common ancestor. These lateral transfer events observed within O-antigen gene clusters may occur as part of the evolution of the pathogenic clones.     

Ribosomal protein gene sequence changes in erythromycin-resistant mutants of Escherichia coli.

The genes for ribosomal proteins L4 and L22 from two erythromycin-resistant mutants of Escherichia coli have been isolated and sequenced. In the L4 mutant, an A-to-G transition in codon 63 predicted a Lys-to-Glu change in the protein. In the L22 strain, a 9-bp deletion removed codons 82 to 84, eliminating the sequence Met-Lys-Arg from the protein. Consistent with these DNA changes, in comparison with wild-type proteins, both mutant proteins had reduced first-dimension mobilities in two-dimensional polyacrylamide gels. Complementation of each mutation by a wild-type gene on a plasmid vector resulted in increased erythromycin sensitivity in the partial-diploid strains. The fraction of ribosomes containing the mutant form of the protein was increased by growth in the presence of erythromycin. Erythromycin binding was increased by the fraction of wild-type protein present in the ribosome population. The strain with the L4 mutation was found to be cold sensitive for growth at 20 degrees C, and 50S-subunit assembly was impaired at this temperature. The mutated sequences are highly conserved in the corresponding proteins from a number of species. The results indicate the participation of these proteins in the interaction of erythromycin with the ribosome.     

Thiol-sensitive genes of Escherichia coli.

The effect of 1-thioglycerol on the expression of genes of Escherichia coli was investigated. Pulse-labeled proteins from aerobically growing, 1-thioglycerol-treated E. coli were separated by two-dimensional gel electrophoresis, and their radioactivities were compared with those of identical proteins from nontreated cells. The first 10 min of exposure to thiol stimulated the synthesis of 10% of the observed proteins and inhibited the production of 16% of the proteins. After 30 min of growth with thiol, the synthesis of 44% of the observed proteins was inhibited and synthesis of 18% of the proteins was stimulated. In general, the expression of genes of carbohydrate metabolism, amino acid metabolism, and protein biosynthesis were inhibited, while nucleic acid synthetic and repair gene expressions showed mixed responses. Synthesis of transport proteins was not affected. Transient stimulation of oxidative-stress proteins and sustained stimulation of the expressions of trxB, ompA, and ompB genes and those of several unidentified gene products were also observed. Whether these complex responses merely reflect adjustments by cellular subsystems to a suddenly reducing environment or whether they are manifestations of a reductive-stress regulon will have to await genetic analysis of this phenomenon.     

Polarity of amber mutations in ribosomal protein genes of Escherichia coli.

Two amber mutations have been mapped inside the spcA-strA region (now called rpsE-rpsL) on the bacterial genome. Derivatives of the transducing phage lambda fus3 carrying each mutation were constructed and assayed in ultraviolet-irradiated bacteria to identify the mutated genes and measure the polarity of the mutations. The data indicated that both mutations, 3162(Am) and 3161(Am), affect genes coding for ribosomal proteins: rplC (L3) and rpsN (S14), respectively. It was shown also that each mutation exerts, inside of its respective operon (S10 and spc units), a relatively strong polar effect on genes distal to the mutated locus.     

Detection of protein similarities using nucleotide sequence databases.

A simple procedure is described for finding similarities between proteins using nucleotide sequence databases. The approach is illustrated by several examples of previously unknown correspondences with important biological implications: Drosophila elongation factor Tu is shown to be encoded by two genes that are differently expressed during development; a cluster of three Drosophila genes likely encode maltases; a flesh-fly fat body protein resembles the hypothesized Drosophila alcohol dehydrogenase ancestral protein; an unknown protein encoded at the multifunctional E. coli hisT locus resembles aspartate beta-semialdehyde dehydrogenase; and the E. coli tyrR protein is related to nitrogen regulatory proteins. These and other matches were discovered using a personal computer of the type available in most laboratories collecting DNA sequence data. As relatively few sequences were sampled to find these matches, it is likely that much of the existing data has not been adequately examined.     

The significance of protein sequence similarities

A general method of assessing the significance of scored bestlocal alignments, particularly suited to protein sequence comparisons,is described. The method establishes the parameters describingthe distribution of the best results from any search program,provided that the set is sufficiently large and the majorityof the alignments arise from unrelated sequences. The expectedfrequency of occurrence of any score can then be calculated,together with the number of standard deviations above expectation.These provide sensible measures of significance without additionalsearch operations. However the biological significance of anyalignment or set of alignments does not solely depend on theimprobability of the alignment, but on all relevant factorsknown to the biologist. Received on August 9, 1987; accepted on November 17, 1987     

Escherichia coli in disguise: molecular origins of Shigella

Shigella, which still stands as a genus with four species today, in reality belongs to the extremely diverse species Escherichia coli. There are several lineages of Shigella strains derived through independent acquisition of the pINV virulence plasmid. The chromosomally determined phenotypic properties of Shigella result from convergent evolution during niche adaptation, most due to loss of function, some from negative selection pressure.     

Amino acid sequence of the ribosomal protein L21 of Escherichia coli.

The primary structure of protein L21 from the 50S subunit of Escherichia coli ribosomes has been completely determined by sequencing the peptides obtained by digestion of L21 with trypsin before and after modification of the arginine residues with 1,2-cyclohexanedione, Staphylococcus aureus protease, thermolysin, and pepsin. Automated Edman degradation using a liquid-phase sequenator was carried out on the intact protein as well as on a fragment arising from cleavage with cyanogen bromide. Protein L21 consists of a single polypeptide chain of 103 amino acids of molecular weight 11 565. An estimation of the secondary structure of protein L21 and a comparison with other E. coli ribosomal protein sequences are presented.     

Translocation of nascent non-signal sequence protein in heated Escherichia coli

Exposure of Escherichia coli to heat resulted in 1) selective inhibition of protein synthesis, 2) synthesis of heat shock proteins, and 3) altered subcellular distribution of newly synthesized proteins. Either 5 min or 1 h at 48 degrees C increases outer membrane proteins of Coomassie Blue-stained gels. After 1 h, there was a loss of stained proteins from the soluble fraction. Much greater changes in the distribution of radiolabeled (newly synthesized) proteins were observed, with marked increases in the number of outer membrane protein species and a corresponding loss of soluble fraction proteins. Three major species of radiolabeled proteins from heat-treated cells remain in the soluble fraction; these proteins have apparent Mr 56,000, 69,200, and 79,400. Cells were labeled with L-[35S] methionine at either 37 or 48 degrees C and chased with non-radiolabeled methionine before a temperature shift to either 48 or 37 degrees C, respectively. Only proteins synthesized at elevated temperature participated in translocation. It is suggested that heat disordering of membrane lipids promotes interlipidic connections between the inner and outer membrane providing pathways for protein movement to the outer membrane and may be the mechanism whereby a cell quickly responds to environmental temperature stress. The response does not require but may trigger synthesis of mRNA.     

Probing the DNA sequence specificity of Escherichia coli RECA protein

Escherichia coli RecA protein catalyzes the central DNA strand-exchange step of homologous recombination, which is essential for the repair of double-stranded DNA breaks. In this reaction, RecA first polymerizes on single-stranded DNA (ssDNA) to form a right-handed helical filament with one monomer per 3 nt of ssDNA. RecA generally binds to any sequence of ssDNA but has a preference for GT-rich sequences, as found in the recombination hot spot Chi (5′-GCTGGTGG-3′). When this sequence is located within an oligonucleotide, binding of RecA is phased relative to it, with a periodicity of three nucleotides. This implies that there are three separate nucleotide-binding sites within a RecA monomer that may exhibit preferences for the four different nucleotides. Here we have used a RecA coprotease assay to further probe the ssDNA sequence specificity of E.coli RecA protein. The extent of self-cleavage of a λ repressor fragment in the presence of RecA, ADP-AlF₄ and 64 different trinucleotide-repeating 15mer oligonucleotides was determined. The coprotease activity of RecA is strongly dependent on the ssDNA sequence, with TGG-repeating sequences giving by far the highest coprotease activity, and GC and AT-rich sequences the lowest. For selected trinucleotide-repeating sequences, the DNA-dependent ATPase and DNA-binding activities of RecA were also determined. The DNA-binding and coprotease activities of RecA have the same sequence dependence, which is essentially opposite to that of the ATPase activity of RecA. The implications with regard to the biological mechanism of RecA are discussed.     

Transcription of ribosomal protein genes carried on F'' plasmids of Escherichia coli.

Summary Spontaneous mutants of the petite-negative yeast Kluyveromyces lactis, resistant to the antibiotics chloramphenicol and oligomycin, were isolated and genetically characterized.Three chloramphenicol-resistant mutants showed non-Mendelian inheritance when crossed to sensitive parents.Of 5 oligomycin-resistant strains studied, three exhibited resistance due to the presence of an extrachromosomal mutation. The resistance of the other two deriving from a nuclear and recessive mutation.When two factor crosses in trans configuration were performed between two of the chloramphenicol and the five oligomycin-resistant mutants a polarity in recombination was observed with a predominance of sensitive (O^SC^S) over resistant (O^RC^R) reciprocal recombinants.Allelism tests carried out among the oligomycin-resistant mutants indicated the presence of at least two distinct extrachromosomal regions responsible for the resistance.