Coli Genetic Map Research Articles

Investigation of the Tus-ter Blocking Efficacy during the Chromosome Replication of Live Escherichia Coli Cells

In E. coli, a circular chromosome is replicated in a bi-directional manner by two replisome complexes that assemble at a single origin of replication (oriC). The two replication forks are thought to terminate in the termination region, which is flanked by 10 ter sites. Each of these sites is bound by a Termination utilization substance (Tus) protein, thereby forming Tus-ter complexes. In vitro studies have shown that the replication forks are blocked when they approach a Tus-ter complex from the non-permissive side, but not from the permissive side. However, the blocking efficacy of the Tus-ter complex on a replication fork approaching from the non-permissive side, and the subsequent dynamics, have not been examined in live cells. To shed light on these processes in vivo, we utilize quantitative fluorescence microscopy combined with microfluidics to study four different E. coli strains that possess either a normal oriC or only an ectopic copy (oriZ) (inserted 344 kb in the E. coli genetic map along the chromosome), and have either wildtype Tus present or knocked-out (Δtus). In the oriZ strain, the clockwise replication fork (labelled via its sliding clamps) encounters the Tus-ter complex (via its non-permissive face) earlier than the counter-clockwise fork does. On tracking the progression of the replication forks in oriZ strain, we find that the presence of the Tus-ter complex reduces the rate of replication. By monitoring the fluorescence intensity of the fork as well as the duplication of a chromosomal locus, we can determine that a replication fork can nonetheless bypass the non-permissive side of a Tus-ter complex. The oriZ-Δtus strain exhibits replisome dynamics similar to that of oriC strain, whereas the chromosome dynamics differ substantially where the oriZ locus moves mostly towards cell-pole.

Pulsed field gel electrophoresis to rapidly detect the presence of IncJ conjugative transposon-like elements

To develop a screening method to detect the presence of the IncJ group of integrating conjugative transposon-like elements upon transfer to Escherichia coli. The unique insertion site of known IncJ elements, the prfC gene, is located in a region of the E. coli chromosome between 98.5 and 100 min on the E. coli genetic map. Using pulsed field gel electrophoresis and the rare cutting restriction enzymes SfiI and XbaI insertions of IncJ elements and an estimate of their size could be determined physically. This method allows initial screening of putative IncJ conjugative transposon-like elements by physical determination of their integration. IncJ-like elements, which appear to be highly homologous to the prototype IncJ element R391, have been found associated with recent epidemic outbreaks of cholera in a number of locations worldwide. Because of their integrative biology this method provides the first initial screening method to physically determine their presence upon transfer to E. coli.

31P NMR and Genetic Analysis Establish hinT as the Only Escherchia coli Purine Nucleoside Phosphoramidase and as Essential for Growth under High Salt Conditions

Eukaryotic cells encode AMP-lysine (AMP-N-epsilon-(N-alpha-acetyl lysine methyl ester) 5'-phosphoramidate) hydrolases related to the rabbit histidine triad nucleotide-binding protein 1 (Hint1) sequence. Bacterial and archaeal cells have Hint homologs annotated in a variety of ways, but the enzymes have not been characterized, nor have phenotypes been described due to loss of enzymatic activity. We developed a quantitative (31)P NMR assay to determine whether Escherichia coli possesses an adenosine phosphoramidase activity. Indeed, soluble lysates prepared from wild-type laboratory E. coli exhibited activity on the model substrate adenosine 5'-monophosphoramidate (AMP-NH(2)). The E. coli Hint homolog, which had been comprehensively designated ycfF and is here named hinT, was cloned, overexpressed, purified, and characterized with respect to purine nucleoside phosphoramidate substrates. Bacterial hinT was several times more active than human or rabbit Hint1 on five model substrates. In addition, bacterial and mammalian enzymes preferred guanosine versus adenosine phosphoramidates as substrates. Analysis of the lysates from a constructed hinT knock-out strain of E. coli demonstrated that all of the cellular purine nucleoside phosphoramidase activity is due to hinT. Physiological analysis of this mutant revealed that the loss of hinT results in failure to grow in media containing 0.75 m KCl, 0.9 m NaCl, 0.5 m NaOAc, or 10 mm MnCl(2). Thus, cation-resistant bacterial cell growth may be dependent on the hydrolysis of adenylylated and/or guanylylated phosphoramidate substrates by hinT.

The RNase Z Homologue Encoded by Escherichia coli elaC Gene Is RNase BN

In eukaryotes, archaea, and in some eubacteria, removal of 3' precursor sequences during maturation of tRNA is catalyzed by an endoribonuclease, termed RNase Z. In contrast, in Escherichia coli, a variety of exoribonucleases carry out final 3' maturation. Yet, E. coli retains an RNase Z homologue, ElaC, whose function is under active study. We have overexpressed and purified to homogeneity His-tagged ElaC and show here that it is, in fact, the previously described enzyme, RNase BN. Thus, purified ElaC displays structural and catalytic properties identical to those ascribed to RNase BN. In addition, an elaC mutant strain behaves identically to a known RNase BN- strain, CAN. Finally, we show that wild type elaC can complement the mutation in strain CAN and that the elaC gene in strain CAN carries a nonsense mutation that results in loss of RNase BN activity. These data correct a previous misassignment for the gene encoding RNase BN. Based on the fact that the original RNase BN mutation has now been identified, we propose that the elaC gene be renamed rbn.

Escherichia coli RNA Polymerase Is the Target of the Cyclopeptide Antibiotic Microcin J25

Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded, cyclic peptide antibiotic consisting of 21 unmodified amino acid residues. It is primarily active on gram-negative bacteria related to the producer strain, inducing cell filamentation in an SOS-independent way. A mutation causing resistance to MccJ25 was isolated. Genetic analysis indicated that it resided in the rpoC gene, encoding the beta' subunit of RNA polymerase, at 90 min on the E. coli genetic map. The mutation was genetically crossed on to a plasmid containing the wild-type rpoC gene. The presence of the recombinant plasmid conferred complete resistance to otherwise sensitive strains. Nucleotide sequencing of the plasmid-borne, mutant rpoC gene revealed a ACC (Thr)-to-ATC (Ile) change at codon 931, within homology block G, an evolutionarily conserved region in the large subunits of all RNA polymerases. MccJ25 decreased RNA synthesis both in vivo and in vitro. These results point to the RNA polymerase as the target of microcin action. We favor the possibility that the filamentous phenotype induced by MccJ25 results from impaired transcription of genes coding for cell division proteins. As far as we know, MccJ25 is the first peptide antibiotic shown to affect RNA polymerase.

Involvement of RNase G in in vivo mRNA metabolism in Escherichia coli

Escherichia coli rng gene (previously called cafA) encodes a novel RNase, named RNase G, which is involved in the 5' end-processing of 16S rRNA. In rng mutant cells, a precursor form of 16S rRNA, 16.3S rRNA, is accumulated. Here we report a role of RNase G in the in vivo mRNA metabolism. We found that rng:cat mutant strains overproduced a protein of about 100 kDa. N-terminal amino acid sequencing of this protein showed that it was identical to the fermentative alcohol dehydrogenase, the product of the adhE gene located at 28 min on the E. coli genetic map. The level of adhE mRNA was significantly higher in the rng:cat mutant strain than that in its parental strain, while such differences were not seen in other genes we examined. A rifampicin-chase experiment revealed that the half-life of adhE mRNA was 2.5-fold longer in the rng:cat disruptant than in the wild-type. These results indicate that, in addition to rRNA processing, RNase G is involved in in vivo mRNA degradation in E. coli.

6] Escherichia coli ribonuclease G

Publisher Summary The cafA gene, a name used previously for the rng gene encoding Escherichia coli RNase G, was originally found as a putative open reading frame (ORF) of unknown function during analysis of mecillinam-resistant shape determination mutants. Significant sequence similarity is reported between the CafA protein and the N-terminal half of RNase E, which is encoded by the rne gene at 24 min on the E. coli genetic map. This chapter outlines the accumulation of Precursor Molecule of 16S rRNA in cafA :: cat mutant and a two-step maturation of 5′ end of 16S rRNA by RNase E and RNase G. The biological role, if any, of the cytoplasmic axial filament formed by the RNase G protein remains to be determined. When histidine-tagged RNase G proteins were constructed for the purpose of purification, it is observed that some of the constructs did not form cytoplasmic axial filaments by overproduction, but still could complement the defect of 16S rRNA maturation in the rng::cat mutant. This suggests that RNase activity and the ability to form filaments are distinguishable.

Control of division gene expression in Escherichia coli

Duplication of the Escherichia coli bacterial cell culminates in the formation of a division septum that splits the progenitor cell into two identical daughter cells. Invagination of the cell envelope is brought about by the co-ordinated interplay of a family of septation-specific proteins that act locally at mid-cell at a specific time in the cell cycle. The majority of the genes known to be required for septum formation are found within the large mra cluster located at 2 min on the E. coli genetic map (nucleotides 89 552–107 474). Examination of the controls exerted on the mra operon shows that E. coli uses an extraordinary range of strategies to co-ordinate the expression of the cell division genes with respect to each other and to the cell cycle.

Control of division gene expression in Escherichia coli.

Duplication of the Escherichia coli bacterial cell culminates in the formation of a division septum that splits the progenitor cell into two identical daughter cells. Invagination of the cell envelope is brought about by the co-ordinated interplay of a family of septation-specific proteins that act locally at mid-cell at a specific time in the cell cycle. The majority of the genes known to be required for septum formation are found within the large mra cluster located at 2 min on the E. coli genetic map (nucleotides 89552-107474). Examination of the controls exerted on the mra operon shows that E. coli uses an extraordinary range of strategies to co-ordinate the expression of the cell division genes with respect to each other and to the cell cycle.

Micado--a network-oriented database for microbial genomes.

We created Micado, a database for managing genomic information, as part of the Bacillus subtilis genome programs. Its content will be progressively extended to the whole microbial world. A relational schema is defined for selective queries. It links eubacterial and archaeal sequences, genetic maps for Bacillus subtilis and Escherichia coli, and information on mutants. The latter comes from a new functional analysis project of unknown genes in B subtilis, and the database allows the community to curate information. To help queries from users, a graphical interface is built on SQL access to the database and provided through the WWW. We have automated imports of microbial sequences, and E. coli genetic map, by programming parsers of flat file distributions. These ensure smooth updates from molecular biology repositories on the Internet. Hyperlinks are created as a complement, to reference other general and specialized related information resources.

Genetic control of the resistance to phage C1 of Escherichia coli K-12.

Escherichia coli K-12 lytic phage C1 was earlier isolated in our laboratory. Its adsorption is controlled by at least three bacterial genes: dcrA, dcrB, and btuB. Our results provide evidence that the dcrA gene located at 60 min on the E. coli genetic map is identical to the sdaC gene. This gene product is an inner membrane protein recently identified as a putative specific serine transporter. The dcrB gene, located at 76.5 min, encodes a 20-kDa processed periplasmic protein, as determined by maxicell analysis, and corresponds to a recently determined open reading frame with a previously unknown function. The btuB gene product is known to be an outer membrane receptor protein responsible for adsorption of BF23 phage and vitamin B12 uptake. According to our data the DcrA and DcrB proteins are not involved in these processes. However, the DcrA protein probably participates in some cell division steps.

The tolZ gene of Escherichia coli is identified as the ftsH gene.

Escherichia coli tolZ mutants are tolerant to colicins E2, E3, D, Ia, and Ib (Tol-), can grow on glucose but not on succinate or other nonfermentable carbon sources (Nfc-), and show temperature-sensitive growth (Ts). A 1.8-kb DNA fragment that complemented the tolZ mutation was cloned. The DNA fragment was sequenced, and one open reading frame was found. This frame was identical to a part of the E. coli FtsH protein, an ATP-dependent metalloprotease that binds to the cytoplasmic membrane. The tolZ gene was located at 69 min on the E. coli genetic map, and the mutation was complemented by a plasmid carrying the ftsH gene, indicating that the tolZ gene is identical to the ftsH gene. The mutated tolZ21 gene was also cloned and sequenced and was found to have a single base change that caused an amino acid alteration of His-418 to Tyr in the FtsH protein. The tolZ21 mutant showed Hfl- (high frequency of lysogenization) and Std- (stop transfer-defective) pheno-types, both of which are due to a mutation in the ftsH (hflB) gene. However, the ftsH1, ftsH101, and hflB29 mutants did not show Tol- and Nfc phenotypes. The tolZ21 mutant was found to have a suppressor mutation, named sfhC, which allowed cells to survive. The sfhC mutation alone caused no Tol-, Nfc-, Ts, or Hfl- phenotypes in the tolZ21 mutant.

A novel member of the cspA family of genes that is induced by cold shock in Escherichia coli

Escherichia coli contains a major cold shock protein, CspA (or CS7.4), whose production is predominantly induced at low temperatures. This bacterium is known to possess five additional genes, each encoding a protein highly similar to CspA (referred to as the CspA family). Here we identified a gene that encodes a cold-shock-inducible analog of CspA and CspB. This newly cloned cspG gene is located at 22 min on the E. coli genetic map, apart from the other cspA family genes. Its gene product (70 amino acids) is 73 and 77% identical to CspA (70 amino acids) and CspB (71 amino acids), respectively. Analyses of a cspG-lacZ transcriptional fusion and Northern (RNA) hybridization revealed that cspG is a low-temperature-responsive gene. Its low-temperature-inducible promoters were determined, and the results indicated that the cspG sequence is highly similar to both the cspA and cspB sequences not only in the coding regions but also in the 5'-upstream noncoding regions surrounding their own promoters.

Purification of H-NS protein and its regulatory effect on transcription in vitro

This chapter discusses the purification of H-NS Protein and its regulatory effect o transcription in vitro . Subsets of Escherichia Coli DNA-binding proteins, which have been collectively termed histone-like proteins, are implicated in the compact organization of chromosomal DNA (i.e., the nucleoid structure). The best characterized are HU, H-NS, and IHF. Its coding gene, HNS, was sequenced and mapped at 27 min on the E. coli genetic map. H-NS is an abundant, neutral protein, composed of 137 amino acids, and appears to be homodimeric in a solution. Although this protein has been characterized as a relatively nonspecific DNA-binding protein, notable features are that it preferentially recognizes curved DNA sequences and induces strong condensation of DNA in vitro . H-NS can be categorized as a transcriptional regulator, which affects, either directly or indirectly, the expression of a number of unlinked genes in E. coli or Salmonella typhimurium . In an in vitro transcription assay, the H-NS protein was demonstrated previously to function as a transcriptional repressor directly and selectively for certain promoters.

The cobalamin (coenzyme B12) biosynthetic genes of Escherichia coli

The enteric bacterium Escherichia coli synthesizes cobalamin (coenzyme B12) only when provided with the complex intermediate cobinamide. Three cobalamin biosynthetic genes have been cloned from Escherichia coli K-12, and their nucleotide sequences have been determined. The three genes form an operon (cob) under the control of several promoters and are induced by cobinamide, a precursor of cobalamin. The cob operon of E. coli comprises the cobU gene, encoding the bifunctional cobinamide kinase-guanylyltransferase; the cobS gene, encoding cobalamin synthetase; and the cobT gene, encoding dimethylbenzimidazole phosphoribosyltransferase. The physiological roles of these sequences were verified by the isolation of Tn10 insertion mutations in the cobS and cobT genes. All genes were named after their Salmonella typhimurium homologs and are located at the corresponding positions on the E. coli genetic map. Although the nucleotide sequences of the Salmonella cob genes and the E. coli cob genes are homologous, they are too divergent to have been derived from an operon present in their most recent common ancestor. On the basis of comparisons of G+C content, codon usage bias, dinucleotide frequencies, and patterns of synonymous and nonsynonymous substitutions, we conclude that the cob operon was introduced into the Salmonella genome from an exogenous source. The cob operon of E. coli may be related to cobalamin synthetic genes now found among non-Salmonella enteric bacteria.

SbmC, a stationary-phase induced SOS Escherichia coli gene, whose product protects cells from the DNA replication inhibitor microcin B17.

Microcin B17 (MccB17) is a ribosomally synthesized peptide antibiotic of 43 amino acids that induces double-strand breaking of DNA in a DNA gyrase-dependent reaction. As a consequence, the SOS regulon is induced and massive DNA degradation occurs. In this work we have characterized an Escherichia coli gene, sbmC, that in high copy number determines high cell resistance to MccB17. sbmC encodes a cytoplasmic polypeptide of 157 amino acids (M(r), 18,095) that has been visualized in SDS-polyacrylamide gels. The gene is located at min 44 of the E. coli genetic map, close to the sbcB gene. sbmC expression is induced by DNA-damaging agents and, also, by the entry of cells into the stationary growth phase. A G-->T transversion at the fifth nucleotide of the quasicanonical LexA-box preceding the gene makes recA cells 16-fold more resistant to exogenous MccB17. The gene product, SbmC, also blocks MccB17 export from producing cells. Altogether, our results suggest that SbmC recognizes and sequesters MccB17 in a reversible way.

Characterization of the carbon starvation-inducible and stationary phase-inducible gene slp encoding an outer membrane lipoprotein in Escherichia coli.

Escherichia coli induces the expression of more than 50 proteins in response to starvation for a carbon source. Strains MC7 (csi7::phoA) and MC19 (csi19::phoA) contain fusions of a signal peptide-deficient phoA reporter sequence to a csi (carbon starvation-inducible) gene. PhoA expression increased when these strains were deprived of a carbon source or entered stationary phase but did not when the cells were deprived of a nitrogen source or subjected to osmotic, oxidative or thermal stress. Mapping and sequence analysis of the cloned phoA fusions in strains MC7 and MC19 indicated that they had occurred in different locations within the same previously unidentified gene. The wild-type allele of this gene was cloned and the encoded protein was found to be a new lipoprotein. Therefore we propose to call this locus slp (starvation lipoprotein). The 22 kDa Slp protein is associated with the outer membrane fraction. The slp gene was located at 78.6 centisomes on the E. coli genetic map. The -10 and -35 regions upstream of the mRNA start site were characteristic of a sigma 70 promoter. The major transcript from this promoter was sufficiently large to contain slp sequences but not the downstream open reading frame. Induction of beta-galactosidase activity from a slp::lacZ translational fusion during carbon starvation or stationary phase was independent of cAMP, RpoS (KatF) and DnaK, all of which are known to affect the expression of certain starvation-inducible or stationary phase-inducible proteins.

Structural analysis of the rhlE gene of Escherichia coli.

The E. coli chromosome is known to carry at least five genes, each of which codes for a "D-E-A-D" box protein that is presumed to possess an ATP-dependent RNA helicase activity. Four of such genes (srmB, deaD, dbpA and rhlB) were already mapped on the E. coli chromosome and their DNA sequences determined. We here report the complete nucleotide sequence of the remaining rhlE gene located at about 17.8 min on the E. coli genetic map. RhlE protein possesses all of the motifs (I to VI) conserved among prokaryotic and eukaryotic "D-E-A-D" proteins and has an arginine-rich carboxyl-terminal region. A null mutant of the rhlE gene was constructed by a new method with a ColE1 plasmid mutant that replicates in RNAse HI-deficient bacterial strains, but not in the wild-type strains. The delta rhlE mutant can grow normally, implying that the rhlE gene product is nonessential for bacterial cell growth.

The expression and regulation of hsdK genes after conjugative transfer.

The type I restriction and modification genes of Escherichia coli can be transferred to other non-modified strains by conjugation without killing the recipient, implying that the restriction function must be regulated. In this study, two isogenic F' plasmids (r+K and r-K) served as donors in quantitative conjugation experiments with various restriction-deficient strains of E. coli and Salmonella typhimurium. Conjugation studies with hsd::lacZ operon fusions in F' plasmids indicate that both of the hsdK promoters, p(res) and pmod, express simultaneously following conjugative transfer. Thus these genes do not appear to be regulated at the transcriptional level. A spontaneous mutant of E. coli C was discovered that is presumably killed upon conjugative transfer of the hsdK genes (defined as a Crc- phenotype). The gene that is defective in the mutant was tentatively designated hsdC (control). Hfr gene replacement studies led to the localization of the putative hsdC gene between 6 and 16 min on the E. coli genetic map.

Novel members of the two-component signal transduction genes in Escherichia coli.

A variety of adaptive response systems in prokaryotes often involve two families (two components) of signal transduction proteins, namely, sensory kinases and response-regulators. To extend the list of such sensor/regulator genes for Escherichia coli, we adopted a random screening method in this study. In particular, we isolated a series of recombinant plasmids that are able phenotypically to suppress mutational lesions of both the envZ and phoR/creC genes, each of which encodes a well-characterized sensory-kinase. Among the recombinant plasmids thus isolated, two clones (named pSN11 and pSN25) were subjected to characterization in detail. These analyses allowed us to identify the genetic loci specifying novel members of the sensor/regulator families. One (pSN11) is located around 45 min on the E. coli genetic map, that contains two adjacent coding-sequences (baeS and baeR). The other (pSN25) is located around 93 min of the genetic map, that also comprises two adjacent coding-sequences (basS and basR). These two pairs of gene-products, thus newly identified, were revealed to belong to typical members of the sensor/regulator families. Furthermore, they were demonstrated to exhibit the in vitro phosphotransfer reaction in the presence of ATP, that is also a characteristic of the sensory-kinase and response-regulator proteins.