Molecular analysis of the cryptic and functional phn operons for phosphonate use in Escherichia coli K-12.

We cloned the cryptic phn operon of a K-12 strain, phn(EcoK), and analyzed the nucleotide sequence of the phn region (11,672 bp). An mRNA start site upstream of the phnC gene was identified by S1 nuclease mapping. The pho regulon activator PhoB protects a pho box region near the mRNA start in DNase I footprinting and methylation protection experiments. The sequence of the cryptic phn(EcoK) operon was very similar to that of the functional phn operon of an Escherichia coli B strain, phn(EcoB) (C.-M. Chen, Q.-Z. Ye, Z. Zhu, B. L. Wanner, and C. T. Walsh, J. Biol. Chem. 265:4461-4471, 1990). The phnE(EcoK) gene has an 8-bp insertion, absent from the phnE(EcoB) gene, which causes a frameshift mutation. The spontaneous activation of the cryptic phn(EcoK) operon is accompanied by loss of this additional 8-bp insertion. Studies of the structure, regulation, and function of the phn region suggest that the phosphate starvation-inducible phn operon consists of 14 cistrons from phnC to phnP.     

Involvement of the Escherichia coli phn (psiD) gene cluster in assimilation of phosphorus in the form of phosphonates, phosphite, Pi esters, and Pi.

The phn (psiD) gene cluster is induced during Pi limitation and is required for the use of phosphonates (Pn) as a phosphorus (P) source. Twelve independent Pn-negative (Pn-) mutants have lesions in the phn gene cluster which, as determined on the basis of recombination frequencies, is larger than 10 kbp. This distance formed the basis for determining the complete DNA sequence of a 15.6-kbp BamHI fragment, the sequences of which suggested an operon with 17 open reading frames, denoted (in alphabetical order) the phnA to phnQ genes (C.-M. Chen, Q.-Z. Ye, Z. Zhu, B. L. Wanner, and C. T. Walsh, J. Biol. Chem. 265:4461-4471, 1990) Ten Pn- lesions lie in the phnD, phnE, phnH, phnJ, phnK, phnO, and phnP genes. We propose a smaller gene cluster with 14 open reading frames, phnC to phnP, which probably encode transporter and regulatory functions, in addition to proteins needed in Pn biodegradation. On the basis of the effects on phosphite (Pt), Pi ester, and Pi use, we propose that PhnC, PhnD, and PhnE constitute a binding protein-dependent Pn transporter which also transports Pt, Pi esters, and Pi. We propose that PhnO has a regulatory role because a phnO lesion affects no biochemical function, except for those due to polarity. Presumably, the 10 other phn gene products mostly act in an enzyme complex needed for breaking the stable carbon-phosphorus bond. Interestingly, all Pn- mutations abolish the use not only of Pn but also of Pt, in which P is in the +3 oxidation state. Therefore, Pn metabolism and Pt metabolism are related, supporting a biochemical mechanism for carbon-phosphorus bond cleavage which involves redox chemistry at the P center. Furthermore, our discovery of Pi-regulated genes for the assimilation of reduced P suggests that a P redox cycle may be important in biology.     

Mapping and disruption of the chpB locus in Escherichia coli.

The chpB locus is a chromosomal homolog of the pem locus, which is responsible for stable maintenance of plasmid R100 within the host cells. Like pem, chpB codes for two genes, chpBK and chpBI, encoding a growth inhibitor and a suppressor for the killing action of the ChpBK protein, respectively. Here, we determined the precise location of the chpB locus, which is linked to ileR and ppa in the order ileR-chpB-ppa, at 95.7 min on the map of Escherichia coli. We then constructed mutants with an insertion of a (cat) fragment within chpBK or chpBI on the E. coli chromosome. These mutants grew normally, indicating that chpB is dispensable for cell growth.     

Involvement of the phosphate regulon and the psiD locus in carbon-phosphorus lyase activity of Escherichia coli K-12.

Escherichia coli K-12 can readily mutate to use methylphosphonic acid as the sole phosphorus source by a direct carbon-to-phosphorus (C-P) bond cleavage activity that releases methane and Pi. The in vivo C-P lyase activity is both physiologically and genetically regulated as a member of the phosphate regulon. Since psiD::lacZ(Mu d1) mutants cannot metabolize methylphosphonic acid, psiD may be the structural gene(s) for C-P lyase.     

Mapping of the fabD locus for fatty acid biosynthesis in Escherichia coli.

fabD mutants of Escherichia coli contain a thermolabile malonyl-coenzyme A-acyl carrier protein transacylase which causes defective fatty acid synthesis and temperature-sensitive growth. By conjugation and P1 transduction the fabD locus has now been mapped at min 24, between pyrC and purB and close to cat. The order of sites is tentatively given as pyrC, cat, fabD, and purB, though the orientation of cat and fabD could be reversed. The possible relationship of fabD with another mutation lying in this region and also affecting acid synthesis is discussed. In the course of these studies we also confirmed the location of the fabA gene, determined that poaA lies between fabA and pyrC, and inadvertently found that the pyr mutation in strain AT3143 is probably pyrF and not pyrC.     

Mapping of a locus (mdoA) that affects the biosynthesis of membrane-derived oligosaccharides in Escherichia coli.

Mutants of Escherichia coli defective in the newly discovered mdoA locus are blocked at an early stage in the biosynthesis of membrane-derived oligosaccharides. The mutation has now been mapped and found to be located near 23 min on the E. coli chromosome between putA and pyrC. The mdoA mutants are defective in the membrane-localized component of the glucosyl transferase system described by Weissborn and Kennedy (A. C. Weissborn and E. P. Kennedy, Fed. Proc. 42:2122, 1983).     

Mapping of a new genetic locus responsible for ampicillin resistance in Escherichia coli.

The ampicillin resistance locus of three different ampicillin-resistant, temperature-sensitive Escherichia coli mutants was mapped between proC and purE and does not correspond to any of the known genes in this region. The mutant gene responsible for the temperature sensitivity and consequent morphological changes in each mutant strain was not located in the same 5-min region, even though the two mutants characteristics co-reverted at a very high frequency.     

Escherichia coli thymidylate kinase: molecular cloning, nucleotide sequence, and genetic organization of the corresponding tmk locus.

Thymidylate kinase (dTMP kinase; EC 2.7.4.9) catalyzes the phosphorylation of dTMP to form dTDP in both de novo and salvage pathways of dTTP synthesis. The nucleotide sequence of the tmk gene encoding this essential Escherichia coli enzyme is the last one among all the E. coli nucleoside and nucleotide kinase genes which has not yet been reported. By subcloning the 24.0-min region where the tmk gene has been previously mapped from the lambda phage 236 (E9G1) of the Kohara E. coli genomic library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987), we precisely located tmk between acpP and holB genes. Here we report the nucleotide sequence of tmk, including the end portion of an upstream open reading frame (ORF 340) of unknown function that may be cotranscribed with the pabC gene. The tmk gene was located clockwise of and just upstream of the holB gene. Our sequencing data allowed the filling in of the unsequenced gap between the acpP and holB genes within the 24-min region of the E. coli chromosome. Identification of this region as the E. coli tmk gene was confirmed by functional complementation of a yeast dTMP kinase temperature-sensitive mutant and by in vitro enzyme assay of the thymidylate kinase activity in cell extracts of E. coli by use of tmk-overproducing plasmids. The deduced amino acid sequence of the E. coli tmk gene showed significant similarity to the sequences of the thymidylate kinases of vertebrates, yeasts, and viruses as well as two uncharacterized proteins of bacteria belonging to Bacillus and Haemophilus species.     

Molecular cloning and expression of a locus (mdoA) implicated in the biosynthesis of membrane-derived oligosaccharides in Escherichia coli

Mutants of Escherichia coli defective in the mdoA locus are blocked at an early stage in the biosynthesis of membrane-derived oligosaccharides. The mdoA locus has now been cloned into multicopy plasmids. A 5 kb DNA fragment is necessary to complement mdoA mutations. Cells harbouring the mdoA+ plasmid produced three to four times more MDO than wild-type cells. MDO overproduction did not affect the degree of MDO substitution with sn-1-phosphoglycerol residues. The biosynthesis of MDO remained under osmotic control in overproducing strains.     

Mapping of the plasmid (pLQ3) from Achromobacter and cloning of its cephalosporinase gene in Escherichia coli

We have constructed a physical map of the plasmid pLQ3 which was originally isolated from Achromobacter and which codes for a beta-lactamase. The enzyme specified by pLQ3 is expressed in Escherichia coli and is unusual in that it is a cephalosporinase, an enzyme usually coded for by chromosome. Plasmid pLQ3 is 12.4 kb in length and has a unique Bam HI site and two BglII sites. From a BamHI + BglII double digest of pLQ3, we have constructed a "shortened" plasmid, pLQ10, in which a 2.96-kb fragment is deleted. We have constructed a clone, pLQ22, in which a 3.27-kb fragment of pLQ3, carrying the beta-lactamase gene, is inserted into the BamHI site of pACYC184. By "comparative mapping" of single and multiple digests of each of these plasmids, we have been able to locate the cleavage sites for PstI, which makes seven cuts in pLQ3.     

The recR locus of Escherichia coli K-12: molecular cloning, DNA sequencing and identification of the gene product.

The recR gene of Escherichia coli, which is associated with recBC-independent mechanisms of recombination and DNA repair, has been located between dnaZX and htpG on a 6.4 kb EcoRI fragment of DNA that has been cloned and analysed in lambda and plasmid vectors. Nucleotide sequencing of this interval revealed two open reading frames that constitute an operon lying immediately downstream of dnaZX. The second of these two reading frames was identified as recR. It encodes a polypeptide with a predicted molecular weight of 21,965 Daltons that migrates on SDS gels as a 26 kDa protein. The first gene of the operon encodes a polypeptide of 12,015 daltons. Its function is not known.     

大肠杆菌膦酸酯代谢途径中phnF基因的研究

大肠杆菌的ｐｈｎ操纵子与膦酸酯（Ｐｎ）的利用密切相关。实验利用ＰＣＲ扩增、ＴＡ克隆等方法,获得了大肠杆菌ｐｈｎ操纵子中ｐｈｎＥ、ｐｈｎＦ和ｐｈｎＧ基因的亚克隆,并进行了序列测定。通过Ｐ１噬菌体转导,构建了ｐｈｎＦ的ＴｎｐｈｏＡ’９转座子插入突变体ＪＷ１９,该突变仅对大肠杆菌的ＡＥＰｎ同化有微弱的影响,而利用ｐｈｎＥ和ｐｈｎＧ序列与染色体重组构建的ｐｈｎＦ全缺失突变株ＪＷ６７,则几乎不能在ＡＥＰｎ培养基上生长。通过ｐｈｎＦ基因的诱导高表达,用亲和柱层析分离纯化了ＰｈｎＦ蛋白,达到电泳纯。并且用凝胶延滞的方法观察到,ＰｈｎＦ蛋白与ｐｈｎ操纵子ＤＮＡ片段相互作用后,可使凝胶图谱类型发生改变。     

Genetic analysis and molecular cloning of the Escherichia coli ruv gene

Summary The genetic organisation of the ruv gene, a component of the SOS system for DNA repair and recombination in Escherichia coli, was investigated. New point mutations as well as insertions and deletions were generated using transposon Tn10 inserted in eda as a linked marker for site specific mutagenesis, or directly as a mutagen. The mutations were ordered with respect to one another and previously isolated ruv alleles by means of transductional crosses. The direction of chromosome mobilization from ruv:: Mud(Ap^R lac)strains carrying F42lac ⁺ established that ruv is transcribed in a counterclockwise direction. Recombinant phages able to restore UV resistance to ruv mutants were identified, and the ruv ⁺ region was subcloned into a low copy number plasmid. The ruv ⁺ plasmid was able to correct the extreme radiation sensitivity and recombination deficiency of ruv recBC sbcB strains.     

Mapping and cloning of gldA, the structural gene of the Escherichia coli glycerol dehydrogenase.

gldA, the structural gene for the NAD(+)-dependent glycerol dehydrogenase, was mapped at 89.2 min on the Escherichia coli linkage map, cotransducible with, but not adjacent to, the glpFKX operon encoding the proteins for the uptake and phosphorylation of glycerol. gldA was cloned, and its position on the physical map of E. coli was determined. The expression of gldA was induced by hydroxyacetone under stationary-phase growth conditions.     

PfkA locus of Escherichia coli.

pfkA was know, on the basis of three mutants, as the likely locus of phosphofructokinase in Escherichia coli, and the unlinked pfkB1 mutation suppressed these mutations by restoring some enzyme activity (Morrissey and Fraenkel, 1972). We now report a new search for the complete inactivation of pfkA (e.g., by deletion or amber mutation), done to assess whether the pfkB1 suppression is by an independent enzyme, phosphofructokinase activity 2 (Fraenkel, Kotlarz, and Buc, 1973). Ten new phosphofructokinase mutants all were at pfkA, rather than at pfkB or pfkC. One of them (pfkA9) gave temperature-sensitive reverants with heat-labile enzyme. Another (pfkA11) proved genetically to be a nonsense mutation, but showed no restored activity when suppressed by supF. However, even unsuppressed it was found to contain an enzyme related to phosphofructokinase activity 1 kinetically (more allosteric), physically (almot identical subunit), and antigenically. All the pfkA mutants apparently contained cross-reacting material to activity 1. All (including pfkA11) were suppressed by the pfkB1 mutation. Several results support the idea that pfkA is the structural gene for the main phosphofructokinase of E. coli (activity 1), but that there is some restriction to its complete inactivation.     

Molecular cloning and characterization of genes required for ribose transport and utilization in Escherichia coli K-12.

We isolated spontaneous and transposon insertion mutants of Escherichia coli K-12 that were specifically defective in utilization or in high-affinity transport of D-ribose (or in both). Cotransduction studies located all of the mutations near ilv, at the same position as previously identified mutations causing defects in ribokinase ( rbsK ) or ribose transport ( rbsP ). Plasmids that complemented the rbs mutations were isolated from the collection of ColE1 hybrid plasmids constructed by Clarke and Carbon. Analysis of those plasmids as well as of fragments cloned into pBR322 and pACYC184 allowed definition of the rbs region. Products of rbs genes were identified by examination of the proteins produced in minicells containing various rbs plasmids. We identified four rbs genes: rbsB , which codes for the 29-kilodalton ribose-binding protein; rbsK , which codes for the 34-kilodalton ribokinase ; rbsA , which codes for a 50-kilodalton protein required for high-affinity transport; and rbsC , which codes for a 27-kilodalton protein likely to be a transport system component. Our studies showed that these genes are transcribed from a common promoter in the order rbsA rbsC rbsB rbsK . It appears that the high-affinity transport system for ribose consists of the three components, ribose-binding protein, the 50-kilodalton RbsA protein, and the 27-kilodalton RbsC protein, although a fourth, unidentified component could exist. Mutants defective in this transport system, but normal for ribokinase , are able to grow normally on high concentrations of the sugar, indicating that there is at least a second, low-affinity transport system for ribose in E. coli K-12.