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1.
Virology ; 468-470: 274-282, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25213407

ABSTRACT

The Escherichia coli bacteriophage P1 packages host chromosome separately from phage DNA, and transfers it to recipient cells at low frequency in a process called generalized transduction. Phage genomes are packaged from concatemers beginning at a specific site, pac. To increase transduction rate, we have inserted pac into the chromosome at up to five equally spaced positions; at least this many are fully tolerated in the absence of P1 infection. A single chromosomal pac greatly increases transduction of downstream markers without decreasing phage yields; 3.5 × as much total chromosomal DNA is packaged. Additional insertions decrease phage yield by > 90% and also decrease phage DNA synthesis, although less dramatically. Packaging of chromosomal markers near to and downstream of each inserted pac site is, at the same time, increased by greater than 10 fold. Transduction of markers near an inserted pac site can be increased by over 1000-fold, potentially allowing identification of such transductants by screening.


Subject(s)
Bacteriophage P1/physiology , Chromosomes, Artificial, P1 Bacteriophage/physiology , Chromosomes, Bacterial/genetics , DNA, Bacterial/genetics , Escherichia coli/genetics , Chromosomes, Artificial, P1 Bacteriophage/genetics , DNA, Viral/analysis , DNA, Viral/physiology , Nucleic Acid Hybridization , Transduction, Genetic
2.
Res Microbiol ; 160(4): 267-77, 2009 May.
Article in English | MEDLINE | ID: mdl-19393741

ABSTRACT

A brief summary of the role of DnaK and GroE chaperones in protein folding precedes a discussion of the role of GroE in Escherichia coli. We consider its obligate substrates, the 8 that are both obligate and essential, and the prospects for constructing a mutant that could survive without it. Structural features of GroE-dependent polypeptides are also considered.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli/metabolism , Heat-Shock Proteins/chemistry , Protein Folding , Escherichia coli/chemistry , Escherichia coli Proteins/physiology , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/physiology , Heat-Shock Proteins/physiology , Stress, Physiological , Substrate Specificity
3.
J Bacteriol ; 188(23): 8317-20, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16980444

ABSTRACT

We found that a new mutant with a deletion/replacement of the Escherichia coli K-12 htrC gene, a gene previously reported to be required for growth at elevated temperatures, is not temperature sensitive. Furthermore, the original mutants, kindly provided by the original authors, although temperature sensitive, do not have mutations in the open reading frame designated htrC. We found that htrC requires RpoS for enhanced expression in the early stationary phase and is expressed at very low levels until then. The growth of our htrC mutant slowed during the early stationary phase, and the mutant was replaced by its parent in mixed cultures. Since we cannot assign a function or distinctive phenotype to htrC, we suggest that this open reading frame should be given a positional designation, yjaZ, until a specific function is identified.


Subject(s)
Escherichia coli K12/physiology , Escherichia coli Proteins/physiology , Genes, Bacterial , Open Reading Frames/physiology , Bacterial Proteins/genetics , Escherichia coli K12/growth & development , Escherichia coli Proteins/genetics , Heat-Shock Response , Mutation , Open Reading Frames/genetics , Sigma Factor/genetics
4.
FEMS Microbiol Lett ; 235(1): 131-7, 2004 Jun 01.
Article in English | MEDLINE | ID: mdl-15158272

ABSTRACT

Dihydropicolinate synthase (DHDPS; E.C. 4.2.1.52) catalyses the first committed step of lysine biosynthesis in plants and bacteria. Plant DHDPS enzymes, which are responsible solely for lysine biosynthesis, are strongly inhibited by lysine (I0.5 =10 microM), whereas the bacterial enzymes which are less responsive or insensitive to lysine inhibition have the additional function of meso-diaminopimelate biosynthesis which is required for cell wall formation. Previous studies have suggested that expression of the Escherichia coli dapA gene, encoding DHDPS, is unregulated. We show here that this is not the case and that expression of LacZ from the dapA promoter (PdapA) increases in response to diaminopimelic acid limitation in E. coli K-12.


Subject(s)
Diaminopimelic Acid/metabolism , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Hydro-Lyases/genetics , Promoter Regions, Genetic , Bacterial Proteins/genetics , Cell Division , Chaperonins , Escherichia coli/enzymology , Escherichia coli/metabolism , Escherichia coli Proteins , Gene Deletion , Gene Expression Regulation, Enzymologic , Heat-Shock Proteins/genetics , Hydro-Lyases/metabolism , Transcriptional Activation
5.
J Bacteriol ; 185(21): 6415-24, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14563877

ABSTRACT

The can (previously yadF) gene of Escherichia coli encodes a beta-class carbonic anhydrase (CA), an enzyme which interconverts CO(2) and bicarbonate. Various essential metabolic processes require either CO(2) or bicarbonate and, although carbon dioxide and bicarbonate spontaneously equilibrate in solution, the low concentration of CO(2) in air and its rapid diffusion from the cell mean that insufficient bicarbonate is spontaneously made in vivo to meet metabolic and biosynthetic needs. We calculate that demand for bicarbonate is 10(3)- to 10(4)-fold greater than would be provided by uncatalyzed intracellular hydration and that enzymatic conversion of CO(2) to bicarbonate is therefore necessary for growth. We find that can expression is ordinarily required for growth in air. It is dispensable if the atmospheric partial pressure of CO(2) is high or during anaerobic growth in a closed vessel at low pH, where copious CO(2) is generated endogenously. CynT, the single E. coli Can paralog, can, when induced with azide, replace Can; also, the gamma-CA from Methanosarcina thermophila can at least partially replace it. Expression studies showed that can transcription does not appear to respond to carbon dioxide concentration or to be autoregulated. However, can expression is influenced by growth rate and the growth cycle; it is expressed best in slow-growing cultures and at higher culture densities. Expression can vary over a 10-fold range during the growth cycle and is also elevated during starvation or heat stress.


Subject(s)
Carbonic Anhydrases/metabolism , Escherichia coli/enzymology , Adaptation, Physiological , Aerobiosis , Anion Transport Proteins/genetics , Anion Transport Proteins/metabolism , Carbon Dioxide/metabolism , Carbonic Anhydrases/genetics , Escherichia coli/growth & development , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Hot Temperature
6.
J Bacteriol ; 184(19): 5513-7, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12218041

ABSTRACT

We report that the genes abc, yaeC, and yaeE comprise metD, an Escherichia coli locus encoding a DL-methionine uptake system. MetD is an ABC transporter with Abc the ATPase, YaeE the permease, and YaeC the likely substrate binding protein. Expression of these genes is regulated by L-methionine and MetJ, a common repressor of the methionine regulon. We propose to rename abc, yaeE, and yaeC as metN, metI, and metQ, respectively.


Subject(s)
ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Multigene Family , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Amino Acid Transport Systems/genetics , Amino Acid Transport Systems/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Escherichia coli/growth & development , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Gene Deletion , Gene Expression Regulation, Bacterial , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Methionine/metabolism , Molecular Sequence Data , Transcription, Genetic
7.
J Bacteriol ; 184(16): 4573-81, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12142427

ABSTRACT

Despite the power of sequencing and of emerging high-throughput technologies to collect data rapidly, the definitive functional characterization of unknown genes still requires biochemical and genetic analysis in case-by-case studies. This often involves the deletion of target genes and phenotypic characterization of the deletants. We describe here modifications of an existing deletion method which facilitates the deletion process and enables convenient analysis of the expression properties of the target gene by replacing it with an FRT-lacZ-aph-P(lac)-FRT cassette. The lacZ gene specifically reports the activity of the deleted gene and therefore allows the determination of the conditions under which it is actively expressed. The aph gene, encoding resistance to kanamycin, provides a selectable means of transducing a deleted locus between strains so that the deletion can be combined with other relevant mutations. The lac promoter helps to overcome possible polar effects on downstream genes within an operon. Because the cassette is flanked by two directly repeated FRT sites, the cassette can be excised by the Flp recombinase provided in trans. Removing the cassette leaves an in-frame deletion with a short scar which should not interfere with downstream expression. Replacements of yacF, yacG, yacH, yacK (cueO), yacL, ruvA, ruvB, yabB, and yabC made with the cassette were used to verify its properties.


Subject(s)
Escherichia coli/genetics , Mutagenesis, Insertional/methods , Alleles , Gene Deletion , Gene Expression Regulation, Bacterial , Genes, Bacterial , Phenotype , Plasmids/genetics
8.
Mol Microbiol ; 44(5): 1287-98, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12068810

ABSTRACT

Expression of the gene pcnB, encoding the dispensable Escherichia coli poly(A) polymerase (PAPI), which is toxic when overproduced, was investigated. Its promoter was identified and found to be moderately strong when used to express a beta-galactosidase reporter. Expression levels were not affected by increasing or decreasing PcnB concentration. Translation of pcnB was found to initiate from the non-canonical initiation codon AUU. The only other coli gene reported to use AUU as initiation codon is infC, which encodes the initiation factor IF-3. AUU, in common with other rarely used initiation codons, is discriminated against by IF-3, resulting in the aborting of most AUU-promoted initiation events. This enables AUU to form part of an autoregulatory circuit controlling IF-3 production. We show that InfC discrimination reduces PcnB production fivefold. This is the first instance of this mechanism being used to limit severely the production of a potentially toxic product.


Subject(s)
Codon, Initiator/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Polynucleotide Adenylyltransferase/genetics , Promoter Regions, Genetic , Protein Biosynthesis , Base Sequence , Gene Expression Regulation, Bacterial , Genes, Bacterial , Molecular Sequence Data , Mutagenesis, Site-Directed , Polynucleotide Adenylyltransferase/metabolism , RNA, Messenger/genetics
9.
Microbiology (Reading) ; 145 ( Pt 11): 3089-3100, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10589716

ABSTRACT

RNAI is a short RNA, 108 nt in length, which regulates the replication of the plasmid ColE1. RNAI turns over rapidly, enabling plasmid replication rate to respond quickly to changes in plasmid copy number. Because RNAI is produced in abundance, is easily extracted and turns over quickly, it has been used as a model for mRNA in studying RNA decay pathways. The enzymes polynucleotide phosphorylase, poly(A) polymerase and RNase E have been demonstrated to have roles in both messenger and RNAI decay; it is reported here that these enzymes can work independently of one another to facilitate RNAI decay. The roles in RNAI decay of two further enzymes which facilitate mRNA decay, the exonuclease RNase II and the endonuclease RNase III, are also examined. RNase II does not appear to accelerate RNAI decay but it is found that, in the absence of RNase III, polyadenylated RNAI, unprocessed by RNase E, accumulates. It is also shown that RNase III can cut RNAI near nt 82 or 98 in vitro. An RNAI fragment corresponding to the longer of these can be found in extracts of an mc+ pcnB strain (which produces RNase III) but not of an rnc pcnB strain, suggesting that RNAI may be a substrate for RNase III in vivo. A possible pathway for the early steps in RNAI decay which incorporates this information is suggested.


Subject(s)
Bacterial Proteins , Endoribonucleases/physiology , Escherichia coli Proteins , Plasmids/physiology , RNA, Antisense/metabolism , Blotting, Northern , DNA Replication/drug effects , Endoribonucleases/genetics , Escherichia coli/enzymology , Exoribonucleases/genetics , Exoribonucleases/physiology , Mutation , Polymerase Chain Reaction , Polynucleotide Adenylyltransferase/genetics , Polynucleotide Adenylyltransferase/physiology , Protein Processing, Post-Translational , RNA, Antisense/pharmacology , RNA, Bacterial/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering , Ribonuclease III
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