The lytB gene of Escherichia coli is essential and specifies a product needed for isoprenoid biosynthesisThe lytB gene of Escherichia coli is essential and specifies a product needed for isoprenoid biosynthesis' Journal of Bacteriology, vol 183, no. 24, pp. 7403-7. General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim.

The Life of Professor Robert Hugh Pritchard, pp. 89-91 (2017) No Access16: Bacterial and Plasmid Replication: Some Memories of the Early DaysMillicent MastersMillicent MastersEdinburgh University, UKhttps://doi.org/10.1142/9789813203754_0016Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: In 1965 I joined Bill Hayes' Bacterial Genetics Unit at Hammersmith Hospital supported by an NIH Postdoctoral Fellowship. My PhD work at UC Berkeley and Princeton was concerned with gene expression during the Bacillus subtilis cell cycle. At Princeton we interacted with the Sueoka group in which Hiroshi Yoshikawa was engaged in the elegant demonstration that B. subtilis had a fixed origin of chromosome replication. This was shown by calculating recombination frequency ratios for a variety of markers, after transformation with DNA extracted from cultures growing in supplemented minimal medium (g = approx. 30 min) or stationary phase cultures. The result was a 2:1 gradient in transformant frequency ratio. This approach was based on the hypothesis, supported by their results, that replication was sequential from a fixed start point or points and proceeded at a fixed rate in the growing cultures, while all chromosomes were fully replicated in the stationary ones (Proc Natl Acad Sci USA 49: 559–566, 1963)… FiguresReferencesRelatedDetails The Life of Professor Robert Hugh PritchardMetrics History PDF download

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.

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.

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.

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.

ABSTRACT The can (previously yadF ) gene of Escherichia coli encodes a β-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 γ-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.

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.

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.

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.

LytB and GcpE, because they are codistributed with other pathway enzymes, have been predicted to catalyze unknown steps in the nonmevalonate pathway for isoprenoid biosynthesis. We constructed a conditional Escherichia coli lytB mutant and found that LytB is essential for survival and that depletion of LytB results in cell lysis, which is consistent with a role for this protein in isoprenoid biosynthesis. Alcohols which can be converted to pathway intermediates beyond the hypothesized LytB step(s) support limited growth of E. coli lytB mutants. An informatic analysis of protein structure suggested that GcpE is a globular protein of the TIM barrel class and that LytB is also a globular protein. Possible biochemical roles for LytB and GcpE are suggested.

Chaperonins participate in the facilitated folding of a variety of proteins in vivo. To see whether the same spectrum of target proteins can be productively folded by the double-ring prokaryotic chaperonin GroEL-GroES and its single-ring human mitochondrial homolog, Hsp60-Hsp10, we expressed the latter in an Escherichia coli strain engineered so that the groE operon is under strict regulatory control. We found that expression of Hsp60-Hsp10 restores viability to cells that no longer express GroEL-GroES, formally demonstrating that Hsp60-Hsp10 can carry out all essential in vivo functions of GroEL-GroES.

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.

Chaperone proteins help other proteins to fold. GroEL, the Escherichia coli form of the ubiquitous Cpn60 chaperonins, has a multimeric barrel-shaped structure with a central cavity, within which almost any protein can fold in vitro1. But what does GroE (GroEL plus its co-chaperone GroES) fold in the cell? Why is it needed for cell survival2? We report here the first definite identification of an essential, GroE-dependent E. coli protein, dihydropicolinate synthase (DapA), without which cell-wall synthesis fails.

ABSTRACT Deletion of ftsK results in the inhibition of cell division, but this inhibition can be reversed by a plasmid carrying only the first ∼17% of ftsK. The division block can be suppressed in most mutants by deletion of dacA, which codes for the d-alanine:d-alanine carboxypeptidase PBP5, or in all mutants by overexpression of ftsN. Overexpression of ftsK inhibits cell division and the formation of FtsZ rings. This division block is not due to the induction of either the SOS or the heat shock regulons.

The expression of dnaA is autoregulated, in that transcription of the gene increases when DnaA is inactivated (and initiation of replication prevented) and decreases when DnaA is supplied in excess. However, the inactivation of DnaA does not necessarily lead to increased DnaA production, as dnaA(Ts; temperature sensitive) strains which are integratively suppressed by derivatives of the plasmid R1 do not show temperature-induced derepression. Several possible explanations for this unanticipated behaviour were considered and ruled out. We suggest here that the completion of a critical step in initiation may prevent dnaA derepression: although DnaA would be required to complete this step at oriC, DnaA(Ts) would be sufficient at the R1 origin. Autoregulation of dnaA has been attributed to the binding of DnaA at a consensus binding site in the dnaA promoter region. We show here, using reporter systems, that this DnaA-binding site is not required for the autoregulatory response. We find, further, that replacement of the chromosomal dnaA gene with one containing a mutated binding site causes no demonstrable phenotypic change: cells with the mutant gene show no disadvantage in competition with dnaA(+) cells.

The replication frequency of plasmid R1 is controlled by an unstable antisense RNA, CopA, which, by binding to its complementary target, blocks translation of the replication rate‐limiting protein RepA. Since the degree of inhibition is directly correlated with the intracellular concentration of CopA, factors affecting CopA turnover can also alter plasmid copy number. We show here that PcnB (PAP I — a poly(A)polymerase of Escherichia coli ) is such a factor. Previous studies have shown that the copy number of ColE1 is decreased in pcnB mutant strains because the stability of the RNase E processed form of RNAI, the antisense RNA regulator of ColE1 replication, is increased. We find that, analogously, the twofold reduction in R1 copy number caused by a pcnB lesion is associated with a corresponding increase in the stability of the RNase E‐generated 3′ cleavage product of CopA. These results suggest that CopA decay is initiated by RNase E cleavage and that PcnB is involved in the subsequent rapid decay of the 3′ CopA stem‐loop segment. We also find that, as predicted, under conditions in which CopA synthesis is unaffected, pcnB mutation reduces RepA translation and increases CopA stability to the same extent.