The nlpA lipoprotein gene is located near the selC tRNA gene on the Escherichia coli chromosome.
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Bacterial Proteins↗
Biomedical subjects
K E Rudd
Publications and source records attributed to K E Rudd.
Physical map location of the argFGH operon of Escherichia coli.
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Arginine↗
Physical mapping of repetitive extragenic palindromic sequences in Escherichia coli and phylogenetic distribution among Escherichia coli strains and other enteric bacteria.
Repetitive extragenic palindromic (REP) sequences are highly conserved inverted repeat sequences originally discovered in Escherichia coli and Salmonella typhimurium. We have physically mapped these sequences in the E. coli genome by using Southern hybridization of an ordered phage bank of E. coli (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) with generic REP probes derived from the REP consensus sequence. The set of REP probe-hybridizing clones was correlated with a set of clones expected to contain REP sequences on the basis of computer searches. We also show that a generic REP probe can be used in Southern hybridization to analyze genomic DNA digested with restriction enzymes to determine genetic relatedness among natural isolates of E. coli. A search for these sequences in other members of the family Enterobacteriaceae shows a consistent correlation between both the number of occurrences and the hybridization strength and genealogical relationship.
Base Sequence↗
Mutations causing aminotriazole resistance and temperature sensitivity reside in gyrB, which encodes the B subunit of DNA gyrase.
Certain mutations in gyrA and gyrB, the genes encoding the two subunits of DNA gyrase, are known to influence expression of the his operon (K. E. Rudd and R. Menzel, Proc. Natl. Acad. Sci. USA 84:517-521, 1987). Such mutations lead to a decrease in tRNA(His) levels and consequently to an attenuator-dependent increase in his operon expression. This effect presumably is due to the dependence of the hisR promoter (hisR encodes tRNA(His) on supercoiling for maximal activity. We used a relaxed (Rel-) strain of Escherichia coli to isolate gyrB mutants by selecting for resistance to the histidine antimetabolite 3-amino-1,2,4-triazole and then screening for temperature-sensitive growth on rich medium. Rel- mutants, which generally have lower basal levels of ppGpp (a positive regulator of his operon transcription), are more sensitive than wild-type E. coli to aminotriazole. The chance of isolating spoT mutants, which can be selected with a similar procedure, was decreased by selecting in the presence of a multicopy plasmid that carries the wild-type spoT gene. Under these conditions, gyrB mutants were isolated preferentially. This scheme selects for loss of function of DNA gyrase, rather than for its alteration due to resistance to specific gyrase inhibitors, and thus a greater variety of gyrase mutations might be obtainable.
Amitrole↗
Mapping sequenced E.coli genes by computer: software, strategies and examples.
Methods are presented for organizing and integrating DNA sequence data, restriction maps, and genetic maps for the same organism but from a variety of sources (databases, publications, personal communications). Proper software tools are essential for successful organization of such diverse data into an ordered, cohesive body of information, and a suite of novel software to support this endeavor is described. Though these tools automate much of the task, a variety of strategies is needed to cope with recalcitrant cases. We describe such strategies and illustrate their application with numerous examples. These strategies have allowed us to order, analyze, and display over one megabase of E. coli DNA sequence information. The integration task often exposes inconsistencies in the available data, perhaps caused by strain polymorphisms or human oversight, necessitating the application of sound biological judgment. The examples illustrate both the level of expertise required of the database curator and the knowledge gained as apparent inconsistencies are resolved. The software and mapping methods are applicable to the study of any genome for which a high resolution restriction map is available. They were developed to support a weakly coordinated sequencing effort involving many laboratories, but would also be useful for highly orchestrated sequencing projects.
Base Sequence↗
Improved algorithms for searching restriction maps.
We present algorithms for searching a DNA restriction enzyme map for a region that best matches a shorter 'probe' map. Our algorithms utilize a new model of map alignments, and extensive experiments prove our model superior to earlier approaches for certain applications. Let M be the number of map sites and P be the number of probe sites. Our first algorithm, which optimizes only over a restricted class of alignments, requires O(MP log P) worst-case time and O(M + P) space. Our second algorithm, which optimizes over all alignments, runs in O(MP3) time and O(M + P2) space, under reasonable assumptions about the distribution of restriction enzyme cleavage sites. Combining the algorithms gives a map-searching method that optimizes over all alignments in O(MP log P) time in practice. The algorithms' effectiveness is illustrated by searches involving a genomic restriction map of Escherichia coli.
Algorithms↗
deaD, a new Escherichia coli gene encoding a presumed ATP-dependent RNA helicase, can suppress a mutation in rpsB, the gene encoding ribosomal protein S2.
We have cloned and sequenced a new gene from Escherichia coli which encodes a 64-kDa protein. The inferred amino acid sequence of the protein shows remarkable similarity to eIF4A, a murine translation initiation factor that has an ATP-dependent RNA helicase activity and is a founding member of the D-E-A-D family of proteins (characterized by a conserved Asp-Glu-Ala-Asp motif). Our new gene, called deaD, was cloned as a gene dosage-dependent suppressor of temperature-sensitive mutations in rpsB, the gene encoding ribosomal protein S2. We suggest that the DeaD protein plays a hitherto unknown role in translation in E. coli.
Amino Acid Sequence↗
Localization of the Escherichia coli amtA gene to 95.8 minutes.
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Chromosomes, Bacterial↗
Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map.
We use the extensive published information describing the genome of Escherichia coli and new restriction map alignment software to align DNA sequence, genetic, and physical maps. Restriction map alignment software is used which considers restriction maps as strings analogous to DNA or protein sequences except that two values, enzyme name and DNA base address, are associated with each position on the string. The resulting alignments reveal a nearly linear relationship between the physical and genetic maps of the E. coli chromosome. Physical map comparisons with the 1976, 1980, and 1983 genetic maps demonstrate a better fit with the more recent maps. The results of these alignments are genomic kilobase coordinates, orientation and rank of the alignment that best fits the genetic data. A statistical measure based on extreme value distribution is applied to the alignments. Additional computer analyses allow us to estimate the accuracy of the published E. coli genomic restriction map, simulate rearrangements of the bacterial chromosome, and search for repetitive DNA. The procedures we used are general enough to be applicable to other genome mapping projects.
Amino Acid Sequence↗
An algorithm for searching restriction maps.
This paper presents an algorithm that searches a DNA restriction enzyme map for regions that approximately match a shorter 'probe' map. Both the map and the probe consist of a sequence of address-enzyme pairs denoting restriction sites, and the algorithm penalizes a potential match for undetected or missing sites and for discrepancies in the distance between adjacent sites. The algorithm was designed specifically for comparing relatively short DNA sequences with a long restriction map, a problem that will become increasing common as large physical maps are generated. The algorithm has been used to extract information from a restriction map of the entire Escherichia coli genome.
Algorithms↗
Characterization of the spoT gene of Escherichia coli.
The Escherichia coli spoT gene encodes a guanosine-3',5'-bispyrophosphate (ppGpp) 3'-pyrophosphohydrolase known to be responsible for cellular (ppGpp) degradation. The DNA sequence of the spoT region is presented. The spoT gene is deduced to be 702 codons long, with a probable UUG initiation codon, and a deduced mass of 79,342 daltons. Two spoT mutations (spoT202 and spoT203) have been localized to an open reading frame by complementation of function as well as by genetic marker rescue. The ability to overexpress the spoT gene is limited, but enough ppGppase activity can be made to reverse ppGpp accumulation during the stringent response to amino acid starvation. The spoT gene is located within a larger spo operon and is flanked by two smaller genes. The first gene in the operon encodes omega, a protein that copurifies with RNA polymerase (Gentry, D. R., and Burgess, R. R. (1986) Gene (Amst.) 48, 33-40). The spoT gene is the second gene in the operon; it is followed by a third open reading frame deduced to encode a protein with a mass of 25,343 daltons. Insertion of a kanamycin resistance gene in the omega gene reduces spoT gene expression as judged by lowered ppGppase activity, relA-dependent reduction of growth rate, and abolition of spoT mutant complementation activity. These effects are reversed by expression of the spoT gene, but not the omega gene, in trans. Transcription of the spo operon occurs in a clockwise direction on the E. coli chromosome and is probably directed by at least two promoters.
Alleles↗
Basal ppGpp level adjustment shown by new spoT mutants affect steady state growth rates and rrnA ribosomal promoter regulation in Escherichia coli.
This work describes an approach towards analyzing the regulatory effects of variation of guanosine 3',5'-bispyrophosphate (ppGpp) basal levels in Escherichia coli during steady state growth. A series of strains was derived by mutating the spoT gene (which encodes the major cellular ppGppase) so as to obtain systematic increments in ppGpp basal levels. These strains differ genetically at the spoT locus and, in some cases, also at the relA locus because of the severity of spoT mutant alleles. Measurements of ppGpp revealed a ten-fold range of basal levels during growth on minimal medium. The empirical relationship between ppGpp concentration and growth rate is a simple linear inverse correlation. Tandem rrnA ribosomal RNA promoters, present on a multicopy plasmid, are shown to be differentially regulated over this range of basal levels. The upstream P1 promoter activity shows an inverse exponential relation to ppGpp concentration whereas the downstream P2 promoter is only weakly affected. We conclude that there are systematic regulatory consequences associated with small changes in ppGpp basal levels during steady state growth that probably are part of a continuum with more dramatic effects observed during the stringent response to amino acid deprivation.
Cell Division↗
his operons of Escherichia coli and Salmonella typhimurium are regulated by DNA supercoiling.
The hisW mutations of Salmonella typhimurium are highly pleiotropic mutations that elevate his operon expression, reduce ilv gene expression, alter stable RNA metabolism, and confer defective growth properties. The hisW mutations are highly linked to a naladixic acid-resistant gyrA mutation of S. typhimurium. Multicopy recombinant plasmids containing the Escherichia coli gyrA gene are able to complement both the growth defects and the elevated his operon expression associated with the hisW mutations. We conclude that hisW mutations are alleles of the gyrA gene. The hisU1820 mutant of S. typhimurium exhibits many of the same phenotypes as hisW mutants. Several lines of evidence, including high transduction linkage to recF, suggest that hisU1820 is an allele of gyrB. Finally, well-characterized gyrA and gyrB alleles of E. coli are also his regulatory mutations. We propose that a wild-type degree of chromosomal superhelicity is required for maximal production of histidyl-tRNA and normal his operon regulation.
Alcohol Oxidoreductases↗
Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression.
The spoT gene of Salmonella typhimurium has been identified. Mutations in spoT map between gltC and pyrE at 79 min. The spoT1 mutant has elevated levels of guanosine 5'-diphosphate-3'-diphosphate (ppGpp) during steady-state growth and exhibits a slower than normal decay of ppGpp after reversal of amino acid starvation. The spoT1 mutation elevates his operon expression but is distinct from known his regulatory mutations. Elevated his operon expression in spoT mutants causes resistance to the histidine analogs, 1,2,4-triazole-3-alanine and 3-amino-1,2,4-triazole. These properties of spoT mutants allowed us to identify and characterize additional spoT mutants. Approximately 40% of these mutants are temperature sensitive for growth on minimal medium, suggesting that the spoT function is essential or that excessive accumulation of ppGpp is lethal.
Alcohol Oxidoreductases↗
RNA polymerase of Myxococcus xanthus: purification and selective transcription in vitro with bacteriophage templates.
DNA-dependent RNA polymerase from vegetative cells of the gram-negative, fruiting bacterium Myxococcus xanthus was purified more than 300-fold by a modified Burgess procedure (Lowe et al., Biochemistry 18:1344-1352, 1979), using Polymin P precipitation, 40 to 65% saturated ammonium sulfate fractional precipitation, double-stranded DNA cellulose chromatography, A5m gel filtration chromatography, and single-stranded DNA agarose chromatography. The last step separated the RNA polymerase into a core fraction and an enriched holoenzyme fraction. The core enzyme showed a subunit structure similar to that of the Escherichia coli polymerase, as follows: beta' and beta (145,000 and 140,000 daltons, respectively) and alpha (38,000 daltons). A comparison of the core enzyme and the holoenzyme implicated two polypeptides as possible sigma subunits. These polypeptides were closely related, as indicated by peptide analysis. M. xanthus RNA polymerase was capable of transcribing DNAs from E. coli phages T7, T4, and lambda, Bacillus subtilis phage phi 29, and M. xanthus phages Mx1, Mx4, and Mx8. Transcription of T7 and phi 29 DNAs was stimulated by KCl, whereas transcription of Mx1, Mx4, and Mx8 DNAs was inhibited by KCl. Magnesium ion dependence, rifampin and heparin sensitivities, and spermidine stimulation of M. xanthus RNA polymerase activity were similar to those found with E. coli RNA polymerase. The pH optimum of M. xanthus RNA polymerase activity was more basic than that of E. coli polymerase. M. xanthus RNA polymerase was capable of selective transcription in vitro when DNAs from phages T7 delta 111, phi 29, and Mx1 were used. The molecular weights of the resulting phage RNA transcripts made by M. xanthus RNA polymerase (as determined by agarose-acrylamide slab gel electrophoresis) were the same as the molecular weights of the transcripts synthesized by E. coli RNA polymerase. No discrete transcripts were detected as the in vitro RNA products of M. xanthus phage Mx4 and Mx8 DNA transcription. Southern transcript synthesized by M. xanthus RNA polymerase. Three transcripts (transcripts A, B, and C; molecular weights, 2.55 X 10(6), 1.95 X 10(6), and 1.56 X 10(6), respectively) were identified as in vitro RNA products of M. xanthus phage Mx1 DNA transcription when either E. coli or M. xanthus RNA polymerase was used. A Southern blot hybridization analysis indicated that the E. coli RNA polymerase and the M. xanthus RNA polymerase transcribe common SalI restriction fragments of Mx1 DNA.
DNA, Viral↗
Novel intergenic repeats of Escherichia coli K-12.
An online catalog of intergenic DNA repeat sequence elements is added to the EcoGene Escherichia coli K-12 genome sequence annotation and analysis project (bmb.med.miami.edu/EcoGene). A library of noncoding (intergenic) DNA sequences depleted of known intergenic repeat classes was searched for DNA sequence similarities to identify novel DNA repeat sequence classes.
Base Sequence↗