Project description:The flavodoxins are flavin mononucleotide-containing electron transferases. Flavodoxin I has been presumed to be the only flavodoxin of Escherichia coli, and its gene, fldA, is known to belong to the soxRS (superoxide response) oxidative stress regulon. An insertion mutation of fldA was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (fldA(+)) allele could carry it. A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene, fldB. Unlike the fldA mutant, an fldB insertion mutant is a viable prototroph in the presence or absence of oxygen. A high-copy-number fldB(+) plasmid did not complement the fldA mutation. Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I. An fldB-lacZ fusion was not induced by H(2)O(2) and is therefore not a member of the oxyR regulon. However, it displayed a soxS-dependent induction by paraquat (methyl viologen), and the fldB gene is preceded by two overlapping regions that resemble known soxS binding sites. The fldB insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a soxS-lacZ fusion. Therefore, fldB is a new member of the soxRS (superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds. However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.
Project description:Bacterial siderophores are a group of chemically diverse, virulence-associated secondary metabolites whose expression exerts metabolic costs. A combined bacterial genetic and metabolomic approach revealed differential metabolomic impacts associated with biosynthesis of different siderophore structural families. Despite myriad genetic differences, the metabolome of a cheater mutant lacking a single set of siderophore biosynthetic genes more closely approximate that of a non-pathogenic K12 strain than its isogenic, uropathogen parent strain. Siderophore types associated with greater metabolomic perturbations are less common among human isolates, suggesting that metabolic costs influence success in a human population. Although different siderophores share a common iron acquisition function, our analysis shows how a metabolomic approach can distinguish their relative metabolic impacts in E. coli.
Project description:We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).
Project description:Escherichia coli (E. coli) amine oxidase (ECAO) encoded by tynA gene has been one of the model enzymes to study the mechanism of oxidative deamination of amines to the corresponding aldehydes by amine oxidases. The biological roles of ECAO have been less addressed. Therefore we have constructed a gene deletion Escherichia coli K-12 strain, E. coli tynA-, and used the microarray technique to address its function by comparing the total RNA gene expression to the one of the wt. Our results suggest that tynA is a reserve gene for stringent environmental conditions and its gene product ECAO a growth advantage compared to other bacteria due to H2O2 production.
Project description:Wild-type Escherichia coli K12 strain W3110 was irradiated by 10 keV nitrogen ions. Specifically, irradiation was performed six times by N(+) ions, followed by the selection of lac constitutive mutants, and each time a stable S55 mutant was produced. By sequencing the whole genome, the fine map of S55 was completed. Compared with reference sequences, a total of eighteen single nucleotide polymorphisms (SNPs), two insertions and deletions (Indels), and nine structural variations (SVs) were found in the S55 genome. Among the 18 SNPs, 11 are transversional from A, T or C to G, accounting for 55.6% of point mutations. GCCA insertion occurs in the target gene lacI. Four SNPs, including three in rlpB and one in ygbN, are connected with cell envelope and transport. All nine structural variations of S55 are deletions and contain insertion sequence (IS) elements. Six deleted SVs contain disrupted ISs, nonfunctional pseudogenes, and one more 23 252 bp SV in the Rac prophage region. Overall, our results show that deletion bias observed in E. coli K12 genome evolution is generally related to the deletion of some nonfunctional regions. Furthermore, since ISs are unstable factors in a genome, the multi-ion irradiations that caused these deleted fragments in S55 turn out to be beneficial to genome stability, generating a wider mutational spectrum. Thus, it is possible that the mutation of these genes increases the ability of the E. coli genome to resist etch and damage caused by ion irradiation.
Project description:The bacterial SOS regulon is strongly induced in response to DNA damage from exogenous agents such as UV radiation and nalidixic acid. However, certain mutants with defects in DNA replication, recombination, or repair exhibit a partially constitutive SOS response. These mutants presumably suffer frequent replication fork failure, or perhaps they have difficulty rescuing forks that failed due to endogenous sources of DNA damage. In an effort to understand more clearly the endogenous sources of DNA damage and the nature of replication fork failure and rescue, we undertook a systematic screen for Escherichia coli mutants that constitutively express the SOS regulon. We identified mutant strains with transposon insertions in 42 genes that caused increased expression from a dinD1::lacZ reporter construct. Most of these also displayed significant increases in basal levels of RecA protein, confirming an effect on the SOS system. As expected, this collection includes genes, such as lexA, dam, rep, xerCD, recG, and polA, which have previously been shown to cause an SOS constitutive phenotype when inactivated. The collection also includes 28 genes or open reading frames that were not previously identified as SOS constitutive, including dcd, ftsE, ftsX, purF, tdcE, and tynA. Further study of these SOS constitutive mutants should be useful in understanding the multiple causes of endogenous DNA damage. This study also provides a quantitative comparison of the extent of SOS expression caused by inactivation of many different genes in a common genetic background.
Project description:The higher affinity of Cd(2+) for sulfur compounds than for nitrogen and oxygen led to the theoretical consideration that cadmium toxicity should result mainly from the binding of Cd(2+) to sulfide, thiol groups, and sulfur-rich complex compounds rather than from Cd(2+) replacement of transition-metal cations from nitrogen- or oxygen-rich biological compounds. This hypothesis was tested by using Escherichia coli for a global transcriptome analysis of cells synthesizing glutathione (GSH; wild type), gamma-glutamylcysteine (DeltagshB mutant), or neither of the two cellular thiols (DeltagshA mutant). The resulting data, some of which were validated by quantitative reverse transcription-PCR, were sorted using the KEGG (Kyoto Encyclopedia of Genes and Genomes) orthology system, which groups genes hierarchically with respect to the cellular functions of their respective products. The main difference among the three strains concerned tryptophan biosynthesis, which was up-regulated in wild-type cells upon cadmium shock and strongly up-regulated in DeltagshA cells but repressed in DeltagshB cells containing gamma-glutamylcysteine instead of GSH. Overall, however, all three E. coli strains responded to cadmium shock similarly, with the up-regulation of genes involved in protein, disulfide bond, and oxidative damage repair; cysteine and iron-sulfur cluster biosynthesis; the production of proteins containing sensitive iron-sulfur clusters; the storage of iron; and the detoxification of Cd(2+) by efflux. General energy conservation pathways and iron uptake were down-regulated. These findings indicated that the toxic action of Cd(2+) indeed results from the binding of the metal cation to sulfur, lending support to the hypothesis tested.
Project description:Environmental fluctuations lead to a rapid adjustment of the physiology of Escherichia coli, necessitating changes on every level of the underlying cellular and molecular network. Thus far, the majority of global analyses of E. coli stress responses have been limited to just one level, gene expression. Here, we incorporate the metabolite composition together with gene expression data to provide a more comprehensive insight on system level stress adjustments by describing detailed time-resolved E. coli response to five different perturbations (cold, heat, oxidative stress, lactose diauxie, and stationary phase). The metabolite response is more specific as compared with the general response observed on the transcript level and is reflected by much higher specificity during the early stress adaptation phase and when comparing the stationary phase response to other perturbations. Despite these differences, the response on both levels still follows the same dynamics and general strategy of energy conservation as reflected by rapid decrease of central carbon metabolism intermediates coinciding with downregulation of genes related to cell growth. Application of co-clustering and canonical correlation analysis on combined metabolite and transcript data identified a number of significant condition-dependent associations between metabolites and transcripts. The results confirm and extend existing models about co-regulation between gene expression and metabolites demonstrating the power of integrated systems oriented analysis.