scholarly journals The nac (Nitrogen Assimilation Control) Gene from Escherichia coli

1998 ◽  
Vol 180 (5) ◽  
pp. 1166-1173 ◽  
Author(s):  
Wilson B. Muse ◽  
Robert A. Bender
Keyword(s):  
Escherichia Coli ◽  
Glutamate Dehydrogenase ◽  
Southern Blotting ◽  
Nitrogen Assimilation ◽  
Reverse Genetics ◽  
Predicted Amino Acid Sequence ◽  
Control Gene ◽  
Limited Growth ◽  
E Coli ◽  
Constitutive Mutant

ABSTRACT The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonella typhimurium by Southern blotting, using a probe from theKlebsiella aerogenes nac (nacK ) gene. The E. coli nac gene (nacE ) was isolated from a cosmid clone by complementation of anac mutation in K. aerogenes. nacE was fully functional in this complementation assay. DNA sequence analysis showed considerable divergence betweennacE and nacK , with a predicted amino acid sequence identity of only 79% and most of the divergence in the C-terminal half of the protein sequence. The total predicted size of NACE is 305 amino acids, the same as for NACK. A null mutation, nac-28, was generated by reverse genetics. Mutants bearing nac-28 have a variety of phenotypes related to nitrogen metabolism, including slower growth on cytosine, faster growth on arginine, and suppression of the failure of an Ntr-constitutive mutant to grow with serine as sole nitrogen source. In addition to a loss of nitrogen regulation of histidase formation,nac-28 mutants also showed a loss of a weak repression of glutamate dehydrogenase formation. This repression was unexpected because it is balanced by a NAC-independent activation of glutamate dehydrogenase formation during nitrogen-limited growth. Attempts to purify NACE by using methods established for NACK failed, and NACE appears to be degraded with a half-life at 30°C as short as 15 min during inhibition of protein synthesis.

scholarly journals The lytB Gene of Escherichia coli Is Essential and Specifies a Product Needed for Isoprenoid Biosynthesis

2001 ◽  
Vol 183 (24) ◽  
pp. 7403-7407 ◽  
Author(s):  
Sean McAteer ◽  
Andrew Coulson ◽  
Neil McLennan ◽  
Millicent Masters
Keyword(s):  
Escherichia Coli ◽  
Protein Structure ◽  
Cell Lysis ◽  
Isoprenoid Biosynthesis ◽  
Globular Protein ◽  
Limited Growth ◽  
E Coli ◽  
Nonmevalonate Pathway ◽  
Tim Barrel

ABSTRACT 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.

Growth of Selected Cross-Contaminating Bacterial Pathogens on Beef and Fish at 15 and 35°C+

1994 ◽  
Vol 57 (4) ◽  
pp. 337-340 ◽  
Author(s):  
AJIBOLA O. FAPOHUNDA ◽  
KENNETH W. MCMILLIN ◽  
DOUGLAS L. MARSHALL ◽  
W. M. WAITES
Keyword(s):  
Escherichia Coli ◽  
Clostridium Perfringens ◽  
Aeromonas Hydrophila ◽  
Growth Response ◽  
Cross Contamination ◽  
Limited Growth ◽  
E Coli ◽  
Fish Samples ◽  
Conditioning Temperature ◽  
The Tropics

Isolates of Escherichia coli and Clostridium perfringens from beef and Aeromonas hydrophila from fish were examined for their ability to survive and grow as cross-contaminates on nonnative tissues at simulated ambient (35°C) and aging/conditioning (15°C) temperatures of handling and retailing found in the tropics. Growth of all isolates over a 10-h period was greater (P < 0.05) on their native tissues at both temperatures. The aging/conditioning temperature effectively limited growth of E. coli and A. hydrophila to less than l-logl0 CFU/g and prevented growth of C. perfringens on beef and fish samples. All three isolates demonstrated characteristic mesophilic growth response on both tissues at 35°C during the 10-h retail period. The study suggests that two muscle food products could be jointly handled to efficiently use available storage/haulage capacity in tropical countries. Potential savings in space, labor and energy would be made if cross-contamination between the two products is minimized by available packaging and sanitizing technologies.

scholarly journals Complete DNA Sequence of a ColBM Plasmid from Avian Pathogenic Escherichia coli Suggests that It Evolved from Closely Related ColV Virulence Plasmids

2006 ◽  
Vol 188 (16) ◽  
pp. 5975-5983 ◽  
Author(s):  
Timothy J. Johnson ◽  
Sara J. Johnson ◽  
Lisa K. Nolan
Keyword(s):  
Escherichia Coli ◽  
Southern Blotting ◽  
Virulence Genes ◽  
Complete Sequence ◽  
Economic Losses ◽  
Avian Pathogenic Escherichia Coli ◽  
Prevalence Studies ◽  
E Coli ◽  
Virulence Plasmids ◽  
Pathogenic Escherichia Coli

ABSTRACT Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli causing colibacillosis in birds, is responsible for significant economic losses for the poultry industry. Recently, we reported that the APEC pathotype was characterized by possession of a set of genes contained within a 94-kb cluster linked to a ColV plasmid, pAPEC-O2-ColV. These included sitABCD, genes of the aerobactin operon, hlyF, iss, genes of the salmochelin operon, and the 5′ end of cvaB of the ColV operon. However, the results of gene prevalence studies performed among APEC isolates revealed that these traits were not always linked to ColV plasmids. Here, we present the complete sequence of a 174-kb plasmid, pAPEC-O1-ColBM, which contains a putative virulence cluster similar to that of pAPEC-O2-ColV. These two F-type plasmids share remarkable similarity, except that they encode the production of different colicins; pAPEC-O2-ColV contains an intact ColV operon, and pAPEC-O1-ColBM encodes the colicins B and M. Interestingly, remnants of the ColV operon exist in pAPEC-O1-ColBM, hinting that ColBM-type plasmids may have evolved from ColV plasmids. Among APEC isolates, the prevalence of ColBM sequences helps account for the previously observed differences in prevalence between genes of the “conserved” portion of the putative virulence cluster of pAPEC-O2-ColV and those genes within its “variable” portion. These results, in conjunction with Southern blotting and probing of representative ColBM-positive strains, indicate that this “conserved” cluster of putative virulence genes is primarily linked to F-type virulence plasmids among the APEC isolates studied.

scholarly journals Metabolic Context and Possible Physiological Themes of ς54-Dependent Genes in Escherichia coli

2001 ◽  
Vol 65 (3) ◽  
pp. 422-444 ◽  
Author(s):  
Larry Reitzer ◽  
Barbara L. Schneider
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Nitrogen Assimilation ◽  
Physiological Function ◽  
Sigma Factors ◽  
Energy Resources ◽  
Transcription Start ◽  
E Coli ◽  
Adverse Conditions ◽  
Single Organism

SUMMARY ς54 has several features that distinguish it from other sigma factors in Escherichia coli: it is not homologous to other ς subunits, ς54-dependent expression absolutely requires an activator, and the activator binding sites can be far from the transcription start site. A rationale for these properties has not been readily apparent, in part because of an inability to assign a common physiological function for ς54-dependent genes. Surveys of ς54-dependent genes from a variety of organisms suggest that the products of these genes are often involved in nitrogen assimilation; however, many are not. Such broad surveys inevitably remove the ς54-dependent genes from a potentially coherent metabolic context. To address this concern, we consider the function and metabolic context of ς54-dependent genes primarily from a single organism, Escherichia coli, in which a reasonably complete list of ς54-dependent genes has been identified by computer analysis combined with a DNA microarray analysis of nitrogen limitation-induced genes. E. coli appears to have approximately 30 ς54-dependent operons, and about half are involved in nitrogen assimilation and metabolism. A possible physiological relationship between ς54-dependent genes may be based on the fact that nitrogen assimilation consumes energy and intermediates of central metabolism. The products of the ς54-dependent genes that are not involved in nitrogen metabolism may prevent depletion of metabolites and energy resources in certain environments or partially neutralize adverse conditions. Such a relationship may limit the number of physiological themes of ς54-dependent genes within a single organism and may partially account for the unique features of ς54 and ς54-dependent gene expression.

scholarly journals Speed versus Efficiency in Microbial Growth and the Role of Parallel Pathways

2002 ◽  
Vol 184 (4) ◽  
pp. 1041-1045 ◽  
Author(s):  
Robert B. Helling
Keyword(s):  
Escherichia Coli ◽  
Glutamate Dehydrogenase ◽  
Microbial Growth ◽  
Atp Synthesis ◽  
Limited Growth ◽  
Atp Production ◽  
Parallel Pathways ◽  
Glutamate Synthesis

ABSTRACT Many microorganisms have sets of parallel pathways for ATP production in respiration and for ATP utilization in glutamate synthesis. The alternatives differ in efficiency of ATP production and utilization. The choice among these parallel pathways has been hypothesized to control the speed and efficiency of growth. Thus, the organism should be able to alleviate (or exaggerate) deficiency in one pathway by deleting another. I show here that in Escherichia coli the effect of lack of the glutamate-synthesizing enzyme glutamate dehydrogenase on glucose-limited growth is altered predictably by ndh, cyo, and cyd mutations affecting parallel pathways leading to ATP synthesis in respiration.

scholarly journals C4-dicarboxylate metabolons: Interaction of C4-dicarboxylate transporters of Escherichia coli with cytosolic enzymes and regulators

2021 ◽  
Author(s):  
Christopher Schubert ◽  
Gottfried Unden
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Assimilation ◽  
Concerted Action ◽  
Phosphotransferase System ◽  
Regulatory Pathways ◽  
E Coli ◽  
Fumarate Respiration ◽  
Genes Encoding ◽  
Two Hybrid

Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here the interaction of enzymes fumarase FumB and aspartase AspA with the C4-DC transporters DcuA and DcuB of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography (mSPINE). DcuB interacted strongly with FumB and AspA, and DcuA with AspA. The fumB-dcuB and the dcuA-aspA genes encoding the respective proteins are known for their colocalization on the genome and the production of co-transcripts. The data consistently suggest the formation of DcuB/FumB, DcuB/AspA and DcuA/AspA metabolons in fumarate respiration for the uptake of L-malate, or L-aspartate, conversion to fumarate and excretion of succinate after reduction. The DcuA/AspA metabolon catalyzes L-Asp uptake and fumarate excretion in concerted action also to provide ammonia for nitrogen assimilation. The aerobic C4-DC transporter DctA interacted with the regulator EIIAGlc of the E. coli glucose phosphotransferase system. It is suggested that EIIAGlc inhibits C4-DC uptake by DctA in the presence of the preferred substrate glucose.

scholarly journals Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

2003 ◽  
Vol 69 (7) ◽  
pp. 3791-3797 ◽  
Author(s):  
Nobuyuki Horinouchi ◽  
Jun Ogawa ◽  
Takafumi Sakai ◽  
Takako Kawano ◽  
Seiichiro Matsumoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Specific Activity ◽  
Cell Extract ◽  
Predicted Amino Acid Sequence ◽  
Enzymatic Reactions ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli

ABSTRACT The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2′-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.

scholarly journals Emergence and Comparative Genomics Analysis of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Carrying mcr-1 in Fennec Fox Imported from Sudan to China

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Chunyan Feng ◽  
Peipei Wen ◽  
Hao Xu ◽  
Xiaohui Chi ◽  
Shuang Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Southern Blotting ◽  
Wild Animals ◽  
Content Type ◽  
E Coli ◽  
Extended Spectrum ◽  
Broth Microdilution Method ◽  
High Prevalence ◽  
Esbl Producers

ABSTRACT The aim of this study was to investigate the occurrence and genomic characteristics of extended-spectrum-β-lactamase-producing Escherichia coli (ESBL-EC) in fennec fox imported from Sudan to China. We screened 88 fecal samples from fennec fox for ESBL-EC, using cefotaxime- and meropenem-supplemented selective medium. Antimicrobial susceptibility testing was performed by the agar dilution method except for colistin and tigecycline; for colistin and tigecycline, testing was conducted by the broth microdilution method. ESBL-EC bacteria were sequenced, and their genomes were characterized. Plasmid conjugation, S1 nuclease pulsed-field gel electrophoresis (PFGE), and Southern blotting were performed for a MCR-1-producing isolate. The genetic environment of mcr-1 and ESBL genes was also investigated. A total of 29 ESBL-EC bacteria were isolated from 88 fennec fox (32.9%), while no carbapenemase producers were found. The most prevalent genotypes were the blaCTX-M-55 and blaCTX-M-14 genes, followed by blaCTX-M-15 and blaCTX-M-64. We detected nine sequence types among 29 ESBL-EC. Furthermore, the mcr-1 gene was detected in isolate EcFF273. Conjugation analysis confirmed that the mcr-1 gene was transferable. S1 PFGE, Southern blotting, and whole-genome sequencing revealed that mcr-1 and blaCTX-M-64 were both located on a 65-kb IncI2 plasmid. This study reports for the first time the occurrence of ESBL-EC in fennec fox. The high prevalence of ESBL producers and the occurrence of MCR-1 producer in fennec fox imported into China from Sudan are unexpected. In addition, it clearly demonstrated that commensal E. coli strains can be reservoirs of blaCTX-M and mcr-1, potentially contributing to the dissemination and transfer of such genes to pathogenic bacteria among fennec fox. Our results support the implication of fennec fox as a biological vector for ESBL-producing members of the Enterobacteriaceae family. IMPORTANCE The extended-spectrum-β-lactamase (ESBL)-producing members of the Enterobacteriaceae family are a global concern for both animal and human health. There is some information indicating a high prevalence of ESBL producers in food animals. Moreover, there have been an increasing number of reports on ESBL-producing strains resistant to the last-resort antibiotic colistin with the global dissemination of the plasmid-mediated mcr-1 gene, which is believed to have originated in animal breeding. However, little is known regarding the burden of ESBL-producing Enterobacteriaceae on wild animals. No data were available on the prevalence of antimicrobial resistance (AMR) among wild animals imported into China. This is the first study to investigate the microbiological and genomics surveillance investigation of ESBL colonization among fennec fox (Vulpes zerda) imported from Sudan to China, and we uncovered a high prevalence of ESBL-EC. Furthermore, the underlying mechanism of colistin resistance in an isolate that harbored mcr-1 was also investigated. Results of characterization and analysis of 29 ESBL-producing E. coli may have important implications on our understanding of the transmission dynamics of these bacteria. We emphasize the importance of improved multisectoral surveillance for colistin-resistant E. coli in this region.

scholarly journals Identification of an Operon Required for Ferrichrome Iron Utilization in Vibrio cholerae

2000 ◽  
Vol 182 (8) ◽  
pp. 2350-2353 ◽  
Author(s):  
Marc B. Rogers ◽  
Jessica A. Sexton ◽  
G. Joel DeCastro ◽  
Stephen B. Calderwood
Keyword(s):  
Escherichia Coli ◽  
Vibrio Cholerae ◽  
Predicted Amino Acid Sequence ◽  
Gene Fusions ◽  
Gene Encoding ◽  
E Coli ◽  
Atp Binding Cassette Transporter ◽  
Genes Encoding ◽  
Iron Utilization

ABSTRACT Mutagenesis of Vibrio cholerae with TnphoA, followed by screening for fusions that were activated under low-iron conditions, led to the identification of seven independent fusion strains, each of which was deficient in the ability to utilize ferrichrome as a sole iron source for growth in a plate bioassay and had an insertion in genes encoding products homologous toEscherichia coli FhuA or FhuD. Expression of the gene fusions was independent of IrgB but regulated by Fur. We report here a map of the operon and the predicted amino acid sequence of FhuA, based on the nucleotide sequence. Unlike those of the E. coli fhuoperon, the V. cholerae ferrichrome utilization genes are located adjacent and opposite in orientation to a gene encoding an ATP-binding cassette transporter homolog, but this gene, if disrupted, does not affect the utilization of ferrichrome in vitro.

scholarly journals Nitrogen Regulation of the codBA (Cytosine Deaminase) Operon from Escherichia coli by the Nitrogen Assimilation Control Protein, NAC

2003 ◽  
Vol 185 (9) ◽  
pp. 2920-2926 ◽  
Author(s):  
Wilson B. Muse ◽  
Christopher J. Rosario ◽  
Robert A. Bender
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Assimilation ◽  
Cytosine Deaminase ◽  
In Vitro Transcription ◽  
Dependent Manner ◽  
E Coli ◽  
Different Response ◽  
Control Protein

ABSTRACT Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. β-Galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position −59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions −120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions −83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.

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