scholarly journals Transcriptional Organization and Regulation of a Polycistronic Cold Shock Operon in Sinorhizobium meliloti RM1021 Encoding Homologs of the Escherichia coli Major Cold Shock Gene cspA and Ribosomal Protein GenerpsU

2000 ◽  
Vol 66 (1) ◽  
pp. 392-400 ◽  
Author(s):  
Kevin P. O'Connell ◽  
Michael F. Thomashow
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Protein ◽  
Sinorhizobium Meliloti ◽  
Cold Shock ◽  
Open Reading Frames ◽  
Mrna Accumulation ◽  
Transcription Analysis ◽  
E Coli ◽  
Shock Gene ◽  
Shock Locus

ABSTRACT A homolog of the major eubacterial cold shock gene cspAwas identified in Sinorhizobium meliloti RM1021 byluxAB reporter transposon mutagenesis. Here we further characterize the organization and regulation of this locus. DNA sequence analysis indicated that the locus includes three open reading frames (ORFs) encoding homologs corresponding to CspA, a novel 10.6-kDa polypeptide designated ORF2, and a homolog of the Escherichia coli ribosomal protein S21. Transcription analysis indicated that this locus produced two different-sized cspA-hybridizing transcripts upon cold shock, a 400-nucleotide (nt) RNA encodingcspA alone and a 1,000-nt transcript encodingcspA-ORF2-rpsU. The sizes of the transcripts agreed with the location of the transcription start site determined by primer extension and the locations of two putative transcriptional terminators. The promoter of the cspA-ORF2-rpsU locus had −10 and −35 elements similar to the E. coliς70 consensus promoter and, like the cspAlocus of E. coli, included an AT-rich region upstream of the −35 hexamer. The promoter of the S. meliloti cspAlocus was found to impart cold shock-induced mRNA accumulation. In addition, the 5′-untranslated region (5′ UTR) was found to increase the fold induction of cspA transcripts after cold shock and depressed the level of luxAB mRNA prior to cold shock, another feature similar to cspA regulation in E. coli. No “cold box” was identified upstream of the S. meliloti cspA gene, however, and there was no other obvious sequence identity between the S. meliloti 5′ UTR and that of E. coli. DNA hybridization analysis indicated that outside the cspA-ORF2-rpsU cold shock locus there are several additional cspA-like genes and a secondrpsU homolog.

scholarly journals A Stress-Induced Bias in the Reading of the Genetic Code in Escherichia coli

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Adi Oron-Gottesman ◽  
Martina Sauert ◽  
Isabella Moll ◽  
Hanna Engelberg-Kulka
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Ribosomal Protein ◽  
Genetic Code ◽  
Open Reading Frames ◽  
Translation Machinery ◽  
E Coli ◽  
Universal Characteristic ◽  
Living Organisms ◽  
Reading Frames

ABSTRACT Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a stress-induced translation machinery (STM), composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we further characterized the STM system, finding that MazF cleaves only ACA sites located in the open reading frames of processed mRNAs, while out-of-frame ACAs are resistant. This in-frame ACA cleavage of MazF seems to depend on MazF binding to an extracellular-death-factor (EDF)-like element in ribosomal protein bS1 (bacterial S1), apparently causing MazF to be part of STM ribosomes. Furthermore, due to the in-frame MazF cleavage of ACAs under stress, a bias occurs in the reading of the genetic code causing the amino acid threonine to be encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA. IMPORTANCE The genetic code is a universal characteristic of all living organisms. It defines the set of rules by which nucleotide triplets specify which amino acid will be incorporated into a protein. Our results represent the first existing report on a stress-induced bias in the reading of the genetic code. We found that in E. coli , under stress, the amino acid threonine is encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA. This is because under stress, MazF generates a stress-induced translation machinery (STM) in which MazF cleaves in-frame ACA sites of the processed mRNAs.

scholarly journals Identification of Cold Shock Gene Loci inSinorhizobium meliloti by Using a luxABReporter Transposon

2000 ◽  
Vol 66 (1) ◽  
pp. 401-405 ◽  
Author(s):  
Kevin P. O'Connell ◽  
Ann M. Gustafson ◽  
M. Deane Lehmann ◽  
Michael F. Thomashow
Keyword(s):  
Escherichia Coli ◽  
Sinorhizobium Meliloti ◽  
Cold Shock ◽  
Insertion Site ◽  
Temperature Shift ◽  
Rrna Genes ◽  
23S Rrna ◽  
Shock Gene ◽  
23S Rrna Genes ◽  
Transposon Insertions

ABSTRACT Using a luxAB reporter transposon, seven mutants ofSinorhizobium meliloti were identified as containing insertions in four cold shock loci. LuxAB activity was strongly induced (25- to 160-fold) after a temperature shift from 30 to 15°C. The transposon and flanking host DNA from each mutant was cloned, and the nucleic acid sequence of the insertion site was determined. Unexpectedly, five of the seven luxAB mutants contained transposon insertions in the 16S and 23S rRNA genes of two of the threerrn operons of S. meliloti. Directed insertion of luxAB genes into each of the three rrnoperons revealed that all three operons were similarly affected by cold shock. Two other insertions were found to be located downstream of a homolog of the major Escherichia coli cold shock gene,cspA. Although the cold shock loci were highly induced in response to a shift to low temperature, none of the insertions resulted in a statistically significant decrease in growth rate at 15°C.

scholarly journals Identification of a novel NADH-specific aldo-keto reductase using sequence and structural homologies

2006 ◽  
Vol 400 (1) ◽  
pp. 105-114 ◽  
Author(s):  
Eric Di Luccio ◽  
Robert A. Elling ◽  
David K. Wilson
Keyword(s):  
Escherichia Coli ◽  
Sinorhizobium Meliloti ◽  
Thermotoga Maritima ◽  
Xylose Reductase ◽  
Sulfolobus Solfataricus ◽  
Sequence Comparisons ◽  
E Coli ◽  
Fluorescence Measurements ◽  
Dual Specificity ◽  
Substrate Dependence

The AKRs (aldo-keto reductases) are a superfamily of enzymes which mainly rely on NADPH to reversibly reduce various carbonyl-containing compounds to the corresponding alcohols. A small number have been found with dual NADPH/NADH specificity, usually preferring NADPH, but none are exclusive for NADH. Crystal structures of the dual-specificity enzyme xylose reductase (AKR2B5) indicate that NAD+ is bound via a key interaction with a glutamate that is able to change conformations to accommodate the 2′-phosphate of NADP+. Sequence comparisons suggest that analogous glutamate or aspartate residues may function in other AKRs to allow NADH utilization. Based on this, nine putative enzymes with potential NADH specificity were identified and seven genes were successfully expressed and purified from Drosophila melanogaster, Escherichia coli, Schizosaccharomyces pombe, Sulfolobus solfataricus, Sinorhizobium meliloti and Thermotoga maritima. Each was assayed for co-substrate dependence with conventional AKR substrates. Three were exclusive for NADPH (AKR2E3, AKR3F2 and AKR3F3), two were dual-specific (AKR3C2 and AKR3F1) and one was specific for NADH (AKR11B2), the first such activity in an AKR. Fluorescence measurements of the seventh protein indicated that it bound both NADPH and NADH but had no activity. Mutation of the aspartate into an alanine residue or a more mobile glutamate in the NADH-specific E. coli protein converted it into an enzyme with dual specificity. These results show that the presence of this carboxylate is an indication of NADH dependence. This should allow improved prediction of co-substrate specificity and provide a basis for engineering enzymes with altered co-substrate utilization for this class of enzymes.

scholarly journals Escherichia coli O157:H7 Strain EDL933 Harbors Multiple Functional Prophage-Associated Genes Necessary for the Utilization of 5-N-Acetyl-9-O-Acetyl Neuraminic Acid as a Growth Substrate

2016 ◽  
Vol 82 (19) ◽  
pp. 5940-5950 ◽  
Author(s):  
Nadja Saile ◽  
Anja Voigt ◽  
Sarah Kessler ◽  
Timo Stressler ◽  
Jochen Klumpp ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Large Intestine ◽  
Substrate Utilization ◽  
Neuraminic Acid ◽  
Open Reading Frames ◽  
Dose Effect ◽  
Content Type ◽  
E Coli ◽  
P Proteins ◽  
Homologous Orfs

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) O157:H7 strain EDL933 harbors multiple prophage-associated open reading frames (ORFs) in its genome which are highly homologous to the chromosomalnanSgene. The latter is part of thenanCMSoperon, which is present in mostE. colistrains and encodes an esterase which is responsible for the monodeacetylation of 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2). Whereas one prophage-borne ORF (z1466) has been characterized in previous studies, the functions of the othernanS-homologous ORFs are unknown. In the current study, thenanS-homologous ORFs of EDL933 were initially studiedin silico. Due to their homology to the chromosomalnanSgene and their location in prophage genomes, we designated themnanS-p and numbered the differentnanS-p alleles consecutively from 1 to 10. The two allelesnanS-p2 andnanS-p4 were selected for production of recombinant proteins, their enzymatic activities were investigated, and differences in their temperature optima were found. Furthermore, a function of these enzymes in substrate utilization could be demonstrated using anE. coliC600ΔnanSmutant in a growth medium with Neu5,9Ac2as the carbon source and supplementation with the different recombinant NanS-p proteins. Moreover, generation of sequential deletions of allnanS-p alleles in strain EDL933 and subsequent growth experiments demonstrated a gene dose effect on the utilization of Neu5,9Ac2. Since Neu5,9Ac2is an important component of human and animal gut mucus and since the nutrient availability in the large intestine is limited, we hypothesize that the presence of multiple Neu5,9Ac2esterases provides them a nutrient supply under certain conditions in the large intestine, even if particular prophages are lost.IMPORTANCEIn this study, a group of homologous prophage-bornenanS-p alleles and two of the corresponding enzymes of enterohemorrhagicE. coli(EHEC) O157:H7 strain EDL933 that may be important to provide alternative genes for substrate utilization were characterized.

scholarly journals afa-8 Gene Cluster Is Carried by a Pathogenicity Island Inserted into the tRNAPhe of Human and Bovine Pathogenic Escherichia coli Isolates

2001 ◽  
Vol 69 (2) ◽  
pp. 937-948 ◽  
Author(s):  
Lila Lalioui ◽  
Chantal Le Bouguénec
Keyword(s):  
Escherichia Coli ◽  
Direct Repeat ◽  
Pathogenicity Island ◽  
Genomic Region ◽  
Open Reading Frames ◽  
Blood Isolate ◽  
Distinctive Features ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
K 12

ABSTRACT We recently described a new afimbrial adhesin, AfaE-VIII, produced by animal strains associated with diarrhea and septicemia and by human isolates associated with extraintestinal infections. Here, we report that the afa-8 operon, encoding AfaE-VIII adhesin, from the human blood isolate Escherichia coli AL862 is carried by a 61-kb genomic region with characteristics typical of a pathogenicity island (PAI), including a size larger than 10 kb, the presence of an integrase-encoding gene, the insertion into a tRNA locus (pheR), and the presence of a small direct repeat at each extremity. Moreover, the G+C content of the afa-8 operon (46.4%) is lower than that of the E. coli K-12/MG1655 chromosome (50.8%). Within this PAI, designated PAI IAL862, we identified open reading frames able to code for products similar to proteins involved in sugar utilization. Four probes spanning these sequences hybridized with 74.3% of pathogenicafa-8-positive E. coli strains isolated from humans and animals, 25% of human pathogenic afa-8-negativeE. coli strains, and only 8% of fecal strains (P = 0.05), indicating that these sequences are strongly associated with the afa-8 operon and that this genetic association may define a PAI widely distributed among human and animal afa-8-positive strains. One of the distinctive features of this study is that E. coli AL862 also carries another afa-8-containing PAI (PAI IIAL862), which appeared to be similar in size and genetic organization to PAI IAL862 and was inserted into the pheV gene. We investigated the insertion sites of afa-8-containing PAI in human and bovine pathogenic E. coli strains and found that this PAI preferentially inserted into the pheV gene.

scholarly journals Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

1999 ◽  
Vol 181 (13) ◽  
pp. 3981-3993 ◽  
Author(s):  
Sylvia A. Denome ◽  
Pamela K. Elf ◽  
Thomas A. Henderson ◽  
David E. Nelson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Binding Proteins ◽  
Kanamycin Resistance ◽  
Open Reading Frames ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Peptidoglycan Biosynthesis ◽  
Kanamycin Resistance Cassette ◽  
Genes Encoding

ABSTRACT The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology ofEscherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two ressites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via λ phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bpres site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, thedacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the β-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.

scholarly journals Rhizobium (Sinorhizobium)meliloti phn Genes: Characterization and Identification of Their Protein Products

1999 ◽  
Vol 181 (2) ◽  
pp. 389-395 ◽  
Author(s):  
George F. Parker ◽  
Timothy P. Higgins ◽  
Timothy Hawkes ◽  
Robert L. Robson
Keyword(s):  
Escherichia Coli ◽  
Sinorhizobium Meliloti ◽  
Insertional Mutagenesis ◽  
Biochemical Characterization ◽  
Polyclonal Antibodies ◽  
E Coli ◽  
New Information ◽  
Phosphorus Sources ◽  
Carbon Phosphorus Lyase

ABSTRACT In Escherichia coli, the phn operon encodes proteins responsible for the uptake and breakdown of phosphonates. The C-P (carbon-phosphorus) lyase enzyme encoded by this operon which catalyzes the cleavage of C-P bonds in phosphonates has been recalcitrant to biochemical characterization. To advance the understanding of this enzyme, we have cloned DNA fromRhizobium (Sinorhizobium) melilotithat contains homologues of the E. coli phnG, -H, -I, -J, and -Kgenes. We demonstrated by insertional mutagenesis that the operon from which this DNA is derived encodes the R. meliloti C-P lyase. Furthermore, the phenotype of this phn mutant shows that the C-P lyase has a broad substrate specificity and that the organism has another enzyme that degrades aminoethylphosphonate. A comparison of the R. meliloti and E. coli phngenes and their predicted products gave new information about C-P lyase. The putative R. meliloti PhnG, PhnH, and PhnK proteins were overexpressed and used to make polyclonal antibodies. Proteins of the correct molecular weight that react with these antibodies are expressed by R. meliloti grown with phosphonates as sole phosphorus sources. This is the first in vivo demonstration of the existence of these hitherto hypothetical Phn proteins.

scholarly journals Genome Sequence of a T4-like Phage, Escherichia Phage vB_EcoM-Sa45lw, Infecting Shiga Toxin-Producing Escherichia coli Strains

2019 ◽  
Vol 8 (32) ◽  
Author(s):  
Yen-Te Liao ◽  
Yujie Zhang ◽  
Alexandra Salvador ◽  
Vivian C. H. Wu
Keyword(s):  
Escherichia Coli ◽  
Surface Water ◽  
Genome Size ◽  
Shiga Toxin ◽  
Open Reading Frames ◽  
Content Type ◽  
E Coli ◽  
Double Stranded Dna ◽  
A Genome ◽  
Reading Frames

Escherichia phage vB_EcoM-Sa45lw, a new member of the T4-like phages, was isolated from surface water in a produce-growing area. The phage, containing double-stranded DNA with a genome size of 167,353 bp and 282 predicted open reading frames (ORFs), is able to infect generic Escherichia coli and Shiga toxin-producing E. coli O45 and O157 strains.

scholarly journals Identification of Candidates for a Subunit Vaccine against Extraintestinal Pathogenic Escherichia coli

2006 ◽  
Vol 75 (4) ◽  
pp. 1916-1925 ◽  
Author(s):  
Lionel Durant ◽  
Arnaud Metais ◽  
Coralie Soulama-Mouze ◽  
Jean-Marie Genevard ◽  
Xavier Nassif ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Subunit Vaccine ◽  
Iron Acquisition ◽  
Passive Immunization ◽  
Open Reading Frames ◽  
Lethal Challenge ◽  
E Coli ◽  
Protective Antigens ◽  
K 12 ◽  
Genomic Regions

ABSTRACT Extraintestinal pathogenic Escherichia coli (ExPEC) strains cause a large spectrum of infections. The majority of ExPEC strains are closely related to the B2 or the D phylogenetic group. The aim of our study was to develop a protein-based vaccine against these ExPEC strains. To this end, we identified ExPEC-specific genomic regions, using a comparative genome analysis, between the nonpathogenic E. coli strain K-12 MG1655 and ExPEC strains C5 (meningitis isolate) and CFT073 (urinary tract infection isolate). The analysis of these genomic regions allowed the selection of 40 open reading frames, which are conserved among B2/D clinical isolates and encode proteins with putative outer membrane localization. These genes were cloned, and recombinant proteins were purified and assessed as vaccine candidates. After immunization of BALB/c mice, five proteins induced a significant protective immunity against a lethal challenge with a clinical E. coli strain of the B2 group. In passive immunization assays, antigen-specific antibodies afforded protection to naive mice against a lethal challenge. Three of these antigens were related to iron acquisition metabolism, an important virulence factor of the ExPEC, and two corresponded to new, uncharacterized proteins. Due to the large number of genetic differences that exists between commensal and pathogenic strains of E. coli, our results demonstrate that it is possible to identify targets that elicit protective immune responses specific to those strains. The five protective antigens could constitute the basis for a preventive subunit vaccine against diseases caused by ExPEC strains.

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