scholarly journals A GC-Rich Prophage-Like Genomic Region of Mycoplasma bovirhinis HAZ141_2 Carries a Gene Cluster Encoding Resistance to Kanamycin and Neomycin

2020 ◽  
Author(s):  
Inna Lysnyansky ◽  
Ilya Borovok
Keyword(s):  
Gene Cluster ◽  
Field Isolate ◽  
Gc Content ◽  
Bioinformatic Analysis ◽  
Genomic Region ◽  
Transcription Analysis ◽  
E Coli ◽  
Functional Defect ◽  
Rapid Amplification ◽  
Carboxy Terminal

Recently, a complete genome of Mycoplasma bovirhinis HAZ141_2 has been published showing presence of 54-kB prophage-like region. Bioinformatic analysis revealed that this region has a more than 40-% GC content and a chimeric organization with three structural elements – a prophage continuous region, a restriction-modification cassette, and a highly transmittable aadE-sat4-aphA-3 gene cluster found in both Gram-positive and Gram-negative bacteria. It is known that aadE confers resistance to streptomycin, sat4 - resistance to streptothricin/nourseothricin, and aphA-3 - resistance to kanamycin and structurally related antibiotics. An aadE-like (aadE*) gene of strain HAZ141_2 encodes a 228 amino acid (aa) polypeptide whose carboxy-terminal domain (44-206) is almost identical to that of a functional 302 aa AadE (140-302). Transcription analysis of the aadE*-sat4-aphA-3 genes showed their co-transcription in M. bovirhinis HAZ141_2. Moreover, a common promoter for aadE*-sat4-aphA-3 was mapped upstream of the aadE* using 5′ rapid amplification of cDNA ends analysis. Determination of MICs to aminoglycosides and nourseothricin revealed that M. bovirhinis HAZ141_2 is highly resistant to kanamycin and neomycin (≥512 μg/ml). However, MICs to streptomycin (64 μg/ml) and nourseothricin (16-32 μg/ml) were similar to these identified in the prophageless M. bovirhinis type strain PG43 and Israeli field isolate 316981. We cloned the aadE*-sat4-aphA-3 genes into a low-copy number vector and transferred them into antibiotic-sensitive Escherichia coli cells. While the obtained E. coli transformants were highly resistant to kanamycin, neomycin as well as to nourseothricin (MICs ≥256 μg/ml), there were no changes in MICs to streptomycin suggesting a functional defect of the aadE*.

scholarly journals The genomic organization of the region containing the Drosophila melanogaster rpL7a (Surf-3) gene differs from those of the mammalian and avian Surfeit loci.

1995 ◽  
Vol 15 (5) ◽  
pp. 2367-2373 ◽  
Author(s):  
N Armes ◽  
M Fried
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Cluster ◽  
Ribosomal Protein Gene ◽  
Single Copy ◽  
Gene Organization ◽  
Genomic Region ◽  
Divergent Evolution ◽  
Close Proximity ◽  
Rapid Amplification ◽  
Rna Blots

The Surf-3 gene of the unusually tight mouse Surfeit locus gene cluster has been identified as the highly conserved ribosomal protein gene L7a (rpL7a). The topography and juxtaposition of the Surfeit locus genes are conserved for the 600 million years of divergent evolution between mammals and birds. This suggests cis interaction and/or coregulation of the genes and suggests that, within this locus, gene organization plays an important role in gene expression. The further evolutionary conservation of the organization of the Surfeit locus was investigated. A cDNA encoding the Drosophila melanogaster homolog of the Surf-3/rpL7a gene was cloned, was shown to be present as a single copy, and was expressed constitutively at high levels throughout development. Genomic cosmid clones encompassing the gene and its surrounding DNA were isolated. The gene was determined to have five introns, of which two were located in the 5' untranslated region of the gene. The remaining three introns had splice sites at positions equivalent to those found in the Surf-3/rpL7a mammalian homologs. S1 analysis and 5' rapid amplification of cDNA ends both confirmed the start of transcription to occur in a polypyrimidine tract in the absence of a TATA box in the promoter. The genomic region around the Surf-3/rpL7a gene was analyzed by low-stringency hybridization with murine Surfeit gene probes, by partial sequence analysis, and by hybridization of fragments to Northern (RNA) blots. No homologs of other members of the Surfeit gene cluster were detected in close proximity to the D. melanogaster Surf-3/rpL7a gene. However, a gene which was detected directly 3' to the Surf-3/rpL7a gene was shown to encode a homolog of a mammalian serine-pyruvate aminotransferase.

scholarly journals The Escherichia coli O111 and Salmonella enterica O35 Gene Clusters: Gene Clusters Encoding the Same Colitose-Containing O Antigen Are Highly Conserved

2000 ◽  
Vol 182 (18) ◽  
pp. 5256-5261 ◽  
Author(s):  
Lei Wang ◽  
Peter R. Reeves
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Salmonella Enterica ◽  
Gc Content ◽  
Gene Clusters ◽  
Gram Negative Bacteria ◽  
Normal Range ◽  
E Coli ◽  
O Antigen ◽  
Antigen Structure

ABSTRACT O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria. Escherichia coli andSalmonella enterica each have many forms of O antigen, but only three are common to the two species. It has been found that, in general, O-antigen genes are of low GC content. This deviation in GC content from that of typical S. enterica or E. coli genes (51%) is thought to indicate that the O-antigen DNA originated in species other than S. enterica or E. coli and was captured by lateral transfer. The O-antigen structure of Salmonella enterica O35 is identical to that of E. coli O111, commonly found in enteropathogenicE. coli strains. This O antigen, which has been shown to be a virulence factor in E. coli, contains colitose, a 3,6-dideoxyhexose found only rarely in theEnterobacteriaceae. Sequencing of the O35-antigen gene cluster of S. enterica serovar Adelaide revealed the same gene order and flanking genes as in E. coli O111. The divergence between corresponding genes of these two gene clusters at the nucleotide level ranges from 21.8 to 11.7%, within the normal range of divergence between S. enterica and E. coli. We conclude that the ancestor of E. coli andS. enterica had an O antigen identical to the O111 and O35 antigens, respectively, of these species and that the gene cluster encoding it has survived in both species.

Synthesis and Reactivity of Precolibactin 886

2019 ◽  
Author(s):  
Seth Herzon ◽  
Alan R. Healy ◽  
kevin wernke ◽  
Chung Sub Kim ◽  
Nicholas Lees ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Gene Cluster ◽  
Amino Alcohol ◽  
Biomimetic Synthesis ◽  
Cross Linking ◽  
E Coli ◽  
Hplc Purification ◽  
Structural Assignment ◽  
Biosynthetic Precursor ◽  
Double Oxidation

<div>The clb gene cluster encodes the biosynthesis of metabolites known as precolibactins and colibactins. The clb pathway is found in gut commensal E. coli, and clb metabolites are thought to initiate colorectal cancer via DNA cross-linking. Precolibactin 886 (1) is one of the most complex isolated clb metabolites; it contains a 15-atom macrocycle and an unusual 5-hydroxy-3-oxazoline ring. Here we report confirmation of the structural assignment via a biomimetic synthesis of precolibactin 886 (1) proceeding through the amino alcohol 9. Double oxidation of 9 afforded the unstable α-ketoimine 2 which underwent macrocyclization to precolibactin 886 (1) upon HPLC purification (3% from 9). Studies of the putative precolibactin 886 (1) biosynthetic precursor 2, the model α-ketoimine 25, and the α-dicarbonyl 26 revealed that these compounds are susceptible to nucleophilic rupture of the C36–C37 bond. Moreover, cleavage of 2 produces other known clb metabolites or biosynthetic intermediates. This unexpected reactivity explains the difficulties in isolating full clb metabolites and accounts for the structure of a recently identified colibactin–adenine adduct. The colibactin peptidase ClbP deacylates synthetic precolibactin 886 (1) to form a non-genotoxic pyridone, suggesting precolibactin 886 (1) lies off-path of the major biosynthetic route.</div>

scholarly journals Epidemiological and phylogenetic analysis reveals Flavobacteriaceae as potential ancestral source of tigecycline resistance gene tet(X)

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Rong Zhang ◽  
Ning Dong ◽  
Zhangqi Shen ◽  
Yu Zeng ◽  
Jiauyue Lu ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Resistance Gene ◽  
Gc Content ◽  
Zhejiang Province ◽  
Bacterial Strains ◽  
Gram Negative ◽  
E Coli ◽  
Transmission Mechanisms ◽  
Low Prevalence ◽  
Potential Origin

Abstract Emergence of tigecycline-resistance tet(X) gene orthologues rendered tigecycline ineffective as last-resort antibiotic. To understand the potential origin and transmission mechanisms of these genes, we survey the prevalence of tet(X) and its orthologues in 2997 clinical E. coli and K. pneumoniae isolates collected nationwide in China with results showing very low prevalence on these two types of strains, 0.32% and 0%, respectively. Further surveillance of tet(X) orthologues in 3692 different clinical Gram-negative bacterial strains collected during 1994–2019 in hospitals in Zhejiang province, China reveals 106 (2.7%) tet(X)-bearing strains with Flavobacteriaceae being the dominant (97/376, 25.8%) bacteria. In addition, tet(X)s are found to be predominantly located on the chromosomes of Flavobacteriaceae and share similar GC-content as Flavobacteriaceae. It also further evolves into different orthologues and transmits among different species. Data from this work suggest that Flavobacteriaceae could be the potential ancestral source of the tigecycline resistance gene tet(X).

scholarly journals Mutational Analysis of the ompA Promoter from Flavobacterium johnsoniae

2007 ◽  
Vol 189 (14) ◽  
pp. 5108-5118 ◽  
Author(s):  
Shicheng Chen ◽  
Michael Bagdasarian ◽  
Michael G. Kaufman ◽  
Adam K. Bates ◽  
Edward D. Walker
Keyword(s):  
Promoter Activity ◽  
Mutational Analysis ◽  
Core Promoter ◽  
Gc Content ◽  
Pronounced Effect ◽  
Promoter Elements ◽  
Spacer Length ◽  
Promoter Sequences ◽  
E Coli ◽  
Flavobacterium Johnsoniae

ABSTRACT Sequences that mediate the initiation of transcription in Flavobacterium species are not well known. The majority of identified Flavobacterium promoter elements show homology to those of other members of the phylum Bacteroidetes, but not of proteobacteria, and they function poorly in Escherichia coli. In order to analyze the Flavobacterium promoter structure systematically, we investigated the −33 consensus element, −7 consensus element, and spacer length of the Flavobacterium ompA promoter by measuring the effects of site-directed mutations on promoter activity. The nonconserved sequences in the spacer region and in regions close to the consensus motifs were randomized in order to determine their importance for promoter activity. Most of the base substitutions in these regions caused large decreases in promoter activity. The optimal −33/−7 motifs (TTTG/TANNTTTG) were identical to Bacteroides fragilis σABfr consensus −33/−7 promoter elements but lacked similarity to the E. coli σ70 promoter elements. The length of the spacer separating the −33 and −7 motifs of the ompA promoter also had a pronounced effect on promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, the GC content of the core promoter sequences had a pronounced effect on Flavobacterium promoter activity. This information was used to conduct a scan of the Flavobacterium johnsoniae and B. fragilis genomes for putative promoters, resulting in 188 hits in B. fragilis and 109 hits in F. johnsoniae.

scholarly journals Contribution of Membrane-Binding and Enzymatic Domains of Penicillin Binding Protein 5 to Maintenance of Uniform Cellular Morphology of Escherichia coli

2002 ◽  
Vol 184 (13) ◽  
pp. 3630-3639 ◽  
Author(s):  
David E. Nelson ◽  
Anindya S. Ghosh ◽  
Avery L. Paulson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Site Directed Mutagenesis ◽  
Outer Face ◽  
Penicillin Binding Protein ◽  
Membrane Anchor ◽  
E Coli ◽  
Membrane Bound ◽  
Carboxy Terminal ◽  
Membrane Anchoring ◽  
Β Sheet

ABSTRACT Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar dd-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other dd-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal β-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the dd-carboxypeptidase enzymatic core.

scholarly journals Identification of the Biosynthetic Gene Cluster for 3-Methylarginine, a Toxin Produced by Pseudomonas syringae pv. syringae 22d/93

2010 ◽  
Vol 76 (8) ◽  
pp. 2500-2508 ◽  
Author(s):  
S. D. Braun ◽  
J. Hofmann ◽  
A. Wensing ◽  
M. S. Ullrich ◽  
H. Weingart ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Biosynthetic Gene Cluster ◽  
Pentanoic Acid ◽  
Promising Candidate ◽  
Biosynthesis Gene Cluster ◽  
E Coli ◽  
Highly Active ◽  
Putative Aminotransferase

ABSTRACT The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (Km , 7 mM; k cat, 85 min−1). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.

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 A Bacterial Microcompartment Is Used for Choline Fermentation byEscherichia coli536

2018 ◽  
Vol 200 (10) ◽  
Author(s):  
Taylor I. Herring ◽  
Tiffany N. Harris ◽  
Chiranjit Chowdhury ◽  
Sujit Kumar Mohanty ◽  
Thomas A. Bobik
Keyword(s):  
Electron Microscopy ◽  
Heart Disease ◽  
Gene Cluster ◽  
Transcriptional Regulator ◽  
Human Gut ◽  
Content Type ◽  
E Coli ◽  
Bacterial Microcompartment ◽  
New Type ◽  
Insight Into

ABSTRACTBacterial choline degradation in the human gut has been associated with cancer and heart disease. In addition, recent studies found that a bacterial microcompartment is involved in choline utilization byProteusandDesulfovibriospecies. However, many aspects of this process have not been fully defined. Here, we investigate choline degradation by the uropathogenEscherichia coli536. Growth studies indicatedE. coli536 degrades choline primarily by fermentation. Electron microscopy indicated that a bacterial microcompartment was used for this process. Bioinformatic analyses suggested that the choline utilization (cut) gene cluster ofE. coli536 includes two operons, one containing three genes and a main operon of 13 genes. Regulatory studies indicate that thecutXgene encodes a positive transcriptional regulator required for induction of the maincutoperon in response to choline supplementation. Each of the 16 genes in thecutcluster was individually deleted, and phenotypes were examined. ThecutX,cutY,cutF,cutO,cutC,cutD,cutU, andcutVgenes were required for choline degradation, but the remaining genes of thecutcluster were not essential under the conditions used. The reasons for these varied phenotypes are discussed.IMPORTANCEHere, we investigate choline degradation inE. coli536. These studies provide a basis for understanding a new type of bacterial microcompartment and may provide deeper insight into the link between choline degradation in the human gut and cancer and heart disease. These are also the first studies of choline degradation inE. coli536, an organism for which sophisticated genetic analysis methods are available. In addition, thecutgene cluster ofE. coli536 is located in pathogenicity island II (PAI-II536) and hence might contribute to pathogenesis.

scholarly journals Biosynthesis of Chlorinated Lactylates in Sphaerospermopsis sp. LEGE 00249

2020 ◽  
Author(s):  
Kathleen Abt ◽  
Raquel Castelo-Branco ◽  
Pedro Leao
Keyword(s):  
Gene Cluster ◽  
Building Blocks ◽  
Bioinformatic Analysis ◽  
Biosynthetic Gene Cluster ◽  
Pathway Engineering ◽  
Dodecanoic Acid ◽  
Biosynthetic Gene ◽  
Precursor Feeding ◽  
Alkyl Chains ◽  
Important Group

Lactylates are an important group of molecules in the food and cosmetic industries. A series of natural halogenated 1-lactylates – chlorosphaerolactyaltes (<b>1</b>-<b>4</b>) – were recently reported from <i>Sphaerospermopsis</i> sp. LEGE 00249. Here, we identify the <i>cly</i> biosynthetic gene cluster, containing all the necessary functionalities to generate and release the natural lactylates. Using a combination of stable isotope-labeled precursor feeding and bioinformatic analysis, we propose that dodecanoic acid and pyruvate are the key building blocks in the biosynthesis of <b>1</b>-<b>4</b>. We additionally report minor analogues of these molecules<b> </b>with varying alkyl chains. The discovery of the <i>cly</i> gene cluster paves the way to accessing industrially-relevant lactylates through pathway engineering.

Sign in / Sign up

Export Citation Format

Share Document