scholarly journals Multiple Chromosomes in Bacteria: The Yin and Yang of trp Gene Localization in Rhodobacter sphaeroides 2.4.1

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 525-538
Author(s):  
Chris Mackenzie ◽  
Adrian E Simmons ◽  
Samuel Kaplan
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Biosynthetic Pathway ◽  
Southern Blotting ◽  
Physical Map ◽  
Functional Integration ◽  
Bacterial Genome ◽  
Single Copy ◽  
Gene Localization ◽  
Yin And Yang ◽  
Trp Genes

Abstract The existence of multiple chromosomes in bacteria has been known for some time. Yet the extent of functional solidarity between different chromosomes remains unknown. To examine this question, we have surveyed the well-described genes of the tryptophan biosynthetic pathway in the multichromosomal photosynthetic eubacterium Rhodobacter sphaeroides 2.4.1. The genome of this organism was mutagenized using Tn5, and strains that were auxotrophic for tryptophan (Trp-) were isolated. Pulsed-field gel mapping indicated that Tn5 insertions in both the large (3 Mb CI) and the small (0.9 Mb CII) chromosomes created a Trp- phenotype. Sequencing the DNA flanking the sites of the Tn5 insertions indicated that the genes trpE-yibQ-trpGDC were at a locus on CI, while genes trpF-aroR-trpB were at locus on CII. Unexpectedly, trpA was not found downstream of trpB. Instead, it was placed on the CI physical map at a locus 1.23 Mb away from trpE-yibQ-trpGDC. To relate the context of the R. sphaeroides trp genes to those of other bacteria, the DNA regions surrounding the trp genes on both chromosomes were sequenced. Of particular significance was the finding that rpsA1, which encodes ribosomal protein S1, and cmkA, which encodes cytidylate monophosphate kinase, were on CII. These genes are considered essential for translation and chromosome replication, respectively. Southern blotting suggested that the trp genes and rpsA1 exist in single copy within the genome. To date, this topological organization of the trp “operon” is unique within a bacterial genome. When taken with the finding that CII encodes essential housekeeping functions, the overall impression is one of close regulatory and functional integration between these chromosomes.

scholarly journals Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution.

Genetics ◽  
1991 ◽  
Vol 127 (4) ◽  
pp. 681-698 ◽  
Author(s):  
A J Link ◽  
M V Olson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Hybridization ◽  
Physical Map ◽  
Single Copy ◽  
Restriction Endonucleases ◽  
Yeast Genome ◽  
Hybridization Probes ◽  
Copy Dna ◽  
Single Copy Dna

Abstract A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.

scholarly journals High prevalence and diversity of beta-lactamase-encoding bacteria in cryosoils and ancient permafrost

2021 ◽  
Author(s):  
Sofia Rigou ◽  
Eugene Christo-Foroux ◽  
Sebastien Santini ◽  
Artemiy Goncharov ◽  
Jens Strauss ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Bacterial Genome ◽  
Environmental Dna ◽  
Single Copy ◽  
The Arctic ◽  
Human Society ◽  
Beta Lactamase ◽  
Extended Spectrum Beta Lactamase ◽  
Arctic Regions ◽  
Global Threat

Background: Antimicrobial resistance is one of the major challenges affecting public health. It is mostly due to the continuous emergence of extended-spectrum beta-lactamase from various environments followed by their rapid dissemination and selection in clinical settings. The warming of Earth' s climate is the other global threat facing human society, in particular with the Arctic regions experiencing a twice faster warming than the global average and permafrost affected by widespread thawing. A potentially dreadful combination of these two threats would be the release and dispersion of harmful microbes that have remained confined to largely uninhabited Arctic regions, or are stored dormant in permafrost. Methods: Environmental DNA was isolated from 12 soil samples from various Arctic and subarctic pristine regions in Siberia (Yakutia and Kamchatka), including 9 permafrost samples collected at various depths. The large datasets obtained from high throughput sequencing was assembled in contigs and their protein-gene contents predicted. We used exhaustive similarity searches to perform taxonomical assignments of bacterial, archaeal, and eukaryotic organisms, as well as DNA viruses. In addition, we specifically identified beta-lactamase genes and their prevalence per bacterial genome estimated through the detection of two universal single copy genes. Findings: A total of 9.217 1011 bp were exploited, leading to a total of 525,313 contigs at least 5kb in size. The DNA content of the various samples was found to be highly variable, not strictly correlated with the depth or radio-carbon-based deposit age, and most likely linked to the global density of microbes trapped in the corresponding permafrost layers. Bacteria account for more than 90% of the contigs in most samples, followed by Eukaryotes and Archaea (always lower than 10%). Viruses represented less than 2% of all contigs in all samples. The taxonomic profiles of surface cryosoils and deep permafrost samples exhibited a high diversity, including between permafrost samples originating from various depths in the same borehole. In all samples, bacterial contigs carrying different beta-lactamases from class A to D were identified. Interpretation: No clear common taxonomic feature could be found shared by surface cryosoils or ancient permafrost layers. However, most samples (9/12) exhibited a high frequency of beta-lactamase genes, with an estimated average close to 1 copy/bacterial genome. In addition to the well-documented reactivation of infectious ancient pathogens (bacteria, viruses, protozoa), we show now that global warming could contribute to the emergence of new antibiotic resistances through the mobilization by contemporary bacteria of ancient DNA released from thawing permafrost.

scholarly journals In Vivo Analysis of Cobinamide Salvaging in Rhodobacter sphaeroides Strain 2.4.1

2009 ◽  
Vol 191 (12) ◽  
pp. 3842-3851 ◽  
Author(s):  
Michael J. Gray ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Rhodobacter Sphaeroides ◽  
High Performance ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Nutritional Analysis ◽  
In Vivo Analysis ◽  
Essential Enzyme ◽  
Necessary And Sufficient ◽  
Coenzyme B

ABSTRACT The genome of Rhodobacter sphaeroides encodes the components of two distinct pathways for salvaging cobinamide (Cbi), a precursor of adenosylcobalamin (AdoCbl, coenzyme B12). One pathway, conserved among bacteria, depends on a bifunctional kinase/guanylyltransferase (CobP) enzyme to convert adenosylcobinamide (AdoCbi) to AdoCbi-phosphate (AdoCbi-P), an intermediate in de novo AdoCbl biosynthesis. The other pathway, of archaeal origin, depends on an AdoCbi amidohydrolase (CbiZ) enzyme to generate adenosylcobyric acid (AdoCby), which is converted to AdoCbi-P by the AdoCbi-P synthetase (CobD) enzyme. Here we report that R. sphaeroides strain 2.4.1 synthesizes AdoCbl de novo and that it salvages Cbi using both of the predicted Cbi salvaging pathways. AdoCbl produced by R. sphaeroides was identified and quantified by high-performance liquid chromatography and bioassay. The deletion of cobB (encoding an essential enzyme of the de novo corrin ring biosynthetic pathway) resulted in a strain of R. sphaeroides that would not grow on acetate in the absence of exogenous corrinoids. The results from a nutritional analysis showed that the presence of either CbiZ or CobP was necessary and sufficient for Cbi salvaging, that CbiZ-dependent Cbi salvaging depended on the presence of CobD, and that CobP-dependent Cbi salvaging occurred in a cbiZ + strain. Possible reasons why R. sphaeroides maintains two distinct pathways for Cbi salvaging are discussed.

scholarly journals Use of the Riboflavin Synthase Gene (ribC) as a Model for Development of an Essential Gene Disruption and Complementation System for Haemophilus influenzae

2004 ◽  
Vol 70 (7) ◽  
pp. 4136-4143 ◽  
Author(s):  
Amna Saeed-Kothe ◽  
Wei Yang ◽  
Scott D. Mills
Keyword(s):  
Haemophilus Influenzae ◽  
Gene Disruption ◽  
Biosynthetic Pathway ◽  
Selectable Marker ◽  
Essential Gene ◽  
Tetracycline Resistance ◽  
Single Copy ◽  
Multiple Cloning Site ◽  
Chloramphenicol Resistance ◽  
Reliable Assessment

ABSTRACT We have developed a system for rapid and reliable assessment of gene essentiality in Haemophilus influenzae Rd strain KW20. We constructed two “suicide” complementation vectors (pASK5 and pASK6) containing 5′ and 3′ regions of the nonessential ompP1 gene flanking a multiple cloning site and a selectable marker (a chloramphenicol resistance gene or a tetracycline resistance cassette). Transformation of H. influenzae with the complementation constructs directs chromosomal integration of a gene of interest into the ompP1 locus, where the strong, constitutive ompP1 promoter drives its expression. This single-copy, chromosome-based complementation system is useful for confirming the essentiality of disrupted genes of interest. It allows genetic analysis in a background free of interference from any upstream or downstream genetic elements and enables conclusive assignment of essentiality. We validated this system by using the riboflavin synthase gene (ribC), a component of the riboflavin biosynthetic pathway. Our results confirmed the essentiality of ribC for survival of H. influenzae Rd strain KW20 and demonstrated that a complementing copy of ribC placed under control of the ompP1 promoter reverses the lethal phenotype of a strain with ribC deleted.

Correlating the Genetic and Physical Map of Barley Chromosome 3H Revealed Limitations of the FISH-Based Mapping of Nearby Single-Copy Probes Caused by the Dynamic Structure of Metaphase Chromosomes

2017 ◽  
Vol 152 (2) ◽  
pp. 90-96 ◽  
Author(s):  
Fernanda O. Bustamante ◽  
Lala Aliyeva-Schnorr ◽  
Jörg Fuchs ◽  
Sebastian Beier ◽  
Andreas Houben
Keyword(s):  
Dna Sequences ◽  
Physical Map ◽  
Dynamic Structure ◽  
Single Copy ◽  
Physical Distance ◽  
Genetic Distances ◽  
Genetic Maps ◽  
Metaphase Chromosomes ◽  
Physical Maps

Genetic maps are based on the recombination frequency of molecular markers which often show different positions in comparison to the corresponding physical maps. To decipher the position and order of DNA sequences genetically mapped to terminal and interstitial regions of barley (Hordeum vulgare) chromosome 3H, fluorescence in situ hybridization (FISH) on mitotic metaphase chromosomes was performed with 16 genomic single-copy probes derived from fingerprinted BAC contigs. Long genetic distances at subterminal regions translated into short physical distances, confirming that recombination events occur more often at distal regions of chromosome 3H. Nonoverlapping FISH signals were frequently obtained for probes with a physical distance of at least 30-60 kb. Only 8% of the analyzed chromosomes showed a symmetric order of FISH signals on both sister chromatids. Due to the dynamic packing of metaphase chromatin, the order of 2 adjacent single-copy signals along the chromosome arms outside the (peri)centromeric region can only reliably be determined if the cytological distance is approximately 3%, corresponding to 21.6 Mb.

scholarly journals Molecular characterization of the duplicated meristem identity genes HvAP1a and HvAP1b in barley

Genome ◽  
2005 ◽  
Vol 48 (5) ◽  
pp. 905-912 ◽  
Author(s):  
Liuling Yan ◽  
Jarislav von Zitzewitz ◽  
Jeffrey S Skinner ◽  
Patrick M Hayes ◽  
Jorge Dubcovsky
Keyword(s):  
Structural Changes ◽  
Growth Habit ◽  
Physical Map ◽  
Single Copy ◽  
Bacterial Artificial Chromosomes ◽  
Vernalization Gene ◽  
Artificial Chromosomes ◽  
Physical Maps ◽  
Meristem Identity

The vernalization gene VRN-1 has been identified as a MADS-box transcription factor orthologous to the meristem identity gene APETALA1 (AP1). A single copy of this gene was found in diploid wheat, but 2 copies were reported in barley. In this study, we present a detailed characterization of these 2 copies to understand their respective roles in the vernalization response. We identified 2 groups of barley bacterial artificial chromosomes (BACs), each containing 1 AP1 copy designated hereafter as HvAP1a and HvAP1b. A physical map of the VRN-H1 region showed that the HvAP1a BACs were part of the VRN-H1 region but that the HvAP1b BACs were not. Numerous structural changes were observed between the barley and wheat VRN-1 physical maps. In a population segregating for VRN-H1, the HvAP1a gene cosegregated with growth habit, suggesting that HvAP1a is the barley vernalization gene VRN-H1. The other copy, HvAP1b, was mapped on the centromeric region of chromosome 1H, the chromosome where vernalization gene VRN-H3 was previously mapped. We developed a mapping population segregating for VRN-H3 and showed that 2 molecular makers flanking HvAP1b locus were not linked to growth habit. The HvAP1b copy has a complete deletion of the first 2 exons, suggesting that it is a truncated pseudogene and not a candidate for VRN-H3. In summary, this study contributed a detailed physical map of the barley VRN-H1 region, showed several structural differences with the orthologous wheat region, and clarified the identity of the barley VRN-H1 gene.Key words: barley, vernalization, Vrn-1, physical map.

scholarly journals Chloroplast Genome Evolution in the Genus Avena

Genetics ◽  
1987 ◽  
Vol 116 (4) ◽  
pp. 613-621
Author(s):  
Koji Murai ◽  
Koichiro Tsunewaki
Keyword(s):  
Chloroplast Genome ◽  
Restriction Site ◽  
Physical Map ◽  
Fragment Size ◽  
Large Subunit ◽  
Single Copy ◽  
Rbcl Gene ◽  
Type I ◽  
Bisphosphate Carboxylase ◽  
Chloroplast Genomes

ABSTRACT The genus Avena contains five different chloroplast genomes, I-V. A physical map of chloroplast (ct) DNA of Avena sativa (type I chloroplast genome) was constructed using three restriction endonucleases, PstI, SalI and SmaI. This genome is ca. 135.5 kbp in size, and contains two inverted repeats of ca. 22.5 kbp each, separated by a large (ca. 79.0 kbp) and small (ca. 12.5 kbp) single copy region. The rbcL gene which codes for the large subunit of ribulose 1,5-bisphosphate carboxylase, was located in the map. Restriction fragment patterns of all five chloroplast genomes were compared, and among them five fragment size and five restriction site mutations were disclosed. Four site mutations were found in two or more chloroplast genomes, the other site and five fragment size mutations were specific to one or another of the chloroplast genomes. A dendrogram showing phylogenetic relationships among the five chloroplast genomes, based on the distribution of the common and specific mutations among them, indicates that chloroplast genome divergence characterized by three restriction site mutations occurred first between two diploid groups, each carrying A and C genome (nuclear), respectively, followed by further speciation in each group.

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