The layout of a bacterial genome

Képès, François; Jester, Brian C.; Lepage, Thibaut; Rafiei, Nafiseh; Rosu, Bianca; Junier, Ivan

doi:10.1016/j.febslet.2012.03.051

Cited by 28 publications

(17 citation statements)

References 88 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Bacterial genomes are very flexible with respect to their gene repertoire but display highly conserved organizational features at the sequence level that deeply impact cell physiology [ 12 , 13 ]. An important organizational characteristic is the existence of a single origin of replication ( oriC ), where DNA duplication begins, proceeding unidirectionally through two equally sized replichores up to the chromosomal terminal region [ 12 ].…”

Section: Introductionmentioning

confidence: 99%

Genomic Location of the Major Ribosomal Protein Gene Locus Determines Vibrio cholerae Global Growth and Infectivity

et al. 2015

View full text Add to dashboard Cite

The effects on cell physiology of gene order within the bacterial chromosome are poorly understood. In silico approaches have shown that genes involved in transcription and translation processes, in particular ribosomal protein (RP) genes, localize near the replication origin (oriC) in fast-growing bacteria suggesting that such a positional bias is an evolutionarily conserved growth-optimization strategy. Such genomic localization could either provide a higher dosage of these genes during fast growth or facilitate the assembly of ribosomes and transcription foci by keeping physically close the many components of these macromolecular machines. To explore this, we used novel recombineering tools to create a set of Vibrio cholerae strains in which S10-spec-α (S10), a locus bearing half of the ribosomal protein genes, was systematically relocated to alternative genomic positions. We show that the relative distance of S10 to the origin of replication tightly correlated with a reduction of S10 dosage, mRNA abundance and growth rate within these otherwise isogenic strains. Furthermore, this was accompanied by a significant reduction in the host-invasion capacity in Drosophila melanogaster. Both phenotypes were rescued in strains bearing two S10 copies highly distal to oriC, demonstrating that replication-dependent gene dosage reduction is the main mechanism behind these alterations. Hence, S10 positioning connects genome structure to cell physiology in Vibrio cholerae. Our results show experimentally for the first time that genomic positioning of genes involved in the flux of genetic information conditions global growth control and hence bacterial physiology and potentially its evolution.

show abstract

Section: Introductionmentioning

confidence: 99%

Genomic Location of the Major Ribosomal Protein Gene Locus Determines Vibrio cholerae Global Growth and Infectivity

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Some basic microbial applications of Cas3 include studying chromosome biology (e.g. replichore asymmetry 41 ), virulence factors 42 , and the impact of the mobilome.…”

Section: Discussionmentioning

confidence: 99%

A minimal CRISPR-Cas3 system for genome engineering

Csörgő

León

Chau-Ly

et al. 2019

Preprint

View full text Add to dashboard Cite

15 CRISPR-Cas technologies have provided programmable gene editing tools that have 16 revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for 17 programmed genetic manipulation, however, they are limited for genome-scale interventions. 18Here, we utilized a Cas3-based system featuring a processive nuclease, expressed 19 endogenously or heterologously, for genome engineering purposes. Using an optimized and 20 minimal CRISPR-Cas3 system (Type I-C) programmed with a single crRNA, large deletions 21 ranging from 7 -424 kb were generated in Pseudomonas aeruginosa with high efficiency and 22speed. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated 23 deletion boundaries were variable in the absence of a homology-directed repair (HDR) template, 24and successfully and efficiently specified when present. The minimal Cas3 system is also 25 portable; large deletions were induced with high efficiency in Pseudomonas syringae and 26Escherichia coli using an "all-in-one" vector. Notably, Cas3 generated bi-directional deletions 27originating from the programmed cut site, which was exploited to iteratively reduce a P. 28aeruginosa genome by 837 kb (13.5%) using 10 distinct crRNAs. We also demonstrate the utility 29 of endogenous Cas3 systems (Type I-C and I-F) and develop an "anti-anti-CRISPR" strategy to 30 circumvent endogenous CRISPR-Cas inhibitor proteins. CRISPR-Cas3 could facilitate rapid 31 strain manipulation for synthetic biological and metabolic engineering purposes, genome 32 minimization, and the analysis of large regions of unknown function. 33 34 65Results 66 Implementation and optimization of genome editing with CRISPR-Cas3 67Type I-C CRISPR-Cas systems utilize just three cas genes to produce the crRNA-guided 68Cascade surveillance complex that can recruit Cas3: cas5, cas8, and cas7 ( Figure 1A), making it 69 a minimal system 24,25 . A previously constructed Pseudomonas aeruginosa PAO1 strain (PAO1 IC ) 70with inducible cas genes and crRNAs 26 was used here to conduct targeted genome manipulation. 71The expression of a crRNA targeting the genome caused a transient growth delay (Figure 1B), 72but survivors were isolated after extended growth. By targeting phzM, a gene required for 73 production of a blue-green pigment (pyocyanin), we observed yellow cultures ( Figure 1C) for 16 74 out of 36 (44%) biological replicates (18 recovered isolates from two independent phzM targeting 75

show abstract

“…Although we can synthesize a genetic program from scratch relatively easily [24], our ability to design it de novo is still limited [25]. One challenge lies in understanding the physical constraints of the genome such as organization, codon bias, and conformation [26]. Furthermore, we should not forget that any engineered cellular chassis will need safety valves to control osmotic pressure, transporters for discarding useless metabolic products, and the ability to cope with leftovers resulting from macromolecule degradation, all of which are indispensable functions for cellular maintenance and robust growth [27].…”

Section: Gene Persistence As a Metric Of Functional Essentialitymentioning

confidence: 99%

From essential to persistent genes: a functional approach to constructing synthetic life

et al. 2013

View full text Add to dashboard Cite

A central undertaking in synthetic biology (SB) is the quest for the ‘minimal genome’. However, ‘minimal sets’ of essential genes are strongly context-dependent and, in all prokaryotic genomes sequenced to date, not a single protein-coding gene is entirely conserved. Furthermore, a lack of consensus in the field as to what attributes make a gene truly essential adds another aspect of variation. Thus, a universal minimal genome remains elusive. Here, as an alternative to defining a minimal genome, we propose that the concept of gene persistence can be used to classify genes needed for robust long-term survival. Persistent genes, although not ubiquitous, are conserved in a majority of genomes, tend to be expressed at high levels, and are frequently located on the leading DNA strand. These criteria impose constraints on genome organization, and these are important considerations for engineering cells and for creating cellular life-like forms in SB.

show abstract

The layout of a bacterial genome

Cited by 28 publications

References 88 publications

Genomic Location of the Major Ribosomal Protein Gene Locus Determines Vibrio cholerae Global Growth and Infectivity

Genomic Location of the Major Ribosomal Protein Gene Locus Determines Vibrio cholerae Global Growth and Infectivity

A minimal CRISPR-Cas3 system for genome engineering

From essential to persistent genes: a functional approach to constructing synthetic life

Contact Info

Product

Resources

About