Periodic Variation of Mutation Rates in Bacterial Genomes Associated with Replication Timing

ABSTRACT The causes and consequences of spatiotemporal variation in mutation rates remain to be explored in nearly all organisms. Here we examine relationships between local mutation rates and replication timing in three bacterial species whose genomes have multiple chromosomes: Vibrio fischeri, Vibrio cholerae, and Burkholderia cenocepacia. Following five mutation accumulation experiments with these bacteria conducted in the near absence of natural selection, the genomes of clones from each lineage were sequenced and analyzed to identify variation in mutation rates and spectra. In lineages lacking mismatch repair, base substitution mutation rates vary in a mirrored wave-like pattern on opposing replichores of the large chromosomes of V. fischeri and V. cholerae, where concurrently replicated regions experience similar base substitution mutation rates. The base substitution mutation rates on the small chromosome are less variable in both species but occur at similar rates to those in the concurrently replicated regions of the large chromosome. Neither nucleotide composition nor frequency of nucleotide motifs differed among regions experiencing high and low base substitution rates, which along with the inferred ~800-kb wave period suggests that the source of the periodicity is not sequence specific but rather a systematic process related to the cell cycle. These results support the notion that base substitution mutation rates are likely to vary systematically across many bacterial genomes, which exposes certain genes to elevated deleterious mutational load. IMPORTANCE That mutation rates vary within bacterial genomes is well known, but the detailed study of these biases has been made possible only recently with contemporary sequencing methods. We applied these methods to understand how bacterial genomes with multiple chromosomes, like those of Vibrio and Burkholderia, might experience heterogeneous mutation rates because of their unusual replication and the greater genetic diversity found on smaller chromosomes. This study captured thousands of mutations and revealed wave-like rate variation that is synchronized with replication timing and not explained by sequence context. The scale of this rate variation over hundreds of kilobases of DNA strongly suggests that a temporally regulated cellular process may generate wave-like variation in mutation risk. These findings add to our understanding of how mutation risk is distributed across bacterial and likely also eukaryotic genomes, owing to their highly conserved replication and repair machinery.

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Periodic variation of mutation rates in bacterial genomes associated with replication timing

10.1101/195818 ◽

2017 ◽

Author(s):

Marcus M. Dillon ◽

Way Sung ◽

Michael Lynch ◽

Vaughn S. Cooper

Keyword(s):

Vibrio Fischeri ◽

Bacterial Species ◽

Replication Timing ◽

Small Chromosome ◽

Burkholderia Cenocepacia ◽

Mutation Rates ◽

Base Substitution ◽

Large Chromosome ◽

Bacterial Genomes ◽

Substitution Mutation

ABSTRACTThe causes and consequences of spatiotemporal variation in mutation rates remains to be explored in nearly all organisms. Here we examine relationships between local mutation rates and replication timing in three bacterial species whose genomes have multiple chromosomes:Vibrio fischeri, Vibrio cholerae, andBurkholderia cenocepacia. Following five evolution experiments with these bacteria conducted in the near-absence of natural selection, the genomes of clones from each lineage were sequenced and analyzed to identify variation in mutation rates and spectra. In lineages lacking mismatch repair, base-substitution mutation rates vary in a mirrored wave-like pattern on opposing replichores of the large chromosome ofV. fischeriandV. cholerae, where concurrently replicated regions experience similar base-substitution mutation rates. The base-substitution mutation rates on the small chromosome are less variable in both species but occur at similar rates as the concurrently replicated regions of the large chromosome. Neither nucleotide composition nor frequency of nucleotide motifs differed among regions experiencing high and low base-substitution rates, which along with the inferred ~800 Kb wave period suggests that the source of the periodicity is not sequence-specific but rather a systematic process related to the cell cycle. These results support the notion that base-substitution mutation rates are likely to vary systematically across many bacterial genomes, which exposes certain genes to elevated deleterious mutational load.

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Modulating Pathogenesis with Mobile-CRISPRi

Journal of Bacteriology ◽

10.1128/jb.00304-19 ◽

2019 ◽

Vol 201 (22) ◽

Cited By ~ 7

Author(s):

Jiuxin Qu ◽

Neha K. Prasad ◽

Michelle A. Yu ◽

Shuyan Chen ◽

Amy Lyden ◽

...

Keyword(s):

Gene Expression ◽

Pathogenic Bacteria ◽

Bacterial Species ◽

Effector Proteins ◽

Infection Model ◽

Secretion Systems ◽

Bacterial Genomes ◽

Gene Encoding ◽

Content Type ◽

Animal Infection

ABSTRACT Conditionally essential (CE) genes are required by pathogenic bacteria to establish and maintain infections. CE genes encode virulence factors, such as secretion systems and effector proteins, as well as biosynthetic enzymes that produce metabolites not found in the host environment. Due to their outsized importance in pathogenesis, CE gene products are attractive targets for the next generation of antimicrobials. However, the precise manipulation of CE gene expression in the context of infection is technically challenging, limiting our ability to understand the roles of CE genes in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of CRISPR interference-based gene knockdown tools that are transferred by conjugation and stably integrate into bacterial genomes that we call Mobile-CRISPRi. Here, we show the efficacy of Mobile-CRISPRi in controlling CE gene expression in an animal infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid nonspecific toxicity. As a proof of principle, we demonstrate that knock down of a CE gene encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of CE genes across many bacterial species and pathogenesis models. IMPORTANCE Antibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically, these genetic drivers have been difficult to manipulate precisely, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study conditionally essential virulence genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.

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MITEAba12, a Novel Mobile Miniature Inverted-Repeat Transposable Element Identified inAcinetobacter baumanniiATCC 17978 and Its Prevalence across theMoraxellaceaeFamily

mSphere ◽

10.1128/mspheredirect.00028-19 ◽

2019 ◽

Vol 4 (1) ◽

Author(s):

Felise G. Adams ◽

Melissa H. Brown

Keyword(s):

Inverted Repeat ◽

Bacterial Species ◽

Consensus Sequence ◽

Target Site ◽

The Novel ◽

Bacterial Genomes ◽

Content Type ◽

Promoter Sequences ◽

Moraxella Osloensis ◽

Genome Level

ABSTRACTInsertion sequences (IS) are fundamental mediators of genome plasticity with the potential to generate phenotypic variation with significant evolutionary outcomes. Here, a recently active miniature inverted-repeat transposon element (MITE) was identified in a derivative ofAcinetobacter baumanniiATCC 17978 after being subjected to stress conditions. Transposition of the novel element led to the disruption of thehnsgene, resulting in a characteristic hypermotile phenotype. DNA identity shared between the terminal inverted repeats of this MITE and coresident ISAba12elements, together with the generation of 9-bp target site duplications, provides strong evidence that ISAba12elements were responsible for mobilization of the MITE (designated MITEAba12) within this strain. A wider genome-level survey identified MITEAba12in 30 additionalAcinetobactergenomes at various frequencies and oneMoraxella osloensisgenome. Ninety MITEAba12copies could be identified, of which 40% had target site duplications, indicating recent transposition events. Elements ranged between 111 and 114 bp; 90% were 113 bp in length. Using the MITEAba12consensus sequence, putative outward-facingEscherichia coliσ70 promoter sequences in both orientations were identified. The identification of transcripts originating from the promoter in one direction supports the proposal that the element can influence neighboring host gene transcription. The location of MITEAba12varied significantly between and within genomes, preferentially integrating into AT-rich regions. Additionally, a copy of MITEAba12was identified in a novel 8.5-kb composite transposon, Tn6645, in theM. osloensisCCUG 350 chromosome. Overall, this study shows that MITEAba12is the most abundant nonautonomous element currently found inAcinetobacter.IMPORTANCEOne of the most important weapons in the armory ofAcinetobacteris its impressive genetic plasticity, facilitating rapid genetic mutations and rearrangements as well as integration of foreign determinants carried by mobile genetic elements. Of these, IS are considered one of the key forces shaping bacterial genomes and ultimately evolution. We report the identification of a novel nonautonomous IS-derived element present in multiple bacterial species from theMoraxellaceaefamily and its recent translocation into thehnslocus in theA. baumanniiATCC 17978 genome. The latter finding adds new knowledge to only a limited number of documented examples of MITEs in the literature and underscores the plastic nature of thehnslocus inA. baumannii. MITEAba12, and its predicted parent(s), may be a source of substantial adaptive evolution within environmental and clinically relevant bacterial pathogens and, thus, have broad implications for niche-specific adaptation.

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Bactopia: a Flexible Pipeline for Complete Analysis of Bacterial Genomes

mSystems ◽

10.1128/msystems.00190-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Robert A. Petit ◽

Timothy D. Read

Keyword(s):

Open Source ◽

Genome Analysis ◽

Bacterial Species ◽

Bacterial Genome ◽

Complete Analysis ◽

Comparative Genomic ◽

Data Sets ◽

Bacterial Genomes ◽

Data Set ◽

Content Type

ABSTRACT Sequencing of bacterial genomes using Illumina technology has become such a standard procedure that often data are generated faster than can be conveniently analyzed. We created a new series of pipelines called Bactopia, built using Nextflow workflow software, to provide efficient comparative genomic analyses for bacterial species or genera. Bactopia consists of a data set setup step (Bactopia Data Sets [BaDs]), which creates a series of customizable data sets for the species of interest, the Bactopia Analysis Pipeline (BaAP), which performs quality control, genome assembly, and several other functions based on the available data sets and outputs the processed data to a structured directory format, and a series of Bactopia Tools (BaTs) that perform specific postprocessing on some or all of the processed data. BaTs include pan-genome analysis, computing average nucleotide identity between samples, extracting and profiling the 16S genes, and taxonomic classification using highly conserved genes. It is expected that the number of BaTs will increase to fill specific applications in the future. As a demonstration, we performed an analysis of 1,664 public Lactobacillus genomes, focusing on Lactobacillus crispatus, a species that is a common part of the human vaginal microbiome. Bactopia is an open source system that can scale from projects as small as one bacterial genome to ones including thousands of genomes and that allows for great flexibility in choosing comparison data sets and options for downstream analysis. Bactopia code can be accessed at https://www.github.com/bactopia/bactopia. IMPORTANCE It is now relatively easy to obtain a high-quality draft genome sequence of a bacterium, but bioinformatic analysis requires organization and optimization of multiple open source software tools. We present Bactopia, a pipeline for bacterial genome analysis, as an option for processing bacterial genome data. Bactopia also automates downloading of data from multiple public sources and species-specific customization. Because the pipeline is written in the Nextflow language, analyses can be scaled from individual genomes on a local computer to thousands of genomes using cloud resources. As a usage example, we processed 1,664 Lactobacillus genomes from public sources and used comparative analysis workflows (Bactopia Tools) to identify and analyze members of the L. crispatus species.

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Loss of O-Linked Protein Glycosylation in Burkholderia cenocepacia Impairs Biofilm Formation and Siderophore Activity and Alters Transcriptional Regulators

mSphere ◽

10.1128/msphere.00660-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 3

Author(s):

Cameron C. Oppy ◽

Leila Jebeli ◽

Miku Kuba ◽

Clare V. Oates ◽

Richard Strugnell ◽

...

Keyword(s):

Biofilm Formation ◽

Protein Modification ◽

Bacterial Species ◽

Protein Glycosylation ◽

Burkholderia Cenocepacia ◽

Luciferase Reporter ◽

Content Type ◽

Bacterial Physiology ◽

Transcriptional Alterations ◽

Siderophore Activity

ABSTRACT O-linked protein glycosylation is a conserved feature of the Burkholderia genus. The addition of the trisaccharide β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc to membrane exported proteins in Burkholderia cenocepacia is required for bacterial fitness and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. Using proteomics, luciferase reporter assays, and DNA cross-linking, we demonstrate the loss of glycosylation leads to changes in transcriptional regulation of multiple proteins, including the repression of the master quorum CepR/I. These proteomic and transcriptional alterations lead to the abolition of biofilm formation and defects in siderophore activity. Surprisingly, the abundance of most of the known glycosylated proteins did not significantly change in the glycosylation-defective mutants, except for BCAL1086 and BCAL2974, which were found in reduced amounts, suggesting they could be degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, biofilm formation, or siderophore activity. Together, our results show that loss of glycosylation in B. cenocepacia results in a global cell reprogramming via alteration of the transcriptional regulatory systems, which cannot be explained by the abundance changes in known B. cenocepacia glycoproteins. IMPORTANCE Protein glycosylation is increasingly recognized as a common posttranslational protein modification in bacterial species. Despite this commonality, our understanding of the role of most glycosylation systems in bacterial physiology and pathogenesis is incomplete. In this work, we investigated the effect of the disruption of O-linked glycosylation in the opportunistic pathogen Burkholderia cenocepacia using a combination of proteomic, molecular, and phenotypic assays. We find that in contrast to recent findings on the N-linked glycosylation systems of Campylobacter jejuni, O-linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that loss of glycosylation in B. cenocepacia strains leads to global proteome and transcriptional changes, including the repression of the quorum-sensing regulator cepR (BCAM1868) gene. These alterations lead to dramatic phenotypic changes in glycosylation-null strains, which are paralleled by both global proteomic and transcriptional alterations, which do not appear to directly result from the loss of glycosylation per se. This research unravels the pleiotropic effects of O-linked glycosylation in B. cenocepacia, demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome.

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GC-content evolution in bacterial genomes: the biased gene conversion hypothesis expands.

10.1101/011023 ◽

2014 ◽

Author(s):

Florent Lassalle ◽

Séverine Périan ◽

Thomas Bataillon ◽

Xavier Nesme ◽

Laurent Duret ◽

...

Keyword(s):

Gene Conversion ◽

Bacterial Species ◽

Gc Content ◽

Base Substitution ◽

Human Populations ◽

Bacterial Genomes ◽

Evolutionary Forces ◽

Evolutionary Force ◽

Biased Gene Conversion ◽

Genomic Regions

The characterization of functional elements in genomes relies on the identification of the footprints of natural selection. In this quest, taking into account neutral evolutionary processes such as mutation and genetic drift is crucial because these forces can generate patterns that may obscure or mimic signatures of selection. In mammals, and probably in many eukaryotes, another such confounding factor called GC-Biased Gene Conversion (gBGC) has been documented. This mechanism generates patterns identical to what is expected under selection for higher GC-content, specifically in highly recombining genomic regions. Recent results have suggested that a mysterious selective force favouring higher GC-content exists in Bacteria but the possibility that it could be gBGC has been excluded. Here, we show that gBGC is probably at work in most if not all bacterial species. First we find a consistent positive relationship between the GC-content of a gene and evidence of intra-genic recombination throughout a broad spectrum of bacterial clades. Second, we show that the evolutionary force responsible for this pattern is acting independently from selection on codon usage, and could potentially interfere with selection in favor of optimal AU-ending codons. A comparison with data from human populations shows that the intensity of gBGC in Bacteria is comparable to what has been reported in mammals. We propose that gBGC is not restricted to sexual Eukaryotes but also widespread among Bacteria and could therefore be an ancestral feature of cellular organisms. We argue that if gBGC occurs in bacteria, it can account for previously unexplained observations, such as the apparent non-equilibrium of base substitution patterns and the heterogeneity of gene composition within bacterial genomes. Because gBGC produces patterns similar to positive selection, it is essential to take this process into account when studying the evolutionary forces at work in bacterial genomes.

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A Broad-Host-Range Tailocin from Burkholderia cenocepacia

Applied and Environmental Microbiology ◽

10.1128/aem.03414-16 ◽

2017 ◽

Vol 83 (10) ◽

Cited By ~ 7

Author(s):

Guichun W. Yao ◽

Iris Duarte ◽

Tram T. Le ◽

Lisa Carmody ◽

John J. LiPuma ◽

...

Keyword(s):

Host Range ◽

Burkholderia Cepacia ◽

Bacterial Species ◽

Burkholderia Cenocepacia ◽

Mass Spectrometry Analysis ◽

Chromosome 1 ◽

Broad Host Range ◽

Core Oligosaccharide ◽

Content Type ◽

Spectrometry Analysis

ABSTRACT The Burkholderia cepacia complex (Bcc) consists of 20 closely related Gram-negative bacterial species that are significant pathogens for persons with cystic fibrosis (CF). Some Bcc strains are highly transmissible and resistant to multiple antibiotics, making infection difficult to treat. A tailocin (phage tail-like bacteriocin), designated BceTMilo, with a broad host range against members of the Bcc, was identified in B. cenocepacia strain BC0425. Sixty-eight percent of Bcc representing 10 species and 90% of non-Bcc Burkholderia strains tested were sensitive to BceTMilo. BceTMilo also showed killing activity against Pseudomonas aeruginosa PAO1 and derivatives. Liquid chromatography-mass spectrometry analysis of the major BceTMilo proteins was used to identify a 23-kb tailocin locus in a draft BC0425 genome. The BceTMilo locus was syntenic and highly similar to a 24.6-kb region on chromosome 1 of B. cenocepacia J2315 (BCAL0081 to BCAL0107). A close relationship and synteny were observed between BceTMilo and Burkholderia phage KL3 and, by extension, with paradigm temperate myophage P2. Deletion mutants in the gene cluster encoding enzymes for biosynthesis of lipopolysaccharide (LPS) in the indicator strain B. cenocepacia K56-2 conferred resistance to BceTMilo. Analysis of the defined mutants in LPS biosynthetic genes indicated that an α-d-glucose residue in the core oligosaccharide is the receptor for BceTMilo. IMPORTANCE BceTMilo, presented in this study, is a broad-host-range tailocin active against Burkholderia spp. As such, BceTMilo and related or modified tailocins have potential as bactericidal therapeutic agents against plant- and human-pathogenic Burkholderia.

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Comprehensive genome data analysis establishes a triple whammy of carbapenemases, ICEs and multiple clinically relevant bacteria

Microbial Genomics ◽

10.1099/mgen.0.000424 ◽

2020 ◽

Vol 6 (10) ◽

Author(s):

João Botelho ◽

Joana Mourão ◽

Adam P. Roberts ◽

Luísa Peixe

Keyword(s):

Type Species ◽

Bacterial Species ◽

Carbapenem Resistance ◽

Beta Lactamase ◽

Bacterial Genomes ◽

Content Type ◽

Integrative And Conjugative Elements ◽

Link Type ◽

Genome Data Analysis ◽

Clinically Relevant Bacteria

Carbapenemases inactivate most β-lactam antibiotics, including carbapenems, and have frequently been reported among Enterobacteriaceae , Acinetobacter spp. and Pseudomonas spp. Traditionally, the horizontal gene transfer of carbapenemase-encoding genes (CEGs) has been linked to plasmids. However, given that integrative and conjugative elements (ICEs) are possibly the most abundant conjugative elements among prokaryotes, we conducted an in silico analysis to ascertain the likely role of ICEs in the spread of CEGs among all bacterial genomes (n=182 663). We detected 17 520 CEGs, of which 66 were located within putative ICEs among several bacterial species (including clinically relevant bacteria, such as Pseudomonas aeruginosa , Klebsiella pneumoniae and Escherichia coli ). Most CEGs detected within ICEs belong to the IMP, NDM and SPM metallo-beta-lactamase families, and the serine beta-lactamase KPC and GES families. Different mechanisms were likely responsible for acquisition of these genes. The majority of CEG-bearing ICEs belong to the MPFG, MPFT and MPFF classes and often encode resistance to other antibiotics (e.g. aminoglycosides and fluoroquinolones). This study provides a snapshot of the different CEGs associated with ICEs among available bacterial genomes and sheds light on the underappreciated contribution of ICEs to the spread of carbapenem resistance globally.

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Low base-substitution mutation rate in the germline genome of the ciliate Tetrahymena thermophila

10.1101/025536 ◽

2015 ◽

Author(s):

Hongan Long ◽

David J. Winter ◽

Allan Y.-C Chang ◽

Way Sung ◽

Steven H. Wu ◽

...

Keyword(s):

Mutation Rate ◽

Tetrahymena Thermophila ◽

Mutation Rates ◽

Base Substitution ◽

Paramecium Tetraurelia ◽

Epigenetic Factors ◽

Detection Approach ◽

Evolution Experiment ◽

Substitution Mutations ◽

Substitution Mutation

AbstractMutation is the ultimate source of all genetic variation and is, therefore, central to evolutionary change. Previous work on Paramecium tetraurelia found an unusually low germline base-substitution mutation rate in this ciliate. Here, we tested the generality of this result among ciliates using Tetrahymena thermophila. We sequenced the genomes of 10 lines of T. thermophila that had each undergone approximately 1,000 generations of mutation accumulation (MA). We applied an existing mutation-calling pipeline and developed a new probabilistic mutation detection approach that directly models the design of an MA experiment and accommodates the noise introduced by mismapped reads. Our probabilistic mutation-calling method provides a straightforward way of estimating the number of sites at which a mutation could have been called if one was present, providing the denominator for our mutation rate calculations. From these methods, we find that T. thermophila has a germline base-substitution mutation rate of 7.61 × 10−12 per site, per cell division, which is consistent with the low base-substitution mutation rate in P. tetraurelia. Over the course of the evolution experiment, genomic exclusion lines derived from the MA lines experienced a fitness decline that cannot be accounted for by germline base-substitution mutations alone, suggesting that other genetic or epigenetic factors must be involved. Because selection can only operate to reduce mutation rates based upon the “visible” mutational load, asexual reproduction with a transcriptionally silent germline may allow ciliates to evolve extremely low germline mutation rates.

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Expression of theEscherichia coli ftsZgene: trials and tribulations of gene fusion studies

Genetics Research ◽

10.1017/s0016672300031050 ◽

1993 ◽

Vol 61 (1) ◽

pp. 1-8

Author(s):

Aline Robin ◽

Richard D'Ari

Keyword(s):

Gene Fusion ◽

Open Reading Frame ◽

Mutation Rates ◽

Base Substitution ◽

Reading Frame ◽

E Coli ◽

New Gene ◽

Cell Division Protein ◽

Selection For ◽

Substitution Mutation

SummaryTheftsZgene ofEscherichia coli, which codes for an essential cell division protein, is subjected to multiple regulation, as shown in part with studies using anftsZ::lacZoperon fusion located on phage λJFLIOO. Using this same fusion, we sought to isolate regulatory mutants overexpressingftsZby selecting mutants able to grow on lactose. One Lac+mutant was obtained which overexpressed theftsZ::lacZfusion 70-fold. The mutation responsible for the overexpression lies in a new gene,cot, located near 56 min on theE. coligenetic map. Thecotmutation probably affects the transcription of a chromosomal open reading frame, 0RF1, lying downstream of thebioAgene and adjacent to theftzZ::lacZfusion of the λJFL100 prophage integrated atattλ. Using anftsZ84(Ts) strain, in which there was a double selection for overexpression of bothftsZ::lacZandftsZ+, no Lac+Tr mutants were obtained from 3·6 × 1010bacteria; the introduction of amutLallele, increasing spontaneous base substitution mutation rates 75-fold, did not permit us to isolate such a mutant. We conclude that Lac+ftsZ-constitutive mutations cannot be obtained in λJFL100 lysogens by a single base substitution.

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