An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35

ABSTRACTStreptomyces iranensisHM 35 is an alternative rapamycin producer toStreptomyces rapamycinicus. Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation inS. rapamycinicus.The approach depends upon efficient conjugational plasmid transfer fromEscherichia colitoStreptomyces, and the failure of this step has frustrated its application toStreptomyces iranensisHM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca2+ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions inS. iranensisHM 35. Among them wererapK, which generates the starter unit for rapamycin biosynthesis, andhutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants ofStreptomycesspecies that are genetically difficult to manipulate.IMPORTANCEThe developed protocol of conjugational plasmid transfer fromEscherichia colitoStreptomyces iranensismay allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable,Streptomycesspecies.

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The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00066-12 ◽

2012 ◽

Vol 56 (6) ◽

pp. 3216-3223 ◽

Cited By ~ 20

Author(s):

Tina Manzhu Kang ◽

Jessica Yuan ◽

Angelyn Nguyen ◽

Elinne Becket ◽

Hanjing Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Bacillus Anthracis ◽

Gene Knockout ◽

Aminoglycoside Antibiotic ◽

Double Strand Breaks ◽

Repair Capacity ◽

Content Type ◽

Strand Breaks ◽

Knockout Mutants ◽

Increased Sensitivity

ABSTRACTThe distribution of mutants in the Keio collection ofEscherichia coligene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ,holC,holD, andpriAknockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded byrecA,recB, andrecC, among others. Additionally, caffeine partially protects cells of bothEscherichia coliandBacillus anthracisfrom killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

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Draft Genome Sequences of Salmonella Lysozyme Gene Knockout Mutants

Genome Announcements ◽

10.1128/genomea.00519-17 ◽

2017 ◽

Vol 5 (23) ◽

Author(s):

Narine Arabyan ◽

Bihua C. Huang ◽

Bart C. Weimer

Keyword(s):

Gene Deletion ◽

Gene Knockout ◽

Draft Genome ◽

Deletion Mutants ◽

Genome Sequences ◽

Content Type ◽

Knockout Mutants ◽

Serovar Typhimurium ◽

Encoding Gene ◽

Glucoside Hydrolases

ABSTRACT Lysozyme enzymes hydrolyze the β-1,4-glycosidic bond in oligosaccharides. These enzymes are part of a broad group of glucoside hydrolases that are poorly characterized; however, they are important for growth and are being recognized as emerging virulence factors. This is the release of four lysozyme-encoding-gene-deletion mutants in Salmonella enterica serovar Typhimurium LT2.

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Combinatorial Strategies for Improving Multiple-Stress Resistance in Industrially Relevant Escherichia coli Strains

Applied and Environmental Microbiology ◽

10.1128/aem.01542-14 ◽

2014 ◽

Vol 80 (19) ◽

pp. 6223-6242 ◽

Cited By ~ 23

Author(s):

Rebecca M. Lennen ◽

Markus J. Herrgård

Keyword(s):

Escherichia Coli ◽

Stress Resistance ◽

Gene Deletion ◽

Elevated Temperatures ◽

Insertion Site ◽

Stress Conditions ◽

Dependent Manner ◽

Gene Deletions ◽

Content Type ◽

Strain Background

ABSTRACTHigh-cell-density fermentation for industrial production of chemicals can impose numerous stresses on cells due to high substrate, product, and by-product concentrations; high osmolarity; reactive oxygen species; and elevated temperatures. There is a need to develop platform strains of industrial microorganisms that are more tolerant toward these typical processing conditions. In this study, the growth of six industrially relevant strains ofEscherichia coliwas characterized under eight stress conditions representative of fed-batch fermentation, and strains W and BL21(DE3) were selected as platforms for transposon (Tn) mutagenesis due to favorable resistance characteristics. Selection experiments, followed by either targeted or genome-wide next-generation-sequencing-based Tn insertion site determination, were performed to identify mutants with improved growth properties under a subset of three stress conditions and two combinations of individual stresses. A subset of the identified loss-of-function mutants were selected for a combinatorial approach, where strains with combinations of two and three gene deletions were systematically constructed and tested for single and multistress resistance. These approaches allowed identification of (i) strain-background-specific stress resistance phenotypes, (ii) novel gene deletion mutants inE. colithat confer single and multistress resistance in a strain-background-dependent manner, and (iii) synergistic effects of multiple gene deletions that confer improved resistance over single deletions. The results of this study underscore the suboptimality and strain-specific variability of the genetic network regulating growth under stressful conditions and suggest that further exploration of the combinatorial gene deletion space in multiple strain backgrounds is needed for optimizing strains for microbial bioprocessing applications.

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Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

Applied and Environmental Microbiology ◽

10.1128/aem.01704-15 ◽

2015 ◽

Vol 81 (20) ◽

pp. 6953-6963 ◽

Cited By ~ 5

Author(s):

Zhe Zhao ◽

Lauren J. Eberhart ◽

Lisa H. Orfe ◽

Shao-Yeh Lu ◽

Thomas E. Besser ◽

...

Keyword(s):

Escherichia Coli ◽

Atp Synthase ◽

Disulfide Bonds ◽

Gene Knockout ◽

Single Gene ◽

Site Directed Mutagenesis ◽

Content Type ◽

E Coli ◽

Synthase Inhibitor ◽

Loss Of Susceptibility

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

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Efficient Generation of Unmarked Deletions inLegionella pneumophila

Applied and Environmental Microbiology ◽

10.1128/aem.02904-10 ◽

2011 ◽

Vol 77 (7) ◽

pp. 2545-2548 ◽

Cited By ~ 15

Author(s):

Andrew Bryan ◽

Kaoru Harada ◽

Michele S. Swanson

Keyword(s):

Escherichia Coli ◽

Legionella Pneumophila ◽

Natural Transformation ◽

Exonuclease Activity ◽

Gene Deletions ◽

Site Specific ◽

Efficient Generation ◽

Λ Red

ABSTRACTUnmarked gene deletions facilitate studies ofLegionella pneumophilamulticomponent processes, such as motility and exonuclease activity. For this purpose,FRT-flanked alleles constructed inEscherichia coliusing λ-Red recombinase were transferred toL. pneumophilaby natural transformation. Resistance cassettes were then efficiently excised using the Flp site-specific recombinase encoded on a plasmid that is readily lost.

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Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01444-15 ◽

2015 ◽

Vol 81 (16) ◽

pp. 5650-5659 ◽

Cited By ~ 4

Author(s):

Shan Goh ◽

Angela Hohmeier ◽

Timothy C. Stone ◽

Victoria Offord ◽

Francisco Sarabia ◽

...

Keyword(s):

Escherichia Coli ◽

Target Gene ◽

Essential Gene ◽

Transcript Level ◽

Essential Genes ◽

Content Type ◽

Reading Frame ◽

Knockout Mutants ◽

Bacterial Genes ◽

Relationship Of

ABSTRACTEssential bacterial genes located within operons are particularly challenging to study independently because of coordinated gene expression and the nonviability of knockout mutants. Essentiality scores for many operon genes remain uncertain. Antisense RNA (asRNA) silencing or in-frame gene disruption of genes may help establish essentiality but can lead to polar effects on genes downstream or upstream of the target gene. Here, theEscherichia coliribF-ileS-lspA-fkpB-ispHoperon was used to evaluate the possibility of independently studying an essential gene using expressed asRNA and target gene overexpression to deregulate coupled expression. The gene requirement for growth in conditional silencing strains was determined by the relationship of target mRNA reduction with growth inhibition as the minimum transcript level required for 50% growth (MTL50). Mupirocin and globomycin, the protein inhibitors of IleS and LspA, respectively, were used in sensitization assays of strains containing both asRNA-expressing and open reading frame-expressing plasmids to examine deregulation of the overlappingileS-lspAgenes. We found upstream and downstream polar silencing effects when eitherileSorlspAwas silenced, indicating coupled expression. Weighted MTL50values (means and standard deviations) ofribF,ileS, andlspAwere 0.65 ± 0.18, 0.64 ± 0.06, and 0.76 ± 0.10, respectively. However, they were not significantly different (P= 0.71 by weighted one-way analysis of variance). The gene requirement forispHcould not be determined due to insufficient growth reduction. Mupirocin and globomycin sensitization experiments indicated thatileS-lspAexpression could not be decoupled. The results highlight the inherent challenges associated with genetic analyses of operons; however, coupling of essential genes may provide opportunities to improve RNA-silencing antimicrobials.

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Increased Transfer of a Multidrug Resistance Plasmid in Escherichia coli Biofilms at the Air-Liquid Interface

Applied and Environmental Microbiology ◽

10.1128/aem.00090-11 ◽

2011 ◽

Vol 77 (15) ◽

pp. 5079-5088 ◽

Cited By ~ 61

Author(s):

Jaroslaw E. Król ◽

Hung Duc Nguyen ◽

Linda M. Rogers ◽

Haluk Beyenal ◽

Stephen M. Krone ◽

...

Keyword(s):

Escherichia Coli ◽

Multidrug Resistance ◽

Gene Transfer ◽

Flow Cell ◽

Plasmid Transfer ◽

Liquid Interface ◽

Transfer Efficiency ◽

Content Type ◽

Resistance Plasmid ◽

Air Liquid Interface

ABSTRACTAlthough biofilms represent a common bacterial lifestyle in clinically and environmentally important habitats, there is scant information on the extent of gene transfer in these spatially structured populations. The objective of this study was to gain insight into factors that affect transfer of the promiscuous multidrug resistance plasmid pB10 inEscherichia colibiofilms. Biofilms were grown in different experimental settings, and plasmid transfer was monitored using laser scanning confocal microscopy and plate counting. In closed flow cells, plasmid transfer in surface-attached submerged biofilms was negligible. In contrast, a high plasmid transfer efficiency was observed in a biofilm floating at the air-liquid interface in an open flow cell with low flow rates. A vertical flow cell and a batch culture biofilm reactor were then used to detect plasmid transfer at different depths away from the air-liquid interface. Extensive plasmid transfer occurred only in a narrow zone near that interface. The much lower transfer frequencies in the lower zones coincided with rapidly decreasing oxygen concentrations. However, when anE. colicsrAmutant was used as the recipient, a thick biofilm was obtained at all depths, and plasmid transfer occurred at similar frequencies throughout. These results and data from separate aerobic and anaerobic matings suggest that oxygen can affect IncP-1 plasmid transfer efficiency, not only directly but also indirectly, through influencing population densities and therefore colocalization of donors and recipients. In conclusion, the air-liquid interface can be a hot spot for plasmid-mediated gene transfer due to high densities of juxtaposed donor and recipient cells.

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Rapid Accumulation of Motility-Activating Mutations in Resting Liquid Culture ofEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.00259-19 ◽

2019 ◽

Vol 201 (19) ◽

Cited By ~ 2

Author(s):

Darren J. Parker ◽

Pınar Demetci ◽

Gene-Wei Li

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Liquid Medium ◽

Regulatory Networks ◽

Gene Knockout ◽

Single Gene ◽

Point Mutations ◽

Content Type ◽

E Coli ◽

Keio Collection

ABSTRACTExpression of motility genes is a potentially beneficial but costly process in bacteria. Interestingly, many isolate strains ofEscherichia colipossess motility genes but have lost the ability to activate them under conditions in which motility is advantageous, raising the question of how they respond to these situations. Through transcriptome profiling of strains in theE. colisingle-gene knockout Keio collection, we noticed drastic upregulation of motility genes in many of the deletion strains compared to levels in their weakly motile parent strain (BW25113). We show that this switch to a motile phenotype is not a direct consequence of the genes deleted but is instead due to a variety of secondary mutations that increase the expression of the major motility regulator, FlhDC. Importantly, we find that this switch can be reproduced by growing poorly motileE. colistrains in nonshaking liquid medium overnight but not in shaking liquid medium. Individual isolates after the nonshaking overnight incubations acquired distinct mutations upstream of theflhDCoperon, including different insertion sequence (IS) elements and, to a lesser extent, point mutations. The rapidity with which genetic changes sweep through the populations grown without shaking shows that poorly motile strains can quickly adapt to a motile lifestyle by genetic rewiring.IMPORTANCEThe ability to tune gene expression in times of need outside preordained regulatory networks is an essential evolutionary process that allows organisms to survive and compete. Here, we show that upon overnight incubation in liquid medium without shaking, populations of largely nonmotileEscherichia colibacteria can rapidly accumulate mutants that have constitutive motility. This effect contributes to widespread secondary mutations in the single-gene knockout library, the Keio collection. As a result, 49/71 (69%) of the Keio strains tested exhibited various degrees of motility, whereas their parental strain is poorly motile. These observations highlight the plasticity of gene expression even in the absence of preexisting regulatory programs and should raise awareness of procedures for handling laboratory strains ofE. coli.

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