Generation of Bacteriophage-Insensitive Mutants ofStreptococcus thermophilusvia an Antisense RNA CRISPR-Cas Silencing Approach

ABSTRACTPredation of starter lactic acid bacteria such asStreptococcus thermophilusby bacteriophages is a persistent and costly problem in the dairy industry. CRISPR-mediatedbacteriophageinsensitivemutants (BIMs), while straightforward to generate and verify, can quickly be overcome by mutant phages. The aim of this study was to develop a tool allowing the generation of derivatives of commercialS. thermophilusstrains which are resistant to phage attack through a non-CRISPR-mediated mechanism, with the objective of generating BIMs exhibiting stable resistance against a range of isolated lyticS. thermophilusphages. To achieve this, standard BIM generation was complemented by the use of the wild-type (WT) strain which had been transformed with an antisense mRNA-generating plasmid (targeting a crucial CRISPR-associated [cas] gene) in order to facilitate the generation of non-CRISPR-mediated BIMs. Phage sensitivity assays suggest that non-CRISPR-mediated BIMs exhibit some advantages compared to CRISPR-mediated BIMs derived from the same strain.IMPORTANCEThe outlined approach reveals the presence of a powerful host-imposed barrier for phage infection inS. thermophilus. Considering the detrimental economic consequences of phage infection in the dairy processing environment, the developed methodology has widespread applications, particularly where other methods may not be practical or effective in obtaining robust, phage-tolerantS. thermophilusstarter strains.

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Draft Genome Sequences of Three VirulentStreptococcus thermophilusBacteriophages Isolated from the Dairy Environment in the Veneto Region of Italy

Genome Announcements ◽

10.1128/genomea.00045-18 ◽

2018 ◽

Vol 6 (10) ◽

Cited By ~ 2

Author(s):

Vinícius da Silva Duarte ◽

Sabrina Giaretta ◽

Laura Treu ◽

Stefano Campanaro ◽

Pedro M. Pereira Vidigal ◽

...

Keyword(s):

Draft Genome ◽

Streptococcus Thermophilus ◽

Phage Infection ◽

Veneto Region ◽

Genome Sequences ◽

Content Type ◽

And Control ◽

New Strategies

ABSTRACTStreptococcus thermophilus, a very important dairy species, is constantly threatened by phage infection. We report the genome sequences of threeS. thermophilusbacteriophages isolated from a dairy environment in the Veneto region of Italy. These sequences will be used for the development of new strategies to detect and control phages in dairy environments.

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Host Exopolysaccharide Quantity and Composition Impact Erwinia amylovora Bacteriophage Pathogenesis

Applied and Environmental Microbiology ◽

10.1128/aem.00067-13 ◽

2013 ◽

Vol 79 (10) ◽

pp. 3249-3256 ◽

Cited By ~ 26

Author(s):

Dwayne R. Roach ◽

David R. Sjaarda ◽

Alan J. Castle ◽

Antonet M. Svircev

Keyword(s):

Population Growth ◽

Erwinia Amylovora ◽

Phage Infection ◽

Wild Type ◽

Artificial Medium ◽

Content Type ◽

Phage Growth ◽

Phage Population

ABSTRACTErwinia amylovorabacteriophages (phages) belonging to theMyoviridaeandPodoviridaefamilies demonstrated a preference for either high-exopolysaccharide-producing (HEP) or low-exopolysaccharide-producing (LEP) bacterial hosts when grown on artificial medium without or with sugar supplementation.Myoviridaephages produced clear plaques on LEP hosts and turbid plaques on HEP hosts. The reverse preference was demonstrated by mostPodoviridaephages, where clear plaques were seen on HEP hosts. Efficiency of plating (EOP) was determined by comparing phage growth on the original isolation host to the that on the LEP or HEP host. Nine of 10Myoviridaephages showed highest EOPs on LEP hosts, and 8 of 11Podoviridaephages had highest EOPs on HEP hosts. Increasing the production of EPS on sugar-supplemented medium or decreasing production by knocking out the synthesis of amylovoran or levan, the two EPSs produced byE. amylovora, indicated that these components play crucial roles in phage infection. Amylovoran was virtually essential for proliferation of mostPodoviridaephages when phage population growth was compared to the wild type. Decreased levan production resulted in a significant reduction of progeny from phages in theMyoviridaefamily. Thus,Podoviridaephages are adapted to hosts that produce high levels of exopolysaccharides and are dependent on host-produced amylovoran for pathogenesis.Myoviridaephages are adapted to hosts that produce lower levels of exopolysaccharides and host-produced levan.

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Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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The Serratia marcescens Siderophore Serratiochelin Is Necessary for Full Virulence during Bloodstream Infection

Infection and Immunity ◽

10.1128/iai.00117-20 ◽

2020 ◽

Vol 88 (8) ◽

Cited By ~ 1

Author(s):

Danelle R. Weakland ◽

Sara N. Smith ◽

Bailey Bell ◽

Ashootosh Tripathi ◽

Harry L. T. Mobley

Keyword(s):

Serratia Marcescens ◽

Bloodstream Infection ◽

Iron Acquisition ◽

Gene Clusters ◽

Clinical Isolates ◽

Wild Type ◽

Serratia Plymuthica ◽

Content Type ◽

Cas Assay ◽

Essential Nutrient

ABSTRACT Serratia marcescens is a bacterium frequently found in the environment, but over the last several decades it has evolved into a concerning clinical pathogen, causing fatal bacteremia. To establish such infections, pathogens require specific nutrients; one very limited but essential nutrient is iron. We sought to characterize the iron acquisition systems in S. marcescens isolate UMH9, which was recovered from a clinical bloodstream infection. Using RNA sequencing (RNA-seq), we identified two predicted siderophore gene clusters (cbs and sch) that were regulated by iron. Mutants were constructed to delete each iron acquisition locus individually and in conjunction, generating both single and double mutants for the putative siderophore systems. Mutants lacking the sch gene cluster lost their iron-chelating ability as quantified by the chrome azurol S (CAS) assay, whereas the cbs mutant retained wild-type activity. Mass spectrometry-based analysis identified the chelating siderophore to be serratiochelin, a siderophore previously identified in Serratia plymuthica. Serratiochelin-producing mutants also displayed a decreased growth rate under iron-limited conditions created by dipyridyl added to LB medium. Additionally, mutants lacking serratiochelin were significantly outcompeted during cochallenge with wild-type UMH9 in the kidneys and spleen after inoculation via the tail vein in a bacteremia mouse model. This result was further confirmed by an independent challenge, suggesting that serratiochelin is required for full S. marcescens pathogenesis in the bloodstream. Nine other clinical isolates have at least 90% protein identity to the UMH9 serratiochelin system; therefore, our results are broadly applicable to emerging clinical isolates of S. marcescens causing bacteremia.

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CHROMOSOMAL BASIS OF THE MEROZYGOSITY IN A PARTIALLY DIPLOID MUTANT OF PNEUMOCOCCUS

Genetics ◽

10.1093/genetics/80.4.667 ◽

1975 ◽

Vol 80 (4) ◽

pp. 667-678

Author(s):

Mary Lee S Ledbetter ◽

Rollin D Hotchkiss

Keyword(s):

High Efficiency ◽

Point Mutations ◽

Resistant Mutant ◽

Chromosomal Marker ◽

Structural Abnormality ◽

C Cells ◽

Wild Type ◽

Chromosomal Locus ◽

Low Efficiency ◽

Derivatives Of

ABSTRACT A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.

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MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

mBio ◽

10.1128/mbio.00236-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 28

Author(s):

Ryan W. Bogard ◽

Bryan W. Davies ◽

John J. Mekalanos

Keyword(s):

Amino Acid ◽

Vibrio Cholerae ◽

Virulence Factor ◽

Current Knowledge ◽

Intestinal Colonization ◽

Wild Type ◽

Pathogenic Potential ◽

Content Type ◽

Mouse Intestine

ABSTRACTLysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel ofV. choleraeEl Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed thatglyA1andmetJwere also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation ofglyA1, indicating that misregulation ofglyA1is likely responsible for the colonization defect observed in themetRmutant. TheglyA1mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.IMPORTANCEVibrio choleraecontinues to be a severe cause of morbidity and mortality in developing countries. Identification ofV. choleraefactors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons inV. choleraeto be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required byV. choleraeto colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements ofV. choleraewithin the mouse intestine.

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South Africa’s economic response to monetary policy uncertainty

Journal of Economic Studies ◽

10.1108/jes-07-2015-0131 ◽

2017 ◽

Vol 44 (2) ◽

pp. 282-293 ◽

Cited By ~ 1

Author(s):

Mehmet Balcilar ◽

Rangan Gupta ◽

Charl Jooste

Keyword(s):

Monetary Policy ◽

Interest Rates ◽

Stochastic Volatility ◽

Stochastic Volatility Model ◽

Economic Consequences ◽

Policy Uncertainty ◽

Dsge Model ◽

Content Type ◽

Volatility Model ◽

Central Bankers

Purpose The purpose of this paper is to study the evolution of monetary policy uncertainty and its impact on the South African economy. Design/methodology/approach The authors use a sign restricted SVAR with an endogenous feedback of stochastic volatility to evaluate the sign and size of uncertainty shocks. The authors use a nonlinear DSGE model to gain deeper insights about the transmission mechanism of monetary policy uncertainty. Findings The authors show that monetary policy volatility is high and constant. Both inflation and interest rates decline in response to uncertainty. Output rebounds quickly after a contemporaneous decrease. The DSGE model shows that the size of the uncertainty shock matters – high uncertainty can lead to a severe contraction in output, inflation and interest rates. Research limitations/implications The authors model only a few variables in the SVAR – thus missing perhaps other possible channels of shock transmission. Practical implications There is a lesson for monetary policy: monetary policy uncertainty, in isolation from general macroeconomic uncertainty, often creates unintended adverse consequences and can perpetuate a weak economic environment. The tasks of central bankers are incredibly difficult. Their models project output and inflation with relatively large uncertainty based on many shocks emanating from various sources. It matters how central bankers react to these expectations and how they communicate the underlying risks associated with setting interest rates. Originality/value This is the first study that looks into monetary policy uncertainty into South Africa using a stochastic volatility model and a nonlinear DSGE model. The results should be very useful for the Central Bank as it highlights how uncertainty, that they create, can have adverse economic consequences.

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Large-Scale Synonymous Substitutions in Cucumber Mosaic Virus RNA 3 Facilitate Amino Acid Mutations in the Coat Protein

Journal of Virology ◽

10.1128/jvi.01007-18 ◽

2018 ◽

Vol 92 (22) ◽

Cited By ~ 1

Author(s):

Tomofumi Mochizuki ◽

Rie Ohara ◽

Marilyn J. Roossinck

Keyword(s):

Amino Acid ◽

Secondary Structure ◽

Viral Genome ◽

Large Scale ◽

Viral Rna ◽

Wild Type ◽

Competitive Fitness ◽

Content Type ◽

Synonymous Substitutions ◽

Cp Gene

ABSTRACTThe effect of large-scale synonymous substitutions in a small icosahedral, single-stranded RNA viral genome on virulence, viral titer, and protein evolution were analyzed. The coat protein (CP) gene of the Fny stain of cucumber mosaic virus (CMV) was modified. We created four CP mutants in which all the codons of nine amino acids in the 5′ or 3′ half of the CP gene were replaced by either the most frequently or the least frequently used synonymous codons in monocot plants. When the dicot host (Nicotiana benthamiana) was inoculated with these four CP mutants, viral RNA titers in uninoculated symptomatic leaves decreased, while all mutants eventually showed mosaic symptoms similar to those for the wild type. The codon adaptation index of these four CP mutants against dicot genes was similar to those of the wild-type CP gene, indicating that the reduction of viral RNA titer was due to deleterious changes of the secondary structure of RNAs 3 and 4. When two 5′ mutants were serially passaged inN. benthamiana, viral RNA titers were rapidly restored but competitive fitness remained decreased. Although no nucleic acid changes were observed in the passaged wild-type CMV, one to three amino acid changes were observed in the synonymously mutated CP of each passaged virus, which were involved in recovery of viral RNA titer of 5′ mutants. Thus, we demonstrated that deleterious effects of the large-scale synonymous substitutions in the RNA viral genome facilitated the rapid amino acid mutation(s) in the CP to restore the viral RNA titer.IMPORTANCERecently, it has been known that synonymous substitutions in RNA virus genes affect viral pathogenicity and competitive fitness by alteration of global or local RNA secondary structure of the viral genome. We confirmed that large-scale synonymous substitutions in the CP gene of CMV resulted in decreased viral RNA titer. Importantly, when viral evolution was stimulated by serial-passage inoculation, viral RNA titer was rapidly restored, concurrent with a few amino acid changes in the CP. This novel finding indicates that the deleterious effects of large-scale nucleic acid mutations on viral RNA secondary structure are readily tolerated by structural changes in the CP, demonstrating a novel part of the adaptive evolution of an RNA viral genome. In addition, our experimental system for serial inoculation of large-scale synonymous mutants could uncover a role for new amino acid residues in the viral protein that have not been observed in the wild-type virus strains.

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Thymidine kinase-mediated killing of rat brain tumors

Journal of Neurosurgery ◽

10.3171/jns.1993.79.5.0729 ◽

1993 ◽

Vol 79 (5) ◽

pp. 729-735 ◽

Cited By ~ 101

Author(s):

David Barba ◽

Joseph Hardin ◽

Jasodhara Ray ◽

Fred H. Gage

Keyword(s):

Gene Therapy ◽

Brain Tumors ◽

Tumor Cells ◽

Wild Type ◽

Cns Disorders ◽

Content Type ◽

Potential Applications ◽

Ganciclovir Treatment ◽

The Brain ◽

Fine Print

✓ Gene therapy has many potential applications in central nervous system (CNS) disorders, including the selective killing of tumor cells in the brain. A rat brain tumor model was used to test the herpes simplex virus (HSV)-thymidine kinase (TK) gene for its ability to selectively kill C6 and 9L tumor cells in the brain following systemic administration of the nucleoside analog ganciclovir. The HSV-TK gene was introduced in vitro into tumor cells (C6-TK and 9L-TK), then these modified tumor cells were evaluated for their sensitivity to cell killing by ganciclovir. In a dose-response assay, both C6-TK and 9L-TK cells were 100 times more sensitive to killing by ganciclovir (median lethal dose: C6-TK, 0.1 µg ganciclovir/ml; C6, 5.0 µg ganciclovir/ml) than unmodified wild-type tumor cells or cultured fibroblasts. In vivo studies confirmed the ability of intraperitoneal ganciclovir administration to kill established brain tumors in rats as quantified by both stereological assessment of brain tumor volumes and studies of animal survival over 90 days. Rats with brain tumors established by intracerebral injection of wild-type or HSV-TK modified tumor cells or by a combination of wild-type and HSV-TK-modified cells were studied with and without ganciclovir treatments. Stereological methods determined that ganciclovir treatment eliminated tumors composed of HSV-TK-modified cells while control tumors grew as expected (p < 0.001). In survival studies, all 10 rats with 9L-TK tumors treated with ganciclovir survived 90 days while all untreated rats died within 25 days. Curiously, tumors composed of combinations of 9L and 9L-TK cells could be eliminated by ganciclovir treatments even when only one-half of the tumor cells carried the HSV-TK gene. While not completely understood, this additional tumor cell killing appears to be both tumor selective and local in nature. It is concluded that HSV-TK gene therapy with ganciclovir treatment does selectively kill tumor cells in the brain and has many potential applications in CNS disorders, including the treatment of cancer.

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A Novel CO-Responsive Transcriptional Regulator and Enhanced H2Production by an Engineered Thermococcus onnurineus NA1 Strain

Applied and Environmental Microbiology ◽

10.1128/aem.03019-14 ◽

2014 ◽

Vol 81 (5) ◽

pp. 1708-1714 ◽

Cited By ~ 25

Author(s):

Min-Sik Kim ◽

Ae Ran Choi ◽

Seong Hyuk Lee ◽

Hae-Chang Jung ◽

Seung Seob Bae ◽

...

Keyword(s):

Production Rate ◽

Gene Cluster ◽

Type Strain ◽

Wild Type Strain ◽

Regulatory System ◽

Transcriptional Regulator ◽

Bioreactor Culture ◽

Wild Type ◽

Content Type ◽

Transcriptional Regulatory

ABSTRACTGenome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism inThermococcus onnurineusNA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes ofThermococcusspecies and “CandidatusKorarchaeum cryptofilum” OPF8. In-frame deletion of eithercorQorcorRcaused a severe impairment in CO-dependent growth and H2production. WhencorQandcorRdeletion mutants were complemented by introducing thecorQRgenes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integratedcorQR(ΔCorR/corQR↑) compared with those in the wild-type strain. In addition, the ΔCorR/corQR↑strain exhibited a much higher H2production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2production rate (191.9 mmol liter−1h−1) and the specific H2production rate (249.6 mmol g−1h−1) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that thecorQRgenes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2production.

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