scholarly journals Induction of L1 and L2 β-Lactamases of Stenotrophomonas maltophilia

2007 ◽  
Vol 52 (3) ◽  
pp. 1198-1200 ◽  
Author(s):  
Rouh-Mei Hu ◽  
Kuang-Jay Huang ◽  
Lii-Tzu Wu ◽  
Ying-Ju Hsiao ◽  
Tsuey-Ching Yang
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Gene Knockout ◽  
Gene Replacement ◽  
Induction Kinetics ◽  
Knockout Mutants ◽  
L1 And L2 ◽  
Kinetics Of ◽  
Dioxygenase Activity

ABSTRACT Isogenic L1 and L2 gene knockout mutants of Stenotrophomonas maltophilia KJ (KJΔL1 and KJΔL2, respectively) were constructed by xylE gene replacement. Induction kinetics of the L1 and L2 genes were evaluated by testing catechol 2,3-dioxygenase activity in the mutants. The results suggested that the induction of the L1 and L2 genes was differentially regulated.

scholarly journals Specific interaction of an RNA-binding protein with the 3′-UTR of its target mRNA is critical to oomycete sexual reproduction

2021 ◽  
Vol 17 (10) ◽  
pp. e1010001
Author(s):  
Hui Feng ◽  
Chuanxu Wan ◽  
Zhichao Zhang ◽  
Han Chen ◽  
Zhipeng Li ◽  
...  
Keyword(s):  
Sexual Reproduction ◽  
Rna Binding ◽  
Gene Knockout ◽  
Binding Protein ◽  
Mrna Level ◽  
Specific Interaction ◽  
Rna Binding Protein ◽  
Pythium Ultimum ◽  
Knockout Mutants ◽  
Oospore Formation

Sexual reproduction is an essential stage of the oomycete life cycle. However, the functions of critical regulators in this biological process remain unclear due to a lack of genome editing technologies and functional genomic studies in oomycetes. The notorious oomycete pathogen Pythium ultimum is responsible for a variety of diseases in a broad range of plant species. In this study, we revealed the mechanism through which PuM90, a stage-specific Puf family RNA-binding protein, regulates oospore formation in P. ultimum. We developed the first CRISPR/Cas9 system-mediated gene knockout and in situ complementation methods for Pythium. PuM90-knockout mutants were significantly defective in oospore formation, with empty oogonia or oospores larger in size with thinner oospore walls compared with the wild type. A tripartite recognition motif (TRM) in the Puf domain of PuM90 could specifically bind to a UGUACAUA motif in the mRNA 3′ untranslated region (UTR) of PuFLP, which encodes a flavodoxin-like protein, and thereby repress PuFLP mRNA level to facilitate oospore formation. Phenotypes similar to PuM90-knockout mutants were observed with overexpression of PuFLP, mutation of key amino acids in the TRM of PuM90, or mutation of the 3′-UTR binding site in PuFLP. The results demonstrated that a specific interaction of the RNA-binding protein PuM90 with the 3′-UTR of PuFLP mRNA at the post-transcriptional regulation level is critical for the sexual reproduction of P. ultimum.

scholarly journals Generating and characterizing single- and multigene mutants of the Rubisco small subunit family in Arabidopsis

2020 ◽  
Vol 71 (19) ◽  
pp. 5963-5975 ◽  
Author(s):  
Panupon Khumsupan ◽  
Marta A Kozlowska ◽  
Douglas J Orr ◽  
Andreas I Andreou ◽  
Naomi Nakayama ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Catalytic Efficiency ◽  
Small Subunit ◽  
Multiple Gene ◽  
Functional Roles ◽  
Knockout Mutants ◽  
Dna Insertion ◽  
Low Levels ◽  
Rbcs Genes ◽  
Viable Approach

Abstract The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] and T-DNA insertion lines to generate a suite of single and multiple gene knockout mutants for the four members of the rbcS family in Arabidopsis, including two novel mutants 2b3b and 1a2b3b. 1a2b3b contained very low levels of Rubisco (~3% relative to the wild-type) and is the first example of a mutant with a homogenous Rubisco pool consisting of a single SSU isoform (1B). Growth under near-outdoor levels of light demonstrated Rubisco-limited growth phenotypes for several SSU mutants and the importance of the 1A and 3B isoforms. We also identified 1a1b as a likely lethal mutation, suggesting a key contributory role for the least expressed 1B isoform during early development. The successful use of CRISPR/Cas here suggests that this is a viable approach for exploring the functional roles of SSU isoforms in plants.

Ontogeny of carnitine biosynthesis in Sus scrofa domesticus, inferred from γ-butyrobetaine hydroxylase (dioxygenase) activity and substrate inhibition

2020 ◽  
Vol 319 (1) ◽  
pp. R43-R49
Author(s):  
Xi Lin ◽  
Pasha A. Lyvers Peffer ◽  
Jason Woodworth ◽  
Jack Odle
Keyword(s):  
Sus Scrofa ◽  
Specific Activity ◽  
Substrate Inhibition ◽  
Biosynthesis Pathway ◽  
Fresh Tissue ◽  
Tissue Homogenates ◽  
Liver And Kidney ◽  
Sus Scrofa Domesticus ◽  
Kinetics Of ◽  
Dioxygenase Activity

γ-Butyrobetaine hydroxylase (γ-BBH) is the last limiting enzyme of the l-carnitine biosynthesis pathway and plays an important role in catalyzing the hydroxylation of γ-butyrobetaine (γ-BB) to l-carnitine. To study the developmental effect of substrate concentration on the enzyme’s specific activity, kinetics of γ-BBH were measured in liver and kidney from newborn and 1-, 7-, 21-, 35-, 56-, and 210-day-old domestic pigs. Fresh tissue homogenates were assayed under nine concentrations of γ-BB from 0 to 1.5 mM. Substrate inhibition associated with age was observed at ≥0.6 mM of γ-BB. Hepatic activity was low at birth but increased after 1 day. By 21 days, the activity rose by 6.6-fold ( P < 0.05) and remained constant after 56 days. Renal activity was higher than in liver at birth but remained constant through 35 days. By 56 days, the velocity increased by 44% over the activity at birth ( P < 0.05). The apparent Km for γ-BB at birth on average was 2.8-fold higher than at 1 day. The Km value was 60% higher in kidney than liver during development but showed no difference in adult pigs. The total organ enzyme activity increased by 130-fold for liver and 18-fold for kidney as organ weight increased from birth to 56 days. In conclusion, age and substrate affect γ-BBH specific activity and Km for γ-BB in liver and kidney. Whereas the predominant organ for carnitine synthesis is likely the kidney at birth, the liver appears to predominate after the pig exceeds 7 days of age.

scholarly journals Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii

2014 ◽  
Vol 13 (11) ◽  
pp. 1465-1469 ◽  
Author(s):  
Wenzhi Jiang ◽  
Andrew J. Brueggeman ◽  
Kempton M. Horken ◽  
Thomas M. Plucinak ◽  
Donald P. Weeks
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Transient Expression ◽  
Gene Knockout ◽  
Nuclease Activity ◽  
Gene Replacement ◽  
Target Site ◽  
Gene Encoding ◽  
Content Type ◽  
Guide Rna ◽  
Eukaryotic Organisms

ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii , failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii , we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10 9 cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii . The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing.

scholarly journals Loss of the F-Actin Binding and Vesicle-Associated Protein Comitin Leads to a Phagocytosis Defect

2002 ◽  
Vol 1 (6) ◽  
pp. 906-914 ◽  
Author(s):  
Thomas Schreiner ◽  
Martina R. Mohrs ◽  
Rosemarie Blau-Wasser ◽  
Alfred von Krempelhuber ◽  
Michael Steinert ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Gene Replacement ◽  
Actin Binding ◽  
Wild Type ◽  
Hyperosmotic Shock ◽  
Latex Beads ◽  
Knockout Mutants ◽  
Mutant Cells

ABSTRACT Comitin is an F-actin binding and membrane-associated protein from Dictyostelium discoideum, which is present on Golgi and vesicle membranes and changes its localization in response to agents affecting the cytoskeleton. To investigate its in vivo functions we have generated knockout mutants by gene replacement. Based on comitin's in vitro functions we examined properties related to vesicular transport and microfilament function. Whereas cell growth, pinocytosis, secretion, chemotaxis, motility, and development were unaltered, comitin-lacking cells were impaired in the early steps of phagocytosis of Saccharomyces cerevisiae particles and of Escherichia coli, whereas uptake of latex beads was unaffected. Furthermore, the lack of comitin positively affected survival of pathogenic bacteria. Mutant cells also showed an altered response to hyperosmotic shock in comparison to the wild type. The redistribution of comitin during hyperosmotic shock in wild-type cells and its presence on early phagosomes suggest a direct involvement of comitin in these processes.

Degradation kinetics of carbendazim by Klebsiella oxytoca, Flavobacterium johnsoniae, and Stenotrophomonas maltophilia strains

2020 ◽  
Vol 27 (23) ◽  
pp. 28518-28526 ◽  
Author(s):  
María Luisa Alvarado-Gutiérrez ◽  
Nora Ruiz-Ordaz ◽  
Juvencio Galíndez-Mayer ◽  
Everardo Curiel-Quesada ◽  
Fortunata Santoyo-Tepole
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Degradation Kinetics ◽  
Klebsiella Oxytoca ◽  
Flavobacterium Johnsoniae ◽  
Kinetics Of

scholarly journals Sequence analysis and enzyme kinetics of the L2 serine beta-lactamase from Stenotrophomonas maltophilia.

1997 ◽  
Vol 41 (7) ◽  
pp. 1460-1464 ◽  
Author(s):  
T R Walsh ◽  
A P MacGowan ◽  
P M Bennett
Keyword(s):  
Amino Acids ◽  
Sequence Analysis ◽  
Clavulanic Acid ◽  
Active Site ◽  
Stenotrophomonas Maltophilia ◽  
Leader Peptide ◽  
Beta Lactamase ◽  
Class A ◽  
Cloned Gene ◽  
Kinetics Of

The L2 serine active-site beta-lactamase from Stenotrophomonas maltophilia has been classified as a clavulanic acid-sensitive cephalosporinase. The gene encoding this enzyme from S. maltophilia 1275 IID has been cloned on a 3.3-kb fragment into pK18 under the control of a Ptac promoter to generate recombinant plasmid pUB5840; when expressed in Escherichia coli, this gene confers resistance to cephalosporins and penicillins. Sequence analysis has revealed an open reading frame (ORF) of 909 bp with a GC content of 71.6%, comparable to that of the L1 metallo-beta-lactamase gene (68.4%) from the same bacterium. The ORF encodes an unmodified protein of 303 amino acids with a predicted molecular mass of 31.5 kDa, accommodating a putative leader peptide of 27 amino acids. Comparison of the amino acid sequence with those of other beta-lactamases showed it to be most closely related (54% identity) to the BLA-A beta-lactamase from Yersinia enterocolitica. Sequence identity is most obvious near the STXK active-site motif and the SDN loop motif common to all serine active-site penicillinases. Sequences outside the conserved regions display low homology with comparable regions of other class A penicillinases. Kinetics of the enzyme from the cloned gene demonstrated an increase in activity with cefotaxime but markedly less activity with imipenem than previously reported. Hence, the S. maltophilia L2 beta-lactamase is an inducible Ambler class A beta-lactamase which would account for the sensitivity to clavulanic acid.

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