Constructing S. aureus Lactate Mutants

The Nebraska Transposon Mutant Library (NTML) was constructed in LAC JE2^{28 (link)}. Δddh, Δldh1, Δldh2, and ΔlctP1 Tn mutants were moved to the USA300 LAC 13c background by transduction with bacteriophage φ11^{28 (link)} (Extended Data 1a). Lactate double (Δldh1/ldh2) and triple (Δddh/ldh1/ldh) mutants were constructed from each single Tn mutant after switching antibiotic resistance cassettes associated with the Tn mutant. Transposon insertions were confirmed by PCR using chromosomal primers flanking the gene containing the Tn insertion. All S. aureus strains were transduced with a GFP plasmid (pCM13)^{91 (link)} and imaged using a Zeiss 710 META laser scanning microscope (Carl Zeiss) after 4 days of biofilm growth to evaluate biofilm structure.

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Heim C.E., Bosch M.E., Yamada K.J., Aldrich A.L., Chaudhari S.S., Klinkebiel D., Gries C.M., Alqarzaee A.A., Li Y., Thomas V.C., Seto E., Karpf A.R, & Kielian T. (2020). Lactate production by Staphylococcus aureus biofilm inhibits HDAC11 to reprogram the host immune response during persistent infection. Nature microbiology, 5(10), 1271-1284.

Publication 2020

Zeiss 710 META laser scanning microscope

Antibiotic resistance Bacteriophage Biofilm Chromosomal Gene Lactate Laser scanning microscope Ldh1 Ldh2 Library Plasmid Primers S aureus Strains Transposon

Corresponding Organization :

Other organizations : University of Nebraska Medical Center, George Washington University

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Variable analysis

independent variables

Deletion of ddh, ldh1, ldh2, and lctP1 genes using transposon (Tn) mutagenesis
Construction of lactate double (Δldh1/ldh2) and triple (Δddh/ldh1/ldh2) mutants

dependent variables

Biofilm structure evaluated by imaging using a Zeiss 710 META laser scanning microscope after 4 days of biofilm growth

control variables

All S. aureus strains were transduced with a GFP plasmid (pCM13)

controls

Positive control: None mentioned
Negative control: None mentioned

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