Vitamin B ₁₂ -Mediated Restoration of Defective Anaerobic Growth Leads to Reduced Biofilm Formation in Pseudomonas aeruginosa

Abstract

<jats:title>ABSTRACT</jats:title><jats:p><jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Pseudomonas aeruginosa</jats:named-content>undergoes cell elongation and forms robust biofilms during anaerobic respiratory growth using nitrate (NO<jats:sub arrange="stagger">3</jats:sub><jats:sup arrange="stagger">−</jats:sup>) as an alternative electron acceptor. Understanding the mechanism of cell shape change induced upon anaerobiosis is crucial to the development of effective treatments against<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">P. aeruginosa</jats:named-content>biofilm infection. Here, we uncovered the molecular basis of anaerobiosis-triggered cell elongation and identified vitamin B<jats:sub>12</jats:sub>to be a molecule that can reinstate defective anaerobic growth of<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">P. aeruginosa</jats:named-content>. The ratio of total cellular DNA content to protein content was significantly decreased in the PAO1 strain grown under anaerobic conditions, indicating that DNA replication is impaired during anaerobic growth. Anaerobic growth of PAO1 reached a higher cell density in the presence of vitamin B<jats:sub>12</jats:sub>, an essential coenzyme of class II ribonucleotide reductase. In addition, cell morphology returned to a normal rod shape and transcription of stress-response genes was downregulated under the same anaerobic growth conditions. These results suggest that vitamin B<jats:sub>12</jats:sub>, the production of which was suppressed during anaerobic growth, can restore cellular machineries for DNA replication and therefore facilitate better anaerobic growth of<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">P. aeruginosa</jats:named-content>with normal cell division. Importantly, biofilm formation was substantially decreased when grown with vitamin B<jats:sub>12</jats:sub>, further demonstrating that anaerobiosis-induced cell elongation is responsible for robust biofilm formation. Taken together, our data reveal mechanistic details of a morphological change that naturally occurs during anaerobic growth of<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">P. aeruginosa</jats:named-content>and illustrates the ability of vitamin B<jats:sub>12</jats:sub>to modulate the biofilm-forming capacity of<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">P. aeruginosa</jats:named-content>under such condition.</jats:p>

cbb 3 -Type Cytochrome c Oxidases, Aerobic Respiratory Enzymes, Impact the Anaerobic Life of Pseudomonas aeruginosa PAO1

“…4A), suggesting that cbb 3 oxidases inhibit RNR II activity, presumably through NO. Vitamin B 12 , a coenzyme of RNR II, has been reported to restore defective anaerobic growth caused by NO (19). In the absence of vitamin B 12 , the ⌬ccoN1 ⌬ccoN2 mutant grew better than the WT.…”

“…NO derived from denitrification inhibits DNA synthesis by ribonucleotide reductase (RNR), especially class II-type RNR (RNR II), during anaerobic growth of P. aeruginosa PAO1 (19). Because RNR catalyzes the formation of deoxyribonucleotides from ribonucleotides (29), cellular dNTP levels in the WT and the ⌬ccoN1 ⌬ccoN2 mutant were quantified by a DPA assay.…”

“…Quantification of the cellular levels of deoxynucleoside triphosphates (dNTPs) in cell extracts was performed based on a previously described method (19). Anoxic incubation of P. aeruginosa was performed using butyl-rubber-sealed 500-ml Erlenmeyer flasks containing 80 ml of LBN medium at 37°C with shaking at 200 rpm.…”

Cholera Toxin Production during Anaerobic Trimethylamine N-Oxide Respiration Is Mediated by Stringent Response in Vibrio cholerae

“…Flow Cytometry Analysis-The average protein or DNA content per single bacterial cell was measured following procedures described previously (29).…”

Functional Screening of a Metagenomic Library Reveals Operons Responsible for Enhanced Intestinal Colonization by Gut Commensal Microbes

“…The quantity of purified DNA was assessed spectrophotometrically, and 400 ng of DNA was used as a template to amplify the E. coli 16S rRNA gene, Bacteroidetes phylum 16S rRNA gene, ORF20 in the 10a operon, and ORF13 in the 25b operon. The real-time PCR was performed as described previously (20), and primers used for the PCR are listed in the supplemental material.…”