RETRACTED ARTICLE: A switch in the poly(dC)/RmlB complex regulates bacterial persister formation

Bacterial persisters are phenotypic variants that tolerate exposure to lethal antibiotics. These dormant cells are responsible for chronic and recurrent infections. Multiple mechanisms have been linked to persister formation. Here, we report that a complex, consisting of an extracellular poly(dC) and its membrane-associated binding protein RmlB, appears to be associated with persistence of the opportunistic pathogen Pseudomonas aeruginosa. Environmental stimuli triggers a switch in the complex physiological state (from poly(dC)/RmlB to P-poly(dC)/RmlB or RmlB). In response to the switch, bacteria decrease proton motive force and intracellular ATP levels, forming dormant cells. This alteration in complex status is linked to a (p)ppGpp-controlled signaling pathway that includes inorganic polyphosphate, Lon protease, exonuclease VII (XseA/XseB), and the type III secretion system. The persistence might be also an adaptive response to the lethal action of the dTDP-l-rhamnose pathway shutdown, which occurs due to switching of poly(dC)/RmlB.

Labelling the common binding site with P-poly(dC 12 )* or poly(dC 12 )* Label with P-poly(dC 12 )*: Washed (s) WT cells were resuspended in an equal volume phosphate buffer saline (PBS) plus excessive P-poly(dC 12 )* at room temperature for 20 min. After washing off free P-poly(dC 12 )*, the cells were resuspended in an equal volume PBS plus 1 % formaldehyde at 37 °C for 15 min. Cross-linking was terminated by addition of 0.125 M Glycine at 37 °C for 5 min.
Label with poly(dC 12 )*: Washed (s) WT cells were resuspended in an equal volume PBS plus excessive poly(dC 12 )* at room temperature for 20 min. The mixture was incubated with T4 polynucleotide kinase at 37 °C for 1 h for transforming poly(dC 12 )* to Ppoly(dC 12 )*. After washing off free P-poly(C 12 )*, the cells were resuspended in an equal volume PBS plus 1 % formaldehyde at 37 °C for 15 min. Cross-linking was terminated by addition of 0.125 M Glycine at 37 °C for 5 min.

2D-PAGE
Total membrane protein was analyzed by conventional 2D-PAGE. The gels were stained with silver nitrate for detection of separated protein. Meanwhile, the gels were scanned by Typhoon Trio-Variable Mode Imager (GE Healthcare) under the excitation of 650 nm for detection of fluorescent labeled protein. After comparative analysis of the digitized gel and the silver staining images, the fluorescent labeled spots were manually picked.

Expression and purification of His-tagged XseA
The xseA gene was amplified by PCR using primer pairs PF xseA1 to which was added an EcoR I site (underlined) (5'-G GAA TTC ATG CGT AAC GAT CCC TTC CAA CGG-3') and PR xseA1 to which was added a Xho I site (underlined) (5'-CCG CTC GAG GTC CAG CAG CGA CAG GGT CAC CGG-3'). The amplified fragment was ligated into pET29a and the resulting plasmid pET29a-xseA was transformed into E. coli BL21 (DE3) pLysS. 1.0 mM IPTG was used to induce expression at 28 °C for 4 h. His-tagged XseA was purified by a 2-ml volume of NTA-Ni 2+ agarose (Qiagen) and stored in 10 mM Tris-HCl buffer (pH 7.5) plus 10 % glycerol at 4 °C.

Expression and purification of His-tagged XseB
The xseB gene was amplified by PCR using primer pairs PF xseB to which was added a Nde I site (underlined) (5'-G GAA TTC CAT ATG ATG GCC CGC AAG AAA ACC CTC  and PR xseB to which was added a Xho I site (underlined) (5'-CCG CTC GAG TGC CTC GTC GCC TTC CGC ATC GAA-3'). The amplified fragment was ligated into pET29a and the resulting plasmid pET29a-xseB was transformed into E. coli BL21 (DE3) pLysS. 1.0 mM IPTG was used to induce expression at 28 °C for 2 h. His-tagged XseB was purified by a 2-ml volume of NTA-Ni 2+ agarose (Qiagen), desalinated by Ultracel PLAC (1-kDa cutoff size), and stored in 10 mM Tris-HCl buffer (pH 7.5) plus 10 % glycerol at 4 °C.

Expression and purification of His-tagged Lon
The lon gene was amplified by PCR using primer pairs PF lon to which was added an EcoR I site (underlined) (5'-G GAA TTC ATG AAA ACA CTC GTC GAA TT-3') and PR lon to which was added a Hind III site (underlined) (5'-CCC AAG CTT ATG CGT GCT AAT TCG CTC CTT-3'). The amplified fragment was ligated into pET29a and the resulting plasmid pET29alon was transformed into E. coli BL21 (DE3) pLysS. 1.0 mM IPTG was used to induce expression at 28 °C for 4 h. His-tagged Lon was purified by a 2-ml volume of NTA-Ni 2+ agarose (Qiagen) and stored in 10 mM Tris-HCl buffer (pH 7.5) plus 10 % glycerol at 4 °C.

Construction of pBBR1MCS3-borne fusion of rmlB with mCherry (RFP)
For insertion of a Hpa I site into the mob region of pBBR1MCS3, the plasmid sequence was amplified using primer pairs PF HpaI to which was added a Hpa I site (underlined) (5′-GTT AAC TGT GCG CCT TCT CGA AGA ACG CCG CCT GCT-3′) and PR HpaI (5′-AGT GGC TGG CGG ACA AGT ACG GGG CGG ATC-3′) and PrimeSTAR Max DNA polymerase (Takara). The amplified fragment was phosphorylated and self-ligated to form plasmid pBBR1MCS3a. The rmlB gene along with the 500 bp upstream of its start codon including the promoter was amplified from P. aeruginosa PAO1 genome using primer pairs PF rmlB2 to which was added a Xho I site (underlined) (5′-CCG CTC GAG TCC ACC ATC GAC TAC TTC ACC ATG CTC-3′) and PR rmlB2 to which was added a Xba I site (underlined) (5′-GC TCT AGA TGC GTA CTG CTT ACC CAC CCA CTC GCG-3′). The amplified fragment was ligated into pBBR1MCS3a to form plasmid pBBR1MCS3a-rmlB.
The mCherry (rfp) gene was amplified from plasmid pYC12-mCherry using primer pairs PF rfp to which was added a Xba I site (underlined) (5′-GC TCT AGA ATG GTG AGC AAG GGC GAG GAG GAT AAC-3′) and PR rfp to which was added a Sac I site (underlined) (5′-C GAG CTC CTA CTT GTA CAG CTC GTC CAT GCC GCC-3′). The amplified fragment was ligated into pBBR1MCS3a-rmlB to form plasmid pBBR1MCS3a-rmlB-rfp.

Construction of a rmlB asRNA expression cassette
A 218 bp fragment containing a 38 bp paired termini (PT) sequence 2,3 , a 44 bp upstream of the start codon of rmlB and the first 76 bp of the open reading frame itself was synthesized and inserted into the multiple cloning site between Pst I and Hind III of pBAD-His A to form plasmid pBAD-rmlBPTasRNA. The rmlB asRNA expression cassette containing araC gene, araBAD promoter, rmlBPTasRNA, and rrnB transcription termination region was amplified from the plasmid using primer pairs PF cassette1 (5′-ACA CCC GCT GAC GCG CCC TGA CGG GCT-3′) and PR cassette1 (5′-AAG GCC CAG TCT TTC GAC TGA GCC TTT-3′). The pBBR1MCS3a-rmlB-rfp was digested with Hpa I and then dephosphorylated by alkaline phosphatase (calf intestine). The amplified cassette was ligated with the linear pBBR1MCS3a-rmlB-rfp and the resulting plasmid pBBR1MCS3a-X1 was transformed into P. aeruginosa PAO1.
(b) 2D-PAGE of total membrane protein extracts (left). The fluorescent labeled protein spots were shown by red circles (right). As a control, a non-neutralizing ssDNA* (the poly(dC 12    , Significance increase or decrease in gene expression was determined by the value of more than 1.5 or less than 0.7. Abundance ratio was calculated by using the averaged spectrum counts from biological triplicates.  a , Since DNA phosphorylation or tailor (from 5' terminus) can in-situ switch poly(dC) to P-poly(dC), the protein responsible for the switching should be a phosphorylase, a nucleotide kinase, or a deoxyribonuclease. b , Candidate protein screening criteria: I) The molecular weight (MW) of candidate protein is less than about 10-kDa.

Supplementary
Although the nominal MW limit of Ultracel-3K Centrifugal Filter Units is 3-kDa, some proteins of 3 to 10-kDa cannot be trapped actually due to molecular shape or other reasons. Large proteins (MW more than 10-KDa) can be trapped completely by Ultracel-3K Units. Thus, detection of peptide fragments of large protein in filtrates does not mean the presence of integral large protein in filtrates. These peptide fragments may be derived from the natural degradation of large protein. II) The candidate protein is only present in the filtrate from Ultracel-3K-treated (d) WT supernatant rather than in the filtrates from Ultracel-3K-treated (e) and (s) WT supernatants.