TgpA, a Protein with a Eukaryotic-Like Transglutaminase Domain, Plays a Critical Role in the Viability of <em>Pseudomonas aeruginosa</em>

<div><p>The Gram-negative bacterium <em>Pseudomonas aeruginosa</em> is an important opportunistic pathogen in compromised individuals, such as patients with cystic fibrosis, severe burns or impaired immunity. In this work we aimed to screen novel essential genes of <em>P. aeruginosa</em> by shotgun antisense identification, a technique that was developed a decade ago for the Gram-positive bacterium <em>Staphylococcus aureus</em> and was under-used in Gram-negative bacteria for a considerable period of time. Following antisense screenings in the PAO1 strain of <em>P. aeruginosa</em>, we focused on a <em>locus</em>, PA2873, which was targeted by an antisense RNA construct that can impair cell growth. The PA2873 gene product was annotated as a hypothetical membrane protein endowed with a periplasmic region harbouring a structural domain belonging to the transglutaminase-like superfamily, a group of archaeal, bacterial and eukaryotic proteins homologous to animal transglutaminases. In this study, we show that the periplasmic portion of the PA2873 protein, which we named TgpA, does possess transglutaminase activity <em>in vitro</em>. This is the first report of transglutaminase activity in <em>P. aeruginosa</em>. In addition, we have provided strong evidences that TgpA plays a critical role in the viability of <em>P. aeruginosa</em>.</p> </div

Analysis of the growth impairment elicited by the M4G6 insert resulting from SALs screenings.

<p><i>E. coli</i> donors harbouring the pVI533-based vectors pVI-M4G6 and pVI-M4G6i, which carry downstream <i>P_BAD</i> promoter the M4G6 insert in antisense and sense orientation, respectively, were mated with <i>P. aeruginosa</i> PAO1 and exconjugants were spotted onto PIA to counterselect <i>E. coli</i> cells. The medium was also supplemented with carbenicillin to select for pVI533 maintenance. As a control, empty pVI533 was transferred to PAO1 with the same procedure. Induction of <i>P_BAD</i> promoter was achieved through the addition of 7.5 mM arabinose (ara). A similar protocol, with the only variant of M9-citrate for donor counterselection, was used for the transfer to PAO1 of pVLT31-based vectors pVLT31-M4G6 and pVLT31-M4G6i, and empty pVLT31. Induction of pVLT31 <i>P_tac</i> promoter was achieved through the addition of 1 mM IPTG.</p

Predicted domain organization and transglutaminase activity of TgpA protein.

<p>(A) Map of the predicted domains DUF3488 (PF11992) and TG (PF01841) along the primary sequence of the PA2873 gene product, called TgpA. The sequence of TgpA spanning aa 396 to 467 of the TG domain is highlighted and aligned to homologous functional TG domains of human coagulation Factor XIII, fish-derived transglutaminase (FTG) and WbmE protein from <i>B. bronchiseptica</i>. Conserved aminoacids of catalytic triad are indicated by an asterisk. (B) Colorimetric assay of transglutaminase activity of purified TgpA TG_180–544 domain by Transglutaminase Colorimetric Microassay Kit (TCM kit; Covalab). TCM kit uses immobilized N-carbobenzoxy(CBZ)-Gln-Gly as the amine acceptor and biotin-conjugated cadaverine as the amine donor. The indicated amounts of purified TgpA TG_180–544 (stock: 2.7 mg/ml, 95% purity) were incubated in 96-well microtiter plate coated with CBZ-Gln-Gly at 37°C for 15 min with calcium, DTT and biotinylated cadaverine, both in the presence and the absence of EDTA supplied in the kit. As a reference for TGase activity, the indicated amounts of kit-included purified guinea pig TGase with specific activity of 0.1 U/mg were incubated under the same conditions. The wells were washed extensively and filled with streptavidin-labelled horseradish peroxidase (HRP) to assay the formation of immobilized γ-glutamyl-cadaverine-biotin by OD₄₅₀ measurement of HRP activity using H₂O₂ as substrate and tetramethyl benzidine as electron acceptor (chromogen).</p

Mutagenesis analysis of the PA2875-2874- <i>tgpA</i> gene cluster.

<p>(A) Each indicated <i>locus</i> was targeted for knock-out by homologous recombination-mediated cointegration of the suicide vector pDM4 carrying chloramphenicol resistance (Cm^R). The <i>dnaG</i> gene for DNA primase and the <i>algR</i> gene for a LytTR-type two-component response regulator were used respectively as positive and negative controls of essentiality. Cointegration targeting was achieved by cloning internal 600–800 bp fragments of PA2875, PA2874, <i>tgpA</i>, <i>dnaG</i> and <i>algR</i>, respectively, into pDM4. The resulting constructs were transferred from <i>E. coli</i> S17-λpir to PAO1 by conjugation, selecting cointegration events by plating the conjugation mixtures on PIA supplemented with chloramphenicol. Three independent conjugation experiments were performed. Efficiency of cointegration in a given <i>locus</i> is expressed as a percentage of Cm^R ex-conjugant colonies relative to the negative control <i>algR</i>. (B) The rhamnose inducible/glucose repressible promoter <i>P_rhaB</i> was inserted upstream to <i>tgpA</i> giving rise to PAO1 <i>P_rhaB</i>::<i>tgpA</i> strain. To test the repression effects of glucose on growth rate, overnight cultures of PAO1 <i>P_rhaB</i>:: <i>tgpA</i> in M9-citrate supplemented with rhamnose were diluted to OD₆₀₀ = 10⁻⁶ and inoculated in microtiter wells filled with 200 µl of M9-citrate supplemented with either rhamnose or glucose. Culture growth at 37° with stirring was monitored in real-time by OD₆₀₀ measurement in a microtiter reader for 21 hrs. Specificity of glucose/rhamnose effects on the growth of PAO1 <i>P_rhaB</i>::<i>tgpA</i> was assessed by monitoring the PAO1 cultures in M9-citrate supplemented with either rhamnose or glucose. Note the opposite effects of glucose on growth of PAO1 <i>P_rhaB</i>::PA2873 and PAO1, respectively. In the insert, overnight cultures of PAO1 <i>P_rhaB</i>::<i>tgpA</i> in M9-citrate supplemented with rhamnose were also tested for growth on solid M9-citrate supplemented with either rhamnose or glucose, by spotting 2 µl of 10-fold serial dilutions, from OD₆₀₀ = 1 (left) to OD₆₀₀ = 10⁻⁶ (right). (C) During the first 7 hrs from inoculum, a time window in which growth rate was undetectable by microtiter reader, the growth of PAO1 <i>P_rhaB</i>::<i>tgpA</i> in liquid M9-citrate supplemented with either rhamnose or glucose was monitored by titration of colony-forming units per ml (CFU/ml) on LB plates.</p

Genetic organization and transcription analysis of the genomic region including PA2873 <i>locus</i> (<i>tgpA</i>).

<p>(A) PA2875-2874-2873-2872 gene cluster is represented according to visualization by GBrowse in Pseudomonas Genome Database. Locations of fragments amplified by the oligo pairs (Roman numbers) used in RT-PCR-based transcription analysis (B) are shown along the region map. The position of plasmid pDM4 cointegration in PAO1 PA2875::pDM4 strain is indicated. (B) Total RNA was extracted from PAO1 and PAO1 PA2875::pDM4 cells in both exponential (E) and stationary (S) phases, and analyzed by RT-PCR. RT untreated samples (RT−) as controls of genomic DNA contamination were included in the analysis.</p