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

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

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

Determination of the Molecular Weight of Low-Molecular-Weight Heparins by Using High-Pressure Size Exclusion Chromatography on Line with a Triple Detector Array and Conventional Methods

The evaluation of weight average molecular weight (Mw) and molecular weight distribution represents one of the most controversial aspects concerning the characterization of low molecular weight heparins (LMWHs). As the most commonly used method for the measurement of such parameters is high performance size exclusion chromatography (HP-SEC), the soundness of results mainly depends on the appropriate calibration of the chromatographic columns used. With the aim of meeting the requirement of proper Mw standards for LMWHs, in the present work the determination of molecular weight parameters (Mw and Mn) by HP-SEC combined with a triple detector array (TDA) was performed. The HP-SEC/TDA technique permits the evaluation of polymeric samples by exploiting the combined and simultaneous action of three on-line detectors: light scattering detectors (LALLS/RALLS); refractometer and viscometer. Three commercial LMWH samples, enoxaparin, tinzaparin and dalteparin, a γ-ray depolymerized heparin (γ-Hep) and its chromatographic fractions, and a synthetic pentasaccharide were analysed by HP-SEC/TDA. The same samples were analysed also with a conventional HP-SEC method employing refractive index (RI) and UV detectors and two different chromatographic column set, silica gel and polymeric gel columns. In both chromatographic systems, two different calibration curves were built up by using (i) γ-Hep chromatographic fractions and the corresponding Mw parameters obtained via HP-SEC/TDA; (ii) the whole γ-Hep preparation with broad Mw dispersion and the corresponding cumulative distribution function calculated via HP-SEC/TDA. In addition, also a chromatographic column calibration according to European Pharmacopoeia indication was built up. By comparing all the obtained results, some important differences among Mw and size distribution values of the three LMWHs were found with the five different calibration methods and with HP-SEC/TDA method. In particular, the detection of the lower molecular weight components turned out to be the most critical aspect. Whereas HP-SEC/TDA may underestimate species under 2 KDa when present in low concentration, other methods appeared to emphasize their content

Naked Bacterium: Emerging Properties of a Surfome-Streamlined Pseudomonas putida Strain

Environmental bacteria are most often endowed with native surface-attachment programs that frequently conflict with efforts to engineer biofilms and synthetic communities with given tridimensional architectures. In this work, we report the editing of the genome of Pseudomonas putida KT2440 for stripping the cells of most outer-facing structures of the bacterial envelope that mediate motion, binding to surfaces, and biofilm formation. To this end, 23 segments of the P. putida chromosome encoding a suite of such functions were deleted, resulting in the surface-naked strain EM371, the physical properties of which changed dramatically in respect to the wild type counterpart. As a consequence, surface-edited P. putida cells were unable to form biofilms on solid supports and, because of the swimming deficiency and other alterations, showed a much faster sedimentation in liquid media. Surface-naked bacteria were then used as carriers of interacting partners (e.g., Jun–Fos domains) ectopically expressed by means of an autotransporter display system on the now easily accessible cell envelope. Abstraction of individual bacteria as adhesin-coated spherocylinders enabled rigorous quantitative description of the multicell interplay brought about by thereby engineered physical interactions. The model was then applied to parametrize the data extracted from automated analysis of confocal microscopy images of the experimentally assembled bacterial flocks for analyzing their structure and distribution. The resulting data not only corroborated the value of P. putida EM371 over the parental strain as a platform for display artificial adhesins but also provided a strategy for rational engineering of catalytic communities.This work was funded by the SETH (RTI2018-095584-B-C42) (MINECO/FEDER), SyCoLiM (ERA-COBIOTECH 2018—PCI2019-111859-2) Project of the Spanish Ministry of Science and Innovation. It was also funded by MADONNA (H2020-FET-OPEN-RIA-2017-1-766975), BioRoboost (H2020-NMBP-BIO-CSA-2018-820699), SynBio4Flav (H2020-NMBP-TR-IND/H2020-NMBP-BIO-2018-814650), and MIX-UP (MIX-UP H2020-BIO-CN-2019-870294) Contracts of the European Union and the InGEMICS-CM (S2017/BMD-3691) Project of the Comunidad de Madrid—European Structural and Investment Funds (FSE, FECER)