Microbiology (2011), 157, 3079–3093 DOI 10.1099/mic.0.052449-0 The Bdellovibrio bacteriovorus twin-arginine transport system has roles in predatory and prey-independent growth Chien-Yi Chang,1 Laura Hobley,1 Rob Till,1 Michael Capeness,1 Machi Kanna,23 William Burtt,1 Pratik Jagtap,34 Shin-Ichi Aizawa2 and R. Elizabeth Sockett1 Correspondence 1Institute of Genetics, School of Biology, University of Nottingham Medical School, R. Elizabeth Sockett Nottingham NG7 2UH, UK [email protected] 2Prefectural University of Hiroshima, 562 Nanatsuka, Shobara, Hiroshima 727-0023, Japan 3Max-Planck Institute for Developmental Biology, 72076 Tu¨bingen, Germany Bdellovibrio bacteriovorus grows in one of two ways: either (i) predatorily [in a host-dependent (HD) manner], when it invades the periplasm of another Gram-negative bacterium, exporting into the prey co-ordinated waves of soluble enzymes using the prey cell contents for growth; or (ii) in a host-independent (HI) manner, when it grows (slowly) axenically in rich media. Periplasmic invasion potentially exposes B. bacteriovorus to extremes of pH and exposes the need to scavenge electron donors from prey electron transport components by synthesis of metalloenzymes. The twin-arginine transport system (Tat) in other bacteria transports folded metalloenzymes and the B. bacteriovorus genome encodes 21 potential Tat-transported substrates and Tat transporter proteins TatA1, TatA2 and TatBC. GFP tagging of the Tat signal peptide from Bd1802, a high-potential iron–sulfur protein (HiPIP), revealed it to be exported into the prey bacterium during predatory growth. Mutagenesis showed that the B. bacteriovorus tatA2 and tatC gene products are essential for both HI and HD growth, despite the fact that they partially complement (in SDS resistance assays) the corresponding mutations in Escherichia coli where neither TatA nor TatC are essential for life. The essentiality of B. bacteriovorus TatA2 was surprising given that the B. bacteriovorus genome encodes a second tatA homologue, tatA1. Transcription of tatA1 was found to be induced upon entry to the bdelloplast, and insertional inactivation of tatA1 showed that it significantly slowed the rates of both HI and HD growth. Received 22 June 2011 B. bacteriovorus is one of a few bacterial species that are reliant on a functional Tat system and Revised 24 August 2011 where deletion of a single tatA1 gene causes a significant growth defect(s), despite the presence Accepted 1 September 2011 of its tatA2 homologue. INTRODUCTION system in bacteria have a consensus twin arginine motif S/T-R-R-X-F-L-K (where X is any polar amino acid), a The twin-arginine translocation (Tat) system transports hydrophobic region and an A-x-A cleavage recognition folded proteins across the cytoplasmic membrane and is motif in their N-terminal leading sequence (Berks, 1996; found in a wide variety of bacteria, whilst homologous Stanley et al., 2000). Tat-transported proteins are typically systems are found in both archaea and eukaryotes (Sargent, involved in metabolism, redox reactions, metal acquisition 2007a; Yuan et al., 2010). Proteins transported by the Tat and cell envelope maintenance (Berks et al., 2005; Palmer et al., 2005) and are involved in pathogenesis, symbiosis, 3Present address: Faculty of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima-shi, Hiroshima 739-8527, Japan. quorum sensing and motility (Sargent, 2007a; Stevenson et al., 2007). 4Present address: Minnesota Supercomputing Institute, University of Minnesota, 117 Pleasant Street SE, Minneapolis, MN 55455, USA. In Escherichia coli, a functional Tat system consists of Abbreviations: eGFP, enhanced GFP; HD, host-dependent; HI, host- TatABC (the genes for which are encoded in an operon) independent. and the TatA homologue TatE (Sargent et al., 1998; Weiner Two supplementary tables and two supplementary figures are available et al., 1998). TatB and TatC form a complex in E. coli with the online version of this paper. (Bolhuis et al., 2001) which recognizes the signal motif of a Downloaded from www.microbiologyresearch.org by 052449 G 2011 SGM Printed in Great Britain 3079 IP: 54.70.40.11 On: Mon, 15 Oct 2018 23:12:00 C.-Y. Chang and others Tat substrate, whereupon the TatA proteins associate to systems is higher than for other free-living bacteria. A form a channel complex of 12–35 monomers, associated systematic review by Barabote et al. (2007) predicted at with TatBC (Alami et al., 2003; Dabney-Smith et al., 2006; least four types of cytoplasmic membrane transportation Gohlke et al., 2005; Holzapfel et al., 2007). The Tat systems and five types of outer membrane secretion systems. substrate is transported across the membrane via the TatA Genes encoding the Tat system have been predicted in the channel and released by proteolytic cleavage at the B. bacteriovorus genome (Barabote et al., 2007; Rendulic C-terminal end of the signal peptide. TatA then disas- et al., 2004), including tatB and tatC as well as two tatA sociates from the TatBC complex to reset the process homologues. In this study we show that B. bacteriovorus (Berks et al., 2005; Lee et al., 2006; Sargent, 2007a). Un- or expresses a functional Tat system required for both mis-folded proteins are rejected then targeted for either predatory [prey/host-dependent (HD)] and axenic [prey/ degradation or refolding (DeLisa et al., 2002; Lee et al., host-independent (HI)] growth and that at least one protein 2006; Sargent, 2007a). containing a Tat signal motif is exported from B. bacteriovorus into the cytoplasm of the prey bdelloplast Deletion of tatC in E. coli abolishes Tat system function during predatory growth. We show that deletion of tatA1 (Bogsch et al., 1998) and whilst TatA and TatB share 25 % slows both predatory and axenic growth, delays gene sequence identity, each is essential for full Tat function, expression of some potential Tat substrates and stops the suggesting that they play different roles (Sargent et al., export of Tat substrate protein Bd1802 into the prey during 2001). Alterations in the E. coli TatA protein sequence predation. This shows that B. bacteriovorus TatA1 and TatA2 allow it to function as both TatA and TatB in a TatBE are not interchangeable and are responsible for the transport deletion strain (Blaudeck et al., 2005). TatE has 60 % of different substrates, and that Tat-transported substrates identity with TatA (Lee et al., 2006) and in aerobically can be further exported beyond the B. bacteriovorus outer grown E. coli, tatE is expressed at very low levels compared membrane into the surrounding bdelloplast, possibly with tatA and the deletion of tatE has no noticeable effect contributing to predatory processes. on Tat substrate transport or viability of planktonically grown cells (Jack et al., 2001). However, in E. coli biofilms, tatE transcription is induced up to 2.12 times more than in METHODS planktonic cultures (Beloin et al., 2004). Only in an E. coli tatA tatE double mutant was the Tat pathway blocked in all Bacterial strains, plasmids and culture conditions. All bacterial conditions (Sargent et al., 1998); but TatE in Pseudomonas strains and plasmids used and created in this study are listed in Supplementary Table S1 (available with the online version of this stutzeri is specifically required to transport proteins for paper). E. coli strains were grown in yeast extract and tryptone (YT) denitrification (Heikkila¨ et al., 2001). This and other broth (Lambert & Sockett, 2008) at 29 uC with shaking at 200 r.p.m. studies suggest that second TatA homologues (TatEs), in for 16 h to give late-exponential-phase cultures for use as prey. E. coli diverse bacteria, play an important role in transportation tat mutants containing expression plasmids were grown in the 2 under certain growth conditions (Bru¨ser, 2007). presence of 50 ml ampicillin ml 1 and 100 nM IPTG. E. coli S17-1 (Simon et al., 1983) was routinely used as prey for growth of B. Bdellovibrio bacteriovorus (Stolp & Starr, 1963) is a small bacteriovorus in predatory cultures at 29 uC with shaking at 200 r.p.m. Gram-negative bacterium which preys upon other Gram- (Hobley et al., 2006), which typically contained 50 ml Ca/HEPES negative bacteria. B. bacteriovorus attaches to, penetrates buffer (2 mM CaCl2, 25 mM HEPES pH 7.6), 3 ml of a late- through the outer layers of and grows within the periplasm exponential-phase E. coli culture and 1 ml of a B. bacteriovorus predatory culture (which had completed prey lysis and contained of prey bacteria. B. bacteriovorus modifies the prey cell wall typically 2.56108 p.f.u. ml21). Antibiotic-resistant insertion-mutant B. forming an osmotically stable structure called the bdello- bacteriovorus were grown with E. coli S17-1 carrying an appropriate plast, which is bounded by an intact prey outer membrane. plasmid as prey with the antibiotics at the following concentrations: Inside this, B. bacteriovorus delivers enzymes both to the kanamycin sulphate, 50 mgml21, S17-1 pZMR100 (Rogers et al., 1986); 2 prey periplasm and (at early stages of predation) via the apramycin sulphate, 50 mgml 1, S17-1 pUC19:apraR; both antibiotics, (still intact) prey cell inner membrane to the cytoplasm to S17-1 pLH003. HI derivatives of B. bacteriovorus strains were isolated and grown at 29 uC on peptone and yeast extract (PY) plates or in PY degrade the cytoplasmic contents of the prey cell. Prey- broth with shaking at 200 r.p.m. as described previously (Evans et al., derived nutrients facilitate B. bacteriovorus growth and 2007; Shilo & Bruff, 1965). reproduction and B. bacteriovorus elongates into a filamen- tous cell. After consuming the bdelloplast interior, the B. bacteriovorus predation assays using E. coli MC4100 and filament divides into multiple progeny B. bacteriovorus B1LK0-P (DtatC) as prey. E. coli strains MC4100 (Casadaban & which become flagellate and escape through the bdelloplast Cohen, 1979) and B1LK0-P (Buchanan et al., 2002) were grown as outer membrane to carry out further predation (Sockett, described above to late exponential phase, then diluted (in cell-free supernatant from the same cultures) and matched to OD600 1.5.