Functional & Integrative Genomics

, Volume 9, Issue 2, pp 153–166

Bacterial intein-like domains of predatory bacteria: a new domain type characterized in Bdellovibrio bacteriovorus

  • Mally Dori-Bachash
  • Bareket Dassa
  • Ofer Peleg
  • Silvia A. Pineiro
  • Edouard Jurkevitch
  • Shmuel Pietrokovski
Original Paper

DOI: 10.1007/s10142-008-0106-7

Cite this article as:
Dori-Bachash, M., Dassa, B., Peleg, O. et al. Funct Integr Genomics (2009) 9: 153. doi:10.1007/s10142-008-0106-7

Abstract

We report a new family of bacterial intein-like domains (BILs) identified in ten proteins of four diverse predatory bacteria. BILs belong to the HINT (Hedgehog/Intein) superfamily of domains that post-translationally self-process their protein molecules by protein splicing and self-cleavage. The new, C-type, BILs appear with other domains, including putative predator-specific domain 1 (PPS-1), a new domain typically appearing immediately upstream of C-type BILs. The Bd2400 protein of the obligate predator Bdellovibrio bacteriovorus includes a C-type BIL and a PPS-1 domains at its C-terminal part, and a signal peptide and two polycystic kidney disease domains at its N-terminal part. We demonstrate the in vivo transcription, translation, secretion, and processing of the B. bacteriovorus protein, and the in vitro autocatalytic N-terminal cleavage activity of its C-type BIL. Interestingly, whereas the Bd2400 gene is constitutively expressed, its protein product is differentially processed throughout the dimorphic life cycle of the B. bacteriovorus predator. The modular structure of the protein, its localization, and complex processing suggest that it may be involved in the interaction between the predator and its prey.

Keywords

ModificationsProtein dynamicsBioinformaticsComparative genomics

Supplementary material

10142_2008_106_MOESM1_ESM.doc (46 kb)
Supplementary Table 1Sequences and masses of Bd2400 peptides identified by LC–MS/MS. Masses include modifications on cysteine or methionine residues. Spots from the 2D analysis of the extracellular fraction were picked and grouped together in eleven groups according to their molecular weight (MW) and their intensity. Each group was submitted individually to in-gel proteolysis and liquid chromatography tandem mass spectrometry (LC–MS/MS) that were performed at the Smoler Proteomics Center of the Technion, Israel. Proteins were in-gel digested with trypsin and dehydrated with acetonitrile, then rehydrated with 10% acetonitrile in 10 mM ammonium bicarbonate containing trypsin [modified trypsin (Promega) at a 1:100 enzyme-to-substrate ratio]. Gel pieces were incubated overnight at 37°C and the resulting peptides were recovered and analyzed. Tryptic peptides were resolved by reverse-phase chromatography on 0.1× 200-mm fused silica capillaries (J&W, 100 μm ID) packed with Everest reversed phase material (Grace Vydac). The peptides were eluted with linear 50 min gradients of 5% to 95% of acetonitrile with 0.1% formic acid in water at flow rates of 0.4 μl/min. Mass spectrometry was performed by an ion-trap MS (LCQ-DecaXP, Finnigan) in a positive mode using repetitively full MS scan followed by collision induces dissociation (CID) of the three most dominant ions selected from the first MS scan. The MS data were clustered and analyzed using the Pep-Miner searching the bacteria database (Beer et al. 2004). (DOC 45.5 KB)
10142_2008_106_MOESM2_ESM.doc (21 kb)
Supplementary Table 2Primers used in this study. (DOC 20.5 KB)
10142_2008_106_MOESM3_ESM.doc (33 kb)
Supplementary Table 3Proteins detected by LC–MS/MS from isolated spots reacting with an anti-BIL antibody in a two-dimensional gel electrophoresis. (DOC 30.0 KB)
10142_2008_106_MOESM4_ESM.ppt (198 kb)
Supplementary Figure 1Bioinformatic analysis of BIL domains. (A) Conserved motifs of HINT protein domains of the three known BIL families. Each row shows conserved motifs of a BIL family. Motifs are ordered left to right in the N′ to C′ positions along the protein sequences. Similar motifs are vertically aligned, unique motifs are marked with dashed boxes. The motifs are shown as sequence logos, where the height of amino acids are proportional to their conservation in each position. N1, N2, N3, N4, C1 and C2 are HINT protein motifs, as described in (Pietrokovski 2001). (B) Phylogenetic tree of the three known BIL families. Generated by PhyML using the default parameters (1,000 bootstraps), from the conserved BIL sequence motifs (54 aligned amino acids) of 143 sequences. (PPT 198 KB)
10142_2008_106_MOESM5_ESM.ppt (287 kb)
Supplementary Figure 2Conserved sequence motifs of PPS-1 domains in predatory bacteria. The PPS-1 conserved cysteine positions are marked with asterisks. Identical residues are shown in black and similar (80%) residues in grey. Sequence coordinates are marked at the right. Species notations of the proteins are Bdellovibrio bacteriovorus (Bd), Bacteriovorax marinus (BMS), Myxococcus xanthus (MXAN), Stigmatella aurantica DW4 3-1 (STIAU). (PPT 729 KB)
10142_2008_106_MOESM6_ESM.ppt (174 kb)
Supplementary Figure 3Schematic diagram of the experimental design in this work. B. bacteriovorus wt and HI-6 cultures were sampled at different life-cycle stages and further analyzed. All lines represent cellular samples, unless indicated otherwise. Since growth kinetics differ, the T1, T2 and T3 samples were not taken at the same time in the wt and the HI-6 cultures (see materials and methods). (PPT 729 KB)
10142_2008_106_MOESM7_ESM.ppt (136 kb)
Supplementary Figure 4Changes in RNA content during the growth cycle and RNA degradation by RNase I. (A) RNA was extracted from the growth phase of the wt strain at 1 (lane 1), 2 (lane 2) and 3 (lane 3) h after mixing the B. bacteriovorus predator and its E. coli prey in a synchronous culture. Lane 4: 1 kb DNA ladder. The same volume of extract was loaded in each lane to show that more RNA is present in the early bdelloplasts. As the cycle proceeds, prey RNA is degraded and its concentration decreases. (B) Confirmation that the massive smear observed in (A, lane 1) is RNA. The sample (1.5 mg) was incubated with 5 units RNase I (Promega) for 20 min at 37°C. Lane 1: 1 Kb DNA ladder; lane 2: bdelloplast, 1 h after mixing predator and prey in a synchronous culture, no RNase I added; lane 3: bdelloplast, 1 h after mixing in the presence of RNase I. (PPT 729 KB)
10142_2008_106_MOESM8_ESM.ppt (217 kb)
Supplementary Figure 5PCR analysis of in-frame Bd2400 deletion mutant. Following the construction of an in-frame deletion of the gene BD2400, merodiploids were first grown under kanamycin selection and excisants were selected on 5% sucrose media and then plated. Fourteen plaques were analyzed with three sets of primers, amplifying (A) the BIL domain, using primers Bd2400-8 and Bd2400-9, (B) the flanking region of the in-frame deletion, using primers Flank_F and Flank_R, and (C) the SacB gene, using primers SBF and SBR (Steyert and Pineiro 2007). (PPT 729 KB)

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Mally Dori-Bachash
    • 1
  • Bareket Dassa
    • 2
  • Ofer Peleg
    • 3
  • Silvia A. Pineiro
    • 4
  • Edouard Jurkevitch
    • 1
  • Shmuel Pietrokovski
    • 2
  1. 1.Department of Plant Pathology and Microbiology, Faculty of Agriculture, Food and EnvironmentThe Hebrew UniversityRehovotIsrael
  2. 2.Department of Molecular GeneticsWeizmann Institute of ScienceRehovotIsrael
  3. 3.Genaphora LTDTel AvivIsrael
  4. 4.Department of Medical and Research Technology, School of MedicineUniversity of MarylandBaltimoreUSA