Bdellovibrio bacteriovorusStrains Produce a Novel Major Outer Membrane Protein during Predacious Growth in the Periplasm of Prey Bacteria

Abstract

Bdellovibrio bacteriovorus is a predatory bacterium that is capable of invading a number of gram-negative bacteria. The life cycle of this predator can be divided into a nonreproductive phase outside the prey bacteria and a multiplication phase in their periplasm. It was suggested that during the reproduction phase, B. bacteriovorus reutilizes unmodified components of the prey's cell wall. We therefore examined the outer membranes of B. bacteriovorus strains HD100 (DSM 50701) and HD114 (DSM 50705) by using Escherichia coli, Yersinia enterocolitica, and Pseudomonas putida as prey organisms. The combined sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometric analyses revealed novel and innate major outer membrane proteins (OMPs) of B. bacteriovorus strains. An incorporation of prey-derived proteins into the cell wall of B. bacteriovorus was not observed. The corresponding genes of the B. bacteriovorus strains were elucidated by a reverse-genetics approach, and a leader peptide was deduced from the gene sequence and confirmed by Edman degradation. The host-independent mutant strain B. bacteriovorus HI100 (DSM 12732) growing in the absence of prey organisms possesses an OMP similar to the major OMPs of the host-dependent strains. The similarity of the primary structure of the OMPs produced by the three Bdellovibrio strains is between 67 and 89%. The leader peptides of all OMPs have a length of 20 amino acids and are highly conserved. The molecular sizes of the mature proteins range from 34.9 to 37.6 kDa. Secondary-structure predictions indicate preferential ␣-helices and little ␤-barrel structures.

Fate of Predator and Prey Proteins during Growth of Bdellovibrio bacteriovorus on Escherichia coli and Pseudomonas syringae Prey

“…As other proteins of the predator, including the strongly expressed cytoplasm-located elongation factor Tu (17), flagellin, and other molecules focusing in different parts of the gels were not detected, we are confident in ruling out contamination by a low level of remaining bdellovibrio attack-phase cells in the ghost envelope fraction. The polypeptide identified in this study is highly homologous to a new, recently described OMP from B. bacteriovorus type strain 100 (2).…”

“…4a), suggesting that it originated from the bdellovibrio predator. Extraction and mass spectrometric analysis of this polypeptide revealed that it was derived from the predator ( Table 1) and was related to a recently described OMP from B. bacteriovorus type strain 100 (2).…”

“…The gene coding for the OMPlike protein identified in ghost envelopes was amplified from B. bacteriovorus cells with primers based on the published sequences from type strain 100 and strain 114 (2). The genes of strains 109J-1 and 100 and of strains 109J-1 and 114 exhibited 86 and 69% identity, respectively, which translated into 82.4% identity (291 out 353 residues) and 86% similarity (307 out 353 residues) and 55% identity (210 out of 376 residues) and 66% similarity (253 out 376 residues), respectively, at the amino acid level.…”

“…Moreover, centrifugation on a Percoll cushion enabled the separation of ghost envelopes and bdellovibrio attack-phase cells. Prey OMPs were identified in (2). Some workers argued that the ability to acquire prey OMPs was greatly dependent upon the prey itself and that the history of the bdellovibrio strain employed also played a role in this ability (32).…”

“…However, the nature of the imported proteins and even the existence of such a capacity has been a matter of contention (2,7,19).…”