The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site

Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (O$_{\rm{R}}$3) in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (O$_{\rm{R}}$3) upstream of the prophage repressor gene. The O$_{\rm{R}}$3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages

Activity of Bdellovibrio Hit Locus Proteins, Bd0108 and Bd0109, Links Type IVa Pilus Extrusion/Retraction Status to Prey-Independent Growth Signalling

Bdellovibrio bacteriovorus are facultatively predatory bacteria that grow within gram-negative prey, using pili to invade their periplasmic niche. They also grow prey-independently on organic nutrients after undergoing a reversible switch. The nature of the growth switching mechanism has been elusive, but several independent reports suggested mutations in the hit (host-interaction) locus on the Bdellovibrio genome were associated with the transition to preyindependent growth. Pili are essential for prey entry by Bdellovibrio and sequence analysis of the hit locus predicted that it was part of a cluster of Type IVb pilus-associated genes, containing bd0108 and bd0109. In this study we have deleted the whole bd0108 gene, which is unique to Bdellovibrio, and compared its phenotype to strains containing spontaneous mutations in bd0108 and the common natural 42 bp deletion variant of bd0108. We find that deletion of the whole bd0108 gene greatly reduced the extrusion of pili, whereas the 42 bp deletion caused greater pilus extrusion than wild-type. The pili isolated from these strains were comprised of the Type IVa pilin protein; PilA. Attempts to similarly delete gene bd0109, which like bd0108 encodes a periplasmic/secreted protein, were not successful, suggesting that it is likely to be essential for Bdellovibrio viability in any growth mode. Bd0109 has a sugar binding YD- repeat motif and an N-terminus with a putative pilin-like fold and was found to interact directly with Bd0108. These results lead us to propose that the Bd0109/Bd0108 interaction regulates pilus production in Bdellovibrio (possibly by interaction with the pilus fibre at the cell wall), and that the presence (and possibly retraction state) of the pilus feeds back to alter the growth state of the Bdellovibrio cell. We further identify a novel small RNA encoded by the hit locus, the transcription of which is altered in different bd0108 mutation background

Attack-Phase Bdellovibrio bacteriovorus Responses to Extracellular Nutrients Are Analogous to Those Seen During Late Intraperiplasmic Growth

Bdellovibrio bacteriovorus is a predatory bacterium which lives by invading the periplasm of gram-negative bacteria and consuming them from within. This predator was thought to be dependent upon prey for nutrients since it lacks genes encoding for critical enzymes involved in amino acid biosynthesis. This study, however, found that planktonic attack-phase predators are not just dependent upon prey for nutrients, but rather, they respond to nutrients in the surrounding medium and, subsequently, synthesize and secrete proteases in a nutrient-dependent manner. The major secreted proteases were identified through mass spectrometry analyses. Subsequent RT-qPCR analyses found that the nutrient-induced proteases are similar to those expressed within the prey periplasm during the late intraperiplasmic growth phase. Furthermore, RNA sequencing found that incubating the planktonic attack-phase cells in a nutritious environment for a short period of time (4 h) changes its gene expression pattern to a status that is akin to the late intraperiplasmic phase, with more than 94% of the genes previously identified as being late intraperiplasmic-specific also being induced by nutrient broth in this study. This strong correlation between the gene expression patterns hints that the availability of hydrolyzed prey cell components to the predator is likely the stimulus controlling the expression of late intraperiplasmic B. bacteriovorus genes during predation

Emerging horizons for industrial applications of predatory bacteria

26 p.-4 fig.-2 tab. The Ecology of Predation at the Microscale pp 173-194This chapter reviews the potential of the predatory bacteria Bdellovibrio bacteriovorus, an obligate predator of other gram-negative bacteria, as a biotechnological tool. Due to the unique lifestyle and the different applications, predatory bacteria have awakened interest to be developed as a lytic tool. The lack of physiological and metabolic information makes difficult this development. However, in the last years, different approaches have been described in order to understand the physiology, morphology, and metabolism of the predators, as well as the population dynamics of the prey-predator interactions. Besides its potential of “living antibiotic”, predatory bacteria have been proposed as a biocontrol agent in the food industry or aquaculture. A recent work using B. bacteriovorus as a biological lytic tool for the recovery of intracellular bioproducts highlighted the potential use of predators in industrial bioprocesses. The bottlenecks of using other Bdellovibrio and like organisms (BALOs) have been also considered and discussed during this chapter.This work was supported by the European Union’s Horizon 2020 Research and Innovation Programme, grant agreement no. 760994-2 (ENGICOIN), the Spanish Ministry of Science, Innovation and Universities (BIO2017-83448-R) and the Community of Madrid (P2018/ NMT4389).Peer reviewe