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Predation structures food webs, influences energy flow, and alters rates and pathways of nutrient cycling through ecosystems, effects that are well documented for macroscopic predators. In the microbial world, predatory bacteria are common, yet little is known about their rates of growth and roles in energy flows through microbial food webs, in part because these are difficult to quantify. Here, we show that growth and carbon uptake were higher in predatory bacteria compared to nonpredatory bacteria, a finding across 15 sites, synthesizing 82 experiments and over 100,000 taxon-specific measurements of element flow into newly synthesized bacterial DNA. Obligate predatory bacteria grew 36% faster and assimilated carbon at rates 211% higher than nonpredatory bacteria. These differences were less pronounced for facultative predators (6% higher growth rates, 17% higher carbon assimilation rates), though high growth and carbon assimilation rates were observed for some facultative predators, such as members of the genera Lysobacter and Cytophaga, both capable of gliding motility and wolf-pack hunting behavior. Added carbon substrates disproportionately stimulated growth of obligate predators, with responses 63% higher than those of nonpredators for the Bdellovibrionales and 81% higher for the Vampirovibrionales, whereas responses of facultative predators to substrate addition were no different from those of nonpredators. This finding supports the ecological theory that higher productivity increases predator control of lower trophic levels. These findings also indicate that the functional significance of bacterial predators increases with energy flow and that predatory bacteria influence element flow through microbial food webs.IMPORTANCE The word "predator" may conjure images of leopards killing and eating impala on the African savannah or of great white sharks attacking elephant seals off the coast of California. But microorganisms are also predators, including bacteria that kill and eat other bacteria. While predatory bacteria have been found in many environments, it has been challenging to document their importance in nature. This study quantified the growth of predatory and nonpredatory bacteria in soils (and one stream) by tracking isotopically labeled substrates into newly synthesized DNA. Predatory bacteria were more active than nonpredators, and obligate predators, such as Bdellovibrionales and Vampirovibrionales, increased in growth rate in response to added substrates at the base of the food chain, strong evidence of trophic control. This work provides quantitative measures of predator activity and suggests that predatory bacteria-along with protists, nematodes, and phages-are active and important in microbial food webs.

Predation structures food webs, influences energy flow, and alters rates and pathways of nutrient cycling through ecosystems, effects that are well documented for macroscopic predators. In the microbial world, predatory bacteria are common, yet little is known about their rates of growth and roles in energy flows through microbial food webs, in part because these are difficult to quantify. Here, we show that growth and carbon uptake were higher in predatory bacteria compared to nonpredatory bacteria, a finding across 15 sites, synthesizing 82 experiments and over 100,000 taxon-specific measurements of element flow into newly synthesized bacterial DNA. Obligate predatory bacteria grew 36% faster and assimilated carbon at rates 211% higher than nonpredatory bacteria. These differences were less pronounced for facultative predators (6% higher growth rates, 17% higher carbon assimilation rates), though high growth and carbon assimilation rates were observed for some facultative predators, such as members of the genera Lysobacter and Cytophaga, both capable of gliding motility and wolf-pack hunting behavior. Added carbon substrates disproportionately stimulated growth of obligate predators, with responses 63% higher than those of nonpredators for the Bdellovibrionales and 81% higher for the Vampirovibrionales, whereas responses of facultative predators to substrate addition were no different from those of nonpredators. This finding supports the ecological theory that higher productivity increases predator control of lower trophic levels. These findings also indicate that the functional significance of bacterial predators increases with energy flow and that predatory bacteria influence element flow through microbial food webs.IMPORTANCE The word "predator" may conjure images of leopards killing and eating impala on the African savannah or of great white sharks attacking elephant seals off the coast of California. But microorganisms are also predators, including bacteria that kill and eat other bacteria. While predatory bacteria have been found in many environments, it has been challenging to document their importance in nature. This study quantified the growth of predatory and nonpredatory bacteria in soils (and one stream) by tracking isotopically labeled substrates into newly synthesized DNA. Predatory bacteria were more active than nonpredators, and obligate predators, such as Bdellovibrionales and Vampirovibrionales, increased in growth rate in response to added substrates at the base of the food chain, strong evidence of trophic control. This work provides quantitative measures of predator activity and suggests that predatory bacteria-along with protists, nematodes, and phages-are active and important in microbial food webs.

The ImpX transporters of the drug/metabolite transporter superfamily were first proposed to transport riboflavin (RF; vitamin B2) based on findings of a cis-regulatory RNA element responding to flavin mononucleotide (an FMN riboswitch). Bdellovibrio exovorous JSS has a homolog belonging to this superfamily. It has 10 TMSs and shows 30% identity to the previously characterized ImpX transporter from Fusobacterium nucleatum. However, the ImpX homolog is not regulated by an FMN-riboswitch. In order to test the putative function of the ImpX homolog from B. exovorous (BexImpX), we cloned and heterologously expressed its gene. We used functional complementation, growth inhibition experiments, direct uptake experiments and inhibition studies, suggesting a high degree of specificity for RF uptake. The EC50 for growth with RF was estimated to be in the range 0.5-1 µM, estimated from the half-maximal RF concentration supporting the growth of a RF auxotrophic Escherichia coli strain, but the Khalf for RF uptake was 20 µM. Transport experiments suggested that the energy source is the proton motive force but that NaCl stimulates uptake. Thus, members of the ImpX family members are capable of RF uptake, not only in RF prototrophic species such as F.  nucleatum, but also in the B2 auxotrophic species, B. exovorous.

Bdellovibrio, δ-proteobacteria, including B. bacteriovorus (Bba) and B. exovorus (Bex), are obligate predators of other Gram-negative bacteria. While Bba grows in the periplasm of the prey cell, Bex grows externally. We have analyzed and compared the transport proteins of these 2 organisms based on the current contents of the Transporter Classification Database (TCDB; www.tcdb.org). Bba has 103 transporters more than Bex, 50% more secondary carriers, and 3 times as many MFS carriers. Bba has far more metabolite transporters than Bex as expected from its larger genome, but there are 2 times more carbohydrate uptake and drug efflux systems, and 3 times more lipid transporters. Bba also has polyamine and carboxylate transporters lacking in Bex. Bba has more than twice as many members of the Mot-Exb family of energizers, but both may have energizers for gliding motility. They use entirely different types of systems for iron acquisition. Both contain unexpectedly large numbers of pseudogenes and incomplete systems, suggesting that they are undergoing genome size reduction. Interestingly, all 5 outer-membrane receptors in Bba are lacking in Bex. The 2 organisms have similar numbers and types of peptide and amino acid uptake systems as well as protein and carbohydrate secretion systems. The differences observed correlate with and may account, in part, for the different lifestyles of these 2 bacterial predators.

Bdellovibrio, δ-proteobacteria, including B. bacteriovorus (Bba) and B. exovorus (Bex), are obligate predators of other Gram-negative bacteria. While Bba grows in the periplasm of the prey cell, Bex grows externally. We have analyzed and compared the transport proteins of these 2 organisms based on the current contents of the Transporter Classification Database (TCDB; www.tcdb.org). Bba has 103 transporters more than Bex, 50% more secondary carriers, and 3 times as many MFS carriers. Bba has far more metabolite transporters than Bex as expected from its larger genome, but there are 2 times more carbohydrate uptake and drug efflux systems, and 3 times more lipid transporters. Bba also has polyamine and carboxylate transporters lacking in Bex. Bba has more than twice as many members of the Mot-Exb family of energizers, but both may have energizers for gliding motility. They use entirely different types of systems for iron acquisition. Both contain unexpectedly large numbers of pseudogenes and incomplete systems, suggesting that they are undergoing genome size reduction. Interestingly, all 5 outer-membrane receptors in Bba are lacking in Bex. The 2 organisms have similar numbers and types of peptide and amino acid uptake systems as well as protein and carbohydrate secretion systems. The differences observed correlate with and may account, in part, for the different lifestyles of these 2 bacterial predators.

The affordability of next generation sequencing (NGS) is transforming the field of mutation analysis in bacteria. The genetic basis for phenotype alteration can be identified directly by sequencing the entire genome of the mutant and comparing it to the wild-type (WT) genome, thus identifying acquired mutations. A major limitation for this approach is the need for an a-priori sequenced reference genome for the WT organism, as the short reads of most current NGS approaches usually prohibit de-novo genome assembly. To overcome this limitation we propose a general framework that utilizes the genome of relative organisms as mediators for comparing WT and mutant bacteria. Under this framework, both mutant and WT genomes are sequenced with NGS, and the short sequencing reads are mapped to the mediator genome. Variations between the mutant and the mediator that recur in the WT are ignored, thus pinpointing the differences between the mutant and the WT. To validate this approach we sequenced the genome of Bdellovibrio bacteriovorus 109J, an obligatory bacterial predator, and its prey-independent mutant, and compared both to the mediator species Bdellovibrio bacteriovorus HD100. Although the mutant and the mediator sequences differed in more than 28,000 nucleotide positions, our approach enabled pinpointing the single causative mutation. Experimental validation in 53 additional mutants further established the implicated gene. Our approach extends the applicability of NGS-based mutant analyses beyond the domain of available reference genomes.