Antisense Gene Silencing and Bacteriophages as Novel Disinfection Processes for Engineered Systems

The growth and proliferation of invasive bacteria in engineered systems is an ongoing problem. While there are a variety of physical and chemical processes to remove and inactivate bacterial pathogens, there are many situations in which these tools are no longer effective or appropriate for the treatment of a microbial target. For example, certain strains of bacteria are becoming resistant to commonly used disinfectants, such as chlorine and UV. Additionally, the overuse of antibiotics has contributed to the spread of antibiotic resistance, and there is concern that wastewater treatment processes are contributing to the spread of antibiotic resistant bacteria.

Due to the continually evolving nature of bacteria, it is difficult to develop methods for universal bacterial control in a wide range of engineered systems, as many of our treatment processes are static in nature. Still, invasive bacteria are present in many natural and engineered systems, where the application of broad acting disinfectants is impractical, because their use may inhibit the original desired bioprocesses. Therefore, to better control the growth of treatment resistant bacteria and to address limitations with the current disinfection processes, novel tools that are both specific and adaptable need to be developed and characterized.

In this dissertation, two possible biological disinfection processes were investigated for use in controlling invasive bacteria in engineered systems. First, antisense gene silencing, which is the specific use of oligonucleotides to silence gene expression, was investigated. This work was followed by the investigation of bacteriophages (phages), which are viruses that are specific to bacteria, in engineered systems.


For the antisense gene silencing work, a computational approach was used to quantify the number of off-targets and to determine the effects of off-targets in prokaryotic organisms. For the organisms of Escherichia coli K-12 MG1655 and Mycobacterium tuberculosis H37Rv the mean number of off-targets was found to be 15.0 + 13.2 and 38.2 + 61.4, respectively, which results in a reduction of greater than 90% of the effective oligonucleotide concentration. It was also demonstrated that there was a high variability in the number of off-targets over the length of a gene, but that on average, there was no general gene location that could be targeted to reduce off-targets. Therefore, this analysis needs to be performed for each gene in question. It was also demonstrated that the thermodynamic binding energy between the oligonucleotide and the mRNA accounted for 83% of the variation in the silencing efficiency, compared to the number of off-targets, which explained 43% of the variance of the silencing efficiency. This suggests that optimizing thermodynamic parameters must be prioritized over minimizing the number of off-targets. In conclusion for the antisense work, these results suggest that off-target hybrids can account for a greater than 90% reduction in the concentration of the silencing oligonucleotides, and that the effective concentration can be increased through the rational design of silencing targets by minimizing off-target hybrids.

Regarding the work with phages, the disinfection rates of bacteria in the presence of phages was determined. The disinfection rates of E. coli K12 MG1655 in the presence of coliphage Ec2 ranged up to 2 h-1, and were dependent on both the initial phage and bacterial concentrations. Increasing initial phage concentrations resulted in increasing disinfection rates, and generally, increasing initial bacterial concentrations resulted in increasing disinfection rates. However, disinfection rates were found to plateau at higher bacterial and phage concentrations. A multiple linear regression model was used to predict the disinfection rates as a function of the initial phage and bacterial concentrations, and this model was able to explain 93% of the variance in the disinfection rates. The disinfection rates were also modeled with a particle aggregation model. The results from these model simulations suggested that at lower phage and bacterial concentrations there are not enough collisions to support active disinfection rates, which therefore, limits the conditions and systems where phage based bacterial disinfection is possible. Additionally, the particle aggregation model over predicted the disinfection rates at higher phage and bacterial concentrations of 108 PFU/mL and 108 CFU/mL, suggesting other interactions were occurring at these higher concentrations. Overall, this work highlights the need for the development of alternative models to more accurately describe the dynamics of this system at a variety of phage and bacterial concentrations. Finally, the minimum required hydraulic residence time was calculated for a continuous stirred-tank reactor and a plug flow reactor (PFR) as a function of both the initial phage and bacterial concentrations, which suggested that phage treatment in a PFR is theoretically possible.

In addition to determining disinfection rates, the long-term bacterial growth inhibition potential was determined for a variety of phages with both Gram-negative and Gram-positive bacteria. It was determined, that on average, phages can be used to inhibit bacterial growth for up to 24 h, and that this effect was concentration dependent for various phages at specific time points. Additionally, it was found that a phage cocktail was no more effective at inhibiting bacterial growth over the long-term than the best performing phage in isolation.

Finally, for an industrial application, the use of phages to inhibit invasive Lactobacilli in ethanol fermentations was investigated. It was demonstrated that phage 8014-B2 can achieve a greater than 3-log inactivation of Lactobacillus plantarum during a 48 h fermentation. Additionally, it was shown that phages can be used to protect final product yields and maintain yeast viability. Through modeling the fermentation system with differential equations it was determined that there was a 10 h window in the beginning of the fermentation run, where the addition of phages can be used to protect final product yields, and after 20 h no additional benefit of the phage addition was observed.

In conclusion, this dissertation improved the current methods for designing antisense gene silencing targets for prokaryotic organisms, and characterized phages from an engineering perspective. First, the current design strategy for antisense targets in prokaryotic organisms was improved through the development of an algorithm that minimized the number of off-targets. For the phage work, a framework was developed to predict the disinfection rates in terms of the initial phage and bacterial concentrations. In addition, the long-term bacterial growth inhibition potential of multiple phages was determined for several bacteria. In regard to the phage application, phages were shown to protect both final product yields and yeast concentrations during fermentation. Taken together, this work suggests that the rational design of phage treatment is possible and further work is needed to expand on this foundation.

Inactivation of Human beta-Defensins 2 and 3 by Elastolytic Cathepsins

beta-Defensins are antimicrobial peptides that contribute to the innate immune responses of eukaryotes. At least three defensins, human beta-defensins 1, 2, and 3 (HBD-1, -2, and -3), are produced by epithelial cells lining the respiratory tract and are active toward Gram-positive (HBD-3) and Gram-negative (HBD-1, -2, and -3) bacteria. It has been postulated that the antimicrobial activity of defensins is compromised by changes in airway surface liquid composition in lungs of patients with cystic fibrosis (CF), therefore contributing to the bacterial colonization of the lung by Pseudomonas and other bacteria in CF. In this report we demonstrate that HBD-2 and HBD-3 are susceptible to degradation and inactivation by the cysteine proteases cathepsins B, L, and S. In addition, we show that all three cathepsins are present and active in CF bronchoalveolar lavage. Incubation of HBD-2 and -3 with CF bronchoalveolar lavage leads to their degradation, which can be completely (HBD-2) or partially (HBD-3) inhibited by a cathepsin inhibitor. These results suggest that beta-defensins are susceptible to degradation and inactivation by host proteases, which may be important in the regulation of beta-defensin activity. In chronic lung diseases associated with infection, overexpression of cathepsins may lead to increased degradation of HBD-2 and -3, thereby favoring bacterial infection and colonization.

Development of a novel DNA microarray to detect bacterial pathogens in patients with chronic obstructive pulmonary disease (COPD)

A novel microarray was constructed with DNA PCR product probes targeting species specific functional genes of nine clinically significant respiratory pathogens, including the Gram-positive organisms (Streptococcus pneumoniae, Streptococcus pyogenes), the Gram-negative organisms (Chlamydia pneumoniae, Coxiella burnetii Haemophilus spp., Legionella pneumophila, Moraxella catarrhalis, and Pseudomonas aeruginosa), as well as the atypical bacterium, Mycoplasma pneumoniae. In a "proof-of-concept" evaluation of the developed microarray, the microarray was compared with real-time PCR from 14 sputum specimens from COPD patients. All of the samples positive for bacterial species in real-time PCR were also positive for the same bacterial species using the microarray. This study shows that a microarray using PCR probes is a potentially useful method to monitor the populations of bacteria in respiratory specimens and can be tailored to specific clinical needs such as respiratory infections of particular patient populations, including patients with cystic fibrosis and bronchiectasis. (C) 2010 Elsevier B.V. All rights reserved.

A system for the construction of targeted unmarked gene deletions in the genus Burkholderia

Burkholderia species are extremely multidrug resistant, environmental bacteria with extraordinary bioremediation and biocontrol properties. At the same time, these bacteria cause serious opportunistic infections in vulnerable patient populations while some species can potentially be used as bioweapons. The complete DNA sequence of more than 10 Burkholderia genomes provides an opportunity to apply functional genomics to a collection of widely adaptable environmental bacteria thriving in diverse niches and establishing both symbiotic and pathogenic associations with many different organisms. However, extreme multidrug resistance hampers genetic manipulations in Burkholderia. We have developed and evaluated a mutagenesis system based on the homing endonuclease I-SceI to construct targeted, non-polar unmarked gene deletions in Burkholderia. Using the cystic fibrosis pathogen Burkholderia cenocepacia K56-2 as a model strain, we demonstrate this system allows for clean deletions of one or more genes within an operon and also the introduction of multiple deletions in the same strain. We anticipate this tool will have widespread environmental and biomedical applications, facilitating functional genomic studies and construction of safe strains for bioremediation and biocontrol, as well as clinical applications such as live vaccines for Burkholderia and other Gram-negative bacterial species.

Burkholderia cenocepacia C5424 produces a pigment with antioxidant properties using a homogentisate intermediate

Burkholderia cenocepacia is a gram-negative opportunistic pathogen that belongs to the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly within phagocytic cells, and some epidemic strains produce a brown melanin-like pigment that can scavenge free radicals, resulting in the attenuation of the host cell oxidative burst. In this work, we demonstrate that the brown pigment produced by B. cenocepacia C5424 is synthesized from a homogentisate (HGA) precursor. The disruption of BCAL0207 (hppD) by insertional inactivation resulted in loss of pigmentation. Steady-state kinetic analysis of the BCAL0207 gene product demonstrated that it has 4-hydroxyphenylpyruvic acid dioxygenase (HppD) activity. Pigmentation could be restored by complementation providing hppD in trans. The hppD mutant was resistant to paraquat challenge but sensitive to H2O2 and to extracellularly generated superoxide anions. Infection experiments in RAW 264.7 murine macrophages showed that the nonpigmented bacteria colocalized in a dextran-positive vacuole, suggesting that they are being trafficked to the lysosome. In contrast, the wild-type strain did not localize with dextran. Colocalization of the nonpigmented strain with dextran was reduced in the presence of the NADPH oxidase inhibitor diphenyleneiodonium, and also the inducible nitric oxide inhibitor aminoguanidine. Together, these observations suggest that the brown pigment produced by B. cenocepacia C5424 is a pyomelanin synthesized from an HGA intermediate that is capable of protecting the organism from in vitro and in vivo sources of oxidative stress.

Characterization of SodC, a periplasmic superoxide dismutase from Burkholderia cenocepacia

Burkholderia cenocepacia is a gram-negative, non-spore-forming bacillus and a member of the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly in phagocytic cells and can produce at least one superoxide dismutase (SOD). The inability of O2- to cross the cytoplasmic membrane, coupled with the periplasmic location of Cu,ZnSODs, suggests that periplasmic SODs protect bacteria from superoxide that has an exogenous origin (for example, when cells are faced with reactive oxygen intermediates generated by host cells in response to infection). In this study, we identified the sodC gene encoding a Cu,ZnSOD in B. cenocepacia and demonstrated that a sodC null mutant was not sensitive to a H2O2, 3-morpholinosydnonimine, or paraquat challenge but was killed by exogenous superoxide generated by the xanthine/xanthine oxidase method. The sodC mutant also exhibited a growth defect in liquid medium compared to the parental strain, which could be complemented in trans. The mutant was killed more rapidly than the parental strain was killed in murine macrophage-like cell line RAW 264.7, but killing was eliminated when macrophages were treated with an NADPH oxidase inhibitor. We also confirmed that SodC is periplasmic and identified the metal cofactor. B. cenocepacia SodC was resistant to inhibition by H2O2 and was unusually resistant to KCN for a Cu,ZnSOD. Together, these observations establish that B. cenocepacia produces a periplasmic Cu,ZnSOD that protects this bacterium from exogenously generated O2- and contributes to intracellular survival of this bacterium in macrophages.

Surface localisation of photosensitisers on intraocular lens biomaterials for prevention of infectious endophthalmitis and retinal protection

Cataract surgery is one of the most commonly-practiced surgical procedures in Western medicine, and, while complications are rare, the most serious is infectious postoperative endophthalmitis. Bacteria may adhere to the implanted intraocular lens (IOL) and subsequent biofilm formation can lead to a chronic, difficult to treat infection. To date, no method to reduce the incidence of infectious endophthalmitis through bacterial elimination, while retaining optical transparency, has been reported. In this study we report a method to optimise the localisation of a cationic porphyrin at the surface of suitable acrylate copolymers, which is the first point of contact with potential pathogens. The porphyrin catalytically generates short-lived singlet oxygen, in the presence of visible light, which kills adherent bacteria indiscriminately. By restricting the photosensitiser to the surface of the biomaterial, reduction in optical transparency is minimised without affecting efficacy of singlet oxygen production. Hydrogel IOL biomaterials incorporating either methacrylic acid (MAA) or methyl methacrylate (MMA) co-monomers allow tuning of the hydrophobic and anionic properties to optimise the localisation of porphyrin. Physiochemical and antimicrobial properties of the materials have been characterised, giving candidate materials with self-generating, persistent anti-infective character against Gram-positive and Gram-negative organisms. Importantly, incorporation of porphyrin can also serve to protect the retina by filtering damaging shortwave visible light, due to the Soret absorption (?max) 430 nm). © 2012 Elsevier Ltd. All rights reserved.

Detection of intrastrain antigenic variation of Bacteroides fragilis surface polysaccharides by monoclonal antibody labelling

Bacteroides fragilis is a constituent of the normal resident microbiota of the human intestine and is the gram-negative obligately anaerobic bacterium most frequently isolated from clinical infection. Surface polysaccharides are implicated as potential virulence determinants. We present evidence of within strain immunochemical variation of surface polysaccharides in populations that are noncapsulate by light microscopy as determined by monoclonal antibody labelling. Expression of individual epitopes can be enriched from a population of an individual strain by use of immunomagnetic beads. Also, individual colonies in which either >94% or 94% of the bacteria carry a given epitope, there is no enrichment for other epitopes recognized by different polysaccharide-specific monoclonal antibodies. This intrastrain variation has important implications for the development of potential vaccines or immunodiagnostic tests.

Evidence for a non-replicative intracellular stage of nontypable Haemophilus influenzae in epithelial cells

Nontypable Haemophilus influenzae (NTHi) is a Gram-negative, non-capsulated human bacterial pathogen, a major cause of a repertoire of respiratory infections, and intimately associated with persistent lung bacterial colonization in patients suffering from chronic obstructive pulmonary disease (COPD). Despite its medical relevance, relatively little is known about its mechanisms of pathogenicity. In this study, we found that NTHi invades the airway epithelium by a distinct mechanism, requiring microtubule assembly, lipid rafts integrity, and activation of phosphatidylinositol 3-kinase (PI3K) signalling. We found that the majority of intracellular bacteria are located inside an acidic subcellular compartment, in a metabolically active and non-proliferative state. This NTHi-containing vacuole (NTHi-CV) is endowed with late endosome features, co-localizing with LysoTracker, lamp-1, lamp-2, CD63 and Rab7. The NTHi-CV does not acquire Golgi- or autophagy-related markers. These observations were extended to immortalized and primary human airway epithelial cells. By using NTHi clinical isolates expressing different amounts of phosphocholine (PCho), a major modification of NTHi lipooligosaccharide, on their surfaces, and an isogenic lic1BC mutant strain lacking PCho, we showed that PCho is not responsible for NTHi intracellular location. In sum, this study indicates that NTHi can survive inside airway epithelial cells.

NO-loaded Zn2+-exchanged zeolite materials: A potential bifunctional anti-bacterial strategy

Nitric oxide (NO) is important for the regulation of a number of diverse biological processes, including vascular tone, neurotransmission, inflammatory cell responsiveness, defence against invading pathogens and wound healing. Transition metal exchanged zeolites are nanoporous materials with high-capacity storage properties for gases such as NO. The NO stores are liberated upon contact with aqueous environments, thereby making them ideal candidates for use in biological and clinical settings. Here, we demonstrate the NO release capacity and powerful bactericidal properties of a novel NO-storing Zn2+-exchanged zeolite material at a 50 wt.% composition in a polytetrafluoroethylene polymer. Further to our published data showing the anti-thrombotic effects of a similar NO-loaded zeolite, this study demonstrates the antibacterial properties of NO-releasing zeolites against clinically relevant strains of bacteria, namely Gram-negative Pseudomonas aeruginosa and Gram-positive methicillin-sensitive and methicillin-resistant Staphylococcus aureus and Clostridium difficile. Thus our study highlights the potential of NO-loaded zeolites as biocompatible medical device coatings with anti-infective properties. (C) 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Lactobacillus strains isolated from infant faeces possess potent inhibitory activity against intestinal alpha- and beta-glucosidases suggesting anti-diabetic potential

Purpose: Inhibitors of intestinal alpha-glucosidases are used therapeutically to treat type 2 diabetes mellitus. Bacteria such as Actinoplanes sp. naturally produce potent alpha-glucosidase inhibitor compounds, including the most widely available drug acarbose. It is not known whether lactic acid bacteria (LAB) colonising the human gut possess inhibitory potential against glucosidases. Hence, the study was undertaken to screen LABs having inherent alpha- and beta-glucosidase inhibitory potential. Methods: This study isolated, screened, identified and extracted Lactobacillus strains (Lb1–15) from human infant faecal samples determining their inhibitory activity against intestinal maltase, sucrase, lactase and amylase. Lactobacillus reference strains (Ref1–7), a Gram positive control (Ctrl1) and two Gram negative controls (Ctrl2–3), were also analysed to compare activity. Results: Faecal isolates were identified by DNA sequencing, with the majority identified as unique strains of Lactobacillus plantarum. Some strains (L. plantarum, L. fermentum, L. casei and L. rhamnosus) had potent and broad spectrum inhibitory activities (up to 89 %; p < 0.001; 500 mg/ml wet weight) comparable to acarbose (up to 88 %; p < 0.001; 30 mg/ml). Inhibitory activity was concentration-dependent and was freely available in the supernatant, and was not present in other bacterial genera (Bifidobacterium bifidum and Escherichia coli or Salmonella typhimurium). Interestingly, the potency and spectrum of inhibitory activity across strains of a single species (L. plantarum) differed substantially. Some Lactobacillus extracts had broader spectrum activities than acarbose, effectively inhibiting beta-glucosidase activity (lactase) as well as alpha-glucosidase activities (maltase, sucrase and amylase). Anti-diabetic potential was indicated by the fact that oral gavage with a L. rhamnosus extract (1 g/kg) was able to reduce glucose excursions (Area under curve; 22 %; p < 0.05) in rats during a carbohydrate challenge (starch; 2 g/kg). Conclusion: These results definitively demonstrate that Lactobacillus strains present in the human gut have alpha- and beta-glucosidase inhibitory activities and can reduce blood glucose responses in vivo. Although the potential use of LAB such as Lactobacillus as a dietary supplement, medicinal food or biotherapeutic for diabetes is uncertain, such an approach might offer advantages over drug therapies in terms of broader spectrum activities and fewer unpleasant side effects. Further characterisation of this bioactivity is warranted, and chronic studies should be undertaken in appropriate animal models or diabetic subjects.

Topological analysis of the Escherichia coli WcaJ protein reveals a new conserved configuration for the polyisoprenyl-phosphate hexose-1-phosphate transferase family

WcaJ is an Escherichia coli membrane enzyme catalysing the biosynthesis of undecaprenyl-diphosphate-glucose, the first step in the assembly of colanic acid exopolysaccharide. WcaJ belongs to a large family of polyisoprenyl-phosphate hexose-1-phosphate transferases (PHPTs) sharing a similar predicted topology consisting of an N-terminal domain containing four transmembrane helices (TMHs), a large central periplasmic loop, and a C-terminal domain containing the fifth TMH (TMH-V) and a cytosolic tail. However, the topology of PHPTs has not been experimentally validated. Here, we investigated the topology of WcaJ using a combination of LacZ/PhoA reporter fusions and sulfhydryl
labelling by PEGylation of novel cysteine residues introduced into a cysteine-less WcaJ. The results showed that the large central loop and the C-terminal tail both reside in the cytoplasm and are separated by TMH-V, which does not fully span the membrane, likely forming a "hairpin" structure. Modelling of TMH-V revealed that a highly conserved proline might contribute to a helix-break-helix structure in all PHPT members. Bioinformatic analyses show that all of these features are conserved in PHPT homologues from
Gram-negative and Gram-positive bacteria. Our data demonstrate a novel topological configuration for PHPTs, which is proposed as a signature for all members of this enzyme family

Dual functional ionic liquids as antimicrobials and plasticisers for medical grade PVCs

Two ionic liquids, 1-ethylpyridinium docusate (IL1) and tri-n-butyl(2-hydroxyethyl)phosphonium docusate (IL2), were designed and synthesized with the explicit intention of imparting a combination of plasticization and antimicrobial efficacy when incorporated into medical grade poly(vinyl chloride)s (PVCs). The glass transition (T-g) of PVC can be reduced by >20 degrees C on addition of 15 wt% IL2. Both IL1 and IL2 leached to varying extents from the base PVC resins rendering the surface of the PVCs hydrophilic. The antimicrobial activity of both ILs is related to the presence and concentration of both cationic and anionic component of the ILs leached from the PVC and inversely proportional to the extent of PVC gelation. Blends of the PVCs with IL1 displayed antibacterial activity against almost all Gram-positive bacteria tested, including coagulase-negative Staphylococci (CoNS) and methicillin-resistant Staphylococcus aureus (MRSA), but not with IL2 at low concentration in contrast to our previous study when high concentrations of IL2 were used. The more hydrophilic IL1 when added to PVC retards biofilm formation.

The Bdellovibrio bacteriovorus twin-arginine transport system has roles in predatory and prey-independent growth

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. B. bacteriovorus is one of a few bacterial species that are reliant on a functional Tat system and where deletion of a single tatA1 gene causes a significant growth defect(s), despite the presence of its tatA2 homologue.

Three motAB stator gene products in Bdellovibrio bacteriovorus contribute to motility of a single flagellum during predatory and prey-independent growth

The predatory bacterium Bdellovibrio bacteriovorus uses flagellar motility to locate regions rich in Gram-negative prey bacteria, colliding and attaching to prey and then ceasing flagellar motility. Prey are then invaded to form a "bdelloplast" in a type IV pilus-dependent process, and prey contents are digested, allowing Bdellovibrio growth and septation. After septation, Bdellovibrio flagellar motility resumes inside the prey bdelloplast prior to its lysis and escape of Bdellovibrio progeny. Bdellovibrio can also grow slowly outside prey as long flagellate host-independent (HI) cells, cultured on peptone-rich media. The B. bacteriovorus HD100 genome encodes three pairs of MotAB flagellar motor proteins, each of which could potentially form an inner membrane ion channel, interact with the FliG flagellar rotor ring, and produce flagellar rotation. In 2004, Flannagan and coworkers (R. S. Flannagan, M. A. Valvano, and S. F. Koval, Microbiology 150:649-656, 2004) used antisense RNA and green fluorescent protein (GFP) expression to downregulate a single Bdellovibrio motA gene and reported slowed release from the bdelloplast and altered motility of the progeny. Here we inactivated each pair of motAB genes and found that each pair contributes to motility, both predatorily, inside the bdelloplast and during HI growth; however, each pair was dispensable, and deletion of no pair abolished motility totally. Driving-ion studies with phenamil, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and different pH and sodium conditions indicated that all Mot pairs are proton driven, although the sequence similarities of each Mot pair suggests that some may originate from halophilic species. Thus, Bdellovibrio is a "dedicated motorist," retaining and expressing three pairs of mot genes.