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Bacteriovorax marinus SJ is a predatory delta-proteobacterium isolated from a marine environment. The genome sequence of this strain provides an interesting contrast to that of the terrestrial predatory bacterium Bdellovibrio bacteriovorus HD100. Based on their predatory lifestyle, Bacteriovorax were originally designated as members of the genus Bdellovibrio but subsequently were re-assigned to a new genus and family based on genetic and phenotypic differences. B. marinus attaches to Gram-negative bacteria, penetrates through the cell wall to form a bdelloplast, in which it replicates, as shown using microscopy. Bacteriovorax is distinct, as it shares only 30% of its gene products with its closest sequenced relatives. Remarkably, 34% of predicted genes over 500 nt in length were completely unique with no significant matches in the databases. As expected, Bacteriovorax shares several characteristic loci with the other delta-proteobacteria. A geneset shared between Bacteriovorax and Bdellovibrio that is not conserved among other delta-proteobacteria such as Myxobacteria (which destroy prey bacteria externally via lysis), or the non-predatory Desulfo-bacteria and Geobacter species was identified. These 291 gene orthologues common to both Bacteriovorax and Bdellovibrio may be the key indicators of host-interaction predatory-specific processes required for prey entry. The locus from Bdellovibrio bacteriovorus is implicated in the switch from predatory to prey/host-independent growth. Although the locus is conserved in B. marinus , the sequence has only limited similarity. The results of this study advance understanding of both the similarities and differences between Bdellovibrio and Bacteriovorax and confirm the distant relationship between the two and their separation into different families.

Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 -- a Nudix hydrolase from Bdellovibrio bacteriovorus-that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases.

Studying potential dietary exposure to antimicrobial drug residues via meat and dairy products is essential to ensure human health and consumer safety. When studying how antimicrobial residues in food impact the development of antimicrobial drug resistance and disrupt normal bacteria community structure in the intestine, there are diverse methodological challenges to overcome. In this study, traditional cultures and molecular analysis techniques were used to determine the effects of tetracycline at chronic subinhibitory exposure levels on human intestinal microbiota using an in vitro continuous flow bioreactor. Six bioreactor culture vessels containing human fecal suspensions were maintained at 37 °C for 7 days. After a steady state was achieved, the suspensions were dosed with 0, 0.015, 0.15, 1.5, 15, or 150 µg/mL tetracycline, respectively. Exposure to 150 µg/mL tetracycline resulted in a decrease of total anaerobic bacteria from 1.9 × 107 ± 0.3 × 107 down to 2 × 106 ± 0.8 × 106 CFU/mL. Dose-dependent effects of tetracycline were noted for perturbations of tetB and tetD gene expression and changes in acetate and propionate concentrations. Although no-observed-adverse-effect concentrations differed, depending on the traditional cultures and the molecular analysis techniques used, this in vitro continuous flow bioreactor study contributes to the knowledge base regarding the impact of chronic exposure of tetracycline on human intestinal microbiota.

Distillers grains are co-products of the corn ethanol industry widely used in animal feed. We examined the effects of erythromycin, penicillin, and virginiamycin at low concentrations reflective of those detected in distillers grains on bacterial resistance selection. At 0.1 µg/ml erythromycin, macrolide-resistant mutants were induced in one Campylobacter coli and one Enterococcus faecium strain, while these strains plus three additional C . coli , one additional E . faecium , and one C . jejuni also developed resistance when exposed to 0.25 µg/ml erythromycin. At 0.5 µg/ml erythromycin, a total of eight strains (four Campylobacter and four Enterococcus ) obtained macrolide-resistant mutants, including two strains from each genus that were not selected at lower erythromycin concentrations. For penicillin, three of five E . faecium strains but none of five Enterococcus faecalis strains consistently developed resistance at all three selection concentrations. Virginiamycin at two M 1 :S 1 ratios did not induce resistance development in four out of five E . faecium strains; however, increased resistance was observed in the fifth one under 0.25 and 0.5 µg/ml virginiamycin selections. Although not yet tested in vivo , these findings suggest a potential risk of stimulating bacterial resistance development in the animal gut when distillers grains containing certain antibiotic residues are used in animal feed.

Residual concentrations of erythromycin in food could result in gastrointestinal tract exposure that potentially poses a health-hazard to the consumer, affecting intestinal epithelial permeability, barrier function, microbiota composition, and antimicrobial resistance. We investigated the effects of erythromycin after acute (48 h single treatment with 0.03 μg/mL to 300 μg/mL) or chronic (repeated treatment with 0.3 µg/mL and 300 µg/mL erythromycin for five days) exposures on the permeability of human colonic epithelial cells, a model that mimics a susceptible intestinal surface devoid of commensal microbiota. Transepithelial electrical resistance (TER) measurements indicated that erythromycin above 0.3 µg/mL may compromise the epithelial barrier. Acute exposure increased cytotoxicity, while chronic exposure decreased the cytotoxicity. Quantitative PCR analysis revealed that only ICAM1 (intercellular adhesion molecule 1) was up-regulated during 0.3 μg/mL acute-exposure, while ICAM1, JAM3 (junctional adhesion molecule 3), and ITGA8 (integrin alpha 8), were over-expressed in the 300 μg/mL acute treatment group. However, during chronic exposure, no change in the mRNA expression was observed at 0.3 μg/mL, and only ICAM2 was significantly up-regulated after 300 μg/mL. ICAM1 and ICAM2 are known to be involved in the formation of extracellular matrices. These gene expression changes may be related to the immunoregulatory activity of erythromycin, or a compensatory mechanism of the epithelial cells to overcome the distress caused by erythromycin due to increased permeability.

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.

The Bdellovibrionaceae are predatory, intraperiplasmic bacteria that prey upon a variety of Gram-negative bacteria. The prey susceptibility pattern is frequently used to characterize new isolates. The objective in this study was to isolate and characterize predators from the Great Salt Lake (GSL) by prey susceptibility testing. To recover the predators, water samples were inoculated into an enrichment medium with Vibrio parahaemolyticus as prey. After several days of incubation, the predators were isolated, pure DNA was extracted, and partial 16S rDNA gene was sequenced. Water samples were also plated for isolation of heterotrophic bacteria. The susceptibility of bacterial isolates from the lake and other sources to each predator isolate was determined. The results revealed that there are predators in the GSL, and they preferentially prey on bacteria from the lake. This is the first report of the isolation of Bdellovibrionaceae from GSL and the predators showing preferences for bacteria from the same habitat.

A promoter vector pACPR33 for Escherichia coli based on the promotorless ampicillin-resistance gene from pBR322 has been constructed. The promoter of the ampicillin-resistance gene was deleted and replaced by a suitable multiple cloning site. Molecular cloning of promoters into the polylinker resulted in activation of the ampicillin resistance in E. coli. The plasmid contains a functional origin of DNA replication and a tetracycline resistance gene for E. coli, and a chloramphenicol resistance gene for S. aureus. The vector permitted direct detection of promoter activity, especially strong promoters, by easy iodometric determination of beta-lactamase activity in liquid or solid media.

Monitoring the number of bacterial colony-forming units is an important step in assuring compliance with the recommendation that water from dental units contain <200 CFU mL(-1). Media that have been used for this purpose include R2A, a standard plate counting medium for water samples, and the Millipore HPC Sampler device, designed to facilitate sampling in dental offices. Discrepancies between the two media have been observed. This study tested the hypothesis that differences in counts on the two media were due to the failure of some bacteria to grow on the HPC sampler or to grow at less efficiency than on R2A. Of four different bacterial colony phenotypes tested in three independent experimental trials, one phenotype did not grow on the HPC device, and another grew inconsistently and at lower efficiency. These results confirmed the hypothesis. From these findings, users of the HPC sampler should be aware that microbial undercounts may occur.

Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 -- a Nudix hydrolase from Bdellovibrio bacteriovorus-that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Aa C alpha RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the alpha -[Beta]- alpha NDPSase fold differentiates NDPSases from ADPRases.