Michael S. DuBow

Antimicrobial drug discovery through bacteriophage genomics

Over evolutionary time bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Here, we apply this concept of phage-mediated bacterial growth inhibition to antibiotic discovery. We sequenced 26 Staphylococcus aureus phages and identified 31 novel polypeptide families that inhibited growth upon expression in S. aureus. The cellular targets for some of these polypeptides were identified and several were shown to be essential components of the host DNA replication and transcription machineries. The interaction between a prototypic pair, ORF104 of phage 77 and DnaI, the putative helicase loader of S. aureus, was then used to screen for small molecule inhibitors. Several compounds were subsequently found to inhibit both bacterial growth and DNA synthesis. Our results suggest that mimicking the growth-inhibitory effect of phage polypeptides by a chemical compound, coupled with the plethora of phages on earth, will yield new antibiotics to combat infectious diseases.

Comparative Genomic Analysis of 18 Pseudomonas aeruginosa Bacteriophages

A genomic analysis of 18 P. aeruginosa phages, including nine newly sequenced DNA genomes, indicates a tremendous reservoir of proteome diversity, with 55% of open reading frames (ORFs) being novel. Comparative sequence analysis and ORF map organization revealed that most of the phages analyzed displayed little relationship to each other.

Identification of Tn10 insertions in the rfaG, rfaP, and galU genes involved in lipopolysaccharide core biosynthesis that affect Escherichia coli adhesion.

Escherichia coli was used as a model to study initial adhesion and early biofilm development to abiotic surface. Tn10 insertion mutants of Escherichia coli K-12 W3110 were selected for altered abilities to adhere to a polystyrene surface. Seven insertion mutants that showed a decrease in adhesion harbored insertions in genes involved in lipopolysaccharide (LPS) core biosynthesis. Two insertions were located in the rfaG gene, two in the rfaP gene, and three in the galU gene. These adhesion mutants were found to exhibit a deep-rough phenotype and to be reduced, at different levels, in type 1 fimbriae production and motility. The loss of adhesion exhibited by these mutants was associated with either the affected type 1 fimbriae production and/or the dysfunctional motility. Apart from the pleiotropic effect of the mutations affecting LPS on type 1 fimbriae and flagella biosynthesis, no evidence for an involvement of the LPS itself in adhesion to polystyrene surface could be observed.

A chromosomal ars operon homologue of Pseudomonas aeruginosa confers increased resistance to arsenic and antimony in Escherichia coli.

Operons encoding homologous arsenic-resistance determinants (ars) have been discovered in bacterial plasmids from Gram-positive and Gram-negative organisms, as well as in the Escherichia coli chromosome. However, evidence for this arsenic-resistance determinant in the medically and environmentally important bacterial species Pseudomonas aeruginosa is conflicting. Here the identification of a P. aeruginosa chromosomal ars operon homologue via cloning and complementation of an E. coli ars mutant is reported. The P. aeruginosa chromosomal ars operon contains three potential ORFs encoding proteins with significant sequence similarity to those encoded by the arsR, arsB and arsC genes of the plasmid-based and E. coli chromosomal ars operons. The cloned P. aeruginosa chromosomal ars operon confers augmented resistance to arsenic and antimony oxyanions in an E. coli arsB mutant and in wild-type P. aeruginosa. Expression of the operon was induced by arsenite at the mRNA level. DNA sequences homologous with this operon were detected in some, but not all, species of the genus Pseudomonas, suggesting that its conservation follows their taxonomic-based evolution.

Assessment of phylogenetic diversity of bacterial microflora in drinking water using serial analysis of ribosomal sequence tags.

We examined chlorinated drinking water samples from three different surface water treatment plants for bacterial 16S rDNA diversity using the serial analysis of V6 ribosomal sequence tag (SARST-V6) method. A considerable degree of diversity was observed in each sample, with an estimated richness ranging from 173 to 333 phylotypes. The community structure shows that there are differences in bacterial evenness between sampled sites. The taxonomic composition of the microbial communities was found to be dominated by members of the Proteobacteria (57.2-77.4%), broadly distributed among the classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria. Additionally, a large proportion of sequences (6.3-36.5%) were found to be distantly related to database sequences of unknown phylogenetic affiliation. Given the apparent significance of this bacterial group in drinking water, a 16S rDNA analysis was performed and confirmed their presence and phylogeny. Notwithstanding the potential under-representation of certain bacterial phyla using the SARST-V6 primer pairs, as revealed by a refined computer algorithm, our results suggest that 16S rDNA corresponding to a variety of eubacterial groups can be detected in finished drinking water, suggesting that this water may contain a higher level of bacterial diversity than previously observed.

An examination of the bacteriophages and bacteria of the Namib desert

Bacteria and their viruses (called bacteriophages, or phages), have been found in virtually every ecological niche on Earth. Arid regions, including their most extreme form called deserts, represent the single largest ecosystem type on the Earth’s terrestrial surface. The Namib desert is believed to be the oldest (80 million years) desert. We report here an initial analysis of bacteriophages isolated from the Namib desert using a combination of electron microscopy and genomic approaches. The virus-like particles observed by electron microscopy revealed 20 seemingly different phage-like morphologies and sizes belonging to the Myoviridae and Siphoviridae families of tailed phages. Pulsed-field gel electrophoresis revealed a majority of phage genomes of 55∼65 kb in length, with genomes of approximately 200, 300, and 350 kb also observable. Sample sequencing of cloned phage DNA fragments revealed that approximately 50% appeared to be of bacterial origin. Of the remaining DNA sequences, approximately 50% displayed no significant match to any sequence in the databases. The majority of the 16S rDNA sequences amplified from DNA extracted from the sand displayed considerable (94∼98%) homology to members of the Firmicutes, and in particular to members of the genus Bacillus, though members of the Bacteroidetes, Planctomycetes, Chloroflexi, and 8-Proteobacteria groups were also observed.

Specific detection of organotin compounds with a recombinant luminescent bacteria

Organotin compounds are widely used as biocides in marine and terrestrial environments. Several currently used techniques allow either the measurement of the chemicals or their effects on living organisms. Our current research focuses on the development of a complementary method based on a bacterial bioluminescence-based bioassay for the specific detection of organotin compounds. The performance of the bioassay was assessed. The Escherichia coli bacterial strain used in this study is specific for TBT and DBT (with Cl, Br or I as the halogen group) with the central tin atom important for light production. The assay is conducted after overnight culture of the bacterial strain, followed by 60 min of contact time with the organotin compound for significant light production. The detection limits were found to be 0.08 microM for TBT (26 microgl(-1)) and 0.0001 microM for DBT (0.03 microgl(-1)) with a linear range of one logarithm. The repeatability of the bioassay is 8% and the reproducibility for TBT and DBT was approximately 14%. Lyophilization of the strains did not significantly modify the detection limit as well as the range of detection. Applications of the bioassay to environmental samples are discussed.

Variations of bacterial 16S rDNA phylotypes prior to and after chlorination for drinking water production from two surface water treatment plants

We examined the variations of bacterial populations in treated drinking water prior to and after the final chlorine disinfection step at two different surface water treatment plants. For this purpose, the bacterial communities present in treated water were sampled after granular activated carbon (GAC) filtration and chlorine disinfection from two drinking water treatment plants supplying the city of Paris (France). Samples were analyzed after genomic DNA extraction, polymerase chain reaction (PCR) amplification, cloning, and sequencing of a number of 16S ribosomal RNA (rRNA) genes. The 16S rDNA sequences were clustered into operational taxonomic units (OTUs) and the OTU abundance patterns were obtained for each sample. The observed differences suggest that the chlorine disinfection step markedly affects the bacterial community structure and composition present in GAC water. Members of the Alphaproteobacteria and Betaproteobacteria were found to be predominant in the GAC water samples after phylogenetic analyses of the OTUs. Following the chlorine disinfection step, numerous changes were observed, including decreased representation of Proteobacteria phylotypes. Our results indicate that the use of molecular methods to investigate changes in the abundance of certain bacterial groups following chlorine-based disinfection will aid in further understanding the bacterial ecology of drinking water treatment plants (DWTPs), particularly the disinfection step, as it constitutes the final barrier before drinking water distribution to the consumer’s tap.

Use of a luminescent bacterial biosensor for biomonitoring and characterization of arsenic toxicity of chromated copper arsenate (CCA).

The increase of arsenic (As) concentration on the earth’s surface is due both to natural sources, such as volcanic activity and weathering processes, and to anthropogenic sources, such as mining activities, agricultural and forestry applications. One example of anthropogenic arsenic contamination in the environment is the use of arsenic-containing wood preservatives. The most extensively used wood preservative is chromated copper arsenate (CCA), which is pressurised into the wood through a process called ‘Wolmanizing’ [1]. Wood intended for marine uses receives 24–40 kg CCA per cubic meter of wood to prevent its destruction by bacteria, fungi and insects. Each of the three chemicals in CCA is known to be toxic to aquatic biota at concentrations above trace levels, and found to be leached from the treated wood in both fresh and sea water [2–4]. Chemicals leached from CCA-treated wood can affect organisms that grow on the wood itself and those that live adjacent to the CCA-treated bulkheads, and also be adsorbed onto sediments, where they can be slowly released or taken up by benthic organisms [4–6]. The rate of metal accumulation in sediments and in benthos differs with each chemical in the order: Cu>As>Cr, and decreases with distance and time [1, 7]. Benthic organisms living near CCA-treated bulkheads were found to contain elevated levels of Cu and As. The number of individuals, as well as the species diversity, were also decreased at sites adjacent to CCA-treated bulkheads [1]. Pathologic and genotoxic effects have also been observed in oysters (Crassostrea virginica) living on CCA-treated wood [8]. In addition, CCA was shown to affect the growth of PCP-degrading bacterial species and their ability to degrade PCP [9, 10]. Thus, monitoring of bioavailable amounts of CCA released by the treated wood, is important in order to detect and rectify its toxic effects. Among the three chemical constituents of CCA, arsenic is the most abundant in the environment, and known to have carcinogenic and teratogenic effects on humans upon chronic exposure [11, 12]. Therefore, the focus of this study is on biomonitoring of arsenic toxicity.

Characterization of the effects of aluminum on luciferase biosensors for the detection of ecotoxicity

Luciferase-based biosensors are becoming increasingly used for environmental monitoring. A transcriptional fusion of the Vibrio harveyi luxAB genes (encoding bacterial luciferase) to the fliC gene of Escherichia coli was constructed and luminescence shown to be induced (in liquid media) in the presence of 1-10 micrograms/ml aluminum, but not copper, iron or nickel. Moreover, luminescence is markedly increased at pH 5.5, where aluminum is more soluble than at pH 7.0. However, aluminum also stimulated luciferase activity when the luxAB genes were located in the xyl operon. This suggests that aluminum stimulates luciferase enzyme activity in vivo. These results are specific to E. coli, as no such aluminum stimulation was observed in the luminescent bacterium V. harveyi. These results have important implications in the generalized use of these clones for environmental monitoring, where aluminum can be present at elevated concentrations.