Scholarly Work - Center for Biofilm Engineering
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/9335
Browse
17 results
Search Results
Item DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology(2019-09) Pratt, Shawna L.; Zath, Geoffrey K.; Williamson, Kelly S.; Franklin, Michael J.; Chang, Connie B.The physiological heterogeneity of cells within a microbial population imparts resilience to stresses such as antimicrobial treatments and nutrient limitation. This resilience is partially due to a subpopulation of cells that can survive such stresses and regenerate the community. Microfluidic approaches now provide a means to study microbial physiology and bacterial heterogeneity at the single cell level, improving our ability to isolate and examine these subpopulations. Drop-based microfluidics provides a high-throughput approach to study individual cell physiology within bacterial populations. Using this approach, single cells are isolated from the population and encapsulated in growth medium dispersed in oil using a 15 μm diameter drop making microfluidic device. The drops are arranged as a packed monolayer inside a polydimethylsiloxane (PDMS) microfluidic device. Growth of thousands of individual cells in identical microenvironments can then be imaged using confocal laser scanning microscopy (CLSM). A challenge for this approach has been the maintenance of drop stability during extended time-lapse imaging. In particular, the drops do not maintain their volume over time during incubation in PDMS devices, due to fluid transport into the porous PDMS surroundings. Here, we present a strategy for PDMS device preparation that stabilizes drop position and volume within a drop array on a microfluidic chip for over 20 h. The stability of water-in-oil drops is maintained by soaking the device in a reservoir containing both water and oil in thermodynamic equilibrium. This ensures that phase equilibrium of the drop emulsion fluids within the porous PDMS material is maintained during drop incubation and imaging. We demonstrate the utility of this approach, which we label DropSOAC (DropStabilization On AChip), for time-lapse studies of bacterial growth. We characterize growth of Pseudomonas aeruginosa and its Δhpf mutant derivative during resuscitation and growth following starvation. We demonstrate that growth rate and lag time heterogeneity of hundreds of individual bacterial cells can be determined starting from single isolated cells. The results show that the DropSOAC capsule provides a high-throughput approach toward studies of microbial physiology at the single cell level, and can be used to characterize physiological differences of cells from within a larger population.Item Metagenomic Profiling of Microbial Pathogens in the Little Bighorn River, Montana(2019-03) Hamner, Steve; Brown, Bonnie L.; Hasan, Nur A.; Franklin, Michael J.; Doyle, John T.; Eggers, Margaret J.; Colwell, Rita R.; Ford, Tim E.The Little Bighorn River is the primary source of water for water treatment plants serving the local Crow Agency population, and has special significance in the spiritual and ceremonial life of the Crow tribe. Unfortunately, the watershed suffers from impaired water quality, with high counts of fecal coliform bacteria routinely measured during run-off events. A metagenomic analysis was carried out to identify potential pathogens in the river water. The Oxford Nanopore MinION platform was used to sequence DNA in near real time to identify both uncultured and a coliform-enriched culture of microbes collected from a popular summer swimming area of the Little Bighorn River. Sequences were analyzed using CosmosID bioinformatics and, in agreement with previous studies, enterohemorrhagic and enteropathogenic Escherichia coli and other E. coli pathotypes were identified. Noteworthy was detection and identification of enteroaggregative E. coli O104:H4 and Vibrio cholerae serotype O1 El Tor, however, cholera toxin genes were not identified. Other pathogenic microbes, as well as virulence genes and antimicrobial resistance markers, were also identified and characterized by metagenomic analyses. It is concluded that metagenomics provides a useful and potentially routine tool for identifying in an in-depth manner microbial contamination of waterways and, thereby, protecting public health.Item Expression and regulation of the Pseudomonas aeruginosa hibernation promoting factor(2018-10) Akiyama, Tatsuya; Williamson, Kerry S.; Franklin, Michael J.Bacterial biofilms contain subpopulations of cells that are dormant and highly tolerant to antibiotics. While dormant, the bacteria must maintain the integrity of macromolecules required for resuscitation. Previously, we showed that hibernation promoting factor (HPF) is essential for protecting Pseudomonas aeruginosa from ribosomal loss during dormancy. In this study, we mapped the genetic components required for hpf expression. Using 5ʹ‐RACE and fluorescent protein reporter fusions, we show that hpf is expressed as part of the rpoN operon, but that hpf also has a second promoter (Phpf) within the rpoN gene. Phpf is active when the cells enter stationary phase, and expression from Phpf is modulated, but not eliminated, in mutant strains impaired in stationary phase transition (ΔdksA2, ΔrpoS and ΔrelA/ΔspoT mutants). The results of reporter gene studies and mRNA folding predictions indicated that the 5ʹ end of the hpf mRNA may also influence hpf expression. Mutations that opened or that stabilized the mRNA hairpin loop structures strongly influenced the amount of HPF produced. The results demonstrate that hpf is expressed independently of rpoN, and that hpf regulation includes both transcriptional and post‐transcriptional processes, allowing the cells to produce sufficient HPF during stationary phase to maintain viability while dormant.Item Resuscitation of Pseudomonas aeruginosa from dormancy requires hibernation promoting factor (PA4463) for ribosome preservation(2017-03) Akiyama, Tatsuya; Williamson, Kerry S.; Schaefer, Robert; Pratt, Shawna; Chang, Connie B.; Franklin, Michael J.Pseudomonas aeruginosa biofilm infections are difficult to treat with antibiotic therapy in part because the biofilms contain subpopulations of dormant antibiotic-tolerant cells. The dormant cells can repopulate the biofilms following alleviation of antibiotic treatments. While dormant, the bacteria must maintain cellular integrity, including ribosome abundance, to reinitiate the de novo protein synthesis required for resuscitation. Here, we demonstrate that the P. aeruginosa gene PA4463 [hibernation promoting factor (HPF)], but not the ribosome modulation factor (PA3049), is required for ribosomal NA preservation during prolonged nutrient starvation conditions. Single-cell–level studies using fluorescence in situ hybridization (FISH) and growth in microfluidic drops demonstrate that, in the absence of hpf, the rRNA abundances of starved cells decrease to levels that cause them to lose their ability to resuscitate from starvation, leaving intact nondividing cells. P. aeruginosa defective in the stringent response also had reduced ability to resuscitate from dormancy. However, FISH analysis of the starved stringent response mutant showed a bimodal response where the individual cells contained either abundant or low ribosome content, compared with the wild-type strain. The results indicate that ribosome maintenance is key for maintaining the ability of P. aeruginosa to resuscitate from starvation-induced dormancy and that HPF is the major factor associated with P. aeruginosa ribosome preservation.Item Heterogeneous rpoS and rhlR mRNA levels and 16S rRNA/rDNA ratios within Pseudomonas aeruginosa biofilms, sampled by laser capture microdissection(2010-03) Perez-Osorio, Ailyn C.; Williamson, Kerry S.; Franklin, Michael J.The local environmental conditions in biofilms are dependent on the impinging aqueous solution, chemical diffusion, and the metabolic activities of cells within the biofilms. Chemical gradients established in biofilms lead to physiological heterogeneities of bacterial gene expression. Previously, we used laser capture microdissection (LCM) and quantitative RT-PCR to target defined biofilm subpopulations for gene expression studies. Here, we combined that approach with quantitative PCR of bacterial DNA to normalize gene expression per cell. By comparing the ratio of 16S rRNA to 16S rDNA, we demonstrate that cells at the top of thick Pseudomonas aeruginosa biofilms have 16S rRNA/genome ratios similar to cells in a transition between exponential and stationary phase. Cells in the middle and bottom layers of these biofilms have ratios that are not significantly different from stationary phase planktonic cultures. Since much of the biofilm appeared to be in a stationary phase-like state, we analyzed local amounts of the stationary phase sigma factor, rpoS, and a quorum sensing regulator, rhlR, per cell. Surprisingly, the amount of rpoS mRNA was greatest at the top of these biofilms at the air-biofilm interface. Less than one rpoS mRNA transcript per cell was observed in the middle or base of the biofilms. The rhlR mRNA content was also greatest at the top of these biofilms, with little detectable rhlR expression at the middle or bottom of the biofilms. While cell density is slightly greater at the bottom of the biofilms, expression of this quorum sensing regulator occurs primarily at the top of the biofilms, where cell metabolic activity is greatest, as indicated by the local expression of the housekeeping gene, acpP and by expression from a constitutive Ptrc promoter. The results indicate that in thick P. aeruginosa biofilms, cells in the 30 µm adjacent to the air-biofilm interface actively express genes associated with stationary phase, while cells in the interior portions do not express these genes, and therefore are in a late stationary phase-like state and are possibly dormant.Item Biosynthesis of the Pseudomonas aeruginosa extracellular polysaccharides, alginate, Pel, and Psl(2011-08) Franklin, Michael J.; Nivens, David E.; Weadge, J. T.; Howell, P. L.Pseudomonas aeruginosa thrives in many aqueous environments and is an opportunistic pathogen that can cause both acute and chronic infections. Environmental conditions and host defenses cause differing stresses on the bacteria, and to survive in vastly different environments, P. aeruginosa must be able to adapt to its surroundings. One strategy for bacterial adaptation is to self-encapsulate with matrix material, primarily composed of secreted extracellular polysaccharides. P. aeruginosa has the genetic capacity to produce at least three secreted polysaccharides; alginate, Psl, and Pel. These polysaccharides differ in chemical structure and in their biosynthetic mechanisms. Since alginate is often associated with chronic pulmonary infections, its biosynthetic pathway is the best characterized. However, alginate is only produced by a subset of P. aeruginosa strains. Most environmental and other clinical isolates secrete either Pel or Psl. Little information is available on the biosynthesis of these polysaccharides. Here, we review the literature on the alginate biosynthetic pathway, with emphasis on recent findings describing the structure of alginate biosynthetic proteins. This information combined with the characterization of the domain architecture of proteins encoded on the Psl and Pel operons allowed us to make predictive models for the biosynthesis of these two polysaccharides. The results indicate that alginate and Pel share certain features, including some biosynthetic proteins with structurally or functionally similar properties. In contrast, Psl biosynthesis resembles the EPS/CPS capsular biosynthesis pathway of Escherichia coli, where the Psl pentameric subunits are assembled in association with an isoprenoid lipid carrier. These models and the environmental cues that cause the cells to produce predominantly one polysaccharide over the others are subjects of current investigation.Item Genotypic and phenotypic variation in Pseudomonas aeruginosa reveals signatures of secondary infection and mutator activity in certain Cystic Fibrosis patients with chronic lung infections(2011-09) Warren, A. E.; Boulianne-Larsen, C. M.; Chandler, C. B.; Chlotti, K.; Kroll, E.; Miller, S. R.; Taddei, F.; Sermet-Gaudelus, I.; Ferroni, A.; McInnerney, Kathleen; Franklin, Michael J.; Rosenzweig, F.Evolutionary adaptation of Pseudomonas aeruginosa to the cystic fibrosis lung is limited by genetic variation, which depends on rates of horizontal gene transfer and mutation supply. Because each may increase following secondary infection or mutator emergence, we sought to ascertain the incidence of secondary infection and genetic variability in populations containing or lacking mutators. Forty-nine strains collected over 3 years from 16 patients were phenotyped for antibiotic resistance and mutator status and were genotyped by repetitive-sequence PCR (rep-PCR), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Though phenotypic and genetic polymorphisms were widespread and clustered more strongly within than between longitudinal series, their distribution revealed instances of secondary infection. Sequence data, however, indicated that interlineage recombination predated initial strain isolation. Mutator series were more likely to be multiply antibiotic resistant, but not necessarily more variable in their nucleotide sequences, than nonmutators. One mutator and one nonmutator series were sequenced at mismatch repair loci and analyzed for gene content using DNA microarrays. Both were wild type with respect to mutL, but mutators carried an 8-bp mutS deletion causing a frameshift mutation. Both series lacked 126 genes encoding pilins, siderophores, and virulence factors whose inactivation has been linked to adaptation during chronic infection. Mutators exhibited loss of severalfold more genes having functions related to mobile elements, motility, and attachment. A 105-kb, 86-gene deletion was observed in one nonmutator that resulted in loss of virulence factors related to pyoverdine synthesis and elements of the multidrug efflux regulon. Diminished DNA repair activity may facilitate but not be absolutely required for rapid evolutionary change.Item Heterogeneity in Pseudomonas aeruginosa biofilms includes expression of ribosome hibernation factors in the antibiotic-tolerant subpopulation and hypoxia-induced stress response in the metabolically active population(2012-02) Williamson, Kerry S.; Richards, Lee A.; Perez-Osorio, Ailyn C.; Pitts, Betsey; McInnerney, Kathleen; Stewart, Philip S.; Franklin, Michael J.Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptome responses of Pseudomonas aeruginosa growing in biofilms by using a microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNA levels for these housekeeping genes were low in cells at the bottom of the biofilms. Selective green fluorescent protein (GFP) labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNA levels for genes regulated by the hypoxia-induced regulator Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-term anoxia. Transcripts for ribosomal proteins were associated primarily with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (the rmf and PA4463 genes) at the bottom of the biofilms. The dormant biofilm cells of a P. aeruginosa Δrmf strain had decreased membrane integrity, as shown by propidium iodide staining. Using selective GFP labeling and cell sorting, we show that the dividing cells are more susceptible to killing by tobramycin and ciprofloxacin. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state.Item Microbial and algal alginate gelation characterized by magnetic resonance(2012-10) Fabich, H. T.; Vogt, Sarah J.; Sherick, Matthew L.; Seymour, Joseph D.; Brown, Jennifer R.; Franklin, Michael J.; Codd, Sarah L.Advanced magnetic resonance (MR) relaxation and diffusion correlation measurements and imaging provide a means to non-invasively monitor gelation for biotechnology applications. In this study, MR is used to characterize physical gelation of three alginates with distinct chemical structures; an algal alginate, which is not O-acetylated but contains poly guluronate (G) blocks, bacterial alginate from Pseudomonas aeruginosa, which does not have poly-G blocks, but is O-acetylated at the C2 and/or C3 of the mannuronate residues, and alginate from a P. aeruginosa mutant that lacks O-acetyl groups. The MR data indicate that diffusion-reaction front gelation with Ca2+ ions generates gels of different bulk homogeneities dependent on the alginate structure. Shorter spin–spin T2 magnetic relaxation times in the alginate gels that lack O-acetyl groups indicate stronger molecular interaction between the water and biopolymer. The data characterize gel differences over a hierarchy of scales from molecular to system size.Item Draft genome sequence and description of Janthinobacterium sp. strain CG3, a psychrotolerant antarctic Supraglacial stream bacterium(2013-11) Smith, Heidi J.; Akiyama, Tatsuya; Foreman, Christine M.; Franklin, Michael J.; Woyke, Tanja; Teshima, H; Davenport, K.; Daligault, H.; Erkkila, T.; Goodwin, L. A.; Gu, W.; Xu, Yan; Chain, P. S.Here we present the draft genome sequence of Janthinobacterium sp. strain CG3, a psychrotolerant non-violacein-producing bacterium that was isolated from the Cotton Glacier supraglacial stream. The genome sequence of this organism will provide insight into the mechanisms necessary for bacteria to survive in UV-stressed icy environments.