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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 Gene expression and protein levels of the stationary phase sigma factors, RpoS, in continuously-fed Pseudomonas aeruginosa biofilms(2001-05) Xu, Karen D.; Franklin, Michael J.; Park, C-H; McFeters, Gordon A.; Stewart, Philip S.Bacteria growing in biofilms experience gradients of environmental conditions, including varying levels of nutrients and oxygen. Therefore, bacteria within biofilms may enter distinct physiological states, depending on the surrounding conditions. In this study, rpoS expression and RpoS levels were measured as indicators of stationary phase growth within thick continuously-fed Pseudomonas aeruginosa biofilms. The level of rpoS expression in a 3-day-old biofilm was found to be three-fold higher than the average expression in stationary phase planktonic cultures. In planktonic cultures, oxygen limitation did not lead to increased levels of RpoS, suggesting that oxygen limitation was not the environmental signal causing increased expression of rpoS. These results suggest that bacteria within P. aeruginosa biofilms may exhibit stationary phase characteristics even when cultured in flow conditions that continually replenish nutrients.Item Compromised host defense on Pseudomonas aeruginosa biofilms: Characterization of neutrophil and biofilm interactions(2003-10) Jesaitis, A. J.; Franklin, Michael J.; Berglund, Deborah L.; Sasaki, Maiko; Lord, Connie I.; Bleazard, Justin Brock; Duffy, James E.; Beyenal, Haluk; Lewandowski, ZbigniewPseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on tissues and other surfaces. We characterized the interaction of purified human neutrophils with P. aeruginosa, growing in biofilms, with regard to morphology, oxygen consumption, phagocytosis, and degranulation. Scanning electron and confocal laser microscopy indicated that the neutrophils retained a round, unpolarized, unstimulated morphology when exposed to P. aeruginosa PAO1 biofilms. However, transmission electron microscopy demonstrated that neutrophils, although rounded on their dorsal side, were phagocytically active with moderate membrane rearrangement on their bacteria-adjacent surfaces. The settled neutrophils lacked pseudopodia, were impaired in motility, and were enveloped by a cloud of planktonic bacteria released from the biofilms. The oxygen consumption of the biofilm/neutrophil system increased 6- and 8-fold over that of the biofilm alone or unstimulated neutrophils in suspension, respectively. H(2)O(2) accumulation was transient, reaching a maximal measured value of 1 micro M. Following contact, stimulated degranulation was 20-40% (myeloperoxidase, beta-glucuronidase) and 40-80% (lactoferrin) of maximal when compared with formylmethionylleucylphenylalanine plus cytochalasin B stimulation. In summary, after neutrophils settle on P. aeruginosa biofilms, they become phagocytically engorged, partially degranulated, immobilized, and rounded. The settling also causes an increase in oxygen consumption of the system, apparently resulting from a combination of a bacterial respiration and escape response and the neutrophil respiratory burst but with little increase in the soluble concentration of H(2)O(2). Thus, host defense becomes compromised as biofilm bacteria escape while neutrophils remain immobilized with a diminished oxidative potential.Item Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin(2003-01) Walters, Marshall C., III; Roe, Frank L.; Bugnicourt, Amandine; Franklin, Michael J.; Stewart, Philip S.The roles of slow antibiotic penetration, oxygen limitation, and low metabolic activity in the tolerance of Pseudomonas aeruginosa in biofilms to killing by antibiotics were investigated in vitro. Tobramycin and ciprofloxacin penetrated biofilms but failed to effectively kill the bacteria. Bacteria in colony biofilms survived prolonged exposure to either 10 micro g of tobramycin ml(-1)or 1.0 micro g of ciprofloxacin ml(-1). After 100 h of antibiotic treatment, during which the colony biofilms were transferred to fresh antibiotic-containing plates every 24 h, the log reduction in viable cell numbers was only 0.49 +/- 0.18 for tobramycin and 1.42 +/- 0.03 for ciprofloxacin. Antibiotic permeation through colony biofilms, indicated by a diffusion cell bioassay, demonstrated that there was no acceleration in bacterial killing once the antibiotics penetrated the biofilms. These results suggested that limited antibiotic diffusion is not the primary protective mechanism for these biofilms. Transmission electron microscopic observations of antibiotic-affected cells showed lysed, vacuolated, and elongated cells exclusively near the air interface in antibiotic-treated biofilms, suggesting a role for oxygen limitation in protecting biofilm bacteria from antibiotics. To test this hypothesis, a microelectrode analysis was performed. The results demonstrated that oxygen penetrated 50 to 90 micro m into the biofilm from the air interface. This oxic zone correlated to the region of the biofilm where an inducible green fluorescent protein was expressed, indicating that this was the active zone of bacterial metabolic activity. These results show that oxygen limitation and low metabolic activity in the interior of the biofilm, not poor antibiotic penetration, are correlated with antibiotic tolerance of this P. aeruginosa biofilm system.Item Evidence that the AlgI/AlgJ gene cassette, required for O-acetylation of Pseudomonas aeruginosa alginate, evolved by lateral gene transfer(2004-07) Franklin, Michael J.; Douthit, Stephanie Ann; McClure, Marcella A.Pseudomonas aeruginosa strains, isolated from chronically infected patients with cystic fibrosis, produce the O-acetylated extracellular polysaccharide, alginate, giving these strains a mucoid phenotype. O acetylation of alginate plays an important role in the ability of mucoid P. aeruginosa to form biofilms and to resist complement-mediated phagocytosis. The O-acetylation process is complex, requiring a protein with seven transmembrane domains (AlgI), a type II membrane protein (AlgJ), and a periplasmic protein (AlgF). The cellular localization of these proteins suggests a model wherein alginate is modified at the polymer level after the transport of O-acetyl groups to the periplasm. Here, we demonstrate that this mechanism for polysaccharide esterification may be common among bacteria, since AlgI homologs linked to type II membrane proteins are found in a variety of gram-positive and gram-negative bacteria. In some cases, genes for these homologs have been incorporated into polysaccharide biosynthetic operons other than for alginate biosynthesis. The phylogenies of AlgI do not correlate with the phylogeny of the host bacteria, based on 16S rRNA analysis. The algI homologs and the gene for their adjacent type II membrane protein present a mosaic pattern of gene arrangement, suggesting that individual components of the multigene cassette, as well as the entire cassette, evolved by lateral gene transfer. AlgJ and the other type II membrane proteins, although more diverged than AlgI, contain conserved motifs, including a motif surrounding a highly conserved histidine residue, which is required for alginate O-acetylation activity by AlgJ. The AlgI homologs also contain an ordered series of motifs that included conserved amino acid residues in the cytoplasmic domain CD-4; the transmembrane domains TM-C, TM-D, and TM-E; and the periplasmic domain PD-3. Site-directed mutagenesis studies were used to identify amino acids important for alginate O-acetylation activity, including those likely required for (i) the interaction of AlgI with the O-acetyl precursor in the cytoplasm, (ii) the export of the O-acetyl group across the cytoplasmic membrane, and (iii) the transfer of the O-acetyl group to a periplasmic protein or to alginate. These results indicate that AlgI belongs to a family of membrane proteins required for modification of polysaccharides and that a mechanism requiring an AlgI homolog and a type II membrane protein has evolved by lateral gene transfer for the esterification of many bacterial extracellular polysaccharides.Item Stratified growth in Pseudomonas aeruginosa biofilms(2004-10) Werner, Erin M.; Roe, Frank L.; Bugnicourt, Amandine; Franklin, Michael J.; Heydorn, Arne; Molin, Søren; Pitts, Betsey; Stewart, Philip S.In this study, stratified patterns of protein synthesis and growth were demonstrated in Pseudomonas aeruginosa biofilms. Spatial patterns of protein synthetic activity inside biofilms were characterized by the use of two green fluorescent protein (GFP) reporter gene constructs. One construct carried an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible gfpmut2 gene encoding a stable GFP. The second construct carried a GFP derivative, gfp-AGA, encoding an unstable GFP under the control of the growth-rate-dependent rrnBp1 promoter. Both GFP reporters indicated that active protein synthesis was restricted to a narrow band in the part of the biofilm adjacent to the source of oxygen. The zone of active GFP expression was approximately 60 µm wide in colony biofilms and 30 µm wide in flow cell biofilms. The region of the biofilm in which cells were capable of elongation was mapped by treating colony biofilms with carbenicillin, which blocks cell division, and then measuring individual cell lengths by transmission electron microscopy. Cell elongation was localized at the air interface of the biofilm. The heterogeneous anabolic patterns measured inside these biofilms were likely a result of oxygen limitation in the biofilm. Oxygen microelectrode measurements showed that oxygen only penetrated approximately 50 µm into the biofilm. P. aeruginosa was incapable of anaerobic growth in the medium used for this investigation. These results show that while mature P. aeruginosa biofilms contain active, growing cells, they can also harbor large numbers of cells that are inactive and not growing.Item Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms(2005-06) Sarkisova, S. A.; Patrauchan, Marianna A.; Berglund, Deborah L.; Nivens, David E.; Franklin, Michael J.Pseudomonas aeruginosa colonizes the pulmonary tissue of patientswith cystic fibrosis (CF), leading to biofilm-associated infections.The pulmonary fluid of CF patients usually contains elevated concentrations of cations and may contain the P. aeruginosa redox-active pigment pyocyanin, which is known to disrupt calcium homeostasis of host cells. Since divalent cations are important bridging ions for bacterial polysaccharides and since they may play regulatory roles in bacterial gene expression, we investigated the effect of calcium ions on the extracellular matrix constituents of P. aeruginosa biofilms. For mucoid strain P. aeruginosa FRD1,calcium addition (1.0 and 10 mM as CaCl2) resulted in biofilmsthat were at least 10-fold thicker than biofilms without added calcium. Scanning confocal laser microscopy showed increased spacing between cells for the thick biofilms, and Fourier transform infrared spectroscopy revealed that the material between cells is primarily alginate. An algD transcriptional reporter demonstrated that calcium addition caused an eightfold increase in alg gene expression in FRD1 biofilms. Calcium addition also resulted in increased amounts of three extracellular proteases (AprA, LasB, and PrpL). Immunoblots of the biofilm extracellular material established that AprA was harbored within the biofilm extracellular matrix. An aprA deletion mutation and a mutation in gene for a putative P. aeruginosa calmodulin-like protein did not significantly affect calcium-induced biofilm structure. Two-dimensional gel electrophoresis showed increased amounts of phenazine biosynthetic proteins in FRD1 biofilms and in calcium-amended planktonic cultures. Spectrochemical analyses showed that the calcium addition causes a three- to fivefold increase in pyocyanin production. These results demonstrate that calcium addition affects the structure and extracellular matrix composition of mucoid P. aeruginosa biofilms, through increased expression and stability of bacterial extracellular products. The calcium-induced extracellular matrix of mucoid P. aeruginosa consists primarily of the virulence factor alginate and also harbors extracellular proteases and perhaps pyocyanin, a biomolecule that may further disrupt cellular calcium levels.Item Calcium influences cellular and extracellular product formation during biofilm-associated growth of a marine Pseudoalteromonas sp.(2005-09) Patrauchan, Marianna A.; Sarkisova, S. A.; Sauer, K.; Franklin, Michael J.Bacteria undergo a variety of physiological changes following a switch from planktonic growth to surface-associated biofilm growth. Here, it is shown that biofilm development of a marine isolate, Pseudoalteromonas sp. 1398, results in global changes in its cytosolic and extracellular proteomes. Calcium influences these proteome responses, and affects the amount of surface-associated biomass and extracellular matrix material produced by Pseudoalteromonassp. 1398. Four extracellular proteins, characterized by N-terminalsequencing, showed increased abundances, while one protein, flagellin, showed reduced abundance at higher [Ca2+]. Immunoblottingand transmission-electron-microscopy analysis confirmed that higher [Ca2+] and surface-associated growth results in the repressionof flagella production. Two-dimensional gel electrophoresis (2DGE) studies combined with cluster analysis of global proteome responses demonstrated that Ca2+ had a greater regulatory influenceon Pseudoalteromonas sp. growing in biofilms than on planktoniccultures. Approximately 22% of the total cytosolic proteins resolved by 2DGE had differing abundances in response to a switch from planktonic growth to surface-associated growth when the cells were cultivated in 1 mM Ca2+. At higher [Ca2+] this number increased to 38%. Fifteen cellular proteins that were differentially expressed in response to biofilm growth and/or Ca2+ were analysed by N-terminal sequencing and/or MS/MS. These proteins were identified as factors involved in cellular metabolic functions, putative proteases and transport proteins, although there were several proteins that had not been previously characterized. These results indicate that Ca2+ causes global changes in matrix material, as well as in cellular and extracellular protein profiles of Pseudoalteromonas sp. 1398. These changes are more pronounced when the bacterium grows in biofilms than when it grows in planktonic culture.Item Epimerase active domain of Pseudomonas aeruginosa AlgG, a protein that contains a right-handed ß-helix(2005-06) Douthit, Stephanie Ann; Dlakic, Mensur; Ohman, Dennis E.; Franklin, Michael J.The polysaccharide alginate forms a protective capsule for Pseudomonas aeruginosa during chronic pulmonary infections. The structure of alginate, a linear polymer of ß1-4-linked O-acetylated D-mannuronate (M) and L-guluronate (G), is important for its activity as a virulence factor. Alginate structure is mediated by AlgG, a periplasmic C-5 mannuronan epimerase. AlgG also plays a role in protecting alginate from degradation by the periplasmic alginate lyase AlgL. Here, we show that the C-terminal region of AlgG contains a right-handed ß-helix (RHßH) fold, characteristic of proteins with the carbohydrate-binding and sugar hydrolase (CASH) domain. When modeled based on pectate lyase C of Erwinia chrysanthemi, the RHßH of AlgG has a long shallow groove that may accommodate alginate, similar to protein/polysaccharide interactions of other CASH domain proteins. The shallow groove contains a 324-DPHD motif that is conserved among AlgG and the extracellular mannuronan epimerases of Azotobacter vinelandii. Point mutations in this motif disrupt mannuronan epimerase activity but have no effect on alginate secretion. The D324A mutation has a dominant negative phenotype, suggesting that the shallow groove in AlgG contains the catalytic face for epimerization. Other conserved motifs of the epimerases, 361-NNRSYEN and 381-NLVAYN, are predicted to lie on the opposite side of the RHßH from the catalytic center. Point mutations N362A, N367A, and V383A result in proteins that do not protect alginate from AlgL, suggesting that these mutant proteins are not properly folded or not inserted into the alginate biosynthetic scaffold. These motifs are likely involved in asparagine and hydrophobic stacking, required for structural integrity of RHßH proteins, rather than for mannuronan catalysis. The results suggest that the AlgG RHßH protects alginate from degradation by AlgL by channeling the alginate polymer through the proposed alginate biosynthetic scaffold while epimerizing approximately every second D-mannuronate residue to L-guluronate along the epimerase catalytic face.Item Sampling and quantification of biofilms in food processing and other environments(2009) Nivens, David E.; Co, B. M.; Franklin, Michael J.In the food industry, assessment of food contact surfaces is necessary to determine whether equipment is properly cleaned and/or sanitized and whether living problematic microorganisms are present. Existing quantitative detection technologies are limited by the inability to directly detect living cells in sporadically dispersed biofilms on large surface areas. Thus, precise and accurate sampling strategies must be coupled with detection technology. This chapter discusses sampling methods and standard (e.g., plating and ATP-bioluminescence) and emerging (e.g., spectrometry, immunosensor, and nucleic acid-based) quantitative techniques to detect biofilms on food contact surfaces with a survey of function, analytical performance, and limitations.
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