Marc B. Habash

Disparate Subcellular Localization Patterns of Pseudomonas aeruginosa Type IV Pilus ATPases Involved in Twitching Motility

The opportunistic pathogen Pseudomonas aeruginosa expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be uncoupled. Truncated fusions that retained polar localization maintained their distinctive distribution patterns. To dissect the cellular factors involved in establishing polarity, fusion protein localization was monitored with a panel of TFP mutants. The localization of yellow fluorescent protein (YFP)-PilT and YFP-PilU was independent of the subunit PilA, other TFP ATPases, and TFP-associated proteins previously shown to be associated with the membrane or exhibiting polar localization. In contrast, YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB. Finally, localization studies performed with fluorescent ATPase chimeras of PilT and PilU demonstrated that information responsible for the characteristic localization patterns of the ATPases likely resides in their N termini.

Genetic improvement of native xylose-fermenting yeasts for ethanol production.

Lignocellulosic substrates are the largest source of fermentable sugars for bioconversion to fuel ethanol and other valuable compounds. To improve the economics of biomass conversion, it is essential that all sugars in potential hydrolysates be converted efficiently into the desired product(s). While hexoses are fermented into ethanol and some high-value chemicals, the bioconversion of pentoses in hydrolysates remains inefficient. This remains one of the key challenges in lignocellulosic biomass conversion. Native pentose-fermenting yeasts can ferment both glucose and xylose in lignocellulosic biomass to ethanol. However, they perform poorly in the presence of hydrolysate inhibitors, exhibit low ethanol tolerance and glucose repression, and ferment pentoses less efficiently than the main hexoses glucose and mannose. This paper reviews classical and molecular strain improvement strategies applied to native pentose-fermenting yeasts for improved ethanol production from xylose and lignocellulosic substrates. We focus on Pachysolen tannophilus, Scheffersomyces (Candida) shehatae, Scheffersomyces (Pichia) stipitis, and Spathaspora passalidarum which are good ethanol producers among the native xylose-fermenting yeasts. Strains obtained thus far are not robust enough for efficient ethanol production from lignocellulosic hydrolysates and can benefit from further improvements.

Synergy of Silver Nanoparticles and Aztreonam against Pseudomonas aeruginosa PAO1 Biofilms

ABSTRACT Pathogenic bacterial biofilms, such as those found in the lungs of patients with cystic fibrosis (CF), exhibit increased antimicrobial resistance, due in part to the inherent architecture of the biofilm community. The protection provided by the biofilm limits antimicrobial dispersion and penetration and reduces the efficacy of antibiotics that normally inhibit planktonic cell growth. Thus, alternative antimicrobial strategies are required to combat persistent infections. The antimicrobial properties of silver have been known for decades, but silver and silver-containing compounds have recently seen renewed interest as antimicrobial agents for treating bacterial infections. The goal of this study was to assess the efficacy of citrate-capped silver nanoparticles (AgNPs) of various sizes, alone and in combination with the monobactam antibiotic aztreonam, to inhibit Pseudomonas aeruginosa PAO1 biofilms. Among the different sizes of AgNPs examined, 10-nm nanoparticles were most effective in inhibiting the recovery of P. aeruginosa biofilm cultures and showed synergy of inhibition when combined with sub-MIC levels of aztreonam. Visualization of biofilms treated with combinations of 10-nm AgNPs and aztreonam indicated that the synergistic bactericidal effects are likely caused by better penetration of the small AgNPs into the biofilm matrix, which enhances the deleterious effects of aztreonam against the cell envelope of P. aeruginosa within the biofilms. These data suggest that small AgNPs synergistically enhance the antimicrobial effects of aztreonam against P. aeruginosa in vitro, and they reveal a potential role for combinations of small AgNPs and antibiotics in treating patients with chronic infections.

A Temporal Examination of the Planktonic and Biofilm Proteome of Whole Cell Pseudomonas aeruginosa PAO1 Using Quantitative Mass Spectrometry

Chronic polymicrobial lung infections are the chief complication in patients with cystic fibrosis. The dominant pathogen in late-stage disease is Pseudomonas aeruginosa, which forms recalcitrant, structured communities known as biofilms. Many aspects of biofilm biology are poorly understood; consequently, effective treatment of these infections is limited, and cystic fibrosis remains fatal. Here we combined in-solution protein digestion of triplicate growth-matched samples with a high-performance mass spectrometry platform to provide the most comprehensive proteomic dataset known to date for whole cell P. aeruginosa PAO1 grown in biofilm cultures. Our analysis included protein–protein interaction networks and PseudoCAP functional information for unique and significantly modulated proteins at three different time points. Secondary analysis of a subgroup of proteins using extracted ion currents validated the spectral counting data of 1884 high-confidence proteins. In this paper we demonstrate a greater representation of proteins related to metabolism, DNA stability, and molecular activity in planktonically grown P. aeruginosa PAO1. In addition, several virulence-related proteins were increased during planktonic growth, including multiple proteins encoded by the pyoverdine locus, uncharacterized proteins with sequence similarity to mammalian cell entry protein, and a member of the hemagglutinin family of adhesins, HecA. Conversely, biofilm samples contained an uncharacterized protein with sequence similarity to an adhesion protein with self-association characteristics (AidA). Increased levels of several phenazine biosynthetic proteins, an uncharacterized protein with sequence similarity to a metallo-beta-lactamase, and lower levels of the drug target gyrA support the putative characteristics of in situ P. aeruginosa infections, including competitive fitness and antibiotic resistance. This quantitative whole cell approach advances the existing P. aeruginosa subproteomes and provides a framework for identifying and studying entire pathways critical to biofilm biology in this model pathogenic organism. The identification of novel protein targets could contribute to the development of much needed antimicrobial therapies to treat the chronic infections found in patients with cystic fibrosis.

Single-Residue Changes in the C-Terminal Disulfide-Bonded Loop of the Pseudomonas aeruginosa Type IV Pilin Influence Pilus Assembly and Twitching Motility

PilA, the major pilin subunit of Pseudomonas aeruginosa type IV pili (T4P), is a principal structural component. PilA has a conserved C-terminal disulfide-bonded loop (DSL) that has been implicated as the pilus adhesinotope. Structural studies have suggested that DSL is involved in intersubunit interactions within the pilus fiber. PilA mutants with single-residue substitutions, insertions, or deletions in the DSL were tested for pilin stability, pilus assembly, and T4P function. Mutation of either Cys residue of the DSL resulted in pilins that were unable to assemble into fibers. Ala replacements of the intervening residues had a range of effects on assembly or function, as measured by changes in surface pilus expression and twitching motility. Modification of the C-terminal P-X-X-C type II beta-turn motif, which is one of the few highly conserved features in pilins across various species, caused profound defects in assembly and twitching motility. Expression of pilins with suspected assembly defects in a pilA pilT double mutant unable to retract T4P allowed us to verify which subunits were physically unable to assemble. Use of two different PilA antibodies showed that the DSL may be an immunodominant epitope in intact pili compared with pilin monomers. Sequence diversity of the type IVa pilins likely reflects an evolutionary compromise between retention of function and antigenic variation. The consequences of DSL sequence changes should be evaluated in the intact protein since it is technically feasible to generate DSL-mimetic peptides with mutations that will not appear in the natural repertoire due to their deleterious effects on assembly.

Characterization of sources and loadings of fecal pollutants using microbial source tracking assays in urban and rural areas of the Grand River Watershed, Southwestern Ontario

Sources of fecal water pollution were assessed in the Grand River and two of its tributaries (Ontario, Canada) using total and host-specific (human and bovine) Bacteroidales genetic markers in conjunction with reference information, such as land use and weather. In-stream levels of the markers and culturable Escherichia coli were also monitored during multiple rain events to gain information on fecal loadings to catchment from diffuse sources. Elevated human-specific marker levels were accurately identified in river water impacted by a municipal wastewater treatment plant (WWTP) effluent and at a downstream site in the Grand River. In contrast, the bovine-specific marker showed high levels of cattle fecal pollution in two tributaries, both of which are characterized as intensely farmed areas. The bovine-specific Bacteroidales marker increased with rainfall in the agricultural tributaries, indicating enhanced loading of cattle-derived fecal pollutants to river from non-point sources following rain events. However, rain-triggered fecal loading was not substantiated in urban settings, indicating continuous inputs of human-originated fecal pollutants from point sources, such as WWTP effluent. This study demonstrated that the Bacteroidales source tracking assays, in combination with land use information and hydrological data, may provide additional insight into the spatial and temporal distribution of source-specific fecal contamination in streams impacted by varying land uses. Using the approach described in this study may help to characterize impacted water sources and to design targeted land use management plans in other watersheds in the future.

Optimization, validation, and application of a real-time PCR protocol for quantification of viable bacterial cells in municipal sewage sludge and biosolids using reporter genes and Escherichia coli

Abstract Biosolids result from treatment of sewage sludge to meet jurisdictional standards, including pathogen reduction. Once government regulations are met, materials can be applied to agricultural lands. Culture-based methods are used to enumerate pathogen indicator microorganisms but may underestimate cell densities, which is partly due to bacteria existing in a viable but non-culturable physiological state. Viable indicators can also be quantified by real-time polymerase chain reaction (qPCR) used with propidium monoazide (PMA), a dye that inhibits amplification of DNA found extracellularly or in dead cells. The objectives of this study were to test an optimized PMA-qPCR method for viable pathogen detection in wastewater solids and to validate it by comparing results to data obtained by conventional plating. Reporter genes from genetically marked Pseudomonas sp. UG14Lr and Agrobacterium tumefaciens 542 cells were spiked into samples of primary sludge, and anaerobically digested and Lystek-treated biosolids as cell-free DNA, dead cells, viable cells, and mixtures of live and dead cells, followed by DNA extraction with and without PMA, and qPCR. The protocol was then used for Escherichia coli quantification in the three matrices, and results compared to plate counts. PMA-qPCR selectively detected viable cells, while inhibiting signals from cell-free DNA and DNA found in membrane-compromised cells. PMA-qPCR detected 0.5–1 log unit more viable E. coli cells in both primary solids and dewatered biosolids than plate counts. No viable E. coli was found in Lystek-treated biosolids. These data suggest PMA-qPCR may more accurately estimate pathogen cell numbers than traditional culture methods.

The dynamic nature of bacterial surfaces: implications for metal-membrane interaction.

Bacterial envelopes are chemically complex, diverse structures. Chemical and physical influences from cellular microenvironments force lipids, proteins, and sugars to organize dynamically. This constant reorganization serves to maintain compartmentalization and function, but also affects the influence of charged functional groups that drive electrochemical interactions with metal ions. The interactions of metal species with cell walls are of particular interest because (i) metals must be taken up or excluded to maintain cell function, and (ii) electrochemical interactions between charged metals and anionic ligands are inevitable. In this review we explore the associations of metals with metal-reactive ligands found within bacterial envelopes, and outward to include those within biofilm matrics. The mechanisms that underpin metal binding to these ligands have not been well considered with respect to the dynamic organization of the biological structures themselves. Bacteria respond sensitively and rapidly to growth environment with de novo syntheses of chemical constituents, which can impact metal interactions. We discuss causes of membrane chemical variability as observed in laboratory experiments, and offer consequences for this adaptability in natural settings. The structural impacts of metal ion associations with bacterial envelopes are often overlooked. This review explores how dynamic bacterial surface chemistry influences metal binding and, in turn, how metal ions impact membrane organization in laboratory and natural conditions.

Monitoring Bacteroides spp. markers, nutrients, metals and Escherichia coli in soil and leachate after land application of three types of municipal biosolids.

A lysimeter-based field study was done to monitor the transfer of culturable Escherichia coli, general (ALLBAC), human (Hf183) and swine (PIG-BAC-1) specific 16S rRNA Bacteroides spp. markers, nutrients and metals through soils and leachate over time following land application of a CP1/Class A as well as two CP2/Class B municipal biosolids (MBs). Hf183 markers were detected up to six days following application in soils receiving dewatered and liquid MBs, but not in leachate, suggesting their use in source tracking is better suited for recent pollution events. The CP2/Class B biosolids and swine manure contributed the highest microbial load with E. coli loads (between 2.5 and 3.7 log CFU (100 mL)(-1)) being greater than North American concentration recommendations for safe recreational water. ALLBAC persisted in soils and leachate receiving all treatments and was detected prior to amendment application demonstrating its unsuitability for identifying the presence of fecal pollution. A significant increase in NO₃-N (for Lystek and dewatered MBs) and total-P (for dewatered and liquid MBs) in leachate was observed in plots receiving the CP1/Class A and CP2/Class B type MBs which exceeded North American guidelines, suggesting impact to surface water. Metal (As, Cd, Cr, Co, Cu, Pb, Mo, Ni, Se, Zn and Hg) transfer was negligible in soil and leachate samples receiving all treatments. This study is one of the first to examine the fate of E. coli and Bacteroides spp. markers in situ following the land application of MBs where surface runoff does not apply.

Bacterial Reductive Dehalogenases

Toxic halogenated organic compounds can originate from both natural (Gribble 1994, 1996) and industrial sources (Fetzner 1998; Mohn and Tiedje 1992). Many industrially produced polyhalogenated organic compounds such as pentachlorophenol (PCP), polychlorinated biphenyls (PCBs), perchloroethylene (PCE), trichloroetheylene (TCE) and dichloromethane (DCM) are significant environmental and human health problems due to their toxicity and persistence in the environment. However, despite their toxicity, some bacteria have evolved mechanisms to metabolize these compounds. A key group of bacterial enzymes that initiate the degradation of halo-organic compounds is the dehalogenases. Many types of dehalogenases have been described. They include monooxygenases, dioxygenases, dehydrohalogenases, reductive dehalogenases, hydrolytic dehalogenases, thiolytic dehalogenases, and methyl transferases (Fetzner 1998). This chapter examines bacterial reductive dehalogenases that catalyze the substitution of a halogen substituent, typically a chlorine, bromine or iodine atom, with a hydrogen atom.