Planktonic Conditions Research Articles

The planktonic foraminifera of the Jurassic. Part II: Stratigraphy, palaeoecology and palaeobiogeography

The stratigraphy, palaeoecology and palaeobiogeography are outlined for two genera and ten species of Jurassic planktonic foraminifera described in Gradstein et al. (Swiss J Palaeontol 2017. doi: 10.1007/s13358-017-0131-z). The two genera are Globuligerina and Conoglobigerina. Globuligerina probably appeared in late Toarcian (late Early Jurassic) and Conoglobigerina first occurred in Middle Oxfordian (early Late Jurassic). Within the two genera ten species are defined, including from older to younger: Globuligerina dagestanica (Morozova), G. avariformis (Kasimova), G. balakhmatovae (Morozova), G. oxfordiana (Grigelis), G. bathoniana (Pazdrowa), G. jurassica (Hofman), G. oxfordiana (Grigelis) calloviensis Kuznetsova emended, G. tojeiraensis Gradstein, Conoglobigerina helvetojurassica (Haeusler), C. grigelisi Gradstein and C. gulekhensis (Gorbachik and Poroshina). Globuligerina balakhmatovae, G. oxfordiana and G. bathoniana are longer ranging, although there are subtle evolutionary trends in these taxa that may refine stratigraphic usage. Other taxa have shorter stratigraphic ranges. Using stratigraphic trends, test morphology and wall texture changes, the species are tentatively assembled in four evolutionary groups, termed A–D, and in six zones from Late Toarcian through Tithonian. The greater species diversity and possibly also specimen abundance took place in the Kimmeridgian, a time of high global sea level and greenhouse palaeoclimatic conditions. Jurassic planktonic foraminifera preferred marine continental margin conditions instead of the distal open ocean and spread from offshore Eastern Canada to offshore Western Australia along the margins of the Tethys Ocean. The group did not venture into austral or boreal realms, likely reasons why migration probably failed to the west coasts of South and North America.

Antimicrobial Activity and Biocompatibility of the Psidium cattleianum Extracts for Endodontic Purposes.

Psidium cattleianum (PC) has been displaying inhibitory effect against a variety of microorganisms, but this effect has not yet been tested against endodontic pathogens. The aim of this study was to evaluate the antimicrobial activity and biocompatibility of the aqueous (PCAE) and hydroethanolic (PCHE) extracts from Psidium cattleianum (PC) leaves. Minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) were determined using the microdilution broth method in order to analyze the antimicrobial effect against Enterococcus faecalis, Pseudomonas aeruginosa, Actinomyces israelii and Candida albicans in planktonic conditions. Biofilm assays were conducted only with the extracts that were able to determine the MLC for microorganisms in planktonic conditions. Immediate and late tissue reactions against PC extracts were evaluated using edemogenic test and histological analysis of subcutaneous implants in Wistar rats. The results showed that the MIC and MLC values ranged between 0.25 and 4 mg/mL. The MLC obtained for PCHE inhibited 100% growth of all the tested strains, except for C. albicans. PCAE had the same effect for E. faecalis and P. aeruginosa. Both PC extracts were able to eliminate E. faecalis biofilms and only the PCHE eliminated P. aeruginosa biofilms. The positive controls inhibited the growth of all tested strains in MIC and MLC essays, but no CHX tested concentrations were able to eliminate A. israelii biofilm. PCAE caused a discrete increase in the edema over time, while PCHE caused a higher initial edema, which decreased progressively. Both PCAE and PCHE extracts were biocompatible, but PCHE showed better results with slight levels of inflammation at 28 days. In conclusion, PCHE was biocompatible and presented better antimicrobial effect against important pathogens associated with persistent endodontic infections.

Zinc oxide nanoparticles hinder fungal biofilm development in an ancient Egyptian tomb

Nanoparticles (NPs) have been proposed as an innovative strategy to prevent fungal colonization of cultural heritage, but until now their efficacy has mainly been proved against fungi in planktonic conditions. Four fungi from microbial alterations of the royal tomb of Tausert and Setnakht in the Valley of the Kings, Thebes, West Bank, Modern Luxor, Egypt, were isolated and identified as Alternaria alternata, Aspergillus niger, Penicillium chrysogenum and P. pinophilum. This work deals with four developing fungal biofilms grown with a colony biofilm approach, and exposed to two concentrations of zinc oxide NPs (0.25 and 0.5%) for 10 days. A significant reduction in the biofilm growth was observed in presence of 0.5% NPs for all fungi, except A. niger. Moreover, the morphology of the fungal biofilm exposed to NPs differed from that of the control, and there was a different polysaccharide to protein ratio in the matrices, and earlier production of coloured compounds and spores. Despite the success of the zinc oxide NPs, this early metabolite production needs to be monitored as a possible source of substances affecting cultural heritage surfaces.

Suppression of Enteric Bacteria by Bacteriophages: Importance of Phage Polyvalence in the Presence of Soil Bacteria.

Bacteriophages are widely recognized for their importance in microbial ecology and bacterial control. However, little is known about how phage polyvalence (i.e., broad host range) affects bacterial suppression and interspecies competition in environments harboring enteric pathogens and soil bacteria. Here we compare the efficacy of polyvalent phage PEf1 versus coliphage T4 in suppressing a model enteric bacterium (E. coli K-12) in mixtures with soil bacteria (Pseudomonas putida F1 and Bacillus subtilis 168). Although T4 was more effective than PEf1 in infecting E. coli K-12 in pure cultures, PEf1 was 20-fold more effective in suppressing E. coli under simulated multispecies biofilm conditions because polyvalence enhanced PEf1 propagation in P. putida. In contrast, soil bacteria do not propagate coliphages and hindered T4 diffusion through the biofilm. Similar tests were also conducted under planktonic conditions to discern how interspecies competition contributes to E. coli suppression without the confounding effects of restricted phage diffusion. Significant synergistic suppression was observed by the combined effects of phages plus competing bacteria. T4 was slightly more effective in suppressing E. coli in these planktonic mixed cultures, even though PEf1 reached higher concentrations by reproducing also in P. putida (7.2 ± 0.4 vs 6.0 ± 1.0 log10PFU/mL). Apparently, enhanced suppression by higher PEf1 propagation was offset by P. putida lysis, which decreased stress from interspecies competition relative to incubations with T4. In similar planktonic tests with more competing soil bacteria species, P. putida lysis was less critical in mitigating interspecies competition and PEf1 eliminated E. coli faster than T4 (36 vs 42 h). Overall, this study shows that polyvalent phages can propagate in soil bacteria and significantly enhance suppression of co-occurring enteric species.

Microbial flora, probiotics, Bacillus subtilis and the search for a long and healthy human longevity.

Probiotics are live microorganisms that have beneficial effects on host health, including extended lifespan, when they are administered or present in adequate quantities. However, the mechanisms by which probiotics stimulate host longevity remain unclear and very poorly understood. In a recent study (Nat. Commun. 8, 14332 (2017) doi: 10.1038/ncomms14332), we used the spore-forming probiotic bacterium Bacillus subtilis and the model organism Caenorhabditis elegans to study the mechanism by which a probiotic bacterium affects host longevity. We found that biofilm-proficient B. subtilis colonized the C. elegans gut and extended the worm lifespan significantly longer than did biofilm-deficient isogenic strains. In addition to biofilm proficiency, the quorum-sensing pentapeptide CSF and nitric oxide (NO) represent the entire B. subtilis repertoire responsible for the extended longevity of C. elegans. B. subtilis grown under biofilm-supporting conditions synthesized higher levels of NO and CSF than under planktonic growth conditions, emphasizing the key role of the biofilm in slowing host aging. Significantly, the prolongevity effect of B. subtilis was primarily due to a downregulation of the insulin-like signaling system that precisely is a key partaker in the healthy longevity of human centenarians. These findings open the possibility to test if the regular consumption of B. subtilis incorporated in foods and beverages could significantly extend human life expectancy and contribute to stop the development of age-related diseases.

Environmental fluctuation governs selection for plasticity in biofilm production.

Bacteria can grow in a free-swimming state, as planktonic cells, or in surface-attached communities, termed biofilms. The planktonic and biofilm growth modes differ dramatically with respect to spatial constraints, nutrient access, population density and cell-cell interactions. Fitness trade-offs underlie how successfully bacteria compete in each of these environments. Accordingly, some bacteria have evolved to be specialists in biofilm formation, while others specialize in planktonic growth. There are species, however, that possess flexible strategies: they can transition between the molecular programs required for biofilm formation and for planktonic growth. Such flexible strategies often sacrifice competitive ability against specialists in a given habitat. There is little exploration of the ecological conditions favoring the evolution of the flexible biofilm production strategy for bacteria in competition with specialist biofilm producers or specialist non-producers. Here, we study the human pathogen Vibrio cholerae, a flexible biofilm-former, as well as constitutive biofilm-producing and non-producing mutants. We assess the fitness of these strains under biofilm conditions, planktonic conditions and conditions that demand the ability to transition between the two growth modes. We show that, relative to the specialists, the wild type is superior at dispersal from biofilms to the planktonic phase; however, this capability comes at the expense of reduced competitive fitness against constitutive biofilm producers on surfaces. Wild-type V. cholerae can outcompete the constitutive biofilm producers and non-producers if habitat turnover is sufficiently frequent. Thus, selection for phenotypic flexibility in biofilm production depends on the frequency of environmental fluctuations encountered by bacteria.

Transcriptional characterization of Vibrio fischeri during colonization of juvenile Euprymna scolopes.

The marine bacterium Vibrio fischeri is the monospecific symbiont of the Hawaiian bobtail squid, Euprymna scolopes, and the establishment of this association involves a number of signaling pathways and transcriptional responses between both partners. We report here the first full RNA-Seq dataset representing host-associated V. fischeri cells from colonized juvenile E. scolopes, as well as comparative transcriptomes under both laboratory and simulated marine planktonic conditions. These data elucidate the broad transcriptional changes that these bacteria undergo during the early stages of symbiotic colonization. We report several previously undescribed and unexpected transcriptional responses within the early stages of this symbiosis, including gene expression patterns consistent with biochemical stresses inside the host, and metabolic patterns distinct from those reported in associations with adult animals. Integration of these transcriptional data with a recently developed metabolic model of V. fischeri provides us with a clearer picture of the metabolic state of symbionts within the juvenile host, including their possible carbon sources. Taken together, these results expand our understanding of the early stages of the squid-vibrio symbiosis, and more generally inform the transcriptional responses underlying the activities of marine microbes during host colonization.

Bacillus subtilis biofilm extends Caenorhabditis elegans longevity through downregulation of the insulin-like signalling pathway

Beneficial bacteria have been shown to affect host longevity, but the molecular mechanisms mediating such effects remain largely unclear. Here we show that formation of Bacillus subtilis biofilms increases Caenorhabditis elegans lifespan. Biofilm-proficient B. subtilis colonizes the C. elegans gut and extends worm lifespan more than biofilm-deficient isogenic strains. Two molecules produced by B. subtilis — the quorum-sensing pentapeptide CSF and nitric oxide (NO) — are sufficient to extend C. elegans longevity. When B. subtilis is cultured under biofilm-supporting conditions, the synthesis of NO and CSF is increased in comparison with their production under planktonic growth conditions. We further show that the prolongevity effect of B. subtilis biofilms depends on the DAF-2/DAF-16/HSF-1 signalling axis and the downregulation of the insulin-like signalling (ILS) pathway.

Effects of meropenem exposure in persister cells of Acinetobacter calcoaceticus-baumannii.

To evaluate the influence of meropenem in the Acinetobacter calcoaceticus-baumannii (ACB) persister levels. Persister levels in planktonic and biofilm cultures from ACB isolates were evaluated after exposure to different meropenem concentrations. A high variability of persister fractions was observed among the isolates cultured under planktonic and biofilm conditions. Meropenem concentration did not influence persister fractions, even when far above the MIC. No correlation was found between persister levels and biofilm biomass. The magnitude of persister levels from ACB planktonic and, particularly, biofilm cultures exposed to meropenem was independent of the antibiotic concentration, dosing regimen and biofilm biomass. These findings, in a context of meropenem failure to treat chronic infections, strengthen the importance of understanding persister behavior.

A novel chimeric lysin with robust antibacterial activity against planktonic and biofilm methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most threatening pathogens due to its multi-drug resistance (MDR) and strong biofilm-forming capacity. Here, we described the screening of a novel chimeolysin (ClyF) that was active against planktonic and biofilm MRSA. Biochemical tests showed that ClyF was active against all S. aureus clinical isolates tested under planktonic and biofilm conditions. Structure analysis revealed that ClyF has an enhanced thermostability and pH tolerance than its parental lysin Pc by forming a hydrophobic cleft in the catalytic domain and an Ig-like structure in the cell-wall binding domain. A single intraperitoneally or topically administration of ClyF showed good MRSA removing efficacy in mouse models of bacteremia and burn wound infection, respectively. Our data collectively demonstrated that ClyF has good bactericidal activity against planktonic and biofilm MRSA both in vitro and in vivo, and therefore represents a useful antibacterial to combat MDR S. aureus.

Determination of NO and CSF Levels Produced by Bacillus subtilis.

The cell-to-cell communication and division of labour that occurs inside a beneficial biofilm produce significant differences in gene expression compared with the gene expression pattern of cells grew under planktonic conditions. In this sense, the levels of NO (nitric oxide) and CSF (Competence Sporulation Stimulating Factor) produced in Bacillus subtilis cultures have been measured only under planktonic growth conditions. We sought to determine whether NO and/or CSF production is affected in B. subtilis cells that develop as a biofilm. To measure the production levels of the two prolongevity molecules, we grew B. subtilis cells under planktonic and biofilm supporting condition.

Comparison of S. aureus Proteomic Profiles from Biofilm and Planktonic Growth Conditions using 2D- Gel Electrophoresis.

Bacteria growing as biofilms are distinct from the same bacteria growing as planktonic cells. Biofilms cells show increased resistance to antimicrobial, immunological, predatory, and chemical attack than planktonic cells. Most studies on bacterial diseases use planktonic bacteria. The objective of this study was to identify expressed proteins that are unique to Staphylococcus aureus biofilm mode of growth. S. aureus was grown in tryptic soy broth and Dulbecco's modified eagle medium at biofilm and planktonic growth conditions. Protein samples were cleaned up and separated according to their electrophoretic mobility using 7 cm IPG strips (pH 3–10 and pH 4–7) on 2D gel electrophoresis. Expressed proteins of both growth conditions were compared. Data analysis revealed that the expression of S. aureus proteins from planktonic and biofilm was higher in TSB media than DMEM media. Biofilm growth condition showed higher intensity of expressed proteins and new expressed proteins were observed. One protein was found to be upregulated in planktonic growth condition. Additionally, the majority of the proteins were clustered in the area of acidic region (pH 4–7). 2D-gel electrophoresis is a powerful and widely used method for the proteomic analysis. Biofilms represent a realistic representation of bacterial behavior and organisms are capable of altering their physiology in the surrounded environments. The results could help to illustrate the differences in pathogenesis between biofilm and planktonic cells in any model of disease. This will identify biological markers to improve the diagnostics, treatment, and prevention of S. aureus biofilms.

Synergistic in vitro activity of sodium houttuyfonate with fluconazole against clinical Candida albicans strains under planktonic growing conditions

Context: Fluconazole resistance is an intractable problem of treating Candida albicans, calling for more antifungal agents to enhance the activity of fluconazole. Objective: This work investigates the anti-C. albicans activities of sodium houttuyfonate (SH) and/or fluconazole and the associated mechanism. Materials and methods: The minimum inhibitory concentrations (MICs) of SH and fluconazole both ranging from 0.5 to 1024 μg/mL were determined by broth microdilution method in 19 C. albicans isolates, and their fractional inhibitory concentration index (FICI) was evaluated by checkerboard assay. After MICSH and/or MICfluconazole treatments, the expressions of IFD6, PHR1, ZAP1, ADH5, BGL2, XOG1 and FKS1 were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in C. albicans 1601. Results and conclusion: The MICs of SH alone ranged from 32 to 256 μg/mL and decreased 2–16-fold in combination. SH showed strong synergism with fluconazole with FICI <0.13–0.5. In C. albicans 1601, we observed that (i) the expression of the seven genes increased notably in a range between 3.71- and 12.63-fold (p < 0.05) when SH was used alone, (ii) the combined use of SH and fluconazole slightly inhibited the expression of IFD6 and PHR1 by 1.23- and 1.35-fold (p > 0.05), but promoted evidently the expression of ZAP1, ADH5, XOG1 and FKS1 by 1.98-, 3.56-, 4.10- and 2.86-fold (p < 0.05). The results suggested SH to be a potential synergist to enhance the antifungal activity of fluconazole in C. albicans resistant isolates, and the underlying mechanism may be associated with β-1,3-glucan synthesis and transportation.

Antimicrobial photodynamic therapy using Indocyanine green and near-infrared diode laser in reducing Entrerococcus faecalis

BackgroundThe use of Antimicrobial Photodynamic Therapy (aPDT) has been suggested as an adjuvant method to eliminate facultative bacteria during root canal disinfection. The purpose of this preliminary in vitro study was to determine whether the light-activated antimicrobial agent, Indocyanine green (ICG), could be used as photosensitizer and kill Enterococcus faecalis strain under planktonic conditions when irradiated with near-infared (NIR) diode laser emitting in 810nm wavelength. MethodsPlanktonic suspension containing Enterococcus faecalis strain was divided into nine experimental groups: (1) aPDT with ICG and laser (medium energy fluence), (2) aPDT with ICG and laser (high energy fluence), (3) only ICG without laser activation, (4) only laser emission without ICG (5) 2.5% Sodium hypochlorite (NaOCl) as irrigant, (6) 2.5% NaOCl and aPDT with ICG and laser, (7) 2.0% Chlorhexidine gluconate (CHX) as irrigant (8) No treatment (positive control), (9) No bacterial biofilm growth (negative control). The samples were incubated for 7days and colony-forming units (CFUs) were determined to evaluate bacterial viability. ResultsThe microbiological test revealed that aPDT groups, regardless the overall power, showed significant lower mean log10 CFU levels, than groups 3, 4 and 7 (p<0.01). The irrigation with 2.5% NaOCl and the combination of PDT and NaOCl achieved total elimination of bacteria. ConclusionThese preliminary in vitro findings imply that the combination of ICG and NIR diode laser may be a novel supplement in aPDT and provide better disinfection during endodontic treatment.

Geochemical imprints of genotypic variants of Globigerina bulloides in the Arabian Sea

Planktonic foraminifera record oceanic conditions in their shell geochemistry. Many palaeoenvironmental studies have used fossil planktonic foraminifera to constrain past seawater properties by defining species based on their shell morphology. Recent genetic studies, however, have identified ecologically distinct genotypes within traditionally recognized morphospecies, signaling potential repercussions for palaeoclimate reconstructions. Here we demonstrate how the presence of Globigerina bulloides cryptic genotypes in the Arabian Sea may influence geochemical signals of living and fossil assemblages of these morphospecies. We have identified two distinct genotypes of G. bulloides with either cool water (type-II) or warm water (type-I) temperature preferences in the Western Arabian Sea. We accompany these genetic studies with analyses of Mg/Ca and stable oxygen isotope (δ18O) compositions of individual G. bulloides shells. Both Mg/Ca and δ18O values display bimodal distribution patterns. The distribution of Mg/Ca values cannot be simply explained by seawater parameters, and we attribute it to genotype-specific biological controls on the shell geochemistry. The wide range of δ18O values in the fossil assemblage also suggests that similar controls likely influence this proxy in addition to environmental parameters. However, the magnitude of this effect on the δ18O signals is not clear from our data set, and further work is needed to clarify this. We also discuss current evidence of potential genotype-specific geochemical signals in published data on G. bulloides geochemistry and other planktonic foraminiferal species. We conclude that significant caution should be taken when utilizing G. bulloides geochemistry for paleoclimate reconstruction in the regions with upwelling activity or oceanographic fronts.

Differences in Biofilm Mass, Expression of Biofilm-Associated Genes, and Resistance to Desiccation between Epidemic and Sporadic Clones of Carbapenem-Resistant Acinetobacter baumannii Sequence Type 191.

Understanding the biology behind the epidemicity and persistence of Acinetobacter baumannii in the hospital environment is critical to control outbreaks of infection. This study investigated the contributing factors to the epidemicity of carbapenem-resistant A. baumannii (CRAB) sequence type (ST) 191 by comparing the differences in biofilm formation, expression of biofilm-associated genes, and resistance to desiccation between major epidemic (n = 16), minor epidemic (n = 12), and sporadic (n = 12) clones. Biofilm mass was significantly greater in the major epidemic than the minor epidemic and sporadic clones. Major and minor epidemic clones expressed biofilm-associated genes, abaI, bap, pgaABCD, and csuA/BABCDE, higher than the sporadic clones in sessile conditions. The csuC, csuD, and csuE genes were more highly expressed in the major epidemic than minor epidemic clones. Interestingly, minor epidemic clones expressed more biofilm-associated genes than the major epidemic clone under planktonic conditions. Major epidemic clones were more resistant to desiccation than minor epidemic and sporadic clones on day 21. In conclusion, the epidemic CRAB ST191 clones exhibit a higher capacity to form biofilms, express the biofilm-associated genes under sessile conditions, and resist desiccation than sporadic clones. These phenotypic and genotypic characteristics of CRAB ST191 may account for the epidemicity of specific CRAB ST191 clones in the hospital.

Cytotoxicity and the effect of cationic peptide fragments against cariogenic bacteria under planktonic and biofilm conditions

This study evaluated the cytotoxicity and effect of fragments derived from three oral cationic peptides (CP): LL-37, D6-17 and D1-23 against cariogenic bacteria under planktonic and biofilm conditions. For cytotoxicity analysis, two epithelial cell lines were used. The minimum inhibitory concentration and the minimal bactericidal concentration were determined for the CP fragments and the control (chlorhexidine-CHX) against cariogenic bacteria. The fractional inhibitory concentration was obtained for the combinations of CP fragments on Streptococcus mutans. Biofilm assays were conducted with the best antimicrobial CP fragment against S. mutans. The results indicated that D6-17 was not cytotoxic. D1-23, LL-37 and CHX were not cytotoxic in low concentrations. D1-23 presented the best bactericidal activity against S. mutans, S. mitis and S. salivarius. Combinations of CP fragments did not show a synergic effect. D1-23 presented a higher activity against S. mutans biofilm than CHX. It was concluded that D1-23 showed a substantial effect against cariogenic bacteria and low cytotoxicity.

RsmW, Pseudomonas aeruginosa small non-coding RsmA-binding RNA upregulated in biofilm versus planktonic growth conditions.

BackgroundBiofilm development, specifically the fundamentally adaptive switch from acute to chronic infection phenotypes, requires global regulators and small non-coding regulatory RNAs (sRNAs). This work utilized RNA-sequencing (RNA-seq) to detect sRNAs differentially expressed in Pseudomonas aeruginosa biofilm versus planktonic state.ResultsA computational algorithm was devised to detect and categorize sRNAs into 5 types: intergenic, intragenic, 5′-UTR, 3′-UTR, and antisense. Here we report a novel RsmY/RsmZ-type sRNA, termed RsmW, in P. aeruginosa up-transcribed in biofilm versus planktonic growth. RNA-Seq, 5’-RACE and Mfold predictions suggest RsmW has a secondary structure with 3 of 7 GGA motifs located on outer stem loops. Northern blot revealed two RsmW binding bands of 400 and 120 bases, suggesting RsmW is derived from the 3’-UTR of the upstream hypothetical gene, PA4570. RsmW expression is elevated in late stationary versus logarithmic growth phase in PB minimal media, at higher temperatures (37 °C versus 28 °C), and in both gacA and rhlR transposon mutants versus wild-type. RsmW specifically binds to RsmA protein in vitro and restores biofilm production and reduces swarming in an rsmY/rsmZ double mutant. PA4570 weakly resembles an RsmA/RsmN homolog having 49 % and 51 % similarity, and 16 % and 17 % identity to RsmA and RsmN amino acid sequences, respectively. PA4570 was unable to restore biofilm and swarming phenotypes in ΔrsmA deficient strains.ConclusionCollectively, our study reveals an interesting theme regarding another sRNA regulator of the Rsm system and further unravels the complexities regulating adaptive responses for Pseudomonas species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0771-y) contains supplementary material, which is available to authorized users.

Pseudomonas aeruginosa Cell Membrane Protein Expression from Phenotypically Diverse Cystic Fibrosis Isolates Demonstrates Host-Specific Adaptations.

Pseudomonas aeruginosa is a Gram-negative, nosocomial, highly adaptable opportunistic pathogen especially prevalent in immuno-compromised cystic fibrosis (CF) patients. The bacterial cell surface proteins are important contributors to virulence, yet the membrane subproteomes of phenotypically diverse P. aeruginosa strains are poorly characterized. We carried out mass spectrometry (MS)-based proteome analysis of the membrane proteins of three novel P. aeruginosa strains isolated from the sputum of CF patients and compared protein expression to the widely used laboratory strain, PAO1. Microbes were grown in planktonic growth condition using minimal M9 media, and a defined synthetic lung nutrient mimicking medium (SCFM) limited passaging. Two-dimensional LC-MS/MS using iTRAQ labeling enabled quantitative comparisons among 3171 and 2442 proteins from the minimal M9 medium and in the SCFM, respectively. The CF isolates showed marked differences in membrane protein expression in comparison with PAO1 including up-regulation of drug resistance proteins (MexY, MexB, MexC) and down-regulation of chemotaxis and aerotaxis proteins (PA1561, PctA, PctB) and motility and adhesion proteins (FliK, FlgE, FliD, PilJ). Phenotypic analysis using adhesion, motility, and drug susceptibility assays confirmed the proteomics findings. These results provide evidence of host-specific microevolution of P. aeruginosa in the CF lung and shed light on the adaptation strategies used by CF pathogens.

Identification of Haloferax volcanii Pilin N-Glycans with Diverse Roles in Pilus Biosynthesis, Adhesion, and Microcolony Formation

N-Glycosylation is a post-translational modification common to all three domains of life. In many archaea, the oligosacharyltransferase (AglB)-dependent N-glycosylation of flagellins is required for flagella assembly. However, whether N-glycosylation is required for the assembly and/or function of the structurally related archaeal type IV pili is unknown. Here, we show that of six Haloferax volcanii adhesion pilins, PilA1 and PilA2, the most abundant pilins in pili of wild-type and ΔaglB strains, are modified under planktonic conditions in an AglB-dependent manner by the same pentasaccharide detected on H. volcanii flagellins. However, unlike wild-type cells, which have surfaces decorated with discrete pili and form a dispersed layer of cells on a plastic surface, ΔaglB cells have thick pili bundles and form microcolonies. Moreover, expressing PilA1, PilA2, or PilA6 in ΔpilA[1-6]ΔaglB stimulates microcolony formation compared with their expression in ΔpilA[1-6]. Conversely, expressing PilA3 or PilA4 in ΔpilA[1-6] cells results in strong surface adhesion, but not microcolony formation, and neither pilin stimulates surface adhesion in ΔpilA[1-6]ΔaglB cells. Although PilA4 assembles into pili in the ΔpilA[1-6]ΔaglB cells, these pili are, unlike wild-type pili, curled, perhaps rendering them non-functional. To our knowledge, this is the first demonstration of a differential effect of glycosylation on pilus assembly and function of paralogous pilins. The growth of wild-type cells in low salt media, a condition that decreases AglB glycosylation, also stimulates microcolony formation and inhibits motility, supporting our hypothesis that N-glycosylation plays an important role in regulating the transition between planktonic to sessile cell states as a response to stress.