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The hair-like cell appendages denoted as type IV pili are crucial for biofilm formation in diverse eubacteria. The protein complex responsible for type IV pilus assembly is homologous with the type II protein secretion complex. In the cyanobacterium Synechococcus elongatus PCC 7942, the gene Synpcc7942_2071 encodes an ATPase homologue of type II/type IV systems. Here, we report that inactivation of Synpcc7942_2071 strongly affected the suite of proteins present in the extracellular milieu (exo-proteome) and eliminated pili observable by electron microscopy. These results support a role for this gene product in protein secretion as well as in pili formation. As we previously reported, inactivation of Synpcc7942_2071 enables biofilm formation and suppresses the planktonic growth of S. elongatus. Thus, pili are dispensable for biofilm development in this cyanobacterium, in contrast to their biofilm-promoting function in type IV pili-producing heterotrophic bacteria. Nevertheless, pili removal is not required for biofilm formation as evident by a piliated mutant of S. elongatus that develops biofilms. We show that adhesion and timing of biofilm development differ between the piliated and non-piliated strains. The study demonstrates key differences in the process of biofilm formation between cyanobacteria and well-studied type IV pili-producing heterotrophic bacteria., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Laboratory-scale reverse osmosis (RO) flat-sheet systems were used with two parallel flow cells, one treated with cleaning agents and a control (ie undisturbed). The cleaning efforts increased the affinity of extracellular polymeric substances (EPS) to the RO membrane and altered the biofilm surface structure. Analysis of the membrane biofilm community composition revealed the dominance of Proteobacteria. However, within the phylum Proteobacteria, γ-Proteobacteria dominated the cleaned membrane biofilm, while β-Proteobacteria dominated the control biofilm. The composition of the fungal phyla was also altered by cleaning, with enhancement of Ascomycota and suppression of Basidiomycota. The results suggest that repeated cleaning cycles select for microbial groups that strongly attach to the RO membrane surface by producing rigid and adhesive EPS that hampers membrane performance.

We investigated the influence of feed-water shear rate during reverse-osmosis (RO) desalination on biofouling with respect to microbial community composition developed on the membrane surface. The RO membrane biofilm's microbial community profile was elucidated during desalination of tertiary wastewater effluent in a flat-sheet lab-scale system operated under high (555.6 s(-1)), medium (370.4 s(-1)), or low (185.2 s(-1)) shear rates, corresponding to average velocities of 27.8, 18.5, and 9.3 cm s(-1), respectively. Bacterial diversity was highest when medium shear was applied (Shannon-Weaver diversity index H' = 4.30 ± 0.04) compared to RO-membrane biofilm developed under lower and higher shear rates (H' = 3.80 ± 0.26 and H' = 3.42 ± 0.38, respectively). At the medium shear rate, RO-membrane biofilms were dominated by Betaproteobacteria, whereas under lower and higher shear rates, the biofilms were dominated by Alpha- and Gamma- Proteobacteria, and the latter biofilms also contained Deltaproteobacteria. Bacterial abundance on the RO membrane was higher at low and medium shear rates compared to the high shear rate: 8.97 × 10(8) ± 1.03 × 10(3), 4.70 × 10(8) ± 1.70 × 10(3) and 5.72 × 10(6) ± 2.09 × 10(3) copy number per cm(2), respectively. Interestingly, at the high shear rate, the RO-membrane biofilm's bacterial community consisted mainly of populations known to excrete high amounts of extracellular polymeric substances. Our results suggest that the RO-membrane biofilm's community composition, structure and abundance differ in accordance with applied shear rate. These results shed new light on the biofouling phenomenon and are important for further development of antibiofouling strategies for RO membranes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A number of techniques are used for testing the anti-biofouling activity of surfaces, yet the correlation between different results is often questionable. In this report, the correlation between initial bacterial deposition (fast tests, reported previously) and biofilm growth (much slower tests) was analyzed on a pristine and a surface-modified reverse osmosis membrane ESPA-1. The membrane was modified with grafted hydrophilic polymers bearing negatively charged, positively charged and zwitter-ionic moieties. Using three different bacterial strains it was found that there was no general correlation between the initial bacterial deposition rates and biofilm growth on surfaces, the reasons being different for each modified surface. For the negatively charged surface the slowest deposition due to the charge repulsion was eventually succeeded by the largest biofilm growth, probably due to secretion of extracellular polymeric substances (EPS) that mediated a strong attachment. For the positively charged surface, short-term charge attraction by quaternary amine groups led to the fastest deposition, but could be eventually overridden by their antimicrobial activity, resulting in non-consistent results where in some cases a lower biofilm formation rate was observed. The results indicate that initial deposition rates have to be used and interpreted with great care, when used for assessing the anti-biofouling activity of surfaces. However, for a weakly interacting 'low-fouling' zwitter-ionic surface, the positive correlation between initial cell deposition and biofilm growth, especially under flow, suggests that for this type of coating initial deposition tests may be fairly indicative of anti-biofouling potential.

Streptococcus gordonii is an important member of the oral biofilm community. As an oral commensal streptococcus, S. gordonii is considered beneficial in promoting biofilm homeostasis. CcpA is known as the central regulator of carbon catabolite repression in Gram-positive bacteria and is also involved in the control of virulence gene expression. To further establish the role of CcpA as central regulator in S. gordonii, the effect of CcpA on biofilm formation and natural competence of S. gordonii was investigated. These phenotypic traits have been suggested to be important to oral streptococci in coping with environmental stress. Here we demonstrate that a CcpA mutant was severely impaired in its biofilm-forming ability, showed a defect in extracellular polysaccharide production and reduced competence. The data suggest that CcpA is involved in the regulation of biofilm formation and competence development in S. gordonii., (© 2011 John Wiley & Sons A/S.)

Streptococcus sanguinis is an oral commensal bacterium and endogenous pathogen in the blood, which is generally naturally competent to take up extracellular DNA. Regarded as a stress response, competence development enables S. sanguinis to acquire new genetic material. The sequenced reference strain SK36 encodes and expresses the genes required for competence (com) and uptake of DNA. Isolated from blood cultures of a confirmed case of infective endocarditis, strain 133-79 encodes all necessary com genes but is not transformable under conditions permissive for competence development in SK36. Using synthetic competence-stimulating peptides (sCSP) based on sequences of SK36 and 133-79 comC, both strains developed competence at similar frequencies in cross-transformation experiments. Furthermore, downstream response pathways are similar in strains SK36 and 133-79 because platelet aggregation and biofilm formation appeared unaffected by CSP. Collectively, the data indicate that strains SK36 and 133-79 respond to CSP similarly, strongly suggesting that endogenous production or release of CSP from 133-79 is impaired., (© 2011 John Wiley & Sons A/S.)

Extracellular polymeric substances (EPS) have major impact on biofouling of reverse osmosis (RO) membranes. On one hand, EPS can reduce membrane permeability and on the other, EPS production by the primary colonizers may influence their deposition and attachment rate and subsequently affect the biofouling propensity of the membrane. The role of bacterial exopolysaccharides in bacterial deposition followed by the biofouling potential of an RO membrane was evaluated using an alginate overproducing (mucoid) Pseudomonas aeruginosa. The mucoid P. aeruginosa PAOmucA22 was compared with its isogenic nonmucoid prototypic parent PAO1 microscopically in a radial stagnation point flow (RSPF) system for their bacterial deposition characteristics. Then, biofouling potential of PAO1 and PAOmucA22 was determined in a crossflow rectangular plate-and-frame membrane cell, in which the strains were cultivated on a thin-film composite, polyamide, flat RO membrane coupon (LFC-1) under laminar flow conditions. In the RSPF system, the observed deposition rate of the mucoid strain was between 5- and 10-fold lower than of the wild type using either synthetic wastewater medium (with ionic strength of 14.7 mM and pH 7.4) or 15 mM KCl solution (pH of 6.2). The slower deposition rate of the mucoid strain is explained by 5- to 25-fold increased hydrophilicity of the mucoid strain as compared to the isogenic wild type, PAO1. Corroborating with these results, a significant delay in the onset of biofouling of the RO membrane was observed when the mucoid strain was used as the membrane colonizer, in which the observed time for the induced permeate flux decline was delayed (ca. 2-fold). In conclusion, the lower initial cell attachment of the mucoid strain decelerated biofouling of the RO membrane. Bacterial deposition and attachment is a critical step in biofilm formation and governed by intimate interactions between outer membrane proteins of the bacteria and the surface. Shielding these interactions by a hydrated and hydrophilic alginate capsule is shown to dramatically lessen the biofouling potential of the membrane colonizers.

Biofilm formation of Pseudomonas aeruginosa on the surface of a reverse osmosis (RO) membrane was studied using a synthetic wastewater medium to simulate conditions relevant to reclamation of secondary wastewater effluent. P. aeruginosa biofilm physiology and spatial activity were analyzed following growth on the membrane using a short-life green fluorescent protein derivative expressed in a growth-dependent manner. As a consequence of the limiting carbon source prevailing in the suspended culture of the RO unit, a higher distribution of active cells was observed in the biofilm close to the membrane surface, likely due to the higher nutrient levels induced by concentration polarization effects. The faster growth of the RO-sessile cells compared to the planktonic cells in the RO unit was reflected by the transcriptome of the two cultures analyzed with DNA microarrays. In contrast to the findings recently reported in gene expression studies of P. aeruginosa biofilms, in the RO system, genes related to stress, adaptation, chemotaxis and resistance to antibacterial agents were induced in the planktonic cells. In agreement with the findings of previous P. aeruginosa biofilm studies, motility- and attachment-related genes were repressed in the RO P. aeruginosa biofilm. Supported by the microarray data, an increase in both motility and chemotaxis phenotypes was observed in the suspended cells. The increase in nutrient concentration in close proximity to the membrane is suggested to enhance biofouling by chemotaxis response of the suspended cells and their swimming toward the membrane surface.

Eight Escherichia coli strains were studied in minimal medium with a continuous flow system using confocal microscopy. K12 wild-type strains ATCC 25404 and MG1655 formed the best biofilms ( approximately 43 microm thick, 21 to 34% surface coverage). JM109, DH5alpha, and MG1655 motA formed intermediate biofilms ( approximately 13 microm thick, 41 to 58% surface coverage). BW25113, MG1655 qseB, and MG1655 fliA had poor biofilms (surface coverage less than 5%). The best biofilm-formers, ATCC 25404 and MG1655, displayed the highest motility, whereas the worst biofilm former, BW25113, was motility-impaired. The differences in motility were due to differences in expression of the motility loci qseB, flhD, fliA, fliC, and motA (e.g., qseB expression in MG1655 was 139-fold higher than BW25113 and 209-fold higher than JM109). Motility affected the biofilm architecture as those strains which had poor motility (E. coli JM109, E. coli MG1655 motA, and DH5alpha) formed flatter microcolonies compared with MG1655 and ATCC 25404, which had more dramatic vertical structures as a result of their enhanced motility. The presence of flagella was also found to be important as qseB and fliA mutants (which lack flagella) had less biofilm than the isogenic paralyzed motA strain (threefold less thickness and 15-fold less surface coverage).

YdgG is an uncharacterized protein that is induced in Escherichia coli biofilms. Here it is shown that deletion of ydgG decreased extracellular and increased intracellular concentrations of autoinducer 2 (AI-2); hence, YdgG enhances transport of AI-2. Consistent with this hypothesis, deletion of ydgG resulted in a 7,000-fold increase in biofilm thickness and 574-fold increase in biomass in flow cells. Also consistent with the hypothesis, deletion of ydgG increased cell motility by increasing transcription of flagellar genes (genes induced by AI-2). By expressing ydgG in trans, the wild-type phenotypes for extracellular AI-2 activity, motility, and biofilm formation were restored. YdgG is also predicted to be a membrane-spanning protein that is conserved in many bacteria, and it influences resistance to several antimicrobials, including crystal violet and streptomycin (this phenotype could also be complemented). Deletion of ydgG also caused 31% of the bacterial chromosome to be differentially expressed in biofilms, as expected, since AI-2 controls hundreds of genes. YdgG was found to negatively modulate expression of flagellum- and motility-related genes, as well as other known products essential for biofilm formation, including operons for type 1 fimbriae, autotransporter protein Ag43, curli production, colanic acid production, and production of polysaccharide adhesin. Eighty genes not previously related to biofilm formation were also identified, including those that encode transport proteins (yihN and yihP), polysialic acid production (gutM and gutQ), CP4-57 prophage functions (yfjR and alpA), methionine biosynthesis (metR), biotin and thiamine biosynthesis (bioF and thiDFH), anaerobic metabolism (focB, hyfACDR, ttdA, and fumB), and proteins with unknown function (ybfG, yceO, yjhQ, and yjbE); 10 of these genes were verified through mutation to decrease biofilm formation by 40% or more (yfjR, bioF, yccW, yjbE, yceO, ttdA, fumB, yjiP, gutQ, and yihR). Hence, it appears YdgG controls the transport of the quorum-sensing signal AI-2, and so we suggest the gene name tqsA.

To show that an adsorbing biofilm carrier (GAC) can be advantageous for atrazine bioremediation over a non-adsorbing carrier, fluidized bed (FB) reactors were operated under atrazine limiting concentrations using Pseudomonas sp. strain ADP as the atrazine degrading bacteria. The following interrelated subjects were investigated: 1) atrazine adsorption to GAC under conditions of atrazine partial penetration in the biofilm, 2) differences in atrazine degradation rates and 3) stability of atrazine biodegradation under non-sterile anoxic conditions in the GAC reactor versus a reactor with a non-adsorbing biofilm carrier. Results from batch adsorption tests together with modeling best described the biofilm as patchy in nature with covered and non-biofilm covered areas. Under conditions of atrazine partial penetration in the biofilm, atrazine adsorption occurs in the non-covered areas and is consequently desorbed at the base of the biofilm substantially increasing the active biofilm surface area. The double flux of atrazine to the biofilm in the GAC reactor results in lower effluent atrazine concentrations as compared to a FB reactor with a non-adsorbing carrier. Moreover, under non-sterile denitrification conditions, atrazine degradation stability was found to be much higher (several months) using GAC as a biofilm carrier while non-adsorbing carrier reactors showed sharp deterioration within 30 days due to contamination of non-atrazine degrading bacteria.

An adsorbing biofilm carrier, like granular activated carbon (GAC), can be the source of an extra flux of pollutant to the biofilm in addition to the bulk liquid. This double flux can improve the performance of a biological GAC (BGAC) reactor as compared to a nonabsorbing carrier reactor but only under conditions of pollutant partial penetration in the biofilm. Pollutant partial penetration in a biofilm often occurs in treatment processes where very low effluent concentrations are required. However, under these conditions, adsorption in BGAC reactors is questionable and requires the existence of biofilm free areas on the GAC carrier. The purpose of this investigation is to prove that under normal BGAC fluidized bed reactor operational conditions patchy biofilm coverage with exposed areas of GAC develops. Adsorption and desorption through these exposed areas can explain the widely debated advantage of BGAC reactors regarding higher biofilm activity. The patchy-like nature of the biofilm coverage on the GAC particles was verified using experimental and modeling tools. Comparison between a nonadsorbing granular carbon carrier and a GAC carrier with an atrazine degrading biofilm (Pseudomonas ADP) under conditions of atrazine partial penetration in the biofilm showed higher biodegradation and lower effluent atrazine concentrations in the BGAC reactor.

Methionine sulphoxide reductase maintains adhesin function during oxidative stress. Using Streptococcus gordonii as a model, we now show the mechanistic basis of adhesin maintenance provided by MsrA. In biofilms, S. gordonii selectively expresses the msrA gene. When the wild-type strain was grown with exogenous hydrogen peroxide (HO), msrA-specific mRNA expression significantly increased, while acid production was unaffected. In the presence of HO, a msrA-deletion mutant (ΔMsrA) showed a 6 h delay in lag phase growth, a 30% lower yield of HO, significantly greater inhibition by HO on agar plates (reversed by complementation), 30% less adhesion to saliva-coated hydroxyapatite, 87% less biofilm formation and an altered electrophoretic pattern of SspAB protein adhesins. Using mass spectrometry, methionine residues in the Met-rich central region of SspB were shown to be oxidized by HO and reduced by MsrA. In intact wild-type cells, MsrA colocalized with a cell wall-staining dye, and MsrA was detected in both cell wall and cytosolic fractions. To maintain normal adhesion and biofilm function of S. gordonii in response to exogenous oxidants therefore msrA is upregulated, methionine oxidation of adhesins and perhaps other proteins is reversed, and adhesion and biofilm formation is maintained. [ABSTRACT FROM AUTHOR]