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Biofilms are dynamic microbial communities in which transitions between planktonic and sessile modes of growth occur interchangeably in response to different environmental cues. In the last decade, early events associated with C. albicans biofilm formation have received considerable attention. However, very little is known about C. albicans biofilm dispersion or the mechanisms and signals that trigger it. This is important because it is precisely C. albicans cells dispersed from biofilms that are the main culprits associated with candidemia and establishment of disseminated invasive disease, two of the gravest forms of candidiasis. Using a simple flow biofilm model recently developed by our group, we have performed initial investigations into the phenomenon of C. albicans biofilm dispersion, as well as the phenotypic characteristics associated with dispersed cells. Our results indicate that C. albicans biofilm dispersion is dependent on growing conditions, including carbon source and pH of the media used for biofilm development. C. albicans dispersed cells are mostly in the yeast form and display distinct phenotypic properties compared to their planktonic counterparts, including enhanced adherence, filamentation, biofilm formation and, perhaps most importantly, increased pathogenicity in a murine model of hematogenously disseminated candidiasis, thus indicating that dispersed cells are armed with a complete arsenal of \"virulence factors\" important for seeding and establishing new foci of infection. In addition, utilizing genetically engineered strains of C. albicans (tetO-UME6 and tetO-PES1) we demonstrate that C. albicans biofilm dispersion can be regulated by manipulating levels of expression of these key genes, further supporting the evidence for a strong link between biofilms and morphogenetic conversions at different stages of the C. albicans biofilm developmental cycle. Overall, our results offer novel and important insight into the phenomenon of C. albicans biofilm dispersion, a key part of the biofilm developmental cycle, and provide the basis for its more detailed analysis.

Abstract The lag phase is a temporary, nonreplicative period observed when a microbial population is introduced to a new, nutrient-rich environment. Although the theoretical concept of growth phases is clear, the practical application of methods for estimating lag lengths is often challenging. In fact, there are two distinct assumptions: (i) that cells do not divide at all during the lag phase or (ii) that they divide but at a suboptimal rate. Therefore, the choice of method should consider not only technical limitations but also consistency with the biological context. Here, we investigate the performance of the most common lag estimation methods, using empirical and simulated datasets. We apply different biological scenarios and simulate curves with varying parameters (i.e. growth rate, noise level, and frequency of measurements) to test their impact on the estimated lag phase duration. Our validation shows that infrequent measurements, low growth rate, longer lag phases, or higher level of noise in the measurements result in higher bias and higher variance of lag estimation. Additionally, in case of noisy data, the methods relying on model fitting perform best. The effect of method assumptions (i.e. no cell divisions or divisions at a suboptimal rate) on the estimated lag phase lengths was investigated using empirical and simulated growth curves.

Echinocandins are a new generation of novel antifungal agent that inhibit cell wall beta(1,3)-glucan synthesis and are normally cidal for the human pathogen Candida albicans. Treatment of C. albicans with low levels of echinocandins stimulated chitin synthase (CHS) gene expression, increased Chs activity, elevated chitin content and reduced efficacy of these drugs. Elevation of chitin synthesis was mediated via the PKC, HOG, and Ca(2+)-calcineurin signalling pathways. Stimulation of Chs2p and Chs8p by activators of these pathways enabled cells to survive otherwise lethal concentrations of echinocandins, even in the absence of Chs3p and the normally essential Chs1p, which synthesize the chitinous septal ring and primary septum of the fungus. Under such conditions, a novel proximally offset septum was synthesized that restored the capacity for cell division, sustained the viability of the cell, and abrogated morphological and growth defects associated with echinocandin treatment and the chs mutations. These findings anticipate potential resistance mechanisms to echinocandins. However, echinocandins and chitin synthase inhibitors synergized strongly, highlighting the potential for combination therapies with greatly enhanced cidal activity.

With the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing based on homoserine lactones was found to influence biofilm formation. Here we discern a mechanism by which quorum sensing controls biofilm formation by screening 5850 transposon mutants of P. aeruginosa PA14 for altered biofilm formation. This screen identified the PA3885 mutant, which had 147-fold more biofilm than the wild-type strain. Loss of PA3885 decreased swimming, abolished swarming, and increased attachment, although this did not affect production of rhamnolipids. The PA3885 mutant also had a wrinkly colony phenotype, formed pronounced pellicles, had substantially more aggregation, and had 28-fold more exopolysaccharide production. Expression of PA3885 in trans reduced biofilm formation and abolished aggregation. Whole transcriptome analysis showed that loss of PA3885 activated expression of the pel locus, an operon that encodes for the synthesis of extracellular matrix polysaccharide. Genetic screening identified that loss of PelABDEG and the PA1120 protein (which contains a GGDEF-motif) suppressed the phenotypes of the PA3885 mutant, suggesting that the function of the PA3885 protein is to regulate 3,5-cyclic diguanylic acid (c-di-GMP) concentrations as a phosphatase since c-di-GMP enhances biofilm formation by activating PelD, and c-di-GMP inhibits swarming. Loss of PA3885 protein increased cellular c-di-GMP concentrations; hence, PA3885 protein is a negative regulator of c-di-GMP production. Purified PA3885 protein has phosphatase activity against phosphotyrosine peptides and is translocated to the periplasm. Las-mediated quorum sensing positively regulates expression of the PA3885 gene. These results show that the PA3885 protein responds to AHL signals and likely dephosphorylates PA1120, which leads to reduced c-di-GMP production. This inhibits matrix exopolysaccharide formation, which leads to reduced biofilm formation; hence, we provide a mechanism for quorum sensing control of biofilm formation through the pel locus and suggest PA3885 should be named TpbA for tyrosine phosphatase related to biofilm formation and PA1120 should be TpbB.

The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer via persulfide to a range of acceptor proteins involved in Fe-S cluster assembly, tRNA modifications, and sulfur-containing cofactor biosynthesis. Several IscS-interacting partners including IscU, a scaffold for Fe-S cluster assembly; TusA, the first member of a sulfur relay leading to sulfur incorporation into the wobble uridine of several tRNAs; ThiI, involved in tRNA modification and thiamine biosynthesis; and rhodanese RhdA are sulfur acceptors. Other proteins, such as CyaY/frataxin and IscX, also bind to IscS, but their functional roles are not directly related to sulfur transfer. We have determined the crystal structures of IscS-IscU and IscS-TusA complexes providing the first insight into their different modes of binding and the mechanism of sulfur transfer. Exhaustive mutational analysis of the IscS surface allowed us to map the binding sites of various partner proteins and to determine the functional and biochemical role of selected IscS and TusA residues. IscS interacts with its partners through an extensive surface area centered on the active site Cys328. The structures indicate that the acceptor proteins approach Cys328 from different directions and suggest that the conformational plasticity of a long loop containing this cysteine is essential for the ability of IscS to transfer sulfur to multiple acceptor proteins. The sulfur acceptors can only bind to IscS one at a time, while frataxin and IscX can form a ternary complex with IscU and IscS. Our data support the role of frataxin as an iron donor for IscU to form the Fe-S clusters.

Sporulation in Bacillus subtilis involves two cells that follow separate but coordinately regulated developmental programs. Late in sporulation, the developing spore (the forespore) resides within a mother cell. The regulation of the forespore transcription factor sigma(G) that acts at this stage has remained enigmatic. sigma(G) activity requires eight mother-cell proteins encoded in the spoIIIA operon and the forespore protein SpoIIQ. Several of the SpoIIIA proteins share similarity with components of specialized secretion systems. One of them resembles a secretion ATPase and we demonstrate that the ATPase motifs are required for sigma(G) activity. We further show that the SpoIIIA proteins and SpoIIQ reside in a multimeric complex that spans the two membranes surrounding the forespore. Finally, we have discovered that these proteins are all required to maintain forespore integrity. In their absence, the forespore develops large invaginations and collapses. Importantly, maintenance of forespore integrity does not require sigma(G). These results support a model in which the SpoIIIA-SpoIIQ proteins form a novel secretion apparatus that allows the mother cell to nurture the forespore, thereby maintaining forespore physiology and sigma(G) activity during spore maturation.

The use of fungi to obtain chitin and chitosan has advantages such as handling, extraction, and production control, thus allowing to generate high quality chitosan. This study aimed to isolate and identify strains of Cunninghamella spp., and assess the production of biomass, chitin, and chitosan. We determined the macroscopic and microscopic phenotypic aspects of the superiors, mycelial growth rate index, consumption of glucose and nitrogen, as well as the pH variation, and the production of biomass, chitin, and chitosan. After assessing the mycelial growth speed index, considered fast, we found that the UFT-Ce08 and UFT-Ce09 isolates showed a higher growth speed. Therefore, we assessed the sporulation of the isolates, and all of them reached the concentration of 106 spores mL-1 in the period of 96 hours. The YPD was considered ideal for biomass production because it promotes an efficient consumption and synthesis of organic compounds by microorganisms. The assessment of the biomass production and the chitin and chitosan yield of nine isolates and the reference strain showed that the UFT-Ce08 isolate had the highest amount of biomass, the UFT-Ce11 isolate had the highest chitin yield, and despite having the second smallest biomass, the UFT-Ce09 isolate had the highest chitosan yield. Seven isolates showed higher chitosan yield than the reference strain. Therefore, chitosan production from this fungus can be further optimized to improve yield on a large-scale production. Chitosan production from this fungus can be further optimized to improve yield in large-scale production

Increased root exudation under elevated atmospheric CO₂ and the contrasting environments in soil macro- and microaggregates could affect microbial growth strategies. We investigated the effect of elevated CO₂ on the contribution of fast- (r-strategists) and slow-growing (K-strategists) microorganisms in soil macro- and microaggregates. We fractionated the bulk soil from the ambient and elevated (for 5 years) CO₂ treatments of FACE-Hohenheim (Stuttgart) into large macro- (>2 mm), small macro- (0.25-2.00 mm), and microaggregates (<0.25 mm) using 'optimal moist' sieving. Microbial biomass (Cmic), the maximum specific growth rate (μ), growing microbial biomass (GMB) and lag-period (tlag) were estimated by the kinetics of CO₂ emission from bulk soil and aggregates amended with glucose and nutrients. Although Corg and Cmic were unaffected by elevated CO₂, μ values were significantly higher under elevated than ambient CO₂ for bulk soil, small macroaggregates, and microaggregates. Substrate-induced respiratory response increased with decreasing aggregate size under both CO₂ treatments. Based on changes in μ, GMB and lag period, we conclude that elevated atmospheric CO₂ stimulated the r-selected microorganisms, especially in soil microaggregates. Such an increase in r-selected microorganisms indicates acceleration of available C mineralization in soil, which may counterbalance the additional C input by roots in soils in a future elevated atmospheric CO₂ environment.

We discovered previously that the small Escherichia coli proteins Hha (hemolysin expression modulating protein) and the adjacent, poorly-characterized YbaJ are important for biofilm formation; however, their roles have been nebulous. Biofilms are intricate communities in which cell signaling often converts single cells into primitive tissues. Here we show that Hha decreases biofilm formation dramatically by repressing the transcription of rare codon tRNAs which serves to inhibit fimbriae production and by repressing to some extent transcription of fimbrial genes fimA and ihfA. In vivo binding studies show Hha binds to the rare codon tRNAs argU, ileX, ileY, and proL and to two prophage clusters D1P12 and CP4-57. Real-time PCR corroborated that Hha represses argU and proL, and Hha type I fimbriae repression is abolished by the addition of extra copies of argU, ileY, and proL. The repression of transcription of rare codon tRNAs by Hha also leads to cell lysis and biofilm dispersal due to activation of prophage lytic genes rzpD, yfjZ, appY, and alpA and due to induction of ClpP/ClpX proteases which activate toxins by degrading antitoxins. YbaJ serves to mediate the toxicity of Hha. Hence, we have identified that a single protein (Hha) can control biofilm formation by limiting fimbriae production as well as by controlling cell death. The mechanism used by Hha is the control of translation via the availability of rare codon tRNAs which reduces fimbriae production and activates prophage lytic genes. Therefore, Hha acts as a toxin in conjunction with co-transcribed YbaJ (TomB) that attenuates Hha toxicity.

onstructed wetland applications have been limited by a large land requirement and capital investment. This study aimed to improve a shallow pond water hyacinth system by incorporating the advantages of engineered attached microbial growth technique (termed Bio-hedge) for on-site domestic wastewater treatment. A laboratory scale continuous-flow system consists of the mesh type matrix providing an additional biofilm surface area of 54 m2/m3. Following one year of experimentation, the process showed more stability and enhanced performance in removing organic matter and nutrients, compared to traditional water hyacinth (by lowering 33%–67% HRT) and facultative (by lowering 92%–96% HRT) ponds. The wastewater exposed plants revealed a relative growth rate of 1.15% per day, and no anatomical deformities were observed. Plant nutrient level averaged 27 ± 1.7 and 44 ± 2.3 mg N/g dry weight, and 5 ± 1.4 & 9±1.2 mg P/g dry weight in roots and shoots, respectively. Microorganisms immobilized on Bio-hedge media (4.06 × 107 cfu/cm2) and plant roots (3.12 × 104 cfu/cm) were isolated and identified (a total of 23 strains). The capital cost was pre-estimated for 1 m3/d wastewater at 78 US $/m3inflow and 465 US$ /kg BOD5 removed. This process is a suitable ecotechnology due to improved biofilm formation, reduced footprint, energy savings, and increased quality effluent.