A Cyanobacterial Component Required for Pilus Biogenesis Affects the Exoproteome

ABSTRACT Protein secretion as well as the assembly of bacterial motility appendages are central processes that substantially contribute to fitness and survival. This study highlights distinctive features of the mechanism that serves these functions in cyanobacteria, which are globally prevalent photosynthetic prokaryotes that significantly contribute to primary production. Our studies of biofilm development in the cyanobacterium Synechococcus elongatus uncovered a novel component required for the biofilm self-suppression mechanism that operates in this organism. This protein, which is annotated as “hypothetical,” is denoted EbsA (essential for biofilm self-suppression A) here. EbsA homologs are highly conserved and widespread in diverse cyanobacteria but are not found outside this clade. We revealed a tripartite complex of EbsA, Hfq, and the ATPase homolog PilB (formerly called T2SE) and demonstrated that each of these components is required for the assembly of the hairlike type IV pili (T4P) appendages, for DNA competence, and affects the exoproteome in addition to its role in biofilm self-suppression. These data are consistent with bioinformatics analyses that reveal only a single set of genes in S. elongatus to serve pilus assembly or protein secretion; we suggest that a single complex is involved in both processes. A phenotype resulting from the impairment of the EbsA homolog in the cyanobacterium Synechocystis sp. strain PCC 6803 implies that this feature is a general cyanobacterial trait. Moreover, comparative exoproteome analyses of wild-type and mutant strains of S. elongatus suggest that EbsA and Hfq affect the exoproteome via a process that is independent of PilB, in addition to their involvement in a T4P/secretion machinery. IMPORTANCE Cyanobacteria, environmentally prevalent photosynthetic prokaryotes, contribute ∼25% of global primary production. Cyanobacterial biofilms elicit biofouling, thus leading to substantial economic losses; however, these microbial assemblages can also be beneficial, e.g., in wastewater purification processes and for biofuel production. Mechanistic aspects of cyanobacterial biofilm development were long overlooked, and genetic and molecular information emerged only in recent years. The importance of this study is 2-fold. First, it identifies novel components of cyanobacterial biofilm regulation, thus contributing to the knowledge of these processes and paving the way for inhibiting detrimental biofilms or promoting beneficial ones. Second, the data suggest that cyanobacteria may employ the same complex for the assembly of the motility appendages, type 4 pili, and protein secretion. A shared pathway was previously shown in only a few cases of heterotrophic bacteria, whereas numerous studies demonstrated distinct systems for these functions. Thus, our study broadens the understanding of pilus assembly/secretion in diverse bacteria and furthers the aim of controlling the formation of cyanobacterial biofilms.

The Impact of Shear Forces and Surface Hydrophobicity on Coccoid Cyanobacterial Biofilm Development

<p>Biofouling is a natural process in marine environments with associated economic and ecological problems. Thus, understanding the conditions that affect cyanobacterial biofilm development is crucial to develop new antibiofouling strategies and decrease the impact of biofilms in the marine environment. In this study, we investigated the relative importance of shear forces and surface hydrophobicity on biofilm development by two coccoid cyanobacteria with different biofilm formation capacities. The strong biofilm-forming <em>Synechocystis salina</em> was used along with the weaker biofilm-forming <em>Cyanobium</em> sp. Biofilms were developed in defined hydrodynamic conditions using glass (a model hydrophilic surface) and a polymeric epoxy coating (a hydrophobic surface) as substrates. Biofilms developed in both surfaces at lower shear conditions contained a higher number of cells and presented higher values for wet weight, thickness, and chlorophyll <em>a</em> content. The impact of hydrodynamics on biofilm development was generally stronger than the impact of surface hydrophobicity, but a combined effect of these two parameters strongly affected biofilm formation for the weaker biofilm-producing organism. The antibiofilm performance of the polymeric coating was confirmed at the hydrodynamic conditions prevailing in ports. Shear forces were shown to have a profound impact on biofilm development in marine settings regardless of the fouling capacity of the existing flora and the hydrophobicity of the surface.</p>

A Storable Mediatorless Electrochemical Biosensor for Herbicide Detection

A novel mediatorless photo-bioelectrochemical sensor operated with a biofilm of the cyanobacterium Synechocystis PCC6803 wt. for herbicide detection with long term stability (>20 days) was successfully developed and tested. Photoanodic current generation was obtained in the absence of artificial mediators. The inhibitory effect on photocurrent of three commonly used herbicides (i.e., atrazine, diuron, and paraquat) was used as a means of measuring their concentrations in aqueous solution. The injection of atrazine and diuron into the algal medium caused an immediate photocurrent drop due to the inhibition of photosynthetic electron transport. The detected concentrations were suitable for environmental analysis, as revealed by a comparison with the freshwater quality benchmarks set by the Environmental Protection Agency of the United States (US EPA). In contrast, paraquat caused an initial increase (~2 h) of the photocurrent effect of about 200%, as this compound can act as a redox mediator between the cells and the anode. A relatively long-term stability of the biosensor was demonstrated, by keeping anodes colonized with cyanobacterial biofilm in the dark at 4 °C. After 22 days of storage, the performance in terms of the photocurrent was comparable with the freshly prepared biosensor. This result was confirmed by the measurement of chlorophyll content, which demonstrated preservation of the cyanobacterial biofilm. The capacity of this biosensor to recover after a cold season or other prolonged environmental stresses could be a key advantage in field applications, such as in water bodies and agriculture. This study is a step forward in the biotechnological development and implementation of storable mediatorless electrochemical biosensors for herbicide detection.

An AlgaSORB Column for the Quantitative Sorption of Arsenic(III) from Water Samples

This study examines a new Scytonema-based biosorbent (AlgaSORB-scy) using column chromatography for the biosorption of total inorganic arsenic [As(III) and As(V)] from aquatic samples. The AlgaSORB-scy was prepared by immobilizing a cyanobacterial biofilm (Scytonema-dimethyl-formamide slurry) over a polymer-modified silica gel and then characterized for stability and sorption/elution conditions under dynamic equilibrations followed by differential pulse polarographic detection. The sorbent exhibited a 100% affinity for As(III) in a multi-elemental solution [in the absence of As(V)] at pH 6.9 and a flow rate of 1 mL min-1 giving rise to a preconcentration factor as high as 44-fold. The interference caused by As(V) in the sorption of As(III) on AlgaSORB-scy necessitated the pre-reduction of As(V) into As(III) by sodium sulfite. The biosorbent was found to be suitable for total arsenic enrichment in the form of As(III) species and demonstrated a better endurance with a recyclability of up to 59 cycles of sorption-elution. The polymeric spacer between the cyanobacterial biofilm and the silica gel enabled the biofilm monolayer to be held firmly onto a solid support and be accessible for quantitative arsenic uptake without encountering any problems of interfacial overlapping, re-adsorption, or matrix effects in the drinking water supplied.

Agromyces subbeticus sp. nov., isolated from a cave in southern Spain

An actinomycete, strain Z33T, was isolated from a cyanobacterial biofilm in the Cave of Bats, near Zuheros (Cordoba, southern Spain). 16S rRNA gene sequence analysis showed that strain Z33T formed a distinct phyletic line within the genus Agromyces. This isolate could be readily distinguished from representatives of all recognized Agromyces species on the basis of a broad range of phenotypic characteristics and DNA–DNA relatedness data. Genotypic and phenotypic properties indicate that strain Z33T represents a novel species, for which the name Agromyces subbeticus sp. nov. is proposed. The type strain is Z33T (=HKI 0340T=DSM 16689T=NCIMB 14025T).