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.

Transcriptomic and phenomic investigations reveal elements in biofilm repression and formation in the cyanobacteriumSynechococcus elongatusPCC 7942

“…The sets of similarly up- or down-regulated sDEGs in DF2 and DF5 are enriched for genes related to defense mechanism, motility, and proteins with predicted secretion signals (Fig. S6B,E, File S3 Sheets 10 & 18), consistent with known alterations in the exoproteome of PilB ::Tn 5 22,23,27 .…”

“…1B,C,D & S2, File S3 Sheet 6). While it is reasonable that tsaP, pilD , and the second pilT genes do not have notable impacts on biofilm formation due to degeneracy or their encoded proteins’ roles as accessories to the pilus, the genes of the Hfq-EbsA-PilB tripartite complex 23 or the EbfE-PteB 26 maturation-secretion system play important roles that likely act at post-transcriptional levels 23,55 . These results demonstrate the limitations of using changes in expression level as a proxy for contribution to a particular phenotype.…”

“…Gene categorical information, including known biofilm genes from previous S. elongatus PCC 7942 biofilm publications 22,23,26,27,55 , pili genes described by Taton, et al 24 , essentiality and conservation data from Rubin, et al 32 , functional categories from the JGI IGM 56 and CyanoBase 57 databases, and signal peptide and secretion predictions 58–63 , were amassed for all genes in the S. elongatus PCC 7942 genome from appropriate sources and are provided in Supplemental Tables S1-3. Significant enrichments of genes in each interest group identified for RNA-Seq and RB-TnSeq data comparison sets for each of the informational categories were determined using custom R scripts to perform two-sided Fisher’s exact tests, with FDR-adjusted p-values ≤ 0.05 being designated as significant.…”

“…In contrast, our studies have demonstrated that biofilm formation in Synechococcus elongatus PCC 7942, which is by default constitutively suppressed in the laboratory 21 , does not require pili 22 . In fact, most of the mutations discovered to date that relieve suppression and enable biofilm formation (Supplemental Table S1) block formation of the Type IV pilus or its assembly apparatus, including the pilin-encoding pilA1 and pilA2 genes; the pilus assembly and Type II secretion proteins encoded by pilB (formerly referred to as t2sE ), pilC , and pilN ; and two proteins encoded by the genes hfq and ebsA that form a tripartite complex with PilB 21–23 . For the majority of these mutants, a biofilm containing 90 – 95% of the cell population forms 3 – 4 days following inoculation in bubbling tubes.…”

“…Electron microscopy has confirmed that the biofilm cells lack pili. Furthermore, they have markedly diminished or altered exoproteomes, suggesting that a single Type IV/II pilus and secretion system fills these dual roles in S. elongatus 21–23 . Removal of this complex is pleiotropic – impeding other ecologically-relevant behaviors such as natural competence and phototaxis-directed twitching motility 24,25 .…”