The role of FraI in cell-cell communication and differentiation in the hormogonia-forming cyanobacterium Nostoc punctiforme.

Multicellular cyanobacteria, like Nostoc punctiforme, rely on septal junctions for cell-cell communication, which is crucial for coordinating various physiological processes including differentiation of N2-fixing heterocysts, spore-like akinetes, and hormogonia-short, motile filaments important for dispersal. In this study, we functionally characterize a protein, encoded by gene Npun_F4142, which in a random mutagenesis approach, initially showed a motility-related function. The reconstructed Npun_F4142 knockout mutant exhibits further distinct phenotypic traits, including altered hormogonia formation with significant reduced motility, inability to differentiate heterocysts and filament fragmentation. For that reason, we named the protein FraI (fragmentation phenotype). The mutant displays severely impaired cell-cell communication, due to almost complete absence of the nanopore array in the septal cell wall, which is an essential part of the septal junctions. Despite lack of communication, hormogonia in the ΔfraI mutant maintain motility and phototactic behavior, even though less pronounced than the wild type (WT). This suggests an alternative mechanism for coordinated movement beyond septal junctions. Our study underscores the significance of FraI in nanopore formation and cell differentiation, and provides additional evidence for the importance of septal junction formation and communication in various differentiation traits of cyanobacteria. The findings contribute to a deeper understanding of the regulatory networks governing multicellular cyanobacterial behavior, with implications for broader insights into microbial multicellularity. IMPORTANCE: The filament-forming cyanobacterium Nostoc punctiforme serves as a valuable model for studying cell differentiation, including the formation of nitrogen-fixing heterocysts and hormogonia. Hormogonia filaments play a crucial role in dispersal and plant colonization, providing a nitrogen source through atmospheric nitrogen fixation, thus holding promise for fertilizer-free agriculture. The coordination among the hormogonia cells enabling uniform movement toward the positive signal remains poorly understood. This study investigates the role of septal junction-mediated communication in hormogonia differentiation and motility, by studying a ΔfraI mutant with significantly impaired communication. Surprisingly, impaired communication does not abolish synchronized filament movement, suggesting an alternative coordination mechanism. These findings deepen our understanding of cyanobacterial biology and have broader implications for multicellular behavior in prokaryotes.

Accelerating the discovery of alkyl halide-derived natural products using halide depletion.

Even in the genomic era, microbial natural product discovery workflows can be laborious and limited in their ability to target molecules with specific structural features. Here we leverage an understanding of biosynthesis to develop a workflow that targets the discovery of alkyl halide-derived natural products by depleting halide anions, a key biosynthetic substrate for enzymatic halogenation, from microbial growth media. By comparing the metabolomes of bacterial cultures grown in halide-replete and deficient media, we rapidly discovered the nostochlorosides, the products of an orphan halogenase-encoding gene cluster from Nostoc punctiforme ATCC 29133. We further found that these products, a family of unusual chlorinated glycolipids featuring the rare sugar gulose, are polymerized via an unprecedented enzymatic etherification reaction. Together, our results highlight the power of leveraging an understanding of biosynthetic logic to streamline natural product discovery.

Identification of a morphogene required for tapered filament termini in filamentous cyanobacteria.

Although the photosynthetic cyanobacteria are monophyletic, they exhibit substantial morphological diversity across species, and even within an individual species due to phenotypic plasticity in response to life cycles and environmental signals. This is particularly prominent among the multicellular filamentous cyanobacteria. One example of this is the appearance of tapering at the filament termini. However, the morphogenes controlling this phenotype and the adaptive function of this morphology are not well defined. Here, using the model filamentous cyanobacterium Nostoc punctiforme ATCC29133 (PCC73102), we identify tftA, a morphogene required for the development of tapered filament termini. The tftA gene is specifically expressed in developing hormogonia, motile trichomes where the tapered filament morphology is observed, and encodes a protein containing putative amidase_3 and glucosaminidase domains, implying a function in peptidoglycan hydrolysis. Deletion of tftA abolished filament tapering inidcating that TftA plays a role in remodelling the cell wall to produce tapered filaments. Genomic conservation of tftA specifically in filamentous cyanobacteria indicates this is likely to be a conserved mechanism among these organisms. Finally, motility assays indicate that filaments with tapered termini migrate more efficiently through dense substratum, providing a plausible biological role for this morphology.

Hormogonium Development and Motility in Filamentous Cyanobacteria.

Filamentous cyanobacteria exhibit some of the greatest developmental complexity observed in the prokaryotic domain. This includes the ability to differentiate nitrogen-fixing cells known as heterocysts, spore-like akinetes, and hormogonia, which are specialized motile filaments capable of gliding on solid surfaces. Hormogonia and motility play critical roles in several aspects of the biology of filamentous cyanobacteria, including dispersal, phototaxis, the formation of supracellular structures, and the establishment of nitrogen-fixing symbioses with plants. While heterocyst development has been investigated extensively at the molecular level, much less is known about akinete or hormogonium development and motility. This is due, in part, to the loss of developmental complexity during prolonged laboratory culture in commonly employed model filamentous cyanobacteria. In this review, recent progress in understanding the molecular level regulation of hormogonium development and motility in filamentous cyanobacteria is discussed, with a focus on experiments performed using the genetically tractable model filamentous cyanobacterium Nostoc punctiforme, which retains the developmental complexity of field isolates.

A DnaK(Hsp70) Chaperone System Connects Type IV Pilus Activity to Polysaccharide Secretion in Cyanobacteria.

Surface motility powered by type IV pili (T4P) is widespread among bacteria, including the photosynthetic cyanobacteria. This form of movement typically requires the deposition of a motility-associated polysaccharide, and several studies indicate that there is complex coregulation of T4P motor activity and polysaccharide production, although a mechanistic understanding of this coregulation is not fully defined. Here, using a combination of genetic, comparative genomic, transcriptomic, protein-protein interaction, and cytological approaches in the model filamentous cyanobacterium N. punctiforme, we provided evidence that a DnaK-type chaperone system coupled the activity of the T4P motors to the production of the motility-associated hormogonium polysaccharide (HPS). The results from these studies indicated that DnaK1 and DnaJ3 along with GrpE comprised a chaperone system that interacted specifically with active T4P motors and was required to produce HPS. Genomic conservation in cyanobacteria and the conservation of the protein-protein interaction network in the model unicellular cyanobacterium Synechocystis sp. strain PCC 6803 imply that this system is conserved among nearly all motile cyanobacteria and provides a mechanism to coordinate polysaccharide secretion and T4P activity in these organisms. IMPORTANCE Many bacteria, including photosynthetic cyanobacteria, exhibit type IV pili (T4P) driven surface motility. In cyanobacteria, this form of motility facilitates dispersal, phototaxis, the formation of supracellular structures, and the establishment of nitrogen-fixing symbioses with eukaryotes. T4P-powered motility typically requires the deposition of motility-associated polysaccharides, and previous studies indicate that T4P activity and polysaccharide production are intimately linked. However, the mechanism by which these processes are coupled is not well defined. Here, we identified and characterized a DnaK(Hsp70)-type chaperone system that coordinates these two processes in cyanobacteria.

The primary transcriptome of hormogonia from a filamentous cyanobacterium defined by cappable-seq.

Hormogonia are motile filaments produced by many filamentous cyanobacteria that function in dispersal, phototaxis and the establishment of nitrogen-fixing symbioses. The gene regulatory network promoting hormogonium development is initiated by the hybrid histidine kinase HrmK, which in turn activates a sigma factor cascade consisting of SigJ, SigC and SigF. In this study, cappable-seq was employed to define the primary transcriptome of developing hormogonia in the model filamentous cyanobacterium Nostoc punctiforme ATCC 29133 in both the wild-type, and sigJ, sigC and sigF mutant strains 6 h post-hormogonium induction. A total of 1544 transcriptional start sites (TSSs) were identified that are associated with protein-coding genes and are expressed at levels likely to lead to biologically relevant transcripts in developing hormogonia. TSS expression among the sigma-factor deletion strains was highly consistent with previously reported gene expression levels from RNAseq experiments, and support the current working model for the role of these genes in hormogonium development. Analysis of SigJ-dependent TSSs corroborated the presence of the previously identified J-Box in the -10 region of SigJ-dependent promoters. Additionally, the data presented provides new insights on sequence conservation within the -10 regions of both SigC- and SigF-dependent promoters, and demonstrates that SigJ and SigC coordinate complex co-regulation not only of hormogonium-specific genes at different loci, but within an individual operon. As progress continues on defining the hormogonium gene regulatory network, this data set will serve as a valuable resource.

The cyanobacterial taxis protein HmpF regulates type IV pilus activity in response to light.

Motility is ubiquitous in prokaryotic organisms including the photosynthetic cyanobacteria where surface motility powered by type 4 pili (T4P) is common and facilitates phototaxis to seek out favorable light environments. In cyanobacteria, chemotaxis-like systems are known to regulate motility and phototaxis. The characterized phototaxis systems rely on methyl-accepting chemotaxis proteins containing bilin-binding GAF domains capable of directly sensing light, and the mechanism by which they regulate the T4P is largely undefined. In this study we demonstrate that cyanobacteria possess a second, GAF-independent, means of sensing light to regulate motility and provide insight into how a chemotaxis-like system regulates the T4P motors. A combination of genetic, cytological, and protein-protein interaction analyses, along with experiments using the proton ionophore carbonyl cyanide m-chlorophenyl hydrazine, indicate that the Hmp chemotaxis-like system of the model filamentous cyanobacterium Nostoc punctiforme is capable of sensing light indirectly, possibly via alterations in proton motive force, and modulates direct interaction between the cyanobacterial taxis protein HmpF, and Hfq, PilT1, and PilT2 to regulate the T4P motors. Given that the Hmp system is widely conserved in cyanobacteria, and the finding from this study that orthologs of HmpF and T4P proteins from the distantly related model unicellular cyanobacterium Synechocystis sp. strain PCC6803 interact in a similar manner to their N. punctiforme counterparts, it is likely that this represents a ubiquitous means of regulating motility in response to light in cyanobacteria.

Identification of a hormogonium polysaccharide-specific gene set conserved in filamentous cyanobacteria.

Cyanobacteria comprise a phylum defined by the capacity for oxygenic photosynthesis. Members of this phylum are frequently motile as well. Strains that display gliding or twitching motility across semisolid surfaces are powered by a conserved type IV pilus system (T4P). Among the filamentous, heterocyst-forming cyanobacteria, motility is usually confined to specialized filaments known as hormogonia, and requires the deposition of an associated hormogonium polysaccharide (HPS). The genes involved in assembly and export of HPS are largely undefined, and it has been hypothesized that HPS exits the outer membrane via an atypical T4P-driven mechanism. Here, several novel hps loci, primarily encoding glycosyl transferases, are identified. Mutational analysis demonstrates that the majority of these genes are essential for both motility and production of HPS. Notably, most mutant strains accumulate wild-type cellular levels of the major pilin PilA, but not extracellular PilA, indicating dysregulation of the T4P motors, and, therefore, a regulatory interaction between HPS assembly and T4P activity. A co-occurrence analysis of Hps orthologs among cyanobacteria identified an extended set of putative Hps proteins comprising most components of a Wzx/Wzy-type polysaccharide synthesis and export system. This implies that HPS may be secreted through a more canonical pathway, rather than a T4P-mediated mechanism.

The Hybrid Histidine Kinase HrmK Is an Early-Acting Factor in the Hormogonium Gene Regulatory Network.

Filamentous, heterocyst-forming cyanobacteria belonging to taxonomic subsections IV and V are developmentally complex multicellular organisms capable of differentiating an array of cell and filament types, including motile hormogonia. Hormogonia exhibit gliding motility that facilitates dispersal, phototaxis, and the establishment of nitrogen-fixing symbioses. The gene regulatory network (GRN) governing hormogonium development involves a hierarchical sigma factor cascade, but the factors governing the activation of this cascade are currently undefined. Here, using a forward genetic approach, we identified hrmK, a gene encoding a putative hybrid histidine kinase that functions upstream of the sigma factor cascade. The deletion of hrmK produced nonmotile filaments that failed to display hormogonium morphology or accumulate hormogonium-specific proteins or polysaccharide. Targeted transcriptional analyses using reverse transcription-quantitative PCR (RT-qPCR) demonstrated that hormogonium-specific genes both within and outside the sigma factor cascade are drastically downregulated in the absence of hrmK and that hrmK may be subject to indirect, positive autoregulation via sigJ and sigC Orthologs of HrmK are ubiquitous among, and exclusive to, heterocyst-forming cyanobacteria. Collectively, these results indicate that hrmK functions upstream of the sigma factor cascade to initiate hormogonium development, likely by modulating the phosphorylation state of an unknown protein that may serve as the master regulator of hormogonium development in heterocyst-forming cyanobacteria.IMPORTANCE Filamentous cyanobacteria are morphologically complex, with several representative species amenable to routine genetic manipulation, making them excellent model organisms for the study of development. Furthermore, two of the developmental alternatives, nitrogen-fixing heterocysts and motile hormogonia, are essential to establish nitrogen-fixing symbioses with plant partners. These symbioses are integral to global nitrogen cycles and could be artificially recreated with crop plants to serve as biofertilizers, but to achieve this goal, detailed understanding and manipulation of the hormogonium and heterocyst gene regulatory networks may be necessary. Here, using the model organism Nostoc punctiforme, we identify a previously uncharacterized hybrid histidine kinase that is confined to heterocyst-forming cyanobacteria as the earliest known participant in hormogonium development.

Role of BGS13 in the Secretory Mechanism of Pichia pastoris.

The methylotrophic yeast Pichia pastoris has been utilized for heterologous protein expression for over 30 years. Because P. pastoris secretes few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, some recombinant proteins intended for secretion are retained within the cell. A mutant strain isolated in our laboratory, containing a disruption of the BGS13 gene, displayed elevated levels of secretion for a variety of reporter proteins. The Bgs13 peptide (Bgs13p) is similar to the Saccharomyces cerevisiae protein kinase C 1 protein (Pkc1p), but its specific mode of action is currently unclear. To illuminate differences in the secretion mechanism between the wild-type (wt) strain and the bgs13 strain, we determined that the disrupted bgs13 gene expressed a truncated protein that had reduced protein kinase C activity and a different location in the cell, compared to the wt protein. Because the Pkc1p of baker's yeast plays a significant role in cell wall integrity, we investigated the sensitivity of the mutant strain's cell wall to growth antagonists and extraction by dithiothreitol, determining that the bgs13 strain cell wall suffered from inherent structural problems although its porosity was normal. A proteomic investigation of the bgs13 strain secretome and cell wall-extracted peptides demonstrated that, compared to its wt parent, the bgs13 strain also displayed increased release of an array of normally secreted, endogenous proteins, as well as endoplasmic reticulum-resident chaperone proteins, suggesting that Bgs13p helps regulate the unfolded protein response and protein sorting on a global scale.IMPORTANCE The yeast Pichia pastoris is used as a host system for the expression of recombinant proteins. Many of these products, including antibodies, vaccine antigens, and therapeutic proteins such as insulin, are currently on the market or in late stages of development. However, one major weakness is that sometimes these proteins are not secreted from the yeast cell efficiently, which impedes and raises the cost of purification of these vital proteins. Our laboratory has isolated a mutant strain of Pichia pastoris that shows enhanced secretion of many proteins. The mutant produces a modified version of Bgs13p. Our goal is to understand how the change in the Bgs13p function leads to improved secretion. Once the Bgs13p mechanism is illuminated, we should be able to apply this understanding to engineer new P. pastoris strains that efficiently produce and secrete life-saving recombinant proteins, providing medical and economic benefits.

A Tripartite, Hierarchical Sigma Factor Cascade Promotes Hormogonium Development in the Filamentous Cyanobacterium Nostoc punctiforme.

Cyanobacteria are prokaryotes capable of oxygenic photosynthesis, and frequently, nitrogen fixation as well. As a result, they contribute substantially to global primary production and nitrogen cycles. Furthermore, the multicellular filamentous cyanobacteria in taxonomic subsections IV and V are developmentally complex, exhibiting an array of differentiated cell types and filaments, including motile hormogonia, making them valuable model organisms for studying development. To investigate the role of sigma factors in the gene regulatory network (GRN) controlling hormogonium development, a combination of genetic, immunological, and time-resolved transcriptomic analyses were conducted in the model filamentous cyanobacterium Nostoc punctiforme, which, unlike other common model cyanobacteria, retains the developmental complexity of field isolates. The results support a model where the hormogonium GRN is driven by a hierarchal sigma factor cascade, with sigJ activating the expression of both sigC and sigF, as well as a substantial portion of additional hormogonium-specific genes, including those driving changes to cellular architecture. In turn, sigC regulates smaller subsets of genes for several processes, plays a dominant role in promoting reductive cell division, and may also both positively and negatively regulate sigJ to reinforce the developmental program and coordinate the timing of gene expression, respectively. In contrast, the sigF regulon is extremely limited. Among genes with characterized roles in hormogonium development, only pilA shows stringent sigF dependence. For sigJ-dependent genes, a putative consensus promoter was also identified, consisting primarily of a highly conserved extended -10 region, here designated a J-Box, which is widely distributed among diverse members of the cyanobacterial lineage.IMPORTANCE Cyanobacteria are integral to global carbon and nitrogen cycles, and their metabolic capacity coupled with their ease of genetic manipulation make them attractive platforms for applications such as biomaterial and biofertilizer production. Achieving these goals will likely require a detailed understanding and precise rewiring of these organisms' GRNs. The complex phenotypic plasticity of filamentous cyanobacteria has also made them valuable models of prokaryotic development. However, current research has been limited by focusing primarily on a handful of model strains which fail to reflect the phenotypes of field counterparts, potentially limiting biotechnological advances and a more comprehensive understanding of developmental complexity. Here, using Nostoc punctiforme, a model filamentous cyanobacterium that retains the developmental range of wild isolates, we define previously unknown definitive roles for a trio of sigma factors during hormogonium development. These findings substantially advance our understanding of cyanobacterial development and gene regulation and could be leveraged for future applications.

A partner-switching regulatory system controls hormogonium development in the filamentous cyanobacterium Nostoc punctiforme.

Filamentous cyanobacteria exhibit developmental complexity, including the transient differentiation of motile hormogonia in many species. Using a forward genetic approach, a trio of genes unique to filamentous cyanobacteria encoding a putative Rsb-like partner-switching regulatory system (PSRS) was implicated in regulating hormogonium development in the model filamentous cyanobacterium Nostoc punctiforme. Analysis of in-frame deletion strains indicated that HmpU (putative serine phosphatase) and HmpV (STAS domain) enhance, while HmpW (putative serine kinase) represses motility and persistence of the hormogonium state. Protein-protein interaction studies demonstrated specificity between HmpW and HmpV. Epistasis analysis between hmpW and hmpV was consistent with HmpV acting as the downstream effector of the system, rather than regulation of a sigma factor by HmpW. Deletion of hmpU or hmpV reduced accumulation of extracellular PilA and hormogonium polysaccharide (HPS), and expression of type IV pilus- and HPS-specific genes was reduced in the ΔhmpV strain. Expression of the Hmp PSRS is induced in hormogonia, and the cytoplasmic localization of HmpV-GFPuv implies that its downstream target is probably cytoplasmic as well. Collectively, these results support a model where HmpU and HmpW antagonistically regulate the phosphorylation state of HmpV, and subsequently, unphosphorylated HmpV positively regulates an undefined downstream target to affect hormogonium-specific gene expression.

Dynamic localization of HmpF regulates type IV pilus activity and directional motility in the filamentous cyanobacterium Nostoc punctiforme.

Many cyanobacteria exhibit surface motility powered by type 4 pili (T4P). In the model filamentous cyanobacterium Nostoc punctiforme, the T4P systems are arrayed in static, bipolar rings in each cell. The chemotaxis-like Hmp system is essential for motility and the coordinated polar accumulation of PilA on cells in motile filaments, while the Ptx system controls positive phototaxis. Using transposon mutagenesis, a gene, designated hmpF, was identified as involved in motility. Synteny among filamentous cyanobacteria and the similar expression patterns for hmpF and hmpD imply that HmpF is part of the Hmp system. Deletion of hmpF produced a phenotype distinct from other hmp genes, but indistinguishable from pilB or pilQ. Both an HmpF-GFPuv fusion protein, and PilA, as assessed by in situ immunofluorescence, displayed coordinated, unipolar localization at the leading pole of each cell. Reversals were modulated by changes in light intensity and preceded by the migration of HmpF-GFPuv to the lagging cell poles. These results are consistent with a model where direct interaction between HmpF and the T4P system activates pilus extension, the Hmp system facilitates coordinated polarity of HmpF to establish motility, and the Ptx system modulates HmpF localization to initiate reversals in response to changes in light intensity.

A Putative O-Linked ß-N-Acetylglucosamine Transferase Is Essential for Hormogonium Development and Motility in the Filamentous Cyanobacterium Nostoc punctiforme.

Most species of filamentous cyanobacteria are capable of gliding motility, likely via a conserved type IV pilus-like system that may also secrete a motility-associated polysaccharide. In a subset of these organisms, motility is achieved only after the transient differentiation of hormogonia, which are specialized filaments that enter a nongrowth state dedicated to motility. Despite the fundamental importance of hormogonia to the life cycles of many filamentous cyanobacteria, the molecular regulation of hormogonium development is largely undefined. To systematically identify genes essential for hormogonium development and motility in the model heterocyst-forming filamentous cyanobacterium Nostoc punctiforme, a forward genetic screen was employed. The first gene identified using this screen, designated ogtA, encodes a putative O-linked ß-N-acetylglucosamine transferase (OGT). The deletion of ogtA abolished motility, while ectopic expression of ogtA induced hormogonium development even under hormogonium-repressing conditions. Transcription of ogtA is rapidly upregulated (1 h) following hormogonium induction, and an OgtA-GFPuv fusion protein localized to the cytoplasm. In developing hormogonia, accumulation of PilA but not HmpD is dependent on ogtA Reverse transcription-quantitative PCR (RT-qPCR) analysis indicated equivalent levels of pilA transcript in the wild-type and ΔogtA mutant strains, while a reporter construct consisting of the intergenic region in the 5' direction of pilA fused to gfp produced lower levels of fluorescence in the ΔogtA mutant strain than in the wild type. The production of hormogonium polysaccharide in the ΔogtA mutant strain is reduced compared to that in the wild type but comparable to that in a pilA deletion strain. Collectively, these results imply that O-GlcNAc protein modification regulates the accumulation of PilA via a posttranscriptional mechanism in developing hormogonia.IMPORTANCE Filamentous cyanobacteria are among the most developmentally complex prokaryotes. Species such as Nostoc punctiforme develop an array of cell types, including nitrogen-fixing heterocysts, spore-like akinetes, and motile hormogonia, that function in dispersal as well as the establishment of nitrogen-fixing symbioses with plants and fungi. These symbioses are major contributors to global nitrogen fixation. Despite the fundamental importance of hormogonia to the life cycle of filamentous cyanobacteria and the establishment of symbioses, the molecular regulation of hormogonium development is largely undefined. We employed a genetic screen to identify genes essential for hormogonium development and motility in Nostoc punctiforme The first gene identified using this screen encodes a eukaryotic-like O-linked ß-N-acetylglucosamine transferase that is required for accumulation of PilA in hormogonia.

Differential secretion pathways of proteins fused to the Escherichia coli maltose binding protein (MBP) in Pichia pastoris.

The Escherichia coli maltose binding protein (MBP) is an N-terminal fusion partner that was shown to enhance the secretion of some heterologous proteins from the yeast Pichia pastoris, a popular host for recombinant protein expression. The amount of increase in secretion was dependent on the identity of the cargo protein, and the fusions were proteolyzed prior to secretion, limiting its use as a purification tag. In order to overcome these obstacles, we used the MBP as C-terminal partner for several cargo peptides. While the Cargo-MBP proteins were no longer proteolyzed in between these two moieties when the MBP was in this relative position, the secretion efficiency of several fusions was lower than when MBP was located at the opposite end of the cargo protein (MBP-Cargo). Furthermore, fluorescence analysis suggested that the MBP-EGFP and EGFP-MBP proteins followed different routes within the cell. The effect of several Pichia pastoris beta-galactosidase supersecretion (bgs) strains, mutants showing enhanced secretion of select reporters, was also investigated on both MBP-EGFP and EGFP-MBP. While the secretion efficiency, proteolysis and localization of the MBP-EGFP was influenced by the modified function of Bgs13, EGFP-MBP behavior was not affected in the bgs strain. Taken together, these results indicate that the location of the MBP in a fusion affects the pathway and trans-acting factors regulating secretion in P. pastoris.

The non-metabolizable sucrose analog sucralose is a potent inhibitor of hormogonium differentiation in the filamentous cyanobacterium Nostoc punctiforme.

Nostoc punctiforme is a filamentous cyanobacterium which forms nitrogen-fixing symbioses with several different plants and fungi. Establishment of these symbioses requires the formation of motile hormogonium filaments. Once infected, the plant partner is thought to supply a hormogonium-repressing factor (HRF) to maintain the cyanobacteria in a vegetative, nitrogen-fixing state. Evidence implies that sucrose may serve as a HRF. Here, we tested the effects of sucralose, a non-metabolizable sucrose analog, on hormogonium differentiation. Sucralose inhibited hormogonium differentiation at a concentration approximately one-tenth that of sucrose. This result implies that: (1) sucrose, not a sucrose catabolite, is perceived by the cell and (2) inhibition is not due to a more general osmolarity-dependent effect. Additionally, both sucrose and sucralose induced the accrual of a polysaccharide sheath which bound specifically to the lectin ConA, indicating the presence of α-D-mannose and/or α-D-glucose. A ConA-specific polysaccharide was also found to be expressed in N. punctiforme colonies from tissue sections of the symbiotically grown hornwort Anthoceros punctatus. These findings imply that plant-derived sucrose or sucrose analogs may have multiple effects on N. punctiforme, including both repression of hormogonia and the induction of a polysaccharide sheath that may be essential to establish and maintain the symbiotic state.

Evidence that a modified type IV pilus-like system powers gliding motility and polysaccharide secretion in filamentous cyanobacteria.

In filamentous cyanobacteria, the mechanism of gliding motility is undefined but posited to be driven by a polysaccharide secretion system known as the junctional pore complex (JPC). Recent evidence implies that the JPC is a modified type IV pilus-like structure encoded for in part by genes in the hps locus. To test this hypothesis, we conducted genetic, cytological and comparative genomics studies on hps and pil genes in Nostoc punctiforme, a species in which motility is restricted to transiently differentiated filaments called hormogonia. Inactivation of most hps and pil genes abolished motility and abolished or drastically reduced secretion of hormogonium polysaccharide, and the subcellular localization of several Pil proteins in motile hormogonia corresponds to the site of the junctional pore complex. The non-motile ΔhpsE-G strain, which lacks three glycosyltransferases that synthesize hormogonium polysaccharide, could be complemented to motility by the addition of medium conditioned by wild-type hormogonia. Based on this result, we speculate that secretion of hormogonium polysaccharide facilitates but does not provide the motive force for gliding. Both the Hps and Pil homologs characterized in this study are almost universally conserved among filamentous cyanobacteria, with the Hps homologs rarely found in unicellular strains. These results support the theory that Hps and Pil proteins compose the JPC, a type IV pilus-like nanomotor that drives motility and polysaccharide secretion in filamentous cyanobacteria.

Genetic analysis reveals the identity of the photoreceptor for phototaxis in hormogonium filaments of Nostoc punctiforme.

In cyanobacterial Nostoc species, substratum-dependent gliding motility is confined to specialized nongrowing filaments called hormogonia, which differentiate from vegetative filaments as part of a conditional life cycle and function as dispersal units. Here we confirm that Nostoc punctiforme hormogonia are positively phototactic to white light over a wide range of intensities. N. punctiforme contains two gene clusters (clusters 2 and 2i), each of which encodes modular cyanobacteriochrome-methyl-accepting chemotaxis proteins (MCPs) and other proteins that putatively constitute a basic chemotaxis-like signal transduction complex. Transcriptional analysis established that all genes in clusters 2 and 2i, plus two additional clusters (clusters 1 and 3) with genes encoding MCPs lacking cyanobacteriochrome sensory domains, are upregulated during the differentiation of hormogonia. Mutational analysis determined that only genes in cluster 2i are essential for positive phototaxis in N. punctiforme hormogonia; here these genes are designated ptx (for phototaxis) genes. The cluster is unusual in containing complete or partial duplicates of genes encoding proteins homologous to the well-described chemotaxis elements CheY, CheW, MCP, and CheA. The cyanobacteriochrome-MCP gene (ptxD) lacks transmembrane domains and has 7 potential binding sites for bilins. The transcriptional start site of the ptx genes does not resemble a sigma 70 consensus recognition sequence; moreover, it is upstream of two genes encoding gas vesicle proteins (gvpA and gvpC), which also are expressed only in the hormogonium filaments of N. punctiforme.

Genetic characterization of the hmp locus, a chemotaxis-like gene cluster that regulates hormogonium development and motility in Nostoc punctiforme.

Filamentous cyanobacteria are capable of gliding motility, but the mechanism of motility is not well defined. Here we present a detailed characterization of the hmp locus from Nostoc punctiforme, which encodes chemotaxis-like proteins. Deletions of hmpB, C, D and E abolished differentiation of hormogonia under standard growth conditions, but, upon addition of a symbiotic partner exudate, the mutant strains differentiated hormogonium-like filaments that lacked motility and failed to secrete hormogonium specific polysaccharide. The hmp locus is expressed as two transcripts, one originating 5' of hmpA and encompassing the entire hmp locus, and the other 5' of hmpB and encompassing hmpBCDE. The CheA-like HmpE donates phosphate to its own C-terminal receiver domain, and to the CheY-like HmpB, but not to the PatA family CheY-like HmpA. A GFP-tagged variant of each hmp locus protein localized to a ring adjacent to the septum on each end of the rod-shaped cell. Immunofluorescence demonstrated that PilA localizes to a ring at the junction between cells. The phenotype of the deletion strains, and the localization of the Hmp proteins and the putative PilA protein to rings at the cell junctions are consistent with the hypothesis that these proteins are part of the junctional pore complex observed in a number of filamentous cyanobacteria.

Comparative transcriptomics with a motility-deficient mutant leads to identification of a novel polysaccharide secretion system in Nostoc punctiforme.

Many filamentous cyanobacteria are capable of gliding motility by an undefined mechanism. Within the heterocyst-forming clades, some strains, such as the Nostoc spp. and Fisherella spp., are motile only as specialized filaments termed hormogonia. Here we report on the phenotype of inactivation of a methyl-accepting chemotaxis-like protein in Nostoc punctiforme, designated HmpD. The gene hmpD was found to be essential for hormogonium development, motility and polysaccharide secretion. Comparative global transcriptional profiling of the ΔhmpD strain demonstrated that HmpD has a profound effect on the transcriptional programme of hormogonium development, influencing approximately half of the genes differentially transcribed during differentiation. Utilizing this transcriptomic data, we identified a gene locus, designated here as hps, that appears to encode for a novel polysaccharide secretion system. Transcripts for the genes in the hps locus are upregulated in two steps, with the second step dependent on HmpD. Deletion of hpsA, hpsBCD or hpsEFG resulted in the complete loss of motility and polysaccharide secretion, similar to deletion of hmpD. Genes in the hps locus are highly conserved in the filamentous cyanobacteria, but generally absent in unicellular strains, implying a common mechanism of motility unique to the filamentous cyanobacteria.