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

   https://doi.org/10.1128/JB.00675-19  

   Zuniga, Esthefani G.; Figueroa, Natalie M.; Gonzalez, Alfonso; Pantoja, Adriana P.; Risser, Douglas D. (February 2020, Journal of Bacteriology)

   ABSTRACT 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. 

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

   https://doi.org/10.1128/mSphere.00231-19  

   Gonzalez, Alfonso; Riley, Kelsey W.; Harwood, Thomas V.; Zuniga, Esthefani G.; Risser, Douglas D. (June 2019, mSphere)

   ABSTRACT 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. 

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

   https://doi.org/10.1111/mmi.14566  

   Zuniga, Esthefani G.; Boateng, Kelvin K. A.; Bui, Nhi U.; Kurnfuli, Shadi; Muthana, Sawsan M.; Risser, Douglas D. (July 2020, Molecular Microbiology)

   Abstract 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 novelhpsloci, 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. 

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4. Regulatory small RNA, Qrr2 is expressed independently of sigma factor-54 and can function as the sole Qrr sRNA to control quorum sensing in Vibrio parahaemolyticus

   https://doi.org/10.1128/JB.00350-21  

   Tague, J.G.; Hong, J.; Kalburge, S.S.; Boyd, E.F. (October 2021, Journal of Bacteriology)

   null (Ed.)

   Bacterial cells alter gene expression in response to changes in population density in a process called quorum sensing (QS). In Vibrio harveyi, LuxO, a low cell density activator of sigma factor-54 (RpoN), is required for transcription of five non-coding regulatory sRNAs, Qrr1-Qrr5, which each repress translation of the master QS regulator LuxR. Vibrio parahaemolyticus, the leading cause of bacterial seafood-borne gastroenteritis, also contains five Qrr sRNAs that control OpaR (the LuxR homolog), controlling capsule polysaccharide (CPS), motility, and metabolism. We show that in a Δ luxO deletion mutant, opaR was de-repressed and CPS and biofilm were produced. However, in a Δ rpoN mutant, opaR was repressed, no CPS was produced, and less biofilm production was observed compared to wild type. To determine why opaR was repressed, expression analysis in Δ luxO showed all five qrr genes were repressed, while in Δ rpoN the qrr2 gene was significantly de-repressed. Reporter assays and mutant analysis showed Qrr2 sRNA can act alone to control OpaR. Bioinformatics analysis identified a sigma-70 (RpoD) -35 -10 promoter overlapping the canonical sigma-54 (RpoN) -24 -12 promoter in the qrr2 regulatory region. The qrr2 sigma-70 promoter element was also present in additional Vibrio species indicating it is widespread. Mutagenesis of the sigma-70 -10 promoter site in the Δ rpoN mutant background, resulted in repression of qrr2. Analysis of qrr quadruple deletion mutants, in which only a single qrr gene is present, showed that only Qrr2 sRNA can act independently to regulate opaR . Mutant and expression data also demonstrated that RpoN and the global regulator, Fis, act additively to repress qrr2 . Our data has uncovered a new mechanism of qrr expression and shows that Qrr2 sRNA is sufficient for OpaR regulation. Importance The quorum sensing non-coding sRNAs are present in all Vibrio species but vary in number and regulatory roles among species. In the Harveyi clade, all species contain five qrr genes, and in V. harveyi these are transcribed by sigma-54 and are additive in function. In the Cholerae clade, four qrr genes are present, and in V. cholerae the qrr genes are redundant in function. In V. parahaemolyticus , qrr2 is controlled by two overlapping promoters. In an rpoN mutant, qrr2 is transcribed from a sigma-70 promoter that is present in all V. parahaemolyticus strains and in other species of the Harveyi clade suggesting a conserved mechanism of regulation. Qrr2 sRNA can function as the sole Qrr sRNA to control OpaR. 

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5. Identifying the Gene Regulatory Network of the Starvation-Induced Transcriptional Activator Nla28

   https://doi.org/10.1128/jb.00265-22  

   Ma, Muqing; Garza, Anthony G.; Lemon, David J.; Caro, Eduardo A.; Ritchie, Linnea; Ryan, Charles; Spearing, Victoria M.; Murphy, Kimberly A.; Welch, Roy D. (December 2022, Journal of Bacteriology)

   O'Toole, George (Ed.)

   ABSTRACT Myxococcus xanthus copes with starvation by producing fruiting bodies filled with dormant and stress-resistant spores. Here, we aimed to better define the gene regulatory network associated with Nla28, a transcriptional activator/enhancer binding protein (EBP) and a key regulator of the early starvation response. Previous work showed that Nla28 directly regulates EBP genes that are important for fruiting body development. However, the Nla28 regulatory network is likely to be much larger because hundreds of starvation-induced genes are downregulated in a nla28 mutant strain. To identify candidates for direct Nla28-mediated transcription, we analyzed the downregulated genes using a bioinformatics approach. Nine potential Nla28 target promoters (29 genes) were discovered. The results of in vitro promoter binding assays, coupled with in vitro and in vivo mutational analyses, suggested that the nine promoters along with three previously identified EBP gene promoters were indeed in vivo targets of Nla28. These results also suggested that Nla28 used tandem, imperfect repeats of an 8-bp sequence for promoter binding. Interestingly, eight of the new Nla28 target promoters were predicted to be intragenic. Based on mutational analyses, the newly identified Nla28 target loci contained at least one gene that was important for starvation-induced development. Most of these loci contained genes predicted to be involved in metabolic or defense-related functions. Using the consensus Nla28 binding sequence, bioinformatics, and expression profiling, 58 additional promoters and 102 genes were tagged as potential Nla28 targets. Among these putative Nla28 targets, functions, such as regulatory, metabolic, and cell envelope biogenesis, were assigned to many genes. IMPORTANCE In bacteria, starvation leads to profound changes in behavior and physiology. Some of these changes have economic and health implications because the starvation response has been linked to the formation of biofilms, virulence, and antibiotic resistance. To better understand how starvation contributes to changes in bacterial physiology and resistance, we identified the putative starvation-induced gene regulatory network associated with Nla28, a transcriptional activator from the bacterium Myxoccocus xanthus . We determined the mechanism by which starvation-responsive genes were activated by Nla28 and showed that several of the genes were important for the formation of a highly resistant cell type. 

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