ChIP analysis unravels an exceptionally wide distribution of DNA binding sites for the NtcA transcription factor in a heterocyst-forming cyanobacterium

[Background] The CRP-family transcription factor NtcA, universally found in cyanobacteria, was initially discovered as a regulator operating N control. It responds to the N regime signaled by the internal 2-oxoglutarate levels, an indicator of the C to N balance of the cells. Canonical NtcA-activated promoters bear an NtcA-consensus binding site (GTAN8TAC) centered at about 41.5 nucleotides upstream from the transcription start point. In strains of the Anabaena/Nostoc genera NtcA is pivotal for the differentiation of heterocysts in response to N stress.[Results] In this study, we have used chromatin immunoprecipitation followed by high-throughput sequencing to identify the whole catalog of NtcA-binding sites in cells of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 three hours after the withdrawal of combined N. NtcA has been found to bind to 2,424 DNA regions in the genome of Anabaena, which have been ascribed to 2,153 genes. Interestingly, only a small proportion of those genes are involved in N assimilation and metabolism, and 65% of the binding regions were located intragenically.[Conclusions] The distribution of NtcA-binding sites identified here reveals the largest bacterial regulon described to date. Our results show that NtcA has a much wider role in the physiology of the cell than it has been previously thought, acting both as a global transcriptional regulator and possibly also as a factor influencing the superstructure of the chromosome (and plasmids).This work was supported by grant BFU2010–17980 from Ministerio de Ciencia e Innovación (Spain), co-financed by FEDER, and grant P08-CVI-03838 from Junta de Andalucia (Spain).Peer Reviewe

Diverse roles of the GlcP glucose permease in free-living and symbiotic cyanobacteria

Certain cyanobacteria can form symbiotic associations with plants, where the symbiont supplies the plant partner with nitrogen and in return obtains sugars. We recently showed that in the symbiotic cyanobacterium Nostoc punctiforme, a glucose specific permease, GlcP, is necessary for the symbiosis to be formed. Results presented here from growth yield measurements of mutant strains with inactivated or overexpressing sugar transporters suggest that GlcP could be induced by a symbiosis specific substance. We also discuss that the transporter may have a role other than nutritional once the symbiosis is established, i.e., during infection, and more specifically in the chemotaxis of the symbiont. Phylogenetic analysis shows that the distribution of GlcP among cyanobacteria is likely influenced by horizontal gene transfer, but also that it is not correlated with symbiotic competence. Instead, regulatory patterns of the transporter in Nostoc punctiforme likely constitute symbiosis specific adaptations.Ministerio de Ciencia e Innovación BFU2011–2276

CcpC-dependent regulation of citrate synthase gene expression in Listeria monocytogenes

Citrate synthase, the first and rate-limiting enzyme of the tricarboxylic acid branch of the Krebs cycle, was shown to be required for de novo synthesis of glutamate and glutamine in Listeria monocytogenes. The citrate synthase (citZ) gene was found to be part of a complex operon with the upstream genes lmo 1569 and lmo 1568. The downstream isocitrate dehydrogenase (citC) gene appears to be part of the same operon as well. Two promoters were shown to drive citZ expression, a distal promoter located upstream of lmo 1569 and a proximal promoter located upstream of the lmo 1568 gene. Transcription of citZ from both promoters was regulated by CcpC by interaction with a single site; assays of transcription in vivo and assays of CcpC binding in vitro revealed that CcpC interacts with and represses the proximal promoter that drives expression of the lmo 1568, citZ, and citC genes and, by binding to the same site, prevents read-through transcription from the distal, lmo 1569 promoter. Expression of the lmo 1568 operon was not affected by the carbon source but was repressed during growth in complex medium by addition of glutamine.Public Health Service GM03671

ZipN is an essential FtsZ membrane tether and contributes to the septal localization of SepJ in the flamentous cyanobacterium Anabaena

The organismic unit of heterocyst-forming cyanobacteria is a filament of communicating cells connected by septal junctions, proteinaceous structures bridging the cytoplasms of contiguous cells. This distinct bacterial organization is preserved during cell division. In Anabaena, deletion of the zipN gene could not be segregated. We generated strain CSL109 that expresses zipN from a synthetic regulatable promoter. Under conditions of ZipN depletion, cells progressively enlarged, reflecting restricted cell division, and showed drastic morphological alterations including cell detachment from the filaments, to finish lysing. In contrast to the wild-type localization in midcell Z-rings, FtsZ was found in delocalized aggregates in strain CSL109. Consistently, the proportion of membrane-associated to soluble FtsZ in fractionated cell extracts was lower in CSL109. Bacterial two-hybrid analysis showed that ZipN interacts with FtsZ and other cell-division proteins including cytoplasmic Ftn6 and SepF, and polytopic FtsW, FtsX, FtsQ and FtsI. Additionally, ZipN interacted with the septal protein SepJ, and in CSL109 depletion of ZipN was concomitant with a progressive loss of septal specificity of SepJ. Thus, in Anabaena ZipN represents an essential FtsZ membrane tether and an organizer of the divisome, and it contributes to the conformation of septal structures for filament integrity and intercellular communication.Agencia Estatal de Investigación BFU2013-44686-P, BFU2016-77097-

FtsZ of filamentous, heterocyst-forming cyanobacteria has a conserved N-Terminal peptide required for normal FtsZ polymerization and cell division

Filamentous cyanobacteria grow by intercalary cell division, which should involve distinct steps compared to those producing separate daughter cells. The N-terminal region of FtsZ is highly conserved in the clade of filamentous cyanobacteria capable of cell differentiation. A derivative of the model strain Anabaena sp. PCC 7120 expressing only an FtsZ lacking the amino acids 2-51 of the N-terminal peptide (1N-FtsZ) could not be segregated. Strain CSL110 expresses both 1N-FtsZ, from the endogenous ftsZ gene promoter, and the native FtsZ from a synthetic regulated promoter. Under conditions of 1N-FtsZ predominance, cells of strain CSL110 progressively enlarge, reflecting reduced cell division, and show instances of asymmetric cell division and aberrant Z-structures notably differing from the Z-ring formed by FtsZ in the wild type. In bacterial 2-hybrid assays FtsZ interacted with 1N-FtsZ. However, 1N-FtsZ-GFP appeared impaired for incorporation into Z-rings when expressed together with FtsZ. FtsZ, but not 1N-FtsZ, interacted with the essential protein SepF. Both FtsZ and 1N-FtsZ polymerize in vitro exhibiting comparable GTPase activities. However, filaments of FtsZ show a distinct curling forming toroids, whereas 1N-FtsZ form thick bundles of straight filaments. Thus, the N-terminal FtsZ sequence appears to contribute to a distinct FtsZ polymerization mode that is essential for cell division and division plane location in Anabaena.Agencia Estatal de Investigación BFU2013-44686-P BFU2016-77097-

Transferencia intercelular de nitrógeno en la cianobacteria formadora de heterocistos Anabaena sp. PCC 7120

En este trabajo se han abordado algunos aspectos del metabolismo del nitrógeno en la cianobacteria Anabaena sp. PCC 7120. Se han estudiado, por una parte, sistemas de transporte de aminoácidos y su implicación en el crecimiento a expensas de nitrógeno atmosférico y, por otra parte, la expresión de los genes del metabolismo del a cianoficina y su implicación en el metabolismo diazotrófico de esta cianobacteria. El sistema de transporte de aminoácidos neutros N-I, que es el del tipo ABC, está implicado en el metabolismo diazotrófico de Anabaena. También participan en dicho metabolismo el transporte y degradación de urea, así como el catabolismo de la prolina, que es el único aminoácido transportado exclusivamente por el sistema N-I, Los genes del metabolismo de la cianoficina en Anabaena sp. PCC 7120 presentan una regulación muy compleja que permite a esta cianobacteria mantener una expresión basal relativamente alta de estos genes en todas las condiciones celulares y, por otra parte, permite que dichos genes se expresen diferencialmente según sea el tipo celular y la condición nitrogenada de que se trate. No obstante, la síntesis de cianoficina no es un proceso imprescindible para Anabaena sp., pueda vivir a expensas del nitrógeno atmosférico

CcpC-Dependent Regulation of Citrate Synthase Gene Expression in Listeria monocytogenes▿

Citrate synthase, the first and rate-limiting enzyme of the tricarboxylic acid branch of the Krebs cycle, was shown to be required for de novo synthesis of glutamate and glutamine in Listeria monocytogenes. The citrate synthase (citZ) gene was found to be part of a complex operon with the upstream genes lmo1569 and lmo1568. The downstream isocitrate dehydrogenase (citC) gene appears to be part of the same operon as well. Two promoters were shown to drive citZ expression, a distal promoter located upstream of lmo1569 and a proximal promoter located upstream of the lmo1568 gene. Transcription of citZ from both promoters was regulated by CcpC by interaction with a single site; assays of transcription in vivo and assays of CcpC binding in vitro revealed that CcpC interacts with and represses the proximal promoter that drives expression of the lmo1568, citZ, and citC genes and, by binding to the same site, prevents read-through transcription from the distal, lmo1569 promoter. Expression of the lmo1568 operon was not affected by the carbon source but was repressed during growth in complex medium by addition of glutamine

Catabolic pathway of arginine in Anabaena involves a novel bifunctional enzyme that produces proline from arginine

Arginine participates widely in metabolic processes. The heterocyst-forming cyanobacterium Anabaena catabolizes arginine to produce proline and glutamate, with concomitant release of ammonium, as major products. Analysis of mutant Anabaena strains showed that this catabolic pathway is the product of two genes, agrE (alr4995) and putA (alr0540). The predicted PutA protein is a conventional, bifunctional proline oxidase that produces glutamate from proline. In contrast, AgrE is a hitherto unrecognized enzyme that contains both an N-terminal ¿/ß propeller domain and a unique C-terminal domain of previously unidentified function. In vitro analysis of the proteins expressed in Escherichia coli or Anabaena showed arginine dihydrolase activity of the N-terminal domain and ornithine cyclodeaminase activity of the C-terminal domain, overall producing proline from arginine. In the diazotrophic filaments of Anabaena, ß-aspartyl-arginine dipeptide is transferred from the heterocysts to the vegetative cells, where it is cleaved producing aspartate and arginine. Both agrE and putA were found to be expressed at higher levels in vegetative cells than in heterocysts, implying that arginine is catabolized by the AgrE-PutA pathway mainly in the vegetative cells. Expression in Anabaena of a homolog of the C-terminal domain of AgrE obtained from Methanococcus maripaludis enabled us to identify an archaeal ornithine cyclodeaminase.Peer Reviewe

Transcriptional regulation of development in heterocyst-forming cyanobacteria.

Filamentous, heterocyst-forming cyanobacteria are among the simplest multicellular systems in Nature. In the absence of combined nitrogen, the filaments consist of vegetative cells that fix CO2 through oxygenic photosynthesis and micro-oxic heterocysts specialized for the fixation of N2 in a proportion of about 10 to 1. The development of a heterocyst-containing filament involves differentiation of vegetative cells into heterocysts in a process that requires a distinct gene expression program. Two transcription factors are strictly required, NtcA and HetR. The CRP-family protein NtcA directly activates the expression of multiple genes during heterocyst differentiation – in some cases assisted by coactivators including HetR – and in mature heterocysts, whereas HetR is needed to build high NtcA levels in differentiating heterocysts and directly activates some particular genes. A few other regulators of gene expression participate at specific differentiation steps, and a specific transcription factor, CnfR, activates nif gene expression under the micro-oxic conditions of the heterocyst

Acoustic Characteristics of Voiced Noise Consonants of the Latvian Language

Promocijas darba „Latviešu valodas balsīgo troksneņu akustisks raksturojums” anotācija Kaut gan akustiskā fonētika vairs nav jauna zinātnes nozare Latvijā, šajā jomā joprojām ir maz pētījumu. Latviešu literārās valodas balsīgo troksneņu akustiskais apraksts latviešu valodniecībā ir novatorisks, tā rezultātus var salīdzināt tikai ar citu valodu pētījumiem ārzemēs. Promocijas darba mērķis ir iegūt pēc iespējas pilnīgu latviešu literārās valodas balsīgo troksneņu akustisko aprakstu, tādējādi arī uzkrāt datus latviešu valodas skaņu sistēmas akustiskajam aprakstam un latviešu literārās valodas akustiskajai datu bāzei. Pētījumā iegūtos rezultātus un mērījumu datus varēs izmantot runas automātiskas atpazīšanas sistēmās, fonētiskās un fonoloģiskās sistēmas aprakstā latviešu valodas gramatikā, mācību grāmatās un topošo filologu studijās. Atslēgas vārdi: balsīgie troksneņi, akustiskās pazīmes, balsīgie slēdzeņi, balsīgie spraudzeņi, fonoloģiskā klasifikācijaAnnotation of promotion paper “Acoustic Characteristics of Voiced Noise Consonants of the Latvian Language” The Acoustic Phonetics is not a new branch of science in Latvia anymore; still there have not been many researches in this field. Until now, researches on vowels in the Latvian linguistics have prevailed, so acoustic description of voiced noise consonants is innovative. It could be compared only to researches of other languages abroad. The aim of the promotion paper is to make an acoustic description of voiced noise consonants of the Standard Latvian. These consonants are described by using acoustic features of stop consonants and fricatives, as well as phonological classification based on acoustic and articulary data. Another aim of the promotion paper is to collect data for the acoustic description of the sound system of the Latvian language and to build an acoustic database of the Standard Latvian. The data of measurements and the results of this research can be used for automatic speech recognition, also in description of phonetic and phonology system, in textbooks and in studies of philology. Key words: voiced noise consonants, acoustic characteristics, voiced stops, voiced fricatives, phonological classificatio