life Article Molecular Components of Nitrogen Fixation Gene Cluster and Associated Enzymatic Activities of Non-Heterocystous Thermophilic Cyanobacterium Thermoleptolyngbya sp. Meijin Li 1, Lei Cheng 2 , Jie Tang 3 and Maurycy Daroch 1,* 1 School of Environment and Energy, Peking University Shenzhen Graduate School, 2199 Lishui Rd., Shenzhen 518055, China; 1801213472@pku.edu.cn 2 Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University, Beijing 100048, China; chenglei@btbu.edu.cn 3 School of Food and Bioengineering, Chengdu University, Chengdu 610106, China; tangjie@cdu.edu.cn * Correspondence: m.daroch@pkusz.edu.cn Abstract: Thermoleptolyngbya is a genus of non-heterocystous cyanobacteria that are typical inhabi- tants of hot spring microbial mats. These filamentous cyanobacteria are capable of nitrogen fixation. In this study, we examined the genome sequences of five publicly available Thermoleptolyngbya strains to explore their nitrogen fixation gene cluster. Analysis of the nitrogen-fixation clusters in these extremophilic strains revealed that the cluster is located in a single locus in Thermoleptolyngbyace. The average nucleotide and amino acid identities of the nitrogen-fixation cluster combined with phylogenetic reconstructions support that nitrogen fixation genes in Thermoleptolyngbyaceae are closely related to one another but also heterogeneous within the genus. The strains from Asia, and China Citation: Li, M.; Cheng, L.; Tang, J.; more specifically, generate a separate clade within the genus. Among these strains Thermoleptolyngbya Daroch, M. Molecular Components of sp. PKUAC-SCTB121 has been selected for experimental validation of clade’s nitrogen fixation capac- Nitrogen Fixation Gene Cluster and Associated Enzymatic Activities of ity. The acetylene reduction experiments of that strain shown that the strain can reduce acetylene to Non-Heterocystous Thermophilic ethylene, indicating a fully functional nitrogenase. The activity of nitrogenase has been tested using Cyanobacterium Thermoleptolyngbya different gas compositions across 72 h and exhibited a two-phase trend, high nitrogenase activity at sp. Life 2021, 11, 640. https:// the beginning of the assay that slowed down in the second phase of the analysis. doi.org/10.3390/life11070640 Keywords: biological N2 fixation; Nif genes; gene cluster; genome analysis; Thermoleptolyngbya; Academic Editors: non-heterocystous cyanobacteria Przemyslaw Malec, Rajesh Jha and Pengcheng Fu Received: 1 June 2021 1. Introduction Accepted: 24 June 2021 Published: 30 June 2021 Biological N2 fixation (BNF) is the process in which atmospheric N2 gas is converted into ammonia nitrogen by a nitrogen-fixing microorganism. The nitrogen fixation is driven nif Publisher’s Note: MDPI stays neutral by a cluster of genes products that catalyze this important reaction [1]. The nitroge- with regard to jurisdictional claims in nase complex consists of two metalloprotein components of the molybdenum iron (MoFe) published maps and institutional affil- protein and iron (Fe) protein [2]. For the microorganisms that could perform BNF, cyanobac- iations. teria are the most diverse photosynthetic bacteria, widely distributed in various conditions, and play an essential role in the BNF. A hot spring is a place where warm or hot groundwater springs from the Earth’s crust on a regular basis for at least a predictable part of the year and is significantly above the average temperature. The water emanating from a hot spring is heated by geothermal Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. heat. A previous study showed that genomes and metagenomes of cyanobacteria that This article is an open access article form hot spring communities harbor a conserved nif gene cluster, suggesting that these distributed under the terms and strains have a potential to perform nitrogen fixation [3]. The role of individual microbes conditions of the Creative Commons in nitrogen cycling as environmental conditions vary over space and time. The present ◦ Attribution (CC BY) license (https:// upper-temperature limit for N2 fixation (92 C) was reported in an organism isolated from creativecommons.org/licenses/by/ a marine hydrothermal vent. The upper-temperature limit for N2 fixation in terrestrial ◦ 4.0/). environments is 82 C[4]. Life 2021, 11, 640. https://doi.org/10.3390/life11070640 https://www.mdpi.com/journal/life Life 2021, 11, 640 2 of 12 Thermophilic cyanobacteria isolated from hot springs are increasingly studied due to their environmental and biotechnological importance [5,6]. Cyanobacteria exhibit remark- able diversity in their morphological structures and can be broadly divided into heterocys- tous and non-heterocystous based on their mode of nitrogen fixation. The N2-fixing system of non-heterocystous cyanobacteria markedly differs from that of heterocystous counter- parts. Heterocystous filamentous cyanobacteria, such as Fischerella thermalis, have attracted early attention for their role in the cycling of nitrogen in thermal ecosystems [7]. Their mode of action is relatively well described [8]. The nitrogen-fixing capacity of non-heterocystous cyanobacteria in thermal ecosystems is less studied. To some extent, the mechanisms of non-heterocystous cyanobacteria are more di- verse [9]. This is especially important considering that non-heterocystous cyanobacteria cannot compartmentalize nitrogen fixation; therefore, an alternative mechanism to avoid the nitrogenase inhibition with oxygen need to be implemented in non-heterocystous strains [2,10]. Previous studies showed that there are various strategies to protect the enzyme complex against oxygen. For example, Trichodesmium sp., a marine filamentous cyanobacterium, exhibits exceptional ability to fix atmospheric nitrogen during the day. Whilst the strain does not differentiate into heterocysts, the localization of its Fe-protein of nitrogenase (dinitrogenase reductase) is confined to a limited subset of cells (on average 14%) in the sections randomly distributed across the filaments. The nitrogenase containing cells perform the O2 protective function, but they are not bona fide heterocysts [11,12]. Recently a new genus of filamentous cyanobacteria, Thermoleptolyngbya was delin- eated from the polyphyletic Leptolyngbya, and several species have been proposed within this genus [13,14]. Strains of this genus are non-heterocystous, and they could play an essential role in the nitrogen cycle within thermal ecosystems, as recently suggested by a study on Thermoleptolyngbya sp. O-77, collected from hot springs of Kumamoto, Japan [15]. Thanks to the advances of modern DNA sequencing technology and genomics, analysis of the genetic information of both cyanobacterial isolates and genome assemblies extracted from metagenomic bins created during hot spring community sequencing is now feasi- ble. The increased sequence pool allows for a better understanding of nitrogen fixation in the thermal niches [16]. Simultaneously, as new cyanobacterial strains are isolated from thermal sites worldwide [17], and their genetic modification systems are being de- veloped [18,19], experimental validation of sequence data is also increasingly possible. Interestingly, complete nitrogen fixation gene clusters have been identified in the genomes of Thermoleptolyngbya, which could be particularly interesting considering their ability to thrive in thermal environments. It also suggests that they are capable of providing nitrogen to the microbial mats they are inhabiting. These advances make Thermoleptolyn- gbya genus potentially interesting for studying nitrogen fixation among thermophilic non-heterocystous cyanobacteria and complement the work done on heterocystous thermal strains belonging to Fisherella. In this study, we used the whole genome sequence of five Thermoleptolyngbya strains deposited in the public databases; compared and assessed their nitrogen-fixation gene cluster information. To complement bioinformatic analysis, we have selected an isolate Ther- moleptolyngbya PKUAC-SCTB121, isolated from hot springs of Ganzi prefecture, Sichuan, China, from our culture collection as a representative of the genus and assessed its acetylene reduction capacity to provide experimental evidence of their nitrogen-fixing capability. 2. Materials and Methods 2.1. Genomic Sequence Information All the sequence data used in this study were downloaded from NCBI Database. Table1 shows their Genebank numbers (Table1). Strains PKUAC-SCTB121 and PKUAC- SCTA183 (hereafter B121 and A183, respectively) were sequenced, assembled and annotated by our group using a hybrid sequencing approach [14]. Life 2021, 11, 640 3 of 12 Table 1. Origin and accession numbers of Thermoleptolyngbya sp. genomes. Strain Accession Number Genome Description Strain Origin Thermoleptolyngbya sp. Complete genome 30◦15057” N CP070366.1 PKUAC-SCTB121 sequence 101◦56055” E Thermoleptolyngbya sp. Complete genome 30◦05014” N CP053661 PKUAC-SCTA83 sequence 101◦52024” E Thermoleptolyngbya sp. Complete genome 32◦48011”N AP017367 O-77 sequence 130◦42028”E Thermoleptolyngbya sp. 29◦69000”N JACYLP000000000.1 Metagenomic bin C42_A2020 83◦68000”E Thermoleptolyngbya sp. 39◦51000”S DVEA00000000.1 Metagenomic bin M55_K2018 72◦50000”W 2.2. Nitrogen Fixation Gene Cluster, ANI and AAI Analysis The nitrogen fixation gene cluster containing 27 well-defined genes and 2 unknown hypothetical proteins was extracted from the B121 genome combined with the results published elsewhere [15] and the annotation data of strains