Alicyclobacillus chiapanensis PA2T, a novel species with specific adaptation in a bacterial cell wall isolated from an active volcano

doi:10.21203/rs.3.rs-2464478/v1

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Alicyclobacillus chiapanensis PA2T, a novel species with specific adaptation in a bacterial cell wall isolated from an active volcano

https://doi.org/10.21203/rs.3.rs-2464478/v1

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Aliclyclobacillus is an interesting genus that has been isolated worldwide from extreme environments, such as acid soils, hot springs, volcanoes and acidic drinks. The species of this genera has phenotypic particularities such as the phospholipid in their membranes and the composition of its cell wall. Besides, the phenotypic diversity of the Alicyclobacillus species it has been reported just 30 genomes. We analyzed the genome of a polyextremophilic bacterium isolated from a crater lake in an active volcano. The identity of the strain PA2^T was analyzed with standard molecular markers gyrB, ileS, lepA and 16S rRNA genes and confirmed by the average nucleotide identity ANIm (97.17%,), ANIb (96.65%,), TETRA (Z-score) (0.98) and DDH (75.5%.). These phylogenetic analyses indicated that PA2^T is a new species, which we have named Alicyclobacillus chiapanensis PA2^T. The genome A. chiapanensis PA2^T structure consists of a circular 2.956.928 bp molecule, with an overall G + C of 62.77%. The entire genome has 3,256 genes, a total of 2,952 genes has a putative function, and 144 were considered unique. A comparative genomic analysis of the COG systems of A. chiapanensis PA2^T showed "extra" genes that correspond to dormancy and sporulation (3), transport across the membrane (5), and the cell wall and capsule (6). This isolate shows changes in structure and chemical composition in the bacterial cell wall. The “extra” genes may be associated with the adaptations to polyextremophilic conditions. Particularly, we identify cell wall-associated specific genes, which participate in the D-rhamnose metabolism and can form an outer layer to protect the cell in inhospitable conditions.

Alicyclobacillus chiapanensis

spores

Gram variable

rhamnose

Life on Earth is the result of various evolutionary processes. For thousands of years, extremophile life has dominated the evolutionary history of our planet (Singh et al., 2018). Polyextremophilic microorganisms are interesting because they can grow and develop under extreme conditions (Merino et al., 2019). These organisms have developed interesting strategies to survive in hostile conditions. For example, thermoacidophilic organisms have modified their genomic content to survive thermal and acid stress (Panja et al., 2020), and they have specific genetic information adapted to hostile conditions, including particular processes like sporulation (Paul et al., 2019). Variations in genetic content can be caused by thermal, acid, saline, and arid stress (Arnold et al., 2008; Gottesman, 2019; Simonsen, 2021). Bacterial responses to stress include complex mechanisms involving transcriptional and post-transcriptional regulators (Gottesman, 2019). Stress-specific genes encoding molecules that immediately maintain cytoplasmic pH homeostasis, repair DNA, and activate particular molecular mechanisms to mantein cell wall integrity and its membrane-associated proteins. Therefore, the regulation and expression of genes rely on structure and genomic composition (Arnold et al., 2018).

Phenotypic adaptations have also been described as a result of genomic adaptations. Extreme organisms modify their cytoplasmic membranes to maintain a pH close to neutrality (Siliakus et al., 2017), modify their cell wall structure to develop external layers that protect the cell from stress fluctuations in temperature and pH (Klingl, 2014), adapt their macromolecules (DNA, lipids, and proteins) and cellular components like extracellular matrices, ribosomes, and RNA polymerases (Borroni and Benussi, 2019), and modify the lipid composition of their membranes with new and specific lipids (15, 16-dimethyl-30-glycerylox-triacontanedioic acid) (Siliakus et al., 2017). Furthermore, acidophilic bacteria modify the cytoplasmic membrane components (Dhakar and Pandey, 2016), and change the sugars associated with the membrane (Dhakar and Pandey, 2016). Some bacteria can generate an extracellular matrix of carbohydrates, which can be linked through glycosidic bond to the cell wall, generating a secondary polymer (S layer) that covers the cell completely (Hanger et al., 2018).

Alicyclobacillus species have recently been reported as having many of phenotypic variations, such as the use of different carbon sources (mono-, di- and polysaccharides), different nitrogen sources (organic and inorganic), and a resistance to NaCl, as well as starch and gelatin hydrolysis, nitrate reduction, and catalase and oxidase activities (Bevilacqua et al., 2015, Ciuffreda et al., 2015). Therefore, diversity of the Alicyclobacillus species reported just a reduced number of genomes (30 genomes have been reported).

Two strains of Aliclyclobacillus have been previously isolated from an extreme environment (the El Chichon crater lake). The strain PA2^T has interesting phenotypic characteristics, such as the variability of Gram dye, the ability to grow without carbon or nitrogen sources, and extracellular hydrolytic activity in hostile conditions (Ortiz-Cortés et al., 2021).

Thus, the aim of this work was to analyze the genome of Alicyclobacillus sp. PA2^T with an emphasis on sporulation, dormancy and cell wall genes in order to explain phenotypic variations such as the changes of Gram dye during batch growth.

Cultivation of A.chiapanensis PA2^T

A. chiapanensis PA2 from the ITTG cell collection was cultured in MB medium at pH 5 and then incubated for 120 h, pH 5 at 65°C (Ortiz-Cortés et al., 2021). A. chiapanensis PA2^T used for subsequent genome sequencing and kinetic studies was grown in MB medium. Aliquots of 3 mL were collected every 6 h. Gram dye analysis was carried out according to Hussey (2013).

Spore identification

A. chiapanensis PA2 ^T was cultured in MB medium at pH 5 in the absence of carbon and nitrogen sources and incubated at 65°C. Aliquots of 3 ml were collected every 6 h from a batch culture and heated for 20 min at room temperature. The samples were fixed on a microscope slide and stained with malachite green. The identification of the spore was performed with an Zeiss® Axiolab optical microscope (Terano et al., 2005).

Genome sequencing and assembly

Late-exponential A. chiapanensis PA2 ^T cells were collected by centrifugation (3000 rpm for 5 min at 4°C) for DNA genomic extraction with the ZR Fungal/Bacterial DNA Kit ™ according to the manufacturer's instructions. The genomic sequence of the A. chiapanensis PA2^T culture was obtained with an Illumina HiSeq (Macrogen, Seoul, South Korea). Reads were trimmed and paired using Trimmomatic version 0.30, and the quality was assessed with FastQC version 0.11.8 (Wingett et al., 2021). Paired-end reads were assembled with SPAdes 3.15.4 in default program mode, with only-assembler and default k-mer parameters (Bankevich et al., 2012). The estimation of genome size, contamination, and completeness was probed using CheckM (Parks et al., 2015). Finally, the chromosomal map and circular genome was constructed with the CIRCOS tool (Drori et al., 2017).

Genome annotation

Gene prediction was conducted with Prodigal (Hyatt et al., 2010). Annotation of CDSs were carried out with BLAST (Zhang and Madden, 1997), rapid annotation subsystem technology (RAST) (Aziz et al., 2008), and BLAST Koala (Kanehisa et al., 2016).

Functional annotation of the protein-coding genes was performed by classifying them into clusters of orthologous groups (COG) from the eggNOG database using the eggNOG-mapper (Jensen et al., 2008).

Phylogenetic analysis

The standard molecular markers gyrB, ileS, lepA and 16S rRNA genes were selected as bacterial identity tools. All sequences were aligned using the on-line tool CLUSTALW. This alignment was used for phylogenetic tree construction using the neighbor-joining method and the Kimura two-parameter model with a bootstrap analysis with 1000 replications (Saitou and Nei, 1987) and the MEGA X software package version 10.1.8 (Kumar et al. 2018).

The identity of the bacterium was confirmed by an ANI analysis using the FastANI tool (Han et al., 2016). Additionally, it was confirmed the average nucleotide identity (ANI), based on BLAST (ANIb) and MUMmer (ANIm). As well as the correlation indexes obtained from TETRA-nucleotide signatures (fragmento of DNA with four bases) using the on-line version of JSpecies (Richter et al., 2016). In silico DNA-DNA hybridization (DDH) values were estimated using the genome-to-genome distance calculator (GGDC) (Auch et al., 2010). Four preserved genes (gyrB, ileS, lepA and 16S rRNA) were used as templates or controls to determine the relationship between PA2^T and other Alicyclobacillus species. The gyrB, ileS, lepA, and 16S rRNA gene sequences were aligned using the on-line tool CLUSTALW. This alignment was used for phylogenetic tree construction using the neighbor-joining method and the Kimura two-parameter model using a bootstrap analysis with 1000 replications (Saitou and Nei 1987). These analyses were performed with the MEGA X software package version 10.1.8 (Kumar et al., 2018).

Comparative genomic analyses

The genomes of 12 Alicyclobacillus species are listed below.

(GCF_001552675), Alicyclobacillus sendaiensis

(GCF_000173835), Alicyclobacillus acidocaldarius LAA1

(GCF_000024285), Alicyclobacillus acidocaldarius DSM 446

(GCF_000219875), Alicyclobacillus acidocaldarius Tc-4-1

(GCF_001570745), Alicyclobacillus mali NBRC 102425

(GCF_900156755), Alicyclobacillus vulcanalis DSM 16176

(GCF_900107035), Alicyclobacillus hesperidum DSM 12489

(GCF_000294675), Alicyclobacillus hesperidum URH17-3-68

(GCF_000444055), Alicyclobacillus acidoterrestris ATCC 49025

(GCF_007991715), Alicyclobacillus acidoterrestris NBRC 106287

(GCF_001447355), Alicyclobacillus tengchongensis CGMCC1504

(GCF_001544355), Alicyclobacillus acidiphilus NBRC 100859) All were downloaded from NCBI refSeq (https://www.ncbi.nlm.nih.gov/genome/, July 2021). Furthermore, RAST was used to identify the cell wall, the capsule, and the membrane transport genes. This server is based on BLASTp and HMM (Aziz et al., 2008).

Data availability

The genome sequence of A. chiapanensis PA2 ^T is available in the GenBank databases under the accession.

In silico modeling of unique proteins in the genome of A. chiapanensis PA2^T

Unique genes in the A. chiapanensis PA2^T genome were identified by comparative genomic analysis. The nucleotide sequences were translated into amino acids through the Expasy ProtParam tool (Gasteiger et al., 2003). Prediction of the protein structure was performed using the homology model through SWISS-MODEL Interactive Workspace by choosing the appropriate template (Waterhouse et al 2018).

Genome assembly of A. chiapanensis PA2^T

The Alicyclobacillus chiapanensis PA2^T structure consists of a circular 2,956,928 bp molecule, with an overall G + C of 62,77%. The main genome features are shown in Fig. 1 and Table 1. The genome assembly quality was 98.82% of integrity without contamination. The entire genome has 3,256 genes. Most of them (2,952 genes) have a putative function for hypothetical proteins. A total of 144 were considered unique. J.

Table 1

Statistics of the genome of *Alicyclobacillus chiapanensis* PA2^T
Attribute	Value	%
Genome size (bp)	2,956,928	100
DNA coding region (bp)	2,798,000	94.62
DNA G + C content (bp)	1,856,059	62.77
Total genes	3257	100
RNA genes	61	2.1
rRNA operons	8
Protein-coding genes	3056	93.83
Genes with function prediction	2921	89.68
Genes in paralog clusters	1396	42.86
Genes assigned to COGs	2952	90.64
Genes assigned Pfam domains	3247	99.69
Unique genes	144	4.42

Table 1.

Protein-coding genes were grouped into 25 categories according to COG. A total of 3,056 CDSs were assigned to a COG category. Amino acids and derivatives (20.75%), carbohydrate metabolism (15.75%), cofactors, vitamins, prosthetic groups and pigments (10.41%), protein metabolism (10.25), lipid metabolism (6.66%), and nucleic and nucleotide metabolism (5.66%) were the most abundant categories. Moreover, the cell wall and the capsule, virulence, transposable elements and plasmids, membrane transport, dormancy and sporulation, respiration, stress response, sulfur metabolism, and phosphorus metabolism, as well as other categories, were also present.

Minimum information about a genome sequence (MIGS) recommendation is presented in Table 2. Former results show that A. chiapanensis PA2^T can grow without nitrogen or carbon sources (Ortiz-Cortés et al., 2021). A. chiapanensis PA2^T is a spore forming bacteria. It has some interesting biochemical characteristics, such as acid production from sorbitol and arabinose but no acid production from glucose, mannitol, inositol, rhamnose, sucrose, melibiose, nor amygdalin. It can use citrate like other Alicyclobacillus species (Goto et al., 2003; Jiang et al., 2008). Although we have not determined the motility of the bacteria, we found Fla A, flg B, flgC and FtsI genes, which are associated with flagella as are some Alicyclobacillus species (Goto et al., 2002; Matsubara et al., 2002).

Table 2

Classification and general features of *Alicyclobacillus chiapanensis* PA2^T according to the minimum information about a genome sequence (MIGS) recommendations¹
MIGS ID	Property	Term	Evidence code
	Current classification	Domain Bacteria	TAS¹
		Phylum Firmicutes	TAS²
		Class Bacilli	TAS²
		Order Bacillales	TAS²
		Family Alicyclobacillaceae	TAS²
		Genus Alicyclobacillus	TAS³
		Species Alicyclobacillus chiapanensis	IDA
		Type strain PA2 ^T	IDA
	Gram strain	Gram-variable cells	IDA
	Cell shape	Regular rod/Sreptorod	IDA
	Motility	Not reported (relevant genes missing)	NAS
	Sporulation	Endospore-forming	IDA
	Temperature range	45°C-65°C	IDA
	Optimum temperature	65°C	IDA
	Salinity	0–3% NaCl	IDA
MIGS-22	Oxygen requirement	Aerobic	IDA
	Carbon source	Saccharolytic, lypolitic and CO₂ fixation	IDA
	Energy source	Carbohydrates, lipids and Fe²⁺	IDA
MIGS-6	Habitat	Hot acid spring and lake sediment	IDA
MIGS-15	Biotic relationship	Free living	IDA
MIGS-14	Pathogenicity	None	IDA
	Isolation	Crater lake	IDA
MIGS-4	Geographic location	El Chichon crater lake in Chapultenango, Chiapas, Mexico	IDA
MIGS-5	Sample collection time	19/08/2016	IDA
MIGS-4.1	Latitude	N17°9'51.01"	IDA
MIGS-4.2	Longitude	O93°22'44"	IDA
MIGS-4.3	Depth	Superficial water	IDA
MIGS-4.2	Altitude	1205 m	IDA

Table 2.

2. Functional and physiological post-genome sequencing analysis

In former work (Ortiz Cortés et al., 2021), the A. chiapanensis PA2^T strain belongs to the Alicyclobacillus genera. This genus was proposed by Wisotzkey et al. (1992) to separate 3 Bacillus species with ω-alicyclic fatty acids in their cell membrane and has 30 species. Recently, a few species of this taxa have been isolated from extreme habitats: A. vulcanalis from hot spring water (Simbahan et al., 2004)d ferrooxidans (Jiang et al., 2008); A. pohliae from geothermal soil (Imperio et al., 2008); A. acidocaldarius (Wisotzkey et al., 1992), A. acidiphilus (Matsubara et al., 2002)d acidoterrestris (Deinhard et al., 1987), which was reclassified as A. acidoterrestris in 1992, from acid environments (Wisotzkey et al., 1992).

A. chiapanensis PA2^T has similar metabolic features when compared to other Alicyclobacillus species, such as heterotrophic metabolism, thermoacidophilic metabolism, and spore formation (Goto et al., 2003). Frequently these species can metabolize different carbon sources, such as pentoses, hexoses, and disaccharides (Simbahan et al., 2004), as well as organic and inorganic nitrogen sources (Jiang et al., 2008). Each species, however, has its own physiological and biochemical features, including NaCl tolerance, oxygen demand, pH, and optimum temperature, which allow them to reveal different phenotypes according to the environmental and nutritional conditions (Albuquerque et al., 2000; Simbahan et al., 2004, Bevilacqua et al., 2015).

In A. chiapanensis PA2^T there is an extracellular hydrolytic extremozyme activity. Mixotrophic metabolism is a remarkable feature described in three strains Alicyclobacillus sp. A4 (Jiang et al., 2008), Alicyclobacillus sp. PA1 and Alicyclobacillus sp. PA2^T (Ortiz-Cortés et al., 2021). A. chiapanensis PA2^T has extracellular activities of β galactosidase, protease, lipase, cellulase, and xylanase at 65°C and pH of 3 to 5. These metabolic and biochemical features present in this strain (A. chiapanensis PA2^T) could be due to adaptive mechanisms that allow them to grow in hostile and changing environments, including cyclic fatty acids changes in cellular membranes in acidophilic conditions, chaperonin and protease expression, and spore formation (Gauvry et al., 2017). Evolutive and genetic mechanisms could also be present in the A. chiapanensis PA2^T genome. There are regions in the genome of this strain that could be associated with these mechanisms. Some contigs of interest are C2, C3, C4, C8, C14, C17, C22, C28, C33, C49 and C58 because they contain genes associated with the cell wall and capsule, membrane transport, dormancy and sporulation. These mechanisms allow phenotypic and genotypic plasticity through probable genetic mutation, horizontal gene transfer, and species differentiation and adaptation. Moreover, genome evolution could lead to speciation.

Phylogenomic analysis with molecular markers

The phylogenetic relationship among A. chiapanensis PA2^T and other Alicyclobacillus species was described with 16S ARNr, gyrB, lepA, and pyrG as molecular markers. According to previous work (Ortiz-Cortés, 2021), this strain belongs to Alicyclobacillus, which is 94.73% identical to A. sendaiensis NBRC 100866 with a 16S ARNr gene comparison (100% cover); both strains appear in a monophylogenetic group (Fig. 2).

Phylogenetic analysis with gyrB, lepA and pyrG is similar in ramifications and interrelationship. In addition, it shows that A. chiapanensis PA2^T is related to A. acidocaldarius DSM 446 (isolated from soil and water, South Africa), A. acidocaldarius TC-4-1 (isolated from acid creek, United States), A. hesperidum DSM 12489 (isolated from solfataric soil), A. mali NBRC 102425 (acidic drinks) and A. vulcanalis DSM 16176 (isolated from a hot spring, United States). Therefore, these strains share genetic features and metabolic particularities but could form different phenotypic groups depending on the salt concentration (NaCl 5%), pH, and temperature (Bevilacqua et al., 2015, Ciuffreda et al., 2015), as well as A. chiapanensis PA2^T. On the other hand, genetic divergence among Alicyclobacillus species may be due to prokaryotic genome evolution, insertion and deletion of genome fragments, and genetic mutation (Iranzo et al., 2019). Genetic change is closely linked to homologous recombination as a critical mechanism that leads to genetic cohesion and prevents genomic divergence (Ke et al., 2017).

Although A. chiapanensis PA2^T and A. sendaiensis NBRC 100866 are related and share phenotypic features, both strains are different species. In the 16S ARNr phylogenetic tree, A. sendaiensis NBRC 100866 relates to another group, next to A. acidocaldarius sp. riitmanni DSM 11297T and A. acidocaldarius subsp. riitmanni MR1T. Particularly A. sendaiensis NBRC 100866 is a Gram-negative, spore-forming bacteria, which grows at 55ºC in a pH of 5. It is characterized by having an extracellular thermostable collagenase (Tsuruoka et al., 2003). Conversely, A. chiapanensis PA2^T is a Gram variable, spore-forming, halotolerant bacteria. It can duplicate at 65ºC, and its optimum pH is 5. Furthermore, it has extracellular hydrolytic thermophilic activities (Ortiz-Cortés et al., 2021). Genomic sequences of A. chiapanensis PA2^T and A. sendaiensis NBRC 100866 were compared. The TETRA (Z-score) was 0.98, and the ANI m was 97.17%. In addition, the ANI b was 96.65%, and the DDH was 75.5%.

The separation and phylogenetic arrangement between A. chiapanensis PA2^T and A. sendaiensis NBRC 100866 may be due to the ecological niche. Particularly, A. chiapanensis PA2^T was isolated from an active volcano that is characterized by being a highly dynamic system, presenting constant physicochemical changes in mainly temperature and pH (Armienta et al., 2014). On the other hand, A. sendaiensis NBRC 100866 was isolated from acid soils in Sendai, Japan (Tsuruoka et al., 2003). In addition, the divergence between A. chiapanensis PA2^T and A. sendaiensis NBRC 100866 may be caused by the genomic plasticity that leads to adaptation to different temperatures, pH, and nutrient disposal in extreme conditions (Zuckerkandl and Pauling, 1965; Bevilacqua et al., 2015; Parks et al., 2020).

Consequently, these processes can generate speciation (Iranzo et al., 2019). The Alicyclobacillus species evolved 15 million years ago in different clades and was isolated in hot spring water and acidic environments, particularly in the last 2 million years (Liu et al., 2021).

3. CHANGES IN THE CELL WALL AND EXTRACELLULAR MATRIX DURING A. chiapanensis PA2^T GROWTH

Gram variable

We analyzed changes in the bacterial cell wall of A. chiapanensis PA2^T in a batch culture with Gram stain. Our results showed bacterial cell wall composition and morphological change during growth in MB medium at 65°C and with a pH of 5 in a batch culture. At the early stages of culture growth (from 0 h at 54 h), the isolated were Gram-positive, but in the late growth stage, it was Gram-negative (to 60 h at 120 h) (Fig. 3). The bacterial cell wall is a vital complex structure that defines cell morphology, integrity, and maintenance (Dörr et al., 2019). This outer structure is the first contact with the surroundings, including biotic and abiotic stresses (Ultee et al., 2019).

Most bacteria have a single morphology (Gram stain) of their cell wall, but A. chiapanensis PA2^T, as in Thermus thermophilus (Artmann et al., 2018), modifies its bacterial cell wall according to environmental conditions and growth time. The changes in the Gram stain indicates variation in the structure and composition of the bacterial cell wall. In thermophiles, thermal stress seems to be diminished by these cell wall variations. Environmental conditions induce differential gene expression to synthesize specialized proteins, involved in signal transduction and other physiological and metabolic processes.

Extreme conditions, like acidic pH (5), high temperatures (65°C), and starvation, may induce morphological changes, including in cell walls. The bacterial cell wall is a vital structure and must be rigid enough to withstand hostile conditions, but it must also be flexible enough to allow cell expansion since it must be constantly renewed and readjusted. Gram-stain variation was previously reported in bacteria such as Bacillus, Butyrivibrio, and Clostridium during batch growth, and it was related to bacterial cell wall complexity and the presence of the S envelope (Beveridge, 1990). Recently, the presence of the S envelope has been related to extremophile stress in thermophilic and hyperthermophilic bacteria because proteomes increase (Klingl, 2014). Therefore, the S envelope can be connected to molecular dynamics as an adaptation strategy in extreme stress conditions (Artmann et al., 2018). In the A. chiapanensis PA2^T cell wall, the changes during bacterial growth may be related to morphogenic plasticity or adaptative morphogenesis These changes may be in size, shape, cell wall, cellular composition, and structure. They may result from evolutive selection, which drives the bacterial shapes and sizes that allow them to adapt to hostile conditions (Ultee et al., 2019).

Sporulation

The evaluation of morphological changes in A. chiapanensis PA2^T during hostile conditions (a pH of 5, 65°C, and starvation) in batch culture is presented in Fig. 4. Spore-like structures were observed at 24 h, 72 h, 96 h, and 120 h of cultivation. Differential stains with malachite green revealed a membranous and dark structure around the spore.

A. chiapaniensis PA2^T spore formation could be related to adapting to hostile environments during the batch culture (Gauvry et al., 2017). It has been suggested that bacterial spores can survive for millions of years (Leggett et al., 2012). Sporulation reveals the morphological plasticity of bacteria because cells can adjust from vegetative to dormant and vice versa. The sporulation mechanisms in bacteria result from a complex evolutive history (Paul et al., 2019).

Hostile growth conditions trigger A. chiapanensis PA2^T spore formation. Usually, spore formation begins when bacteria are in thermophilic and acidophilic conditions and deprived of carbon or nitrogen sources (Gauvry et al., 2017). In these conditions, some bacterial cells develop a regulatory net for communication and quorum sensing phenomena (Paul et al., 2019). Transcriptional factors, such as spoIIE, sigH, and ftsZ, induce the expression of over 200 genes to control the sporulation process. In particular, in the A. chiapanensis PA2^T genome, 35 genes associated with spore formation were found, such as SigH RNA polymerase sporulation specific sigma factor SigH, a transcription-repair coupling factor, spore germination protein GerD, spore maturation protein A, spore maturation protein B, sporulation protein YaaT, sporulation protein D (SpoIID), sporulation serine phosphatase for sigma-F (SpoIIE), sporulation protein AA, sporulation protein AB, sporulation protein AC, sporulation protein AD, sporulation AE, sporulation AF, sporulation AG and sporulation AH.

The assembly and layout of the structural compounds in A. chiapanensis PA2^T spores might be important adaptation mechanisms in hostile culture conditions. Spores of other species of Alicyclobacillus are hardy. For example, A. acidoterrestris spores remain stable in temperatures (95°C) and pH (3.0–7.0) changes (Bevilacqua et al., 2021). Noticeably, the ability to induce latent state-like resistant spores is an advantage developed during bacterial evolution.

The crater lake El Chichon is an environment similar to where primitive bacteria emerged. Before the eruption, the water temperature varied between 52 and 58°C, and the pH was extremely acidic (pH 5) with high concentrations of total dissolved solids (34 g/L), boron (433 mg/ L), chloride (24,030 ml/ L) and sulfate (3,550 m L / L (Casadevall et al., 1984). As a consequence of the eruption, probably an active magmatic system was formed over the crater lake, which influenced the lake's variation in temperature, pH, and chemical composition (Armienta and De la Cruz-Reyna, 1995). These fluctuations include change in the cationic profile from CaCl to Na-K, as well as decrease in chemical species derived from volcanic gases such as Cl^− 1, SO₂ ^− 4, and F^− 1 (Armienta and De la Cruz-Reyna, 1995), as well as increases of Na⁺ (of 9 to 904 mg/mL), Ca^+ 2 (of 16 to 355 mg/mL), Mg^+ 2 (of 6 to 87 mg/L), Si (of 52 to 288 mg/L) and B^+ 3 (of 0.3 to 24,3 mg/L) (Armienta et al., 2014). There was also a complex relationship between annual rainfall and lake chemistry (Rouwet et al., 2009). In addition, some factors such as annual rainfall, meteorological, geochemical and biological factors could influence the variations in the physicochemical composition and biogeochemical interactions of this lake (Cuoco et al., 2013; Armienta et al., 2014). Because of the above, the El Chichon volcano is probably the most dynamic lake on Earth (Rouwet et al., 2008).

4. ANALYSIS OF THE CELL WALL, CAPSULE AND DORMANCY GENES IN A. chiapanensis PA2^T COMPARED WITH OTHER Alicyclobacillus

A comparative genomic analysis of COG systems of A. chiapanensis PA2^T with the closest related species, A.sendaiensis NBRC1008666 (Tsuruoka et al., 2003), is shown in Fig. 5. The distribution patterns of both strains are similar, and these "extra" genes correspond to dormancy and sporulation (3), transport across the membrane (5), and the cell wall and capsule (6). Even though both isolates are genetically related, these particular genes’ presence and their phenotypic particularities reveal that they belong to different species.

The membrane transport system genes were divided into ABC transporters, cation transporters, uni-, sym-, and antiporters, protein and nucleoprotein secretion system type IV.

There is unique gene for transporting nickel and cobalt, as well as two singular proteins (twin-arginine translocation protein TatA and twin-arginine translocation protein TatC) in the subsystem´s protein translocation across cytoplasmic membranes. The coding genes for the cell wall and capsule proteins were divided into the subsystem´s polysaccharide deacetylases, murein hydrolases, UDP-N-acetylmuramate from fructose-6-phosphate biosynthesis and the recycling of peptidoglycan amino acids. Interestingly, the A. chiapanensis PA2^T genome also has proteins involved in teichoic and lipoteichoic acid biosynthesis diglucosyl diacylglycerol synthase LTA membrane anchor synthesis, poly glycerol-phosphate alpha-glucosyltransferase (EC 2.4.1.52), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (EC 2.7.7.60), teichoic acid translocation permease protein TagG, N-acetyl mannosaminyl transferase (EC 2.4.1.187) and teichoic acid export ATP-binding protein TagH (EC 3.6.3.40). These genes have been described for Gram-positive bacteria. Additionally, there are proteins related to dTDP-rhamnose synthesis glucose-1-phosphate thymidylyltransferase (EC 2.7.7.24), dTDP-glucose 4,6-dehydratase (EC 4.2.1.46), dTDP-4-dehydrorhamnose reductase (EC 1.1.1.133) and dTDP-RhA:a-D-GlcNAc diphosphoryl polyprenol, L-rhamnosyl transferase WbbL.

All these genes present might be related to the biogenesis of the pseudo-crystalline superficial layer (S layer) (Youssef et al., 2019). The S layer has been described for Gram-negative bacteria, some of them thermophilic and hyperthermophilic (Klingl, 2014). This outer layer conformation has been reported for other sporulating Gram-positive bacteria, such as Thermogemmatispora onikobensis ONI-1 T = JCM 16817 T = KCTC 19768 T and T. foliorum sp. ONI-5 T = JCM 16818 T = KCTC 19767 T. Both bacteria have few amino acids (glutamic acid, serine, histidine, alanine, and ornithine), some lipids (phosphatidylinositol, phosphatidylglycerol and a glycolipid), with rhamnose as the main carbohydrate in the S layer (Yabe et al., 2011). The S layer is also present in Gram-positive bacteria, like Aneurinibacillus thermoaerophilus DSM 10155. The glycoproteins of the S layer of A. thermoaerophilus DSM 10155 are glucan with glycoproteins composed of glycan chains and repeating units of l-rhamnose- and d-glycero-D-manno-heptose. Glycoproteins bind to the S layer polypeptide through core structures and new O-glycosidic bonds (Graninger et al., 2002). Therefore, rhamnose is a critical compound in the bacterial cell wall, particularly in the adaptation to thermal stress.

The comparison of the genes for dormancy and sporulation, membrane transport, the cell wall, and the capsule of A. chiapanensis PA2^T with A. sendaiensis NBRC100866 shows that the A. chiapanensis PA2^T has more genes and is more adaptable to extreme poly environments.

Recently, it was reported that the S layer seems to play a fundamental role in microbial development in polyextremophilic conditions (Youssef et al., 2019). Therefore, the study of the cell wall genes of this type of organism is of interest because the rhamnose metabolism genes could form an extracellular structure similar to the S layer, when this strain is grown in polyextremophilic conditions, and consequently could change from Gram-negative to Gram-positive.

5. A. chiapanensis PA2^T IS A NEW SPECIES

In a previous study, optimal growth conditions for A. chiapanensis PA2^T indicate a phenotype more adapted to thermoacidophilic conditions, compared with A. sendaiensis NBRC 100866. It was also published that the strain PA2 ^T presents an autotrophic, diazotrophic, and halotolerant metabolism not reported for A. sendaiensis NBRC 100866 (Ortiz-Cortés et al., 2021). Conversely, the analysis of 16S rRNA from Alicyclobacillus sp. PA2^T indicated approximately 94.73% similarity to A. sendaiensis NBRC 100866. This means that PA2^T does not belong to this species, confirmed by the TETRA (Z-score) value of 0.98. The values of ANIb (97.17%) and ANIm (96.65%) seem to indicate that A. chiapanensis PA2^T is phylogenetically grouped with the A. sendaiensis strain NBRC 100866; however, the DDH value (75.5%) indicates a new subspecies.

Description

Alicyclobacillus chiapanensis takes its name from the Mexican state of Chiapas, where the type strain was isolated. The A. chiapanensis PA2^T strain is characterized by being rod-shaped, endospore-forming, oligotrophic, diazotrophic, autotrophic and halotolerant. The cell stain is Gram variable. The colonial morphology in solid culture media is smooth and creamy with 2 mm of diameter. The temperature range for growth of A. chiapanensis PA2^T is 45°C- 65°C (optimum 65°C). The pH range for growth is 3–5 (optimum pH 5). Grows in the presence of 3% (w/v) NaCl in MB medium. Can use diverse carbohydrates (lactose, fructose, mannitol, sorbitol, car- boxymethyl cellulose and xylan) and triglycerides (vegetable oils of palm, safflower, canola, jatropha soybean and olive vegetable oils) as carbon sources. Moreover, this strain can use organic and inorganic nitrogen sources. A. chiapanensis PA2^T is positive for β-galactosidase, citrate as only carbon source and urease. Negative for tryptophan, indole test, acetoin, gelatinase and catalase activity (Ortiz-Cortés et al., 2021).

The genome size of the strain is 2,956,928 bp, with an overall G + C of 62,77% and has 3,256 genes. Most of the 2,952 genes have a putative function. Interestingly, 144 unique genes are encoded. In particular, this strain has specific genes associated with dormancy and sporulation (3), transport across the membrane (5), and the cell wall and capsule (6). The phenotypic particularities of this strain can be attributed to these.

The analysis of the complete genome of A. chiapanensis PA2^T shows that the constitution of PA2^T differs from A. sendaiensis NBRC 100866. A. chiapanensis PA2^T is 163,078 bp higher than NBRC 100866. Additionally, A. chiapanensis PA2^T genes could be associated with the Gram variable phenotype in the cell wall and the sporulation process (Fig. 6). Therefore, the cell wall-associated rhamnose subsystem and sporulation genes of A. chiapanensis PA2^T could indicate this organism's genetic diversification, which may lead to the formation of a new species.

ACKNOWLEDGEMENTS

The authors thank Jorge Roberto Velázquez Milanes for bioinformatics support.

FUNDING

Consejo Nacional de Ciencia y Tecnología (National Council of Science and Technology, México) for the scholarship granted 853058. Institute of Science, Technology and Innovation of Chiapas (ICTI-México) and National Technology of Mexico (TecNM)for financial support.

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Alicyclobacillus chiapanensis PA2T, a novel species with specific adaptation in a bacterial cell wall isolated from an active volcano

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Materials And Methods

Genome sequencing and assembly

Genome annotation

Phylogenetic analysis

Comparative genomic analyses

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2. Functional and physiological post-genome sequencing analysis

Phylogenomic analysis with molecular markers

Gram variable

Sporulation

Description

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