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Communication

Insights into the Potential Role of Gordonia alkanivorans Strains in Biotechnologies †

by
Ekaterina Frantsuzova
1,*,
Alexander Bogun
1,
Lidiya Shishkina
2,
Anna Vetrova
1,
Inna Solyanikova
1,3 and
Yanina Delegan
1
1
Institute of Biochemistry and Physiology of Microorganisms, Federal Research Center “Pushchino Scientific Center for Biological Research of Russian Academy of Sciences” (FRC PSCBR RAS), 142290 Pushchino, Russia
2
State Research Center for Applied Microbiology and Biotechnology, 142279 Obolensk, Russia
3
Regional Microbiological Center, Belgorod State University, 308015 Belgorod, Russia
*
Author to whom correspondence should be addressed.
†
Participant of 2nd International Electronic Conference on Processes: Process Engineering—Current State and Future Trends (ECP2023), Online, 17–31 May 2023: https://doi.org/10.3390/ECP2023-14689.
Processes 2023, 11(11), 3184; https://doi.org/10.3390/pr11113184
Submission received: 20 October 2023 / Revised: 6 November 2023 / Accepted: 6 November 2023 / Published: 8 November 2023
(This article belongs to the Special Issue Selected Papers from the 2nd International Electronic Conference on Processes: Process Engineering—Current State and Future Trends (ECP2023))
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Abstract

:
Members of many species of the genus Gordonia are known for their significant metabolic potential, including the ability to utilize compounds of different structures. The aim of the work was to study the ability of nine G. alkanivorans strains to degrade persistent organic pollutants and to analyze the genomic peculiarities of these strains. The genomes of nine Gordonia alkanivorans strains were sequenced and assembled. The utilization of these strains as alkane and benzoate degraders in environmental biotechnologies, coupled with their capacity to potentially produce diverse secondary metabolites, holds promising prospects for both environmental and pharmaceutical applications.

1. Introduction

The genus Gordonia is an aerobic Gram-positive bacteria of the order Mycobacteriales. Currently, this genus comprises 52 validly published and correctly named species [1]. Gordonia species have been isolated from various sources such as soil, marine samples, wastewater, clinical specimens, human skin, and even from the bark of rubber trees [2,3,4,5,6,7,8,9]. It can be said that Gordonia strains are ubiquitous. The studied genus exhibits a broad geographical distribution, with occurrences reported in the USA [10,11], Puerto Rico [12], Colombia [13], Brazil [14], Argentina [15], Israel [16], India [8], Namibia [17], South Africa [18], Germany [19], Russia [20,21], China [22,23,24], Taiwan [25], Thailand [26], South Korea [27,28], and Japan [29]. This diverse geographic range underscores the genus’s ability to adapt to different climatic and ecological conditions.
Their extensive metabolic capabilities due to the remarkable plasticity of the genomes of this genus [30,31,32,33] make Gordonia strains promising for application in the field of environmental biotechnology. Furthermore, it is known that Gordonia strains can adapt to various environmental conditions, including high salt concentrations. For example, Gordonia iterans Co17 is capable of growing in a 12% salt concentration, while its ability to degrade alkanes remains intact within the range of C8 to C32 under a 10% NaCl concentration [2].
Members of many species of the genus Gordonia are known for their ability to utilize compounds of a different structure, including persistent organic pollutants. For instance, the degradation process of petroleum hydrocarbons has been observed in the strain Gordonia sihwaniensis lys 1–3. Furthermore, the authors have, for the first time, analyzed and visualized the pathway degradation process of n-hexadecane by this species [34]. Regarding aromatic hydrocarbons, they constitute a group of primary organic pollutants critical to the environment and public health due to their toxic, carcinogenic properties, and widespread prevalence. They include monocyclic (benzene, toluene) and polycyclic aromatic hydrocarbons (from naphthalene to pyrenes), as well as alkyl-substituted isomers. The literature frequently reports the ability of Gordonia to utilize substances such as anthracene, naphthalene, phenanthrene, pyrene, and benzene [35]. For instance, the G. iterans Co17 strain can degrade aromatic compounds such as naphthalene and anthracene by 55.3% and 63.2%, respectively [2]. The degradation of phenanthrene is a complex metabolic process. However, the ability of the Gordonia sp. SCSIO19801 strain to utilize phenanthrene (100 mg/L) as the sole carbon source at pH 8 and 28 °C has been studied. It was observed that after 7 days of the experiment, the SCSIO19801 strain had utilized over 80% of the phenanthrene through the salicylate metabolic pathway [36].
The strains Gordonia hongkongensis RL-LY01 [37] and Gordonia sp. GZ-YC7 [38] exhibit a strong potential for environmental bioremediation due to their ability to effectively degrade various phthalate esters or phthalic acid esters (PAEs). Hu et al. (2022) reported that Gordonia sp. GZ-YC7 efficiently removed more than 45% of di-(2-ethylhexyl) phthalate with an initial concentration of 500 mg/kg within 5 days, even when soil microbial activity is typically inhibited at lower di-(2-ethylhexyl) phthalate (DEHP) concentrations. For the strain Gordonia sp. GONU, the ability to hydrolyze high-molecular-weight phthalates has been observed. Specifically, DEHP was completely utilized within 24 h and di-n-octyl phthalate (DnOP) within 20 h. Dhar and colaborators (2023) have determined that the DEHP and DnOP degradation pathways are inducible. The information about the regulation of genes involved in the degradation of phthalate diesters forms the basis for further studies on the purification, characterization, and detection of PAEs [39]. Members of the genus Gordonia can also be considered as potential candidates for polyethylene biodegradation. For instance, the strain Gordonia polyisoprenivorans B251 demonstrated the ability to degrade polyethylene (PE), which was confirmed through a 30-day incubation with a PE film by detecting specific chemical changes and surface cracks [40].
This genus is of great importance in removing sulfur from fossil fuels and has been extensively covered by a number of authors [41], where the mechanisms and future prospects for the development of sustainable, environmentally friendly oil recycling technologies have been investigated.
The anabolic capabilities of strains within this genus are also recognized. For instance, several authors have noted the ability of Gordonia representatives to produce L-lysine, various polysaccharides, biosurfactants, carotenoids, and antimicrobial compounds [3,42,43,44,45,46,47,48]. Microbial surfactants are frequently employed in environmental remediation methods to address pollution. For example, the bioemulsans produced by Gordonia sp. BS29 are efficient in eliminating crude oil and PAHs from soil, while also providing a modest boost to the biodegradation of stubborn branched hydrocarbons; however, their most favorable outcomes are achieved when applied in the process of soil washing to remove hydrocarbons [49].
Microbial secondary metabolites, which are one of the main sources of bioactive natural products, are increasingly in demand for various biotechnological applications, with a primary focus on discovering new pharmaceuticals. Ma et al. (2021) isolated the strain Gordonia WA 4-31, capable of synthesizing secondary metabolites, from which the following compounds with antimicrobial activity were extracted: actinomycin D, actinomycin X2, mojavensin A, and cyclic (leucine-leucine) dipeptide. These compounds exhibited both anti-fungal and antiproliferative activities [50]. Additionally, the strain Gordonia sp. VO29-3, isolated from marine sediments, showed moderate antibacterial activity, inhibiting the growth of Staphylococcus aureus and Pseudomonas aeruginosa [51]. Liu and colaborators (2023) also noted the ability of the Gordonia terrae WA8-44 strain to synthesize three active compounds (collismycin A, actinomycin D and actinomycin X2), with the capability of Gordonia to synthesize collismycin A and its potential as an effective anti-filamentous fungi agent being reported for the first time. Based on their research, the authors present collismycin A from Gordonia as a promising candidate for the development of oral medications [52]. Furthermore, the strain Gordonia sp. 647W.R.1a.05 was found to possess the ability to synthesize neuroactive small-molecule natural products [53]. However, the genomes of Gordonia strains often contain BGCs that are not identified in databases with known metabolites [54]. This suggests that there is merit in further advancing research and development in this field.
The species Gordonia alkanivorans was introduced in 1999 due to the isolation of a type strain of this species, HKI 0136T, from tar- and phenol-contaminated soil [19]. Several recent studies have shown that representatives of this species can be used for oil pollution remediation, including those with a high sulfur content, as well as for cleaning natural systems contaminated with pesticides. Research is also being conducted to study the production of secondary metabolites, particularly carotenoids. For example, Silva et al. (2022) investigated the enhancement of carotenoid production using G. alkanivorans strain 1B through the optimization of cultivation conditions, such as the choice of the carbon source, the influence of light, and salt concentration. The study demonstrated that carotenoid production is stimulated by the presence of light and the glucose content in the medium [55].
For the strain Gordonia alkanivorans W33, it has been demonstrated that it can degrade 56.3% of the petroleum in contaminated soil within 45 days [56]. Yang and collaborators (2023) note that the efficient remediation of oil-contaminated soil was achieved by increasing the bacteria concentration through high-density fermentation and using vermiculite powder as a carrier, in addition to the inclusion of sophorolipids and rhamnolipids. It is known that crude oil may contain up to 10% sulfur, with the majority of it existing in an organic form, primarily as condensed thiophenes [57]. Previously, it was thought that thiophene catabolism in G. alkanivorans was only carried out using the dsz operon [58,59]. However, in previous studies, we have shown that there are G. alkanivorans strains that utilize thiophenes without dsz genes [60,61]. In one of the studies (2023), the ability of the strain Gordonia alkanivorans GH-1 to degrade beta-cypermethrin (β-CY), a pesticide whose breakdown products can have adverse effects on human health, was investigated. It was found that the strain not only degrades the substance itself but also its intermediate metabolites (dibutyl phthalate, benzoic acid, and phenol) [62].
The aim of this work is to study the ability of nine G. alkanivorans strains to degrade persistent organic pollutants and to analyze the genomic features of these strains. The results obtained will help to expand the existing knowledge of this species and its contribution to the biodegradation of aliphatic and aromatic compounds and to the biosynthesis of new natural products.

2. Materials and Methods

2.1. Bacterial Strains and Cultivation Conditions

We used 9 bacterial strains (Table 1) isolated from oil-contaminated soils and G. alkanivorans strain 135 [20,60] as a reference to investigate the catabolic properties. During previous studies with G. alkanivorans strains, we observed that representatives of this species exhibit significant biotechnological potential as degraders of pollutants of various chemical natures. Consequently, we became interested in determining whether there are differences in the genomic organization of strains representing the same species (G. alkanivorans) with similar catabolic capabilities.
The ability of the strains to utilize thiophenes as the sole sulfur source was tested using the method described by Delegan (2021) [60]. The growth of bacterial strains was visually assessed based on the turbidity of the medium. The ability of the strains to grow on even alkanes (C6–C20) and aromatic compounds (naphthalene, phenol, benzoate, catechol) was tested using a mineral medium of the following composition: K2HPO4—8.71 g/L; 5 M NH4Cl solution—1 mL/L; 0.1 M Na2SO4 solution—1 mL/L; 62 mM MgCl2 solution—1 mL/L; 1 mM CaCl2 solution—1 mL/L; 0.005 mM of (NH4)6Mo7O24 × 4 H2O solution; micronutrients—1 mL (micronutrient composition in g/l: ZnO—0.41 g; FeCl3 × 6 H2O—5.4 g; MnCl2 × 4 H2O—2 g; CuCl2 × 2 H2O—0.17 g; CoCl2 × 6 H2O—0.48 g; H3BO3—0.06 g), and pH 7.0. The medium is balanced in such a way that Gordonia strains, when grown on it, yield maximum biomass output. The influence of CaCl2 and MgCl2 on biomass growth was described by Yang (2023) [56]. Alkanes were added at 7.5 mL/L, naphthalene, phenol, and benzoate at 1 g/L, and catechol at 0.1 g/L.

2.2. Genome Sequencing and Analysis

The genomic DNA of the strains was isolated from biomass grown on LB [63] agar using a DNeasy Blood and Tissue Kit (QIAGEN, 69506) and processed following a standard protocol as per the manufacturer’s instructions. Whole-genome sequencing (WGS) was performed on a MGI platform (DNBSEQ-G400) using the DNBSEQ-G400RS High-throughput Sequencing Set (FCL PE150) (2 × 150 bp). A paired-end library was prepared with the MGIEasy Universal DNA Library Prep Set in accordance with the protocol. The information of the generated data is presented in Table 2.
The raw reads were filtered using Trimmomatic v. 0.39 [64] with the following parameters: LEADING:15 TRAILING:15 SLIDINGWINDOW:4:15 MINLEN:100; and assembled using SPAdes v. 3.15.4 [65]. Contigs shorter than 500 bp were removed.
To clarify species affiliations, the Average Nucleotide Identity (ANI) value with the type strain G. alkanivorans NBRC16433 (BACI00000000.1) was determined using the EzBioCloud ANI Calculator [66] with the OrthoANIu algorithm, and digital DNA–DNA hybridization (dDDH) was calculated using the Genome-to-Genome Distance Calculator (GGDC) [67]. Some authors present ANI as a more modern approach that correlates with DDH data, thereby eliminating the need for using both methods [68]. Nevertheless, there are instances where the ANI value surpasses the threshold, while the DDH value does not reach it [69]. Furthermore, although ANI offers a more precise assessment of genomic relatedness, the heterogeneity of DDH values can provide valuable insights in certain cases.
The initial annotation was conducted using Prokka [70] and RAST [71] to enhance accuracy in gene function determination while minimizing errors. Subsequently, when the genomes were submitted to the GenBank database, additional annotation was carried out using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) version 4.6 [72]. The functional annotation of the genome was carried out using KEGG [73] and BlastKOALA [74], which is suitable for annotating fully sequenced genomes. The identification and analysis of secondary metabolite biosynthesis gene clusters were carried out using antiSMASH 7.0 [75], which is advantageous for its extensive dataset and analytical tools. For pangenome analysis and unique genes search, we used OrthoVenn3 [76] due to its enhanced accuracy in identifying orthologous clusters and providing clear data visualization.

3. Results

3.1. Identifying the Strains

Based on the results of the whole genome sequencing data, several strains were reidentified (Table 3).
Thus, all the strains reliably belong to Gordonia alkanivorans. The difference in the DDH value between the studied strains and the type of strain G. alkanivorans NBRC 16433 indicates some heterogeneity of the species, but all the strains pass the species threshold by both ANI (>96%) and DDH (>70%) [77]. Both of these methods fall under the OGRI (overall genome related index) category, which is used to determine if a strain belongs to a known species by assessing the relatedness between the genome sequences of the strains and the type strain of a species. When the sequences of two genome sequences are fragmented and the fragments are compared to identify homologous regions, the percentage of nucleotide matches in such a region is referred to as the identity value, and the average identity value is calculated for all aligned fragments, resulting in ANI. DDH values using GGDC are determined through in silico techniques without the necessity of actual DNA hybridization experiments. Similar to ANI, genome sequences of two strains are compared after fragmentation into shorter segments. GGDC computes the intergenomic distances between the fragments of the two genomes and measures the degree of similarity between corresponding fragments in the two genomes. This serves as the basis for employing various mathematical models to generate the DDH hybridization value, offering an alternative to the traditional DDH determination. Heterogeneity of DDH values may indicate evolutionary divergence among different strains within the same species and is often an indicator of the species genomic plasticity, allowing it to adapt to changing environmental conditions.
The genomes of the G. alkanivorans strains used in this work have sizes ranging from 4.9 to 5.1 Mb, with the exception of strain 144 (Table S1). Despite the fact that this strain reliably belongs to the G. alkanivorans species, it has a smaller genome size and a higher number of contigs in the assembly compared to the other strains.

3.2. Physiological and Biochemical Characteristics of Strains

On agarized rich media (LB), all the strains form small round colonies of a pink-orange color. When grown on mineral media with alkanes as a carbon source or thiophenes as a sulfur source, lighter orange colonies are formed. The color change may indicate that when growing on mineral media with poorly accessible energy sources, the microorganisms use energy for basic metabolic processes and substrate utilization, but not for the biosynthesis of secondary metabolites (carotenoids) that give the cells their color.
Crude oil is a complex mixture of components, those being aliphatic and aromatic hydrocarbons and their sulfur-, nitrogen-, and oxygen-containing derivatives. We assumed that the strains isolated from areas contaminated with crude oil would be able to utilize compounds from each of these groups. All strains were able to utilize alkanes from C10 to C20 and benzoate; some strains are able to use dibenzothiophene (DBT) as the sole source of sulfur (Table 4).
Thus, the strains are capable of degrading a wide range of alkanes and some aromatic compounds, such as benzoate; however, none of the strains can utilize PAHs (naphthalene) or their metabolites (catechol).
Gene encoding enzymes of the complete pathway of benzoate degradation, including both subunits of benzoate 1,2-dioxygenase, which is part of the group of enzymes responsible for the initial transformation of persistent organic pollutants and other aromatic compounds, were also found in all strains. The presence of these genes may suggest adaptation to high levels of aromatic compound pollution [78]. For example, chlorinated benzoates are released into the environment through herbicide use or as metabolites and intermediates of other compounds [79]. Furthermore, benzoate is a metabolite of the transformation of polychlorinated biphenyls, which belong to a category of enduring organic contaminants that were extensively utilized in industrial applications and continue to endure in the soil [80]. Therefore, the studied strains can be used as part of a consortium for the remediation of these pollutants.

3.3. Osmoprotectant Metabolism

Bacteria use the accumulation of compatible solutes as a prevalent defense mechanism against the harmful effects of high osmolarity. These compounds, referred to as compatible solutes, can be acquired through uptake systems or biosynthesis. The accumulation of these substances can be achieved both through biosynthesis and by absorption from the surrounding environment. For all examined strains, complete pathways for trehalose biosynthesis have been identified. However, one of the key reasons why uptake systems are crucial to cellular responses to osmotic stress is that they allow the energy-efficient scavenging of osmostress protectants from the environment. Most of the currently known transporters of compatible soluble substances can be assigned to one of two groups of transport systems: ABC transporters and secondary transporters.
Among the binding-protein-dependent ATP-binding cassette (ABC) systems, opu genes were found. Secondary transporters: members of the major facilitator superfamily (MFS)—ProP; members of the betaine-choline-carnitine transport (BCCT) system—BetT; and members of the sodium-solute-symporter (SSS).
The most effective participants in protecting the microorganism from osmotic stress are glycine betaine and ectoine. One of the pathways for betaine biosynthesis occurs through the oxidation of choline in two stages, catalyzed by choline dehydrogenase (BetA) [EC 1.1.99.1] and betaine aldehyde dehydrogenase (BetB) [EC 1.2.1.8]. The functionality of this pathway depends on the presence of the BetT transporter. It should be noted that betA was not found, but codA was identified (Table 5), which encodes choline oxidase [EC 1.1.3.17], an enzyme with a similar function. CodA catalyzes the two-step oxidative process, converting choline into glycine-betaine with betaine aldehyde as an intermediate (Figure 1).
It can use either choline or the intermediate betaine-aldehyde as a substrate [81]. Breisch (2021) demonstrated through mutagenesis that in the absence of betA, choline oxidation to glycine betaine does not occur. Additionally, the betA deletion mutant shows no impairment in utilizing glycine betaine as a compatible solute, as evidenced by the restoration of growth when glycine betaine is added to the medium [82]. Therefore, it can be assumed that in the studied strains, CodA may perform the function of BetA.
The biosynthesis of ectoine begins with aspartate through a series of sequential reactions catalyzed by diaminobutyrate-2-oxoglutarate transaminase EctB [EC:2.6.1.76], L-2,4-diaminobutyric acid acetyltransferase EctA [EC:2.3.1.178] и L-ectoine synthase EctC [EC:4.2.1.108] (Figure 2). The ectABC gene cluster involved in the biosynthesis of ectoine has been found in all strains. Additionally, a stand-alone gene ectD, encoding ectoine hydroxylase [EC:1.14.11.55], which is involved in the conversion of ectoine to hydroxyectoine, has been identified.
Thus, all the studied strains contain the genes of the most important solutes that can protect cell structures and metabolic processes at high salt concentrations. The ability of the strains to grow in a medium with an increased NaCl concentration was experimentally evaluated (Table 6).
Almost all strains are capable of growing at salt concentrations of 1–3%. Strain 96 exhibited the highest resilience to saline environmental conditions, with the capacity to grow in a 10% NaCl solution.

3.4. Pangenome Analysis

The pangenome of the studied strains was analyzed in conjunction with the type strain Gordonia alkanivorans NBRC 16433 (Figure S1). It is represented by 4779 clusters, of which 3744 (78.34%) make up the core. As a result of the GO annotation of the core clusters, the highest number of proteins with known functions were assigned to «trehalose O-mycolyltransferase activity» and «antibiotic biosynthetic process».
Strain 144 contains the highest number of unique protein sequences (97) that are not found in the genomes of the other studied strains. These sequences include proteins with peptidase and hydrolase activity, Acyl-CoA dehydrogenases, as well as sequences involved in targeting and inserting nascent membrane proteins into the cytoplasmic membrane. Additionally, a viral sequence (Caudovirales, Siphoviridae) involved in peptidoglycan catabolic processes was detected. Furthermore, for strain 144, it was found to have the highest number of singletons (genes for which no orthologs could be found in any of the other species) (Table 7). Nevertheless, a substantial portion of unique sequences currently remains unannotated, potentially indicating gaps in existing databases concerning the genetic organization among prokaryotes.
Based on the results of genome analysis and individual unique genes, the strains can be divided into two groups within the species. The representatives of the first group (strains 129, 132, 133, 144) are characterized by unique genes of tyrocidine and gramicidin biosynthesis [83]. The representatives of the second group have a greater catabolic potential: the genomes of the strains contained (1) operons for the biosynthesis of steroid compounds, (2) additional copies of genes involved in dibenzothiophene catabolism, and (3) genes for the catabolism of aromatic compounds, cytochrome P450-pinF2 and phenol hydroxylase P5.
In the first group of 4404 clusters, 4022 are part of the core, constituting 91.32% (Figure S2, Tables S2 and S3). The pangenome of the second group consists of 4369 clusters, with 3905 (89.37%) belonging to the core. Overall, it is evident that the second group is more heterogeneous (Figure S3). In the second group, among the core clusters (Figure S4, Tables S4 and S5), the highest number of proteins with known functions in a single cluster are associated with «antibiotic biosynthetic process», «vancomycin biosynthetic process», and «cholesterol catabolic process». The «steroid biosynthetic» and «steroid metabolic process» GO categories are represented by several clusters each (7 and 5, respectively). In general, both groups of representatives exhibit a large number of diverse clusters annotated as «biosynthetic process».

3.5. Features of the Genetic Organization of G. alkanivorans

3.5.1. Hydrocarbon Degradation Ability

Approximately 45% of the CDS (coding DNA sequences) in all strains were annotated and categorized into various functional groups. Genes responsible for the degradation of different organic pollutants were identified. In the «Degradation of aromatic compounds» category 21 orthologous functional groups (KO) were found for all strains, except for strains 144 and 134, which had 19 and 23 groups, respectively. Additionally, all strains, except for 96 and 142, were found to contain genes for the degradation of dibenzofuran (DF) and dibenzo-p-dioxin (DD), both of which are toxic compounds and are formed as by-products during the synthesis of pesticides and herbicides [84]. In the subcategory «Benzoate degradation» within the «Xenobiotics biodegradation and metabolism» category, 19 KOs were identified for all strains, except for strain 134, which had 20 groups. The ability to use benzoate as a source of carbon and energy is a characteristic feature of representatives of this genus [61,85].
Integral membrane non-heme iron monooxygenases AlkB were absent in the genomes of all examined strains. However, soluble cytochrome P450s, specifically the CYP153 genes, were found in multiple copies (Table 8). Furthermore, each strain contained one copy of alkane hydroxylase for long-chain length alkanes (>C18), known as LadA, which is a thermophilic soluble monooxygenase from Geobacillus [86]. Gene clusters prmDCBA, encoding multiple components of propane monooxygenase, were also discovered in all strains.
Thus, all the G. alkanivorans strains studied lack alkB genes in their genomes; therefore, we assume that the ability to utilize alkanes in these strains is controlled by the CYP153 and LadA genetic systems. The absence of the alkB genetic system has also been noted in our previous studies [60].
The CYP153 hydroxylases have at least a 99% identity between the strains, and they have one amino acid substitution each: one hydroxylase has A/T variants at position 239, the other hydroxylase has A/S variants at position 7, and they are in the first and second strain groups, respectively (Figure 3).

3.5.2. Plasmids

Of the nine strains of G. alkanivorans, two (strains 142 and 152) have plasmid elements. The plasmid of strain 142 (p142) is 67,219 bp in length; the plasmid of strain 152 (p152) is 44,937 bp. We assume that they are circular, since Gordonia is generally not characterized by the maintenance of large linear plasmids as, for example, in rhodococci.
In total, 98% of the entire length of plasmid p142 is a 99% repeat of plasmids pCP89 (CP094666.1) of the Gordonia amicalis G2 strain (percent identity (PI) 99.70%) and pCP86 (CP096597.1) of the G. amicalis 6-1 strain (PI 99.98%) (Figure 4). It is interesting to note this relationship between plasmids whose hosts are strains of different species.
The plasmid of strain 152 has a relationship with the plasmid pG135. The ANI value between the plasmids p142 and p152 is 80.00%.

3.5.3. Biosynthesis of Secondary Metabolites

The examined strains were found to have a significant number of biosynthetic gene clusters (BGCs), the majority of which showed no homology with gene clusters encoding the production of known natural products. Types of clusters that are common to all nine strains include:
  • Terpene (SF2575 biosynthetic gene cluster: polyketide type II; carotenoid);
  • Streptozotocin;
  • Type I polyketide synthase;
  • NRPS (non-ribosomal peptide synthetase);
  • Non-alpha poly-amino acids (ε-Poly-L-lysine);
  • Ectoine;
  • Redox cofactor;
  • NRPS-independent, IucA/IucC-like siderophores;
  • Arylpolyene (primycin like most similar known cluster is found in all strains except for 134, 142, and 144, which do not exhibit similarity to this group).
The colibrimycin biosynthetic gene cluster (present in all strains except 144), nonribosomal peptide metallophores, namely madurastatin (except 96, 144 and 152), depsibosamycin (present only in strains 132, 133 and 144), cadaside (present in 134 and 152), speibonoxamine (present in 96 and 152) were also detected. Clusters identified only in individual strains are presented in Table 9.
The production of carotenoids by Gordonia is widely known and finds applications in various industries, including food, medical, cosmetic, and pharmaceutical [55]. However, many other products, such as streptozotocin [87] or colibrimycin [88], are primarily documented for other genera, particularly Streptomyces. It is noted that ulbactins F and G from Brevibacillus sp exhibit inhibitory effects on tumor cell migration within the submicromolar to micromolar range [89]. Echoside A and echoside B have demonstrated significant potential as antiviral agents. The authors propose that these compounds could be further explored as potential antiviral agents against SARS-CoV-2 [90]. Additionally, the discovery of cahuitamycins provides a critical foundation for future research endeavors aimed at developing drugs to prevent biofilm formation [91]. Of particular interest is the complete similarity of the icosalide A biosynthetic gene cluster from Burkholderia gladioli in strain 152. Icosalide is a lipocyclopeptide antibiotic, which originally was isolated from a fungus of the Aureobasidium species. It has shown activity against entomopathogenic bacteria [92,93]. There is also information of icosalides, which shows certain inhibitory activity against the active stage of Mycobacterium tuberculosis and some cancer cell lines [94]. Therefore, considering the fact that actinomycete genomes often contain «silent» genes [95], members of the genus Gordonia may have significant potential for producing various novel natural products.

4. Discussion

There are many areas with a high salt content, including oil production sites, which are subject to hydrocarbon contamination. In such situations, an effective solution is the use of microorganisms capable of tolerating high salt concentrations. The studied strains show the ability to degrade a variety of aliphatic hydrocarbons and benzoate, while being tolerant to salty conditions. These features make them potential candidates for cleanup and remediation of contaminated areas. In general, we can say that the strains of G. alkanivorans species are similar in terms of their physiological properties and degradative potential. Nevertheless, representatives of this species do not have metabolic pathways for the degradation of naphthalene. Thus, there is still no strain of G. alkanivorans capable of utilizing PAHs (naphthalene) or their metabolites (catechol) known at this time. However, considering the presence of a greater number of catabolic genes and operons in the representatives of the subgroups (strains 96, 134, 142, 12, 152), we can hypothesize that these strains hold promise in the removal of steroid compounds. Our results, together with data from other studies [96], suggest that all currently sequenced Gordonia alkanivorans strains do not have a AlkB genetic system for the utilization of alkanes. Nevertheless, their biodegradation potential of these pollutants is not inferior to representatives of species containing CYP153. Furthermore, for some strains lacking the dsz gene cluster, growth on DBT is observed, which correlates with our previous research on this genus [60,61].
Furthermore, most of the examined strains exhibit the ability to synthesize various secondary metabolites, such as terpenes, saccharides, polyketides, ribosomal, and nonribosomal peptides. The capacity to produce diverse secondary metabolites is of significant importance in the food, feed, and fuel industries. Seeing that the 100% genetic cluster similarity of strain 152 with the known icosalide biosynthesis cluster, this strain may be considered for therapeutic and pharmaceutical purposes after further research. However, it is important to note that under conventional culture conditions, the majority of bioactive metabolites encoded in actinomycete genomes remain inactive. Therefore, to further study the production of new compounds, it is necessary to optimize cultivation conditions.
Moreover, the identified genes for degradation of pollutants, genes for the formation of osmoprotectors and synthesis of secondary metabolites can be used for mutagenesis to create more productive organisms. For example, Goel and collaborators (2011) successfully transferred the bacterial gene coda to create a transgenic plant [97]. Moreover, our earlier data (not published) show that the genetic material of Gordonia is easily transferred to closely related genera such as Rhodococcus.

5. Conclusions

The genomes of nine strains of Gordonia alkanivorans isolated from oil-contaminated soils were sequenced and assembled. Expanding the database of sequenced genomes may be useful for more accurate identification and development of omics studies. The genomes are about 5 Mb in size. Some of the strains contain plasmids, but the functions of these plasmids are currently not fully understood. It is interesting to note that Gordonia plasmids do not appear to be species-specific. The function of plasmid p142 in strain 142 is currently unclear, but the fact that copies of the plasmid have been observed in members of another species (G. amicalis) may indicate the importance of this plasmid for the vital activity of Gordonia. We plan to obtain a plasmid-free eliminant of strain 142 in the future in order to obtain a better understanding of the functions of this plasmid. At this point, we can assume that plasmids such as p142 are required by strains for metal transport and resistance.
As we found out, none of the G. alkanivorans strains studied in this work are capable of utilizing aromatic compounds. In this regard, we assume that in the future, there will be the creation of modified strains carrying gene clusters for PAH catabolism on their basis. Such strains will have greater potential for use in biotechnology.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pr11113184/s1. Figure S1: A diagram showing the results of overlapping clusters of orthologous genes in strains (overlapping clusters containing proteins from different strains, sorted in descending order); Figure S2: GO annotation core clusters for the representatives of the first group (strains 129, 144, 132, 133); Figure S3: The representatives of the first group (a,c); of the second group (b,d): (a), (b)—Venn diagram depicting pangenome of two group strains; (c), (d)—UpSet table representing the number of orthologous clusters in each strains and the number of unique and shared homologous gene clusters; Figure S4: GO annotation core clusters for the representatives of the second group (96, 134, 142, 12, 152); Table S1: Assembly metrics of the strains; Table S2: Biological process list for the representatives of the first group; Table S3: Molecular function list for the representatives of the first group; Table S4: Biological process list for the representatives of the second group; Table S5: Molecular function list for the representatives of the second group.

Author Contributions

Conceptualization, Y.D. and A.V.; methodology, E.F., A.B. and I.S.; software, Y.D.; validation, A.B., Y.D. and I.S.; formal analysis, A.V.; investigation, E.F., L.S. and A.V.; data curation, Y.D.; writing—original draft preparation, E.F. and Y.D.; writing—review and editing, A.B. and A.V.; visualization, E.F.; supervision, Y.D.; project administration, Y.D.; funding acquisition, Y.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Russian Science Foundation, grant number 22-74-10082.

Data Availability Statement

The whole-genome sequences of the strains can be found under BioProject number PRJNA955828, BioSamples SAMN34194808-SAMN34194816.

Conflicts of Interest

The authors declare no conflict of interest.

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Processes 11 03184 g001
Figure 1. Betaine biosynthesis pathway.
Figure 1. Betaine biosynthesis pathway.
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Figure 2. Ectoine biosynthesis pathway.
Figure 2. Ectoine biosynthesis pathway.
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Figure 3. The CYP153 amino acid substitutions position (pink markers).
Figure 3. The CYP153 amino acid substitutions position (pink markers).
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Figure 4. Mauve alignment demonstrating relatedness of the plasmids p142 (strain G. alkanivorans 142), pCP86 (G. amicalis 6-1), and pCP89 (G. amicalis G2). Vertical bars mark boundaries between elements. Colored blocks show regions of a genome aligned with parts of another genome.
Figure 4. Mauve alignment demonstrating relatedness of the plasmids p142 (strain G. alkanivorans 142), pCP86 (G. amicalis 6-1), and pCP89 (G. amicalis G2). Vertical bars mark boundaries between elements. Colored blocks show regions of a genome aligned with parts of another genome.
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Table 1. Information about the strains used in the study.
Table 1. Information about the strains used in the study.
StrainCollection NumberIsolation SourcePreviously Identified As
96IEGM 96crude oil-contaminated soil, UkraineG. rubripertincta
129IEGM 129crude oil-contaminated soil, Ivano-Frankovsk, UkraineG. rubripertincta
132IEGM 132crude oil-contaminated soil, Ivano-Frankovsk, UkraineG. rubripertincta
133IEGM 133crude oil-contaminated soil, Ivano-Frankovsk, UkraineG. rubripertincta
134IEGM 134crude oil-contaminated soil, oilfield, Lvov, UkraineG. rubripertincta
142IEGM 142crude oil-contaminated soil, UkraineG. rubripertincta
144IEGM 144crude oil-contaminated soil, Lvov, UkraineG. terrae
12-oil-polluted soil, Moscow, RussiaG. alkanivorans
152-oil-polluted soil, Moscow, RussiaG. alkanivorans
Table 2. Number of sequencing data before and after filtration.
Table 2. Number of sequencing data before and after filtration.
StrainRead Pairs before
Filtration		Read Pairs after
Filtration		Read Pairs after
Filtration (%)
962,962,8132,835,52195.70
1293,147,9533,003,84895.42
1324,354,9674,162,08895.57
1335,192,4735,019,73796.67
1345,815,5655,558,10795.57
1423,330,9183,171,60895.22
1445,382,8815,150,56195.68
129,985,9638,692,67387.05
1526,985,2864,928,24770.55
Table 3. Species identification of the strains.
Table 3. Species identification of the strains.
StrainANI Value with the Type Strain of G. rubripertincta, %DDH Value with the Type Strain of G. rubripertincta, %ANI Value with the Type Strain of G. alkanivorans, %DDH Value with the Type Strain of G. alkanivorans, %Taxonomic Position of the Strain
9692.5876.2098.4589.70G. alkanivorans
12992.4576.0098.4288.60G. alkanivorans
13292.4076.3098.4288.60G. alkanivorans
13392.4076.1098.3788.50G. alkanivorans
13492.2576.8098.2387.60G. alkanivorans
14292.5476.3098.2391.00G. alkanivorans
14492.4266.8098.6378.10G. alkanivorans
1292.3876.2098.4090.10G. alkanivorans
15292.4876.6098.3488.00G. alkanivorans
Table 4. Substrate specificity profile of strains.
Table 4. Substrate specificity profile of strains.
StrainAlkanes C10–C16Alkanes C18–C20BenzoatePhenolNaphthaleneCatecholDBT
96++++---±
129+++----
132+++---±
133+++----
134+++++----
142+++----
144+++----
12++++---±
152++++----
135+++++---+
++ very good growth, + good growth, ± weak growth, - no growth.
Table 5. The number of copies of genes in certain osmoprotective systems.
Table 5. The number of copies of genes in certain osmoprotective systems.
StrainbetA/codAbetBbetTopuAopuBDopuC
96141122
129-51132
132131122
133131122
134131122
142141122
144131122
12141122
152131122
Table 6. The ability to grow in high salt concentrations.
Table 6. The ability to grow in high salt concentrations.
StrainConcentration of NaCl
1%3%7%10%
96++++
129+±±-
132++--
133++±±
134++--
142++--
144+±±-
12++--
152++±-
Table 7. Number of genes without orthologs.
Table 7. Number of genes without orthologs.
StrainProteinsClustersSingletons
964446431861
1294380425222
132452144041
133452043994
1344587431260
1424530432963
14453904506315
124480452225
1524421418388
NBRC 1643344454155109
Table 8. The number of copies of the CYP153 gene.
Table 8. The number of copies of the CYP153 gene.
StrainCYP153
962
1291
1323
1333
1342
1422
1443
122
1522
Table 9. Similarity with known clusters.
Table 9. Similarity with known clusters.
StrainMost Similar Known ClusterSimilarity
96sarpeptin25%
129ulbactin
echoside		28%
11%
144cahuitamycin12%
12leucomycin3%
152icosalide100%
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Frantsuzova, E.; Bogun, A.; Shishkina, L.; Vetrova, A.; Solyanikova, I.; Delegan, Y. Insights into the Potential Role of Gordonia alkanivorans Strains in Biotechnologies. Processes 2023, 11, 3184. https://doi.org/10.3390/pr11113184

AMA Style

Frantsuzova E, Bogun A, Shishkina L, Vetrova A, Solyanikova I, Delegan Y. Insights into the Potential Role of Gordonia alkanivorans Strains in Biotechnologies. Processes. 2023; 11(11):3184. https://doi.org/10.3390/pr11113184

Chicago/Turabian Style

Frantsuzova, Ekaterina, Alexander Bogun, Lidiya Shishkina, Anna Vetrova, Inna Solyanikova, and Yanina Delegan. 2023. "Insights into the Potential Role of Gordonia alkanivorans Strains in Biotechnologies" Processes 11, no. 11: 3184. https://doi.org/10.3390/pr11113184

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