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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 2  |  Page : 138-143

Mycolicibacterium smegmatis possesses operational agmatinase but contains no detectable polyamines


1 Research Center of Biotechnology of the Russian Academy of Sciences, Bach Institute of Biochemistry, Moscow, Russia
2 Perm State University, Biology Faculty; Perm Federal Research Center of the Ural Branch of the Russian Academy of Sciences, Institute of Ecology and Genetics of Microorganisms, Perm, Russia
3 Perm Federal Research Center of the Ural Branch of the Russian Academy of Sciences, Institute of Ecology and Genetics of Microorganisms, Perm, Russia
4 Shemyakin and Ovchinnikov, Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Department of Genomics and Postgenomic Technologies, Moscow, Russia
5 Federal Research and Clinical Centre of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia

Date of Web Publication29-May-2020

Correspondence Address:
Mikhail Shumkov
Leninsky prospect 33, build 3, Moscow 119071
Russia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_48_20

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  Abstract 


Background: Polyamines are widespread intracellular molecules able to influence antibiotic susceptibility, but almost nothing is known on their occurrence and physiological role in mycobacteria. Methods: here, we analyzed transcriptomic, proteomic and biochemical data and obtained the first evidence for the post-transcriptional expression of some genes attributed to polyamine metabolism and polyamine transport in Mycolicibacterium smegmatis (basionym Mycobacterium smegmatis). Results: in our experiments, exponentially growing cells demonstrated transcription of 21 polyamine-associated genes and possessed 7 enzymes of polyamine metabolism and 2 polyamine transport proteins. Conclusion: Mycolicibacterium smegmatis putrescine synthesizing enzyme agmatinase SpeB was originally shown to catalyze agmatine conversion to putrescine in vitro. Nevertheless, we have not found any polyamines in mycobacterial cells.

Keywords: Agmatine, mycobacteria, Mycolicibacterium smegmatis, polyamines, putrescine


How to cite this article:
Zamakhaev M, Tsyganov I, Nesterova L, Akhova A, Grigorov A, Bespyatykh J, Azhikina T, Tkachenko A, Shumkov M. Mycolicibacterium smegmatis possesses operational agmatinase but contains no detectable polyamines. Int J Mycobacteriol 2020;9:138-43

How to cite this URL:
Zamakhaev M, Tsyganov I, Nesterova L, Akhova A, Grigorov A, Bespyatykh J, Azhikina T, Tkachenko A, Shumkov M. Mycolicibacterium smegmatis possesses operational agmatinase but contains no detectable polyamines. Int J Mycobacteriol [serial online] 2020 [cited 2020 Jul 5];9:138-43. Available from: http://www.ijmyco.org/text.asp?2020/9/2/138/285232




  Introduction Top


Polyamines are aliphatic hydrocarbons possessing two or more amino groups. The most widespread biogenic polyamines include putrescine (1,4-diaminobutane), cadaverine (1,5-diaminopentane), spermidine (N-(3-Aminopropyl)-1,4-diaminobutane), and spermine (N, N′-Bis (3-aminopropyl)-1,4-diaminobutane). The latter is usually a component of eukaryotic organisms and is rarely found in eubacteria. The other three compounds, on the contrary, represent common microbial molecules and are able to perform a spectrum of various physiological functions in bacterial cells.[1]

Thus far, it is known that polyamines can modulate transcription[2] and protein stability,[3] influence porin permeability,[4] change membrane fluidity, and above all, regulate translation through mRNA secondary structure stabilization.[5] The described variety of functions is a direct consequence of the presence of amino groups in polyamine molecules. Under physiological conditions, these groups are protonated and thus positively charged. As a result, they are able to interact with negatively charged cell components, primarily nucleic acids and phospholipids.

One of the most challenging problems of modern medicine is tuberculosis treatment. Mycobacterium tuberculosis (MTB), the disease causative agent, infects 9 million people annually, resulting in nearly 2 million deaths per year.[6] Since polyamines can significantly change bacterial antibiotic susceptibility,[7] it is worth studying their metabolism in mycobacterial cells with the aim to prevent the development of increased tolerance and even the resistance of mycobacteria to antibacterial drugs.

The experiments carried out earlier with exogenous polyamine supplementation have shown that these molecules exert a positive influence on the activity of mycobacterial RNA polymerase.[8],[9] Just like in case of Gram-negative bacteria, they also reduce porin-dependent transport of antibacterial drugs and heavy metals into mycobacterial cells.[10],[11] Moreover, antimycobacterial antibiotic ethambutol resembles spermine by its structure and has been considered to act through polyamine metabolism inhibition.[12],[13] However, hypothetical ethambutol influence on spermidine synthesis entirely contradicts the data on the distribution of polyamines in microorganisms, which featured Mycolicibacterium smegmatis (basionym M. smegmatis) cells to contain very low (if not zero) polyamine concentrations.[14]

Thus, it is still the question if mycobacteria can use polyamines in their physiological processes. The ability of mycobacteria to synthesize and transform polyamines is also not established yet. Here, we analyze transcriptomic, proteomic, and biochemical data to judge if mycobacteria possess operational polyamine transport systems or polyamine metabolism enzymes making these bacteria principally able to contain endogenous polyamines and if there are any polyamines in M. smegmatis cells in fact.


  Methods Top


Cultivation conditions

Single colonies of M. smegmatis MC2 155 strain, grown on either nutrient broth (NB, Himedia, India) or Middlebrook 7H10 (Difco, France) solid medium, were initially taken to obtain the starter bacterial cultures. The cultures were grown at 37°C with agitation (200 rpm) for 20 h in the liquid NB or Middlebrook 7H9 broth, respectively, supplemented with 0.05% Tween 80, were then inoculated into fresh medium (same composition) and further incubated aerobically at 37°C in an orbital thermoshaker in 250 ml flasks containing 50 ml of medium. The cultures' optical density (OD) was measured at 600 nm with the ultraviolet-1280 spectrophotometer (Shimadzu, Japan).

Isolation of RNA, Illumina sequencing

RNA was isolated from three independent M. smegmatis cultures after 30 h of cultivation in NB medium (OD600 = 1.8). Bacterial cultures were rapidly cooled on ice, centrifuged, and total RNA was isolated by phenol-chloroform extraction and cell disruption with BeadBeater (BioSpec Products, USA) as previously described.[15] After isolation, RNA was treated with Turbo DNase (Life Technologies, USA) to remove the traces of genomic DNA. RNA preparations were depleted of 16S and 23S rRNA using the MICROBExpress Bacterial mRNA Enrichment kit (Life Technologies, USA). cDNA for Illumina sequencing was prepared according to the directional mRNA-seq sample preparation guide (Part # 15018460 Rev. A) as previously described.[16] This procedure preserves strand specificity by the ligation of a single-stranded 3′ RNA adapter and 5′ DNA adapter. Sequencing was performed by running 77 cycles on HiSeq 2000 sequencer (Illumina, USA).

Processing of RNA sequencing data

The reads were aligned to M. smegmatis reference sequence MC2 155 (GenBank accession number NC_008596.1) with Bowtie 2[17] setting parameter local. The transcriptional profiles for the forward and reverse strands of the genome representing the counts of overlapping reads for every nucleotide and mapping statistics were generated using HTSeq-count package built into the custom python script. Gene expression was represented in reads per kilobase per million reads measure.

Proteomic studies

Biomass of M. smegmatis MC2 155 strain was collected after 30 h of cultivation in NB medium (OD600 = 1.8) in triplicate. Protein extraction and proteolytic ingel digestion with trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega, USA) were performed as described previously.[18],[19] Liquid chromatography-tandem mass spectrometry (LC-MS)/MS analysis was carried out on a TripleTOF 5600 + mass spectrometer with an iron source of NanoSpray III (AB Sciex, Canada) connected to a nano-high-performance liquid chromatography (HPLC) system of NanoLC Ultra 2D + (Eksigent, Singapore). Raw data (.wiff files) were converted to Mascot Generic Format (.mgf files and peak lists) using the command-line program, AB SCIEX MS Data Converter version 1.3 (AB SCIEX, Framingham, MA, USA). Mascot Server, version 2.5.1 (Matrix Science Ltd., UK) was used for the identification against M. smegmatis MC2 155 sequence database downloaded from the RefSeq database (RefSeq: NC_008596.1).

Polyamine assays

Both free and bound with cellular structures polyamine pools were measured. For the free intracellular polyamine analysis, 1 ml of bacterial culture was pelleted by centrifugation, washed twice with saline, resuspended in 0.4 N HClO4 and disrupted by sonication with CPX 130 Ultrasonic processor (Cole-Parmer, USA). The crude cell extract was neutralized with saturated Na2 CO3, and then derivatized by dansyl chloride (Sigma, Switzerland) treatment and analyzed with thin-layer chromatography (TLC)[20] or HPLC[21] methods as described earlier. For the total (both bound and free) polyamine pool detection, M. smegmatis culture was also centrifuged and washed with saline. The pellet was hydrolyzed in 6M HCl at 110°C for 12 h. After extraction, the supernatants were separated by centrifugation and neutralized with Na2 CO3, derivatized with dansyl chloride and analyzed by the same TLC[20] or HPLC[21] methods. When performing sample hydrolysis and derivatization procedures, standard solution with known polyamine concentrations (0.2 mM each, prepared out of 0.4 N HClO4-dissolved hydrochloride polyamine salts) was also processed and used as a reference. The assay used is based on the earlier studies[22],[23] and is established to be well applied onto both Gram-negative bacteria and eukaryotic cells.

Determination of polyamine-synthesizing enzymes activity

Bacterial culture obtained through cultivation in NB medium was pelleted for 10 min at 4500 g and 0°C, washed twice with ice-cold saline and disrupted by sonication with CP × 130 ultrasonic processor (Cole-Parmer, USA). Cell debris was separated by centrifugation at 16000 g and 0°C for 20 min; the crude cell extract was transferred into enzyme incubation mixture containing 100 mM citrate-phosphate buffer (pH 7.5), 0.04 mM pyridoxal phosphate, 1 mM dithiothreitol and 4 mM MgSO4 extra (in the case of arginine decarboxylase activity measurement) to reach 100 mcg of total protein in 0.5 ml final volume. The enzymatic reaction was initiated by substrate injection (10 mM agmatine, ornithine, or arginine) followed by incubation at 37°C for 240 min. Afterward, it was stopped by supplementation with HClO4 to the final concentration of 0.4 M. The expected reaction end products (putrescine and agmatine) were measured as described in polyamine assays section. Specific enzyme activity was calculated after the polyamine production in nmol/mg of total protein/minute units.


  Results Top


Mycolicibacterium smegmatis transcriptomic profile presumes active polyamine metabolism in mycobacteria

Transcriptomic analysis of active M. smegmatis cells revealed mRNAs of genes annotated in M. smegmatis genome (GenBank accession number NC_008596.1) as potentially connected with polyamine metabolism [Table 1]. The obtained information allowed the reconstructive prediction of possible polyamine metabolism pathways in M. smegmatis.
Table 1: Identified products of polyamine metabolism and transport genes

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Specifically, we found the potential ability of the mycobacterial cells to derive putrescine from arginine. The biosynthetic pathway includes arginine decarboxylase SpeA and agmatine ureohydrolase (agmatinase) SpeB [Figure 1]. In the case of enterobacteria, the pathway is minor as they synthesize putrescine through ornithine decarboxylation with SpeC enzyme primarily.[23],[24] However, no speC homologs were annotated in M. smegmatis genome to date.
Figure 1: Presumable Mycobacterium smegmatis polyamine metabolism

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Among polyamine degradation pathways in enterobacteria, there is a chemical reaction cascade with GabDTP proteins engaged. It is known as γ-aminobutyric acid (GABA) pathway, which begins with putrescine transformation into γ- aminobutyraldehyde (putrescine aminotransferase [PatA]-dependent reaction). The latter is then transformed into GABA (the responsibility of γ-aminobutyraldehyde dehydrogenase), which is, in turn, converted into succinate semialdehyde (GabT enzyme) and succinate (GabD enzyme) entering tricarboxylic acid cycle [Figure 1].

We found mRNAs of almost all the genes of this pathway in M. smegmatis cell extracts, except for the first one PatA [Table 1]. Such expression of presumable putrescine-degrading genes made the functioning of this biochemical way highly plausible.

Apart from the polyamine synthesis and degradation genes, we found mRNAs of polyamine-transporting proteins [Table 1], which suggested the existence of active polyamine transport in mycobacterial cells.

Thus, in the exponentially growing M. smegmatis cells, there are a number of transcriptionally active genes annotated to encode polyamine metabolism and transport proteins.

Proteomic profiling supports the potential presence of polyamines in Mycolicibacterium smegmatis cells

On the protein level in the extracts of active mycobacterial cells, polypeptides of all the metabolic pathways deduced transcriptomically were found. We identified both putrescine biosynthetic (SpeB, agmatinase) and putrescine degrading (4-aminobutyrate aminotransferase, GabT, and succinate semialdehyde dehydrogenase, GabD) enzymes. In addition, polyamine transport system proteins were detected. We found a putrescine importer (MSMEG_0446) and ATP-binding protein of spermidine/putrescine ABC transporter (MSMEG_3281).

Hence, the products of presumable polyamine metabolism and transport genes in M. smegmatis cells could be found both on mRNA and protein levels, though we identified just some polypeptides of those transcriptomically proposed.

Biochemical studies reveal no polyamines in Mycolicibacterium smegmatis cells

In order to establish if there really are any polyamines in mycobacterial cells, polyamine accumulation under cultivation in rich (NB) and minimal (Middlebrook 7H9) media was measured first [Supplementary Figure 1]. Neither free nor bound endogenous polyamines (putrescine, cadaverine, spermidine, or spermine) was found. The result was reproduced both in exponential and stationary phases and was the same with different assay methods used (TLC or HPLC). Moreover, the study on polyamine content in the culture medium revealed none as well. Thus, no detectable polyamine production was observed in M. smegmatis cells under standard cultivation conditions [Supplementary Figure 2].



Next-stage experiments were performed with exogenous polyamines added. Middlebrook 7H9 medium was supplemented with 1, 2, or 5 mM putrescine, cadaverine, spermidine, or spermine. These concentrations were close to those endogenous in enterobacteria, and under cultivation in Luria-Bertani (LB) medium was known to provide a significant accumulation of polyamines in Escherichia coli, for example, when supplemented with 10 mM putrescine (our data), E. coli cells accumulated the compound in a concentration of 130.52±9.8 nmol/mg of dry weight comparing to 17.42 ± 5.71 nmol/mg of dry weight concentration in the control culture (data are represented in mean ± SE format). In M. smegmatis batch cultures under the conditions studied, we observed no established polyamine accumulation in the cytoplasm that proclaims there was no polyamine influx.

The final examination of polyamine content was performed in cultures supplemented with polyamine precursors. In enterobacteria, such an approach resulted in a certain accumulation of the target compounds.[7] However, in M. smegmatis, neither lysine nor ornithine and arginine led to polyamine concentration growth [Supplementary Figure 3]. Agmatine addition did not influence intracellular polyamine content also. Hence, one might conclude there are either no operational polyamine-producing enzymes in M. smegmatis cells, or the transport of both polyamines and their precursors occurs at a dramatically low level.



For the final check of the polyamine-synthesizing enzymes functionality and the exclusion of transport limitations influence, the polyamine production in crude cell extracts supplemented with either amino acids or agmatine was measured in vitro. Both arginine and ornithine decarboxylase activities were not detected, though agmatine ureohydrolase activity was found to reach up to 18.3 ± 2.67 nmol Pt/mg protein/minute [Table 2]. It should be noted that the agmatine ureohydrolase activity was not the same in cells harvested under different cultivation conditions supposing other polyamine metabolic enzymes might have needed special requirements for the emergence of their activity as well.
Table 2: Polyamine synthesizing enzymes activity in M. smegmatis crude cell extracts

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Thus, biochemical data revealed unexpectedly almost no polyamine synthesizing and transport activity in M. smegmatis cells. The only operational polyamine-related protein found was agmatinase (speB gene product). The enzyme was able to realize its activity and perform putrescine production from agmatine in vitro but did not demonstrate its functionality in vivo under conditions studied.


  Discussion Top


It is known that very low (if not zero) polyamine concentrations could be found in mycobacteria under standard conditions.[14] Nevertheless, polyamine metabolism and transport genes were annotated in almost every mycobacteria species studied.[25] They are absent in Mycobacterium leprae and represented in a restricted number in MTB; however, they are highly abundant in Mycobacterium phlei. M. smegmatis genome holds an intermediate position with respect to the number of polyamine genes. In general, it seems the abundance of these genes in the genome is dependent on mycobacterium way of life: whether it is a free-living environmental organism or mostly parasitic one. The fewer polyamine genes could be found in the particular genome; the less is the probability for the organism to live in the environment independently. From here, it could be also assumed polyamines are necessary growth factors that some mycobacteria could synthesize themselves and others take from the environment.

The recent paper demonstrated M. smegmatis polyamine- associated genes to be transcriptionally active.[26] The spectrum of transcripts identified in our work substantiates the earlier observation, although we performed the experiments in LB but not in 7H9 Middlebrook medium.

As it was stated above, just the transcripts of arginine decarboxylase and agmatine ureohydrolase genes were found when speaking on the polyamine synthesis system [Table 1]. This indicates agmatinase pathway is the only possible process for polyamine production in M. smegmatis cells. Unfortunately, just agmatinase enzyme was detected on the protein level, which demonstrated the pathway is incomplete, and polyamines are unlikely to be synthesized this way.

Nonetheless, we have identified mRNAs of almost all the genes of GABA polyamine degradation pathway in M. smegmatis cell extracts [Table 1]. The expression of genes potentially involved in putrescine degradation processes could have made the functioning of GABA shunt remarkably probable. However, this pathway is not unique for polyamine metabolism, so our observation can be interpreted in different ways because of the employment of ”branched” Krebs cycle in MTB and M. smegmatis cells[27] in which α- ketoglutarate may escape from direct transformation into succinate. Instead, it is processed by glutamate dehydrogenase (MSMEG_5442 encoded enzyme) with the resultant glutamate transformation into GABA in glutamate decarboxylase-dependent (GadB) reaction and into succinate thereafter (GabTD enzymes). Moreover, the metabolic pathway deduced transcriptomically is unlikely to be functional in M. smegmatis, since neither mRNA nor the protein of the key PatA enzyme has been identified.

Polyamine transport system does not seem to be operational as well, whereas just two corresponding proteins have been identified [Table 1], and no transport into the cytoplasm has been observed after culture medium supplementation with polyamines or their precursors.

It should be noted, on the protein level, nothing has been known on the expression of polyamine-related genes in mycobacteria to date. We for the first time demonstrated here some polyamine-attributed proteins to be really synthesized in M. smegmatis cells [Table 1]. However, the majority of M. smegmatis genes (GenBank accession number NC_008596.1) was identified using matching to a known protein family; therefore, those genes could have been easily misannotated. Hence, polyamine-associated functions of those proteins should have been further cross checked.

Through biochemical assays, we originally proved that there was an enzyme-converting agmatine to putrescine in M. smegmatis. The reaction is considered to be catalyzed by agmatinase SpeB. Other polyamine synthesizing processes were not shown to take place, just like no corresponding proteins were detected.

To sum up, we found no detectable polyamine levels in M. smegmatis cells under standard cultivation conditions, although our transcriptomic and proteomic data, so as the results of other research groups[28] made the synthesis of these molecules in mycobacteria highly plausible.

The possible reasons for the polyamines not been recovered under the conditions studied are the following. First, the restricted transport of polyamine precursors, as far as, we found no putrescin accumulation after M. smegmatis culture supplementation with agmatine, though agmatinase activity was unambiguously detected in crude cell extracts [Table 2]. The second reason is conditional accumulation, i.e., enzyme induction might require special conditions for its development. In particular, this could shed light on the increased agmatinase activity under the reduced oxygen supply [Table 2]. This seemed to be the real cause, however, no polyamine gene transcription was detected under hypoxia,[29] and no polyamines themselves were identified under oxidative, acid, or starvation conditions in M. smegmatis[30] (as well as in MTB[31]) cells. Finally, there could be an intense polyamine turnover, which means polyamines were transformed into different compounds rapidly. The idea is supported by metabolomics data, which uncovered some polyamine derivatives (N-carbamoylputrescine, N-acetylputrescine, and glutathionylspermine) in M. smegmatis cells,[28] while revealed no polyamines per se.[28],[29] The detected in vitro transformation of agmatine to putrescine substantiates the hypothesis indirectly as well; however, the absence of polyamine derivative-synthesizing enzymes contradicts the assumption.

Besides the physiological reasons, the lack of certain genes (or their corresponding transcripts and proteins) in some of the pathways deduced, or the misannotation of genes purported to be involved in polyamine synthesis could be the cause for the failed polyamine detection as it is stated above. In the first case, it means the biochemical pathways of polyamine synthesis comprise just part of the genes needed for their successful functioning, or the existing genes are not transcribed or translated. The second assumption suggests the genes may be functionally attributed wrong. In other words, a gene might be referred to as arginine decarboxylase, for example, while encoding a different enzyme in fact. According to our omics [Table 1] and enzyme activity [Table 2] experiments, both ”nonphysiological” explanations could be valid.

Hence, no polyamine accumulation was found in our experiments, though there was an enzyme in M. smegmatis cells which was able to convert agmatine to putrescine in vitro. It looks like M. smegmatis possesses no operational polyamine transport systems or complete polyamine metabolism pathways. This minimizes the probability for M. smegmatis to have endogenous polyamines and makes it hard to imagine that polyamines have any physiological significance in mycobacteria. Otherwise, their functions are not connected with the regulation of cell wall permeability, which was proposed based on the experiments with exogenous polyamines added,[10],[11] or RNA polymerase activity[8],[9] or antibiotic susceptibility decrease obviously.[7] They just could be associated with the synthesis of polyamine derivatives that could have been able to help overcome oxidative stress (glutathionylspermine) or participate in amino acid synthesis (N-acetylputrescine). The hypothesis on the correlation between the opportunity for mycobacteria to live in the environment independently and the abundance of polyamine genes in the genome does not work under the studied conditions as well.

Acknowledgments

The authors are very grateful to Prof. A. S. Kaprelyants for his valuable comments on the manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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