Gene multiplicity in glycolytic pathway genes.
Abstract
Streptomycetes exhibit genetic multiplicity, like many other microorganisms, and redundancy occurs in many of the genes involved in carbon metabolism. The enzymes of the glycolytic pathway presenting the greatest multiplicity were phosphofructokinase, fructose 1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase. The genes that encode citrate synthase and subunits of the succinate dehydrogenase complex are the ones that show the greatest multiplicity, while in the phosphoenolpyruvate-pyruvate-oxaloacetate node, only malic enzymes and pyruvate phosphate dikinase present two copies in some Streptomyces. The extra DNA from these multiple gene copies can be more than 50 kb, and the question arises whether all of these genes are transcribed and translated. As far as we know, there is few information about the transcription of these genes in any of this Streptomyces, nor if any of the activities that are encoded by a single gene could be limiting both for growth and for the formation of precursors of the antibiotics produced by these microorganisms. Therefore, it is important to study the transcription and translation of genes involved in carbon metabolism in antibiotic-producing Streptomyces growing on various sugars.
Keywords
- gene multiplicity
- carbon metabolism
- Streptomyces
- antibiotic
- glycolytic pathway
1. Introduction
Gene multiplicity or redundancy is a characteristic of microorganisms and means there are two or more genes coding for proteins that perform the same function. Inactivation of any of these genes does not affect or has little relevance to the biological phenotype [1]. Gene redundancy has been observed in all organisms, including prokaryotes and eukaryotes, and is particularly important for actinobacteria that produce metabolites of industrial interest [2]. The most significant property of
One little-studied area is the carbon metabolism in these organisms. Few studies have examined the presence of genes that participate in the glycolytic pathway, TCA cycle, or phosphoenolpyruvate-pyruvate-oxaloacetate (PEP-PYR-OXA) node. In general, microorganisms metabolize glucose through the glycolysis and hexose monophosphate pathways [6]. Many of the intermediates in these metabolic pathways are used to synthesize other essential bacterial compounds (amino acids, polysaccharides, nucleic acids, lipids, fatty acids, and antibiotics).
The EMP pathway consists of nine reactions, in which the final product is pyruvate. The first reaction consists of the isomerization of glucose 6-phosphate to fructose 6-phosphate catalyzed by phosphoglucose isomerase. Another phosphate with adenosine triphosphate (ATP) as the donor is incorporated into fructose 6-phosphate by phosphofructokinase. The next step is the cleavage of fructose 1,6-diphosphate by aldolase, generating dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, which can be interconverted by triose phosphate isomerase. Glyceraldehyde 3-phosphate is oxidized to 1,3-diphosphoglycerate by glyceraldehyde 3-phosphate dehydrogenase to generate NADH. The phosphoglycerate kinase-catalyzed reaction generates an ATP molecule, an example of substrate-level phosphorylation. The 3-phosphoglycerate is converted to 2-phosphoglycerate by phosphoglycerate mutase and is subsequently dehydrated by enolase. Phosphoenolpyruvate (PEP) is used to generate another molecule of ATP and PYR through a reaction catalyzed by pyruvate kinase. Then, four ATP molecules are generated, and two high-energy phosphate bonds are used; thus, the net gain is two ATPs per oxidized glucose molecule [6].
The TCA cycle is one of the most important metabolic pathways, not only as part of catabolism but also as an important intermediate for amino acid biosynthesis and synthesizing secondary metabolites. Generally, the citric acid cycle is the main oxidation pathway for carbon chains of carbohydrates, fatty acids, and many amino acids to CO2 and water. At each turn of the cycle, two molecules of CO2 are released. Most of the energy generated during oxidation is stored as NADH, FADH, or ATP (or GTP).
Because the intermediates of the TCA cycle are used as precursors in other pathways, they are replenished through anaplerotic reactions. Under normal conditions, the reactions that take intermediates in the cycle and those that replace them are kept in dynamic equilibrium; therefore, their concentrations remain constant. The most common anaplerotic reactions are those in which PYR or PEP are converted to Oxaloacetic acid (OXA) or malate. For example, this first reaction can be mediated by PEP carboxylase in some plants, yeasts, and bacteria or by the malic enzyme (ME), which is widely distributed in prokaryotes and eukaryotes [7].
2. Importance of Streptomyces
Streptomycetes are bacteria that produce the largest amount of commercially used antibiotics worldwide. To date, many
Many antibiotics have precursors that act as intermediates for different metabolic pathways. Gunnarsson et al. (2004) described how central carbon metabolism is linked to producing many different antibiotics [9]; however, little has been achieved to improve the synthesis of inermediates and influence the biosynthesis of antibiotics or other commercially important compounds.
Genome sequencing of important antibiotic-producing
3. Gene multiplicity in glycolytic pathway genes
There are many databases, such as KEGG, to identify how many genes code for the same activity. It is known that nine enzymes participate in the glycolytic pathway, as shown in Table 1, and it was found that three genes encode phosphofructokinase, which catalyzes the phosphorylation of fructose 6-phosphate in most antibiotic-producing
SALS | SAVERM | SCLF | SCO | SGR | SHJGH | SKA | SLAV | SNOUR | SPRI | SVEN | |
---|---|---|---|---|---|---|---|---|---|---|---|
Antibiotic | Salinomycin | Avermectin | Clavulanic acid | Actinorhodin, Undecylprodigiosine | Streptomycin | Rapamycin | Kanamycin | Streptothricin | Nystatine | streptogramin | Chloramphenicol |
Phosphoglucomutase | SAVERM_803 (556) | SCO7443 (546) | SGR_6728 (547) | SHJGH_1760 (546) | |||||||
Phosphoglucose isomerase | SLNWT_5962 (550) | SAVERM_1770 (549) SAVERM_6302 (550) | BB341_22345 (550) | SCO1942 (551) SCO6659 (550) | SGR_5578 (552) | SHJGH_3162 (550) SHJGH_7334 (550) | CP970_33250 (550) | SLAV_27865 (553) | SNOUR_30680 (558) | SPRI_5587 (552) | SVEN_1571 (556) |
Phosphofructokinase | SLNWT_1861 (341) SLNWT_5764 (345) SLNWT_7260 (341) | SAVERM_2822 (341) SAVERM_6083 (342) SAVERM_7123 (341) | BB341_07185 (341) BB341_21445 (342) BB341_25335 (341) | SCO1214 (341) SCO2119 (342) SCO5426 (341) | SGR_2110 (341) SGR_6306 (341) | SHJGH_2417 (341) SHJGH_3367 (342) SHJGH_6262 (341) | CP970_13335 (341) CP970_32070 (342) CP970_41110 (341) | SLAV_12675 (341) SLAV_26605 (343) SLAV_31335 (342) | SNOUR_05765 (341) SNOUR_14515 (341) SNOUR_29760 (342) | SPRI_2465 (341) SPRI_5380 (342) SPRI_6334 (341) | SVEN_0823 (341) SVEN_5078 (341) |
Aldolase | SLNWT_0350 (285) SLNWT_3143 (343) | SAVERM_1445 (301) SAVERM_4523 (340) | BB341_12300 (283) BB341_15265 (340) | SCO3649 (343) SCO5852 (282) | SGR_285 (289) SGR_3418 (343) | SHJGH_2271 (286) SHJGH_5149 (340) | CP970_20370 (343) CP970_37965 (278) | SLAV_00910 (278) SLAV_20325 (340) SLAV_32740 (281) SLAV_38480 (278) | SNOUR_09120 (293) SNOUR_22110 (340) | SPRI_0508 (299) SPRI_3596 (340) | SVEN_3414 (340) SVEN_7329 (278) |
Triose phosphate isomerase | SLNWT_5959 (259) | SAVERM_6298 (258) | BB341_22330 (258) | SCO0578 (259) SCO1945 (258) | SGR_5575 (258) | SHJGH_3166 (258) | CP970_33235 (261) | SLAV_27850 (262) | SNOUR_30660 (258) | SPRI_5584 (262) | SVEN_1574 (259) |
Glyceraldehyde 3P-dehydrogenase | SLNWT_5957 (335) | SAVERM_2990 (334) SAVERM_6296 (335) | BB341_01000 (481) BB341_22320 (335) | SCO1947 (336) SCO7040 (481) SCO7511 (332) | SGR_5573 (336) SGR_936 (481) | SHJGH_1334 (337) SHJGH_1590 (332) SHJGH_1800 (481) SHJGH_3168 (335) | CP970_08935 (481) CP970_33225 (336) | SLAV_27840 (334) SLAV_31635 (481) SLAV_35550 (332) | SNOUR_30650 (334) SNOUR_35775 (481) | SPRI_5582 (336) SPRI_6547 (481) | SVEN_0459 (461) SVEN_1576 336 SVEN_7344 (331) |
Phosphoglycerate kinase | SLNWT_5958 (403) | SAVERM_6297 (403) | BB341_22325 (403) | SCO1946 (403) | SGR_5574 (403) | SHJGH_3167 (403) | CP970_33230 (403) | SLAV_27845 (403) | SNOUR_30655 (403) | SPRI_5583 (403) | SVEN_1575 (403) |
Phosphoglycerate mutase | SLNWT_3230 (511) | SAVERM_3979 (253) | BB341_12505 (252) BB341_19285 (217) | SCO4209 (253) SCO6818 (511) | SGR_4005 (253) | SHJGH_4634 (253) | CP970_23555 (253) | SLAV_17715 (252) | SNOUR_07945 (218) SNOUR_18235 (253) | SPRI_4058 (253) | SVEN_3958 (252) |
Enolase | SLNWT_1793 (427) | SAVERM_3533 (428) | BB341_17295 (426) | SCO3096 (426) SCO7638 (434) | SGR_4439 (426) SGR_6721 (434) | SHJGH_4327 (431) | CP970_25900 (428) | SLAV_05265 (435) SLAV_22580 (426) | SNOUR_24895 (426) | SPRI_0700 (432) SPRI_4452 (428) | SVEN_2899 (428) SVEN_6040 (433) |
Pyruvate kinase | SLNWT_1865 (474) SLNWT_5886 (478) | SAVERM_2825 (476) SAVERM_6217 (478) | BB341_07200 (474) BB341_22070 (477) | SCO2014 (478) SCO5423 (476) | SGR_2113 (476) SGR_5516 (479) | SHJGH_3253 (478) SHJGH_6259 (457) | CP970_13350 (476) CP970_32715 (478) | SLAV_12690 (476) SLAV_27525 (475) | SNOUR_14530 (475) SNOUR_30335 (480) | SPRI_2468 (475) SPRI_5515 (474) | SVEN_1640 (475) SVEN_5075 (476) |
The conversion of fructose 6-phosphate to fructose 1,6-bisphosphate with the concomitant hydrolysis of adenosine triphosphate represents the first irreversible step specific to glycolysis. This reaction catalyzed by phosphofructokinase (PFK; EC 2.7.1.11) is subjected to tight control, thus rendering it a critical regulatory point of the glycolytic flux [10]. The genes that encode PFK present a high multiplicity, and in these 11 antibiotic-producing
The next reaction is performed using aldolase (EC 4.1.2.13), which catalyzes the conversion of fructose 1-6-diphosphate to glyceraldehyde 3-phosphate and dihydroxy-acetone phosphate. Most
Triosephosphate isomerase (EC 5.3.1.1) is an enzyme that converts dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Of all the antibiotic-producing
Another
In all antibiotic-producing
Phosphosphoglycerate mutase (EC 5.4.2.11) performs the internal transfer of a phosphate group from the C-3 carbon to the C-2 carbon, resulting in the isomerization of 3-phosphoglycerate to 2-phosphoglycerate. Only two genes are involved in this activity in
Enolase (EC 4.2.1.11), also known as phosphopyruvate hydratase, is a metalloenzyme responsible for converting 2-phosphoglycerate to PEP.
Pyruvate kinase (EC 2.7.1.40) is a key enzyme involved in the last step of glycolysis that catalyzes the transfer of a phosphate group from PEP to ADP, yielding one molecule of PYR and one molecule of ATP. There are two types: type I and type II, and both enzymes show positive cooperative effects concerning PEP. The type I enzyme is activated by fructose 1,6-bisphosphate (F1,6BP) and the type II by AMP [13]. According to the amino acid sequences of PYKF and PYKA enzymes from
4. TCA cycle
The TCA cycle is a central metabolic pathway of all aerobic organisms and synthesizes many important precursors and molecules [14] and eight reactions are performed for complete glucose oxidation.
The first reaction is citrate synthase (CS, EC 2.3.3.1), which catalyzes the irreversible conversion of OXA and acetyl-CoA into citrate. The proteins are classified into two types: type I and type II, and are encoded by four genes in eight of the selected antibiotic-producing
SALS | SAVERM | SCLF | SCO | SGR | SHJGH | SKA | SLAV | SNOUR | SPRI | SVEN | |
---|---|---|---|---|---|---|---|---|---|---|---|
Citrate synthase | SLNWT_1427 (439) SLNWT_1428 (422) SLNWT_4294 (369) SLNWT_5026 (434) | SAVERM_2427 (388) SAVERM_2428 (418) SAVERM_3859 (366) SAVERM_5330 (429) | BB341_05380 (375) BB341_05385 (432) BB341_11620 (363) BB341_18800 (429) | SCO2736 (429) SCO4388 (366) SCO5831 (421) SCO5832 (390) | SGR_1691 (381) SGR_1692 (432) SGR_3076 (367) SGR_4826 (432) | SHJGH_4003 (433) SHJGH_4518 (221) SHJGH_6697 (421) SHJGH_6698 (388) | CP970_11090 (395) CP970_11095 (417) Cp970_24400 (366) CP970_28100 (451) | SLAV_10725 1 (383) SLAV_10730 2 (416) SLAV_17155 I (368) SLAV_24055 I (433) | SNOUR_17670 (367) SNOUR_26625 (433) | SPRI_4216 (366) SPRI_4812 (429) | SVEN_2535 (433) SVEN_4205 (366) SVEN_5584 (377) |
Aconitase | SLNWT_0836 (904) | SAVERM_2258 (905) | BB341_04765 (908) | SCO5999 (904) | SGR_1506 (911) | SHJGH_6830 (905) | CP970_10270 (904) | SLAV_10035 (904) | SNOUR_12030 (904) | SPRI_1818 (905) | SVEN_5812 (910) |
Isocitrate dehydrogenase | SLNWT_6831 (739) | SAVERM_7214 (739) | BB341_23905 (739) | SCO7000(739) | SGR_1224 (740) | SHJGH_7521 (739) | CP970_35840 (739) | SLAV_07110 (739) | SNOUR_17135 (406) SNOUR_33110 (740) | SPRI_3067 (409) SPRI_6612 (739) | SVEN_0436 (741) |
2-oxoglutarate dehydrogenase E1 | SLNWT_2008 (1276) | SAVERM_2972 (1276) | BB341_08105 (1287) | SCO5281 (1272) | SGR_2226 (1267) | SHJGH_6150 (1170) | CP970_14080 (1294) | SLAV_13205 (1305) | SNOUR_15075 (1262) | SPRI_2599 (1272) | SVEN_4966 (1266) |
2-oxoglutarate dehydrogenase E2 (dihydrolipoamide succinyltransferase) | SLNWT_2008 | BB341_08105 | SCO1268 (372) SCO7123 (417) | SGR_2226 | SHJGH_6150 | SLAV_13205 | SNOUR_15075 | SPRI_2599 | SVEN_4966 | ||
2-oxoglutarate dehydrogenase E2 (dihydrolipoamide dehydrogenase) | SLNWT_5161 (467) | SAVERM_2154 (478) SAVERM_6024 (462) | BB341_21130 (462) | SCO2180 (486) | SGR_5330 (468) | SHJGH_6150 | CP970_31745 (462) CP970_41240 (467) | SLAV_26305 (462) SLAV_35195 (467) | SNOUR_29490 (462) SNOUR_37990 (466) | SPRI_0819 (466) SPRI_1688 (469) SPRI_5319 (462) | SVEN_1842 (501) SVEN_3731 (467) |
2-oxoglutarate ferredoxin oxidoreductase subunit beta | SLNWT_4515 (373) | SAVERM_4876 (359) | BB341_10730 (333) | SCO4594 (352) SCO6269 (350) | SGR_2930 (357) | SHJGH_5497 (353) | CP970_17695 (363) | SLAV_16235 (356) SLAV_39690 (467) | SNOUR_22995 (364) | SPRI_3245 (349) | SVEN_4303 (353) |
2-oxoglutarate/2-oxoacid ferredoxin oxidoreductase subunit alpha | SLNWT_4516 (644) | SAVERM_4877 (642) | BB341_10725 (597) BB341_25540 (597) | SCO4595 (645) SCO6270 (630) | SGR_2929 (654) | SHJGH_5498 (642) | CP970_17690 (643) | SLAV_16230 (649) SLAV_39695 (621) | SNOUR_23000 (650) | SPRI_3244 (648) | SVEN_4304 (653) |
Succiny CoA synthetase subunit β | SLNWT_4730 (392) | SAVERM_1818 (376) SAVERM_3452 (392) | BB341_10035 (391) | SCO4808 (394) SCO6585 (383) | SGR_2723 (393) | SHJGH_5672 (392) SHJGH_7288 (376) | CP970_05375 (374) CP970_16770 (392) | SLAV_15400 (392) | SNOUR_17190 (392) | SPRI_1230 (375) SPRI_3081 (393) | SVEN_4485 (424) |
Succiny CoA synthetase subunit α | SLNWT_4731 (305) | SAVERM_1817 (299) SAVERM_3451 (294) | BB341_10030 (294) | SCO4809 (294) SCO6586 (308) | SGR_2722 (294) | SHJGH_5673 (294) SHJGH_7289 (293) | CP970_05370 (290) CP970_16765 (294) | SLAV_15395 (295) | SNOUR_17185 (294) | SPRI_1229 (296) SPRI_3080 (294) | SVEN_4486 (294) |
Succinate dehydrogenase, iron-sulfur protein | SLNWT_4174 (248) SLNWT_4779 (244) | SAVERM_3182 (258) SAVERM_3398 (257) SAVERM_7309 (249) | BB341_01735 (248) BB341_08890 (255) BB341_09850 (252) | SCO0922 (248) SCO4855 (257) SCO5106 (259) | SGR_2690 (255) SGR_705 (248) | SHJGH_5739 (259) SHJGH_5963 (257) SHJGH_7644 (249) | CP970_03035 (248) CP970_15135 (256) CP970_16460 (255) | SLAV_06165 (246) SLAV_14090 (260) SLAV_15205 (252) | SNOUR_15960 (265) SNOUR_16955 (257) SNOUR_30130 (247) | SPRI_0753 (248) SPRI_2773 (256) SPRI_3031 (252) | SVEN_4531 (252) SVEN_4752 (257) SVEN_6837 (249) |
Succinate dehydrogenase, flavoprotein subunit | SLNWT_4173 (652) SLNWT_4780 (584) | SAVERM_3181 (667) SAVERM_3397 (584) SAVERM_7308 (649) | BB341_08885 (667) BB341_09845 (584) | SCO0923 (649) SCO4856 (584) SCO5107 (653) SCO7109 (576) | SGR_2689 (584) SGR_706 (649) | SHJGH_5740 (584) SHJGH_5964 (651) SHJGH_7643 (649) | CP970_03040 (652) CP970_15130 (656) CP970_16455 (584) | SLAV_06170 (650) SLAV_14085 (633) SLAV_15200 (584) | SNOUR_15955 (640) SNOUR_16950 (584) SNOUR_30135 (648) | SPRI_0754 (647) SPRI_2772 (647) SPRI_3030 (584) | SVEN_4532 (584) SVEN_4753 (636) SVEN_6836 (648) |
Succinate dehydrogenase hydrophobic membrane anchor protein | SLNWT_4782 (161) | SAVERM_3396 (160) | BB341_09840 (158) | SCO4857 (160) | SGR_2688 (160) | SHJGH_5741 (160) | CP970_16450 (163) | SLAV_15195 (160) | SNOUR_16945 (154) | SPRI_3029 (158) | SVEN_4533 (159) |
succinate dehydrogenase cytochrome β-556 subunit | SLNWT_4172 (223) SLNWT_4781 (110) | SAVERM_3395 (126) SAVERM_7307 (235) | BB341_01745 (207) BB341_09835 (144) | SCO0924 (243) SCO4858 (126) | SGR_707 (234) SGR_2687 (126) | SHJGH_5742 (110) SHJGH_7642 (223) | CP970_03045 (235) CP970_16445 (126) | SLAV_06175 (241) SLAV_15190 (126) | SNOUR_16940 (126) SNOUR_30140 (278) | SPRI_0755 (234) SPRI_3028 (126) | SVEN_4534 (126) SVEN_6835 (208) |
Fumarate hydratase Class I | SLNWT_2307 (554) | SAVERM_3218 (558) | BB341_08995 (562) | SCO5044 (558) | SGR_2481 (558) | SHJGH_5903 (534) | CP970_15380 (558) | SLAV_14315 (559) | SNOUR_16110 (558) | SPRI_2823 (555) | SVEN_4713 (556) |
Fumarate hydratase, class II | SLNWT_2312 (473) | SAVERM_3221 (467) | SCO5042 (461) | SGR_2491 (471) | SHJGH_5902 (461) | CP970_15390 (470) | SLAV_14340 (467) | SNOUR_16150 (464) | SVEN_4708 (469) | ||
Malate dehydrogenase | SLNWT_4746 (329) | SAVERM_3436 (329) | BB341_09975 (329) | SCO4827 (329) | SGR_2711 (329) | SHJGH_5712 (329) | CP970_16700 (329) | SLAV_15340 (329) | SNOUR_17130 (329) | SPRI_3065 (329) | SVEN_4498 (329) |
Aconitase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), the E1 component of 2-oxoglutarate dehydrogenase (EC 1.2.4.2), and malate dehydrogenase (EC 1.1.1.37) are encoded by unique genes in all
The 2-oxoglutarate dehydrogenase complex is a central enzyme in aerobic metabolism that catalyzes the oxidative decarboxylation of oxoglutarate, generating NADH [16]. 2-oxoglutarate dehydrogenase is composed of three subunits, E1 (EC 1.2.4.2), E2 (EC 2.3.1.61, dihydrolipoamide succinyltransferase), and E3 (EC 1.8.1.4; dihydrolipoamide dehydrogenase). As previously mentioned, there is a single gene coding for component E1 in all selected antibiotic-producing
Multiple alignments of the amino acid sequences of the E2 subunit of 2-oxoglutarate dehydrogenase showed close resemblance, with many highly conserved amino acids within the conserved domains, such as the YDHR region, which is part of the 2-oxoacid dehydrogenase acyltransferase catalytic domain. The proteins encoded by
There is an alternative way to perform the synthesis of 2-oxoglutarate via 2-oxoglutarate ferredoxin oxidoreductase (EC 1.2.7.11) formed by two subunits called a and b.
The next reaction is catalyzed by succinyl-CoA synthetase (EC 6.2.1.5), which is also composed of two protein subunits: α and β.
Succinate dehydrogenase (SDH; EC 1.3.5.1, EC 1.3.5.4), which catalyzes the oxidation of succinic acid to generate fumaric acid is a four-subunit multimeric enzyme (iron-sulfur protein, flavoprotein subunit, hydrophobic membrane anchor protein, and cytochrome b-556 subunit). The number of genes that code for each subunit varies, and even the same genome
The genes that code for iron-sulfur protein have great redundancy, finding three copies in all antibiotic-producing
It has been found that there are three different flavoprotein proteins, some with 649–652 amino acids with identities greater than 90%, another group of proteins with 584 amino acids and identities between 91 and 93%, and the last group with 633–667 residues and identities greater than 80%. The flavoproteins SCO7109 and SHJGH_5964 do not resemble each other or the proteins of the previous groups.
The hydrophobic membrane anchor proteins are smaller than the previous ones, around 17 kDa, and present identities between 77% and 82%, while there are two types of cytochrome b subunit, one of around 223–243 amino acids with lower identities than the previous ones, between 58% and 71%. The second type includes proteins of smaller size and identity between 74% and89%.
The hydration reaction of fumarate to generate malate is catalyzed by fumarate hydratase (EC 4.2.1.2; fumarase). There are two classes of proteins: fumarase classes I and II. Most antibiotic-producing
The final TCA cycle reaction is catalyzed by malate dehydrogenase, which catalyzes the reversible conversion of malate to OXA using NAD+ or NADP+ as the coenzyme [17], which is only encoded by a single gene in all microorganisms that produce different antibiotics. The multiple sequence alignment of this enzyme showed a high degree of conservation between them, distributed in two main clades from a common ancestor, which was then subdivided into eight subclades (Figure 5). The identity is higher than 90%.
5. PEP-PYR-OXA node
The PEP-PYR-OXA node is a major branch of central carbon metabolism and acts as a connection point between glycolysis, gluconeogenesis, and the TCA cycle [7]. A large variety of enzymes involved in the node have been reported, such as PEP carboxylase (EC 4.1.1.31), pyruvate carboxylase (EC 6.4.1.1), PEP carboxykinase (EC 4.1.1.32), malic enzymes (EC 1.1.1.38), pyruvate kinase (EC 2.7.1.40), and pyruvate phosphate dikinase (EC 2.7.9.1). These enzymes are indispensable for distributing PEP, PYR, and OXA in
This anaplerotic pathway does not present multiplicity in any of the genes that encode the enzymes participating in the PEP-PYR-OXA node, except for malic enzymes and pyruvate phosphate dikinase as shown in Table 3. PYR carboxylase is an enzyme that is present only in
SALS | SAVERM | SCLF | SCO | SGR | SHJGH | SKA | SLAV | SNOUR | SPRI | SVEN | |
---|---|---|---|---|---|---|---|---|---|---|---|
Pyruvate carboxylase | SLNWT_3899 (1124) | SCO0546 (1124) | SHJGH_7997 (1124) | SPRI_1970 (1124) | |||||||
Phosphoenol pyruvate carboxylase | SLNWT_4462 (912) | SAVERM_3566 (910) | BB341_17140 (909) | SCO3127 (911) | SGR_4379 (909) | SHJGH_4361 (910) | CP970_25710 (917) | SLAV_22415 (920) | SNOUR_24730 (921) | SPRI_4413 (909) | SVEN_2951 (909) |
NAD-Malic enzyme | SLNWT_2497 (476) | SAVERM_1514 (587) SAVERM_3870 (573) SAVERM_5126 (477) | BB341_17990 (476) | SCO2951 (471) | SGR_4581 (478) | SHJGH_4187 (471) SHJGH_4528 (570) | CP970_26965 (477) | SLAV_23225 (474) | SNOUR_25740 (477) | SPRI_4590 (477) | SVEN_2718 (476) |
NADP-Malic enzyme | SLNWT_2020 (407) | SAVERM_2981 (407) | BB341_08155 (412) | SCO5261 (409) | SGR_2236 (413) | SHJGH_6136 (407) | CP970_14130 (407) | SLAV_13290 (400) | SNOUR_15125 (409) | SPRI_2612 (403) | SVEN_4951 (403) |
Phosphoenol pyruvate carboxykinase | SLNWT_4964 (609) | SAVERM_3287 (607) | BB341_09370 (621) | SCO4979 (609) | SGR_2556 (608) | SHJGH_5842 (607) | CP970_15680 (623) | SLAV_14715 (613) | SNOUR_16390 (605) | SPRI_2904 (606) | SVEN_4658 (607) |
pyruvate phosphate dikinase | SLNWT_5381 (902) | SAVERM_5654 (916) | BB341_01330 (608) BB341_19830 (905) | SCO0208 (898) SCO2494 (909) | SGR_5048 (903) | SHJGH_3733 (906) SHJGH_8556 (895) | CP970_29700 (896) | SLAV_24965 (933) | SNOUR_27780 (911) | SPRI_5017 (903) | SVEN_2286 (934) |
PEP carboxylase, an enzyme that catalyzes the carboxylation of PEP to OXA, is encoded by a single gene and is present in all antibiotic-producing
PEP carboxykinase, an enzyme with the opposite action to the previous ones, decarboxylates OXA to form PEP. This enzyme could be considered gluconeogenic; however, it is also part of the PEP-PYR-OXA node for distributing the carbon flux between the different central pathways of metabolism [19]. Its molecular weight is approximately 67 kDa, some of which are a few amino acids long. In the same way as the previous enzymes,
MEs catalyzes the oxidative decarboxylation of l-malate to produce PYR and CO2, coupled with the reduction of NAD(P)+ cofactors (EC 1.1.1.38, EC 1.1.1.40). In general, NAD+- dependent MEs function to provide PYR for the TCA cycle, whereas NADP+- dependent MEs function to generate NADPH for anabolic reactions. Bacterial ME isoforms are comparatively understudied and collectively demonstrate greater structural and functional diversity (ranging from “minimal” 40 kDa subunits to much larger 85 kDa multidomain proteins). The greater bacterial ME complexity arises from the need for allosteric regulation owing to the non-compartmentalization of the bacterial cell [20].
The genes coding for MEs in these antibiotic-producing
Pyruvate phosphate dikinase (EC 2.7.11.32) converts PEP, inorganic pyrophosphate, and AMP into PYR, inorganic phosphate, and ATP. This protein is a gluconeogenic and anaplerotic enzyme and, in some bacteria, it plays other important roles. This protein is associated with virulence in
Finally, the genes that encode all enzymes of the glycolytic pathway, the TCA cycle, and the PEP-PYR-OXA node are distributed throughout the genome and represent up to 50 kb that might not be needed. Although many genes are found in the core, others are also found in the arms, and the question arises as to whether all genes are expressed and translated. For example, four genes that encode CS are transcribed to generate functional proteins? Viollier et al. (2001) reported that the deletion of the
As mentioned before, antibiotics are synthesized from precursors that are intermediates from carbon metabolism. By providing an overview of the gene multiplicity and determining which enzymes are encoded by a single gene, it will be possible to design strategies aimed at the sufficient biosynthesis of precursors for the metabolism of microorganisms to satisfy the demand for these same compounds to form antibiotics.
On the other hand, the growth of
6. Conclusions
Genetic multiplicity is present in all antibiotic-producing
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