Adaptation of Lacticaseibacillus rhamnosus CM MSU 529 to Aerobic Growth: A Proteomic Approach

The study describes the effect of aerobic conditions on the proteome of homofermentative lactic acid bacterium Lacticaseibacillus rhamnosus CM MSU 529 grown in a batch culture. Aeration caused the induction of the biosynthesis of 43 proteins, while 14 proteins were downregulated as detected by label-free LC-MS/MS. Upregulated proteins are involved in oxygen consumption (Pox, LctO, pyridoxine 5’-phosphate oxidase), xylulose 5-phosphate conversion (Xfp), pyruvate metabolism (PdhD, AlsS, AlsD), reactive oxygen species (ROS) elimination (Tpx, TrxA, Npr), general stress response (GroES, PfpI, universal stress protein, YqiG), antioxidant production (CysK, DkgA), pyrimidine metabolism (CarA, CarB, PyrE, PyrC, PyrB, PyrR), oligopeptide transport and metabolism (OppA, PepO), and maturation and stability of ribosomal subunits (RbfA, VicX). Downregulated proteins participate in ROS defense (AhpC), citrate and pyruvate consumption (CitE, PflB), oxaloacetate production (AvtA), arginine synthesis (ArgG), amino acid transport (GlnQ), and deoxynucleoside biosynthesis (RtpR). The data obtained shed light on mechanisms providing O2-tolerance and adaptation to aerobic conditions in strain CM MSU 529. The biosynthesis of 39 from 57 differentially abundant proteins was shown to be O2-sensitive in lactic acid bacteria for the first time. To our knowledge this is the first study on the impact of aerobic cultivation on the proteome of L. rhamnosus.


Introduction
Lactic acid bacteria (LAB) are widely used as starter and probiotic cultures in the food industry. Probiotics as functional food ingredients benefit human and animal health. Probiotics have great potential in clinical applications as well, treating allergies, lactose intolerance, dental caries, hypercholesterolemia, gastroenterititis, and inflammatory bowel disease [1]. Among different species of lactobacilli considered as probiotics, Lacticaseibacillus rhamnosus is intensively studied. L. rhamnosus strains are able to survive in a gastrointestinal tract due to adhesion to the epithelial cells of intestines followed by biofilm formation, which subsequently competitively suppresses the attachment and growth of pathogenic microorganisms [2]. L. rhamnosus J10-L and L. rhamnosus GG (LGG) revealed a strong antibacterial activity towards Shigella sonnei in vitro due to the production of the lactic acid [3]. L. rhamnosus strains prevented the gastrointestinal tract colonization by Candida species [4,5]. L. rhamnosus L34 suppressed the production of interleukin-8, a mediator of an inflammatory response, by colonic epithelial cells induced by Clostridium difficile causing hospital-acquired diarrhea and colitis [6]. LGG could modulate M1 macrophage polarization in vitro to prevent Salmonella Typhimurium infection, supporting its role in Fisher Scientific GmbH, Dreieich, Germany) [23]. The cysteines were reduced by 5 mM Tris(2-caboxyethyl)phosphine and incubated for 10 min at 90 • C. The samples were briefly cooled on ice and carbamidomethylated in the presence of 10 mM iodoacetamide for 30 min at 25 • C in the dark.

Protein Digest
Aliquots containing 50 µg of dissolved protein were taken from each sample and diluted with 100 mM ABC to a final concentration of 0.42% DOC. The obtained samples were digested with 1 µg of MS approved trypsin (Serva Electrophoresis GmbH, Heidelberg, Germany) at 30 • C for 12 h. The DOC was then precipitated by acidification with trifluoroacetic acid (TFA) and subsequent centrifugation (15,000 rpm, 20 min, 4 • C).

Data Acquisition
MS spectra were acquired at a resolution of 60,000 over a mass-to-charge range of 350-1650 m/z using a maximum injection time of 25 ms. MS scans were followed by tandem MS/MS scans. To increase the efficiency of MS/MS scans, precursor ions that were unassigned, singly charged or with charge states > 6 were excluded. The dynamic exclusion duration was set to 14 s. Peptide precursors were isolated (isolation window 1.5 m/z), fragmented using HCD collision energy of 27%, and analyzed at a resolution of 15,000.

Peptide Identification, Quantification, and Statistical Analysis
MS data were searched against an in-house protein database using Sequest embedded into Proteome Discoverer 1.4 software (Thermo Fisher Scientific GmbH). The resulted reference was the L. rhamnosus LRHMDP2 proteome (Uniprot proteome ID: UP000009330). The obtained msf-files were combined into a summary table using Scaffold (v. 5.0.1). The identification parameters for peptides were as follows: trypsin digestion with maximum of 2 missed cleavage sites; peptide length of 7-144 amino acids; maximum delta Cn − 0.05; the precursor mass tolerance and fragment mass tolerance were ±10 ppm and ±0.02 Da, respectively; spectrum matching requirements were set to 1 for mass of b and y ions; carbamidomethyl cysteine (+57.021 Da) was set as a static modification; dynamic modifications with oxidation (+15.995 Da) of the Met residues and deamidation (+0.984 Da) of the Asn and Gln residues. Protein and peptide FDR were set to 0.01 (1%) and the minimum peptide number was 1 for the identified proteins.
Furthermore, MS data evaluation and statistical analysis were performed using MaxQuant (v. 2.0.1.0) and SaveQuant (v. 2.3.5). Proteins of score > 75, of fold change by 1.5 times (log 2 fold change ≥ 0.6 or ≤ −0.6) with p and q values ≤ 0.05 were considered to be differentially expressed in response to aeration versus static growth. Protein fold changes were estimated using mean intensities measured in samples of the three independent cultivations (cv ≤ 0.3).

Cell Dry Weight Determination
The 10-15 mL cultures were harvested by centrifugation (4000 rpm, 10 min, 4 • C) and washed 3 times with deionized water. Mass of centrifuge tubes, empty or with biomass pellets, was adjusted to a constant weight at 90 • C. Cell dry weights (or pH values of culture supernatants) are presented as "mean ± standard deviation" calculated using Microsoft Excel (Microsoft, Redmond, WA, USA) on the basis of the data obtained from the three independent cultivations.

Protein-Protein Interaction Analysis
Protein-protein interactions were analyzed using a STRING (v. 11.5) web resource (https://string-db.org/, accessed on 29 November 2022); 53 of 57 differentially abundant proteins of strain CM MSU 529 were identified in LGG proteome to obtain a full STRING network. The following basic settings were applied: The edges indicate both functional and physical protein associations with a confidence cutoff of 0.7; active interaction sources included textmining, experiments, databases, co-expression, neighborhood, gene fusion, and co-occurrence; only associated proteins are shown. Protein-protein network was clustered using MCL inflation parameter of 1.1.

Effect of Aeration on the Proteome of Strain CM MSU 529
The intensive aeration significantly affected the proteomic profile of L. rhamnosus CM MSU 529 at the late exponential growth phase. The level of 43 proteins was higher, whereas the level of 14 proteins was lower during aerobic growth compared to static growth (Tables 1 and S1-S3). The upregulated proteins belonged to the following COG categories: cell cycle control, cell division, chromosome partitioning (D); post-translational modification, protein turnover, and chaperones (O); signal transduction mechanisms (T); defense mechanisms (V); translation, ribosomal structure and biogenesis (J); transcription (K); energy production and conversion (C); amino acid transport and metabolism (E); nucleotide transport and metabolism (F); carbohydrate transport and metabolism (G); coenzyme transport and metabolism (H); lipid transport and metabolism (I); secondary metabolites biosynthesis, transport, and catabolism (Q); general function prediction only (R); function unknown (S). The downregulated proteins were involved in defense mechanisms (V), transcription (K), energy production and conversion (C), amino acid transport and metabolism (E), nucleotide transport and metabolism (F), carbohydrate transport and metabolism (G), and unknown function (S).
The abundance of cell division protein SepF, a cytoplasmic protein forming a division septum in a cytokinesis process by binary fission, increased in aerobic conditions. The production of thioredoxin (TrxA) and its electron acceptor, Tpx-type thiol peroxidase (Tpx), was higher under aerobiosis, while alkyl hydroperoxide reductase C (AhpC), a subunit of an NADH-dependent two-subunit thiol peroxidase complex Ahp, was downregulated upon intensive aeration. The levels of a small stress-induced cytoplasmic ATP-dependent 10-kDa chaperonin (GroES) assisting correct protein folding, a universal stress protein (USP), and a ThiJ/PfpI family protein were increased under aerobiosis. Under aerobic cultivation, the biosynthesis of three proteins involved in translation, ribosomal structure, and biogenesis was upregulated. These are: ribosome-binding factor A (RbfA) assisting in the late steps of the functional core maturation of a free 30S ribosomal subunit, probable tRNA sulfurtransferase (ThiI) catalyzing the ATP-dependent transfer of a sulfur to tRNA to produce 4-thiouridine in tRNAs, and RNA-binding lactamase B domain-containing protein exhibiting exonuclease activity. Two transcriptional regulators of the MarR family were involved in the oxidative stress response in L. rhamnosus CM MSU 529. The synthesis of organic hydroperoxide resistance transcriptional regulator OhrR was upregulated by aeration, whereas the synthesis of transcriptional regulator homologous to the product of gene LRHMDP2_405 was downregulated by O 2 .
Among proteins involved in the amino acid transport and metabolism, the levels of carbamoyl-phosphate synthase (CarAB) participating in L-arginine and pyrimidine biosynthesis; oligopeptide ABC transporter periplasmic oligopeptide-binding protein OppA, a component of transport system for oligopeptides from the environment; neutral endopeptidase (PepO) hydrolyzing internal polypeptide α-peptide bonds; glycine cleavage system H protein (GcvH) exhibiting the reversible oxidation of glycine to a methylene group; and cysteine synthase (CysK) increased under aerobiosis, while acetolactate synthase (AlsS) converting pyruvate into α-acetolactate and CO 2 was overproduced (2.9 log 2 protein fold change). On the contrary, the synthesis of transcriptional regulator, GntR family domain/aspartate aminotransferase (AvtA), involved in production of oxaloacetate from L-aspartate and 2-oxoglutarate; argininosuccinate synthase (ArgG) important for L-arginine biosynthesis; and cell division transporter ATP-binding protein FtsE (GlnQ) with ABC-type glutamine transporter activity was downregulated by aeration.
The abundance of the following enzymes involved in carbohydrate metabolism was higher under aerobiosis: probable phosphoketolase (Xfp) catalyzing the cleavage of xylulose 5-phosphate into glyceraldehyde 3-phosphate and acetyl-phosphate, pyruvate oxidase (Pox), and glycogen biosynthesis protein GlgD with glucose 1-phosphate adenylyltransferase activity. The level of ABC transporter substrate-binding protein containing sugar binding domain was lower during aerobic growth. The production of citrate lyase β-chain (CitE) significantly decreased in aerobic conditions (−2.9 log 2 protein fold change).
In spite of the differences mentioned above in proteomic profiles between static and aerated growth conditions, the biomass production and pH values of culture supernatants during static growth were similar to those observed in our previous study during the aerobic growth of strain CM MSU 529 [22]. The biomass was of 2.1 ± 0.1 g dw cells/l and and pH value of culture supernatant was of 3.92 ± 0.03 after 24 h of cultivation in static conditions.

Protein-Protein Interaction Network
Twenty-two of fifty-three differentially abundant proteins were connected in a STRING network indicating functional and physical protein interactions ( Figure 1, Tables S5-S8). Cluster analysis revealed 4 independent clusters. The cluster 1 (red) was represented by proteins involved in pyruvate metabolism plus LplA highly associated with lipoatedependent PdhD. The cluster 2 (white) included highly associated proteins of nucleotide and amino acid metabolism (F/E) plus glycogen biosynthesis protein GlgD (G). DUF4430 domain-containing protein (LGG_02295) of unknown function is encoded by a gene in neighborhood with an rtpR gene, whereas RtpR and PfpI are encoded by fused genes in the cluster 3 (green). Components of a thiol-specific antioxidant system are connected into the cluster 4 (blue). LGG_01836 (pox), LGG_02356 (lctO), and LGG_02295 (LRHMDP2_2713).

Discussion
Aeration had a pronounced impact on the proteome of L. rhamnosus CM MSU 529. Under intensive aeration, the synthesis of Xfp, a key enzyme of the pentose phosphate pathway, increased ( Figure 2). By contrast, the levels of enzymes producing oxaloacetate, CitE and AvtA, decreased in aerobic conditions. These facts imply that there is a shift in pyruvate production routs from the citrate-oxaloacetate pathway towards the pentose phosphate pathway under aerobiosis compared to static growth in L. rhamnosus CM MSU 529. Aeration increased the production of Xfp and decreased the production of CitE in L. casei N87 as well [20]. Pyruvate is a central intermediate of lactic acid fermentation and can be further converted by lactate dehydrogenase (Ldh) into lactate, by PflB into formate and acetyl-CoA, by Pox into acetyl-phosphate and CO2, by Pdh into acetyl-CoA and NADH, and by AlsS into α-acetolactate and CO2. Pox, PdhD, AlsS, and AlsD were upregulated during the aerobic growth of strain CM MSU 529, while PflB was downregulated by O2. Thus, acetyl-CoA production was mainly due to PflB activity during static growth and due to Pdh, Pox/phosphate acetyltransferase (Pta) or Xfp/Pta activities during aerobic growth. Alternatively in the last two cases, the acetyl-phosphate produced by Pox and Xfp may be further converted to acetate by acetate kinase (AckA) with ATP production. AckA is considered to be important in gaining extra energy under aerobiosis in lactobacilli LGG_01836 (pox), LGG_02356 (lctO), and LGG_02295 (LRHMDP2_2713).

Discussion
Aeration had a pronounced impact on the proteome of L. rhamnosus CM MSU 529. Under intensive aeration, the synthesis of Xfp, a key enzyme of the pentose phosphate pathway, increased ( Figure 2). By contrast, the levels of enzymes producing oxaloacetate, CitE and AvtA, decreased in aerobic conditions. These facts imply that there is a shift in pyruvate production routs from the citrate-oxaloacetate pathway towards the pentose phosphate pathway under aerobiosis compared to static growth in L. rhamnosus CM MSU 529. Aeration increased the production of Xfp and decreased the production of CitE in L. casei N87 as well [20]. Pyruvate is a central intermediate of lactic acid fermentation and can be further converted by lactate dehydrogenase (Ldh) into lactate, by PflB into formate and acetyl-CoA, by Pox into acetyl-phosphate and CO2, by Pdh into acetyl-CoA and NADH, and by AlsS into α-acetolactate and CO2. Pox, PdhD, AlsS, and AlsD were upregulated during the aerobic growth of strain CM MSU 529, while PflB was downregulated by O2. Thus, acetyl-CoA production was mainly due to PflB activity during static growth and due to Pdh, Pox/phosphate acetyltransferase (Pta) or Xfp/Pta activities during aerobic growth. Alternatively in the last two cases, the acetyl-phosphate produced by Pox and Xfp may be further converted to acetate by acetate kinase (AckA) with ATP production. AckA is considered to be important in gaining extra energy under aerobiosis in lactobacilli [13]. Since the abundance of AckA was relatively low in the proteome (score < 50), this reaction does not seem to play a significant role in the energy metabolism of strain CM MSU 529. LGG_01836 (pox), LGG_02356 (lctO), and LGG_02295 (LRHMDP2_2713).

Discussion
Aeration had a pronounced impact on the proteome of L. rhamnosus CM MSU 529. Under intensive aeration, the synthesis of Xfp, a key enzyme of the pentose phosphate pathway, increased ( Figure 2). By contrast, the levels of enzymes producing oxaloacetate, CitE and AvtA, decreased in aerobic conditions. These facts imply that there is a shift in pyruvate production routs from the citrate-oxaloacetate pathway towards the pentose phosphate pathway under aerobiosis compared to static growth in L. rhamnosus CM MSU 529. Aeration increased the production of Xfp and decreased the production of CitE in L. casei N87 as well [20]. Pyruvate is a central intermediate of lactic acid fermentation and can be further converted by lactate dehydrogenase (Ldh) into lactate, by PflB into formate and acetyl-CoA, by Pox into acetyl-phosphate and CO2, by Pdh into acetyl-CoA and NADH, and by AlsS into α-acetolactate and CO2. Pox, PdhD, AlsS, and AlsD were upregulated during the aerobic growth of strain CM MSU 529, while PflB was downregulated by O2. Thus, acetyl-CoA production was mainly due to PflB activity during static growth and due to Pdh, Pox/phosphate acetyltransferase (Pta) or Xfp/Pta activities during aerobic growth. Alternatively in the last two cases, the acetyl-phosphate produced by Pox and Xfp may be further converted to acetate by acetate kinase (AckA) with ATP production. AckA is considered to be important in gaining extra energy under aerobiosis in lactobacilli [13]. Since the abundance of AckA was relatively low in the proteome (score < 50), this reaction does not seem to play a significant role in the energy metabolism of strain CM MSU 529.

Discussion
Aeration had a pronounced impact on the proteome of L. rhamnosus CM MSU 529. Under intensive aeration, the synthesis of Xfp, a key enzyme of the pentose phosphate pathway, increased ( Figure 2). By contrast, the levels of enzymes producing oxaloacetate, CitE and AvtA, decreased in aerobic conditions. These facts imply that there is a shift in pyruvate production routs from the citrate-oxaloacetate pathway towards the pentose phosphate pathway under aerobiosis compared to static growth in L. rhamnosus CM MSU 529. Aeration increased the production of Xfp and decreased the production of CitE in L. casei N87 as well [20]. Pyruvate is a central intermediate of lactic acid fermentation and can be further converted by lactate dehydrogenase (Ldh) into lactate, by PflB into formate and acetyl-CoA, by Pox into acetyl-phosphate and CO2, by Pdh into acetyl-CoA and NADH, and by AlsS into α-acetolactate and CO2. Pox, PdhD, AlsS, and AlsD were upregulated during the aerobic growth of strain CM MSU 529, while PflB was downregulated by O2. Thus, acetyl-CoA production was mainly due to PflB activity during static growth and due to Pdh, Pox/phosphate acetyltransferase (Pta) or Xfp/Pta activities during aerobic growth. Alternatively in the last two cases, the acetyl-phosphate produced by Pox and Xfp may be further converted to acetate by acetate kinase (AckA) with ATP production. AckA is considered to be important in gaining extra energy under aerobiosis in lactobacilli [13]. Since the abundance of AckA was relatively low in the proteome (score < 50), this reaction does not seem to play a significant role in the energy metabolism of strain CM MSU 529.

Discussion
Aeration had a pronounced impact on the proteome of L. rhamnosus CM MSU 529. Under intensive aeration, the synthesis of Xfp, a key enzyme of the pentose phosphate pathway, increased ( Figure 2). By contrast, the levels of enzymes producing oxaloacetate, CitE and AvtA, decreased in aerobic conditions. These facts imply that there is a shift in pyruvate production routs from the citrate-oxaloacetate pathway towards the pentose phosphate pathway under aerobiosis compared to static growth in L. rhamnosus CM MSU 529. Aeration increased the production of Xfp and decreased the production of CitE in L. casei N87 as well [20]. Pyruvate is a central intermediate of lactic acid fermentation and can be further converted by lactate dehydrogenase (Ldh) into lactate, by PflB into formate and acetyl-CoA, by Pox into acetyl-phosphate and CO2, by Pdh into acetyl-CoA and NADH, and by AlsS into α-acetolactate and CO2. Pox, PdhD, AlsS, and AlsD were upregulated during the aerobic growth of strain CM MSU 529, while PflB was downregulated by O2. Thus, acetyl-CoA production was mainly due to PflB activity during static growth and due to Pdh, Pox/phosphate acetyltransferase (Pta) or Xfp/Pta activities during aerobic growth. Alternatively in the last two cases, the acetyl-phosphate produced by Pox and Xfp may be further converted to acetate by acetate kinase (AckA) with ATP production. AckA is considered to be important in gaining extra energy under aerobiosis in lactobacilli [13]. Since the abundance of AckA was relatively low in the proteome (score < 50), this reaction does not seem to play a significant role in the energy metabolism of strain CM MSU 529.

Discussion
Aeration had a pronounced impact on the proteome of L. rhamnosus CM MSU 529. Under intensive aeration, the synthesis of Xfp, a key enzyme of the pentose phosphate pathway, increased ( Figure 2). By contrast, the levels of enzymes producing oxaloacetate, CitE and AvtA, decreased in aerobic conditions. These facts imply that there is a shift in pyruvate production routs from the citrate-oxaloacetate pathway towards the pentose phosphate pathway under aerobiosis compared to static growth in L. rhamnosus CM MSU 529. Aeration increased the production of Xfp and decreased the production of CitE in L. casei N87 as well [20]. Pyruvate is a central intermediate of lactic acid fermentation and can be further converted by lactate dehydrogenase (Ldh) into lactate, by PflB into formate and acetyl-CoA, by Pox into acetyl-phosphate and CO2, by Pdh into acetyl-CoA and NADH, and by AlsS into α-acetolactate and CO2. Pox, PdhD, AlsS, and AlsD were upregulated during the aerobic growth of strain CM MSU 529, while PflB was downregulated by O2. Thus, acetyl-CoA production was mainly due to PflB activity during static growth and due to Pdh, Pox/phosphate acetyltransferase (Pta) or Xfp/Pta activities during aerobic growth. Alternatively in the last two cases, the acetyl-phosphate produced by Pox and Xfp may be further converted to acetate by acetate kinase (AckA) with ATP production. AckA is considered to be important in gaining extra energy under aerobiosis in lactobacilli [13]. Since the abundance of AckA was relatively low in the proteome (score < 50), this reaction does not seem to play a significant role in the energy metabolism of strain CM MSU 529.

Discussion
Aeration had a pronounced impact on the proteome of L. rhamnosus CM MSU 529. Under intensive aeration, the synthesis of Xfp, a key enzyme of the pentose phosphate pathway, increased ( Figure 2). By contrast, the levels of enzymes producing oxaloacetate, CitE and AvtA, decreased in aerobic conditions. These facts imply that there is a shift in pyruvate production routs from the citrate-oxaloacetate pathway towards the pentose phosphate pathway under aerobiosis compared to static growth in L. rhamnosus CM MSU 529. Aeration increased the production of Xfp and decreased the production of CitE in L. casei N87 as well [20]. Pyruvate is a central intermediate of lactic acid fermentation and can be further converted by lactate dehydrogenase (Ldh) into lactate, by PflB into formate and acetyl-CoA, by Pox into acetyl-phosphate and CO2, by Pdh into acetyl-CoA and NADH, and by AlsS into α-acetolactate and CO2. Pox, PdhD, AlsS, and AlsD were upregulated during the aerobic growth of strain CM MSU 529, while PflB was downregulated by O2. Thus, acetyl-CoA production was mainly due to PflB activity during static growth and due to Pdh, Pox/phosphate acetyltransferase (Pta) or Xfp/Pta activities during aerobic growth. Alternatively in the last two cases, the acetyl-phosphate produced by Pox and Xfp may be further converted to acetate by acetate kinase (AckA) with ATP production. AckA is considered to be important in gaining extra energy under aerobiosis in lactobacilli [13]. Since the abundance of AckA was relatively low in the proteome (score < 50), this reaction does not seem to play a significant role in the energy metabolism of strain CM MSU 529.

Discussion
Aeration had a pronounced impact on the proteome of L. rhamnosus CM MSU 529. Under intensive aeration, the synthesis of Xfp, a key enzyme of the pentose phosphate pathway, increased ( Figure 2). By contrast, the levels of enzymes producing oxaloacetate, CitE and AvtA, decreased in aerobic conditions. These facts imply that there is a shift in pyruvate production routs from the citrate-oxaloacetate pathway towards the pentose phosphate pathway under aerobiosis compared to static growth in L. rhamnosus CM MSU 529. Aeration increased the production of Xfp and decreased the production of CitE in L. casei N87 as well [20]. Pyruvate is a central intermediate of lactic acid fermentation and can be further converted by lactate dehydrogenase (Ldh) into lactate, by PflB into formate and acetyl-CoA, by Pox into acetyl-phosphate and CO2, by Pdh into acetyl-CoA and NADH, and by AlsS into α-acetolactate and CO2. Pox, PdhD, AlsS, and AlsD were upregulated during the aerobic growth of strain CM MSU 529, while PflB was downregulated by O2. Thus, acetyl-CoA production was mainly due to PflB activity during static growth and due to Pdh, Pox/phosphate acetyltransferase (Pta) or Xfp/Pta activities during aerobic growth. Alternatively in the last two cases, the acetyl-phosphate produced by Pox and Xfp may be further converted to acetate by acetate kinase (AckA) with ATP production. AckA is considered to be important in gaining extra energy under aerobiosis in lactobacilli [13]. Since the abundance of AckA was relatively low in the proteome (score < 50), this reaction does not seem to play a significant role in the energy metabolism of strain CM MSU 529.

Discussion
Aeration had a pronounced impact on the proteome of L. rhamnosus CM MSU 529. Under intensive aeration, the synthesis of Xfp, a key enzyme of the pentose phosphate pathway, increased (Figure 2). By contrast, the levels of enzymes producing oxaloacetate, CitE and AvtA, decreased in aerobic conditions. These facts imply that there is a shift in pyruvate production routs from the citrate-oxaloacetate pathway towards the pentose phosphate pathway under aerobiosis compared to static growth in L. rhamnosus CM MSU 529. Aeration increased the production of Xfp and decreased the production of CitE in L. casei N87 as well [20]. Pyruvate is a central intermediate of lactic acid fermentation and can be further converted by lactate dehydrogenase (Ldh) into lactate, by PflB into formate and acetyl-CoA, by Pox into acetyl-phosphate and CO2, by Pdh into acetyl-CoA and NADH, and by AlsS into α-acetolactate and CO2. Pox, PdhD, AlsS, and AlsD were upregulated during the aerobic growth of strain CM MSU 529, while PflB was downregulated by O2. Thus, acetyl-CoA production was mainly due to PflB activity during static growth and due to Pdh, Pox/phosphate acetyltransferase (Pta) or Xfp/Pta activities during aerobic growth. Alternatively in the last two cases, the acetyl-phosphate produced by Pox and Xfp may be further converted to acetate by acetate kinase (AckA) with ATP production. AckA is considered to be important in gaining extra energy under aerobiosis in lactobacilli [13]. Since the abundance of AckA was relatively low in the proteome (score < 50), this reaction does not seem to play a significant role in the energy metabolism of strain CM MSU 529.

Discussion
Aeration had a pronounced impact on the proteome of L. rhamnosus CM MSU 529. Under intensive aeration, the synthesis of Xfp, a key enzyme of the pentose phosphate pathway, increased (Figure 2). By contrast, the levels of enzymes producing oxaloacetate, CitE and AvtA, decreased in aerobic conditions. These facts imply that there is a shift in pyruvate production routs from the citrate-oxaloacetate pathway towards the pentose phosphate pathway under aerobiosis compared to static growth in L. rhamnosus CM MSU 529. Aeration increased the production of Xfp and decreased the production of CitE in L. casei N87 as well [20]. Pyruvate is a central intermediate of lactic acid fermentation and can be further converted by lactate dehydrogenase (Ldh) into lactate, by PflB into formate and acetyl-CoA, by Pox into acetyl-phosphate and CO 2 , by Pdh into acetyl-CoA and NADH, and by AlsS into α-acetolactate and CO 2 . Pox, PdhD, AlsS, and AlsD were upregulated during the aerobic growth of strain CM MSU 529, while PflB was downregulated by O 2 . Thus, acetyl-CoA production was mainly due to PflB activity during static growth and due to Pdh, Pox/phosphate acetyltransferase (Pta) or Xfp/Pta activities during aerobic growth. Alternatively in the last two cases, the acetyl-phosphate produced by Pox and Xfp may be further converted to acetate by acetate kinase (AckA) with ATP production. AckA is considered to be important in gaining extra energy under aerobiosis in lactobacilli [13]. Since the abundance of AckA was relatively low in the proteome (score < 50), this reaction does not seem to play a significant role in the energy metabolism of strain CM MSU 529.  (Tables 1, S1-S3), and other identified enzymes are in black (Table S4).
The proteome of L. rhamnosus CM MSU 529 contains two pyruvate oxidases, CidC and Pox. The synthesis of both enzymes was upregulated by oxygen. The former isoform possesses pyruvate:ubiquinone oxidoreductase activity, whereas the latter isoform oxidizes pyruvate into acetyl-phosphate, CO2, and H2O2. Recently we have shown that strain CM MSU 529 realizes respiratory metabolism in a medium supplemented with hemin and menaquinone [22]. CidC is likely to be a component of the ETC functioning in strain CM MSU 529 under respiratory cultivation. Overexpression of CidC under aerobiosis implies that some components of ETC may be activated by O2 in LAB. The expression of the pox gene was activated by O2 in Lp. plantarum Lp80 [26], Lp. plantarum WCFS1 [27], Lt. buchneri CD034 [17], L. rhamnosus N132, Levilactobacillus spicheri LP38 [28], L. casei N87 [10], and Levilactobacillus brevis ATCC 367 [29]. In the last two species, the induction of Pox activity was also observed. Pox5 (PoxB) involved in conversion of pyruvate to acetate was upregulated under aerobiosis versus anaerobiosis in Lp. plantarum WCFS1 [19]. In addition to Pox, the synthesis of the H2O2-producing flavin-dependent oxidases, LctO and predicted PPO, was upregulated by O2 in L. rhamnosus CM MSU 529. LctO oxidizes lactate into pyruvate, rerouting a final product of lactic acid fermentation back to the carbon and energy metabolism. Oxygen induced transcription of the lctO gene in Lt. buchneri CD034 [17].  (Tables 1 and S1-S3), and other identified enzymes are in black (Table S4).
The proteome of L. rhamnosus CM MSU 529 contains two pyruvate oxidases, CidC and Pox. The synthesis of both enzymes was upregulated by oxygen. The former isoform possesses pyruvate:ubiquinone oxidoreductase activity, whereas the latter isoform oxidizes pyruvate into acetyl-phosphate, CO 2 , and H 2 O 2 . Recently we have shown that strain CM MSU 529 realizes respiratory metabolism in a medium supplemented with hemin and menaquinone [22]. CidC is likely to be a component of the ETC functioning in strain CM MSU 529 under respiratory cultivation. Overexpression of CidC under aerobiosis implies that some components of ETC may be activated by O 2 in LAB. The expression of the pox gene was activated by O 2 in Lp. plantarum Lp80 [26], Lp. plantarum WCFS1 [27], Lt. buchneri CD034 [17], L. rhamnosus N132, Levilactobacillus spicheri LP38 [28], L. casei N87 [10], and Levilactobacillus brevis ATCC 367 [29]. In the last two species, the induction of Pox activity was also observed. Pox5 (PoxB) involved in conversion of pyruvate to acetate was upregulated under aerobiosis versus anaerobiosis in Lp. plantarum WCFS1 [19]. In addition to Pox, the synthesis of the H 2 O 2 -producing flavin-dependent oxidases, LctO and predicted PPO, was upregulated by O 2 in L. rhamnosus CM MSU 529. LctO oxidizes lactate into pyruvate, rerouting a final product of lactic acid fermentation back to the carbon and energy metabolism. Oxygen induced transcription of the lctO gene in Lt. buchneri CD034 [17].
The abundance of AlsS and AlsD increased during aerobic growth. The former converts pyruvate into α-acetolactate and CO 2 , while the latter decarboxylates α-acetolactate into acetoin. Alternatively, α-acetolactate may be converted into diacetyl by nonenzymatical decarboxylation in the presence of oxygen. The proteome of strain CM MSU 529 lacks butanediol dehydrogenase and diacetyl reductase. Als was upregulated by O 2 in Leuconostoc gelidum subsp. gelidum TMW2.1618 and in Ln. gelidum subsp. gasicomitatum TMW2.1619 grown in a heme-supplemented medium [21].
L. rhamnosus CM MSU 529 contains two thiol-specific peroxidases, TrxA-dependent Tpx-type and NADH-dependent AhpC, which synthesis was up-and downregulated by O 2 , respectively. The production level of TrxA, a small disulphide-containing protein, was higher under aerobiosis. Since reduced TrxA possesses a Tpx-disulfide reductase activity, it provides the Tpx functioning [13]. Upregulated by O 2 NADH dehydrogenase with FAD/NAD(P) + -binding domain (K8Q7J1) might play a role of TrxA reductase supporting an intracellular disulphide/thiol balance in strain CM MSU 529. In addition to Tpx, production of Npr significantly increased under aerobiosis. Both peroxidases play a key role in the effective elimination of H 2 O 2 formed by oxidases (LctO, Pox, and PPO), which synthesis was induced during aerobic growth. Genes encoding TrxA, Tpx, and Npr are widely spread among lactobacilli [13]. L. rhamnosus lacks genes encoding catalase and superoxide dismutase. Thus, Tpx and Npr participate in detoxification of H 2 O 2 and organic hydroxyperoxides predominantly in aerobic conditions, whereas AhpC carries out this function during the static growth of strain CM MSU 529. The ahpC gene expression was upregulated by cold in Lc. lactis MCC866 cocultivated with bifidobacteria after refrigerated storage in fermented milk [30]. The synthesis of AhpC was cold-inducible in Leuconostoc mesenteroides NH04 [31]. NADH peroxidase Npr2 was upregulated under aerobiosis versus anaerobiosis in Lp. plantarum WCFS1 [19].
The CysK level increased upon intensive aeration in strain CM MSU 529. This observation is in a good agreement with the upregulation of TrxA and Tpx biosynthesis, since these proteins harbor Cys residues in their active centers. In addition, Cys is a strong antioxidant itself [32]. Overexpression of the predicted PPO under aerobiosis is likely to support the biosynthesis of pyridoxal 5'-phosphate-dependent CysK in strain CM MSU 529. The amount of CysK increased in a controlled batch culture of L. casei N87 under aerobiosis versus anaerobiosis [20].
DkgA catalyzes the NADPH-dependent reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid, a key intermediate in the synthesis of L-ascorbic acid. Thus, the higher amount of DkgA under aeration may enhance the antioxidant properties of strain CM MSU 529.
The biosynthesis of proteins involved in the general stress response (GroES, USP, PfpI, and YqiG) was upregulated by O 2 in L. rhamnosus CM MSU 529. The putative heat shock protein GroES was among the proteins whose synthesis was induced in response to acid conditions in Lactobacillus acidophilus CRL 639 [33] and Lactobacillus bulgaricus [34]. The role and the mechanism of action of the USPs in Gram-positive bacteria are still obscure. The usp1 gene was overexpressed during the the phenolic acid stress response in Lp. plantarum [35]. The capacity of Usp1 to inactivate the repressor of the phenolic acid stress response PadR implies that it could be a mediator in the acid stress response mechanism. The ThiJ/PfpI family proteins are suggested to be involved in cellular protection against various environmental stresses [36]. The members of the ThiJ/PfpI family are diverse in structure and function and include heat-shock protein 31 (Hsp31), a chaperone and a peptidase of E. coli [37]; PH1704, a thermophilic protease/peptidase of archaean Pyrococcus horikoshii OT3 [38]; and YhbO involved in the response to hyperosmotic and acid stresses in E. coli [39]. Hsp31 paralog with glyoxalase activity was demonstrated to combat oxidative stress in Saccharomyces cerevisiae [40]. FMN-dependent OYEs reduce a wide range of activated C=C bonds in α,β-unsaturated carbonyl compounds to their saturated counterparts [41] and were reported to participate in stress response. Thus, the expression of ofrA gene encoding OYE of Staphylococcus aureus was induced by hypochlorite, oxidative, and electrophilic stresses [42]. YqiG appears to be important in maintaining an intracellular redox balance reducing the electrophilic carbonyl compounds imposed by intensive aeration in strain CM MSU 529. Upregulated upon aeration NADP + -dependent 3-hydroxyisobutyrate dehydrogenase related β-hydroxyacid dehydrogenase should be also considered as the general stress response protein. The function of 3-hydroxyisobutyrate dehydrogenases/β-hydroxyacid dehydrogenases reducing succinic semialdehyde and glyoxylate to γ-hydroxybutyrate and glycolate, respectively, has been proposed to detoxify both aldehydes during stress response in Arabidopsis [43]. Phylogenetically related glyoxylate reductases/succinic semialdehyde reductases were found in bacteria as well. The purified 3-hydroxyisobutyrate dehydrogenase-type proteins from Geobacter spp. [44] and Gluconobacter oxydans 621H [45] revealed succinic semialdehyde reductase activities. In addition, the NAD(P)H-dependent glyoxylate reductase with high substrate specificity for glyoxylate was recently characterized from Acetobacter aceti JCM20276 [46].
The increased production of RbfA and RNA-binding VicX may be important for the maturation and stability of the free 30S and 70S ribosomal subunit in strain CM MSU 529 under aerobiosis, respectively. ThiI transfers a sulfur to tRNA and to the sulfur carrier protein ThiS, forming ThiS-thiocarboxylate, which is a stage in the synthesis of thiazole in the thiamine diphosphate (TPP) biosynthetic process. Thus, the higher level of the ThiI supports the upregulated synthesis of TPP-dependent enzymes (Pox, AlsS) in L. rhamnosus CM MSU 529 during aerobic growth. Interestingly, the Fe-S biosynthesis domain-containing protein (YkuJ) with unknown function may be involved in the [2Fe-2S] cluster synthesis of ferredoxin needed for sulfurtransferase activity of ThiI.
Two transcriptional regulators of the MarR family changed in amount in L. rhamnosus CM MSU 529 upon aeration. OhrR was involved in the oxidative stress response, while the transcriptional regulator homologous to the gene LRHMDP2_405 product was downregulated upon intensive aeration. The MarR family transcriptional regulators are known to be repressors of genes activating the oxidative stress regulons. OhrR is a transcriptional repressor of the organic hydroperoxide resistance protein (OhrA). The gene ohrR deficient mutant revealed a strong resistance to H 2 O 2 in L. casei IGM394 [47]. OhrA was not detected in this study.
The level of PdhD increased upon aeration in strain CM MSU 529. The amount of PdhD also increased in the proteome of L. casei N87 in a controlled batch culture under aerobiosis compared to anaerobiosis [20]. The expression of pdh genes, as well as Pdh activity, was upregulated by oxygen in Lv. brevis ATCC 367 at the late exponential growth phase [29].
GcvH is a component of the glycine cleavage system catalyzing the reversible oxidative cleavage of glycine to a methylene group, ammonia, and CO 2 . Thus, the increased level of GcvH may maintain pH homeostasis in L. rhamnosus CM MSU 529 under aerobiosis. Another mechanism that prevents the intracellular acidification is the upregulation upon aeration of enzymes of the pyruvate-diacetyl/acetoin pathway, AlsS and AlsD. This pathway contributed to pH homeostasis in acid stress conditions in Lc. lactis [48].
LplA responsible for ATP-dependent protein lipoylation via the exogenous pathway was upregulated upon aeration in strain CM MSU 529. This fact is in good agreement with upregulation of the lipoate-requiring proteins, GcvH and PdhD.
The abundance of CarAB increased under aerobic compared to static conditions, whereas the production of ArgG decreased under intensive aeration in strain CM MSU 529. The arginine and pyrimidine biosynthesis pathways have a common precursor, carbamoyl-phosphate. This implies that there is a shift from L-arginine synthesis towards pyrimidine nucleotides synthesis in lactic acid bacterium under aerobic conditions. The higher levels of PyrE, PyrC, PyrB, and PyrR involved in pyrimidine biosynthesis in aerobic conditions compared to static growth support this conclusion. The carbamoyl-phosphate and uridine 5 -monophosphate (UMP) synthesis depends on concentration of the dissolved form of CO 2 (HCO 3 − ) in a growth medium. A high requirement of CO 2 for arginine and pyrimidine biosynthesis was found in 74 of 207 tested strains of lactobacilli [49]. Therefore, the upregulation of CO 2 -producing enzymes (Pox, AlsS, AlsD, and GcvH) favors the pyrimidine nucleotides biosynthesis in strain CM MSU 529 during aerobic growth. The upregulation of CarAB and PyrECBR in L. rhamnosus CM MSU 529 by O 2 is rather unusual. The expression of the carB and pyrC genes was downregulated by aeration in Lc. lactis [15]. The amount of CarB decreased in a controlled batch culture of L. casei N87 under aerobiosis compared to anaerobiosis [20]. The abundance of PyrC was lower in oxic versus anoxic conditions in Carnobacterium divergens TMW2.1577 grown in a meat simulation medium [21]. The upregulation PyrECBR was not followed by an increase in biomass production of L. rhamnosus CM MSU 529 under aerobiosis. Instead, an increase in the level of GlgD promoted the activation of glycogen biosynthetic process by aeration in strain CM MSU 529.
DeoC reversibly degradates 2-deoxy-D-ribose 5-phosphate to D-glyceraldehyde 3phosphate and acetaldehyde providing both the synthesis and catabolism of nucleosides. In the latter case, D-glyceraldehyde 3-phosphate enters the central carbon metabolism pathways as a carbon and energy source ( Figure 2). E. coli mutants lacking DeoC were not able to catabolize the deoxyribose moiety of deoxyribonucleosides, while there were no significant alterations in the cellular pools of NTPs and dNTPs [50], suggesting a catabolic function of DeoC. The activities of DeoC isolated from Lp. plantarum and a rat liver were highly dependent on the presence of polycarboxylic acids [51,52]. In this connection, the downregulation of CitE upon aeration in strain CM MSU 529 may favor the activation of DeoC by citrate during aerobic growth. Interestingly, the amount of DeoC was lower under aerobiosis in comparison with anaerobiosis in C. divergens TMW2.1577 [21].
Ribonucleosidereductase reduces nucleosides (U, A, G, and C) to the corresponding deoxynucleosides [53]. The adenosylcobalamin-dependent RtpR of strain CM MSU 529 catalyzes this reaction at the level of NDP and NTP using TrxA as an electron donor. The underexpression of RtpR in aerobiosis may help to maintain the high levels of NDPs/NTPs needed for RNA biosynthesis to support the production of the upregulated by aeration enzymes. The downregulation of deoxyadenosine kinase/deoxyguanosine kinase results in decreased concentrations of dAMP/dGMP, the precursors of dATP/dGTP, during the aerobic growth of strain CM MSU 529. This fact is in good agreement with the low level of PstS under aeration.
OppA, a surface located protein in lactobacilli, is a component of the transport system for oligopeptides from the environment for nutrition. The amounts of OppA and PepO increased, while the abundance of GlnQ decreased under aerobiosis, suggesting rerouting of nutrition from amino acids to oligopeptides upon aeration in strain CM MSU 529. The enhanced import of oligopeptides appears to support the biosynthesis of upregulated proteins in lactic acid bacterium under aerobic conditions. The expression of the oppa3 gene was upregulated under aerobic versus anaerobic conditions in Lt. buchneri CD034 [17]. The pepO1 expression was stimulated by aeration compared to static conditions in Lc. lactis MG1363 [18]. The glnQ gene expression was downregulated in Lc. lactis in aerobic conditions compared to static conditions [14].
Proteins of L. rhamnosus CM MSU 529 were identified by comparative analysis with the L. rhamnosus LRHMDP2 proteome. Sequencing of the entire genome of strain CM MSU 529 may lead to a deeper knowledge of its protein profile.

Conclusions
Proteomic analysis is a powerful tool to elucidate the molecular mechanisms of cellular adaptation to various environmental conditions. The significant difference in the proteomic profile of aerobically grown cells compared to statically grown cells of L. rhamnosus CM MSU 529 was observed. Aerobic cultivation caused the induction of enzymes responsible for oxygen consumption, xylulose 5-phosphate and pyruvate conversion, ROS defense, general stress response, antioxidant production, pyrimidine biosynthesis, oligopeptide transport and metabolism, ribosome stabilization, intracellular pH and redox homeostasis. In spite of the fact that intensive aeration impaired the citrate-pyruvate pathway, arginine and DNA biosynthesis, it had no effect on the biomass production. The metabolism of L. rhamnosus CM MSU 529 is therefore precisely tuned to the standing concentration of O 2 in such a way that the strain is able to maintain energy conservation enough for achieving optimal biomass production under intensive aeration. Further elucidation of the influence of aerobic cultivation on probiotic properties, cell survival to a long-term storage and environmental stresses is important in light of the prospect of industrial application of a strain CM MSU 529 aerobic phenotype. The presented results provide new knowledge on the molecular mechanisms employed by LAB in adaptation to aerobic growth conditions.  Data Availability Statement: The mass spectrometry proteomics data were deposited to the Pro-teomeXchange Consortium via the PRIDE (http://www.ebi.ac.uk/pride, accessed on 15 December 2022) partner repository with the dataset identifier PXD038847.