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Molecular and metabolic adaptations of Lactococcus lactis at near-zero growth 1

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Onur Ercan1,2,3,4, Michiel Wels2,4, Eddy J. Smid2,6, Michiel Kleerebezem2,4,5,* 5

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Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 5057, 2600 GA Delft, The 7

Netherlands1; Top Institute Food and Nutrition, Nieuwe Kanaal 9A, 6709 PA Wageningen, The 8

Netherlands2; Laboratory of Microbiology, Wageningen University, P.O. Box 8033, 6700 EJ 9

Wageningen, The Netherlands3; NIZO food research, P.O. Box 20, 6710 BA Ede, The 10

Netherlands4; Host Microbe Interactomics, Wageningen University, P.O. Box 338, 6700 AH 11

Wageningen, The Netherlands5, Laboratory of Food Microbiology, Wageningen University, P.O. 12

Box 17, 6700 AA Wageningen, The Netherlands6 13

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Running Head: Molecular adaptations of L. lactis to zero-growth 15

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Keywords: Lactococcus lactis, zero-growth, transcriptome, amino acids, CodY-binding site, 17

alternative carbon scavenging 18

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*Address correspondence to Michiel Kleerebezem, Host Microbe Interactomics Group, 20

Wageningen University, De Elst 1, 6708 WD Wageningen, The Netherlands. Phone: +31-317-21

486125; Fax: +31-317-483962; E-mail: michiel.kleerebezem@wur.nl 22

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AEM Accepts, published online ahead of print on 24 October 2014Appl. Environ. Microbiol. doi:10.1128/AEM.02484-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 26

This paper describes the molecular and metabolic adaptations of Lactococcus lactis during the 27

transition from a growing to a near-zero growth state using carbon-limited retentostat cultivation. 28

Transcriptomic analyses revealed that metabolic patterns shifted between lactic- and mixed-acid 29

fermentation during retentostat cultivation, which appeared to be controlled at the transcription 30

level of the corresponding pyruvate-dissipation encoding genes. During retentostat cultivation, cells 31

continued to consume several amino acids, but also produced specific amino acids, which may 32

derive from the conversion of glycolytic intermediates. We identify a novel motif containing 33

CTGTCAG, in the upstream regions of several genes related to amino acid conversion, which we 34

propose to be the target site for CodY in Lactococcus lactis KF147. Finally, under extremely low 35

carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic 36

functions were found to be highly expressed, which was confirmed by enhanced initial acidification 37

rates on various sugars in cells obtained from near-zero growth cultures. The present integrated 38

transcriptome and metabolite (amino acids and previously reported fermentation end-products) 39

study provides molecular understanding of the adaptation of Lactococcus lactis to conditions 40

supporting low-growth rates, and expands our earlier analysis of the quantitative physiology of this 41

bacterium at near-zero growth rates towards gene regulation patterns involved in zero-growth 42

adaptation. 43

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INTRODUCTION 49

Fundamental knowledge of microbial physiology and cellular regulation is obtained mainly 50

from studies of microorganisms in batch cultures. However, the pace of life and its associated 51

physiological phases in batch cultivation differ strongly from what is found in natural environments 52

(24). During the early phase of batch cultivations, all nutrients, including carbon and energy sources 53

are usually present in excess, and specific growth rate of the microorganism equals the maximum 54

specific growth rate (4). Thereby, our understanding of microbial energy metabolism originates 55

mostly from microbial population studies performed under laboratory conditions that include rapid 56

growth, high metabolic activity, and high cell density. However, natural microbial communities 57

generally live in relative famine conditions with low specific growth and metabolic rates due to 58

limited supply of nutrients and energy sources (27). Analogously, under specific industrial 59

fermentation conditions, microorganisms may experience strongly restricted access to nutrients for 60

longer periods of time. For example, lactic acid bacteria (LAB) experience long periods of 61

extremely low nutrient availability during the maturing process of dry sausage (25) and cheese (41) 62

productions. Despite these harsh conditions, several LAB succeed to survive in these processes 63

during months of maturation and may continue to contribute to flavor and aroma formation in the 64

product matrix (10, 15, 25). 65

L. lactis is used in food fermentation processes for several products including cheese, sour 66

cream, and other fermented milk products. In these processes, L. lactis converts the available carbon 67

source into lactic acid, resulting in acidification of the food raw-material. In addition, L. lactis is 68

also commonly encountered in diverse natural environments, in particular in decaying plant 69

materials (39). The strain used in this study, L. lactis KF147, was isolated from mung bean sprouts, 70

and its genome sequence reflects many adaptations to the plant-associated habitat, which are in 71

particular apparent from the repertoire of enzymes and pathways predicted to be involved in 72

utilization of plant cell wall polysaccharides. 73

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To study the physiological and genome-wide adaptations of microorganisms to near-zero 74

growth rates, retentostat cultivation or recycling fermenter set-ups have been designed (23). 75

Retentostat cultivation is an adaptation of chemostat cultivation in which a growth-limiting 76

substrate is supplied at a fixed dilution rate, while the complete biomass is retained in the bioreactor 77

by removing the spent medium effluent through an external cross-flow filter. Prolonged retentostat 78

cultivation leads to growth rates that approximate zero while the rate of energy transduction 79

(through substrate consumption and conversion) equals the maintenance energy requirements (e.g., 80

osmoregulation, turnover of damaged cellular components) (14, 47). Therefore, retentostat 81

cultivation comprises a gradual transition from a growing to a near-zero growth state under stable 82

environmental conditions, which sustain high cell-viability. 83

Although retentostat cultivations have been performed to study the fundamental physiology 84

of several microorganisms, including Escherichia coli (7), Bacillus polymyxa (2), Paracoccus 85

denitrificans, Bacillus licheniformis (47), Nitrosomonas europaea and Nitrobacter winogradskyi 86

(43) at low-growth rates, these studies were not consistently complemented with detailed molecular 87

analyses. Exceptions are the retentostat studies performed with Lactobacillus plantarum (22), 88

Aspergillus niger (26), and Saccharomyces cerevisiae (4, 5) that included the analysis of metabolic 89

and transcriptome responses. 90

Previously, we have described how retentostat cultivation allows uncoupling of growth and 91

non-growth-related processes in L. lactis KF147, allowing the investigation of the energy household 92

and quantitative physiology of L. lactis at extremely low-growth rates, exemplified by the estimated 93

specific growth rate of 0.0001 h-1 that corresponds to a doubling time of more than 260 days (14). 94

Despite the imposed extremely low-growth rate, the culture viability was sustained above 90% 95

during prolonged retentostat cultivation (14). This study allowed the accurate calculation of 96

maintenance energy requirements and other quantitative physiology parameters of the strain 97

subjected to retentostat cultivation (14). Furthermore, the calculated values of maintenance-related 98

substrate coefficient (ms) and biomass yield (Ysxmax) revealed that the relative distribution of carbon 99

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source derived energy towards maintenance-associated processes increased from approximately 100

30% to 99% during the transition from growing cultures to prolonged, near zero-growth retentostat 101

cultivation (14). These energy distribution measurements confirmed that the retentostat cultures had 102

reached a typical near-zero growth state after approximately 14 days. Remarkably, the prolonged 103

retentostat culture of L. lactis displayed significant shifts in central carbon metabolism, switching 104

between mixed-acid and lactic-fermentation (14). 105

In the present study, we complement our previous study with an in-depth molecular level 106

analysis of L. lactis KF147 under these retentostat cultivation conditions, including transcriptome, 107

amino acid metabolism and previously obtained fermentation metabolite (14) analyses. Thereby, 108

this study deciphers the molecular adaptation underlying the previously reported physiological 109

observations. The genome-wide transcriptome analyses were in remarkable agreement with the 110

previously observed oscillation of the culture between lactic and mixed-acid fermentation at 111

extremely low-growth rates. Moreover, integrated metabolome and transcriptome analyses created a 112

global view of the interconnected carbon- and nitrogen-metabolism adaptations under these near-113

zero growth conditions. Notably, these adaptations included the progressive relief of carbon 114

catabolite repression, preparing the culture to effectively scavenge alternative carbon sources when 115

they become available. Finally, the transcriptome adaptations are discussed in the context of 116

regulatory circuits that are proposed to govern them, including a predicted role for the central 117

carbon- and nitrogen-metabolism control proteins CcpA and CodY, respectively. 118

MATERIALS AND METHODS 119

Bacterial isolates, media and cultivation conditions. Lactococcus lactis subsp. lactis strain 120

KF147 originates from mung bean sprouts, and its genome sequence was determined (39). Pre-121

cultures for retentostat cultivations (14) were inoculated in 50 ml M17 broth (44) complemented 122

with 0.5% glucose (w/v) and grown overnight at 30°C. Overnight cultures were harvested by 123

centrifugation (6,000 g, 10 min., 4°C) and washed twice with physiological salt solution (0.9% 124

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NaCl in water). Next, the culture was inoculated into chemically defined medium (CDM) 125

containing 0.5% glucose (w/v) for chemostat cultivation. After steady-state had been achieved with 126

six volume changes in chemostat, the fermenters were switched to retentostat mode by withdrawing 127

the effluent through the cross-filter, and pH was controlled at 5.5 through automated 5 M NaOH 128

titration in chemostat and retentostat cultivation (14). To keep the medium composition constant 129

during long-term cultivation, 120-liter batches of medium were prepared, filter sterilized, and used 130

during retentostat cultivations (14). 131

Two independent, carbon source-limited retentostat cultivations were performed under 132

anaerobic conditions, initiating from chemostat cultivation at dilution rates of 0.025 h-1 as 133

previously described (14). Retentostat set-up was assembled with a 1.5-l fermenter (Applikon 134

Biotechnology, Schiedam, The Netherlands) and an autoclavable polyethersulfone cross-flow filter 135

(Spectrum Laboratories, CA, USA). As removal of samples could interrupt biomass accumulation, 136

sample volume and sampling frequency were minimized. 137

Biomass and amino acids determination. During fermentations, culture samples were 138

withdrawn at regular intervals to measure cell dry weight (CDW) and amino acid concentrations. 139

For CDW determination, 5 ml of culture was passed through pre-weighted membrane filters with a 140

pore size 0.45µm (Merck Millipore, Darmstadt, Germany) using a vacuum filtration unit (Sartorius 141

stedim biotech, Gottingen, Germany). Subsequently, membrane filters were dried at 55°C for 24 142

hours, and the biomass collected on the membranes was determined in g/ml. 143

Concentration of amino acids in the culture supernatant and in the medium feed were 144

measured by EZ:fast free amino acid analysis kit (KG0-7165) (Phenomenex, CA, USA). According 145

to the manufacturer’s instructions, the analysis was performed using a gas chromatography (GC) 146

(Thermo Scientific, MA, USA) with a flame ionization detector (FID) (Thermo Scientific, MA, 147

USA). 148

Acidification activity. Acidification profiles were determined in triplicate in 200 µl PBS 149

buffer (initial pH 6) with 0.5% of a chosen carbon source (glucose, ribose, mannitol, sucrose, 150

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fructose, and raffinose) (v/v) at 30°C, inoculated with 5 x 108 cfu/ml of stock cultures of retentostat 151

cultures collected at days 14, 21, 35, and 42, using 96-well microplate HydroPlate® HP96U 152

(PreSens, Regensburg, Germany). These plates encompass an optical pH sensor on the bottom of 153

each well, which can be read-out through the bottom of the plate using a fluorescence reader. 154

According to the manufacturer’s instructions, pH values of cell suspensions in PBS buffers with the 155

chosen carbon source were measured every 10 minutes for 10 hours with a microplate fluorescence 156

reader (Tecan Safire II, Grödig, Austria). The maximum acidification rate (Vmax) value that has an 157

arbitrary and pH-based unit (pH Ux10-3min-1), was calculated on basis of the slope of the pH versus 158

time plot using at least 8 subsequent time-points (regression coefficient > 0.99). 159

RNA isolation and transcriptome analysis. Total L. lactis RNA was isolated from two 160

independent retentostat cultures harvested at days 0, 2, 7, 14, 21, 28, 35, and 42. RNA extraction, 161

reverse transcription, labeling, hybridization, and data analysis were done as previously described 162

(34). Briefly, following methanol quenching, RNA was phenol-chloroform extracted and purified 163

using the High Pure RNA isolation kit (Roche Diagnostics, Mannheim, Germany). Total RNA was 164

used as a template to synthesize cDNA, using the Superscript TMIII reverse transcriptase (RT) 165

enzyme (Invitrogen, Carlsbad, CA), followed by purification by the CyScribe GFX purification kit 166

(GE Healthcare, Buckinghamshire, United Kingdom) and Cy-3 or Cy-5 labeling (Amersham; 167

CyDye postlabeling reactive dye pack; GE Healthcare, Buckinghamshire, United Kingdom). L. 168

lactis KF1471 cDNA was hybridized to OligoWiz (48) designed oligonucleotide DNA microarrays 169

(Agilent Technologies, Santa Clara, CA). After washing (34) slides were scanned at several photo 170

multiplier tube values, and optimal scans were selected on the basis of signal distribution, and data 171

were normalized using the Lowess and inter-slide scaling normalization as available in MicroPrep 172

(46). Median intensities of different probes per gene were taken as absolute gene expression 173

intensities per gene for each condition. The microarray hybridization scheme for the transcriptome 174

analyses at retentostat cultivations consisted of a compound loop design with 26 arrays (Fig. S1). 175

Microarray data and the experimental procedure have been submitted to the NCBI Gene Expression 176

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Omnibus (GEO) under accession number GPL17806 and GSE51494 177

(http://www.ncbi.nlm.nih.gov), respectively. 178

The gene expression intensities were compared and clustered using Short Time-series 179

Expression Minor (STEM) (version 1.3.6, http://www.cs.cmu.edu/~jernst/stem/) (16). The STEM 180

clustering algorithm was used to identify enrichment of Gene Ontology (GO) terms, using 181

Bonferroni correction to determine significance and a maximum number of model profiles of 50. 182

The expressions of genes involved in specific pathways (e.g., glycolysis, pyruvate dissipation, and 183

amino acid metabolism) were projected in heat-maps using the MultiExperiment Viewer (MeV) 184

(http://www.tm4.org/mev/) (37). The correlation of the transcriptome data at each time-point for the 185

two independent retentostat cultivations was calculated by Pearson correlation analysis and 186

displayed as hierarchical clustering using the MeV tool. 187

DNA motif mining. Gene expression data from the significant model profiles identified by 188

STEM were used as data source to identify transcription factor binding sites (TFBSs) in the genome 189

of L. lactis KF147. Binding sites searches were performed using 300 bp upstream regions of each 190

regulated gene using the algorithm for fitting of a mixture model by expectation maximization 191

(MEME) (3), using the parameters mod anr (unlimited number of motifs per upstream sequence), 192

revcomp (allowing motifs to be present on both “+” and “-” strand), and allowing maximally 3 193

motifs to be found in each upstream region without restricting the total number of motifs. The 194

PePPeR database was used as a source of literature based regulon clusters (11). 195

RESULTS 196

Transcriptome data analysis. In our earlier study, metabolic adaptations and physiology of 197

L. lactis KF147 at extreme low-growth rates were studied in anaerobic and carbon-limited 198

retentostat cultivations (sustained for 42 days) (14). During retentostat cultivation biomass 199

accumulated, reaching a plateau level after approximately 14 days and ultimately growth rates 200

declined from 0.025 h-1 to approximately 0.0001 h-1 after 42 days of retentostat cultivation (Fig. 1A; 201

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14). To examine time-resolved transcriptome adaptation of L. lactis KF147 to near- zero growth 202

conditions, samples were taken before starting the retentostat cultivation regime (t = 0 day; 203

chemostat conditions at D = 0.025 h-1), and 2, 7, 14, 21, 28, 35, and 42 days after initiating the 204

retentostat regime, in biologically independent duplicate cultivations. Hierarchical clustering and 205

Pearson correlation analysis illustrated that the transcriptome profiles taken from the two replicates 206

displayed highly similar transcriptome evolutions over time (Pearson correlation > 0.92), supporting 207

the high reproducibility of the experimental set-up (Fig. 1B). In addition, cluster analysis clearly 208

separated transcriptome patterns of samples taken during the growing stages of the experiment 209

(days 0, 2, and 7), from those where growth was stagnating to eventually reach near-zero-growth 210

conditions (days 14, 21, 28, 35, and 42) (Fig. 1B). The discrimination of these two main clusters 211

underpin the separation of the transcriptome signatures related to growth and near-zero growth 212

associated processes. 213

To identify gene expression patterns during the course of the experiment, absolute 214

expression levels of all genes (2533 genes in L. lactis KF147) were subjected to expression cluster 215

analysis using the Short Time-series Expression Minor (STEM) module, which employs a process 216

of statistical clustering of time-series datasets into pre-composed patterns of expression (16). STEM 217

analysis divided the expression patterns into 50 time-resolved model expression profiles, which 218

were sorted on basis of the number of genes assigned to the profile. In total 66.9 % and 64.4 % of 219

the annotated L. lactis KF147 genes in the retentostat duplicates were clustered by STEM into eight 220

and eleven statistically significant model profiles, respectively (Fig. S2A & S2B). Since the 221

congruency between the transcriptome profiles obtained in the replicate experiments was very high, 222

and highly similar STEM profile distributions were obtained for the duplicate retentostat 223

cultivations, the data presented in this study are those obtained from one of the retentostat cultures, 224

which consistently displayed highly congruent expression in the duplicate retentostat cultivation. 225

Since the expression patterns in model profiles 7, 8, 40, and 41 coincided with the carbon 226

metabolism shifts that we described previously (14), we focused on conserved gene sets in these 227

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model profiles (Table 1, Fig. S2 &S4) analyzing the significantly (p-value ≤ 0.05) enriched gene 228

ontology (GO). 229

Cell membrane biosynthesis related processes expression during retentostat cultivation. 230

In STEM-Model profile 8 was enriched for a variety of metabolic processes (Table 1). Genes in this 231

cluster were characterized by progressively reducing expression during the growth associated stages 232

of the retentostat cultivation, followed by a period of stable low expression and subsequent 233

increasing expression during prolonged near-zero growth conditions (Fig. S2 & S3). This cluster 234

also included the category “fatty acid metabolic process” containing the fabDFGHZ and accABCD 235

genes associated with fatty acid biosynthesis (Fig. S4A) of which the expression remained very low 236

also upon prolonged retentostat cultivation. This observation indicates that L. lactis KF147 adapted 237

to near-zero growth by repression of genes related to fatty acid production, thus down regulating the 238

synthesis of one of the main building-blocks of the cell membrane. Model profile 7 clusters genes 239

that were expressed at continuously declining levels during retentostat cultivation (Fig. S2 & S3). In 240

this profile, the overrepresented functional categories related to membrane associated functions 241

(Table 1). These clusters included many ATP-binding cassette (ABC) and phosphotransferase 242

transport systems. In addition, cluster 7 contained genes involved in exopolysaccharide (EPS) 243

synthesis (Fig. S4B & S4C), which is in agreement with the notion that LAB produce and secrete 244

EPSs into their environment only during growth (30). Model profiles 40 and 41 appear to have 245

similar patterns of expression, characterized by initially increasing expression during early stages of 246

retentostat cultivation and remaining stably expressed or slightly declined levels of expression 247

during prolonged retentostat cultivation (Fig. S2 & S3). These model profiles were enriched for 248

functional categories related to RNA synthesis and regulation, transcription and translation (Table 249

1). 250

Central carbon metabolism (CCM) expression during retentostat cultivation. STEM-251

Model profile 8 was enriched for GO terms associated with “organic acid metabolic process” (Table 252

1), and the genes in this class displayed progressively reducing expression during the growth 253

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associated stages of the retentostat cultivation (up to day 7), followed by a period of stable low 254

expression and a recovery of high expression levels during prolonging retentostat conditions and 255

near-zero growth rates (Fig. S2). Thereby, the expression profiles of these genes followed the 256

timing of the carbon and pyruvate dissipation metabolism fluctuations observed during retentostat 257

fermentation (14), which is in good agreement with the “organic acid metabolic process” 258

enrichment. The expression profiles of the genes associated with these processes were visualized 259

using heat-map representation during chemostat (day 0) and retentostat cultivation (Fig. 2). 260

Pyruvate dissipation associated genes displayed a remarkably consistent time-dependent 261

transcription profile during retentostat cultivation. At days 0, 2, and 7, the lactate dehydrogenase 262

encoding gene (ldhL) (involved in lactic acid production) was expressed at a low level, whereas the 263

genes encoding pyruvate dehydrogenase (pdhABCD), alcohol dehydrogenase (adhAE), pyruvate 264

formate lyase (pflA), phosphotransacetylase (eutD), and acetate kinase (ackA1A2), involved in 265

mixed-acid fermentation (production of ethanol, formic acid, and acetate) were highly expressed 266

(Fig. 2B). Subsequently, the transcript level of ldhL was elevated on days 14, 21, and 28, while 267

those of pdhABCD, adhAE, pflA, and eutD, and ackA1A2 were decreased during these stages of 268

cultivation. At the last stage of retentostat cultivation (days 35 and 42), the ldhL gene transcription 269

was again repressed, which coincided with a recovery of the higher expression level of genes 270

involved in mixed-acid fermentation (Fig. 2B). These findings indicate that the fluctuations in 271

pyruvate dissipation behavior during retentostat cultivation (14) accurately reflect the transcriptome 272

profiles of the genes involved in these catabolic pathways, and could explain the mixed-acid and 273

lactic-fermentation switches observed (14). 274

Unlike the regulation of pyruvate-dissipation related genes, the genes of the glycolysis 275

pathway (glk, pgi, pfk, fbaA, tpiA, gapAB, pgk, pmg, enoAB, pyk) were inconsistently regulated in 276

the first week of retentostat cultivation, where all glycolytic genes were repressed or did not change 277

(i.e., < 2-fold change), except the glk, pfk, and fbaA genes (Fig. 2B). However, during subsequent 278

days of retentostat cultivation (days 14 and 21) the expression of glk, pgi, pfk, fbaA, tpiA, gapAB, 279

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pgk, pmg, enoAB, and pyk was consistently up-regulated, whereas their expression was again 280

suppressed during prolonged retentostat cultivation (days 28, 35, and 42), except the gapA, pmg, 281

and enoA genes (Fig. 2B). Moreover, the progressive reduction of the growth rate (Fig. 1A; 14) 282

appears to be paralleled by reduced glycolytic gene expression, although at the time-points that 283

corresponded with increasing lactic acid production, the expression of the glycolytic genes was 284

transiently increased. These findings imply that under retentostat cultivation conditions, the 285

expression pattern of the majority of the glycolytic genes appears to follow the previously described 286

metabolic fluctuations between mixed-acid and lactic-fermentation (14) rather than actual growth 287

rates. The exception appears to be gapA, pmg, and enoA that were continuously expressed at 288

elevated levels after 14 days of retentostat cultivation. Intriguingly, the metabolites associated with 289

these enzymes (substrates and products), also happen to be substrates for the inter-conversion to 290

amino acids biosynthesis, and thereby appear to be consistent with the observed amino acid 291

production at certain stages of the fermentation (see below). 292

Amino acid profiles and amino acid metabolism regulation during retentostat 293

cultivation. Amino acid concentrations were determined (Fig. 3 & S5), at each time-point after 294

initiation of retentostat growth (day 0). The branched chain amino acids (BCAAs) valine, leucine, 295

and isoleucine were constantly consumed during the first week of the retentostat cultivation and 296

subsequently the consumption decreased gradually until days 21 and 28 of the retentostat 297

cultivation, whereas prolonged retentostat cultivation beyond 28 days led to a gradual increase 298

again of the BCAA consumption towards the end of the cultivation (Fig. 3A). Notably, the 299

consumption of the aromatic amino acids (AAAs) phenylalanine, tyrosine, and tryptophan appeared 300

to display similar patterns of concentrations as compared to the BCAAs (Fig. 3B). 301

The GO terms “cellular amino acid metabolic process” and “branched chain family amino 302

acid metabolic process” were overrepresented in transcriptome model profile 8 in both retentostat 303

cultivations (Table 1), and thereby appeared to reflect the pattern of amino acid 304

consumption/production during retentostat cultivation. The ilvABCDH, leuABCD, als, bcaT, and 305

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aspC genes that encode enzymes responsible for BCAAs biosynthesis displayed similar 306

transcription patterns during retentosat cultivation. Their expression fluctuated during the first week 307

of the retentostat cultivation, but consistently decreased after 7 days, followed by increased 308

expression after 35 days of retentostat cultivation (Fig. 4). Notably, genes associated with 309

tryptophan (trpCDFGS) and histidine (hisABDFGHIKZ) syntheses were also clustered in model 310

profile 8 and displayed a similar expression pattern as the BCAA associated genes (Fig. 5). These 311

transcriptional profiles establish a relatively good correspondence between the 312

consumption/production of these amino acids and their biosynthetic pathway encoding genes. 313

The other amino acids appeared to display similar concentration fluctuations as compared to 314

BCAAs and AAAs during retentostat cultivation (Fig. S5). Notably, at certain time-points (days 14 315

and 21) several amino acids appeared to be net-produced by the culture. At these time-points, the 316

genes encoding glutamate synthase, a glycerol-3-phosphate transporter, and a glutamate ABC 317

transporter (gltBDPS), as well as cysteine synthase (cysK), a lysine specific permease (lysQ) and a 318

homoserine kinase and threonine synthase (thrBC) were expressed at low levels, while their 319

expression was higher at stages prior and after the period of net production (Fig. S6). Conversely, 320

the diaminopimelate decarboxylase encoding lysA gene displayed an expression profile that is the 321

inverse of the related lysQ gene (Fig. S6). Taken together, the production of glutamic acid, cysteine, 322

threonine, and lysine coincides with the period that is characterized by enhanced lactic fermentation 323

and could be controlled by the gltBDPS, cysK, thrBC, lysAQ genes, respectively. 324

Identification of a cis-acting DNA-motif potentially involved in near-zero growth gene 325

regulation. Transcriptional regulators strongly control expression levels of genes by binding to 326

TFBSs. To identify candidate DNA-motifs that are potential TFBSs involved in adaptation to near-327

zero growth conditions, we searched for overrepresented DNA sequences in the upstream regions of 328

genes that showed correlated expression. As a result, a highly conserved motif, encompassing the 329

palindromic sequence element 5’-CTGTCAG-3’ (Fig. 6A) was identified in profile 7 and 8. The 330

motif is present upstream of genes related to synthesis of BCAAs (ilvAB, ileS, leuC), histidine 331

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(hisB), cysteine (cysK), arginine (argBF, arcD), serine (serS), tryptophan (trpG), and fatty acid 332

(accD, fabZ); and peptide uptake (dtpT, pepC) (Fig. S7), suggesting that it could play a key role in 333

the adaptation to near-zero growth conditions by its role in nitrogen metabolism regulation of L. 334

lactis KF147 (see discussion). 335

Genome level prediction of enhanced catabolic flexibility of L. lactis KF 147. Model 336

profiles 40 and 41 also encompasses many genes involved in uptake and metabolism of alternative 337

carbon sources such as ribose, mannitol, galacturonate, raffinose, sucrose, and fructose. These genes 338

were relatively lowly expressed during the growth associated stages of retentostat cultivation (up to 339

day 14) and subsequently became gradually higher expressed upon prolonged retentostat 340

cultivation, reaching very high levels of expression during the near-zero growth conditions reached 341

at the end of cultivation (days 35 and 42) (Fig. 7 & S8). For example, the expression of genes 342

associated with uptake and metabolism of ribose (rbsABCDKR) and mannitol (mtlADFR) was 343

increased more than 10-fold during prolonged retentostat cultivation (Fig. 7). These observations 344

illustrate the transcriptional response to prolonged and severe carbon source limitation that is 345

encountered during prolonged retentostat cultivation, leading to the progressive derepression and/or 346

activation of expression of several genes required for the catabolism of alternative carbon sources in 347

L. lactis KF147. These responses raised the question whether these retentostat adapted L. lactis 348

KF147 cultures would display significantly enhanced catabolic flexibility and would more readily 349

and rapidly ferment carbon sources other than glucose. To address this question, the fermentation 350

rate of various carbohydrates by non-growing bacterial suspensions of L. lactis KF147 withdrawn 351

from the retentostat culture on days 14, 21, 35, and 42 were determined. 352

To this end, the maximum acidification rate (Vmax), which was expressed using an arbitrary, 353

pH-based unit (pH Ux10-3min-1; see materials and methods), was determined in L. lactis KF147 354

cell-suspensions derived from the retentostat culture on days 14, 21, 35, and 42 by incubation with 355

glucose, ribose, mannitol, sucrose, fructose, or raffinose as the fermentable substrate. Since the 356

culture biomass did not significantly increase after 14 days of retentostat cultivation and at this 357

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stage the expression level of the relevant genes appeared to be unaffected compared to carbon-358

limited chemostat growth conditions, this sample was used as a reference in these analyses. The 359

highest Vmax values were obtained from the sample taken after 42 days of retentostat cultivation, for 360

all carbon sources used (Table 2). Specifically, the Vmax of the cell suspension derived from the 361

retentostat culture on day 42 was several folds higher than the Vmax of the suspension based on day 362

14 cultures for the carbon sources ribose (3-fold), mannitol (5-fold), sucrose (2-fold), and raffinose 363

(3-fold), respectively (Table 2). Only the acidification rates obtained for fructose and glucose were 364

similar between all time-points evaluated (days 14, 21, 35, and 42), where glucose served as a 365

positive control in these analyses and was consistently associated with the highest acidification rate 366

for all of the suspensions tested (Table 2). These results confirmed that the induced transcription of 367

genes involved in alternative sugar utilization pathways enabled the cells to rapidly adjust to the 368

utilization of alternative carbon sources when these became available. Notably, the progressive 369

derepression of metabolic pathways dedicated to the use of alternative carbon and energy sources 370

clearly exceeds the basal levels of expression that can be seen upon initial carbon limitation 371

conditions, e.g., during carbon-limited chemostat cultivation. 372

DISCUSSION 373

This paper presents the molecular adaptation of L. lactis to near-zero growth rates induced 374

by carbon-limited retentostat cultivation and thereby expands our previous analysis of the 375

quantitative energy household and physiology of this bacterium under these conditions (14). 376

Genome-wide transcriptional data were integrated with metabolite datasets of organic- and amino-377

acid production and consumption under retentostat conditions, allowing the identification of 378

transcription signatures that reflect near-zero growth adaptation inferred by retentostat cultivation. 379

Transcriptome adaptations established the repression of several growth associated functions, 380

specifically those related to the biosynthesis of particular macromolecules, i.e., membrane 381

components and extracellular polysaccharides. Notably the retentostat conditions did not induce 382

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stringency-like responses and genes encoding the components of the machineries for DNA 383

replication, transcription and translation remained relatively highly expressed. 384

Our previous study (14) highlighted that L. lactis KF147 retentostat cultures display 385

intriguing metabolic switches within their central carbohydrate and energy metabolism, fluctuating 386

between mixed-acid and lactic fermentation. Notably, in the present study we show that the 387

fermentation end-product analyses (14) are congruent with the transcriptional patterns of the genes 388

involved in the corresponding pathways, implying that the distribution of pyruvate among the 389

different dissipation pathways is not only controlled by allosteric interactions or redox-balance 390

changes (9, 21), but also controlled at the transcriptional level in L. lactis KF147. Intriguingly, 391

carbon-starved batch cultures of L. lactis strongly suppressed carbon metabolism genes during the 392

stationary phase, but sustained high expression of glycolytic and pyruvate dissipation functions 393

(36). In the current experiments, genes encoding glycolytic enzymes were transiently expressed at 394

elevated levels during early stages of stagnated growth (days 14 to 28), which are proposed to lead 395

to enhanced glycolytic flux that may drive the changes of pyruvate dissipation from mixed-acid 396

towards lactic fermentation, which includes boosted expression of the lactate dehydrogenase. The 397

transcriptional control of glycolytic and pyruvate dissipation pathways appears to be in apparent 398

contradiction with previously proposed regulation by enzyme level control through allosteric 399

interactions with glycolytic intermediates, ATP demand and/or carbon source import rates (8, 21, 400

36, 45). Several glycolytic enzymes have been assigned key roles in the regulation of glycolytic 401

flux in L. lactis as a function of the specific strain of this species and its environmental condition, 402

like the pyruvate kinase and phosphofructokinase in L. lactis MG1363 during batch cultivation on 403

maltose (42, 1) or the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in strain NCDO 2118 404

during batch growth on lactose (18). Although the regulation of glycolytic flux in L. lactis is not 405

entirely understood, it has been shown that its flux is not controlled by an individual and rate-406

limiting glycolytic enzyme nor dictated by sugar import rates (33), although the latter have been 407

proposed to predominantly regulate glycolytic flux under conditions of very low carbon flux (8). 408

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Glycolytic flux also appears to be controlled by the cellular energy state, which could elegantly be 409

shown by decreasing ATP levels by increasing F1F0-H+-ATPase expression, which could drive up 410

to 3-fold increased glycolytic flux but led to declining growth rates and biomass yields (28, 29). 411

Nevertheless, mathematical models using the kinetic parameters of the enzymes that constitute the 412

lactococcal glycolytic pathway demonstrated that glycolytic flux could accurately be predicted with 413

such model. This is quite remarkable since the kinetic parameters of enzymes are commonly 414

obtained from in vitro experiments that in many cases neglect many potentially relevant interactions 415

and modifications, including transcriptional regulation, protein phosphorylation, or allosteric 416

modulation (33). The present study revealed that transcriptional regulation contributes to glycolytic 417

flux and pyruvate dissipation control in L. lactis KF147. This could be specific for lactococci 418

isolated from plant origins, like KF147, which would be supported by the many studies that address 419

metabolic control in dairy L. lactis strains that reach alternative but not necessarily mutually 420

exclusive or consistent control-conclusions. Alternatively, this could also be specific for the 421

conditions employed in this study, and thus depend on the specific metabolic characteristics induced 422

by near-zero growth conditions. 423

The response of L. lactis KF147 to extreme low-growth rates also included some remarkable 424

fluctuations of amino acid metabolism. During the continuous reduction of the growth rate in the 425

first retentostat cultivation period, the overall declining rate of amino acid consumption is most 426

likely explained by the reduced requirement for these biomass building-blocks, which has 427

previously also been shown for batch cultures that enter the stationary phase of growth (36). 428

Intriguingly, the transcription of the genes encoding the glycolytic enzymes GAPDH, 429

phosphoglycerate mutase, and enolase which are involved in the production of the intermediates 3-430

phosphoglycerate (3PG), phosphoenolpyruvate (PEP), and pyruvate, were continuously 431

overexpressed after 14 days of retentostat cultivation. These glycolytic intermediates are known to 432

serve as substrates in reactions that link the CCM (central carbon metabolism) to amino acid 433

synthesis, and are involved in the pathways that lead to Ser, Cys, and Gly; Phe, Trp, and Tyr; Ala, 434

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Ile, Leu, Val, and Thr synthesis, respectively (20, 35). This possible role of these intermediates is 435

supported by the observation that intermediate stages (days 14-28) of retentostat cultivation were 436

associated with the net production of certain amino acids and in parallel expressed their glycolytic 437

genes at elevated levels. 438

In several low-GC Gram-positive bacteria, including L. lactis, the transcriptional regulator 439

CodY controls the expression of degradation of oligopeptides, uptake and metabolism of 440

di/tripeptides and amino acids, especially BCAAs, Asn, Glu, His, and Arg, in response to the 441

availability of amino acids or peptides (6, 12). When lactococcal cultures reach the stationary phase, 442

and nutrients become limited, CodY-dependent repression of peptide and amino acid transporter 443

systems is relieved to maintain nitrogen metabolism in the cells (12). Moreover, CodY has also 444

been shown to modulate functions in carbon metabolism since it has been shown to control the 445

expression of citrate synthase (gltA), isocitrate dehydrogenase (icd), and aconitase (citB) that belong 446

to the incomplete Krebs cycle in L. lactis MG1363(12). Importantly, this incomplete Krebs cycle 447

can support the production of α-ketoglutarate, which can serve as a substrate for glutamate 448

production and as a co-substrate for the first step of BCAA catabolism (12, 51), illustrating how 449

CodY regulation could connect nitrogen- to carbon-metabolism regulation (12). Similarly, the 450

detection of a CodY-like regulatory motif in the near-zero growth regulons of L. lactis KF147, led 451

us to propose CodY mediated control of nitrogen metabolism during these conditions, although the 452

expression of the codY gene itself appeared constitutive under the experimental conditions used in 453

this study. This role could include a connecting regulatory role of CodY in CCM regulation via the 454

glycolytic intermediates 3PG, PEP, and pyruvate (see above). Moreover, the connection of pyruvate 455

metabolism and BCAA biosynthesis (Fig. 4A), also implies that CodY could indirectly influence 456

pyruvate dissipation and thereby may play a role in controlling lactic versus mixed-acid 457

fermentation behavior in L. lactis KF147 under retentostat conditions (Fig. 8). 458

In L. lactis MG1363, a 15-bp cis-element with the consensus AATTTTCWGAAAATT has 459

been identified as a high affinity binding site for CodY (12; Fig. 6B). The proposed CodY binding 460

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motif identified in L. lactis KF147 resembles the CodY motif of strain MG1363 (12), despite 461

differences in the deduced consensus sequences for each strain (Fig. 6C). Notably, the motif 462

identified in strain KF147 encompasses a palindromic element and frequently is encountered in a 463

head-to-tail tandem orientation upstream of the identified genes, expanding the palindromic nature 464

of the composite cis-acting element. Therefore, we propose that the motif we identified represents 465

the CodY target site in L. lactis KF147, and that CodY plays a prominent role in the regulation of 466

nitrogen metabolism adaptations in L. lactis KF147 under extremely low-growth rates, and may 467

possibly also control the typical carbon-metabolism fluctuations observed under these conditions in 468

a more indirect manner (Fig. 8). The relevance of nitrogen metabolism regulation in the context of 469

industrial applications of L. lactis is obvious since it has been well established that flavor formation 470

for example in cheese ripening is driven largely by amino acid conversions that involve nitrogen 471

metabolism associated enzymes (40). 472

This study also established that genes involved in import and utilization of alternative 473

carbon sources (other than glucose) were progressively derepressed during prolonged retentostat 474

cultivation, enabling the bacteria to more rapidly switch to alternative energy and carbon resources 475

upon their availability in the environment. This adaptation may reflect the evolutionary benefit of 476

scavenging trace amounts of nutrients in growth limiting environments, which clearly go beyond 477

the relief of catabolite control protein A (CcpA)-mediated carbon catabolite repression (CCR; 13, 478

19, 31, 38, 49), since CCR relief was already effective in the carbon-limited chemostat culture that 479

was used to initiate the retentostat cultivation. In LAB, CcpA-mediated CCR includes the 480

repression of catabolic operons in response to the availability of a “preferred” carbon source, which 481

commonly is glucose (19). Intriguingly, in L. lactis CcpA also acts as an activator for the las operon 482

that encodes three key-enzymes in the glycolytic pathway, phosphofructokinase (pfk), pyruvate 483

kinase (pyk) and lactate dehydrogenase (ldh), creating a CcpA connection to central metabolism 484

control (17, 32, 42). The role of CcpA and/or its co-regulators (e.g., HPr, ATP/ADP levels etc.) in 485

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the high level induction of alternative carbon utilization systems during near-zero growth conditions 486

remains to be determined (Fig. 8). 487

In conclusion, transcriptome and metabolite adaptations of L. lactis KF147 to near-zero 488

growth rates inferred by retentostat cultivation are clearly distinct from those elicited by starvation- 489

or stationary phase conditions, and includes particular fluctuating metabolic behavior. The 490

regulation of nitrogen metabolism, and indirectly, possibly also the fluctuating mixed-acid and 491

lactic fermentation patterns, might involve CodY (Fig. 8) through the identified cis-acting motif that 492

resembles the previously reported CodY-box. Retentostat cultivation also led to a progressive relief 493

of carbon catabolite repression and the activation of pathways associated with the utilization of 494

alternative substrates, which goes beyond the canonical CcpA-mediated carbon catabolite 495

regulation. Intriguingly, recent work in Bacillus subtilis established that CcpA and CodY can form a 496

complex that interacts with RpoA, underpinning the interactions between the gene-regulation 497

networks involved in carbon- and nitrogen-metabolism regulation (50). The gene regulation profiles 498

identified in this study include several CcpA and CodY target genes and their regulation may 499

involve a similar regulatory complex encompassing both CodY and CcpA. 500

ACKNOWLEDGEMENTS 501

We thank Sacha van Hijum for designing the hybridization scheme, Jan van Riel for 502

technical assistance with GC, and Marjo Starrenburg for her assistance during hybridization and 503

scanning procedures (NIZO food research, Ede, The Netherlands). In addition, we thank our 504

colleagues from Industrial Microbiology Section, Delft University of Technology and Molecular 505

Genetics Group, University of Groningen in the joint zero-growth project group (Kluyver Centre, 506

Netherlands) for invaluable discussions. 507

This project was carried out within the research programme of the Kluyver Centre for 508

Genomics of Industrial Fermentation which is part of the Netherlands Genomics Initiative / 509

Netherlands Organization for Scientific Research. 510

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50. Wünsche, A., Hammer, E., Bartholomae, M., Völker, U., Burkovski, A., Seidel, G., and 641

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TABLES 658

Table 1: Gene Ontology (GO) enrichment analysis. This table contains the GO enrichment results 659

for the set of genes shown in the figure S3A and S3B where the enrichment is computed based on 660

actual size enrichment (p-value ≤ 0.05). 661

Model Profile # GO Category Name

7 Membrane

Integral to membrane Membrane part

8

Cellular amino acid metabolic process Fatty acid metabolic process

Organic acid metabolic process Carboxylic acid biosynthetic process

Lipid metabolic process Primary metabolic process

Glutamine family amino acid metabolic process Branched chain family amino acid metabolic process

Arginine metabolic process

40

Sequence-specific DNA binding transcription factor activity RNA biosynthetic process

Regulation of RNA metabolic process Regulation of cellular biosynthetic process

Regulation of gene expression Regulation of macromolecule metabolic process

41

Regulation of transcription, DNA-dependent Carbohydrate kinase activity

Regulation of RNA biosynthetic process Regulation of biological process

662

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Table 2: Maximum acidification rates on different carbon source of L. lactis KF147 samples taken 664

on days 14, 21, 35, or 42 of the retentostat cultivation period. 665

Carbon sources Vmax (pH Ux10-3min-1)* 14 21 35 42

Glucose 3.9 ± 0.34 2.7 ± 0.69 3.7 ± 0.41 5.6 ± 0.43 Ribose 0.2 ± 0.05 0.1 ± 0.06 0.5 ± 0.05 0.6 ± 0.06

Mannitol 0.2 ± 0.03 0.2 ± 0.04 0.7 ± 0.03 1.0 ± 0.02 Sucrose 0.5 ± 0.02 0.5 ± 0.01 1.1 ± 0.04 1.1 ± 0.03 Fructose 2.3 ± 0.05 1.8 ± 0.06 2.3 ± 0.04 2.5 ± 0.05 Raffinose 0.3 ± 0.01 0.3 ± 0.01 0.6 ± 0.02 0.9 ± 0.02

* R2, regression coefficient > 0.99 666

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FIGURES 682

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692

693

694

Fig. 1: Growth of L. lactis KF147 and hierarchical clustering linkage of transcriptome profiles in 695

retentostat culture. A steady-state anaerobic chemostat culture was switched to retentostat mode at 696

time zero. (A) Data points represent average ± mean deviation of measurements of two independent 697

cultures. Specific growth rate (h-1) (diamonds) and biomass accumulation (g.l-1) (squares) of L. 698

lactis KF147 under retentostat conditions (adapted from Ercan, et al., 2013). (B) Hierarchical 699

clustering linkage of retentostat 1 (R1) and 2 (R2) samples. Complete clustering linkage was 700

performed for samples days on 0, 2, 7, 14, 21, 28, 35, and 42 of duplicate retentostat cultivations 701

based on Pearson correlation with using MeV. 702

703

A

B

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704

705

706

707

708

709

710

711

712

713

714

715

716

717

718

719

720

721

Fig. 2: Overview of central carbon metabolism in L. lactis KF147. (A) Simple scheme of glycolysis 722

and pyruvate dissipation pathways. One arrow represents one metabolic reaction and dashed line 723

arrows correspond to more than one reaction. Genes are indicated beside the arrows. End-products 724

are indicated in ellipses. (B) Heat-map of L. lactis KF147 glycolysis and pyruvate dissipation genes 725

differentially expressed (on log2-scale, p-value ≤ 0.05) during retentostat cultivation over the 726

beginning of the chemostat (day 0) (retentostat 1). Similar transcriptome results obtained in 727

retentostat 2 confirmed the consistency of these results in an independent experiment. 728

729

Glucose

Fructose-1,6-P

3-P-Glycerate

PyruvateLactate

Acetyl-CoA

Formate

Acetyl-PAcetaldehydeEthanol

Acetate

glk pgi pfk

fbaA tpiA gapAB pgk

pmg enoAB pyk

ldhL

pflA pdhABCD

adhE

adhA

eutD

ackA1A2

Substrate

Product

A

B

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730

731

732

733

734

735

736

737

738

739

740

741

Fig. 3: Concentration of branched chain amino acids (BCAAs) (A) and aromatic amino acids 742

(AAAs) (B) in L. lactis KF147 in retentostat culture. Data points represent average ± mean 743

deviation of measurements of two independent cultures. (A) Concentration of Val (diamonds), Leu 744

(squares), and Ile (triangles). (B) Concentration of Phe (diamonds), Tyr (squares), and Trp 745

(triangles). All concentrations in panels A and B are presented as the difference between the 746

measured concentration in the medium feed and the measured concentration in the filter line efflux 747

samples. Negative numbers indicate net-consumption; positive numbers indicate net-production. 748

749

750

A

B

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751

752

753

754

755

756

757

758

759

760

761

762

763

764

Fig. 4: Overview of branched chain amino acids (BCAAs) biosynthesis in L. lactis KF147. (A) 765

Simple scheme Ile, Val, and Leu amino acid production pathways. One arrow represents one 766

metabolic reaction and dashed line arrows correspond to more than one reaction. Genes are 767

indicated beside the arrows. End-products are indicated in ellipses. (B) Heat-map of L. lactis KF147 768

BCAAs biosynthesis genes differentially expressed (on log2-scale, p-value ≤ 0.05) during 769

retentostat cultivation over the beginning of the chemostat (day 0) (retentostat 1). Similar 770

transcriptome results obtained in retentostat 2 confirmed the consistency of these results in an 771

independent experiment. 772

773

774

775

776

A

B

Pyruvate

(S)-3-Methyl-2-oxopentanoate

Isoleucine

(S)-2-Acetolactate

Valine

(S)-2-Aceto-2-hydroxybutanoate

Leucine

2-Oxoisovalerate

(2S)-2-Isopropylmalate

Acetyl-CoA

(R)-2-Methylmalate

2-OxobutanoateThreonine leuB leuC leuD ilvA

als ilvB ilvH

ilvC ilvD

bcaT

leuA

leuB leuC leuD bcaT

Pyruvate metabolism

aspC

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777 778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

793

794

795

796

Fig. 5: Overview of Trp (AAA) and His biosynthesis in L. lactis KF147. (A) Simple scheme Trp 797

and His amino acid production pathways. One arrow represents one metabolic reaction and dashed 798

line arrows correspond to more than one reaction. Genes are indicated beside the arrows. End-799

products are indicated in ellipses. (B) Heat-map of L. lactis KF147 BCAAs Trp and His 800

biosynthesis genes differentially expressed (on log2-scale, p-value ≤ 0.05) during retentostat 801

cultivation over the beginning of the chemostat (day 0) (retentostat 1). Similar transcriptome results 802

obtained in retentostat 2 confirmed the consistency of these results in an independent experiment. 803

hisD

hisGZPRPP Phosphoribosyl-

ATP

Phosphoribosyl-AMP

hisI

Phosphoribosyl-formimino-AICAR-P

Phosphoribulosyl-formimino-AICAR-P

Imidazole-glycerol-3P

Imidazole-acetol-P

Histidinol-P

Histidinol

Histidinal

Histidine

Pentose phosphate pathway

hisI

hisB

hisFH

hisA

hisC

hisK

hisD

A

B

Indole

trpB

Glycolysis Chorismate

Anthranilate

trpEG

trpD

N-(5-phospho-D-ribosyl)

anthranilate

1-(2-carboxyphenylamino)-1-deoxy-D-ribulose

5-phosphate

trpF

Indoleglycerol phosphate

trpC

trpAB

trpA

Tryptophan

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804

805

806

807

808

809

810

811

812

813

814

Fig. 6: Weblogo visualization of the postulated CodY motif in L. lactis KF147 (A) and the 815

experimentally identified CodY motif in L. lactis MG1363 (12) (B); alignment of the CodY motifs 816

from both strains. (A) The postulated CodY binding sequence found in L. lactis KF147. The 817

CTGTCAG palindrome sequence that forms the core of the motif is positioned from nucleotide 2 to 818

8. The thymidine at position 5 appears to be conserved as well. (B) The experimentally verified 819

CodY motif in L. lactis MG1363. (C) The consensus CodY motifs identified in L. lactis MG1363 820

and L. lactis KF147, in which the proposed motif sequence is underlined. 821

822

823

824

825

826

827

828

829

Strain Sequence L. lactis KF147 TCTGTCAGTAAATTT (in this study)

L. lactis MG1363 AATTTTCWGAAAATT (12)

B

A

C

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830

831

832

833

834

835

Fig. 7: Expression graphs of genes involved in (A) ribose and (B) mannitol uptake and metabolism 836

during retentostat cultivation over the beginning of the chemostat (day 0) from model profiles 40 837

and 41 (retentostat 1). Similar transcriptome results obtained in retentostat 2 confirmed the 838

consistency of these results in an independent experiment. 839

840

841

842

843

844

845

846

847

848

849

850

851

852

Fig. 8: Integrated view of adaptive regulation of L. lactis KF147 to near-zero growth rates induced 853

by retentostat cultivation. In rectangular boxes, red and green indicate increased and decreased 854

expression of the mentioned functional categories, respectively. 855

A B

Retentostat mode

Enhanced expression of alternative carbon source systems

Relief of carbon catabolite repression

switch to retentostat mode

Growing state Transition state Near-zero growth state

Nitrogen metabolism control

Glycolytic flux control

Mixed-acid fermentation control

Chemostat

HPr CcpA

CodY

Lactic acid fermentation control

?

7 14 420

Days

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