The Transcriptional Response of Lactobacillus sanfranciscensis DSM20451T and Its tcyB Mutant Lacking a Functional Cystine Transporterto Diamide Stress
Mandy Stetina, Jürgen Behr, Rudi F. Vogel
Technische Universität München, Lehrstuhl für Technische Mikrobiologie, Freising, Germany
As a result of its strong adaptation to wheat and rye sourdoughs, Lactobacillus sanfranciscensis has the smallest genome withinthe genus Lactobacillus. The concomitant absence of some important antioxidative enzymes and the inability to synthesize gluta-thione suggest a role of cystine transport in maintenance of an intracellular thiol balance. Diamide [synonym 1,1=-azobis(N,N-dimethylformamide)] disturbs intracellular and membrane thiol levels in oxidizing protein thiols depending on its initial con-centration. In this study, RNA sequencing was used to reveal the transcriptional response of L. sanfranciscensis DSM 20451T
(wild type [WT]) and its �tcyB mutant with a nonfunctional cystine transporter after thiol stress caused by diamide. Along withthe different expression of genes involved in amino acid starvation, pyrimidine synthesis, and energy production, our resultsshow that thiol stress in the wild type can be compensated through activation of diverse chaperones and proteases whereas the�tcyB mutant shifts its metabolism in the direction of survival. Only a small set of genes are significantly differentially expressedbetween the wild type and the mutant. In the WT, mainly genes which are associated with a heat shock response are upregulatedwhereas glutamine import and synthesis genes are downregulated. In the �tcyB mutant, the whole opp operon was more highlyexpressed, as well as a protein which probably includes enzymes for methionine transport. The two proteins encoded by spxAand nrdH, which are involved in direct or indirect oxidative stress responses, are also upregulated in the mutant. This work em-phasizes that even in the absence of definitive antioxidative enzymes, bacteria with a small genome and a high frequency of geneinactivation and elimination use small molecules such as the cysteine/cystine couple to overcome potential cell damage resultingfrom oxidative stress.
Lactobacillus sanfranciscensis is a highly adapted species, which candominate wheat and rye sourdough microbiota. As a result of this
strong adaptation, it has lost many genes present in other lactobacilliand therefore has the smallest genome currently known within thegenus Lactobacillus (1). Aerobic growth in this strain results in ahigher final cell yield and growth rate than does an anaerobicenvironment. While many lactic acid bacteria (LAB) produce re-active oxygen species (ROS), e.g., via the NADH oxidase 1 reac-tion, L. sanfranciscensis expresses NADH oxidase 2, which directlyproduces water and thus minimizes oxidative stress from that re-action (2). Still, the published genome data of L. sanfranciscensisTMW 1.1304 revealed enzymes which provide the strain’s abilityto cope with the formation of radicals. Genes thus far related tooxidative stress response include those for NADH oxidase, gluta-thione (GSH) reductase, glutaredoxin-like protein, two thiore-doxin reductases, putative thioredoxin peroxidase, three thiore-doxin-like proteins, and a cyst(e)ine transport protein (1).
Cysteine acts in the catalytic site of enzymes, helps in proteinfolding by forming disulfide bonds, and is a precursor of manymolecules (methionine, glutathione, biotin, coenzyme A [CoA],thiamine, etc.) with diverse functions. Because of its reactive SHgroup, proteins in L. sanfranciscensis such as thioredoxin (reduc-tase), thioredoxin-like protein, and glutaredoxin-like protein relyon the reduced form of that sulfur-containing amino acid. Theseknown thiol/disulfide oxidoreductases are involved in regulation ofthe intracellular redox balance in this bacterium. The import of cys-teine is realized directly by ABC transporters or symporters, whichpredominantly import it as cystine. Bacterial cystine transport sys-tems exhibit a high specificity for cystine. The role of cystine trans-porters has been described for Bacillus subtilis (3), Lactobacillus reu-
teri BR11 (earlier classified as Lactobacillus fermentum BR11 [4,5]), and Escherichia coli (6). In B. subtilis, three L-cystine trans-porters (two high- and one low-affinity transporter) exist,whereas in E. coli, two different systems participate in cystinetransport with different substrate specificities and selectivity (3,6). A cyuC mutant in L. reuteri BR11 which lacks the cystine trans-porter was not able to retain high intracellular concentrations ofcysteine which were necessary for aerobic growth (4).
In the L. sanfranciscensis TMW 1.1304 genome, tcyB (LSA_08550) encoding the cystine transporter displays four predictedtransmembrane segments and no signal peptide as analyzed withPhobius (7). The liberation of cystine or cysteine from oligopep-tides would be also possible, especially because lactic acid bacteriaare known for their preference for importing peptides via oligo-peptide transporters.
Previous work indicates the participation of cysteine/cystinetransport in the oxidative stress response. Severe growth defects inmedium without cysteine during aerobiosis and in the presence ofparaquat could be observed for an L. sanfranciscensis DSM 20451T
�gshR mutant and an L. sanfranciscensis DSM 20451T �nox mu-tant, which were also sensitive to diamide treatment (2, 8). Afteraddition of cysteine to the medium, these effects could be re-versed. In agreement with the data for L. sanfranciscensis, the de-letion of the cystine uptake gene in L. reuteri BR11 led to defectivegrowth in the presence of oxygen and increased sensitivity to para-quat (9). The mutant was not able to export sulfhydryl groups andtherefore had a decreased ability to build a reductive environment,which can exhibit a protective barrier. When oxidizing conditionswere used, an increased expression of the cystine transporter bspA(cyuC) was measured in the latter strain, suggesting a role of thistransporter under oxidizing conditions (10). A mutant of the cys-tine transporter of L. reuteri (L. reuteri BR11�cyuC) reachedhigher extracellular thiol levels than did the wild type (WT), al-though the strain was able to accumulate thiols from substratesother than cystine (11).
Diamide [1,1=-azobis(N,N-dimethylformamide)] as a thiol-oxidizing agent has the advantage of penetrating easily throughmembranes, and it acts not via radical formation but through thioloxidation. Depending on the initial diamide concentration, intra-cellular and membrane protein thiols are oxidized, which leads toperturbation of the cell’s “thiol status,” and regeneration dependsmainly on the presence and concentration of reductive com-pounds (12).
To date, no data are available on overall transcriptional re-sponses in L. sanfranciscensis to environmental changes or stresses.In this work, we focused on transcriptional analysis of thiol stressand a possible participation of cysteine/cystine transport to eluci-date whether cystine transport also contributes to intracellularthiol homeostasis through activation or repression of transcriptsof “redox” genes or simply serves nutritional requirements in L.sanfranciscensis DSM 20451T. Apart from the impact on the bac-terium, the oxidation/reduction of thiols in cereal proteins is im-portant to form protein networks and influence proteolysis inwheat and rye sourdoughs, which are the habitat of this species. Sofar, studies of other LAB have focused mainly on growth behaviorin different media with different, mostly oxidizing agents (H2O2,paraquat, and diamide); determination of intracellular and extra-cellular thiol groups; and testing of transport activities/specifici-ties with labeled or unlabeled components. RNA sequencing is arelatively new emerging technology which has the potential toshed light on regulation at the transcriptional level. The broaddynamic range for gene expression level quantification, revelationof different gene isoforms, low background noise, and single-baseresolution indicate some of the advantages of this technology (13).In this work, we outlined the transcriptional stress response of L.sanfranciscensis DSM 20451T (wild type [WT]) and its deletionmutant lacking a functional cyst(e)ine transporter (�tcyB) afterthiol stress induced by diamide treatment.
MATERIALS AND METHODSStrain selection. L. sanfranciscensis TMW 1.53 (isogenic with strain DSM20451T) was chosen for all experiments. In the text, we use the designationDSM 20451T for easier reference.
General molecular techniques and insertional inactivation of thetcyB gene by single crossover integration. Procedures for cloning, DNAand plasmid manipulations, and agarose gel electrophoresis were per-formed as already described (14). For chromosomal DNA isolation, theE.Z.N.A. bacterial DNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA)was used according to the protocol of the supplier with a few adaptations.E. coli plasmid DNA was isolated with the plasmid minikit from Omega
Bio-Tek (Norcross, GA, USA). Restriction endonuclease digestions andligations with T4 DNA ligase were performed as indicated by the supplier(Fermentas, St. Leon-Rot, Germany). PCR was conducted in thermocy-clers (Eppendorf, Wesseling-Berzdorf, Germany) by using Taq polymer-ase, buffer, and deoxynucleoside triphosphates from MP Biomedicals(Santa Ana, CA). PCR products were purified with the QIAquick purifi-cation kit (Qiagen, Hilden, Germany). Sequencing was performed by thecompany GATC Biotech (Constance, Germany). Sequence analysis wascarried out with ChromasPro, and alignments were conducted withthe online tool ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/).Transformations were carried out with a Bio-Rad gene pulser apparatus in0.2-mm cuvettes (Bio-Rad Laboratories, Hercules, CA) with the followingparameters for lactobacilli: 1.2 kV, 25 �F, and 1,000 �.
For insertional inactivation of the tcyB gene, a 592-bp fragment basedon the genome of L. sanfranciscensis was obtained with PCR using primersCys_F_1 and Cys_R_1 (Table 1), carrying BamHI restriction sites. Diges-tion and ligation into plasmid pME-1 resulted in the nonreplicating inte-gration vector pME-1�tcyB. The vector was cloned into E. coli DH5� andisolated with the plasmid minikit (Omega Bio-Tek, Norcross, GA, USA).
For the preparation of electrocompetent L. sanfranciscensis cells, onecolony of the strain was grown on modified de Man-Rogosa-Sharpe(mMRS [modified MRS, containing no vitamin mix but an additional 5g/liter fructose]) medium supplemented with 1% (wt/vol) glycine at 30°Cin a water bath covered with aluminum foil to an optical density at 590 nm(OD590) of 0.6. The cells were centrifuged at 4°C (5,500 � g, 15 min) andwashed three times with 40 ml of 10 mM MgCl2 solution, once withglycerol (10%, vol/vol), and once with glycerol-sucrose solution (10%,vol/vol; 0.5 M). The cells were resuspended in glycerol-sucrose solution,incubated on ice for 20 min, aliquoted to 80 to 100 �l, frozen with liquidnitrogen, and stored at �80°C. All centrifugation steps were carried out at4°C, and all washing and storage solutions were cooled on ice. After elec-troporation, the cells were immediately recovered with prewarmedmMRS medium and incubated in the water bath at 30°C for 5 h prior toplating on mMRS plates with 10 �g/ml erythromycin. Erythromycin-resistant colonies were plated again onto mMRS (plus 10 �g/ml erythro-mycin) plates and cultured also in liquid medium with 5 �g/ml of eryth-romycin added. To verify the insertion of plasmid pME-1�tcyB into thetcyB gene, DNA of the erythromycin-resistant colonies was extracted asdescribed above. PCR was carried out with primers targeting the regionsupstream and downstream of the tcyB gene (ABC_trans_f and pyrP_na_r)and regions on the plasmid from pME-1 (SP6 and eryR) (Table 1). ThePCR products obtained with primers were sequenced. A detailed schemeof the chromosomal insertion into tcyB can be found in Fig. S1 in thesupplemental material.
Amino acid comparison of tcyB genes in the genus Lactobacillus.The L-cystine transport system permease (TcyB) protein sequence of L.sanfranciscensis (GI: 345504578) was analyzed with blastp of the NationalCenter for Biotechnology Information database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) against nonredundant protein sequences (nr) in thegenus Lactobacillus.
Growth experiments on L. sanfranciscensis DSM 20451T and its�tcyB mutant. To determine predicted transport functions of TcyB,growth experiments were performed in chemically defined medium. L.sanfranciscensis DSM 20451T and its �tcyB mutant were grown in mMRS(mMRS plus 5 �g/ml erythromycin) medium till stationary phase. After
the cells were washed twice, cell suspensions of the samples were adjustedto an optical density (OD) of 2 before the prepared chemically definedmedium was inoculated 1/100. Chemically defined medium was preparedaccording to reference 15 and optimized for the growth of L. sanfrancis-censis (10 g liter�1 maltose addition instead of glucose). Stock solutions of0.1 M cystine (prepared in 0.5 M HCl) and 0.57 M cysteine (prepared indistilled water) were added to the sterile-filtered chemically defined me-dium (final concentrations of 72 �M cystine and 285 �M cysteine).Growth was measured in a plate reader at 30°C for 24 h. The experimentwas conducted in triplicate.
For determination of growth under diamide stress, L. sanfranciscensisDSM 20451T and the �tcyB mutant were streaked onto mMRS plates(plus 10 �g/ml erythromycin for the mutant) and incubated anaerobicallyfor 48 h at 30°C. The strains were inoculated in mMRS (plus 5 �g/mlerythromycin for the mutant) medium overnight at 30°C. After centrifu-gation, the pellets were washed two times in Ringer solution before theywere solved in 50 �l Ringer solution. The following concentrations of thethiol-oxidizing agent diamide [synonym 1,1=-azobis(N,N-dimethylfor-mamide); Sigma-Aldrich, Germany] were prepared: 100 mM, 40 mM, 20mM, 10 mM, 5 mM, and 1 mM. The diamide solutions were preparedfreshly on the day of use. The growth of the strains was measured auto-matically in a microplate reader (Tecan, Switzerland) at 30°C for 24 h inwhich the optical density (OD590) was monitored every 60 min. For that,200 �l of mMRS medium/well with diamide and 2 �l of bacterial cells/wellwere mixed. The experiment was performed in triplicate for each dilutionand each strain. The data were analyzed and visualized with SigmaPlot12.0.
The resistance against diamide in L. sanfranciscensis DSM 20451T
(WT) and the �tcyB mutant was tested on agar plates on two differentmedium types (mMRS and mMRS5 without cysteine and fructose) ac-cording to reference 2. To maintain the genomic insertion, 5 to 10 �g/mlof erythromycin was added to agar plates for the mutant. The plates werecovered with 150 �l of overnight cultures of WT and �tcyB mutant. Afterthe plates were dried, sterile Sensi-Discs (BD Diagnostics, Heidelberg,Germany) were supplemented with diamide (final concentrations, 1 Mand 0.5 M) and placed in the middle of the agar plates. To compare thegrowth rates under normal conditions, separate plates with the addition ofdistilled water were used for each strain as positive controls. Plates wereincubated anaerobically for 48 h at 30°C. The growth inhibition was mea-sured as the diameter of growth inhibition expressed in millimeters. Datawere loaded into R (http://www.bioconductor.org), and P values (�5%)were calculated with a two-sample t test indicating significant effects.
Determination of extracellular and intracellular thiol groups afterdiamide treatment. Cultures of WT and �tcyB strains were grown anaer-obically in mMRS medium until the OD reached 0.5. A stock solution ofdiamide (Sigma-Aldrich, Steinheim, Germany) was prepared and sterilefiltered before addition. The final concentration of diamide in the cultureswas approximately 1.67 mM; distilled water was added to the controlcultures. After incubation at 30°C for 1 h, thiol groups were determined asalready described in reference 8 with some modifications. After centrifu-gation, cells were washed twice in KPM solution (0.1 M K2HPO4 adjustedto pH 6.5 with H3PO4 and containing 10 mM MgSO4·7H2O) before beingaerated with nitrogen. A 5-�l volume of a 10 �M L-cystine solution and 10�l of 1 M D-glucose solution were added to KPM cell suspensions. Thetubes were incubated for 1 h at 30°C before the cells were centrifuged, andthe supernatant was transferred into new tubes and placed on ice fordetermination of extracellular thiol groups. The procedure of determina-tion of intracellular thiol groups followed the protocol described above.For quantification of thiol groups, several dilutions of L-cysteine wereprepared in KPM solution. The experiment was conducted in triplicate.
RNA isolation, sequencing, and data analysis. L. sanfranciscensis WTand L. sanfranciscensis �tcyB strains were grown anaerobically at 30°C inmMRS medium with 15 g per liter of maltose and 5 g per liter of fructose.Additionally, 5 �g/ml of erythromycin was added to the medium for themutant to maintain the stability of the genomic insertion. After both
strains reached mid-exponential phase (OD, �0.5), 68.4 mM diamidesolved in distilled water was added to the 40-ml cultures (final concentra-tion of 1.7 mM). The control cultures were treated in the same way, butinstead of adding diamide, distilled water was used. After 35 min of incu-bation at 30°C without shaking, the cultures were treated with �20°C coldmethanol and centrifuged at 6,000 rpm before the pellet was solved inTris-EDTA (TE) buffer. The cells were disrupted with silica beads beforethe RNA midikit (Qiagen, Germany) was used for RNA isolation. Theinstructions of the supplier were followed for isolation. For each condi-tion, two biological replicates were prepared on two individual days. Theresulting RNA was solved in RNase-free water, and the quantity was de-termined with a Nanodrop ND-1000 spectrophotometer. The qualitycheck was carried out with the Agilent 2100 Bioanalyzer. The remainingDNA was removed with the Turbo DNA-free kit (Ambion, Life Technol-ogies, USA) according to the instructions of the supplier. The RNA wasprecipitated with ice-cold 3 M sodium acetate and absolute ethanol. Afterprecipitation, the samples were again checked for quantity and quality.Then, RNA samples were mixed with the RNA stable reagent (Biomatrica,USA) and placed at room temperature under the flow hood for drying. Alldried RNA samples were sent to BGI Hongkong for further preparationand RNA sequencing analysis using Illumina HiSeq 2000 technology.
The data analysis was performed according to the protocol of Trapnelland coworkers (16). The generated paired-end FASTAQ files weremapped using Bowtie (17) onto the only available published genome of L.sanfranciscensis TMW 1.1304 and the plasmids pLS1 and pLS2. The re-sulting sam files were sorted. After filtering with samtools, the generatedbam files were provided as input to cufflinks, which generates assembledtranscript fragments with FPKM (fragments per kilobase of transcript permillion mapped reads) values. These data files were merged into a singletranscript with cuffmerge taking the reference transcriptome annotationdata. The quantification was done with cuffdiff analyzing differential ex-pression. The output data were imported into R and further processedwith the cummeRbund package. The bam files of WT and mutant sampleswere changed into tmp files and loaded together with the FASTA file of thegenome of L. sanfranciscensis TMW 1.1304 into DNAPlotter for visualiza-tion purposes (18).
RESULTSSimilarity of tcyB genes in the genus Lactobacillus. The proteinsequence of tcyB in L. sanfranciscensis is 74% to 76% identical tocysteine ABC transporter permeases of Lactobacillus fructivoransand Lactobacillus florum, which belong, together with L. sanfran-ciscensis, to the same phylogenetic group (19, 20). The L-cystineuptake system in L reuteri BR11, an integral membrane proteinencoded in the bspA locus, shares 55% homology to tcyB. Se-quence similarity accounts for 34% to 69% of other Lactobacillusspecies, suggesting that tcyB of L. sanfranciscensis encodes a cys-tine/cysteine ABC transporter permease.
Dependency of WT and �tcyB growth on availability of cys-tine and cysteine. L. sanfranciscensis DSM 20451T and the �tcyBmutant were grown in chemically defined medium, which selec-tively offered cystine or cysteine. While WT and mutant strainswere able to grow on medium containing cysteine, the mutantvirtually failed to grow on medium with cystine (Fig. 1). Thissuggests that the �tcyB mutant transports cystine preferably overcysteine.
Growth response of WT and �tcyB strains after applicationof diamide. The response of L. sanfranciscensis DSM 20451T and�tcyB strains to thiol stress as induced by diamide is reflected intheir growth behavior, which is reflected in Fig. 2. Without di-amide stress, the WT and �tcyB strains show identical growthbehavior. Distinct growth for the other concentrations (100 mM,40 mM, 20 mM, 10 mM, and 5 mM) could be detected neither for
Stetina et al.
4116 aem.asm.org Applied and Environmental Microbiology
the WT nor for the �tcyB strain and is therefore not included inthe diagram. Based on these results, the growth of the �tcyB mu-tant is already disturbed with 1 mM diamide.
Extracellular and intracellular thiol groups in WT and �tcyBstrains. Extracellular thiol levels in the �tcyB mutant were en-hanced in comparison to the WT while intracellular thiol levelsshowed no significant difference (Fig. 3). Treatment with diamidedid not elicit any changes in the thiol levels.
Sensitivities of WT and �tcyB strains to diamide. The sensi-
tivity to diamide was tested in a plate assay, and results are shownin Table 2. Diamide causes growth reduction in the WT and mu-tant in full medium (mMRS). In the absence of cysteine and fruc-tose, growth of the �tcyB strain is completely inhibited.
Results of mRNA sequencing analysis of WT and �tcyBstrains after diamide treatment. Mapping of the differentiallyexpressed genes of L. sanfranciscensis DSM 20451T revealed theirpresence in the genome of the sequenced strain TMW 1.1304.Mainly, the transcriptional responses of the WT and �tcyB mu-tant under diamide stress do not show any pronounced differencein the overall patterns of the transcriptomic graphs.
A more detailed view of significantly differentially expressedgenes is given in Tables 3 (results of treated versus untreated WT),4 (results of treated WT versus treated �tcyB mutant), and 5 (re-sults of untreated WT versus untreated �tcyB mutant). The tablesinclude the annotated gene names, corresponding FPKM (frag-ments per kilobase of transcript per million mapped reads) values ofthe tested strain and condition, log2 fold changes, and correspondinggene descriptions for further information. The FPKM values are nor-malized by transcript length and total number of fragments se-quenced. Thus, if the FPKM value of condition 1 is higher than that ofcondition 2, the corresponding gene is overexpressed in condition 1and the resulting log2 fold changes are negative.
Significantly differentially expressed gene isoforms are markedin bold in the tables. The isoforms oppD/oppF and glnH/glnM aredisplayed in Fig. 4 and 5, respectively. The FPKM values are plot-ted against the sample names. It seems that the isoformTCONS_00000027 of oppD is much more affected after diamidetreatment than is TCONS_00000026. The same can be statedfor the isoforms of glnM (TCONS_00001438 versus TCONS_00001437).
FIG 1 Growth of L. sanfranciscensis DSM 20451T and �tcyB mutant in chemically defined medium with 72 �M cystine and 285 �M cysteine. Gray diamonds,WT incubated with 285 �M cysteine; black squares, WT incubated with 72 �M cystine; open triangles, �tcyB mutant grown with 285 �M cysteine; open circles,�tcyB mutant incubated with 72 �M cystine. Shown are the OD590 values of triplicate measurements with corresponding standard deviations.
FIG 2 Growth of L. sanfranciscensis DSM 20451T and �tcyB mutant in mMRSmedium with 1 mM diamide. Depicted is the time course of the increase ofoptical density at 590 nm (OD590). Closed and open circles indicate the OD590
values of the WT and the �tcyB mutant, respectively. Mean values of threeindependent cultures are displayed.
The results in Tables 4 and 5 are summarized in Table 6, inwhich genes were classified into three groups (redox, transport,and alternative) for the treated and untreated conditions. Theallocation was conducted according to the description and overallfunctions of the genes. Not-yet-annotated genes are named in theformat LSA_XXXX; that format was maintained throughout thiswork for correct gene identification.
DISCUSSION
In this work, we investigated the transcriptional answer of L. sanfran-ciscensis to (oxidative) thiol stress elicited by diamide and furtherdemonstrated the importance of the cystine/cysteine transporter tcyBin the tolerance of thiol stress. Therefore, transcriptional responseswere compared from (i) the wild type and (ii) the �tcyB mutant withand without thiol stress and (iii) the respective stress responses of thewild type and the �tcyB mutant.
Obviously, this minimalistic bacterium has few alternatives tocope with such stresses. Rather, it is adapted to even high levels ofreductive compounds and tolerates dithiothreitol (DTT), cys-teine, and glutathione (GSH) at concentrations of 100 mM. Thelatter has also been demonstrated for Staphylococcus aureus andStreptococcus pyogenes, which tolerate high levels of reductivecompounds at concentrations which are lethal for other bacteria(21). The application of oxidative substances such as diamide hassevere effects even at concentrations of 1 to 5 mM. It is clear thatthe �tcyB mutant needs time to partially compensate for the thiolstress and the resulting damage caused by 1 mM diamide. It isobservable that the mutant experiences a lag phase of growth be-
tween 2 and 9 h. After 10 h, the effect of thiol stress seems to bepartially resolved so that the focus of the bacteria shifts from sur-vival to growth. Similar effects could be seen for exponentiallygrowing E. coli treated with 2 to 3 mM diamide (22). The authorsstate that the duration of the lag phase depends on the initialdiamide concentration. Based on these data, the experimentaloutline of conducting the RNA sequencing analysis with approx-imately 1 mM diamide seems reasonable. Higher concentrationswould lead to growth arrest and even cell death. The deletion ofthe cystine transporter causes a decrease of the intracellular thiolpool, which strengthens the theory that cystine itself is involved inactivities against oxidative stress in this bacterium. The protectionin L. reuteri BR11 is mediated by cysteine rather than by cystine orother thiols (4). Cysteine in conjunction with Fe2 can have neg-ative effects through formation of ·OH by the Fenton reaction dueto the presence of hydrogen peroxide (23). As cystine and cysteineare interconvertible sulfur-containing amino acids, it cannot beclearly affirmed which substance is more effective, as both containsulfur atoms, which provide sites for redox activity and electrontransfer. The existence of an intracellular pool of cystine and cys-teine in which the two can be converted into one another depend-ing on the redox state of the bacterial cell and the consequentialdemand thereof could also be conceivable. Another possible ex-planation would be that the interchangeability of cysteine andcystine is already determined at the transport levels. The deletionof cystine transport leads to higher expression of cysteine trans-porters, although a direct transport of cysteine, mediated throughother transporters (e.g., LSA_00850) with lower substrate speci-ficities, cannot be excluded.
Therefore, the question arises of how a knockout mutant ofL. sanfranciscensis, which lacks the cystine transporter (tcyB),responds on the transcriptome level to systematically appliedthiol stress through diamide. The high genotypic similarity ofspecific regions between different strains of L. sanfranciscensisand a clear separation from other lactobacilli (24) enabled themapping of transcriptomic data of strain DSM 20451T tothe genome of strain TMW 1.1304. Further, the alignment ofthe bam files (WT versus mutants) with the FASTA file in theIntegrative Genomic Viewer (IGV) showed that most of the
FIG 3 Extracellular and intracellular thiol groups in L. sanfranciscensis DSM 20451T and �tcyB mutant. Mean values (mM) of triplicate measurements withcorresponding standard deviations are plotted. Shown are the concentrations of thiols at an OD of 0.5 after 1 h of diamide treatment. The dark gray bars indicatethe treated samples, whereas the light gray bars are the control samples. Significant effects (P � 0.05) are marked (*) above the corresponding bars.
TABLE 2 Sensitivities of L. sanfranciscensis DSM 20451T (WT) and�tcyB mutant to growth inhibition by 0.5 M and 1 M diamide
reads could be mapped onto the genome and that only a fewsingle-base-pair differences were observed, indicating thatthese two strains are very closely related (25). Also, we have(unpublished) data from a partial genome of strain DSM20451T, which further corroborate this similarity.
Thiol stress response of the WT. Evaluating the RNA sequenc-ing results for the WT as shown in Table 3 indicates that the ma-jority of overexpressed genes resemble the bacterial response afterthiol stress induction. Interestingly, the molecular chaperonegenes dnaJK, the heat shock protein gene grpE, and the heat shockresponse transcriptional regulator gene hrcA, and the chaperonegenes groESL, the ATP-dependent clp protease genes clpEPC, andthe transcriptional regulator gene ctsR, show higher FPKM valuesand are mainly known for their induction under heat stress. Thechaperones encoded by dnaK and dnaJ are involved in the degra-dation of misfolded proteins in E. coli (26) and other bacteria afterapplication of higher temperatures (27–30). Together with hrcAand grpE, the operon structure hrcA-grpE-dnaK-dnaJ is alreadydescribed in B. subtilis (31), Enterococcus faecalis (32), Streptococ-cus mutans (33), and others. In Lactobacillus sakei, the wholeoperon is induced after heat, salt, and ethanol stress (34).
Synthesis of clpP in B. subtilis was induced during heat, salt,oxidative stress, and oxygen and glucose limitation (35), but thisgene has also a distinct role in general protein quality control innonstressed cells (36). The same role is described for clpP and clpEin Lactococcus lactis (37, 38). The regulator ctsR controls clp ex-pression through specific binding to the promoter regions of clpC,-E, and -P in B. subtilis after heat stress; the same finding is alsoproposed for other Gram-positive bacteria (39). In Oenococcusoeni, dnaK and groESL are controlled by ctsR (40). The control ofclp and chaperone expression mediated through ctsR or even alsohrcA as already specified for Streptococcus salivarius (41) seemslikely, as both genes exist in the genome of L. sanfranciscensis, butrequires further investigations.
High-pressure treatment of L. sanfranciscensis DSM 20451T ledto overexpression of proteins which had sequence homologieswith heat shock proteins encoded by dnaK and groEL and withclp-encoded proteases (42). The results of the present study showthat the overexpression of the molecular chaperones and pro-teases responds to diamide stress in a similar way in L. sanfranci-scensis DSM 20451T.
Diamide treatment of the WT resulted further in lower expres-sion of glutamine transport genes (glnHM, -P, and -Q), lowerglutamine synthesis (glnA), and lower expression of the transcrip-tional regulator (glnR). While a connection of thiol stress andglutamine/glutamate metabolism may be considered far from ob-vious, it demonstrates the charm of transcriptomics in the discov-ery of unexpected connections. It is known from other bacteriathat the function of glnR depends on glnA (43). In Streptococcuspneumoniae, glnR is involved in the regulation of transcription ofgenes involved in glutamine and glutamate synthesis and glu-tamine uptake and glnR-dependent regulation depends on theconcentrations of glutamate, glutamine, and ammonium in thegrowth medium (44). It has already been published that ammo-nium transport is also regulated by glnR in Lactobacillus planta-rum, Lactococcus lactis, and other Gram-positive bacteria (43, 45,46). As glutamine can be formed from glutamate and ammoniumby glutamine synthetase (glnA), the downregulation of the ammo-nia channel (amt and ywnH) in L. sanfranciscensis leads togetherwith decreased glutamine import to a lower intracellular glu-tamine level.
Further diamide treatment leads to downregulation of adh2and significant overexpression of glmS (glucosamine-fructose-6-phosphate aminotransferase) in the WT. The decrease of adh2expression possibly reflects a mechanism to balance the NAD/NADH pool during thiol stress. glmS catalyzes the formation ofD-glucosamine 6-phosphate and L-glutamate from D-fructose6-phosphate and L-glutamine. As the reaction goes in both direc-
TABLE 3 Significantly differentially expressed genes for treated WT () versus untreated WT (�)a
tions, the formation of D-fructose 6-phosphate and L-glutamine inthe WT seems to be favored because feedback regulation couldlead to downregulation of genes responsible for glutamine synthe-sis and uptake as discussed above.
Importance of the cystine/cysteine transporter in the �tcyBmutant in tolerance to thiol stress. Evaluating the transcriptomicdata of Table 4 (treated WT versus treated �tcyB mutant) revealsthat the redox-sensitive regulator enzyme gene spxA is slightlyupregulated in the knockout mutant. The spx-encoded protein, as
a member of the arsenate reductase (arsC) family, responds toseveral stresses (low pH, high temperatures, presence of bacteri-cidal antibiotics, detergents, and reactive oxygen species) in such away that it represses or activates the transcription of genes in-volved in different bacterial processes (47, 48). The mode ofaction of this small and conserved protein can already be clar-ified in several low-GC Gram-positive bacteria (47–51). An�spx mutant of E. faecalis could not grow at low pH or highertemperatures or in medium with a high salt concentration (48).
TABLE 4 Significantly differentially expressed genes for treated WT () and treated �tcyB mutant ()a
Gene(s)WT ,FPKM 1
�tcyBmutant ,FPKM 2
Log2 fold change(FPKM 2/FPKM 1) Gene description
oppD, oppF 3,226.82 9,794.01 1.60179 Oligopeptide transport ATP-binding protein OppDoppB 1,740.2 4,865.5 1.48334 Oligopeptide transport system permease OppBoppC 1,610.97 4,592.21 1.51126 Oligopeptide transport system permease OppCoppA 3,515.92 7,098.13 1.01353 Oligopeptide-binding protein OppAgltT 311.406 509.371 0.709921 Proton/sodium-glutamate symport proteinLSA_00850 1,063.21 10,840.1 3.34989 Hypothetical proteinLSA_02330 44.7,525 266.704 2.5752 Hypothetical proteinspxA 549.505 953.57 0.795206 Regulatory protein SpxLSA_03800 652.628 340.516 �0.93854 Hypothetical proteinLSA_03810 445.03 225.341 �0.981793 Hypothetical proteinglnR 302.21 599.889 0.989141 HTHb-type transcriptional regulator GlnRglnA 989.525 1,721.88 0.799175 Glutamine synthetaseLSA_04670 181.256 559.443 1.62596 L-2-Hydroxyisocaproate dehydrogenasepyrB 412.165 185.494 �1.15185 Aspartate carbamoyltransferasepyrC 1,510.67 690.227 �1.13004 PseudogeneLSA_05910 440.341 241.258 �0.868041 PseudogeneyqhL 678.485 1,272.45 0.90722 Hypothetical proteinLSA_00240 43.453 248.435 2.51534 Hypothetical proteinLSA_00590 142.035 708.803 2.31914 Hypothetical proteinyxkA 1,648.85 2,671.09 0.695964 Hypothetical proteinnrdH 5,508.4 2,971.28 �0.890549 Glutaredoxin-like protein NrdHprsA 4,964.1 3,008.27 �0.722599 Foldase protein PrsAdtd 1,175.87 2,186.74 0.89505 Hypothetical proteinrelA 260.218 542.357 1.05952 GTP pyrophosphokinaseLSA_08440 132.533 219.523 0.728016 Hypothetical proteinLSA_08570 375.05 622.749 0.731569 Hypothetical proteinpatA 114.716 457.585 1.99597 Aminotransferase AmnmA 3,137.22 5,048.64 0.68641 tRNA-specific 2-thiouridylase MnmAadhA 4,132.93 8,055.41 0.962792 Alcohol dehydrogenasepyre 652.102 281.127 �1.21388 Orotate phosphoribosyltransferasepyrF 576.405 269.738 �1.09552 Orotidine 5=-phosphate decarboxylaseglnP 276.29 561.852 1.02401 Glutamine ABC transporter permease GlnPglnH, glnM 616.766 1,255.13 1.02504 Glutamine ABC transporter permease GlnMglnQ 389.391 662.733 0.767209 Glutamine transport ATP-binding protein GlnQmnmG 3,529.33 1,909.38 �0.886292 tRNA uridine 5-carboxymethylaminomethyl modification enzyme MnmGmnmE 3,122.92 1,711.74 �0.86743 tRNA modification GTPase MnmEa Depicted are the FPKM (fragments per kilobase of transcript per million mapped reads) values, log2 fold changes, and gene descriptions. Isoforms of the genes oppD and -F andglnH and -M are marked in bold.b HTH, helix-turn-helix.
TABLE 5 Significantly differentially expressed genes for untreated WT and �tcyB mutant (�)a
Gene WT �, FPKM 1�tcyB mutant �,FPKM 2
Log2 fold change(FPKM 2/FPKM 1) Description
LSA_03800 766.695 343.807 �1.15705 Hypothetical proteinLSA_13190 517.663 949.523 0.87519 Hypothetical proteinnrdH 4977.65 2282.22 �1.12502 Glutaredoxin-like protein NrdHa Depicted are the FPKM (fragments per kilobase of transcript per million mapped reads) values, log2 fold changes, and gene descriptions.
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Besides these sensitivities, the mutant showed also decreased tol-erance to oxygen, H2O2, and diamide. These observations overlapthose of studies in B. subtilis, S. aureus, and S. mutans in which spxpositively influences genes which are associated with oxidativestress (50, 52, 53). In the above-mentioned strain, spx is one of theregulators which control cysteine biosynthesis genes and thereforegives clear indications that the spx-encoded protein seems to beconnected with the cysteine/cystine metabolism in L. sanfrancis-censis also.
The fact that spxA is significantly more highly expressed in the�tcyB mutant than in the wild type gives clear indications that thewild type stressed with diamide is better able to tolerate thiolstress. It is known that diamide decreases the intracellular cysteineand methionine pool in B. subtilis and S. aureus (54) and furthercan lead to posttranslational protein modifications such as proteinS-cysteinylation or oxidation of protein thiols (55). The wild typeovercomes the emerging disulfide stress through simply utilizingreductive thiol groups from cysteine or cystine. However, nonex-istence of a cystathionine �- lyase in the minimalistic genome of L.sanfranciscensis TMW 1.1304 suggests another mechanism thanthat described for L. reuteri BR11 (11).
The gene nrdH (glutaredoxin-like protein) is significantlymore highly expressed in the wild type than in the �tcyB mutantindependent of the diamide treatment (Table 5, results of un-treated WT versus untreated �tcyB mutant). Therefore, the ex-pression of nrdH seems to be connected to the mutation in thecystine transporter tcyB gene itself rather than to the action ofdiamide. Rabinovitch and coworkers (56) tested the involvementof the nrdH-encoded protein during oxidative stress in construct-ing an �nrdH mutant which was exposed to hydrogen peroxideand diamide. The hypothesis of a specific role of nrdH duringoxidative stress could not be confirmed. The expression data of thepresent study propose that the nrdH-encoded protein seems to beconnected with the cystine transport and/or metabolism in thebacterial cell, which agrees well with the fact that this protein canbe mainly found in bacteria which lack glutathione. nrdH-en-coded proteins function as efficient reductants for disulfide bondsin low-molecular-weight (LMW) substrates and can be effectiveelectron donors with high specificity for class Ib ribonucleotidereductases (RNR) (56). Assuming that L. sanfranciscensis cantransport only glutathione, an efficient reduction of availableLMW thiols appears intuitive.
FIG 4 Differential expression of oppD/F. Isoforms of oppD/F visualized with the cummeRbund package of R. The FPKM (fragments per kilobase of transcript permillion mapped reads) values are plotted against the samples; the abbreviations WT_plus and Cys_plus reflect the wild type (treated) and the �tcyB mutant(treated).
This Lactobacillus could use cysteine for glutathione synthesis;however, related genes (gshA and gshB) in the genome of L. san-franciscensis TMW 1.1304 are absent, although the transport ofthe tripeptide is possible. Several studies evidenced low-molec-ular-weight (LMW) thiols such as cysteine, coenzyme A (CoA),
and bacillithiol (BSH) which are likewise able to cope with ROS(53, 57).
Looking at transcriptional changes of transporters, the wholeopp operon (oppD, oppF, oppB, oppC, and oppA) is upregulatedwhen L. sanfranciscensis �tcyB is stressed with diamide (Table 4,
FIG 5 Differential expression of glnH/M. Isoforms of glnH/M visualized with cummeRbund package of R. The FPKM (fragments per kilobase of transcript permillion mapped reads) values are plotted against the samples; the abbreviations WT_plus and Cys_plus reflect the wild type (treated) and the �tcyB mutant(treated).
TABLE 6 Summary of the significantly differentially expressed genesa
Test condition and group Gene(s) significantly differentially expressed
Untreated WT and �tcyB mutant (�)Redox nrdHTransport LSA_03800; LSA_13190Alternative
a Genes were divided into three main groups (redox, transport, and alternative) for treated WT/�tcyB mutant () and untreated WT/�tcyB mutant (�). Gene annotations weremaintained.
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WT and treated �tcyB mutant). This upregulation reflects a sim-ple adaptation mechanism in which cystine due to the deletion oftcyB is released through (oligo)peptidases. Increased thiol stresscauses an intracellular oxidative stress response in which the mu-tant requires oligopeptides to a greater extent than does the wildtype. It is suggested that cystine itself is involved in keeping thethiol balance in a specific redox state in the bacterial cell. This issupported by the finding that extracellular thiol levels are in-creased in the �tcyB mutant. L. sanfranciscensis DSM 20451T ap-parently has no functional alternative, because the deletion of tcyBresulted in hardly detectable growth. As the aminopeptidase pepNis located right after the oppA gene, the higher read coverage asretrieved from IGV (25) for the treated than for the untreated�tcyB cells corresponds to the observations for the opp operon. Asimultaneous transcription of opp and pepN is highly probable forcleaving oligopeptides in time into amino acids.
A closer look into gene isoforms and possible regulation is oneof the advantages of RNA sequencing. The isoforms of oppD/oppFand glnH/glnM as shown in Fig. 4 and 5 respond differently tothiol stress of the wild type compared to the �tcyB mutant. Thesecond isoforms (TCONS_00000027 and TCONS_00001438) areinfluenced to a larger extent by thiol stress. However, it is evidentthat the confidence intervals are quite broad for both gene iso-forms; a possible explanation could be the low sequencing depthfor these genes.
The transcription of the gene LSA_00850 is also highly upregu-lated in the �tcyB mutant compared to the WT. A blastp searchindicates 85% homology of a not-yet-characterized lipoprotein,which is predicted to transport methionine in L. florum. The de-pletion of cysteine and methionine in the mutant is further sup-ported by the fact that relA is significantly more highly expressed.An accumulation of (p)ppGpp through relA is known as the alar-mone of the stringent response, which could be observed duringdisulfide stress and which is activated also, for instance, duringamino acid starvation (54). In E. faecalis, (p)ppGpp accumulationmediates stress survival, amino acid synthesis, and antibiotic tol-erance (58). In S. mutans, relA is important to balance growth andsurvival and to control key catabolic pathways dependent on theenvironmental conditions (59). It is known for E. coli that thecobalamin-independent methionine synthase (metE), which is in-volved in methionine biosynthesis, is inactivated by diamide treat-ment. The oxidation of cysteine 645 in the metE protein results ina modulated activity and leads to a limited methionine concentra-tion (60). The same could be found when E. coli was exposed tooxidative stress in general (22). The mechanisms of action includeS thiolations in S. aureus and B. subtilis cells after diamide treat-ment as already mentioned (54). It is comprehensible that oxida-tion of thiol groups in proteins or LMW thiols can have severeimpacts on signal transduction and enzyme activities as outlinedabove for E. coli. The expression of the msrB (methionine sulfox-ide reductase B) gene in the �tcyB strain treated with diamide ishigher than that under the control condition (see Table S1 in thesupplemental material). Oxidized methionine in proteins can bereduced by msrA and msrB, decreasing the accumulation of oxi-dative damage. A similar higher expression of msrB which is sig-nificant could not be observed for the wild type, as this effectseems to be overall compensated in the treated groups. The ex-pression of msrA is significant in neither the wild type nor themutant independent of the conditions.
As L. sanfranciscensis TMW 1.1304 is auxotrophic for cysteine
and methionine, the higher requirement after oxidation appearsat the level of increased methionine reduction through msrB andexpression of methionine transport and not of methionine syn-thesis.
A BLAST search of two proteins, LSA_03800 and LSA_03810,indicates a predicted branched-chain amino acid (BCAA) trans-porter as the two share protein sequence homologies (52 to 79%)to branched-chain amino acid transporters of various other lacto-bacilli. Additionally, the alignment of the nucleotide sequence ofLSA_03800 and an azlC-encoded protein (branched-chain aminoacid transporter, accession no. AJ937238) of L. reuteri LTH5531(61) resulted in 72% homology. Both genes are underexpressedduring thiol stress in the �tcyB strain. In L. sanfranciscensis LSCE1,some genes involved in the catabolism of BCAA were upregulatedduring acid stress (62). Balancing the cellular redox homeostasiscould be a possible explanation. In other Gram-positive bacteria,diamide caused changes in BCAA biosynthesis pathways whichresulted in decreased levels of valine and isoleucine (54). The in-creased expression of relA leads to accumulation of (p)ppGpp asalready mentioned above. In S. mutans, a (p)ppGpp deletion mu-tant (�relAPQ strain) was not able to grow in medium withoutleucine and valine (63). Also, it is discussed elsewhere that a dis-tinct level of (p)ppGpp is necessary for growth in medium withoutthese two amino acids (59). If more relA causes a higher (p)ppGpppool resulting in a sufficient basal level, the expression of BCAAtransporter due to the presence of sufficient BCAA will be de-creased. In B. subtilis, BCAAs activate codY, which acts in repress-ing the genes for BCAA synthesis (64), but no codY homologuecan be found in the genome of L. sanfranciscensis.
The alcohol dehydrogenase gene adhA is more highly ex-pressed in the �tcyB mutant when treated with diamide. As theshutdown of the ethanol branch in the presence of external elec-tron acceptors (oxygen, fructose, and citrate) is very commonamong heterofermentative lactic acid bacteria, the formation ofethanol via alcohol dehydrogenase (adhA) seems counterintuitiveas the growth medium contained fructose. The oxidation of eth-anol to acetaldehyde generates NADH. The use of fructose enablesthe formation of acetate and ATP via an acetate kinase reaction.Emerging thiol stress increases the energy demand within the bac-terial cell, and the allocation of reducing equivalents such asNADH is needed.
The pyr genes for carbamoylphosphate synthase (pyrA), aspar-tate transcarbamylase (pyrB), dihydroorotase (pyrC), dihydro-orotate oxidase (pyrD), orotate phosphoribosyltransferase (pyrE),and orotidine monophosphate decarboxylase (pyrF) are essentialfor the de novo biosynthesis of pyrimidine nucleotides. In the ge-nome of L. sanfranciscensis, the genes pyrDA, -B, and -C form anoperon whereas pyrE and -F are located in another part of thechromosome. The transcription of pyrB, -C, -E, and -F in the�tcyB mutant is reduced compared to that in the wild type. Dur-ing thiol stress, the bacterium has to cope with oxidative damageto ensure survival and therefore shuts down pyrimidine synthesis.In L. plantarum, the pyr operon is regulated by transcription at-tenuation (65). It is said that the transcription initiation of the pyroperon occurs depending on the presence of uracil in the medium(66). In chemically defined medium, L. sanfranciscensis was not ableto grow without additional uracil. The transport of uracil in L. san-franciscensis is accomplished with uracil permease (pyrP), which isnot located within the pyr operon. The growth of this strain also failedin medium without pyrimidine and purine bases. As expression of
pyrE and pyrR could be observed in L. sakei in medium without addeduracil, the transcription of pyrR1BCAaAbDFE could be detected in L.plantarum cells grown in uracil-free medium (66, 67). The RNAbinding regulator pyrR is the first gene within the pyr operon for B.subtilis and L. plantarum (68). The regulator represses the expres-sion of pyr genes and therefore inhibits the de novo synthesis ofpyrimidine nucleotides in the absence of uracil in the medium. InL. sanfranciscensis, the pyrR regulator is not located in the pyroperon which supports the above-mentioned fact that uracil ful-fills other roles besides a possible relevance to the repression ofpyrimidine biosynthesis.
ACKNOWLEDGMENTS
This research project was supported by the German Ministry of Econom-ics and Technology (via AiF) and the FEI (Forschungskreis derErnährungsindustrie e.V. Bonn) project AiF 16907 N.
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