BSGatlas

rhaEW

BSGatlas-gene-3667

BSGatlas

Description	Information
Coordinates	3201860..3203929
Genomic Size	2070 bp
Name	rhaEW
Outside Links	SubtiWiki
	BsubCyc
Strand	-
Type	CDS

SubtiWiki

Description	Information
Alternative Name	rhaEW
	rhaEW
	yulA
	yuwG
Category	SW 2 Metabolism
	SW 2.2 Carbon metabolism
	SW 2.2.2 Utilization of specific carbon sources
	SW 2.2.2.12 Utilization of rhamnose
Description	bifunctional-rhamnulose-phosphate aldolase/L-lactate dehydrogenase
Function	utilizatio of rhamnose and rhamnogalacturonan (pectin)
Is essential?	no
Isoelectric point	5.78
Locus Tag	BSU_31220
Molecular weight	75.8374
Name	rhaEW
Product	bifunctional-rhamnulose-phosphate aldolase/L-lactate dehydrogenase

RefSeq

Description	Information
Alternative Locus Tag	BSU31220
Description	Evidence 1a: Function from experimental evidencesin the studied strain; PubMedId: 24391637, 26712933;Product type e: enzyme
Enzyme Classifications	EC 4.1.2.19: rhamnulose-1-phosphate aldolase
Functions	16.11: Scavenge (Catabolism)
Locus Tag	BSU_31220
Name	rhaE
Title	bifunctional rhamnulose-1-phosphatealdolase/alcohol dehydrogenase
Type	CDS

BsubCyc

Description	Information
Alternative Name	yulA
	yuxG
Citation	Hirooka K;Kodoi Y;Satomura T;Fujita Y Regulation of the rhaEWRBMA Operon Involved in l-Rhamnose Catabolism through Two Transcriptional Factors, RhaR and CcpA, in Bacillus subtilis. J Bacteriol 198(5);830-45 (2015) PUBMED: 26712933
	Rodionova IA;Li X;Thiel V;Stolyar S;Stanton K;Fredrickson JK;Bryant DA;Osterman AL;Best AA;Rodionov DA Comparative genomics and functional analysis of rhamnose catabolic pathways and regulons in bacteria. Front Microbiol 4;407 (2013) PUBMED: 24391637
Comment	RhaEW is a novel (as of 2013) bifunctional enzyme catalyzing two consecutive steps in \|FRAME: L-rhamnose\| catabolism, \|FRAME: EC-4.1.2.19\| and \|FRAME: EC-1.2.1.22\|. The gene encoding the enzyme was identified in diverse bacterial lineages including Actinobacteria, α-proteobacteria, Bacilli, Bacteroidetes, and Chloroflexi \|CITS: [24391637]\|. The N-terminal domain of the enzyme from \|FRAME: TAX-1423\| is 14% identical to RhaD from \|FRAME: TAX-562\|, while the C-terminal domain is homologous to various NADH-and NADPH-dependent sugar dehydrogenases. The \|FRAME: BSU31220\| gene was cloned from \|FRAME: TAX-1423\| with an N-terminal His₆ tags and expressed in \|FRAME: TAX-562\|, and the recombinant protein was purified \|CITS: [24391637]\|. To assay the purified protein, purified recombinant \|FRAME: EC-5.3.1.14\| (RhaA) from \|FRAME: TAX-562\| and \|FRAME: EC-2.7.1.5\| (RhaB) form \|FRAME: TAX-2336\| were pre-incubated with \|FRAME: L-rhamnose\|, producing \|FRAME: RHAMNULOSE-1P\| (which is not available commercially). The RhaEW enzyme was then added, and the reaction was followed by monitoring the reduction of NAD+. The identity of the products was confirmed by GC-MS \|CITS: [24391637]\|. The function of the enzyme was further validated by creating a knockout mutant. The mutant lost the ability to utilize \|FRAME: L-rhamnose\| \|CITS: [24391637]\|.
Description	bifunctional L-rhamnulose 1-phosphate aldolase/lactaldehyde dehydrogenase
Enzyme Classifications	EC 1.2.1.22: lactaldehyde dehydrogenase
	EC 4.1.2.19: rhamnulose-1-phosphate aldolase
Gene Ontology	GO:0008152 metabolic process
	GO:0016491 oxidoreductase activity
	GO:0019301 rhamnose catabolic process
	GO:0055114 oxidation-reduction process
Locus Tag	BSU31220
Molecular weight	76.02
Name	rhaEW

Nicolas et al. predictions

Description	Information
Expression neg. correlated with	BSU28280, BSU28270, new_2896973_2897093_c, BSU15600, new_2897341_2897467_c, BSU_misc_RNA_1, BSU_misc_RNA_3, BSU_misc_RNA_6, BSU_misc_RNA_7, BSU_misc_RNA_8, BSU_misc_RNA_9, BSU_misc_RNA_10, BSU_misc_RNA_11, BSU_misc_RNA_12, BSU_misc_RNA_13, BSU_misc_RNA_14, BSU_misc_RNA_15, BSU_misc_RNA_16, BSU_misc_RNA_17, BSU_misc_RNA_18, BSU_misc_RNA_19, BSU_misc_RNA_20, BSU_misc_RNA_21, BSU_misc_RNA_23, BSU_misc_RNA_24, BSU_misc_RNA_25, BSU_misc_RNA_26, BSU_misc_RNA_27, BSU_misc_RNA_28, BSU_misc_RNA_29, BSU_misc_RNA_31, BSU_misc_RNA_32, BSU_misc_RNA_34, BSU_misc_RNA_36, BSU_misc_RNA_38, BSU_misc_RNA_39, BSU_misc_RNA_40, BSU_misc_RNA_41, BSU_misc_RNA_42, BSU_misc_RNA_44, BSU_misc_RNA_45, BSU_misc_RNA_46, BSU_misc_RNA_47, BSU_misc_RNA_48, BSU_misc_RNA_49, BSU_misc_RNA_50, BSU_misc_RNA_51, BSU_misc_RNA_52, BSU_misc_RNA_53, BSU_misc_RNA_54, BSU_misc_RNA_56, BSU_misc_RNA_59, BSU_misc_RNA_60, BSU_misc_RNA_63, BSU28310, new_1633963_1634060, BSU15610, BSU28260
Expression pos. correlated with	BSU34160, new_630028_630114, BSU05840, BSU31210, BSU40110, new_4122850_4123061_c, BSU05830, BSU40120, BSU34150, BSU05850
Highly expressed condition	(dia0) Diamide was added to an exponentially growing culture (OD600 approx. 0.6) at a sub-lethal concentration(0.5 mM) and growth continued at 37°C with vigorous shaking. Samples were collected 0, 5 and 15 minutes after diamide addition [dia0, dia5 and dia15].
	(G135) Purified spores were obtained by growing cells in DSM medium (P. Schaeffer, J. Millet, J. P. Aubert, Proc Natl Acad Sci U S A 54, 704, Sep, 1965) at 37°C for 48 hours after which they were washed ten times in ice cold distilled waterover a period of 5 days. Purified spores were heat activated at 70°C in Tris 10 mM pH8.4 and germination was initiated by the addition of L-alanine 10 mM (A. Moir, J Bacteriol 146, 1106, Jun, 1981). After incubation for one hour at 37°C, the culture was diluted with an equal volume of 2X LBmedium and germinating cells were harvested at 135, 150 or 180 minutes after addition of L-alanine [G135, G150 and G180].
	(G150) Purified spores were obtained by growing cells in DSM medium (P. Schaeffer, J. Millet, J. P. Aubert, Proc Natl Acad Sci U S A 54, 704, Sep, 1965) at 37°C for 48 hours after which they were washed ten times in ice cold distilled waterover a period of 5 days. Purified spores were heat activated at 70°C in Tris 10 mM pH8.4 and germination was initiated by the addition of L-alanine 10 mM (A. Moir, J Bacteriol 146, 1106, Jun, 1981). After incubation for one hour at 37°C, the culture was diluted with an equal volume of 2X LBmedium and germinating cells were harvested at 135, 150 or 180 minutes after addition of L-alanine [G135, G150 and G180].
	(G180) Purified spores were obtained by growing cells in DSM medium (P. Schaeffer, J. Millet, J. P. Aubert, Proc Natl Acad Sci U S A 54, 704, Sep, 1965) at 37°C for 48 hours after which they were washed ten times in ice cold distilled waterover a period of 5 days. Purified spores were heat activated at 70°C in Tris 10 mM pH8.4 and germination was initiated by the addition of L-alanine 10 mM (A. Moir, J Bacteriol 146, 1106, Jun, 1981). After incubation for one hour at 37°C, the culture was diluted with an equal volume of 2X LBmedium and germinating cells were harvested at 135, 150 or 180 minutes after addition of L-alanine [G135, G150 and G180].
	(HiTm) Cells were grown in Spizizen’s minimal medium (SMM) (C. Anagnostopoulos, J. Spizizen, J Bacteriol 81, 741, May, 1961) with vigorous agitation. The control culture was grown at 37 °C [SMMPr]. For growth at high or low temperatures, pre-cultures were grown at 37 °C, diluted to an OD578nm of 0.1 and subsequently transferred to 51 °C [HiTm] and 16 °C [LoTm], respectively. For the growth at high salinity, the salinity of the medium was adjusted by adding NaCl (5 M stock solution) to produce a final concentration of 1.2 M [HiOs].
	(Mt0) Cells were grown in LB medium at 37°C with vigorous shaking. An exponentially growing culture (O.D.600 approx. 0.25) was divided: one culture acted as the control [no mitomycin C , M0] while mitomycin was added to the second culture to a final concentration of 40 ng/ml [mitomycin, M40]. Samples were harvested at 0, 45 and 90 minutes after mitomycin addition [t0, t45 and t90].
	(T0.30H) Anon-sporulating B. subtilis strain was grown in a modified M9 medium in batch culture (T. Hardiman, K. Lemuth, M. A. Keller, M. Reuss, M. Siemann-Herzberg, J Biotechnol 132, 359, Dec 1, 2007). Glucose was exhausted when the culture reached an OD600 of approx. 10 and this was designated T0 [T0.0H]. 7 samples were harvested at various times before glucose exhaustion [T-5.40H to T-0.40H] and 10 samples at various times after glucose exhaustion [T0.30H to T5.0H].
	(T1.0H) Anon-sporulating B. subtilis strain was grown in a modified M9 medium in batch culture (T. Hardiman, K. Lemuth, M. A. Keller, M. Reuss, M. Siemann-Herzberg, J Biotechnol 132, 359, Dec 1, 2007). Glucose was exhausted when the culture reached an OD600 of approx. 10 and this was designated T0 [T0.0H]. 7 samples were harvested at various times before glucose exhaustion [T-5.40H to T-0.40H] and 10 samples at various times after glucose exhaustion [T0.30H to T5.0H].
	(T1.30H) Anon-sporulating B. subtilis strain was grown in a modified M9 medium in batch culture (T. Hardiman, K. Lemuth, M. A. Keller, M. Reuss, M. Siemann-Herzberg, J Biotechnol 132, 359, Dec 1, 2007). Glucose was exhausted when the culture reached an OD600 of approx. 10 and this was designated T0 [T0.0H]. 7 samples were harvested at various times before glucose exhaustion [T-5.40H to T-0.40H] and 10 samples at various times after glucose exhaustion [T0.30H to T5.0H].
	(T2.0H) Anon-sporulating B. subtilis strain was grown in a modified M9 medium in batch culture (T. Hardiman, K. Lemuth, M. A. Keller, M. Reuss, M. Siemann-Herzberg, J Biotechnol 132, 359, Dec 1, 2007). Glucose was exhausted when the culture reached an OD600 of approx. 10 and this was designated T0 [T0.0H]. 7 samples were harvested at various times before glucose exhaustion [T-5.40H to T-0.40H] and 10 samples at various times after glucose exhaustion [T0.30H to T5.0H].
Lowely expressed condition	(B36) A fresh colony grown on an LB plate was used to inoculate 10 ml of LB and grown for 10 hoursat 30°C. This culture wasused to inoculate 10 ml of MSgg medium (S.S. Branda et al., J Bacteriol 186, 3970, Jun, 2004) and incubated with vigorous shaking. The cultures in MSgg were diluted to the same extent in 96 wells microtiterplates (5 μl for 1.5 ml of medium) and incubated without shaking at 30°C. Cells from the control cultures were harvested after 24 hours of incubation [BT]. Biofilms were harvested from 96 well plates after incubation for 36 hours [B36] and 60 hours [B60].
	(C90) Cellsgrown overnight on LB agar plates at 30°Cwere harvested and used to inoculate pre-warmed minimal medium at OD600 of 0.5 (D. Dubnau, R. Davidoff-Abelson, J Mol Biol 56, 209, Mar 14, 1971). After growth at 37°C with vigorous shaking, cells were diluted ten times in fresh pre-warmed minimal medium and samples were harvested after a period of 30 minutes [C30] , i.e. before maximal induction of competence, and after a period of 90 minutes [C90], i.e. when competence induction was maximal.
	(Etha) Cells were grown in a synthetic medium (J. Stülke, R. Hanschke, M. Hecker, J Gen Microbiol 139, 2041, Sep, 1993) with 0.2 % glucose as carbon source (Belitsky Minimal Medium/BMM) at 37 °C with vigorous shaking. Stress was applied to exponentially growing cultures at OD500nm of 0.4. Samples were harvested before stress [BMM]; after a rapid temperature up-shift from 37 °C to 48 °C [Heat]; after a temperature down-shift from 37 °C to 18 °C [Cold]. Ethanol stress was imposed by adding ethanol to a final concentration of 4 % (v/v) and cells were harvested 10 minutes after ethanol addition [Etha].
	(ferm) Cells were grown in a synthetic medium (E. Härtig, A. Hartmann, M. Schätzle, A. M. Albertini, D. Jahn, Appl Environ Microbiol 72, 5260, 2006) at 37 °C. For aerobic growth, an overnight culture was used to inoculate 100 ml of the synthetic medium to a starting OD578 of 0.05. The culture was then incubated in a 500 ml baffled flask with shaking at 250 rpm [aero]. Anaerobic growth was carried out (i) in the presence of 10 mM potassium nitrate (nitrate respiration) [nit]; or (ii) in the absence of 10 mM postassium nitrate (fermentative growth) [ferm]. The procedure for anaerobic growth was: medium was inoculated to an OD578 nm of 0.1 in flasks completely filled with medium and sealed with rubber stoppers. They were shaken at 100 rpm to minimize cell aggregation. These cultures were inoculated aerobically with an aerobically grown overnight culture. Anaerobic conditions were achieved in the stoppered flasks after a short time through the consumption of residual oxygen. Cells were harvested during the exponential growth phase.
	(HiOs) Cells were grown in Spizizen’s minimal medium (SMM) (C. Anagnostopoulos, J. Spizizen, J Bacteriol 81, 741, May, 1961) with vigorous agitation. The control culture was grown at 37 °C [SMMPr]. For growth at high or low temperatures, pre-cultures were grown at 37 °C, diluted to an OD578nm of 0.1 and subsequently transferred to 51 °C [HiTm] and 16 °C [LoTm], respectively. For the growth at high salinity, the salinity of the medium was adjusted by adding NaCl (5 M stock solution) to produce a final concentration of 1.2 M [HiOs].
	(LoTm) Cells were grown in Spizizen’s minimal medium (SMM) (C. Anagnostopoulos, J. Spizizen, J Bacteriol 81, 741, May, 1961) with vigorous agitation. The control culture was grown at 37 °C [SMMPr]. For growth at high or low temperatures, pre-cultures were grown at 37 °C, diluted to an OD578nm of 0.1 and subsequently transferred to 51 °C [HiTm] and 16 °C [LoTm], respectively. For the growth at high salinity, the salinity of the medium was adjusted by adding NaCl (5 M stock solution) to produce a final concentration of 1.2 M [HiOs].
	(nit) Cells were grown in a synthetic medium (E. Härtig, A. Hartmann, M. Schätzle, A. M. Albertini, D. Jahn, Appl Environ Microbiol 72, 5260, 2006) at 37 °C. For aerobic growth, an overnight culture was used to inoculate 100 ml of the synthetic medium to a starting OD578 of 0.05. The culture was then incubated in a 500 ml baffled flask with shaking at 250 rpm [aero]. Anaerobic growth was carried out (i) in the presence of 10 mM potassium nitrate (nitrate respiration) [nit]; or (ii) in the absence of 10 mM postassium nitrate (fermentative growth) [ferm]. The procedure for anaerobic growth was: medium was inoculated to an OD578 nm of 0.1 in flasks completely filled with medium and sealed with rubber stoppers. They were shaken at 100 rpm to minimize cell aggregation. These cultures were inoculated aerobically with an aerobically grown overnight culture. Anaerobic conditions were achieved in the stoppered flasks after a short time through the consumption of residual oxygen. Cells were harvested during the exponential growth phase.
	(Pyr) A 5 ml aliquot of LB medium was inoculated using frozen culture stocks. After a few hours growth at 37°C, precultures were prepared by inoculating 5 ml of M9 with this LB culture at several different dilutions usually ranging from 500- to 2000-fold. The dilution range was chosen so that one of these precultures had grown to and OD600 of 0.5 - 1.0 after overnight inculation. The chosen M9 medium precultures [at OD600 of 0.5 - 1.0] were used to inoculate 100 mL of M9 medium in 500 mL non-baffled shake flasks to an OD600 of 0.02. Filter-sterilized carbon sources were added separately to the medium M9 at following concentration: D-Glucose 3g/L[Glu], L-Malic acid 4.5g/L[Mal], L-Malic acid + D-Glucose 3 and 2g/L[M+G], D-Fructose 3g/L[Fru], D-Gluconate 4g/L[Glucon], Pyruvate 6g/L[Pyr], Glycerol 6g/L[Gly], Glutamic acid + Succinic acid 2 and 2g/L[G+S]. Where necessary, carbon source solutions were pH neutralized with 4 M NaOH prior to addition to the medium. Cells were harvested during the exponential growth phase.
	(Salt) Cells were grown in Spizizen’s minimal medium (SMM) at 37 °C with vigorous shaking. Salt was added, to a final concentration of 0.4 M to an exponentially growing culture of cells at OD500 of 0.4. Samples were harvested before [SMM] and 10 minutes after [Salt] NaCl addition.
Name	rhaEW

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