Canadian Journal of Microbiology
Genomic Comparison of Facultatively Anaerobic and Obligatory Aerobic Caldibacillus debilis Strains GB1 and Tf Helps Explain Physiological Differences
Journal: Canadian Journal of Microbiology
Manuscript ID cjm-2018-0464.R2
Manuscript Type: Article
Date Submitted by the 18-Dec-2018 Author:
Complete List of Authors: Wushke, Scott; University of Manitoba Froese, Alan; University of Manitoba , Microbiology Fristensky, Brian; University of Manitoba, Plant Science Zhang, Xiang;Draft University of Manitoba, Plant Science Spicer, Victor; University of Manitoba, Physics & Astronomy Krokhin, Oleg; University of Manitoba, Physics & Astronomy Levin, David B.; University of Manitoba, Biosystems Engineering Sparling, Richard; University of Manitoba, Microbiology
Keyword: Thermophile, Fermentation, Genomics
Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :
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1 Genomic Comparison of Facultatively Anaerobic and Obligatory Aerobic Caldibacillus
2 debilis Strains GB1 and Tf Helps Explain Physiological Differences
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4 Scott Wushke1, Alan Froese1, Brian Fristensky2, Xiang Li Zhang2, Vic Spicer3, Oleg V.
5 Krokhin4, David B. Levin 5, Richard Sparling1*
6 1Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
7 2Department Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada
8 3Department Physics & Astronomy, University of Manitoba, Winnipeg, Manitoba, Canada 9 4Department of Internal Medicine & DraftManitoba Centre for Proteomics and Systems Biology, 10 University of Manitoba, Winnipeg, Manitoba, Canada
11 5Department of Biosystems Engineering, University of Manitoba, Winnipeg, Manitoba, Canada
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18 * Corresponding Author: Richard Sparling, Department of Microbiology, University of
19 Manitoba, Winnipeg, Manitoba, Canada, R3T 2N2; Tel: (204) 474-8320; Fax: (204) 474-8320;
20 E-mail: [email protected]
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21 Abstract
22 Caldibacillus debilis strains GB1 and Tf display distinct phenotypes. C. debilis GB1 is capable
23 of anaerobic growth and can synthesize ethanol while C. debilis Tf cannot. Comparison of the
24 GB1 and Tf genome sequences revealed that the genomes were highly similar in gene content
25 and showed a high level of synteny. At the genome scale, there were several large sections of
26 DNA that appeared to be from lateral gene transfer into the GB1 genome. Tf did have unique
27 genetic content but at a much smaller scale; 300 genes in Tf verses 857 genes in GB1 that
28 matched at ≤90% sequence similarity. Gene complement and copy number of genes for the
29 glycolysis, tricarboxylic acid (TCA) cycle, and electron transport chain (ETC) pathways were 30 identical in both strains. While Tf is an Draftobligate aerobe, it possesses the gene complement for an 31 anaerobic lifestyle (ldh, ak, pta, adhE, pfl). As a species, other strains of C. debilis should be
32 expected to have the potential for anaerobic growth. Assaying the whole cell lysate for ADH
33 activity revealed an approximately 2-fold increase in the enzymatic activity in GB1 when
34 compared to Tf
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36 Keywords: Thermophilic, Fermentation, Genomics
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38 Importance
39 This work characterizes the genomes of the thermophilic C. debilis strains GB1 and Tf with
40 regard to core metabolism. Comparison of two highly related but physiologically distinct strains
41 highlights the physiology and genome potential of the single species genus Caldibacullis
42 especially with regards to fermentation. Caldibacillus spp. may be of interest in industrial
43 process, namely biofuel production, a deep understanding of core metabolism is needed in order
44 to optimize these processes.
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46 1.0 Introduction 47 48 Caldibacillus debilis GB1 was isolated from a co-culture found to convey aerotolerance
49 to Clostridium thermocellum when co-cultured aerobically on cellulose (Wushke et al. 2013).
50 The recently defined single species genus Caldibacillus has been the subject of only a few
51 physiological studies in pure culture (Banat et al. 2004; Wushke et al. 2015). Caldibacillus
52 debilis Tf was originally isolated from a cool soil environment in Northern Ireland (Banat et al.
53 2004). Contrary to what was observed with C. debilis GB1, Caldibacillus debilis Tf was unable
54 to create a micro-aerophilic environment suitable for co-culturing with the strict anaerobe
55 Clostridium thermocellum (Wushke et al. 2015). We believe that the inability of C. debilis Tf to 56 grow anaerobically may affect its abilityDraft to create the reduced anaerobic microenvironment 57 needed for C. thermocellum to thrive. Previously, Wushke et al. (2015) compared the
58 physiological profile of two strains of C. debilis, GB1 and Tf. They showed near identical
59 characteristics in growth rate, temperature optimum, substrate utilization profile, and types of
60 end-products produced under aerobic and oxygen-limiting conditions, with the notable exception
61 of ethanol production. A C. debilis Tf draft genome has been generated and annotated by the
62 Joint Genome Institute (JGI) (Grigoriev et al. 2012), but there has not been a detailed genome
63 analysis published. The C. debilis GB1 genome was previously sequenced and its proteome was
64 analyzed with respect to aerobic and anaerobic growth (Wushke et al. 2017). Because of the
65 major physiological differences observed, including anaerobic growth and ethanol synthesis, we
66 hypothesized that a comparison of the genomes sequences of the GB1 and Tf, with a focus on
67 core metabolism, could explain the observed physiological differences. Part of the work
68 presented herein was adapted from the Ph.D. thesis of Scott Wushke (2017).
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69 2.0 Materials and Methods 70
71 2.1 Cell Culturing
72 The methods for growing Caldibacillus debilis strain GB1 DSM 29516 and C. debilis Tf
73 DSM 16016 cultures were described previously (Wushke et al. 2013, 2015). C. debilis strains
74 were grown on modified 1191 (M-1191) medium (Islam et al. 2006) with a lower concentration
75 of yeast extract (0.76 g/L) and the pH was adjusted to 7.2 for all experiments. Sealed Balch tubes
76 (26 mL) from Bellco Glass Inc. (Vineland, NJ) and 1L Corning bottles supplied from Fisher
77 Scientific (Toronto, ON), were used to carry out experiments. Anaerobic and aerobic
78 environments in Balch tubes and Corning bottles were prepared as previously described by
79 Wushke et al. (2015). Draft
80 81 2.2 Genomic Comparison 82 83 The GB1 genome was sequenced and annotated as previously described (Wushke et al.
84 2017). The GB1 and Tf genome sequences can both be accessed through the National Center for
85 Biotechnology Information (NCBI) under the catalogue numbers AZRV00000000 and
86 ARVR00000000, respectively. Joint Genome Institute (JGI) Integrated Microbial Genomes
87 (IMG) Expert Review (IMG/ER) was used to compare the bacterial genomes (Markowitz et al.
88 2008). NUCmer plots were generated through IMG/ER. Gview was used to create genome-scale
89 comparative images (Stothard et al 2017, Petkau et al. 2010). Protein and DNA alignments were
90 done with Multiple Sequence Alignment Tool (MUSCLE) (Edgar et al. 2004). NCBI Blast was
91 used to create a 2D model of domains and key amino acids involved in function (Johnson et al.
92 2008). 3D models of the ADHE proteins were created using RAPTORX (Källberg et al. 2012).
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93 2.3 Protein Extraction 94 95 Samples for protein extraction were collected during early exponential in aerobic growth,
96 OD600 0.2, shaken at 150rpm for both strains GB1 and Tf of C. debilis. Approximately ~200 mg
97 cell pellets were washed twice in phosphate buffered saline (PBS) buffer then re-suspended in 1
98 mL of lysis buffer [1% sodium dodecyl sulfate (SDS), 100 mM dithiothreitol (DTT)] in 100 mM
99 ammonium bicarbonate (ABC). The mixture was vortexed for 30 seconds (sec), then placed in
100 boiling water for 5 minutes (min), and then cooled for 5 min. Samples were then sonicated three
101 times with 15 sec pulses, and 1 min breaks in-between then were centrifuged at 16000 x g for 20
102 min. Supernatants containing the proteins were kept for further processing, and stored at -80 ºC. 103 Aliquots (20 μL) of the protein containingDraft supernatants were diluted with 180 µL of 100 mM 104 ABC buffer. Twenty (20) µL of 500 mM iodoacetic acid (IAA) were added, vortexed for 5 sec
105 and stored in the dark for 45 min to complete alkylation. To quench excess IAA, 33 µL of
106 100mM DTT was added. Then, 150 µL of ABC were added to bring total volume to ~380 µL to
107 reduce the SDS concentration in order to allow digestion. Trypsin (4 µg) was added, and CaCl2
108 was added to a final concentration of 10 mM. Samples were incubated for 12 hours at 37 ºC to
109 allow trypsinization to occur. Samples were then dried using roto-evaporation (Savant Speedvac-
110 sc110), and 200 µL of 3 M KCl was added and vortexed for 10 min, then were spun at 16000 x g
111 for 25 min and supernatants were gently removed from the precipitate (containing the SDS,
112 which is incompatible mass spectrometry methods). The samples were diluted 2 fold with 0.5%
113 trifluoroacetic acid (TFA) then desalted by HPLC using a C18 column as described previously
114 (Wushke et al. 2017).
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115 2.4 Mass Spectrometry Methods 116
117 One-dimensional liquid chromatography followed by mass spectrometry (1D-LC-MS)
118 proteomics were used to identify proteins by their peptide sequences, as previously described by
119 Gungormusler-Yilmaz et al. (2014). Proteomic profiles of the C. debilis strains GB1 and Tf
120 grown to early exponential phase on cellobiose were compared using an in-house data analysis
121 system called UNITY (Table S5), as previously described (Fu et al. 2015). The XML raw output
122 file from an X!tandem search and its corresponding TIC intensities can be found at
123 http://hs2.proteome.ca/thegpm-cgi/plist.pl?path=/gpm/archive/C.debilis_GB1/GB1A-
124 BASERESULTS.xml&proex=-1&npep=0 and http://hs2.proteome.ca/thegpm- 125 cgi/plist.pl?path=/gpm/archive/C.debilis_GB1/GB1B-BASERESULTS.xml&proex=-1&npep=0Draft 126 and http://hs2.proteome.ca/thegpm-cgi/plist.pl?path=/gpm/archive/C.debilis_GB1/DSM1606AX-
127 BASERESULTS.xml&proex=-1&npep=0.
128
129 2.5 Proteogenomics 130 131 To enhance the genome sequence and annotation, a proteogenomic approach was used,
132 on the basis of the proteomes described above. We have previously described the use of
133 proteomic data to identify and correct improperly annotated genes using a naïve assembler
134 (Wushke et al. 2017) using the same methodology as Verbeke et al. (2014). We used these
135 proteomic data to verify unique regions and genes missing in the C. debilis GB1 and Tf genome
136 by analyzing the peptides generated with respect to both the GB1 and Tf genomes.
137
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138 2.6 Whole cell extract enzymatic assays
139 C. debilis cells grown to early exponential (OD600 0.2) on cellobiose were pelleted
140 through centrifugation at 13,000 rpm for 2 minutes. The pelleted cells were resuspended in lysis
141 buffer containing 10 mM MOPS, pH 7.4, 20 mM NaCl, and 5 mM DTT, and lysed using
142 sonication for 6 minutes with 30 seconds on / 30 seconds off cycles. The alcohol dehydrogenase
143 (ADH) assay reaction mixture contained 0.2 mM NADH or NADPH, 10 mM acetaldehyde, 10
144 mM DTT, 100 mM MOPS, and 20 µl cell extract. The final volume was 200 µl, the assay
145 temperature was 50°C, the pH was 7.4, and the assay was started by the addition of acetaldehyde.
146 The ADH reaction was also done in the reverse direction using 1.36 M ethanol, 5 mM NAD+ or
147 NAD(P)+, 10 mM DTT, 100 mM MOPS, and 20 µl cell extract. In the ALDH forward reaction
148 the acetaldehyde in the reaction mixtureDraft was replaced with 0.5 mM acetyl-CoA. In the reverse
149 direction for the ALDH reaction 1 mM CoA was added and the NAD(P)H was replaced with 2.5
150 mM NAD(P)+. Background activity was measured by adding water instead of the usual starter
151 chemical, and was subtracted from the reaction activity recorded. The changes in NAD(P)(H)
152 concentrations were recorded in a BioTek synergy 4 plate reader by monitoring the absorbance at
153 340 nm. All enzymatic assays were done in triplicate.
154
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155 3.0 Results 156 157 3.1 GB1 and Tf Genome Comparison 158 159 A comparision of the GB1 (Wushke et al. 2017) and Tf (Mukherjee et al. 2017)
160 permanent draft genomes containing 49 and 41 contigs respectively can be found in Table 1
161 (complete genome comparison summary in Table S1). CheckM analysis (Parks et al. 2014)
162 indicates 99.42 completeness for Tf and 99.27 for GB1 indicating that these genomes are mostly
163 complete with respect to functional genes. A pairwise ANI of the two genomes is consistent
164 with two strains of the same species (Table S2). The genome sequences of C. debilis strains
165 GB1 and Tf are highly similar in both gene identity and genome synteny (2354 genes in 166 matching cassettes, where a cassette consistsDraft of blocks of associated genes in sets of 2 or more). 167 GB1 has 2570 genes matching Tf with a sequence identity ≥90%. Figure S2 shows a NUCmer
168 plot demonstrating their synteny produced using IMG/ER. When the genome sequences of the
169 two strains were compared, there were 853 genes in GB1 that did not match with a sequence
170 identity greater than or equal to 90% in Tf. Since these strains primarily have highly related
171 genes, matches with ≤90% sequence identity were considered to be unique. Many of the
172 unmatched genes found in GB1 were considered hypothetical (408). Many of the unmatched
173 genes could be attributed to two structural differences: i) the addition of 207 genes on contig 4
174 which appear to form a conjugative element; ii) the addition of the full length genome of a
175 cryptic prophage on contig 16 described elsewhere Wushke et al. (2018), as well as several
176 partial copies of this prophage associated with the end of various other contigs. There were also
177 16 transposase-associated genes dispersed through the genome several of which were near
178 identical copies. Figure 1 provides a graphical representation produced by G-View of the GB1
179 and Tf aligned genome contigs with genes matching at ≤90% sequence similarity highlighted. In
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180 contrast to GB1, Tf has only 300 unique genes that do not match sequences in the GB1 genome
181 at a ≤90% sequence similarity. Unlike GB1 most of the unique genes in Tf were scattered
182 throughout the various contigs and did not appear in any large contiguous sections. The unique
183 genes seen in Tf came from a variety of genes including different transposase-associated genes,
184 different small hypothetical genes, and differences in ABC cassettes. Figure S1 is a Venn
185 diagram of unique genes of GB1 and Tf at a 90% sequence similarity cut off.
186 Sequence analyses of GB1 revealed that there are 24 tRNA genes (Cdeb_01241-
187 Cdeb_01300) on contig 4 that are associated with the conjugative element described above.
188 There are 85 tRNA genes in GB1 compared to only 58 in Tf, as shown in Table S3. 189 Investigation of differences in theDraft gene complement of the Phosphotransferase (PT) 190 systems show that GB1 has several extra subunits associated with putative cellobiose transport
191 specificity (Table S4), consistent with its isolation from cellulose enrichment cultures and plates
192 containing cellobiose as carbon and energy substrate (Wushke et al. 2013).
193 We found two annotation differences between GB1 and Tf during our investigation: i)
194 LSU ribosomal protein L36P (Cdeb_03413), while Tf possessed the exact same DNA as GB1
195 sequence but the ORF was missing in Tf. ii) GB1 has its pyruvate kinase split into two ORF’s
196 (Cdeb_02915, Cdeb_02914) which was annotated as separate but adjacent genes representing the
197 two domains of the pyruvate kinase in GB1 and one gene, a pyruvate kinase, in Tf even though
198 DNA in the coding region is 100% identical.
199 3.2 Proteogenomics 200 201 The general features of the C. debilis GB1 genome have been described previously, and
202 proteogenomics used to correct some annotation start sites (Wushke et al. 2017). In this study we
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203 used a further single shallow proteome for each strain (Table S5), GB1 and Tf (704 and 473
204 proteins detected respectively), during early aerobic growth (Figure S3) which allowed us to: i)
205 further check for genes that were missed due to incomplete assembly of sequences and ii) check
206 whether or not genes in regions which appeared unique to GB1 were expressed. No new genes
207 were found in GB1 or the Tf using proteogenomics that would indicate incomplete sequencing of
208 major transcribed and translated genes. The regions that appeared to be unique to GB1, a cryptic
209 prophage region within contig 16 and conjugation equipment comprising contig 4, resulted in 5
210 and 3 proteins detected respectively. When the Tf proteome was used to check the GB1 genome
211 unique regions on contig 16 and contig 4 no proteins were observed, indicating that these regions
212 were most likely absent in Tf, but unique to GB1.
213 3.4 Pyruvate Fermentation Draft
214 A list of pyruvate metabolism genes and their abbreviations GB1 and Tf can be found in
215 Table 2, the full table with corresponding locus tags can be found in the supplementary (Table
216 S6). When focusing on pyruvate fermentation pathways, small differences were observed in
217 regions within 200bp up stream of a several genes of interest shown in Figure S4. When
218 examining the genes each appeared to have a Pribnow box and transcription start site. In some
219 cases, the differences in the upstream regions between GB1 and Tf were extensive (ldh, pfl, adh),
220 compared to others (ak, pta, adhE, aldh) where differences were observed but were less
221 extensive, as seen in Figure S4. Some of the coding sequences for pyruvate fermentation
222 contained differences (adhE, pfl activating enzyme, one of two aldh, and four of the adh genes),
223 shown in Figure S5, and others (one of two aldh, pfl C-acetyltransferase, ldh and ak) did not, as
224 would be expected in closely related strains. The activity of key enzymes PFL, LDH, AK,
225 ADHE could be surmised by end-product analysis under oxygen-limited conditions, as their
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226 corresponding products were detected, with the exception of ethanol in Tf, even though the
227 genome of strain Tf encodes ADHE. Both strains of C. debilis have a single copy of adhe as well
228 as discrete copies of adh and aldh.
229 While lactic acid production was low in both organisms, differences in lactic acid
230 production were observed between the strains, with GB1 typically producing significantly less
231 than Tf under comparable conditions (Wushke et al. 2015). There are significant differences in
232 upstream regions of the ldh gene between the two strains; these changes may have affected the
233 transcriptional regulation of LDH.
234 When cultured under oxygen-limiting conditions both strains, GB1 and Tf, synthesize 235 formate and acetate as end-products indicatingDraft that both are able to use PFL (Wushke et al. 236 2015). There is a difference in the amount of formate produced by each strain under oxygen-
237 limited conditions; significant differences in the upstream regions of PFL are observed.
238 3.5 Alcohol Aldehyde Dehydrogenase (adhE) fine analysis and Alcohol Aldehyde
239 Dehydrogenase whole lysate assays
240 While the genomes of both strains encoded a putative ADHE enzyme, detailed analyses
241 revealed sequence differences in regions upstream as well as within the coding sequence of the
242 adhE gene. Nucleotide substitutions in the ADHE coding sequence, shown in Figure S6, result in
243 different amino acids: GB1 Tf = 269AE, 482DE, 495VI, 825D A, 829R K,
244 831PH. These amino acid changes, shown graphically in Figure S6 are caused by a single
245 nucleotide polymorphism in all but one case, 495VI where two bp were changed. Changes
246 482DE and 495VI, are most likely benign sense mutations. Two codon changes, 825 DA
247 and 829RK, are seen in other functional ADHE alignments and seem to be normal variations
248 in coding sequence (Extance et al. 2013). However, 269AE is a missense substitution rather
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249 close to a putative catalytic cysteine at position 255. The 831PH codon change is a missense
250 mutation that appears to place a charged amino acid in an α-helix region on the outer shell of the
251 Tf ADHE protein. The changes in the ADH domain of ADHE are not directly within any
252 putative active site, cofactor binding-site, or known allosteric regulation site (Zheng et al. 2015).
253 The Blast domain analysis is shown in Figure S7. These amino acid substitutions affect a small
254 loop connecting an α-helix region (Extance et al. 2013), and therefore could affect overall
255 protein or multimeric protein structure.
256 While there are also differences in regions upstream of adhE, they did not appear to
257 affect the promoter binding site and are not as extensive as the differences observed in ldh and
258 pfl (Figure S4). Draft 259 In order to determine if ADH activity or expression could account for the observed
260 differences in end-product production and anaerobic growth whole cell lysates were assayed.
261 The cell extract ADH activity in the forward direction for GB1 was 2.14 (st. dev = 0.031) U/mg
262 of cell-free extract protein (U = 1 µmol of substrate/min) and for Tf was 1.06 (st. dev. = 0.060)
263 U/mg (Table 3). The ADH activity in the reverse direction for GB1 was 5.75 (st. dev = 0.833)
264 U/mg of cell-free extract protein and for Tf was 0.861 (st. dev. = 0.031) U/mg (Table 3). There
265 was no detectable ADH activity for either GB1 or Tf when using NADPH as the cofactor in the
266 cell extract. The activity of AlDH could not be effectively measured in the reverse direction,
267 possibly due to the presence of an aldh (Cdeb_00412) that only required H2O, acetaldehyde, and
268 NAD+ to form acetate. This enzyme activity masked the CoA dependent activity of aldh, Table
269 S7 shows that activity was the same regardless of the addition of HS-CoA. The forward reaction
270 was not tried as acetyl-CoA is a substrate not only for aldh but also for pyruvate dehydrogenase
271 (PDH) which again would mask results in cell free-extracts.
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272
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273 4.0 Discussion
274 4.1 Genomics
275 The genome comparison confirms that these two organisms are distinct strains of the
276 same species despite their divergent capabilities to grow in the absence of oxygen. While there
277 are some genome structural changes observed, the metabolic potential appears to be nearly
278 identical. Interpreting changes in physiology on the basis of minor changes at the nucleotide
279 level in and surrounding some of the core metabolism genes must be approached with caution as
280 such differences are also consistent with what would be expected in distinct strains (Hayashi et
281 al. 2001). Indeed many of the differences noted between GB1 and Tf would not be expected to 282 affect gene expression or activity (e.g. silentDraft mutations, mutations in non-coding areas). 283 Differences in tRNA complement have the potential to affect gene regulation at a global
284 level at the level of translation. However, all of the tRNA’s unique to GB1 had anticodons that
285 were shared by Tf and GB1, therefore the codon usage ability was not expanded.
286
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287 4.2 Pyruvate Fermentation
288 Oxygen depleting conditions, i.e. conditions that become microaerobic, were the most
289 informative to observe differences in end-product profiles between the 2 strains, Table S8
290 (Wushke et al. 2015). Indeed the end product profile observed previously by Wushke et al.
291 (2015) was key in driving the genomic comparison done in this study.
292 Many wild type thermophilic and mesophilic organisms synthesize lactate as a major
293 fermentative end-product (Narayanan et al. 2004) While there are differences in the proportion
294 of lactate produced for Tf and GB1 and differences in the DNA sequence of the gene, clear
295 reasons for difference in lactic acid production were not apparent despite the difference in LDH 296 sequence. Draft 297 Production of formate and acetate alone from pyruvate cannot act as an electron sink for
298 NADH generated by glycolysis. Under microaerobic conditions where oxygen is present and
299 formate and acetate are being produced, Tf is able to use the electrons generated through
300 glycolysis via the electron transport chain (ETC). If typical glycolysis and PFL are the primary
301 means by which acetyl-CoA is generated, then anaerobic growth is not possible unless an
302 additional reduced compound is produced. Strain GB1 is capable of anaerobic growth because it
303 is also able to synthesize ethanol. Typically, expression of an air-sensitive enzyme, such as PFL,
304 is linked to anaerobic metabolism, including in Bacillus subtilis which has a similar end-product
305 profile as C. debilis GB1 (Nakano et al. 1998). The use of PFL can be important for biosynthesis
306 in other Firmicutes (Rydzak et al. 2015). It is more likely that Tf represents a strain that has lost
307 the ability to produce ethanol, preventing anaerobic growth. Tf still maintains regulation and
308 expression of PFL since formate production is seen under oxygen limiting conditions (Wushke et
309 al. 2015; 2017). Since both strains demonstrate the ability to express PFL, as indicated by the
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310 presences of formate, the focus should be on ethanol production genes, since this is where there
311 is a clear physiological difference.
312 The synthesis of ethanol appears to be the main mechanism used by C. debilis GB1 to
313 dispose of excess electrons from glycolysis under anaerobic conditions, as lactic acid production
314 seems to be limited in both strains under the growth conditions used (Wushke et al. 2015; 2017).
315 Ethanol synthesis may therefore be an anaerobic requirement in C. debilis GB1. ADHE is
316 associated with the bulk of ethanol production in many anaerobic organisms (Carere et al. 2012,
317 Peng et al. 2008).
318 4.3 Lack of ethanol synthesis in Tf 319 The lack of ethanol synthesis in DraftC. debilis Tf may be understood by looking at the 320 sequence of the bifunctional alcohol/aldehyde dehydrogenase (adhE) gene. Differences in adhE
321 gene expression regulation or absence of ADHE enzyme activity could result in the ethanol-
322 minus Tf phenotype and explain the lack of anaerobic growth. There is precedence in
323 Geobacillus thermoglucosidasius, an organism from closely related genus, which produces
324 similar end-products to C. debilis GB1 under both aerobic and anaerobic growth conditions
325 (Extance et al. 2013). G. thermoglucosidasius has a similar core metabolism gene complement to
326 C. debilis GB1 including several ADH genes, a standalone AlDH and a ADHE Extance et al.
327 (2013) noted that gene knock-outs of G. thermoglucosidasius ADHE results in a lack of
328 anaerobic growth and an ethanol-minus phenotype despite the presence of alternate annotated
329 alcohol dehydrogenases and AlDH, very similar to the Tf phenotype. Furthermore, we do
330 observe a ~2 fold increase in acetaldehyde reduction to ethanol activity in C. debilis GB1 cell
331 extracts, and a 5 fold difference in the opposite direction. Taken together, the results suggest that
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332 the phenotype differences observed when comparing GB1 and Tf could be due to the differences
333 in the amino acid sequence of the ADHE.
334 4.4 Respirofermentative Metabolism
335 Previous experiments done by Wushke et al. (2015) characterised end-product production
336 on cellobiose aerobically and anaerobically for strains GB1 and Tf. Under conditions where
337 oxygen concentrations are low Tf and GB1 both utilise incomplete respiration and non-reductive
338 components of fermentative pathways, also referred to as respirofermentative growth (Siso et al.
339 1996, Zigha et al. 2007). Incomplete respiration can happen when TCA cycle flux is less than
340 that of glycolysis (Vemuri et al. 2006). During respirofermentative growth a significant 341 proportion of the electrons produced fromDraft glycolysis are consumed by respiration, but excess 342 electrons from glycolysis are shunted to fermentation products such as ethanol. GB1 can either
343 use its electrons to create ethanol and/or lactate and/or use them in ETC; based on end-product
344 production under oxygen limiting conditions it appears to do all three. Tf, when undergoing
345 respiro-fermentative growth, utilizes the ETC and produces lactate as alternate electron sink.
346 While organism like Lactobacillus balance its electrons anaerobically by producing lactate alone
347 (De Vries et al.1970), Tf does not do this under the growth conditions tested. While Tf and GB1
348 both have additional adh and aldh genes, these may be used primarily for aerobic ethanol
349 oxidation; Wushke et al. (2015) show GB1 is capable of consuming ethanol aerobically.
350
351 4.5 Issues with the current genus description for Caldibacillus
352 Members of the Bacillus and Geobacillus genera appear to fill many niches within both
353 aerobic and facultativly anaerobic environmental communities. The loss of anaerobic
354 metabolism would seem to be a rather large change regarding taxonomical classification, which
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355 would typically lead to different strains being described as different species based on
356 physiological characteristics. However, strains GB1 and Tf do appear to be 2 strains of the same
357 species based on their genome comparison. The inability of Tf to undergo anaerobic metabolism
358 appears to be the result of one or several very small changes in the genomes. The ability of
359 similar strains to fulfill distinct roles within a co-culture has been previously described by
360 Verbeke et al. (2011). However, because of the geographic distance between the sources of these
361 2 strains it is impossible at this point to state whether loss of anaerobic growth capabilities is due
362 to natural microdiversity within the species or post enrichment genetic drift because of
363 differences in growth conditions in laboratory prior to sequencing.
364 Overall C. debilis GB1 and Tf are very similar organisms at the genetic level, in both
365 synteny and gene complement, includingDraft core metabolism pathway genes. Through our genome
366 analysis a gene complement consistent with fermentation (pfl, ldh, ak, pta, and adhE) was
367 observed in Tf and GB1. Tf likely evolved from a strain that could ferment cellobiose and
368 produce ethanol. The ability of C. debilis GB1 to grow under anaerobic conditions and
369 synthesize ethanol clearly distinguishes it from the C. debilis the type strain, Tf. The genus
370 description of Caldibacillus should be amended to include anaerobic growth as a possible
371 characteristic for strains of this genus, since it is observed in at least one strain of the only
372 species currently described.
373 Acknowledgements
374 This research was supported by Genome Canada, through the Applied Genomics Research in
375 Bioproducts or Crops (ABC) program for the grant titled, “Microbial Genomics for Biofuels and
376 Co-Products from Biorefining Processes” and by the Natural Sciences and Engineering Research
377 Council of Canada (NSERC) Discovery grant (RGPIN-06173-2014) held by Dr. Sparling
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498 Figure 1. Unique Genome of C. debilis GB1 (panel A) and Tf (Panel B). Gene bank file (.gbk) 499 comparison of Unique Genome genes that match ≤90% sequence similarity to the 500 corresponding strain are shown in red, ORF’s are shown in brown. This figure is a 501 graphical representation of the differences between the two genomes created by 502 concatenating the contigs of each organism and then comparing them. In panel A, Contig 503 4 is shown with a green arrow, the full putative phage genome shown with orange arrow, 504 plasmid shown with blue arrow. In panel B the areas of uniqueness are much shorter, 505 typically only corresponding to a few genes. The purple arrow points to a divergent ATP 506 binding cassette transporter. 507 508
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A)
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B)
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Table 1. General genome features of C. debilis GB1 and Tf (source: IMG/er) GB1 Tf DNA, total number of bases 3346235 3061346 DNA coding number of bases 2670185 2430336 % G+C content 51.2 51.6
DNA scaffolds 49 41 CRISPR Count 5 8
Genes total number 3374 2896 Protein coding genes 3264 2800 RNA genes 110 96 rRNA genes 8 13 5S rRNA 5 5 16S rRNA 2 5 23S rRNA 1 3 tRNA genes 85 58 Other RNA genes 17 25 Protein coding genes with functionDraft 2450 2212 prediction without function prediction 814 588 Shared genes (<90% sequence similarity) 2571 Unique genes (<90% sequence similarity) 853 300
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Table 2. Pyruvate metabolism gene complement of GB1 and Tf as specified through KEGG maps built in IMG/ER
Pyruvate Metabolism # of gene copies in GB1 (same as Tf) Pyruvate formate lyase (pfl) 2 Bifunctional aldehyde/alcohol dehydrogenase (adhe) 1 Acetate kinase (ak) 1 Phosphotransacetylase (pta) 1 Lactate dehydrogenase (ldh) 1 Aldehyde dehydrogenase (aldh) 2 Alcohol dehydrogenase (adh) 4 Pyruvate Dehydrogenase (pdh) E1 (EC:1.2.4.1) 6 E2 (EC:2.3.1.12) 3 E3 (EC:1.8.1.4) 3
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Table 3. NADH/NAD+ dependent ADH activity in whole cell lysate U/mg Strain added to reaction mixture protein Standard deviation GB1 acetaldehyde 2.033 0.031 Total activity of GB1 ADH in forward direction* 2.137 Tf acetaldehyde 1.281 0.059 Total activity of Tf ADH in forward direction* 1.059 GB1 ethanol 6.59 0.833 Total activity of GB1 in reverse direction* 5.75 Tf ethanol 0.710 0.031 Total activity of Tf in reverse direction* 0.861 *Corrected by subtracting the no carbon substrate control
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