Antonie van Leeuwenhoek (2014) 106:1259–1267 DOI 10.1007/s10482-014-0296-z
ORIGINAL PAPER
Oenococcus alcoholitolerans sp. nov., a lactic acid bacteria isolated from cachac¸a and ethanol fermentation processes Fernanda Badotti • Ana Paula B. Moreira • Luciane A. Chimetto Tonon • Brı´gida T. Luckwu de Lucena • Fa´tima de Ca´ssia O. Gomes • Ricardo Kruger Cristiane C. Thompson • Marcos Antonio de Morais Jr. • Carlos A. Rosa • Fabiano L. Thompson
•
Received: 24 March 2014 / Accepted: 30 September 2014 / Published online: 15 October 2014 Ó Springer International Publishing Switzerland 2014
Abstract Four strains of lactic acid bacteria isolated from cachac¸a and alcohol fermentation vats in Brazil were characterised in order to determine their taxonomic position. Phylogenetic analysis revealed that they belong to the genus Oenococcus and should be distinguished from their closest neighbours. The 16S rRNA gene sequence similarity against the type strains of the other two species of the genus was below 94.76 % (Oenococcus kitaharae) and 94.62 % (Oenococcus oeni). The phylogeny based on pheS gene sequences also confirmed the position of the new taxon. DNA–DNA hybridizations based on in silico
genome-to-genome comparison, Average Amino Acid Identity, Average Nucleotide Identity and Karlin genomic signature confirmed the novelty of the taxon. Distinctive phenotypic characteristics are the ability to metabolise sucrose but not trehalose. The name Oenococcus alcoholitolerans sp. nov. is proposed for this taxon, with the type strain UFRJ-M7.2.18T ( = CBAS474T = LMG27599T). In addition, we have determined a draft genome sequence of the type strain. Keywords Oenococcus alcoholitolerans Cachac¸a Ethanol Genomic taxonomy
Fernanda Badotti and Ana Paula B. Moreira have contributed equally to this work.
Introduction
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0296-z) contains supplementary material, which is available to authorized users.
Brazil is currently the world major producer of ethanol derived from sugar cane. The Organization of the
F. Badotti (&) C. A. Rosa Departamento de Microbiologia, Instituto de Cieˆncias Biolo´gicas (ICB), Universidade Federal de Minas Gerais, C.P. 486, Belo Horizonte, MG 31270-901, Brazil e-mail: [email protected] A. P. B. Moreira L. A. C. Tonon C. C. Thompson Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-599, Brazil e-mail: [email protected] B. T. L. de Lucena Centro de Cieˆncias Biolo´gicas e Sociais Aplicadas, Universidade Estadual da Paraı´ba, Joa˜o Pessoa, Brazil
F. C. O. Gomes Departamento de Quı´mica, Centro Federal de Educac¸a˜o Tecnolo´gica de Minas Gerais, Belo Horizonte, Brazil R. Kruger Laborato´rio de Enzimologia, Departamento de Biologia Celular, Instituto de Biologia, Universidade de Brası´lia, Brası´lia, Brazil M. A. de Morais Jr. Departamento de Gene´tica, Universidade Federal de Pernambuco, Recife, Brazil
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United Nations reports that in 2011, sugar cane production was around 7 million hectares per year. Sugar, cachac¸a and fuel ethanol are important products derived from the sugar cane agroindustry in Brazil. Ethanol production was *21.1 billion liters in 2013, representing 25 % of the alcohol used as fuel globally (FAOSTAT 2013). Cachac¸a is the most popular alcoholic beverage produced in the country and is obtained by the distillation of fermented sugar cane juice. The sugar cane fermentation is a process characterised by the presence of a complex microbial community provided mainly by the substrate and the equipment’s surface. Saccharomyces cerevisiae is responsible for the alcoholic fermentation and predominates during the process. However, non-Saccharomyces yeasts and lactic acid bacteria (LAB) are frequently isolated (Badotti et al. 2012). The abundance and diversity of microorganisms present during the fermentative processes are widely recognised to contribute significantly for the quality and production rate of cachac¸a and ethanol (Lucena et al. 2010; Badotti et al. 2012). In contrast to the dairy industry, where LAB play critical roles in the texture, flavour and quality of products, in cachac¸a and ethanol fermentations this group of microorganisms are regarded as contaminants. LAB are often brought to the fermentation vats with sugar cane juice where they compete with the fermenting yeast S. cerevisiae for the nutrients. They are very abundant in these processes due to their tolerance to ethanol, low pH and high temperatures (Gomes et al. 2010; Lucena et al. 2010; Badotti et al. 2012). Many studies have shown that LAB are the most commonly isolated bacterial group in ethanolic fermentations (Schwan et al. 2001; Skinner and Leathers 2004; Narendranath 2009; Beckner et al. 2011) and Lactobacillus is the predominant genus (Beckner et al. 2011). Lactobacillus fermentum, Lactobacillus vini (Chang et al. 1995; Lucena et al. 2010), Lactobacillus plantarum (Heist 2009), as well as L. delbruckii (Skinner and Leathers 2004), have
F. L. Thompson Instituto de Biologia and Laborato´rio de Sistemas Avanc¸ados de Gesta˜o da Produc¸a˜o - SAGE/ COPPE, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-972, Brazil
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been found as the predominant species during ethanol production. Carvalho-Netto et al. (2008) characterised the bacterial community in cachac¸a production by a cultivation-independent technique and found Lactobacillus hilgardii and L. plantarum as prevalent species. Gomes et al. (2010) identified Lactobacillus casei and L. plantarum as the most abundant cultivatable species involved in the cachac¸a fermentation. The genus Oenococcus currently comprises two species, Oenococcus oeni (Garvie 1967; Dicks et al. 1995) and Oenococcus kitaharae (Endo and Okada 2006), which belongs to the Class Bacillii, Order Lactobacillales, Family Leuconostocaceae. The type species of the genus Oenococcus was formerly described as Leuconostoc oenos, being subsequently reclassified as O. oeni (Dicks et al. 1995). O. oeni is an important bacterium in wine-resident microbiota. It performs the malolactic fermentation (MLF) via the malolactic enzyme (MLE) (Kunkee 1991) decreasing wine sourness, preventing proliferation of undesirable microorganisms, and also ensuring the production of secondary metabolites that confer aroma and flavour (Kunkee 1991; Nielsen et al. 1996). O. kitaharae was isolated from a composting distilled schochu residue (Endo and Okada 2006) and is non-malolactic due to a point mutation that results in a premature stop codon in the gene for the MLE (Borneman et al. 2012). During two independent studies focused on the characterisation of LAB diversity from sugar cane fermentations in Brazil, we obtained four isolates that either differed from the currently recognised Oenococcus species based on the 16S rRNA gene sequences (i.e. UFMG-PEM25 and UFMG-PEM32A) or did not match any Amplified Ribosomal DNA Restriction Analysis (ARDRA) profile (UFRJ-M7.2.18T and UFRJ-JP7.3.6) (Lucena et al. 2010). Ethanol and cachac¸a fermentations are new habitats for Oenococcus members, which may reveal new biochemical and genomic traits of the genus. The aim of this study was to perform the taxonomic characterisation of the new Oenococcus strains. The isolates from cachac¸a and ethanol production were found to form a tight genetic group, which is here proposed as Oenococcus alcoholitolerans sp. nov., with the type strain UFRJ-M7.2.18T ( = LMG27599T = CBAS474T). In our genomic taxonomic approach, we analyzed a draft genome sequence of the type strain UFRJ-M7.2.18T in order to determine genomic and phenotypic features of the new species.
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Materials and methods Bacterial strains Strains UFMG-PEM25 and UFMG-PEM32A were isolated from fermentation vats for cachac¸a production during the 2005 season. Samples were collected from traditional distilleries located in the state of Pernambuco (UFMG-PEM25 from Engenho A´gua Doce distillery and UFMG-PEM32A from Sa˜o Sarue´ distillery). For the isolation of LAB from cachac¸a fermentation, 10-6 and 10-8 dilutions of must were spread onto plates containing Man, Rogosa and Sharp medium (MRS, Difco, USA) supplemented with 100 mg L-1cycloheximide. The plates were incubated in an anaerobic chamber (Forma Scientific Company, USA) at 37 °C for 48 h. Strains UFRJM7.2.18T and UFRJ-JP7.3.6 were isolated from ethanol production plants, from distilleries Miriri and Japungu, respectively, located in Paraiba state, during the harvesting season 2007–2008 as described in Lucena et al. (2010). Phenotypic tests Morphological, physiological and biochemical characteristics were determined by standard methods. The growth of isolates was tested for 49 different carbon sources using Api 50 CH kit (Biome´rieux) according to the manufacturer’s instructions. For analysis of stress factors, fresh cultures were used to inoculate 5 mL of MRS/BHI broth (Endo and Okada 2006). After 24 h, the cultures were adjusted to a turbidity of 1.0 MacFarland standard using sterile saline (0.8 % NaCl). An aliquot (2 mL) of suspension was then used to inoculate the stress media. Four stress factors were studied: pH (4.0, 4.5, 5.0, 6.0, 6.5, 7.0 and 7.5), NaCl (1.0, 2.0, 2.5 and 3.0 % m/v), temperature (15, 20, 25, 30 and 37 °C) and ethanol (5, 7 and 12 % v/v). Stress media were prepared using MRS/BHI broth supplemented with ethanol or NaCl. The pH was adjusted using NaOH or HCl standard solutions. Temperature analyses were performed by incubating the tubes at the above-mentioned temperatures. Fermentation tests were performed using Durham tubes. The motility of strains was evaluated by inoculating the culture in semi-solid MRS with a strap-shaped needle. Nitrate reduction was tested in
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a medium containing 2 % agar, 1.17 % Yeast Carbon Base (YCB, Difco, USA) and 0.078 % potassium nitrate. A negative control was inoculated in parallel containing only 1.17 % YCB. Except for temperature stress tests, all media were incubated at 30 °C. Phylogenetic, fingerprinting and genomic analyses The 16S rRNA genes of strains UFMG-PEM25 and UFMG-PEM32A were amplified as described by Lane (1991) with primers 27F and 1492R. DNA extraction was carried out according to Moreira et al. (2005). The amplified DNA was concentrated, cleaned and sequenced using an ABI3130 (Life Technologies, USA) automated sequencing system at the Veterinary Faculty of the Federal University of Minas Gerais, Brazil. Strains UFRJ-M7.2.18T and UFRJ-JP7.3.6 were identified to genus level as described in Lucena et al. (2010). Briefly, a collection of isolates, from different distilleries, were grouped by ARDRA and identified according to the patterns described by Moreira et al. (2005). M7.2.18 and JP7.3.6, named group BL7, did not match any ARDRA profile. Phylogenetic analysis based on 16S rRNA (1,506 bp) and partial pheS gene sequences (see Naser et al. 2007) revealed the novelty of the group. All sequences were edited with Chromas V. 2.33 (Technelysium Pty Ltd) and compared to known sequences in the NCBI Genbank database using the Local Alignment Search Tool (BLAST) algorithm (Altschul et al. 1990). Sequences were aligned using ClustalW. The phylogenetic relationships were inferred using the Neighbour-Joining (Saitou and Nei 1987) and Maximum Likelihood (ML) (Felsenstein 1981) methods. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches (Felsenstein 1985). The evolutionary distances were computed using the p-distance (Fig. 1) (Nei and Kumar 2000), the Kimura 2-parameter (Fig. 2) (Kimura 1980) and the Jukes-Cantor (Supplementary Figs. 1 and 2) (Jukes and Cantor 1969) methods and are shown in the units of the number of base differences per site. All ambiguous positions were removed for each sequence pair. There were a total of 1,587 nucleotide positions for the 16S rRNA gene and 456 for pheS in the final datasets. Evolutionary
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Fig. 1 Neighbour-joining phylogenetic tree showing the position of the Oenococcus alcoholitolerans sp. nov. strains based on 16S rRNA gene sequences (1,587 bp). Bootstrap values ([70 %) based on 1,000 repetitions are shown. The sequence of Lactococcus taiwanensis 0905C15T was used as outgroup. Bar 2 % estimated sequence divergence
Fig. 2 Neighbour-joining phylogenetic tree showing the position of the Oenococcus alcoholitolerans sp. nov strains based on pheS gene sequences (456 bp). The analysis involved 19 nucleotide sequences. Codon positions included were
1st ? 2nd ? 3rd ? Noncoding. The sequence of Carnobacterium maltaromaticum LMG 6903T was used as outgroup. Bar 5 % estimated sequence divergence
analyses were conducted in MEGA5 (Tamura et al. 2011). Partial 16S rRNA gene and pheS sequences identities were calculated by pairwise comparison using Jalview V.2 (Waterhouse et al. 2009). The type strains’ 16S rRNA gene sequences used were those recommended by the Strainfo (http://www.straininfo. net/) SeqRank, for which the accession numbers are AB681195 and AB221475 (for O. oeni NBRC 100497T and O. kitaharae NRIC 0645T, respectively). Genomic fingerprinting of the four isolates was performed using repetitive sequence-based PCR with the (GTG)5 oligonucleotide (Versalovic et al. 1994). Each 25 lL PCR reaction contained 2.5 lL 10X reaction
buffer, 1.5 lL 1.5 M MgCl, 1.0 lL 10 mM dNTP (2.5 mM each), 2.0 lL 10 pmol (GTG)5, 1.0 lL 200 ng lL-1 DNA, 0.2 lL 1.25 U Taq DNA polymerase. PCR amplifications were performed in an automated thermal cycler (BioCycler MG48G) with an initial denaturation (94 °C, 2 min) followed by 40 cycles of denaturation (93 °C, 45 s), annealing (50 °C, 1 min) and extension (72 °C, 1 min) with a single final extension (72 °C, 6 min). The products were electrophoresed in 1 % agarose gel, stained with gel red and visualized under UV light. The draft genome sequence of the type strain UFRJM7.2.18 T (CBAS474T) was obtained by
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Antonie van Leeuwenhoek (2014) 106:1259–1267 Table 1 Characteristics of the genomes of UFRJM7.2.18T and the other Oenococcus species, provided by RAST
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Characteristics
O. alcoholitolerans UFRJ-M7.2.18T
O. kitaharae DSM17330T
O. oeni PSU-1
GenBank accession
AXCV00000000
AFVZ01000000
CP000411.1
RAST ID
6666666.47730
6666666.48432
6666666.48741
Size (MB) Number of contigs
1.2 698
1.8 2
1.7 1
Median sequence size (bp)
1,296
921,069
1,780,517
DNA G?C (mol%)
39.5
42.7
37.9
Subsystems
162
259
262
Coding sequences
1,431
1,854
1,893
RNAs
22
48
49
Features in subsystems
421 (30 %)
833 (45 %)
855 (46 %)
Non hypothetical
403
807
829
Not in subsystems
1,010 (70 %)
1,021 (55 %)
1,038 (54 %)
Non hypothetical
538
529
532
Subsystems coverage
pyrosequencing using a Roche 454 GS FLX Titanium system (Margulies et al. 2005). All reads were assembled using gsAssembler 2.3. Digital DNA– DNA hybridization (DDH) values between the type strain (UFRJ-M7.2.18T), O. kitaharae DSM 17330T and O. oeni PSU-1 were estimated using in silico genome-to-genome comparison by means of GenomeTo-Genome Distance Calculator V.2 (GGDC) (Auch et al. 2010a, b; Meier-Kolthoff et al. 2013). Average Amino Acid Identity (AAI) was calculated according to Konstantinidis and Tiedje (2005) using BLAST (v. 2.2.29?) (Altschul et al. 1997). The parameters to select conserved CDSs between genome pairs were E value (less than 1e-5), sequence identity (more than 40 %), and the alignable region (more than 70 %). Average Nucleotide Identity (ANI) was calculated according to Goris et al. (2007). The genomic signature was determined by the dinucleotide relative abundance value according to Karlin et al. (1997) and Karlin (1998). Genomic taxonomy followed the assumptions of Thompson et al. (2013a), i.e. species of the same genus will form monophyletic groups on the basis of 16S rRNA gene sequences, and the in silico DDH, AAI, ANI and Karlin genomic signature cut-offs that delineate a microbial species are [70, [95,[95 and \10 %, respectively. Determination of the genome general features, G?C mol% and MLF related gene searches were performed with Rapid Annotation using Subsystem Technology (RAST) (Aziz et al. 2008). A global visualization of the genus
Oenococcus genomes (O. alcoholitolerans sp. nov. UFRJ-M7.2.18T; O. kitaharae DSM 17330T; O. oeni ATCC BAA-1163) has been represented through Genome Atlas based on GeneWiz browser 0.94 server (Hallin et al. 2008). O. oeni PSU-1 was used as a reference genome. The nucleotide sequence (GenBank accession X82326) described by Labarre et al. (1996) and ‘‘OKIT_0114’’ (locus_tag) of O. kitaharae DSM 17330T genome (GenBank accession AFVZ01000000) were employed as template for enzyme searches using BLAST algorithm in RAST environment. Analysis of retrieved sequences was performed using blastn, blastp, and Psi-Blast at The Seed Viewer (Version 2.0). Protein alignment was provided by The Multiple Sequence Alignment Package T-COFFEE, Version 5.31 (Notredame et al. 2000). A protein search using blastx, alignment (CLUSTALO) and identification were also conducted against the Uniprot database (http://www.uniprot.org/). The most similar sequences, derived from complete proteome (only), were used to build a guide tree.
Results and discussion 16S rRNA gene sequence analysis revealed that the novel cluster represented by the designated type strain UFRJM7.2.18T formed a tight monophyletic branch affiliated to the genus Oenococcus, in a robust sub-cluster
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1264 Table 2 Acid production from different carbon sources for the four strains of Oenococcus alcoholitolerans sp. nov. and their closest relatives
Antonie van Leeuwenhoek (2014) 106:1259–1267
Phenotypic characteristic
O. kitaharae NRIC 0645Ta
O. oeni NCDO 1674b
O. alcoholitolerans UFMGPEM25
UFMGPEM32A
UFRJM7.2.18
UFRJJP7.3.6
D-glucose
?
?
?
?
?
?
D-fructose
?
?
?
?
?
?
D-galactose
?
v
?
-
-
-
D-mannose
?
v
?
?
-
?
Maltose
?
-
?
-
-
-
D-melibiose
?
v
?
?
?
?
D-trehalose
?
?
-
-
-
-
D-ribose
w
ND
?
-
-
-
D-gluconate
w
ND
-
-
-
-
L-rhamnose
-
ND
-
-
-
-
D-mannitol
-
-
-
-
-
-
D-raffinose
w
-
?
?
?
?
L-arabinose
-
v
?
-
-
-
D-xylose
-
v
?
-
-
-
D-melezitose
-
ND -
-
-
?
?
D-sorbitol
-
ND
-
-
-
-
Starch
-
ND
-
-
-
-
a-D-lactose ? Grow, - not grow, v variable, w weak, ND not determined a b
Endo and Okada (2006) Dicks et al. (1995)
Sucrose
-
-
?
?
?
?
D-cellobiose
v
v
?
?
-
?
D-salicin
v
v
?
-
?
?
separated from its closest neighbours (Fig. 1, Supplementary Fig. 1). The four isolates shared more than 99 % 16S rRNA gene sequence similarity, however the banding pattern derived from our fingerprinting analysis discriminated them (Supplementary Fig. 3). The sequence similarities towards the closest neighbours were \97 %, the conservative threshold established for delineation of a bacterial species (Stackebrandt and Goebel 1994; Vandamme et al. 1996), and C94.5 %, the recently proposed genus border (Yarza et al. 2014). O. oeni NBRC 100497T showed 94.49 % (UFRJ-M7.2.18T) to 94.62 % (UFMG-PEM25) sequence similarity; and O. kitaharae NRIC 0645T showed 94.51 % (UFRJM7.2.18T) to 94.76 % (UFMG-PEM25). Strain UFRJM7.2.18T shared less than 75.5 and 73.4 % pheS gene sequence similarity with their closest neighbours, O. oeni LMG 9851T and O. kitaharae LMG 24489T, respectively (Fig. 2 and Supplementary Fig. 2). General features of the UFRJ-M7.2.18T genome are supplied in Table 1. In silico DDH (%) values between the genome sequences of UFRJ-M7.2.18T and O. kitaharae DSM 17330T and O. oeni PSU-1 were 20.3 (SD: 2.31) and 21.9 (SD: 2.35),
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respectively. Between O. kitaharae DSM 17330T and O. oeni PSU-1 the result was 17.6 (SD: 2.23). Endo and Okada (2006) determined laboratory DDH values (microplates method; Ezaki et al. 1989) between O. kitaharae strains and O. oeni NRIC 0331T varying from 25 to 30 %, highlighting that DNA–DNA similarities inferred by GGDC are well suited to delineate Oenococcus species, as previously demonstrated for other groups (Thompson et al. 2013b). AAI (%) between the genome sequences of UFRJ-M7.2.18T and O. kitaharae DSM 17330T and O. oeni PSU-1 were 65.4 and 64.7, respectively , both[50 %, the proposed genus boundary for prokaryotes (Qin et al. 2014). AAI between the genome sequences of O. kitaharae DSM 17330T and O. oeni PSU-1 was 68.5. AAI values for these three genome pairs and the value of shared ORFs (62 %) among O. kitaharae DSM 17330T and other O. oeni genomes (n = 3) (Borneman et al. 2012) fell within the range 62–69 %. Two-way ANI (%) between the genome sequences of UFRJ-M7.2.18T and O. kitaharae DSM 17330T and O. oeni PSU-1 were 81.74 (SD: 6.93) and 80.26 (SD: 7.92), respectively. Values between the
Antonie van Leeuwenhoek (2014) 106:1259–1267 Table 3 Growth profiles of Oenococcus alcoholitolerans sp. nov
? Grow, - not grow, w weak, ND not determined a
Endo and Okada (2006)
b
Dicks et al. (1995)
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Growth conditions
O. kitaharae NRIC 0645Ta
O. oeni NCDO 1674b
UFMGPEM25
UFMGPEM32A
UFRJM7.2.18
UFRJJP7.3.6
pH 4
-
?
w
?
w
w
NaCl 1.0 %
?
ND
?
?
?
?
NaCl 2.0 % NaCl 2.5 %
ND -
ND ND
? ?
w w
? ?
? -
NaCl 3.0 %
ND
ND
-
-
-
-
Ethanol 5 %
?
?
?
?
?
?
Ethanol 7 %
ND
?
?
?
?
?
Ethanol 12 %
-
ND
?
w
w
?
Glucose fermentation
?
?
?
w
?
?
genome sequences of O. kitaharae DSM 17330T and O. oeni PSU-1 were 79.94 (SD: 5.70). The ANI results correlated well with the estimated DDH values. Karlin genomic signature dissimilarity between the genome sequences of UFRJ-M7.2.18T and O. kitaharae DSM 17330T and O. oeni PSU-1 were 59 and 45, respectively. Between the genome sequences of O. kitaharae DSM 17330T and O. oeni PSU-1 it was 80, and among O. oeni PSU-1 and other twenty O. oeni genomes available in Genbank ranged from 7 to 10, meaning Karlin genomic signature dissimilarity was also discriminative for members of the genus Oenococcus. All the genomic parameters analyzed are in agreement and robustly indicate that strain UFRJ-M7.2.18T ( = CBAS474T) represents a new Oenococcus species. A comparative analysis of the Oenococcus genomes is shown in Supplementary Fig. 4. The inner genome (O. oeni ATCC BAA-1163) is the most conserved (coloured) compared to the reference sequence (O. oeni PSU-1). There are several regions of low conservation (lightly coloured areas) in the other two genomes (i.e. O. kitaharae DSM 17330T and strain UFRJ-M7.2.18T) illustrating well the GGDC results herein reported. The strain UFRJ-M7.2.18T genome contains the MLE gene located at contig 00329, peg.1004. Partial sequence length is 378 bp. Translated CDS comprises 126 aa and shares 82.4 % identity with the O. oeni PSU-1 orthologue (22 mismatches, 0 gaps). The comparison with the MLE gene of O. kitaharae DSM 17330T reveals that strain UFRJM7.2.18T does not carry the nonsense mutation
associated with absence of MLF in O. kitaharae. We thus conclude that strain UFRJ-M7.2.18T likely has the capacity for MLF. Nucleotide and protein alignments are shown in Supplementary Figs. 5 and 6. A guide tree constructed with the most similar protein sequences, derived from complete proteomes only (n = 26), is shown in Supplementary Fig. 7. The UFRJ-M7.2.18T enzyme clusters with those of O. oeni strains whilst enzymes from members of Lactobacillus and Pediococcus are found in the other branch. Phenotypic characteristics and growth profiles of the isolates are shown in Tables 2 and 3, respectively, and in the species description. Distinctive features are the acid production from sucrose but not from trehalose. Acid production from raffinose further helps distinguish the new taxon from the type strain of O. oeni (Table 3). Based on the phenotypic, genomic and phylogenetic information obtained in this study we propose the new species Oenococcus alcoholitolerans for which the type strain UFRJ-M7.2.18T ( = LMG27599T = CBAS474T) is designated. Accession numbers The GenBank/EMBL/DDBJ sequence accession numbers for sequences determined in this study are as follows: HQ009794-5 (UFRJ-M7.2.18T, UFRJ-JP7.3.6), KF731675-6 (UFMG-PEM25, UFMG-PEM32A) for the 16S rRNA genes; HQ009781-2 (UFRJ-M7.2.18T, UFRJ-JP7.3.6) for pheS genes; and AXCV00000000 for the draft genome of strain UFRJ-M7.2.18T.
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Description of Oenococcus alcoholitolerans Badotti, Moreira, Tonon, Rosa, and Thompson, sp nov. Oenococcus alcoholitolerans (N.L. n. alcohol -olis, alcohol; L. part. adj. tolerans, tolerating; N.L. part. adj. alcoholitolerans, alcohol-tolerating.) Cells are Gram-positive, non-motile, white, smooth, ellipsoidal cocci of approximately 0.9–1.0 lm in diameter. Usually appears in pairs, sometimes in chains of four, less frequently of six, and rarely individually. Facultatively anaerobic and catalasenegative. Growth on agar medium is enhanced under anaerobic conditions. Strains are heterofermentative, produce lactic acid, carbon dioxide and ethanol or acetic acid from D-glucose, although some strains may be slow fermenters. Nitrate is not reduced. Acid is produced from D-glucose, D-fructose, D-melibiose, Draffinose and sucrose. Acid production from D-galactose, D-mannose, D-maltose, D-ribose, L-arabinose, Dxylose, D-lactose, D-cellobiose and D-salicin shows variable reactions within the species. Acid is not formed from D-trehalose, D-gluconate, L-rhamnose, Dmannitol, D-melezitoze, D-sorbitol and starch. Esculin is hydrolyzed. Temperature range for growth is 20–40 °C with an optimum of 30 °C. Does not grow at 15 °C. The pH range for growth is 4.0–7.5 with an optimum of 6.0–6.5. Some strains, including the type strain, show weak growth at pH 4.0. NaCl range for growth is 1–2.5 %, although some strains do not grow in 2.5 %. Grows at 12 % ethanol but growth is weak for some strains, including the type strain. The DNA G?C mol% of the type strain is 39.5. The type strain UFRJ-M7.2.18T ( = LMG27599T = CBAS474T) was isolated from an ethanol production plant. Acknowledgments Financial support came from Fundac¸a˜o de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), Fundac¸a˜o de Amparo a Pesquisa do Estado de Pernambuco (FACEPE), Fundac¸a˜o de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).
References Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410
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Antonie van Leeuwenhoek (2014) 106:1259–1267 Altschul SF, Madden TL, Scha¨ffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402 Auch AF, Klenk H-P, Go¨ker M (2010a) Standard operating procedure for calculating genome-to-genome distances based on high-scoring segment pairs. Stand Genomic Sci 2(1):142–148. doi:10.4056/sigs.541628 Auch AF, Klenk H-P, Go¨ker M (2010b) Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genomic Sci 2(1):117–134. doi:10.4056/sigs.531120 Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008) The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9(1):75. doi:10.1186/ 1471-2164-9-75 Badotti F, Gomes F, Rosa C (2012) Brazilian cachac¸a: fer¨, mentation and production. In: Hui YH, Evranuz EO Hansen AS (eds) Handbook of plant-based fermented food and beverage technology, 2nd edn. CRC Press, Boca Raton, pp 639–648 Beckner M, Ivey ML, Phister TG (2011) Microbial contamination of fuel ethanol fermentations. Lett Appl Microbiol 53(4):387–394 Borneman AR, McCarthy JM, Chambers PJ, Bartowsky EJ (2012) Functional divergence in the genus Oenococcus as predicted by genome sequencing of the newly-described species, Oenococcus kitaharae. PLoS ONE 7(1):e29626 Carvalho-Neto O, Rosa DD, Camargo LEA (2008) Identification of contaminant bacteria in cachac¸a yeast by 16S rDNA gene sequencing. Sci Agric 65:508–515 Chang IS, Kim BH, Shin PK, Lee WK (1995) Bacterial contamination and its effects on ethanol fermentation. J Microbiol Biotechnol 5(6):309–314 Dicks LMT, Dellaglio F, Collins MD (1995) Proposal to reclassify Leuconostoc Oenos as Oenococcus Oeni [corrig] gen-nov, comb-nov. Int J Syst Evol Microbiol 45(2):395–397 Endo A, Okada S (2006) Oenococcus kitaharae sp nov., a nonacidophilic and non-malolactic-fermenting oenococcus isolated from a composting distilled shochu residue. Int J Syst Evol Microbiol 56:2345–2348 Ezaki T, Hashimoto Y, Yabuuchi E (1989) Fluorometric deoxyribonucleic acid deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane-filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39(3):224–229 FAOSTAT (2013) http://www.faostat.fao.org/site/339/default. aspx. Accessed 15/Jul/2014 Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17(6):368–376 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39(4):783–791 Garvie EI (1967) Leuconostoc oenos sp. nov. J Gen Microbiol 48(3):431–438 Gomes FCO, Silva CLC, Vianna CR, Lacerda ICA, Borelli BM, Nunes AC, Franco GR, Mourao MM, Rosa CA (2010)
Antonie van Leeuwenhoek (2014) 106:1259–1267 Identification of lactic acid bacteria associated with traditional cachaca fermentations. Braz J Microbiol 41(2):486–492 Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM (2007) DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57(Pt 1):81–91 Hallin PF, Binnewies TT, Ussery DW (2008) The genome ¨ ˆıa GeneWiz extension for visualization of BLASTatlas, A whole-genome homology. Mol BioSyst 4(5):363–371 Heist P (2009) Identifying, controlling the most common microbial contaminants. Ethanol Producer Magazine. http://www.ethanolproducer.com/articles/5464/ identifying-the-most-common-microbial-contaminants Accessed 15/Jul/2013 Jukes T, Cantor C (1969) Evolution of protein molecules. In: Munro HN (ed) Mammalian protein metabolism. Academic Press, New York, pp 21–132 Karlin S, Mra´zek J, Campbell AM. (1997) Compositional biases of bacterial genomes and evolutionary implications. J Bacteriol 179(12):3899–913 Karlin S. (1998) Global dinucleotide signatures and analysis of genomic heterogeneity. Curr Opin Microbiol 1(5):598–610 Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16(2):111–120 Konstantinidis KT, Tiedje JM (2005) Towards a genome-based taxonomy for prokaryotes. J Bacteriol 187:6258–6264 Kunkee RE (1991) Some roles of malic-acid in the malolactic fermentation in wine-making. FEMS Microbiol Rev 88(1):55–72 Labarre C, Guzzo J, Cavin J-F, Divies C (1996) Cloning and characterization of the genes encoding the malolactic enzyme and the malate permease of Leuconostoc oenos. Appl Environ Microbiol 62(4):1274–1282 Lane D (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 115–175 Lucena BTL, dos Santos BM, Moreira JLS, Moreira APB, Nunes AC, Azevedo V, Miyoshi A, Thompson FL, de Morais Junior MA (2010) Diversity of lactic acid bacteria of the bioethanol process. BMC Microbiol 10:298 Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen ZT, Dewell SB, Du L, Fierro JM, Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Irzyk GP, Jando SC, Alenquer MLI, Jarvie TP, Jirage KB, Kim JB, Knight JR, Lanza JR, Leamon JH, Lefkowitz SM, Lei M, Li J, Lohman KL, Lu H, Makhijani VB, McDade KE, McKenna MP, Myers EW, Nickerson E, Nobile JR, Plant R, Puc BP, Ronan MT, Roth GT, Sarkis GJ, Simons JF, Simpson JW, Srinivasan M, Tartaro KR, Tomasz A, Vogt KA, Volkmer GA, Wang SH, Wang Y, Weiner MP, Yu PG, Begley RF, Rothberg JM (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437(7057):376–380 Meier-Kolthoff JP, Go¨ker M, Spro¨er C, Klenk HP (2013) When should a DDH experiment be mandatory in microbial taxonomy? Arch Microbiol 195(6):413–418. doi:10.1007/ s00203-013-0888-4 Moreira JO, Mota R, Horta M, Teixeira S, Neumann E, Nicoli J, ˚ (2005) Identification to the species level of Nunes A
1267 Lactobacillus isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling. BMC Microbiol 5(1):15 Narendranath NSB (2009) Bacterial contamination and control in ethanol production. In: WM I (ed) The alcohol textbook, vol 4. 5 edn. Nottingham University Press, Nottingham, pp 481–490 Naser SM, Dawyndt P, Hoste B, Gevers D, Vandemeulebroecke K, Cleenwerck I, Vancanneyt M, Swings J (2007) Identification of lactobacilli by pheS and rpoA gene sequence analyses. Int J Syst Evol Microbiol 57(Pt 12):2777–2789 Nei M, Kumar S (2000) Molecular evolution and phylogenetics. Oxford University Press, New York Nielsen JC, Prahl C, LonvaudFunel A (1996) Malolactic fermentation in wine by direct inoculation with freeze-dried Leuconostoc oenos cultures. Am J Enol Vitic 47(1):42–48 Notredame C, Higgins DG, Heringa J (2000) T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302(1):205–217 Qin QL, Xie BB, Zhang XY, Chen XL, Zhou BC, Zhou J, Zhang YZ (2014) A proposed genus boundary for the prokaryotes based on genomic insights. J bacteriol 196(12):2210–2215 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425 Schwan RF, Mendonca AT, da Silva JJ, Rodrigues V, Wheals AE (2001) Microbiology and physiology of Cachaca (Aguardente) fermentations. Antonie Van Leeuwenhoek 79(1):89–96 Skinner KA, Leathers TD (2004) Bacterial contaminants of fuel ethanol production. J Ind Microbiol Biotechnol 31(9):401–408. doi:10.1007/s10295-004-0159-0 Stackebrandt E, Goebel B (1994) Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Evol Microbiol 44:846–849 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739 Thompson CC, Chimetto L, Edwards RA, Swings J, Stackebrandt E, Thompson FL (2013a) a Microbial genomic taxonomy. BMC Genom 14(1):913 Thompson CC, Silva GG, Vieira NM, Edwards R, Vicente ACP, Thompson FL (2013b) b Genomic Taxonomy of the Genus Prochlorococcus. Microb Ecol 66(4):752–762 Vandamme P, Pot B, Gillis M, De Vos P, Kersters K, Swings J (1996) Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 60:407–438 Versalovic J, Schneider M, de Bruijn FJ, Lupski JR (1994) Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol Cell Biol 5:25–40 Waterhouse AM, Procter JB, Martin DM, Clamp Ml, Barton GJ (2009) Jalview version 2, a multiple sequence alignment editor and analysis workbench. Bioinformatics 25(9):1189–1191 Yarza P, Yilmaz P, Pruesse E, Glo¨ckner FO, Ludwig W, Schleifer KH, Rossello´-Mo´ra R (2014) Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol 12(9):635–645
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ORIGINAL PAPER
Oenococcus alcoholitolerans sp. nov., a lactic acid bacteria isolated from cachac¸a and ethanol fermentation processes Fernanda Badotti • Ana Paula B. Moreira • Luciane A. Chimetto Tonon • Brı´gida T. Luckwu de Lucena • Fa´tima de Ca´ssia O. Gomes • Ricardo Kruger Cristiane C. Thompson • Marcos Antonio de Morais Jr. • Carlos A. Rosa • Fabiano L. Thompson
•
Received: 24 March 2014 / Accepted: 30 September 2014 / Published online: 15 October 2014 Ó Springer International Publishing Switzerland 2014
Abstract Four strains of lactic acid bacteria isolated from cachac¸a and alcohol fermentation vats in Brazil were characterised in order to determine their taxonomic position. Phylogenetic analysis revealed that they belong to the genus Oenococcus and should be distinguished from their closest neighbours. The 16S rRNA gene sequence similarity against the type strains of the other two species of the genus was below 94.76 % (Oenococcus kitaharae) and 94.62 % (Oenococcus oeni). The phylogeny based on pheS gene sequences also confirmed the position of the new taxon. DNA–DNA hybridizations based on in silico
genome-to-genome comparison, Average Amino Acid Identity, Average Nucleotide Identity and Karlin genomic signature confirmed the novelty of the taxon. Distinctive phenotypic characteristics are the ability to metabolise sucrose but not trehalose. The name Oenococcus alcoholitolerans sp. nov. is proposed for this taxon, with the type strain UFRJ-M7.2.18T ( = CBAS474T = LMG27599T). In addition, we have determined a draft genome sequence of the type strain. Keywords Oenococcus alcoholitolerans Cachac¸a Ethanol Genomic taxonomy
Fernanda Badotti and Ana Paula B. Moreira have contributed equally to this work.
Introduction
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0296-z) contains supplementary material, which is available to authorized users.
Brazil is currently the world major producer of ethanol derived from sugar cane. The Organization of the
F. Badotti (&) C. A. Rosa Departamento de Microbiologia, Instituto de Cieˆncias Biolo´gicas (ICB), Universidade Federal de Minas Gerais, C.P. 486, Belo Horizonte, MG 31270-901, Brazil e-mail: [email protected] A. P. B. Moreira L. A. C. Tonon C. C. Thompson Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-599, Brazil e-mail: [email protected] B. T. L. de Lucena Centro de Cieˆncias Biolo´gicas e Sociais Aplicadas, Universidade Estadual da Paraı´ba, Joa˜o Pessoa, Brazil
F. C. O. Gomes Departamento de Quı´mica, Centro Federal de Educac¸a˜o Tecnolo´gica de Minas Gerais, Belo Horizonte, Brazil R. Kruger Laborato´rio de Enzimologia, Departamento de Biologia Celular, Instituto de Biologia, Universidade de Brası´lia, Brası´lia, Brazil M. A. de Morais Jr. Departamento de Gene´tica, Universidade Federal de Pernambuco, Recife, Brazil
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United Nations reports that in 2011, sugar cane production was around 7 million hectares per year. Sugar, cachac¸a and fuel ethanol are important products derived from the sugar cane agroindustry in Brazil. Ethanol production was *21.1 billion liters in 2013, representing 25 % of the alcohol used as fuel globally (FAOSTAT 2013). Cachac¸a is the most popular alcoholic beverage produced in the country and is obtained by the distillation of fermented sugar cane juice. The sugar cane fermentation is a process characterised by the presence of a complex microbial community provided mainly by the substrate and the equipment’s surface. Saccharomyces cerevisiae is responsible for the alcoholic fermentation and predominates during the process. However, non-Saccharomyces yeasts and lactic acid bacteria (LAB) are frequently isolated (Badotti et al. 2012). The abundance and diversity of microorganisms present during the fermentative processes are widely recognised to contribute significantly for the quality and production rate of cachac¸a and ethanol (Lucena et al. 2010; Badotti et al. 2012). In contrast to the dairy industry, where LAB play critical roles in the texture, flavour and quality of products, in cachac¸a and ethanol fermentations this group of microorganisms are regarded as contaminants. LAB are often brought to the fermentation vats with sugar cane juice where they compete with the fermenting yeast S. cerevisiae for the nutrients. They are very abundant in these processes due to their tolerance to ethanol, low pH and high temperatures (Gomes et al. 2010; Lucena et al. 2010; Badotti et al. 2012). Many studies have shown that LAB are the most commonly isolated bacterial group in ethanolic fermentations (Schwan et al. 2001; Skinner and Leathers 2004; Narendranath 2009; Beckner et al. 2011) and Lactobacillus is the predominant genus (Beckner et al. 2011). Lactobacillus fermentum, Lactobacillus vini (Chang et al. 1995; Lucena et al. 2010), Lactobacillus plantarum (Heist 2009), as well as L. delbruckii (Skinner and Leathers 2004), have
F. L. Thompson Instituto de Biologia and Laborato´rio de Sistemas Avanc¸ados de Gesta˜o da Produc¸a˜o - SAGE/ COPPE, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-972, Brazil
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been found as the predominant species during ethanol production. Carvalho-Netto et al. (2008) characterised the bacterial community in cachac¸a production by a cultivation-independent technique and found Lactobacillus hilgardii and L. plantarum as prevalent species. Gomes et al. (2010) identified Lactobacillus casei and L. plantarum as the most abundant cultivatable species involved in the cachac¸a fermentation. The genus Oenococcus currently comprises two species, Oenococcus oeni (Garvie 1967; Dicks et al. 1995) and Oenococcus kitaharae (Endo and Okada 2006), which belongs to the Class Bacillii, Order Lactobacillales, Family Leuconostocaceae. The type species of the genus Oenococcus was formerly described as Leuconostoc oenos, being subsequently reclassified as O. oeni (Dicks et al. 1995). O. oeni is an important bacterium in wine-resident microbiota. It performs the malolactic fermentation (MLF) via the malolactic enzyme (MLE) (Kunkee 1991) decreasing wine sourness, preventing proliferation of undesirable microorganisms, and also ensuring the production of secondary metabolites that confer aroma and flavour (Kunkee 1991; Nielsen et al. 1996). O. kitaharae was isolated from a composting distilled schochu residue (Endo and Okada 2006) and is non-malolactic due to a point mutation that results in a premature stop codon in the gene for the MLE (Borneman et al. 2012). During two independent studies focused on the characterisation of LAB diversity from sugar cane fermentations in Brazil, we obtained four isolates that either differed from the currently recognised Oenococcus species based on the 16S rRNA gene sequences (i.e. UFMG-PEM25 and UFMG-PEM32A) or did not match any Amplified Ribosomal DNA Restriction Analysis (ARDRA) profile (UFRJ-M7.2.18T and UFRJ-JP7.3.6) (Lucena et al. 2010). Ethanol and cachac¸a fermentations are new habitats for Oenococcus members, which may reveal new biochemical and genomic traits of the genus. The aim of this study was to perform the taxonomic characterisation of the new Oenococcus strains. The isolates from cachac¸a and ethanol production were found to form a tight genetic group, which is here proposed as Oenococcus alcoholitolerans sp. nov., with the type strain UFRJ-M7.2.18T ( = LMG27599T = CBAS474T). In our genomic taxonomic approach, we analyzed a draft genome sequence of the type strain UFRJ-M7.2.18T in order to determine genomic and phenotypic features of the new species.
Antonie van Leeuwenhoek (2014) 106:1259–1267
Materials and methods Bacterial strains Strains UFMG-PEM25 and UFMG-PEM32A were isolated from fermentation vats for cachac¸a production during the 2005 season. Samples were collected from traditional distilleries located in the state of Pernambuco (UFMG-PEM25 from Engenho A´gua Doce distillery and UFMG-PEM32A from Sa˜o Sarue´ distillery). For the isolation of LAB from cachac¸a fermentation, 10-6 and 10-8 dilutions of must were spread onto plates containing Man, Rogosa and Sharp medium (MRS, Difco, USA) supplemented with 100 mg L-1cycloheximide. The plates were incubated in an anaerobic chamber (Forma Scientific Company, USA) at 37 °C for 48 h. Strains UFRJM7.2.18T and UFRJ-JP7.3.6 were isolated from ethanol production plants, from distilleries Miriri and Japungu, respectively, located in Paraiba state, during the harvesting season 2007–2008 as described in Lucena et al. (2010). Phenotypic tests Morphological, physiological and biochemical characteristics were determined by standard methods. The growth of isolates was tested for 49 different carbon sources using Api 50 CH kit (Biome´rieux) according to the manufacturer’s instructions. For analysis of stress factors, fresh cultures were used to inoculate 5 mL of MRS/BHI broth (Endo and Okada 2006). After 24 h, the cultures were adjusted to a turbidity of 1.0 MacFarland standard using sterile saline (0.8 % NaCl). An aliquot (2 mL) of suspension was then used to inoculate the stress media. Four stress factors were studied: pH (4.0, 4.5, 5.0, 6.0, 6.5, 7.0 and 7.5), NaCl (1.0, 2.0, 2.5 and 3.0 % m/v), temperature (15, 20, 25, 30 and 37 °C) and ethanol (5, 7 and 12 % v/v). Stress media were prepared using MRS/BHI broth supplemented with ethanol or NaCl. The pH was adjusted using NaOH or HCl standard solutions. Temperature analyses were performed by incubating the tubes at the above-mentioned temperatures. Fermentation tests were performed using Durham tubes. The motility of strains was evaluated by inoculating the culture in semi-solid MRS with a strap-shaped needle. Nitrate reduction was tested in
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a medium containing 2 % agar, 1.17 % Yeast Carbon Base (YCB, Difco, USA) and 0.078 % potassium nitrate. A negative control was inoculated in parallel containing only 1.17 % YCB. Except for temperature stress tests, all media were incubated at 30 °C. Phylogenetic, fingerprinting and genomic analyses The 16S rRNA genes of strains UFMG-PEM25 and UFMG-PEM32A were amplified as described by Lane (1991) with primers 27F and 1492R. DNA extraction was carried out according to Moreira et al. (2005). The amplified DNA was concentrated, cleaned and sequenced using an ABI3130 (Life Technologies, USA) automated sequencing system at the Veterinary Faculty of the Federal University of Minas Gerais, Brazil. Strains UFRJ-M7.2.18T and UFRJ-JP7.3.6 were identified to genus level as described in Lucena et al. (2010). Briefly, a collection of isolates, from different distilleries, were grouped by ARDRA and identified according to the patterns described by Moreira et al. (2005). M7.2.18 and JP7.3.6, named group BL7, did not match any ARDRA profile. Phylogenetic analysis based on 16S rRNA (1,506 bp) and partial pheS gene sequences (see Naser et al. 2007) revealed the novelty of the group. All sequences were edited with Chromas V. 2.33 (Technelysium Pty Ltd) and compared to known sequences in the NCBI Genbank database using the Local Alignment Search Tool (BLAST) algorithm (Altschul et al. 1990). Sequences were aligned using ClustalW. The phylogenetic relationships were inferred using the Neighbour-Joining (Saitou and Nei 1987) and Maximum Likelihood (ML) (Felsenstein 1981) methods. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches (Felsenstein 1985). The evolutionary distances were computed using the p-distance (Fig. 1) (Nei and Kumar 2000), the Kimura 2-parameter (Fig. 2) (Kimura 1980) and the Jukes-Cantor (Supplementary Figs. 1 and 2) (Jukes and Cantor 1969) methods and are shown in the units of the number of base differences per site. All ambiguous positions were removed for each sequence pair. There were a total of 1,587 nucleotide positions for the 16S rRNA gene and 456 for pheS in the final datasets. Evolutionary
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Fig. 1 Neighbour-joining phylogenetic tree showing the position of the Oenococcus alcoholitolerans sp. nov. strains based on 16S rRNA gene sequences (1,587 bp). Bootstrap values ([70 %) based on 1,000 repetitions are shown. The sequence of Lactococcus taiwanensis 0905C15T was used as outgroup. Bar 2 % estimated sequence divergence
Fig. 2 Neighbour-joining phylogenetic tree showing the position of the Oenococcus alcoholitolerans sp. nov strains based on pheS gene sequences (456 bp). The analysis involved 19 nucleotide sequences. Codon positions included were
1st ? 2nd ? 3rd ? Noncoding. The sequence of Carnobacterium maltaromaticum LMG 6903T was used as outgroup. Bar 5 % estimated sequence divergence
analyses were conducted in MEGA5 (Tamura et al. 2011). Partial 16S rRNA gene and pheS sequences identities were calculated by pairwise comparison using Jalview V.2 (Waterhouse et al. 2009). The type strains’ 16S rRNA gene sequences used were those recommended by the Strainfo (http://www.straininfo. net/) SeqRank, for which the accession numbers are AB681195 and AB221475 (for O. oeni NBRC 100497T and O. kitaharae NRIC 0645T, respectively). Genomic fingerprinting of the four isolates was performed using repetitive sequence-based PCR with the (GTG)5 oligonucleotide (Versalovic et al. 1994). Each 25 lL PCR reaction contained 2.5 lL 10X reaction
buffer, 1.5 lL 1.5 M MgCl, 1.0 lL 10 mM dNTP (2.5 mM each), 2.0 lL 10 pmol (GTG)5, 1.0 lL 200 ng lL-1 DNA, 0.2 lL 1.25 U Taq DNA polymerase. PCR amplifications were performed in an automated thermal cycler (BioCycler MG48G) with an initial denaturation (94 °C, 2 min) followed by 40 cycles of denaturation (93 °C, 45 s), annealing (50 °C, 1 min) and extension (72 °C, 1 min) with a single final extension (72 °C, 6 min). The products were electrophoresed in 1 % agarose gel, stained with gel red and visualized under UV light. The draft genome sequence of the type strain UFRJM7.2.18 T (CBAS474T) was obtained by
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Antonie van Leeuwenhoek (2014) 106:1259–1267 Table 1 Characteristics of the genomes of UFRJM7.2.18T and the other Oenococcus species, provided by RAST
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Characteristics
O. alcoholitolerans UFRJ-M7.2.18T
O. kitaharae DSM17330T
O. oeni PSU-1
GenBank accession
AXCV00000000
AFVZ01000000
CP000411.1
RAST ID
6666666.47730
6666666.48432
6666666.48741
Size (MB) Number of contigs
1.2 698
1.8 2
1.7 1
Median sequence size (bp)
1,296
921,069
1,780,517
DNA G?C (mol%)
39.5
42.7
37.9
Subsystems
162
259
262
Coding sequences
1,431
1,854
1,893
RNAs
22
48
49
Features in subsystems
421 (30 %)
833 (45 %)
855 (46 %)
Non hypothetical
403
807
829
Not in subsystems
1,010 (70 %)
1,021 (55 %)
1,038 (54 %)
Non hypothetical
538
529
532
Subsystems coverage
pyrosequencing using a Roche 454 GS FLX Titanium system (Margulies et al. 2005). All reads were assembled using gsAssembler 2.3. Digital DNA– DNA hybridization (DDH) values between the type strain (UFRJ-M7.2.18T), O. kitaharae DSM 17330T and O. oeni PSU-1 were estimated using in silico genome-to-genome comparison by means of GenomeTo-Genome Distance Calculator V.2 (GGDC) (Auch et al. 2010a, b; Meier-Kolthoff et al. 2013). Average Amino Acid Identity (AAI) was calculated according to Konstantinidis and Tiedje (2005) using BLAST (v. 2.2.29?) (Altschul et al. 1997). The parameters to select conserved CDSs between genome pairs were E value (less than 1e-5), sequence identity (more than 40 %), and the alignable region (more than 70 %). Average Nucleotide Identity (ANI) was calculated according to Goris et al. (2007). The genomic signature was determined by the dinucleotide relative abundance value according to Karlin et al. (1997) and Karlin (1998). Genomic taxonomy followed the assumptions of Thompson et al. (2013a), i.e. species of the same genus will form monophyletic groups on the basis of 16S rRNA gene sequences, and the in silico DDH, AAI, ANI and Karlin genomic signature cut-offs that delineate a microbial species are [70, [95,[95 and \10 %, respectively. Determination of the genome general features, G?C mol% and MLF related gene searches were performed with Rapid Annotation using Subsystem Technology (RAST) (Aziz et al. 2008). A global visualization of the genus
Oenococcus genomes (O. alcoholitolerans sp. nov. UFRJ-M7.2.18T; O. kitaharae DSM 17330T; O. oeni ATCC BAA-1163) has been represented through Genome Atlas based on GeneWiz browser 0.94 server (Hallin et al. 2008). O. oeni PSU-1 was used as a reference genome. The nucleotide sequence (GenBank accession X82326) described by Labarre et al. (1996) and ‘‘OKIT_0114’’ (locus_tag) of O. kitaharae DSM 17330T genome (GenBank accession AFVZ01000000) were employed as template for enzyme searches using BLAST algorithm in RAST environment. Analysis of retrieved sequences was performed using blastn, blastp, and Psi-Blast at The Seed Viewer (Version 2.0). Protein alignment was provided by The Multiple Sequence Alignment Package T-COFFEE, Version 5.31 (Notredame et al. 2000). A protein search using blastx, alignment (CLUSTALO) and identification were also conducted against the Uniprot database (http://www.uniprot.org/). The most similar sequences, derived from complete proteome (only), were used to build a guide tree.
Results and discussion 16S rRNA gene sequence analysis revealed that the novel cluster represented by the designated type strain UFRJM7.2.18T formed a tight monophyletic branch affiliated to the genus Oenococcus, in a robust sub-cluster
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1264 Table 2 Acid production from different carbon sources for the four strains of Oenococcus alcoholitolerans sp. nov. and their closest relatives
Antonie van Leeuwenhoek (2014) 106:1259–1267
Phenotypic characteristic
O. kitaharae NRIC 0645Ta
O. oeni NCDO 1674b
O. alcoholitolerans UFMGPEM25
UFMGPEM32A
UFRJM7.2.18
UFRJJP7.3.6
D-glucose
?
?
?
?
?
?
D-fructose
?
?
?
?
?
?
D-galactose
?
v
?
-
-
-
D-mannose
?
v
?
?
-
?
Maltose
?
-
?
-
-
-
D-melibiose
?
v
?
?
?
?
D-trehalose
?
?
-
-
-
-
D-ribose
w
ND
?
-
-
-
D-gluconate
w
ND
-
-
-
-
L-rhamnose
-
ND
-
-
-
-
D-mannitol
-
-
-
-
-
-
D-raffinose
w
-
?
?
?
?
L-arabinose
-
v
?
-
-
-
D-xylose
-
v
?
-
-
-
D-melezitose
-
ND -
-
-
?
?
D-sorbitol
-
ND
-
-
-
-
Starch
-
ND
-
-
-
-
a-D-lactose ? Grow, - not grow, v variable, w weak, ND not determined a b
Endo and Okada (2006) Dicks et al. (1995)
Sucrose
-
-
?
?
?
?
D-cellobiose
v
v
?
?
-
?
D-salicin
v
v
?
-
?
?
separated from its closest neighbours (Fig. 1, Supplementary Fig. 1). The four isolates shared more than 99 % 16S rRNA gene sequence similarity, however the banding pattern derived from our fingerprinting analysis discriminated them (Supplementary Fig. 3). The sequence similarities towards the closest neighbours were \97 %, the conservative threshold established for delineation of a bacterial species (Stackebrandt and Goebel 1994; Vandamme et al. 1996), and C94.5 %, the recently proposed genus border (Yarza et al. 2014). O. oeni NBRC 100497T showed 94.49 % (UFRJ-M7.2.18T) to 94.62 % (UFMG-PEM25) sequence similarity; and O. kitaharae NRIC 0645T showed 94.51 % (UFRJM7.2.18T) to 94.76 % (UFMG-PEM25). Strain UFRJM7.2.18T shared less than 75.5 and 73.4 % pheS gene sequence similarity with their closest neighbours, O. oeni LMG 9851T and O. kitaharae LMG 24489T, respectively (Fig. 2 and Supplementary Fig. 2). General features of the UFRJ-M7.2.18T genome are supplied in Table 1. In silico DDH (%) values between the genome sequences of UFRJ-M7.2.18T and O. kitaharae DSM 17330T and O. oeni PSU-1 were 20.3 (SD: 2.31) and 21.9 (SD: 2.35),
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respectively. Between O. kitaharae DSM 17330T and O. oeni PSU-1 the result was 17.6 (SD: 2.23). Endo and Okada (2006) determined laboratory DDH values (microplates method; Ezaki et al. 1989) between O. kitaharae strains and O. oeni NRIC 0331T varying from 25 to 30 %, highlighting that DNA–DNA similarities inferred by GGDC are well suited to delineate Oenococcus species, as previously demonstrated for other groups (Thompson et al. 2013b). AAI (%) between the genome sequences of UFRJ-M7.2.18T and O. kitaharae DSM 17330T and O. oeni PSU-1 were 65.4 and 64.7, respectively , both[50 %, the proposed genus boundary for prokaryotes (Qin et al. 2014). AAI between the genome sequences of O. kitaharae DSM 17330T and O. oeni PSU-1 was 68.5. AAI values for these three genome pairs and the value of shared ORFs (62 %) among O. kitaharae DSM 17330T and other O. oeni genomes (n = 3) (Borneman et al. 2012) fell within the range 62–69 %. Two-way ANI (%) between the genome sequences of UFRJ-M7.2.18T and O. kitaharae DSM 17330T and O. oeni PSU-1 were 81.74 (SD: 6.93) and 80.26 (SD: 7.92), respectively. Values between the
Antonie van Leeuwenhoek (2014) 106:1259–1267 Table 3 Growth profiles of Oenococcus alcoholitolerans sp. nov
? Grow, - not grow, w weak, ND not determined a
Endo and Okada (2006)
b
Dicks et al. (1995)
1265
Growth conditions
O. kitaharae NRIC 0645Ta
O. oeni NCDO 1674b
UFMGPEM25
UFMGPEM32A
UFRJM7.2.18
UFRJJP7.3.6
pH 4
-
?
w
?
w
w
NaCl 1.0 %
?
ND
?
?
?
?
NaCl 2.0 % NaCl 2.5 %
ND -
ND ND
? ?
w w
? ?
? -
NaCl 3.0 %
ND
ND
-
-
-
-
Ethanol 5 %
?
?
?
?
?
?
Ethanol 7 %
ND
?
?
?
?
?
Ethanol 12 %
-
ND
?
w
w
?
Glucose fermentation
?
?
?
w
?
?
genome sequences of O. kitaharae DSM 17330T and O. oeni PSU-1 were 79.94 (SD: 5.70). The ANI results correlated well with the estimated DDH values. Karlin genomic signature dissimilarity between the genome sequences of UFRJ-M7.2.18T and O. kitaharae DSM 17330T and O. oeni PSU-1 were 59 and 45, respectively. Between the genome sequences of O. kitaharae DSM 17330T and O. oeni PSU-1 it was 80, and among O. oeni PSU-1 and other twenty O. oeni genomes available in Genbank ranged from 7 to 10, meaning Karlin genomic signature dissimilarity was also discriminative for members of the genus Oenococcus. All the genomic parameters analyzed are in agreement and robustly indicate that strain UFRJ-M7.2.18T ( = CBAS474T) represents a new Oenococcus species. A comparative analysis of the Oenococcus genomes is shown in Supplementary Fig. 4. The inner genome (O. oeni ATCC BAA-1163) is the most conserved (coloured) compared to the reference sequence (O. oeni PSU-1). There are several regions of low conservation (lightly coloured areas) in the other two genomes (i.e. O. kitaharae DSM 17330T and strain UFRJ-M7.2.18T) illustrating well the GGDC results herein reported. The strain UFRJ-M7.2.18T genome contains the MLE gene located at contig 00329, peg.1004. Partial sequence length is 378 bp. Translated CDS comprises 126 aa and shares 82.4 % identity with the O. oeni PSU-1 orthologue (22 mismatches, 0 gaps). The comparison with the MLE gene of O. kitaharae DSM 17330T reveals that strain UFRJM7.2.18T does not carry the nonsense mutation
associated with absence of MLF in O. kitaharae. We thus conclude that strain UFRJ-M7.2.18T likely has the capacity for MLF. Nucleotide and protein alignments are shown in Supplementary Figs. 5 and 6. A guide tree constructed with the most similar protein sequences, derived from complete proteomes only (n = 26), is shown in Supplementary Fig. 7. The UFRJ-M7.2.18T enzyme clusters with those of O. oeni strains whilst enzymes from members of Lactobacillus and Pediococcus are found in the other branch. Phenotypic characteristics and growth profiles of the isolates are shown in Tables 2 and 3, respectively, and in the species description. Distinctive features are the acid production from sucrose but not from trehalose. Acid production from raffinose further helps distinguish the new taxon from the type strain of O. oeni (Table 3). Based on the phenotypic, genomic and phylogenetic information obtained in this study we propose the new species Oenococcus alcoholitolerans for which the type strain UFRJ-M7.2.18T ( = LMG27599T = CBAS474T) is designated. Accession numbers The GenBank/EMBL/DDBJ sequence accession numbers for sequences determined in this study are as follows: HQ009794-5 (UFRJ-M7.2.18T, UFRJ-JP7.3.6), KF731675-6 (UFMG-PEM25, UFMG-PEM32A) for the 16S rRNA genes; HQ009781-2 (UFRJ-M7.2.18T, UFRJ-JP7.3.6) for pheS genes; and AXCV00000000 for the draft genome of strain UFRJ-M7.2.18T.
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1266
Description of Oenococcus alcoholitolerans Badotti, Moreira, Tonon, Rosa, and Thompson, sp nov. Oenococcus alcoholitolerans (N.L. n. alcohol -olis, alcohol; L. part. adj. tolerans, tolerating; N.L. part. adj. alcoholitolerans, alcohol-tolerating.) Cells are Gram-positive, non-motile, white, smooth, ellipsoidal cocci of approximately 0.9–1.0 lm in diameter. Usually appears in pairs, sometimes in chains of four, less frequently of six, and rarely individually. Facultatively anaerobic and catalasenegative. Growth on agar medium is enhanced under anaerobic conditions. Strains are heterofermentative, produce lactic acid, carbon dioxide and ethanol or acetic acid from D-glucose, although some strains may be slow fermenters. Nitrate is not reduced. Acid is produced from D-glucose, D-fructose, D-melibiose, Draffinose and sucrose. Acid production from D-galactose, D-mannose, D-maltose, D-ribose, L-arabinose, Dxylose, D-lactose, D-cellobiose and D-salicin shows variable reactions within the species. Acid is not formed from D-trehalose, D-gluconate, L-rhamnose, Dmannitol, D-melezitoze, D-sorbitol and starch. Esculin is hydrolyzed. Temperature range for growth is 20–40 °C with an optimum of 30 °C. Does not grow at 15 °C. The pH range for growth is 4.0–7.5 with an optimum of 6.0–6.5. Some strains, including the type strain, show weak growth at pH 4.0. NaCl range for growth is 1–2.5 %, although some strains do not grow in 2.5 %. Grows at 12 % ethanol but growth is weak for some strains, including the type strain. The DNA G?C mol% of the type strain is 39.5. The type strain UFRJ-M7.2.18T ( = LMG27599T = CBAS474T) was isolated from an ethanol production plant. Acknowledgments Financial support came from Fundac¸a˜o de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), Fundac¸a˜o de Amparo a Pesquisa do Estado de Pernambuco (FACEPE), Fundac¸a˜o de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).
References Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410
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Antonie van Leeuwenhoek (2014) 106:1259–1267 Altschul SF, Madden TL, Scha¨ffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402 Auch AF, Klenk H-P, Go¨ker M (2010a) Standard operating procedure for calculating genome-to-genome distances based on high-scoring segment pairs. Stand Genomic Sci 2(1):142–148. doi:10.4056/sigs.541628 Auch AF, Klenk H-P, Go¨ker M (2010b) Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genomic Sci 2(1):117–134. doi:10.4056/sigs.531120 Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008) The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9(1):75. doi:10.1186/ 1471-2164-9-75 Badotti F, Gomes F, Rosa C (2012) Brazilian cachac¸a: fer¨, mentation and production. In: Hui YH, Evranuz EO Hansen AS (eds) Handbook of plant-based fermented food and beverage technology, 2nd edn. CRC Press, Boca Raton, pp 639–648 Beckner M, Ivey ML, Phister TG (2011) Microbial contamination of fuel ethanol fermentations. Lett Appl Microbiol 53(4):387–394 Borneman AR, McCarthy JM, Chambers PJ, Bartowsky EJ (2012) Functional divergence in the genus Oenococcus as predicted by genome sequencing of the newly-described species, Oenococcus kitaharae. PLoS ONE 7(1):e29626 Carvalho-Neto O, Rosa DD, Camargo LEA (2008) Identification of contaminant bacteria in cachac¸a yeast by 16S rDNA gene sequencing. Sci Agric 65:508–515 Chang IS, Kim BH, Shin PK, Lee WK (1995) Bacterial contamination and its effects on ethanol fermentation. J Microbiol Biotechnol 5(6):309–314 Dicks LMT, Dellaglio F, Collins MD (1995) Proposal to reclassify Leuconostoc Oenos as Oenococcus Oeni [corrig] gen-nov, comb-nov. Int J Syst Evol Microbiol 45(2):395–397 Endo A, Okada S (2006) Oenococcus kitaharae sp nov., a nonacidophilic and non-malolactic-fermenting oenococcus isolated from a composting distilled shochu residue. Int J Syst Evol Microbiol 56:2345–2348 Ezaki T, Hashimoto Y, Yabuuchi E (1989) Fluorometric deoxyribonucleic acid deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane-filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39(3):224–229 FAOSTAT (2013) http://www.faostat.fao.org/site/339/default. aspx. Accessed 15/Jul/2014 Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17(6):368–376 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39(4):783–791 Garvie EI (1967) Leuconostoc oenos sp. nov. J Gen Microbiol 48(3):431–438 Gomes FCO, Silva CLC, Vianna CR, Lacerda ICA, Borelli BM, Nunes AC, Franco GR, Mourao MM, Rosa CA (2010)
Antonie van Leeuwenhoek (2014) 106:1259–1267 Identification of lactic acid bacteria associated with traditional cachaca fermentations. Braz J Microbiol 41(2):486–492 Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM (2007) DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57(Pt 1):81–91 Hallin PF, Binnewies TT, Ussery DW (2008) The genome ¨ ˆıa GeneWiz extension for visualization of BLASTatlas, A whole-genome homology. Mol BioSyst 4(5):363–371 Heist P (2009) Identifying, controlling the most common microbial contaminants. Ethanol Producer Magazine. http://www.ethanolproducer.com/articles/5464/ identifying-the-most-common-microbial-contaminants Accessed 15/Jul/2013 Jukes T, Cantor C (1969) Evolution of protein molecules. In: Munro HN (ed) Mammalian protein metabolism. Academic Press, New York, pp 21–132 Karlin S, Mra´zek J, Campbell AM. (1997) Compositional biases of bacterial genomes and evolutionary implications. J Bacteriol 179(12):3899–913 Karlin S. (1998) Global dinucleotide signatures and analysis of genomic heterogeneity. Curr Opin Microbiol 1(5):598–610 Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16(2):111–120 Konstantinidis KT, Tiedje JM (2005) Towards a genome-based taxonomy for prokaryotes. J Bacteriol 187:6258–6264 Kunkee RE (1991) Some roles of malic-acid in the malolactic fermentation in wine-making. FEMS Microbiol Rev 88(1):55–72 Labarre C, Guzzo J, Cavin J-F, Divies C (1996) Cloning and characterization of the genes encoding the malolactic enzyme and the malate permease of Leuconostoc oenos. Appl Environ Microbiol 62(4):1274–1282 Lane D (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 115–175 Lucena BTL, dos Santos BM, Moreira JLS, Moreira APB, Nunes AC, Azevedo V, Miyoshi A, Thompson FL, de Morais Junior MA (2010) Diversity of lactic acid bacteria of the bioethanol process. BMC Microbiol 10:298 Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen ZT, Dewell SB, Du L, Fierro JM, Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Irzyk GP, Jando SC, Alenquer MLI, Jarvie TP, Jirage KB, Kim JB, Knight JR, Lanza JR, Leamon JH, Lefkowitz SM, Lei M, Li J, Lohman KL, Lu H, Makhijani VB, McDade KE, McKenna MP, Myers EW, Nickerson E, Nobile JR, Plant R, Puc BP, Ronan MT, Roth GT, Sarkis GJ, Simons JF, Simpson JW, Srinivasan M, Tartaro KR, Tomasz A, Vogt KA, Volkmer GA, Wang SH, Wang Y, Weiner MP, Yu PG, Begley RF, Rothberg JM (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437(7057):376–380 Meier-Kolthoff JP, Go¨ker M, Spro¨er C, Klenk HP (2013) When should a DDH experiment be mandatory in microbial taxonomy? Arch Microbiol 195(6):413–418. doi:10.1007/ s00203-013-0888-4 Moreira JO, Mota R, Horta M, Teixeira S, Neumann E, Nicoli J, ˚ (2005) Identification to the species level of Nunes A
1267 Lactobacillus isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling. BMC Microbiol 5(1):15 Narendranath NSB (2009) Bacterial contamination and control in ethanol production. In: WM I (ed) The alcohol textbook, vol 4. 5 edn. Nottingham University Press, Nottingham, pp 481–490 Naser SM, Dawyndt P, Hoste B, Gevers D, Vandemeulebroecke K, Cleenwerck I, Vancanneyt M, Swings J (2007) Identification of lactobacilli by pheS and rpoA gene sequence analyses. Int J Syst Evol Microbiol 57(Pt 12):2777–2789 Nei M, Kumar S (2000) Molecular evolution and phylogenetics. Oxford University Press, New York Nielsen JC, Prahl C, LonvaudFunel A (1996) Malolactic fermentation in wine by direct inoculation with freeze-dried Leuconostoc oenos cultures. Am J Enol Vitic 47(1):42–48 Notredame C, Higgins DG, Heringa J (2000) T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302(1):205–217 Qin QL, Xie BB, Zhang XY, Chen XL, Zhou BC, Zhou J, Zhang YZ (2014) A proposed genus boundary for the prokaryotes based on genomic insights. J bacteriol 196(12):2210–2215 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425 Schwan RF, Mendonca AT, da Silva JJ, Rodrigues V, Wheals AE (2001) Microbiology and physiology of Cachaca (Aguardente) fermentations. Antonie Van Leeuwenhoek 79(1):89–96 Skinner KA, Leathers TD (2004) Bacterial contaminants of fuel ethanol production. J Ind Microbiol Biotechnol 31(9):401–408. doi:10.1007/s10295-004-0159-0 Stackebrandt E, Goebel B (1994) Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Evol Microbiol 44:846–849 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739 Thompson CC, Chimetto L, Edwards RA, Swings J, Stackebrandt E, Thompson FL (2013a) a Microbial genomic taxonomy. BMC Genom 14(1):913 Thompson CC, Silva GG, Vieira NM, Edwards R, Vicente ACP, Thompson FL (2013b) b Genomic Taxonomy of the Genus Prochlorococcus. Microb Ecol 66(4):752–762 Vandamme P, Pot B, Gillis M, De Vos P, Kersters K, Swings J (1996) Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 60:407–438 Versalovic J, Schneider M, de Bruijn FJ, Lupski JR (1994) Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol Cell Biol 5:25–40 Waterhouse AM, Procter JB, Martin DM, Clamp Ml, Barton GJ (2009) Jalview version 2, a multiple sequence alignment editor and analysis workbench. Bioinformatics 25(9):1189–1191 Yarza P, Yilmaz P, Pruesse E, Glo¨ckner FO, Ludwig W, Schleifer KH, Rossello´-Mo´ra R (2014) Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol 12(9):635–645
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